Fungal Allergy and Pathogenicity
Chemical Immunology
Vol. 81
Series Editors
Luciano Adorini, Milan
Ken-ichi Arai, Tokyo
Claudia Berek, Berlin
Anne-Marie Schmitt-Verhulst, Marseille
Basel · Freiburg · Paris · London · New York ·
New Delhi · Bangkok · Singapore · Tokyo · Sydney
Fungal Allergy and
Pathogenicity
Volume Editors
Michael Breitenbach, Salzburg
Reto Crameri, Davos
Samuel B. Lehrer, New Orleans, La.
48 figures, 11 in color and 22 tables, 2002
Basel · Freiburg · Paris · London · New York ·
New Delhi · Bangkok · Singapore · Tokyo · Sydney
Chemical Immunology
Formerly published as ‘Progress in Allergy’ (Founded 1939)
Edited by Paul Kallos 1939–1988, Byron H. Waksman 1962–2002
Michael Breitenbach
Professor, Department of Genetics and General Biology, University of Salzburg,
Salzburg
Reto Crameri
Professor, Swiss Institute of Allergy and Asthma Research (SIAF), Davos
Samuel B. Lehrer
Professor, Clinical Immunology and Allergy, Tulane University School of Medicine,
New Orleans, LA
Bibliographic Indices. This publication is listed in bibliographic services, including Current Contents®
and Index Medicus.
Drug Dosage. The authors and the publisher have exerted every effort to ensure that drug selection and
dosage set forth in this text are in accord with current recommendations and practice at the time of
publication. However, in view of ongoing research, changes in government regulations, and the constant flow
of information relating to drug therapy and drug reactions, the reader is urged to check the package insert
for each drug for any change in indications and dosage and for added warnings and precautions. This is
particularly important when the recommended agent is a new and/or infrequently employed drug.
All rights reserved. No part of this publication may be translated into other languages, reproduced or
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© Copyright 2002 by S. Karger AG, P.O. Box, CH–4009 Basel (Switzerland)
www.karger.com
Printed in Switzerland on acid-free paper by Reinhardt Druck, Basel
ISSN 1015–0145
ISBN 3–8055–7391–X
Contents
XII Foreword
M. Breitenbach, Salzburg; R. Crameri, Davos; S.B. Lehrer, New Orleans, La.
1 Introduction
A.M. Chiu, J.N. Fink, Milwaukee, Wisc.
3 References
5 Impact of Current Genome Projects on the Study of Pathogenic and
Allergenic Fungi
M. Breitenbach, Salzburg; R. Crameri, Davos; S.B. Lehrer, New Orleans, La.
6
7
7
8
8
Candida albicans Genome Project
Cryptococcus neoformans Genome Project
Aspergillus fumigatus Genome Project
The Postgenomic Era
References
10 Fungal Aerobiology: Exposure and Measurement
E. Levetin, W.E. Horner, Atlanta, Ga.
11
13
15
19
22
24
25
Sampling Equipment
Analysis
Patterns of Variation
Measurement Problems
Clinical Implications
Conclusions
References
28 Allergy to Basidiomycetes
A. Helbling, K.A. Brander, Bern; W.E. Horner, Atlanta, Ga.;
S.B. Lehrer, New Orleans, La.
28
30
31
31
32
32
33
33
33
34
35
35
35
36
37
37
38
39
40
40
41
44
44
44
Prevalence of Airborne Basidiospores Including Indoor Exposure
Prevalence of Sensitization
Clinical Aspects
Respiratory Allergy
Bronchial and Nasal Challenges
Atopic Eczema
Contact Dermatitis
Food Allergy
Invasive Mycosis
Source Materials
Basidiomycetes as Allergens
B. edulis (cèpe)
Calvatia spp. (Puffballs)
Coprinus spp. (Inky Cap)
Ganoderma spp.
Pleurotus spp. (Oyster Mushroom)
Psilocybe spp.
Cross-Reactivity
Molecular Biological Approaches to Basidiomycete Allergens
Screening of C. comatus Phage Display Library – 7 Putative Allergens
Cop c 1, the First C. comatus Allergen
Cop c 2
Conclusions
References
48 The Allergens of Cladosporium herbarum and Alternaria alternata
M. Breitenbach, B. Simon-Nobbe, Salzburg
49
51
54
54
54
57
59
59
61
62
63
Problems with Reproducibility of Mold Extracts and the Study of Mold Allergens
Experience with Specific Immunotherapy in the Treatment of Mold Allergies
Importance of Molecular Cloning Techniques
Cloning, Analysis, Production and Clinical Testing of the
Allergens of Cladosporium and Alternaria
Cloning Methods
The Major Allergen of A. alternata, Alt a1
The Major Allergen, Cla h1
Enolase, an Important Allergen in C. herbarum and A. alternata,
Exhibits Cross-Reactive Properties
Epitope Mapping of C. herbarum Enolase
Other Allergens
A. alternata
Contents
VI
63
C. herbarum
66
Production of Highly Purified Alt a1 and A. alternata Enolase for Clinical Use
66
Alt a1
66
A. alternata Enolase
67
Clinical Study with Recombinant A. alternata Allergens
68 Discussion and Outlook
68 Acknowledgments
69 References
73 Molecular Cloning of Aspergillus fumigatus Allergens and Their Role
in Allergic Bronchopulmonary Aspergillosis
R. Crameri, Davos
74
75
76
77
82
83
86
87
87
88
Host Defense Mechanisms and A. fumigatus-Related Diseases
A. fumigatus-Related Diseases
Diagnosis and Epidemiology of Allergic Bronchopulmonary Aspergillosis
Cloning and Molecular Characteristics of A. fumigatus Allergens
Clinical and Diagnostic Evaluation of Recombinant A. fumigatus Allergens
The Role of A. fumigatus Allergens in Allergic Bronchopulmonary
Aspergillosis
Utilizing the Allergenic Repertoire of A. fumigatus Identified with Advanced
Technologies
Conclusions
Acknowledgments
References
94 Defense Mechanisms of the Airways against Aspergillus fumigatus:
Role in Invasive Aspergillosis
H.F. Kauffman, J.F.C. Tomee, Groningen
95 Overview of Manifestations of Aspergillosis
95
Nonpathogenic Saprophytic Colonization
96
Aspergilloma
96
Hypersensitivity-Induced Aspergillosis
96
Aspergillus Asthma
97
Allergic Bronchopulmonary Aspergillosis
98
Hypersensitivity Pneumonitis
99
Invasive Pulmonary Aspergillosis
99 Host defense against Aspergillus
99
Innate Defense Strategies of Airways against Fungi
105
The Adaptive Immunological Response
108 Effect of Immunosuppressing Agents on the Innate and Adaptive Defense
Mechanisms
109 Concluding Remarks
110 References
Contents
VII
114 Secreted Proteinases and Other Virulence Mechanisms of
Candida albicans
M. Monod, Lausanne; M. Borg-von Zepelin, Göttingen
115 Site-Directed Mutagenesis and Genomics to Investigate
Virulence Factors
116 Dimorphism
116 Switching System of C. albicans
118 Adherence
119 Secreted Hydrolases
119
Phospholipases
120
Secreted Aspartic Proteinases
121
Aspartic Proteinases in the Adherence Process
123
Aspartic Proteinases in Deep-Seated Candidiasis
124 Acknowledgment
124 References
129 Cutaneous Mycology
T. Hawranek, Salzburg
130 Pathogenesis of Cutaneous Fungal Infection
132 Clinical Mycology
132
Superficial Mycoses
132
Pityriasis versicolor
133
White Piedra
133
Black Piedra
134
Tinea nigra
134
Cutaneous Mycoses
134
Dermatophytosis (Ringworm, Tinea)
134
Special Clinical Patterns
134
Tinea corporis
138
Tinea inguinalis (Tinea cruris)
138
Tinea capitis
141
Tinea barbae
141
Tinea pedis (‘Athlete’s Foot’)
142
Tinea manuum
142
Onychomycosis
143
Skin and Nail Infections by Molds
143
Scytalidium Species
144
Onychomycosis Caused by Scopulariopsis brevicaulis
144
Onychomycosis Caused by Other Molds
144
Tinea incognito
144
‘Superficial’ Candidiasis
145
Oral Candidiasis (Thrush)
146
Pseudomembranous Candidiasis
Contents
VIII
146
146
146
146
146
147
147
147
148
148
149
149
149
150
150
150
150
151
151
151
152
152
152
153
153
153
154
154
155
155
155
155
156
156
156
156
157
157
157
158
159
163
163
Erythematous (Atrophic) Candidiasis
Candida Leukoplakia (Chronic Plaque-Like or
Hyperplastic Candidiasis)
Genital Candidiasis
Candida Balanitis
Vaginal Candidiasis
Scrotal and Perianal Candidiasis
Candida Paronychia and Onychomycosis
Congenital Candidiasis
Chronic Mucocutaneous Candidiasis
Other Forms of Candidiasis
Subcutaneous Mycoses
Sporotrichosis
Chromoblastomycosis
Mycetoma (Madura Foot)
Subcutaneous Zygomycosis
Entomophthoromycosis
Localized Mucormycosis
Lobomycosis
Rhinosporidiosis
Systemic (Deep) Mycoses Caused by Pathogenic Fungi
Coccidioidomycosis
Paracoccidioidomycosis
Histoplasmosis
Blastomycosis
Systemic (Deep) Mycoses Caused by Opportunistic Fungi
Candidiasis
Cryptococcosis
Systemic Zygomycosis
Aspergillosis
Pseudallescheriasis
Rare Mycoses
Hyphomycosis
Pheohyphomycosis
Hyalohyphomycosis
Protothecosis
Penicillium marneffei Infection
Laboratory Diagnosis
Collection of Material
Direct Examination (Potassium Hydroxide Test)
Culture and Additional Tests
Histopathology, Serology, Identification of Yeasts and Molds
Principles of Therapy
References
Contents
IX
167 Toxins of Filamentous Fungi
D. Bhatnagar, J. Yu, K.C. Ehrlich, New Orleans, La.
168
170
170
171
173
173
176
176
177
179
179
181
181
182
182
183
183
184
187
190
191
191
192
193
193
194
196
196
197
Mycotoxicosis
Mycotoxicology
Natural Occurrence of Mycotoxins
History of Mycotoxins
Classification of Mycotoxins
Economic Impact of Mycotoxins
Detection and Screening of Mycotoxins
Selected Mycotoxins
Aflatoxins
Toxic Polyketides Other Than Aflatoxins
Ochratoxins
Cyclopiazonic Acid
Patulin
Penicillic Acid
Citrinin
Sterigmatocystin
Zearalenone
Fumonisins
Trichothecenes
Alternaria Toxins
Neurotropic Mycotoxins
Ergot Alkaloids and Related Toxins
Other Neurotropic Mycotoxins
Management of Mycotoxin Contamination
Preharvest Control
Postharvest Control
Dietary Consideration
Summary
References
207 Phylogeny and Systematics of the Fungi with Special
Reference to the Ascomycota and Basidiomycota
H. Prillinger, K. Lopandic, W. Schweigkofler, Wien; R. Deak, Budapest;
H.J.M. Aarts, Wageningen-UR; R. Bauer, Tübingen, K. Sterflinger, G.F. Kraus, Wien,
A. Maraz, Budapest
210
212
215
224
226
228
231
The Kingdom Mycobionta (Eumycota) or True Fungi
Morphological Differentiation within the Kingdom Mycobionta
Sexual Differentiation within the Kingdom Mycobionta
Phylogenetic Relationships among the Chytridiomycota and Zygomycota
Phylogenetic Relationships among the Ascomycota and Their Anamorphs
Hemiascomycetes
Protomycetes
Contents
X
233
234
235
237
239
242
242
243
244
244
246
248
249
249
253
255
255
256
257
258
258
259
259
260
261
263
264
264
265
267
268
268
269
270
272
275
275
294
Euascomycetes
Chaetothyriales
Eurotiales
Onygenales
Hypocreales
Ophiostomatales
Phyllachorales
Sordariales
Microascales
Dothideales
Pleosporales
Leotiales
Pezizales
Phylogenetic Relationships of the Basidiomycota and Their Anamorphs
Urediniomycetes
Cystobasidiales
Microbotryales
Uredinales
Ustilaginomycetes
Malasseziales
Georgefischerales
Microstromatales
Ustilaginales
Hymenomycetes
Tremellales
Cantharellales
Gomphales
Thelephorales
Polyporales
Hymenochaetales
Russulales
Boletales
Schizophyllales
Agaricales
Genotypic Identification
Acknowledgments
References
Notes added in proof
296 Glossary
302 Author Index
303 Subject Index
Contents
XI
Foreword
Fungal organisms (yeasts and molds) are an increasing public health
problem (Chiu and Fink) worldwide for several reasons. After transplantation
surgery immunosuppressed patients frequently suffer from fungal infections
which can become fatal; the AIDS pandemic, which is still on the increase, also
leads to fungal infections that are difficult to treat and there is an increase in
real and perceived allergic diseases which, in some developed countries,
involve 20–30% of the population; a proportion of these allergies is due to
inhaled fungal spores and other fungal material. Some of the older patients suffering from allergic or ‘nonallergic’ asthma are particularly prone to fungal
infections of the lung. Moreover, fungal toxins (Bhatnagar et al.) still represent
an important health problem both in humans and farm animals.
Exposure to fungal spores is ubiquitous and, therefore, of pivotal importance for the development of mycoses acquired via the respiratory tract. This
situation has also led to increased public awareness of the importance of indoor
air quality and to the emergence of aerobiology, which has established itself as
a major environmental academic discipline (Levetin and Horner).
Fungal infections have led to an increasing demand for antifungal drugs.
Compared to the well-known antibacterial antibiotics, these are generally less
than satisfactory, because it is more difficult to combat eukaryotic than procaryotic pathogens, and severe side effects are still frequent. It is expected that
fungal whole genome sequences (Breitenbach et al.), which are now being
determined, will be very helpful in devising new antifungal drugs. Fungal
genetics plays an increasing role in the study of clinically relevant fungi.
Genome sequences will also lead the way to the discovery of new virulence
factors which are important as drug targets. There are good reasons for hope,
but at present no antifungal drug developed on the basis of fungal genomics is
yet on the market.
Fungi are different from bacterial pathogens also because they are mostly
opportunistic, that is they are present all the time in healthy individuals (for
instance, on the skin), and only become dangerous in certain situations. A variety
XII
of pathological conditions, including impaired immune function, are believed to
cause host susceptibility to fungal infections. The major reason for the increase in
systemic mycoses is undoubtedly related to an increased number of patients with
congenital or acquired immunodeficiency. Therefore, it is important to study not
only the fungal pathogens but also the host factors that contribute to fungal infectivity (Kauffman and Tomee, and Monod and Borg-von Zepelin).
Until recently, the study of fungal allergy was still in its infancy. It is no
exaggeration to say that modern cDNA cloning techniques caused a major
breakthrough in this field. Before the advent of allergen cloning, it was difficult, for several reasons, to identify the relevant fungal allergens unequivocally.
Commercially available fungal extracts were for the most part not satisfactory
for a reliable diagnosis and were not authorized for specific immune therapy
(‘hyposensitization’) in many countries. This situation will improve with the
advent of well-defined pure recombinant fungal allergens (Helbling et al.,
Breitenbach and Simon-Nobbe, and Crameri).
Nearly all major systematic groups of fungi are now known to contain allergenic and/or pathogenic species (Prillinger et al.). Among the Ascomycota, the
new sequence-based phylogeny defines three large groups: the Hemiascomycetes,
the Protomycetes (Archiascomycetes) and the Euascomycetes. Presently there
is some debate on whether the Protomycetes are primitive Ascomycota
(Archiascomycetes) or a derived group of Ascomycota with similarities to the
Basidiomycota (Prillinger et al.). Until recently, some Ascomycota (Aspergillus,
Alternaria, Cladosporium, Penicillium, Candida and others) were called ‘fungi
imperfecti’ (Deuteromycota), but are now recognized as close relatives of ‘perfect’
Ascomycota based on molecular systematics.
The Hemiascomycete, Candida albicans, is a very important pathogen and
allergen, aspects of which are treated by Monod and Borg-von Zepelin. Among
the Euascomycetes, Trichophyton rubrum causes superficial skin infections
(Hawranek); Histoplasma capsulatum is an intracellular parasite of the monocyte/macrophage system occasionally causing fatal infections, and Aspergillus
fumigatus is a very important allergen and pathogen that is able to colonize
the human lungs. Some aspects of Aspergillus infections are the topic of the
chapters by Crameri, and Kauffman and Tomee. Cladosporium herbarum and
Alternaria alternata (Breitenbach and Simon-Nobbe) are Euascomycetes. They
are important causes worldwide of allergies. Pneumocystis carinii, an important pathogenic member of the Protomycetes, is the most common cause of lung
infection in AIDS patients.
Among the major groups of the Basidiomycota, only the Uredinomycetes
contain practically no known pathogens and/or allergens. Rhodotorula mucilaginosa and Rhodotorula glutinis have occasionally been isolated from patients.
Interestingly, Malassezia (previously called Pityrosporum) furfur, a human
Foreword
XIII
pathogen infecting the skin and hair follicles (Hawranek), is a member of the
Ustilaginomycetes, a large group of plant pathogens. The most highly developed
group of the Basidiomycota, the Hymenomycetes, contain a number of recently
recognized important allergy-causing fungi: Coprinus cinereus, Psilocybe
cubensis, and puffballs of the genus Calvatia, among others (Helbling et al.).
Another Hymenomycete, Cryptococcus neoformans, is an important human
pathogen causing the often fatal cryptococcosis of the lung and meningitis.
This volume clearly shows the importance of correct fungal systematics to
understand the clinically relevant fungi. For this reason, we have included an
extensive chapter on the new molecular fungal systematics. This chapter
(Prillinger et al.) should be consulted whenever questions appear as to the correct systematic nomenclature and the older synonyms of a fungal organism. We
are including a glossary because we feel that the highly specialized terminology, especially of clinical mycology and of systematic mycology, should be
explained for nonspecialists.
This book addresses not only clinicians who want to learn more about
clinically important fungi, but also allergologists, mycologists, biologists and
lawyers concerned with the increasing number of lawsuits because of fungal
spores in indoor air, which are claimed to be a major reason for ‘sick building
syndrome’.
We are very grateful to Thomas Nold and the members of the team of
Karger (Basel, Switzerland) for their excellent cooperation and patience during
the time of collecting and editing the chapters of this book. We are also very
grateful to the authors of this book who have spent many days checking and
rechecking every chapter, especially to Birgit Simon-Nobbe and to Hansjörg
Prillinger. Finally, we thank our families for their support and confidence in the
final success of this project.
M. Breitenbach, R. Crameri, S.B. Lehrer
Foreword
XIV
Breitenbach M, Crameri R, Lehrer SB (eds): Fungal Allergy and Pathogenicity.
Chem Immunol. Basel, Karger, 2002, vol 81, pp 1–4
Introduction
Asriani M. Chiu, Jordan N. Fink
Medical College of Wisconsin, Department of Pediatrics and Medicine
(Allergy and Immunology), Milwaukee, Wisc., USA
Allergy is a hypersensitive response of the immune system to inhaled or
ingested proteins, or allergens [1]. Since only some individuals will develop
allergy even though many are exposed, a genetic predisposition along with
environmental exposure is necessary for the atopic state to occur. The allergic
response can involve different organ systems, and symptoms can include nasal
congestion, shortness of breath or skin rash, resulting in rhinoconjunctivitis,
asthma, urticaria and angioedema, or even anaphylaxis and death. Over 20% of
the population has one form of allergic disease or another, and a number of
allergens associated with different forms of allergy have been reported all over
the world [2]. The concentration of environmental allergens depends on many
variables, including climate, vegetation, and air quality. The outdoor allergens
are predominantly plant pollens and fungal spores. The indoor allergens include
proteins from dust mite, cockroach, animal dander, and also fungal spores.
Indoor allergen concentrations are affected by humidity, ventilation, and the
presence or absence of pets, carpets, or houseplants. Avoidance or reduction of
exposure may be important factors in decreasing the development of allergy to
these indoor and outdoor allergens.
Fungi are eukaryotic, unicellular (yeasts), dimorphic or filamentous, and
usually spore-bearing organisms. A number of biochemical and genetic markers
clearly distinguish them from plants and algae. They lack chlorophyll, and
dendrograms derived from rDNA sequence comparisons group them outside the
plants and animals in a kingdom ‘fungi’. Existing as parasites, saprobes or symbionts they depend on outside sources for nourishment. There are currently over
80,000 described species of fungi, including the yeasts and the molds. The yeasts
typically are single-celled-organisms and multiply asexually by budding. The
yeasts can also reproduce sexually by the formation of ascospores or
basidiospores. Molds are comprised of branching tubular (siphonal or trichal)
hyphae, and collectively, all the hyphae of one fungal organism are called the
mycelium. Certain pathogenic fungi are dimorphic and able to exist in nature as
hyphae and convert to either yeasts or spherules after they infected the body of
a human or animal. Under special environmental conditions, some yeasts can
form pseudohyphae as well as blastospores, arthrospores, and chlamydospores,
depending on the genus and species of yeast. Mycotic diseases include infections –
cutaneous, localized, or invasive; mycotoxicoses, which result from the ingestion
of the toxic metabolites of fungi and allergy, which will be the main topic of this
monograph. Fungi can also produce potent carcinogens. The airborne spores of
fungi are usually considered to be etiologic agents of allergic rhinitis and asthma.
The prevalence of respiratory allergy to fungi is estimated to be 20–30% among
atopic individuals, and 6% of the general population in hot and humid areas of the
world. These numbers can be somewhat smaller in other climatic zones.
The major allergic manifestations induced by fungi include asthma [3],
rhinitis, allergic sinusitis, allergic bronchopulmonary mycoses, and hypersensitivity pneumonitis. Allergy to Alternaria is a risk factor for death in patients with
asthma. Allergic bronchopulmonary aspergillosis is a pulmonary disease characterized by immunologic reactions to antigens of Aspergillus fumigatus, and
diagnostic criteria include a history of asthma, elevated total serum IgE, immediate cutaneous reactivity to Aspergillus, elevated serum IgE and/or IgG antibodies to A. fumigatus, and central bronchiectasis. Hypersensitivity pneumonitis
is a syndrome with a spectrum of clinical features related to the immunologic
pulmonary inflammation after the inhalation of a variety of organic dusts, and
can present in an acute, subacute, or chronic form depending on the frequency
and intensity of the exposure. Other disorders that have been attributed to molds
include sick building syndrome [4], and pulmonary hemorrhage and hemosiderosis related to Stachybotrys atra [5]. Sick building syndrome is a term used
to describe a constellation of symptoms that include rhinitis, difficulty breathing, headaches, flu-like symptoms, and watery eyes, and are associated with
poor indoor air quality. S. atra exposure has long been known to cause respiratory symptoms in animals, due to its mycotoxic effects. In 1993–1994, there was
a cluster of 10 cases of pulmonary hemorrhage and hemosiderosis in Cleveland,
Ohio, USA, that was possibly related to exposure to toxigenic S. atra.
Among the groups of fungi, there are three that contain all the species that are
important causes of allergic disease. These are the Zygomycota, which include the
bread molds (e.g. Rhizopus and Mucor); the Ascomycota, many of which are soil
fungi (e.g. Penicillium and Aspergillus) and some that are leaf surface fungi or
endophytes (e.g. Alternaria and Cladosporium); and the third group, the
Basidiomycota, which include the mushrooms, puffballs, plant rusts and smuts.
There have been problems obtaining adequate quantities of fungal extracts
that are qualitatively and quantitatively reproducible in terms of allergen content.
Chiu/Fink
2
There are different considerations when selecting source materials for a fungal
extract, and these include whether fungi should be grown in liquid culture
medium, or harvested from the wild, and whether spores or hyphae should be
used. There also is variability in allergen content and in the amount of allergen
produced from fungal sources, even if grown in the laboratory environment. The
allergenicity of the extract or antigen fraction can be evaluated by skin prick or
intradermal testing of allergic subjects. Specific in vitro tests, radioallergosorbent test or enzyme-linked immunosorbent assay, may also be used. In addition
to standardization difficulties with fungal extracts, it is also often difficult to
identify mold-allergic patients [6]. This may be due to the sizable number of
mold species, and the difficulty in deciding which are clinically relevant and
important, as well as the fact that no allergy-testing materials may be available
for evaluation. Furthermore, cross-reactivity among fungi may make the interpretation of the skin tests more difficult. In the last several years there has been
progress in the area of characterization of fungal allergens. It is now possible to
grow allergenic fungi in synthetically defined media with less variability. The
common fungal allergens have been characterized, and can be cloned and
expressed by molecular biologic techniques [7]. Based on the primary structure
of the fungal allergens, it appears that the majority of the identified allergens are
proteins associated with essential basic fungal metabolism, such as protein
synthesis or glycolysis. Recombinant allergens currently available from fungi
include allergens from Alternaria alternata, including Alt a 1, Cladosporium
herbarum, including Cla h 6, and A. fumigatus, including Asp f 1. T cell and
B cell epitope mapping has been studied with Asp f 1 from A. fumigatus, and
B cell epitope mapping with Asp f 2 and Cla h 6. The increasing knowledge of
the three-dimensional structures and the T cell and B cell epitopes of fungal
allergens may provide information to help develop newer therapeutic strategies
including immunotherapy with synthetic peptides, recombinant mutant allergen
isoforms, or other modified allergens.
As more research is done in the area of molecular biology of fungal
allergens, we will hopefully begin to have better quality and more standardized
allergen extracts, and thus have better ways to diagnose and treat patients. This
text will provide a comprehensive overview of the current knowledge of fungal
allergy and its immunopathogenicity.
References
1
2
Blumenthal MN, Rosenberg A: Definition of an Allergen (Immunobiology); in Lockey RF,
Bukantz SC (eds): Allergen and Allergen Immunotherapy. New York, Dekker 1999, pp 39–51.
Wüthrich B: Epidemiology of allergic disease: Are they really on the increase? Int Arch Allergy
Appl Immun 1989;90:3–10.
Introduction
3
3
4
5
6
7
O’Hollaren MT, Yunginger JW, Offord KP, Somers MJ, O’Connell EJ, Ballard DJ, Sachs MI:
Exposure to an aeroallergen as a possible precipitating factor in respiratory arrest in young
patients with asthma. N Engl J Med 1991;324:359–363.
Cooley JD, Wong WC, Jumper CA, Straus DC: Correlation between the presence of certain fungi
and sick building syndrome. Occup Environ Med 1998;55:579 –584.
Etzel RA, Montana E, Sorenson WG, Kullman GJ, Allan TM, Dearborn DG, Olson DR, Jarvis
BB, Miller JD: Acute pulmonary hemorrhage in infants associated with exposure to Stachybotrys
atra and other fungi. Arch Pediatr Adolesc Med 1998;152:757–762.
Karlsson-Borga A, Jonsson P, Rolfsen W: Specific IgE antibodies to 16 widespread mold genera
in patients with suspected mold allergy. Ann Allergy 1989;63:521–526.
Kurup VP, et al: Molecular biology and immunology of fungal allergens. Indian J Clin Biochem
2000;12(suppl):31–42.
Prof. Jordan N. Fink, MD, Medical College of Wisconsin,
Department of Pediatrics and Medicine, Allergy and Immunology,
Milwaukee, WI 53226 (USA)
Tel. ⫹1 414 266 6840, Fax ⫹1 414 266 6437, E-Mail jfink@chw.org
Chiu/Fink
4
Breitenbach M, Crameri R, Lehrer SB (eds): Fungal Allergy and Pathogenicity.
Chem Immunol. Basel, Karger, 2002, vol 81, pp 5–9
Impact of Current Genome Projects
on the Study of Pathogenic and
Allergenic Fungi
M. Breitenbach a, R. Crameri b, S.B. Lehrer c
a
b
c
Department of Genetics and General Biology, University of Salzburg,
Salzburg, Austria;
Swiss Institute of Allergy and Asthma Research, Davos, Switzerland, and
Department of Medicine, Tulane University Medical Center, New Orleans, La., USA
The present era of biomedical research is characterized by a predominance
of genetic approaches and models to investigate the basis and causes of disease.
Although this tendency is not without dangers (such as underestimating lifestyle and environmental influences on the development of disease or underestimating the importance of biochemical thinking), application of the genetic
paradigm has undeniably been highly successful and will remain so for decades
to come. The chapters in this book testify to this fact.
While modern molecular genetics has been at the center of the biomedical
revolution in research for about 25 years now, its latest development, genomics,
has only just started to influence medical research. It is therefore appropriate to
briefly describe the potential of studying whole genome sequences as a new
tool in biomedical research.
In particular, the ongoing Candida albicans, Aspergillus fumigatus and
Cryptococcus neoformans genome-sequencing projects are very useful examples to illustrate the importance of whole genome sequences. Other genomesequencing projects of pathogenic fungi are also under way or will soon be
started (for instance, Pneumocystis carinii).
Generally speaking, the progress of sequencing technology and of bioinformatics (data management, gene prediction algorithms, functional analysis
in silico) has made feasible the sequencing and analysis of many genomes which
have approximately the size of the genome of Saccharomyces cerevisiae (12.5 millions of base pairs), the first eukaryotic genome to be sequenced completely [1].
The genome sequence of S. cerevisiae, a prototypic and nonpathogenic fungal
organism is therefore of utmost importance for systematic comparison with the
fungal pathogens. Most known fungal genomes are in the maximal range of 15–25
million base pairs and many sequencing projects are under way [2].
Why is a whole genome sequence a useful tool for medical research? What
are the immediate and long-term benefits for our understanding and for the
diagnosis and treatment of disease?
Parasites and pathogens generally have a specialized genetic outfit to
successfully interact with the host. Frequently those genes are not housekeeping genes but specialized genes which (1) are not found in closely related freeliving microorganisms and (2) are transcriptionally induced during interaction
with the host. Knowing these genes is of great importance for understanding the
pathomechanisms, for finding and identifying new antibiotic targets and for the
development of new drugs [3]. The methods used to achieve these goals include
annotation of all genes, genome-wide transcript analysis, comparisons of
closely related pathogenic vs. nonpathogenic organisms (like C. albicans vs.
S. cerevisiae), the systematic deletion and phenotypic analysis of candidate
genes coding for new drug targets, and testing of deletion mutants in an animal
model of the disease. The development of new antifungal drugs is extremely
important as the major classes of currently used antifungal drugs suffer from
numerous problems like development of resistance and/or severe side effects.
The morbidity and mortality rates associated with infections caused by fungal
pathogens are high, and prevention, diagnosis and treatment of these infections
remain quite difficult. At the same time, typical fungal infections are rapidly
increasing due to an increasing number of immune-suppressed patients after
organ transplantation, after HIV infection and as a consequence of certain cancers of the immune system (leukemias). C. albicans is presently considered the
most important fungal pathogen in terms of frequency of infection and total
cost of treatment.
Candida albicans Genome Project
‘Shotgun sequencing of the diploid C. albicans genome … is complete
with the sequencing of 10.4 haploid genome equivalents, which is sufficient
to ensure identification of all of the genes in this organism’ [4]. A complete
(or even partial) annotation of the Candida genes has not been published
at the time of writing. Current information about the ongoing project can be
found at two websites: http://www-sequence.stanford.edu/group/candida and
http://alces.med.umn.edu/Candida.html. The genome sequence has so far
revealed a very close relationship between C. albicans and S. cerevisiae including the discovery of the Candida mating type locus [5] and of many of the
Breitenbach/Crameri/Lehrer
6
genes needed for meiosis and recombination as indicated by close sequence
similarity to the respective S. cerevisiae genes [4]. Low-frequency mating has
been achieved experimentally [6, 7], but meiosis has so far not been observed.
About 80% of the genes found are closely related in sequence to S. cerevisiae,
but the remaining genes have no counterpart in the well-known S. cerevisiae
genome. It can be assumed that these 20% of the genome have to do with the
parasitic lifestyle of C. albicans and are a rich ground for the discovery of new
drug targets and of genes coding for new virulence factors. One aspect of the
virulence of C. albicans that became very clear in recent years is the formation
of hyphae of this dimorphic yeast [8]. The genome project showed that formation of hyphae is under the control of a hierarchy of signaling systems and transcription factors, homologues of which are well known from S. cerevisiae
studies, in particular the MAP-kinase and the cAMP/PKA pathways [Alistair
J.P. Brown, pers. commun.] [9]. The genome of Candida tropicalis, a human
pathogen that is closely related to C. albicans, has been partially sequenced, and
about 1,000 new genes have been found [10].
Cryptococcus neoformans Genome Project
Current information about the ongoing project [11] can be found at the
website http://www-sequence.stanford.edu/group/C.neoformans/index.html.
At the time of writing (December 2001) sequencing of the C. neoformans
serotype D genome is ‘nearing completion’ [12]. A detailed bioinformatic analysis of the C. neoformans genome sequence is not yet available. C. neoformans
is a member of the Basidiomycota and the only known pathogen from this group.
Still, the pathogen-specific genes, for instance genes responsible for the formation of the protective coat that is of prime importance for virulence [12], have no
counterpart in closely related basidiomycetes. This situation is very similar to
the one encountered in C. albicans and its close relative, S. cerevisiae. Formation
of the protective coat is controlled by the cAMP-dependent protein kinase,
PKA [13].
Aspergillus fumigatus Genome Project
Plans for sequencing the complete genome of this important ascomycete
human pathogen were announced in early 2000 [14]. The sequencing project is
a joint effort of the University of Manchester, the Sanger Centre and the Institut
Pasteur. Details about the current progress of this project can be found at their
website http://www.aspergillus.man.ac.uk. A parallel project is under way at the
Genome Projects of Pathogenic and Allergenic Fungi
7
Institute of Genomic Research (TIGR) in the USA (http://www.tigr.org).
Comparison with the genetically well-characterized and nonpathogenic species
Aspergillus nidulans will certainly help in the identification of virulence factors
and drug targets.
The Postgenomic Era
The genome sequence of S. cerevisiae has been completed, providing us
with a powerful tool for comparisons between pathogenic and non-pathogenic
fungi. Sequence projects involving pathogenic fungi have been nearly completed or started recently. Embedded within these genomes are the sequences of
both, essential genes governing the metabolic pathways and survival of the
organisms and genes involved in pathogenesis. One of the central goals of the
postgenome era will be to relate individual genes to specific functions and particularly to identify genes responsible for the pathogenic behavior of certain
fungal organisms. This approach will lead to the identification of target genes
for the development of new generations of antifungal drugs. However, we
should keep in mind that most fungi are opportunistic pathogens and therefore
depend on specific underlying conditions of the human host to be able to
become pathogens. In spite of the progress achieved in fungal genomics, we
should not forget that fungal infections have been shown to be influenced by
host-specific conditions and that many drug responses of single individuals are
influenced by inherited differences in our genes. Clearly, this means that human
genomics and genetics also have a strong impact on the study of fungal pathogenicity. To win the fight against fungal pathogens both, genome projects and
an in-depth investigation of host-pathogen interactions will be required.
References
1
2
3
4
Goffeau A, Barrell BG, Bussey H, Davis RW, Dujon B, Feldmann H, Galibert F, Hoheisel JD, Jacq
C, Johnston M, Louis EJ, Mewes HW, Murakami Y, Philippsen P, Tettelin H, Oliver SG: Life with
6000 genes. Science 1996;274:546–567.
Souciet J, Aigle M, Artiguenave F, Blandin G, Bolotin-Fukuhara M, Bon E, Brottier P, Casaregola S,
de Montigny J, Dujon B, Durrens P, Gaillardin C, Lepingle A, Llorente B, Malpertuy A, Neuveglise
C, Ozier-Kalogeropoulos O, Potier S, Saurin W, Tekaia F, Toffano-Nioche C, Wesolowski-Louvel M,
Wincker P, Weissenbach J: Genomic exploration of the hemiascomycetous yeasts. 1. A set of yeast
species for molecular evolution studies. FEBS Lett 2000;487:3–12.
Black T, Hare R: Will genomics revolutionize antimicrobial drug discovery? Curr Opin Microbiol
2000;3:522–527.
Tzung KW, Williams RM, Scherer S, Federspiel N, Jones T, Hansen N, Bivolarevic V, Huizar L,
Komp C, Surzycki R, Tamse R, Davis RW, Agabian N: Genomic evidence for a complete sexual
cycle in Candida albicans. Proc Natl Acad Sci USA 2001;98:3249–3253.
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8
5
6
7
8
9
10
11
12
13
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Hull CM, Johnson AD: Identification of a mating type-like locus in the asexual pathogenic yeast
Candida albicans. Science 1999;285:1271–1275.
Hull CM, Raisner RM, Johnson AD: Evidence for mating of the ‘asexual’ yeast Candida albicans
in a mammalian host. Science 2000;289:307–310.
Magee BB, Magee PT: Induction of mating in candida albicans by construction of MTLa and
MTLalpha strains. Science 2000;289:310–313.
Madhani HD, Fink GR: The control of filamentous differentiation and virulence in fungi. Trends
Cell Biol 1998;8:348–353.
Whiteway M: Transcriptional control of cell type and morphogenesis in Candida albicans. Curr
Opin Microbiol 2000;3:582–588.
Blandin G, Ozier-Kalogeropoulos O, Wincker P, Artiguenave F, Dujon B: Genomic exploration of
the hemiascomycetous yeasts: 16. Candida tropicalis. FEBS Lett 2000;487:91–94.
Heitman J, Casadevall A, Lodge JK, Perfect JR: The Cryptococcus neoformans genome sequencing project. Mycopathologia 1999;148:1–7.
D’Souza CA, Heitman J: Dismantling the Cryptococcus coat. Trends Microbiol 2001;9:112–113.
D’Souza CA, Alspaugh JA, Yue C, Harashima T, Cox GM, Perfect JR, Heitman J: Cyclic AMPdependent protein kinase controls virulence of the fungal pathogen Cryptococcus neoformans.
Mol Cell Biol 2001;21:3179–3191.
Brookman JL, Denning DW: Molecular genetics in Aspergillus fumigatus. Curr Opin Microbiol
2000;3:468–474.
Dr. M. Breitenbach
University of Salzburg, Department of Genetics and General Biology,
Hellbrunnerstrasse 34, A–5020 Salzburg (Austria)
Tel. ⫹43 662 8044 5786, Fax ⫹43 662 8044 144, E-Mail michael.breitenbach@sbg.ac.at
Genome Projects of Pathogenic and Allergenic Fungi
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Breitenbach M, Crameri R, Lehrer SB (eds): Fungal Allergy and Pathogenicity.
Chem Immunol. Basel, Karger, 2002, vol 81, pp 10–27
Fungal Aerobiology: Exposure
and Measurement
Estelle Levetin a, W. Elliott Horner b
a
b
Faculty of Biological Science, University of Tulsa, Tulsa, Okla., and
Air Quality Sciences, Inc., Atlanta, Ga., USA
The earliest well-known aerobiology experiment was likely Pasteur’s
demonstration of the causes of putrefaction. Pasteur was attacking the paradigm
of spontaneous generation, but also established that airborne dust included
germs – or bioaerosols. Even with the relatively simple equipment available in
the 19th century, some basic principles of aerobiology were established. One of
these was the value of volumetric sampling. As early as 1900, at least one
microbiology textbook stated that settle plates were unreliable indicators of
airborne microbial concentrations because the volume of air sampled was
unknown [1].
Perhaps surprisingly, there were several volumetric air samplers available
even then. These were cumbersome, but useful. Known sample volumes were
collected in one sampler by siphoning a liquid from one container to another.
The displacement of the siphoned liquid drew air into the sampler and the volume of air sampled equaled the volume of the liquid. Quantitative data were
produced without batteries, power sources or calibrations.
Several methods were also available to capture airborne particles. An agarcoated cylinder was the collector in one sampler. After being filled with air and
capped, the spores were allowed to settle onto the inner walls of the cylinder.
Packed cylinders of sterile sand were also used as filters that could then be
plated onto culture media.
Although inventive, these samplers were cumbersome, as a result extensive
sampling was impractical. The renaissance of aerobiology was essentially the
mid 20th century when samplers were developed that made repeat observations
feasible. The two chief samplers developed at this time were the Hirst spore trap
and the Andersen culture impactor [2, 3]. The half-century since their development has seen aerobiology greatly expand and support fields such as plant
pathology, industrial hygiene, palynology and allergy. Numerous other samplers have been developed and improvements on some of these original designs
are available [4]. The more important developments at this time however were
synthetic.
Gregory [5] was involved early on in the renaissance of aerobiology. He
contributed to and also first compiled the fundamental descriptive work of aerobiology and the basic science of the behavior of small particles in fluids such
as air. Later, Lacey’s work was fundamental to the early application of aerobiology sampling and the aerodynamics of biological particles to the problems of
human health associated with exposure to bioaerosols [6]. These are among the
giants upon whose shoulders current workers are standing.
Presently, emphasis is on the application of immunochemical, molecular,
and chemical analyses of air samples. Monoclonal antibodies have been developed for selected agents and these have been used to detect these agents in air
[7]. Gene probes and primers for specific agents have also been developed. In
addition, the improved sensitivity of some chemical analyses from biological
materials show promise of being able to detect biological agents of significance
to human health. Perhaps the most notable of these is endotoxin.
Sampling Equipment
Every exposed surface, either indoors or outdoors, likely is contaminated
with fungal spores from the air. Although the function of most airborne spores
is to colonize new surfaces (substrates), these spores are often remarkably
adapted to staying airborne, which means that effort is required to capture them.
More importantly, different spores are adapted to different ways of being
removed from the air. Spores of leaf surface fungi or foliar pathogens may be
adapted to interception by or impaction onto leaves under turbulent conditions.
Spores of litter decay fungi, on the other hand, may be adapted to settling onto
surfaces in quite still air. Air normally contains spores of both types of fungi,
often in addition to spores adapted to other dispersal mechanisms, such as
water splashes. Thus, capturing a representative sample of spores from air is a
challenge since these spore types differ in their ability to stay airborne, or
conversely differ in their ease of capture.
Passive sampling by collecting spores that settle onto a surface (settle
plate, or gravity plate) has two critical flaws. Unless precautions are taken to
assure perfectly still air over the surface, the volume of air that is sampled cannot be determined and the method is thus nonvolumetric. Volumetric sampling
is essential if a concentration of spores is to be determined, which is crucial for
rigorously comparing results from different samples. More importantly, passive
Fungal Aerobiology: Exposure and Measurement
11
Table 1. Impactor samplers with their advantages and weaknesses
Sampler
Advantage
Weakness
Rotorod
Relatively inefficient for spores
Air-O-Cell
Independent of wind orientation
Formerly was less expensive
No power requirements
Time discrimination
Ease of use
Self-contained fan
Inexpensive, ease of use
Andersen
SAS
Very efficient for spores
Portable
Tauber
Burkard 7 day
Personal Burkard
Not suitable for fungal spores
Expensive
Single grab sample
Variable (user) slide preparation
Single grab sample
Less efficient for small spores
Requires externally powered pump
Expensive
sampling cannot compensate for spores that are more difficult to recover
from air, and hence favors the recovery of spore types that are easier to recover.
This produces a bias in the type of fungi that are recovered, in addition to the
nonvolumetric nature of passive sampling.
Active sampling is any sampling that uses artificial force (not gravity) to
help collect spores from air. This usually means some type of fan or pump to
induce and control airflow through the sampler. If properly calibrated, this can
provide samples from a known volume of air (volumetric) that allows results to
be expressed as concentrations and therefore comparable with other results.
More importantly, an artificial force can largely compensate for differences
among spore types in ease of recovery (resistance to capture). This provides a far
more representative sample of the spore mix that is present in the air sampled.
The samplers most often used for aeroallergen monitoring and for indoor
air studies are impactors and filters, although there are samplers based on other
principles, such as impingers and electrostatic precipitators [4]. Filter samplers
have been used to collect spores as well as antigens from outdoor and indoor
air. Studies using filter samplers helped establish that pollen allergens are
not always associated with pollen grains. Starch grains released from pollen
and other pollen fragments have been shown to contain allergens [8]. Filter
samplers are also used in indoor allergen studies. Although not widely used,
impingers have several advantages. The sample is collected in a liquid, which
can protect spores for culture analysis. The impinger fluid can also be divided
and analyzed in different ways.
The true workhorses of aeroallergen samplers however, are all impactors
(table 1). The rotorod, the Burkard (Hirst-type) spore trap, the personal
Burkard, the Air-O-Cell, and the Andersen (several types) are all impactor
Levetin/Horner
12
samplers. The surface to agar system (SAS) sampler is a portable type of sieve
plate impactor sampler. Each of the samplers mentioned above has a limitation; each has an advantage and a favored use. The Andersen is a sampler for
culturable (sometimes called viable) mold spores, the others sample for particles (spores and/or pollen) without a need for culture. The principle that all
impactors use to capture particles is to sharply deflect an airstream so that particles in the airstream break free due to inertia and impact onto the sampling
surface.
In the rotorod sampler, the surface (sampling rod) is accelerated rapidly
through the air, which is deflected around the rod. With the other samplers
mentioned, a pump or fan is used to direct an airstream through a sharp 90° turn
in front of a sampling surface at a critical (short) distance from the air inlet of
the sampler. As the airstream is forced to turn sharply, any entrained particles
(pollen grains and mold spores) break free of the airstream and impact onto the
sampling surface. Tauber traps are nonvolumetric sedimentary samplers used
by scientists wishing to study the deposition of pollen over weeks, months, or
even years. Pollen collected by Tauber traps is considered analogous to pollen
incorporated into sediments. In general these traps are not suitable for analysis
of airborne fungal spores.
Analysis
Air samples collected by the equipment described above are analyzed by
various methods. Culturing and microscopy are the most common methods;
however, biochemical, immunochemical, and molecular techniques are finding
greater applications.
Culturing is the method of analysis required for several instruments,
such as Andersen samplers, which impact air samples onto an agar surface.
Culturing can also be used with samples from impingers, filter samples, or dust
samples. Although a variety of culture media can be utilized for air sampling,
malt extract agar is generally suggested for mesophilic fungi [9] and DG-18,
which contains dichloran (2,6-dichloro-4-nitroaniline) and 18% glycerol for
xerophilic fungi, especially from dust. Samples are usually incubated for 5–10
days at room temperature. Identification often depends on observing reproductive structures and methods of spore development, which can be seen in culture.
As a result, the major advantage of culture analysis is that many fungi can be
precisely identified provided trained personnel are available. The disadvantage
is that not all fungi are able to grow or reproduce on the culture medium used.
Some fungi are not able to grow at all in culture, while others require specialized media to grow or reproduce. Also, some airborne spores lose viability
Fungal Aerobiology: Exposure and Measurement
13
quickly. Although these dead spores obviously cannot grow in culture, they may
still retain their allergenicity. Consequently, results derived from culture methods only reflect a portion of the air spora.
Samples collected by Burkard (Hirst-type) spore traps, rotorod samplers,
Air-O-Cell samplers, and MK-3 samplers are usually analyzed by microscopy.
This is the most important method of analysis for outdoor air samples, which
usually contain both fungal spores and pollen. Samples are typically stained
with basic fuchsin or phenosafranin to assist in pollen identification although
fungal spores are seldom stained by these chemicals. Wet mounts can be prepared and samples examined immediately; however, many investigators prepare
permanent slides especially for Burkard samples, and this may take up to 24 h
for the mounting medium to harden. The major advantage of this method is that
microscopy permits the visualization and counting of all spores, both viable and
nonviable. The disadvantage of this method is that some spores cannot be identified. Small spherical spores that lack distinctive morphological features cannot be identified. In addition, Penicillium and Aspergillus spores, which can be
common in indoor samples, cannot be distinguished from each other by this
method. Microscopy also requires highly skilled personnel to identify the many
types of spores and pollen found in air samples. Additionally, for accurate
counting and identification, the slide should be analyzed at a magnification of
1,000⫻; this requires considerable time.
Biochemical methods can be used to detect specific fungal compounds,
such as ergosterol or -glucans. Ergosterol is a sterol found in fungal cell membranes, whereas -glucan is a fungal wall carbohydrate. Assays for these compounds can be used with samples collected by an impinger or on filters as well
as dust samples. These assays provide an estimate of total fungal biomass, but
they cannot supply any identification.
Immunochemical and molecular methods are normally used when specific
allergens or pathogens are the focus of study, because only the target organism
will be detected by these techniques [7]. With the large variety of airborne fungi
that occur in the atmosphere it is not practical to use these methods for routine
analysis of outdoor air samples. However, they are of value when the presence
of a specific fungus is suspected in an indoor environment. Air samples collected by an impinger, on filters, or even on spore trap samples can be analyzed
with these procedures as can dust samples. For immunochemical analysis, it is
necessary to have monoclonal or polyclonal antibodies specific for the fungus
under investigation. These antibodies can be used in an ELISA assay or tagged
with a fluorescent marker. The polymerase chain reaction (PCR) is an example
of a molecular method that can also be used to identify specific fungi.
Segments of DNA from an environmental isolate are amplified. Nucleic acid
probes specific for the target organism are added and will bind to the amplified
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DNA if the organism of interest is the same as the environmental isolate. Both
immunochemical and molecular methods are very sensitive and specific, and
results can be obtained in a shorter period of time than through culturing. The
disadvantage is that only predetermined fungi can be detected if present.
Despite this limitation, these methods are finding greater applications for
analysis of air samples [10].
Patterns of Variation
Daily pollen and spore counts provide a snapshot of the atmosphere in a
given area and typically represent the average daily concentration from a single
air sampler. However, a great deal of variability exists within the atmosphere
and physicians should be aware of the clinical implications of this variability.
Major causes of variability relate to the diurnal rhythms of spore discharge,
discharge related to weather events, seasonal effects, and spatial effects. Also
contributing to variability are the ways that different spore types respond to
environmental conditions.
The major groups of fungi important in aerobiology are the Ascomycota
(the ascomycetes or sac fungi), the Basidiomycota (the basidiomycetes or club
fungi) and asexual fungi (deuteromycetes or imperfect fungi). The ascomycetes
include numerous microfungi as well as larger cup fungi and morels. They
produce sexual spores known as ascospores within a sac-like structure called
an ascus. Generally there are eight ascospores within each ascus, and they are
explosively shot from the ascus when moisture is available. The basidiomycetes
include mushrooms, bracket fungi and puffballs. They produce sexual spores
called basidiospores on a club-like structure, which is known as a basidium.
Typically, four basidiospores are produced by each basidium, and numerous
basidia line the gills of mushrooms and the pores of bracket fungi. Like the
ascospores, basidiospores are actively released when moisture is available. Also
included within the basidiomycetes are the smut fungi and the rust fungi, two
groups of plant pathogens that produce enormous numbers of airborne spores.
The asexual fungi reproduce through the formation of asexual spores called
conidia. Although sexual reproduction is rare or absent entirely in this group,
the majority of these fungi are ascomycetes, with a small percent being basidiomycetes. The asexual fungi are extremely successful and are the most abundant microfungi in the environment. Many asexual fungi are leaf surface or soil
saprobes, and on most days their conidia dominate the air spora.
During dry weather, the spores of many asexual fungi show an early to
mid-afternoon peak in spore discharge and an early morning low [5, 11, 12]. The
daily peak usually occurs when temperatures and wind speeds are high, and
Fungal Aerobiology: Exposure and Measurement
15
4
0
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Fig. 1. Diurnal variation in airborne levels of basidiospores from four genera of
basidiomycetes.
humidity is low. These spores are often referred to as the ‘dry air spora’ and
include conidia of Cladosporium, Alternaria, Drechslera, Epicoccum,
Curvularia, Botrytis, and Pithomyces as well as other genera of asexual fungi and
smut spores. Release of these spores is passive and related to the meteorological
conditions, especially wind speed. In temperate areas the dry air spora dominates
the atmosphere from spring through fall except during periods of rain.
Basidiospores usually show a strong diurnal rhythm with peak concentrations during late night to early morning hours and lowest levels in the late afternoon. The spores are actively discharged from the basidium by a mechanism that
requires atmospheric moisture but not rainfall. As a result peak atmospheric levels occur when humidities are high. In a recent study of spores in the Tulsa
atmosphere total basidiospores showed peaks from 4:00 a.m. to 6:00 a.m. [13].
At a second site south of Tulsa, total basidiospore levels also showed a 4:00 a.m.
peak; however, when individual genera of basidiomycetes were examined, the
time of the peak varied with the spore type (fig. 1). Agaricus basidiospores
showed a peak at midnight, Boletus spores at 2:00 a.m., Ganoderma at 4:00 a.m.
and Coprinus basidiospores showed an 8:00 a.m. peak. Although the dispersal of
basidiospores does not depend on rain, basidiomycete fruiting bodies, such as
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Percent of average daily total
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16
mushrooms and puffballs, frequently develop within a few days after rainfall. As
a result, high concentrations of airborne basidiospores released from these fruiting bodies generally occur during seasons when rainfall is frequent [14].
Ascospores also require moisture for discharge. Some types of ascospores
are released during periods of high humidity and show a diurnal rhythm similar to
basidiospores; however, most ascospores are only released during or after rainfall
[15]. Ascomycetes vary in their response to rainfall. Some species begin releasing
spores within 10–15 min after the start of rain, while other species release spores
only when the reproductive structures begin to dry following a rain event [13, 15,
16]. The rainfall requirement for many ascomycetes tends to obscure any diurnal
rhythm; however, when rain is present some ascomycetes show rhythms with
either daytime or nighttime peaks depending on the species [15].
Besides triggering the active release of ascospores, rain can cause the
passive dispersal of other spores by various mechanisms. Members of the dry air
spora can be dispersed by raindrops as they strike leaf surfaces and other
substrates [17]. The initial raindrops that hit the leaf can cause vibrations that
result in a puff of conidia being dislodged from the surface. This mechanism
may explain some anomalous air-sampling data that showed increases of
Cladosporium, Alternaria, and other conidia during rain events [11, 18, 19].
Rain splash is another method of spore dispersal for some fungi [20]. Spores dispersed by rain splash are generally formed in mucilage, which inhibits dispersal
by wind. During rain, the initial drops dissolve the mucilage. This leaves a spore
suspension available for dispersal by subsequent raindrops. Although these
mechanisms describe dispersal during rain, rain also cleanses spores from the
atmosphere, especially members of the dry air spora [21].
In addition to rainfall, other meteorological events can cause variability in
atmospheric spore levels. Wind gusts and other conditions associated with
approaching weather systems can have dramatic effects on the air spora. Although
spore levels in Tulsa are often high, remarkable increases (spore plumes) in spore
levels have occurred on some days. These spore plumes last for a short time, and
hourly concentrations have exceeded 100,000 spores/m3. One of these events
occurred on 30 September 1998. Between 10:00 a.m. and noon on that day, the
hourly concentrations increased from about 27,000 to 144,000 spores/m3. The
spore plume coincided with increases in both wind speed and barometric pressure. The daily average of approximately 40,000 spores/m3 on 30 September 1998
clearly did not reflect variability during the day nor the magnitude of the exposure risk at noon and the early afternoon hours.
Meteorological factors are also responsible for seasonal variability in the
air spora. In most temperate areas, spore concentrations tend to increase in late
spring often showing a significant correlation with increasing temperatures
[13]. Many studies have shown highest levels of airborne spores typically
Fungal Aerobiology: Exposure and Measurement
17
occurring from late spring through early fall [12, 22–26]. In tropical and
subtropical areas, airborne spores may be abundant year round or follow
seasonal rainfall patterns.
Some components of the air spora are produced by plant pathogenic fungi,
and their seasonal peaks may parallel the flowering or harvesting of various
crop plants. The dependence on the life cycle of the host is especially pronounced for smut fungi, which only have a single reproductive phase each year.
Although smut spores are found in the Tulsa atmosphere for at least 6 months
of the year, different smuts are prevalent at different seasons. The most common
types in the spring include smut species that infect Bermuda grass, Johnson
grass, oats, and wheat, while in the fall the most common spores were produced
by the fungus that causes corn smut and those infecting some native grasses
[27]. Pathogenic ascomycetes may also show parallels with the host plant life
cycles. For example, in the spring Venturia ascospores are produced on dead
apple leaves that have overwintered in orchard [28, 29]. The asexual spores of
this pathogen are produced later in the growing season.
The atmosphere contains a heterogeneous mixture of fungal spores from
many different sources. Although there are major similarities in the air spora
within an area, at any one time or place the air spora may be dominated by
nearby sources of spores [30]. Air samples collected from a single roof top
sampler do not reflect the exposure closer to a source. Concentrations of airborne basidiospores from the bracket fungus Ganoderma were compared from
two sites in Tulsa using Burkard spore traps. The central sampling station was
located on the roof of a building on the University of Tulsa campus at 12 m
above ground. The second station was in a residential neighborhood; the intake
orifice at this site was at nose level, 1.5 m above ground. Ganoderma
basidiospores are typically in the atmosphere for 6 months of the year [31].
During the season peak in 1987 (July to September), the average daily concentration from the roof top sampler was 94 spores/m3, while the nose level sampler had a significantly higher mean of 180 spores/m3 (p ⬍ 0.05). Although both
samplers showed parallel day-to-day fluctuations, the nose level sampler had
consistently higher concentrations of Ganoderma spores (fig. 2). By contrast
total basidiospore levels showed little difference during this period [14]. These
data suggest that the higher Ganoderma concentrations from the nose level
sampler reflected nearby sources. Although no fruiting bodies were seen in the
immediate vicinity of the sampler, they were common in the surrounding area.
The atmospheric variability has also been examined in previous studies.
Solomon et al. [32] examined intrasampler variance and found small but consistent differences between the recoveries of four adjacent rotorod samplers and
also between the recoveries of three adjacent rotoslide samplers. Although the
authors suggested that the intrasampler variance reflected minor differences in
Levetin/Horner
18
Spores/m3
1000
900
800
700
600
500
400
300
200
100
0
Site B
7/1/87
Site A
8/1/87
9/1/87
Fig. 2. Average daily concentration of airborne Ganoderma spores from two locations
in Tulsa, Okla., during the months of July, August, September 1987.
the functioning of the samplers, it is possible that the inherent variability of
atmospheric bioaerosols also contributed to the variance. Hall [33] specifically
examined the question of atmospheric variability using an experimental array
of nine Tauber traps located approximately 6 m apart. He found that the mean
annual pollen deposition from the sampler array had a standard deviation of
10.5% [33]. Tauber traps are sedimentary samplers that rely on gravity to assess
the composition of the atmosphere. As a result, the variance seen in that study
could not be attributed to differences in sampler function. While it is not possible to routinely analyze multiple samples, the clinical significance of the atmospheric variability should always be considered.
Measurement Problems
The variability in the atmosphere means that the average spore count does
not apply to all times of the day or to all parts of a city (or region). There are
additional sources of variance not related to the atmosphere that the physician
should also recognize. Although the Burkard (Hirst-type) spore trap is one of
the most widely used air-sampling instruments, there is no standard counting
method used in analyzing the spores or pollen captured by this type of sampler.
The most accurate method of analysis would be to count the entire exposed
surface; however, this is not practical because of time limitations. As a result
only a portion of the daily capture is counted. Because the sampler drum
with attached tape moves at 2 mm per hour by the intake orifice, microscopic
Fungal Aerobiology: Exposure and Measurement
19
a
b
c
Fig. 3. Three counting methods frequently used for analysis of slides from a Burkard
spore trap. a Twelve transverse sweeps. b Single longitudinal sweep. c Four longitudinal
sweeps.
analysis of the slide at 4-mm intervals (perpendicular to the direction of movement) can provide information on the concentrations every 2 h throughout the
day. This allows determination of diurnal rhythms for the various spore or
pollen types. Conversely, one or more longitudinal sweeps parallel to the direction of movement will provide information on the average daily concentration.
Three counting methods are widely used by aerobiologists: 12 transverse
sweeps (fig. 3), a single longitudinal sweep and 3 or 4 longitudinal sweeps. The
accuracy of these counting methods has been addressed in several studies
[34–36]. Each counting method resulted in errors when compared to the totals
for the whole slide. Kapyla and Penttinen [34] found that the 12 transverse
sweep method gave reliable estimates of daily concentrations of airborne
pollen, whereas 1 or 2 longitudinal sweeps gave unreliable estimates. Comtois
et al. [35] showed that both 12 transverse and 4 longitudinal sweeps had lower
percent errors than the single longitudinal sweep. These two studies specifically
addressed pollen counting, but Sterling et al. [36] compared the single longitudinal sweep with the 12 transverse method for the major spore types found in
the atmosphere. They found that the 12 transverse method generally had higher
concentrations, but both methods sensed parallel fluctuations in daily concentration. Comparisons with the counts for the total slide showed that neither
Levetin/Horner
20
method was equivalent to the actual count but the 12 transverse sweep method
gave slightly better approximations. These studies suggest that the values
obtained for pollen and spore concentrations should be used as good indicators
of the outdoor bioaerosol concentrations, but not considered as absolute values.
Physicians should also be aware of the reporting lag often involved with
disseminating air sampling data through the local media. Typically, individuals
involved in daily pollen and spore counting will change the sampler in the
morning and then analyze the sample, which was exposed to the atmosphere for
the previous 24 h. After analysis the sampling data may be reported to the local
media or to a city, county, or state agency. It may be late afternoon or evening
before these day-old counts are available to the general public, and atmospheric concentrations may have changed dramatically since sample collection.
Because of the time involved in analysis of air samples this delay is unavoidable; however, aerobiologists are beginning to make the transition from descriptive aerobiology to predictive aerobiology [37]. Reliable forecasts for airborne
allergens may be widely available in the future.
Measurement problems also occur for air samples analyzed by culturing.
Some widely used culture plate impactors pull air through holes in a ‘sieve plate’
which direct the air stream onto the agar plate. This sampler design constrains
the trapped particles to land only under the sieve holes, which leads to multiple
spores often landing close together. Since culture plate samplers are analyzed
by culturing colonies that result from spores landing on the agar surface, any
juxtaposed colonies will be indistinguishable and counted as one. Statistical corrections are available that will estimate the true count on the plate that is needed
to give an observed number of colonies [38]. When using data from sieve plate
impactors, such as the Andersen, SAS or similar type, it is important to note
whether raw counts or corrected counts were used to calculate the airborne
concentration being measured.
There is an optimal density for counting particles on a surface. Too few
particles give too sketchy a sample of the pollen and spore populations. But too
much of a particle load on the sampling surface will decrease capture efficiency
and also increase analysis errors due to crowding and for culturable analyses,
incur risks of overgrowth and inhibition. Some samplers have a set sampling
rate, so that adjusting the count density is not possible. On other samplers, the
sample density is easily adjusted by varying sample volume, usually by changing the amount of time sampled. Unfortunately, it is often difficult to gauge the
best sample volume until the sample is analyzed. Drawing on experience is
perhaps the most practical way to strike a balance between these concerns.
Culture plates should have at least 10–30 colonies to be representative.
However, if there are more than 60–100 colonies, some colonies will be overgrown or inhibited by other colonies. On spore trap type samplers, sampling
Fungal Aerobiology: Exposure and Measurement
21
should not exceed a point where 50% of the sampling surface is covered, or
additional particles will begin bouncing off previously trapped particles rather
than sticking to open sampling surfaces. Conversely, enough particles should be
trapped to permit a reasonable time of examination to attain counts above 100
particles.
Clinical Implications
Although fungal spores are well-known allergens, only a few epidemiological studies have been conducted that show the direct relationship between
outdoor spore levels and symptoms. Recent studies have focused primarily on
asthma symptoms. Targonski et al. [39] studied asthma deaths in Chicago from
1985 to 1989. They found that airborne spore levels were significantly higher
on days when asthma deaths occurred than on days with no asthma-related
deaths. O’Hollaren et al. [40] studied a group of 11 asthma patients with sudden
respiratory arrest along with a group of 99 control patients with no history of
arrest. They found a significant difference in sensitivity to Alternaria between
the two groups. Their data showed 91% of the patients with respiratory arrest
were sensitive to Alternaria while only 31% of the control patients were sensitive (p ⬍ 0.001). In addition serum IgE antibodies to Alternaria were elevated
in 9 of the 11 patients (the other 2 patients were not tested) and all the respiratory arrest events occurred during the season when airborne Alternaria concentrations were usually high. Neukirch et al. [41] studied the association of
skin test sensitivity with asthma severity. Subjects were tested with 11 common
allergens, and asthmatics were categorized as having mild, moderate, or severe
disease. Using multiple-regression analysis, the investigators found that only
Alternaria was independently associated with severe asthma.
Two studies by Delfino et al. [42, 43] focused on the effects of outdoor
airborne allergens and air pollution on asthma symptoms. Subjects in each study
were asthmatics who self-reported daily symptoms and inhaler use. Although
both studies were carried out in California, the first was conducted in a coastal
area during the fall, and the second was in an inland community in the spring.
Both studies found that outdoor fungal spore levels were significantly associated
with asthma severity. Among the spore types, the greatest effects on symptom
scores were produced by atmospheric basidiospores, especially for children [43].
Similar studies in other parts of the world have also shown correlations between
asthma exacerbation and atmospheric basidiospore levels [44, 45]. Basidiospore
extracts are generally not included in routine skin testing; however, several studies using noncommercial extracts showed levels of sensitivity comparable to
fungi typically used in skin test panels [46]. Also, Horner et al. [47] found
Levetin/Horner
22
that the relative risk of developing asthma was significantly related to
basidiospore sensitivity. These studies clearly illustrate the clinical importance
of basidiospores as well as other fungal spores as triggers of allergic disease.
There is currently great interest and active debate about the health effects
of indoor mold exposure. Over the last 10–15 years, numerous studies documented an association between damp and/or moldy indoor environments and an
increased prevalence of respiratory complaints. A recent review [48] summarized 9 such studies that included some quantitative measure of indoor mold
exposure. Seven of the 9 studies found a positive association between some
exposure index and symptoms, although there were no significant associations
with other exposure indexes.
Interestingly, ‘total culturable counts’ (from either air or dust), i.e. with no
attempt at identification, was prevalent among the exposure measures with no
association. This is not surprising though, since measuring ‘total culturable
mold’ is analogous to counting total pollen levels with no identification. A
study on allergic rhinitis would demand counts of particular pollens to associate with symptoms in a population with known sensitization, rather than
attempting to associate total pollen counts with rhinitis symptoms. Surprisingly,
the allergy literature still tolerates ‘total’ mold counts.
In addition to questions of how best to assess exposure, significant problems remain with the diagnostic reagents for assessing sensitization to fungi.
This has constrained the ability to distinguish between allergic and other, nonallergic mechanisms possibly inducing mold-associated respiratory symptoms.
The debate on mechanisms of mold/damp-associated respiratory complaints is
focused on the indoor rather than the outdoor environment. This is at least
partly due to the recent increased attention to the indoor environment in
general. However, subjects with respiratory complaints that are associated with
damp/moldy indoor environments often are not allergic to other indoor allergens and are skin test negative to the available mold allergen reagents. This
suggests that affected subjects may be reacting to molds through nonallergic
mechanisms.
The principal nonallergic mechanisms that have been proposed involve
nonspecific biologically active mold components such as -glucans or sporeborne mycotoxins. -Glucans have long been known to have immunomodulatory effects [49], particularly on macrophages (cytokine production), but also on
antibody production [50, 51]. Only a few studies have measured their levels, but
increased -glucans have correlated with greater nonspecific respiratory complaints [52]. Numerous molds that are common in damp buildings are capable of
producing mycotoxins [53]. There is currently disagreement on the potential
clinical effects of mycotoxin exposure from indoor molds, and this is unlikely to
be resolved soon due to technical difficulties of measuring mycotoxin levels in
Fungal Aerobiology: Exposure and Measurement
23
indoor environments. A recent synopsis of the available literature, however,
suggests that inhalation is a more potent route of exposure than ingestion, and
the potential effects of mycotoxins should be critically studied rather than
disregarded [54].
Any presumed causal relationship between a building exposure and a clinical
effect should include some evaluation of the building rather than relying only on a
subject’s assertion. It is important to note that there are several methods to sample
indoor environments for dampness and/or mold growth. Source and reservoir sampling are often more valuable than air sampling due to the (often rapid) fluctuations inherent in airborne spore levels [10]. In some cases, a thorough inspection
by an experienced building investigator may be sufficient. There should be a clear
plan, however, to evaluate the building side of the equation, just as a differential
diagnosis for the patient would be pursued.
Debate and research continue on whether allergic reactions, nonspecific
mechanisms, or their interaction underlie the association of respiratory complaints with damp/moldy buildings. Debate also continues on what method of
exposure assessment is most reliable, and there are currently insufficient data
to establish any risk-based guidelines for indoor mold levels. Despite these limitations on understanding the respiratory effects of being in damp/moldy buildings, there is ample evidence that documents the association [48]. Fortunately,
these respiratory complaints are avoidable, since the engineering aspects of
maintaining dry buildings are well known and entirely feasible.
Conclusions
•
•
•
•
•
Aerobiology can be a valuable tool for estimating bioaerosol exposure;
however, it is essential that volumetric sampling be used.
Samples collected by gravity plates or slides (passive samples) are biased
toward larger spores and greatly underestimate the contribution of many
different fungi with small spores.
Culturing and microscopy are standard methods of analysis for routine air
sampling. Both methods require highly skilled individuals for spore identification.
Immunochemical and molecular methods are useful for detecting specific
fungi from air samples. These techniques are finding greater applications
as monoclonal antibodies and DNA probes become available for fungi
with known health effects.
The atmosphere contains a mixture of spores that is heterogeneous both in
morphology and in ecology. Variations in the air spora are due to many factors: the diurnal rhythms of spore release (ecology) that differ for various
Levetin/Horner
24
•
•
•
•
fungi, weather-related factors, and spatial factors. The clinical implications
of this variability should always be considered.
Measurement problems resulting from counting errors, overloading, or
undersampling can all affect the accuracy of air-sampling analyses. As a
result, concentrations obtained from air sampling should not be considered
as absolute values but as relative levels of exposure.
Only a few recent epidemiological studies have shown a direct relationship
between spore levels and asthma symptoms. Although more research is
needed, these studies have illustrated the importance of fungal spores both
indoors and outdoors as triggers of allergic disease.
Indoor mold exposure is considered potentially very serious by some
researchers, and by others to have only minor proven health effects, if any.
Epidemiological studies argue strongly that respiratory complaints
increase in damp or moldy buildings, though.
Indoor mold measures might include air sampling or source sampling.
Interpretation of either should not rely on total counts, but rather on the
types of molds recovered and the rank order of types of mold spores (or
colonies) recovered.
References
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Ball MV: Essentials of Bacteriology, ed 4. Philadelphia, Saunders, 1900.
Hirst JM: An automatic volumetric spore trap. Ann Appl Biol 1952:39:257–265.
Andersen AA: New sampler for the collection, sizing and enumeration of viable airborne particles.
J Bacteriol 1958;76:471–484.
Lacey J, Venette J: Outdoor air sampling techniques; in Cox CS, Wathes CM (eds): Bioaerosols
Handbook. Boca Raton, CRC Press, 1995, pp 407–471.
Gregory PH: The Microbiology of the Atmosphere, ed 2. New York, Halstead Press, 1973.
Lacey J: The aerobiology of conidial fungi; in Cole T, Kendrick WB (eds): The Biology of
Conidial Fungi. New York, Academic Press, 1981, pp 373–416.
Schmechel D, McCartney HA, Halsey K: The development of immunological techniques for the
detection and evaluation of fungal disease inoculum in oilseed rape crops; in Schots A, Dewey
FM, Oliver R (eds): Modern Assays for Plant Pathogenic Fungi: Identification, Detection and
Quantification. Oxford, CAB, 1994, pp 247–253.
Grote M, Vrtala S, Niederberger V, Valenta R, Reichelt R: Expulsion of allergen-containing materials from hydrated rye grass (Lolium perenne) pollen revealed by using immunogold field emission
scanning and transmission electron microscopy. J Allergy Clin Immunol 2000;105:1140–1145.
Burge HA: Monitoring for airborne allergens. Ann Allergy 1992;69:9–18.
Macher J (ed): Bioaerosols: Assessment and Control. American Conference of Government
Industrial Hygienists, Cincinnati, 1999.
Hirst JM: Changes in atmospheric spore content: Diurnal periodicity and the effects of weather.
Trans Br Mycol Soc 1953;36:375–393.
Hamilton ED: Studies on the air spora. Acta Allergol 1959;13:143–175.
Troutt C, Levetin E: Correlation of spring spore concentrations and meteorological conditions in
Tulsa, Oklahoma. Int J Biometeor 2001;45:64–74.
Levetin E: Identification and concentration of airborne basidiospores. Grana 1991;30:123–128.
Ingold CT: Fungal Spores: Their Liberation and Dispersal. Oxford, Clarendon Press, 1971.
Fungal Aerobiology: Exposure and Measurement
25
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
Meredith DS: Significance of spore release and dispersal mechanisms in plant disease epidemiology. Annu Rev Phytopathol 1973;11:313–342.
Lacey J: Spore dispersal – its role in ecology and disease: The British contribution to fungal aerobiology. Mycol Res 1996;100:641–660.
Venables KM, Allitt U, Collier CG, Emberlin J, Grieg JB, Hardaker PJ, Highham JH, Laing-Morton
T, Maynard RL, Murray V, Strachan D, Tee RD: Thunderstorm-related asthma – the epidemic of
24/25 June 1994. Clin Exp Allergy 1997;27:725–736.
Allitt U: Airborne fungal spores and the thunderstorm of 24 June 1994. Aerobiologia 2000;16:
397–406.
Fitt BDL, McCartney HA, Walklate PJ: The role of rain in dispersal of pathogen inoculum. Annu
Rev Phytopathol 1989;27:241–270.
Deacon JW: Modern Mycology, ed 3. Oxford, Blackwell Science, 1997.
Ebner MR, Haselwandter K, Frank A: Seasonal fluctuations of airborne fungal allergens. Mycol
Res 1989;92:170–176.
Hjelmroos M: Relationship between airborne fungal spore presence and weather variables. Grana
1993;32:40–47.
Halwagy M: Seasonal airspora at three sites in Kuwait 1977–1982. Mycol Res 1989;93:208–213.
Cosentino S, Pisano PL, Fadda ME, Palmas F: Pollen and mold allergy: Aerobiologic survey in
the atmosphere of Cagliari, Italy (1986–1988). Ann Allergy 1990;65:393–400.
Anon: 1998 Pollen and Spore Report, American Academy of Allergy, Asthma, and Immunology
(AAAAI), Milwaukee, 1999.
Crotzer V, Levetin E: The aerobiological significance of smut spores in Tulsa, Oklahoma.
Aerobiologia 1996;12:177–184.
Aylor DE: Aerobiology of apple scab. Plant Dis 1998;82:838–849.
Ponti I, Cavanni P: Aerobiology in plant protection. Aerobiologia 1992;8:94–101.
Lacey J: Aerobiology and health: The role of airborne fungal spores in respiratory disease; in
Hawksworth DL (ed): Frontiers in Mycology. Kew, CAB International, 1991.
Craig R, Levetin E: Multiyear study of Ganoderma aerobiology. Aerobiologia 2000;16:79–85.
Solomon WR, Burge HA, Boise JR, Becker M: Comparative particle recoveries by the retracting
rotorod, rotoslide, and Burkard spore trap sampling in a compact array. Int J Biometeor 1980;24:
107–116.
Hall SA: Comparative pollen influx at a nine-trap array in the Grand Prairie of northern Texas.
Texas J Sci 1992;44:469–474.
Kapyla M, Penttinen A: An evaluation of the microscopic counting methods of the tape in HirstBurkard pollen and spore trap. Grana 1981;20:131–141.
Comtois P, Alcazar P, Neron D: Pollen count statistics and its relevance to precision. Aerobiologia
1999;15:19–28.
Sterling M, Rogers C, Levetin E: An evaluation of two methods used for microscopic analysis of
airborne fungal spore concentrations from the Burkard Spore Trap. Aerobiologia 1999;15:9–18.
Van de Water PK, Keever T, Main CE, Levetin E: Forecasting long-distance transport of allergenic
mountain cedar (Juniperus ashei) pollen from source areas in central Texas and south central
Oklahoma. J Allergy Clin Immunol 2000;105:S231.
Willeke K, Macher J: Air Sampling; in Macher J (ed): Bioaerosols: Assessment and Control.
American Conference of Government Industrial Hygienists, Cincinnati, 1999, pp 11.1–11.25.
Targonski PV, Persky VW, Ramekrishnan V: Effect of environmental molds on risk of death from
asthma during the pollen season. J Allergy Clin Immunol 1995;95:955–961.
O’Hollaren MT, Yunginger JW, Offord KP, Somers MJ, O’Connell EJ, Ballard DJ, Sachs MI:
Exposure to an aeroallergen as a possible precipitating factor in respiratory arrest in young patients
with asthma. N Engl J Med 1991;324:359–363.
Neukirch C, Henry C, Leynaert B, Liard R, Bousquet J, Neukirch F: Is sensitization to Alternaria
alternata a risk factor for severe asthma? A population-based study. J Allergy Clin Immunol 1999;
103:709–711.
Delfino RJ, Coate BD, Zieger RS, Seltzer JM, Street DH, Koutrakis P: Daily asthma severity in
relation to personal ozone exposure and outdoor fungal spores. Am J Respir Crit Care Med 1996;
154:633–641.
Levetin/Horner
26
43
44
45
46
47
48
49
50
51
52
53
54
Delfino RJ, Zieger RS, Seltzer JM, Street DH, Matteucci RM, Anderson PR, Koutrakis P: The
effect of outdoor fungal spore concentrations on daily asthma severity. Environm Health Perspect
1997;105:622–635.
Epton MJ, Martin IR, Graham P, Healy PE, Smith H, Balasubramaniam R, Harvey IC, Fountain
DW, Hedley J, Town GI: Climate and aeroallergen levels in asthma: A 12-month prospective study.
Thorax 1997;52:528–534.
Dales RE, Cakmak S, Burnett RT, Judek S, Coates F, Brook JR: Influence of ambient fungal
spores on emergency visits for asthma to a regional children’s hospital. Am J Resp Crit Care Med
2000;162:2087–2090.
Lehrer SB, Hughes JM, Altman LC, Bousquet J, Davies RJ, Gell L, Li J, Lopez M, Malling HJ,
Mathison DA, Sastre J, Schultze-Werninghaus G, Schwartz HJ: Prevalence of basidiomycete
allergy in the USA and Europe and its relationship to allergic respiratory symptoms. Allergy
1994;49:460–465.
Horner WE, Hughes JM, Lopez M, Lehrer SB: Basidiomycete skin test reactivity is a risk factor
for asthma. Pediatr Asthma, Allergy Immunol 2000;14:69–74.
Verhoeff AP, Burge HA: Health risk assessment of fungi in home environments. Ann Allergy
Asthma Immunol 1997;78:544–554.
DiLuzio NR: Update on the immunomodulating activities of glucans. Springer Semin Immunopathol
1985;8:387–400.
Okazaki M, Adachi Y, Ohno N, Yadomae T: Structure-activity relationship of (1→3) beta-Dglucans in the induction of cytokine production from macrophages, in vitro. Biol Pham Bull 1995;
18:1320–1327.
Rylander R, Holt PG: (1→3) Beta-D-glucan and endotoxin modulate immune response to inhaled
allergen. Mediators Inflamm 1998;7:105–110.
Rylander R, Norrhall M, Engdahl U, Tunsater A, Holt PG: Airways inflammation, atopy and
(1→3) beta-D-glucan exposures in two schools. Am J Respir Crit Care Med 1998;158:1685–1687.
Samson RA, Flannigan B, Flannigan ME, Verhoeff AP, Adan OCG, Hoekstra ES (eds): Health
Implications of Fungi in Indoor Environments. Amsterdam, Elsevier, 1994.
Sorenson WG: Fungal spores: Hazardous to health? Environ Health Perspect 1999;107(suppl 3):
469–472.
Estelle Levetin
Faculty of Biological Science,
University of Tulsa, Tulsa OK 74104 (USA)
Tel. ⫹1 918 631 2764, Fax ⫹1 918 631 2762 , E-Mail estelle-levetin@utulsa.edu
Fungal Aerobiology: Exposure and Measurement
27
Breitenbach M, Crameri R, Lehrer SB (eds): Fungal Allergy and Pathogenicity.
Chem Immunol. Basel, Karger, 2002, vol 81, pp 28–47
Allergy to Basidiomycetes
Arthur Helbling a, Karl A. Brander a, W. Elliott Horner b,
Samuel B. Lehrer c
a
Division of Allergology, Clinic for Rheumatology, Clinical Immunology and
Allergology, Inselspital Bern, Switzerland;
b
Air Quality Sciences, Inc., Atlanta, Ga., and
c
Section of Clinical Immunology, Allergy and Rheumatology, Tulane University
School of Medicine, New Orleans, La., USA
For many years, fungal spores have been recognized as potential causes
of respiratory allergies [1–7]. Among the fungi, molds or microfungi, are well
recognized as sources of allergens. Basidiomycetes, another group of fungi, that
are physically the largest and morphologically the most complex fungi, can also
be allergenic (table 1) [5, 7, 8]. There are about 14,000 basidiomycete species,
including mushrooms, bracket fungi, puffballs, toad stools and jelly fungi, as
well as the plant-pathogenic rusts and smuts (table 2) [9].
Prevalence of Airborne Basidiospores Including Indoor Exposure
Spores of basidiomycetes (fig. 1) are abundant throughout many parts of
the world. Amounts range from 5 to 60% of the total spore load [6, 8, 10–16].
In several studies, basidiospores are among the top categories of airborne
spores [10, 11, 13–16]. Atmospheric concentrations of basidiomycete spores
vary according to daily and seasonal patterns [8]. For example, mushroom
spore counts for a given day are usually highest after midnight and during periods of high humidity. In temperate zones, seasonal peaks of basidiospores are
observed in spring and autumn, which coincide with the major periods of mushroom fruiting [11–14, 16]. However, some airborne basidiospores are usually
present during any time of the year with temperatures above freezing and adequate moisture.
Since many basidiomycetes will not readily grow on laboratory culture
media, spore trap samplers such as the Burkard or Lanzoni are typically used to
Table 1. Skin test reactivity to basidiomycetes
Authors1
Locality
Selection
criteria
Extract source
material
Species
tested
Positive skin
test reactors, %
Herxheimer et al.
Gold et al.
Hasnain et al.
Lehrer et al.
Sprenger et al.
Helbling et al.
Cardiff, UK
Ann Arbor, USA
Auckland, NZ
New Orleans, USA
Seattle, USA
Bern, CH
summer asthma
resp. allergy
resp. allergy
resp. allergy
resp. allergy
unselected
spores
spores
spores/tissue
spores
spores
spores/tissue
8
5
26
15
15
3
16
64
10
32
30
3.8
1
Individual references cited in Horner et al. [7, 8].
Table 2. Taxonomic distribution of the subphylum Basidiomycota (following
Hawksworth et al. [9])
Class Basidiomycetes (32 orders, 140 families, 473 genera, 14,000 species)
Phragmobasidiomycetidae
Agarcostilbales (2 spp.)
Atriactiellales (13 spp.)
Auriculariales (16 spp.)
Heterogastridiales (1 spp.)
Tremellales (256 spp.)
Holobasidiomycetidae1
Agaricales (6,000 spp.)
Coprinus, Lentinus, Pleurotus, Psilocybe
Boletales (727 spp.)
Boletus
Ganodermatales (81 spp.)
G. lucidum, G. applanatum
Lycoperdales (272 spp.)
Calvatia
Schizophyllales (46 spp.)
S. commune
Class Teliomycetes (2 orders, 15 families, 167 genera, 7,134 species)
(allergenicity demonstrated by Wittich and Stakman, cited in Horner et al. [7])
Class Ustomycetes (7 orders, 10 families, 63 genera, 1,064 species)
(allergenicity demonstrated by Cadham, cited in Horner et al. [7])
1
There are an additional 22 orders for which there are no allergen data.
sample airborne basidiospores rather than culture-based samplers. Basidiospores
are found in large quantities in Burkard samples taken in the atmosphere of the
city of Bern (Switzerland). For example, in 1997 spores of Ganoderma spp.
were registered from mid June until the end of October with levels as high
as 3,000 spores/m3 [17]. Ganoderma spp. spore load ranked second after
Allergy to Basidiomycetes
29
Fig. 1. Spores of P. pulmonarius (length width 5 10 m).
Cladosporium of all spores identified in this area. Although there is a significant
exposure to basidiomycetes, since basidiospores are often not tallied separately
in aerobiology studies, their allergenic significance may be underestimated.
Basidiomycete antigens have also been demonstrated from indoor house
dusts [18, 19]. In the study of Horner et al. [18], 4 of 9 houses had moderate to
high levels of basidiomycete antigen in dusts collected on two or more sampling visits [18]. This could be readily explained by infiltration of basidiospores
from outdoor sources, as occurs with pollen. Since basidiospores are smaller
than pollen grains, their infiltration is likely even greater than that of pollen.
Also, if wood becomes wet, such as through leaks, basidiomycetes will decay
this wood, which can release basidiospores, often indoors. Mycelial antigens,
can also be released, either as mycelial fragments or as secreted antigens
which might become incorporated into dust. Thus, although basidiomycete
exposure is predominantly outdoors, clearly indoor exposure may be important
as well.
Prevalence of Sensitization
Comparisons of sensitization rates to various basidiomycetes, and between
basidiomycetes and other groups of fungi have been made in the frame of skin
test surveys [20–22]. In a multicenter study in Europe and the USA, it was
shown that sensitization to basidiomycetes was as prevalent as sensitization to
other well-established allergenic molds, such as Alternaria, Cladosporium,
Aspergillus and Fusarium [21]. Generally, 25–30% of subjects with respiratory
Helbling/Brander/Horner/Lehrer
30
allergy were sensitized to at least one basidiomycete extract [8, 20, 21]. Since
October 1996, more than 4,000 patients referred to the outpatient allergy Clinic
in Bern were tested with extracts of the basidiomycetes Boletus edulis, Coprinus
comatus and Pleurotus pulmonarius. Overall, 3.5% of the subjects were sensitized to at least one basidiomycete; 14% of the basidiomycete-positive individuals were mono-sensitized to oyster mushroom (P. pulmonarius), 8% reacted
solely to cèpe (B. edulis) and 2% only to shaggy cap (C. comatus). Sensitization
only to basidiomycetes was detected in 4% of these individuals. Among atopic
subjects, the sensitization rate to basidiomycetes was 9.8% [22]. Thus, it is
important to distinguish this study, which was based on unselected allergy clinic
attendees, from prior studies that generally selected mold-sensitized or asthmatic subjects. It is estimated from these data that in Switzerland (7.5 million
inhabitants) approximately 250,000 individuals are sensitized to at least one of
these three mushrooms.
Clinical Aspects
Respiratory Allergy
Respiratory allergic symptoms, primarily of asthma, have been correlated
with outdoor spore exposures such as Alternaria, Cladosporium or basidiomycetes [23–25]. The focus of basidiospore allergy studies on asthmatics is
justified by the small particle size of basidiospores, many of which are
5–15 m. Many basidiospores are also elliptic or oblong, so their aerodynamic
diameter is closer to the smaller dimension that allows them to easily reach the
lower airways and initiate an asthmatic reaction. The potential of basidiospores
to induce allergic disease (shown in sensitization and provocation studies) is
widely accepted; their significance as allergens is still debated though. The
clinical relevance of basidiospore exposure and asthma was indicated in a study
that correlated several climate and aeroallergen factors with asthma symptoms.
Epton et al. [25] found that among the climate and aeroallergen factors
measured, only elevated basidiospore levels were associated with aggravation
of asthma. Clearly, there is a portion of the asthmatic population for whom
basidiospore exposure is clinically relevant, and important.
Besides respiratory allergies with hay-fever-like symptoms and asthma in
atopic individuals, continuous occupational basidiospore exposure may result
in hypersensitivity pneumonitis or extrinsic allergic alveolitis. The basidiomycetes Lentinus edodes, Pleurotus ostreatus, and Merulius lacrymans have
all been shown to cause hypersensitivity pneumonitis from occupational exposure [26–28]. Actinomycetes in the compost used to grow button mushroom
Allergy to Basidiomycetes
31
(Agaricus bitorquis) also causes hypersensitivity pneumonitis, but basidiospores
of Agaricus are usually not involved in these cases.
Bronchial and Nasal Challenges
Bronchoprovocation and nasal challenges indicate the clinical relevance of
basidiomycete extracts to allergic disease. Lopez et al. [29] showed that 5 out
of 8 skin-prick-test (SPT)-positive asthmatic subjects had a significant, dosedependent decrease in forced expiratory volume in 1 s ranging from 20–47%
after bronchial challenge with basidiospore extracts of Psilocybe cubensis,
Ganoderma lucidum, or Coprinus quadrifidus [29]. More recently, nasal airflow was measured after nasal challenges with three different concentrations of
P. pulmonarius spore extracts (0.1, 1.0, 10.0 mg protein/ml) on 12 skin-testreactive subjects with respiratory allergies (9 asthmatic and 3 rhinitic) [22].
Whereas 6 skin-test-negative controls, including 2 pollen-allergic subjects,
were unaffected, all 12 P. pulmonarius skin-test-reactive subjects had a significant reduction in nasal airflow (mean 73%) with subjective symptoms. In a 41year-old mushroom collector who claimed to recognize Boletus spp. by acute
hay fever symptoms, active anterior rhinomanometry demonstrated complete
obstruction of the nasal air flow with accompanying conjunctivitis. A facial rash
also occurred following nasal challenge with B. edulis extract at a concentration
of 0.1 mg protein/ml [30]. Thus, basidiomycetes are widely accepted potential
sensitizers that can induce clinically relevant respiratory allergic disease.
Atopic Eczema
Patch testing [31] of sensitized subjects supports the relevance of basidiomycete allergens in skin reactions. Atopy patch-test solutions from cap tissue
or extracts of spore containing tissue of C. comatus were applied for 48 h in
test chambers on clinically uninvolved skin on the back of 38 subjects with
atopic dermatitis [32]. Twelve (32%) patients showed a positive skin reaction
either to Coprinus spore extract or ground Coprinus cap in Vaseline. The
lesions that were evoked were consistent by histologic and immunohistochemical findings with acute atopic eczema. A few subjects reacted solely to the
Coprinus extract. Similar results were obtained with P. pulmonarius spore
extracts [30]. These data indicate that basidiomycete allergens can induce
eczematous skin lesions in individuals with atopic dermatitis, although the clinical relevance of positive atopy patch tests to aeroallergens is not completely
elucidated.
Helbling/Brander/Horner/Lehrer
32
Contact Dermatitis
Several reports have described allergic contact dermatitis from handling
mushrooms of various species [33–35]. Although in most cases, extensive
contact with the offending mushroom occurred, there is only 1 case reported
after initial exposure to the mushroom species. This case involved a plant
pathologist, with known contact dermatitis to other mushrooms; this suggests
clinically relevant cross-reactivity among various species [33]. Generally, any
component of the mushroom may cause cutaneous lesions, but in some
cases only particular parts produce symptoms, e.g. elements of the cap but not
spores [33]. Thus, taken together, these reports suggest that basidiomycete
allergens can also induce clinically relevant dermatologic and respiratory
allergic disease.
Food Allergy
In the past, reports of food allergies to mushrooms were largely anecdotal;
however, there are 6 well-documented cases of food allergy to mushrooms
[36–38]. B. edulis (known colloquially as cèpe, boler, steinpilz, porcini, or King
bolete) caused IgE-mediated systemic allergic reactions in 5 occupationally and
nonoccupationally exposed subjects. Symptoms ranged from generalized pruritus, abdominal pain and diarrhea [37] to urticaria, asthma and even anaphylaxis
[36]. The authors are aware of 2 additional subjects with anaphylaxis following
ingestion of Boletus spp. By SDS-PAGE IgE-immunoblotting, digestion-stable
Boletus proteins between 75 and 80 kD were identified by reactivity in a
nonatopic, B. edulis SPT-positive subject [30]. These IgE-binding bands were
not detected in B. edulis skin-test-positive atopic subjects with respiratory
allergies. Another report described a reproducible eosinophilia and gastrointestinal symptoms consistent with eosinophilic gastroenteritis following
chronic shiitake (Lentinus edodes) intake in a 56-year-old male participant in a
cholesterol-lowering study [38]. Eosinophilia caused by ingestion of shiitake
powder was shown in 40% of healthy subjects [38]; this is a concern since
shiitake powder is an increasingly popular dietary supplement.
Invasive Mycosis
Well-documented cases where filamentous basidiomycetes were responsible for mycosis in immunocompromised subjects have recently been reported
[39–41]. Chronic sinusitis was the most frequent clinical symptom, but lung
Allergy to Basidiomycetes
33
lesions, brain abscesses, allergic pulmonary mycosis, ulcerative buccal lesions
of the mucosa and endocarditis have also been described [39–41]. These cases
involved Schizophyllum commune (naturally occurs on decomposing branches),
Ustillago spp. (a plant parasite), and Coprinus spp. (turf, dung, rotting wood).
De Hoog et al. [42] demonstrated the advantage of PCR to identify the correct
species of basidiomycetes in invasive mycosis [42]. These cases emphasize that
basidiomycetes are also involved in nonallergic disease.
Source Materials
Only limited data directly compare the allergenic potential of different
source materials of fungi. Among basidiomycetes, comparisons have been
made for Coprinus, Pleurotus, Ganoderma and Psilocybe [43–46]. In Coprinus
quadrifidus, allergens were present in caps, spores, and stalks; in some cases,
the caps and stalks exhibited greater allergenic activity than did spores. Pleurotus
extracts from washed mycelium (broth-cultured), caps, and spores each contained allergens [44]. Crossed radioimmunoelectrophoresis (CRIE) detected
spore allergens that were not in the other tissues, although the total allergenic
activity of the spore extract was not greater than that of other tissues, and spore
and mycelial extracts of Pleurotus ostreatus were comparable in skin test
activity [44]. These observations that fruiting bodies of Coprinus, Ganoderma,
and Pleurotus all contain allergens support recent reports of food-induced
allergic reactions from eating mushrooms which suggest that edible parts
of the mushroom contain clinically relevant food allergens [36–38]. Thus,
allergens of basidiomycetes may be present throughout the mushroom, but no
data are available on allergen expression in various tissues or developmental
stages.
Spores are the presumed source for sensitization to fungal allergens. In
most skin test surveys, basidiomycete extracts originate from disrupted spores.
Though such extracts favor the release of soluble proteins, spore disruption may
also release components, which are not normally released. Little is known
about the kinetics of allergen release from intact fungal components. Horner
et al. [47] compared allergen release in (control) extracts of homogenized
spores with that released from intact (nonhomogenized) spores. Calvatia
cyathiformis and Psilocybe cubensis both have very thick spore walls. In contrast to the strongly melanized and rather easily wetted walls of P. cubensis,
spores of C. cyathiformis are not heavily melanized but are very hydrophobic.
Intact spores of these species did not release as much allergen as homogenized
spores and generally required at least 24-hour elutions to obtain any significant
release of allergens. Conversely, Lentinus edodes and P. ostreatus both have
Helbling/Brander/Horner/Lehrer
34
thin-walled hyaline spores; spores from these species released substantial allergenic activity rather quickly, with little additional release beyond 1 h. This suggests that allergen release is related to spore wall characteristics, at least in
some species. The spores with thick, or melanized cell walls are well protected
from environmental stress, but also release minimal allergen very slowly. The
spores of Lentinus and Pleurotus are probably not well protected by their cell
wall from environmental stress, and their allergen content is readily released
into the environment.
Basidiomycetes as Allergens
Of the many basidiomycetes to which we are exposed, only 50 or so species
have been tested for allergenicity, and about 25 are recognized as significantly
allergenic [7, 8]. Allergens identified at the DNA or protein level have been
described for six genera of basidiomycetes, and are discussed below.
B. edulis (cèpe) (fig. 2a)
Aqueous extracts of dried (cèpe) mushroom, contain several allergens
from 14 to approximately 90 kD by SDS-PAGE/Western blot [36, 37]. The
serum of a woman with a food allergy to B. edulis identified three IgEbinding bands at 14, 26, and 39 kD [38]. In a nonatopic, B. edulis SPT-positive
subject with recurrent anaphylaxis following the ingestion of B. edulis, a triplet
of IgE-binding proteins of molecular weights between 75 and 80 kD were
detected [30]. An IgE-binding component of 75 kD has been shown to be resistant to pepsin digestion after 60 min. However, this component could not be
detected with sera of Boletus SPT-reactive individuals who otherwise could eat
it without clinical symptoms.
Calvatia spp. (Puffballs) (fig. 2b)
Immunoprints with a panel of 19 sera from SPT-reactive subjects to Calvatia
cyathiformis crude extracts revealed at least 21 different protein-binding bands
[48]. Two of these proteins (pI 9.3 and pI 6.6) reacted with 68 and 63%, respectively, of the sera tested. The most reactive allergen had an estimated isoelectric
point (pI) of 9.3 and was particularly labile [49]. Preparative isoelectric focusing was used to isolate the pI 9.3 allergen, which has a molecular weight
Allergy to Basidiomycetes
35
a
b
c
d
Fig. 2. a B. edulis (photographed by G. Martinelli, Dietikon, Switzerland). b C. cyathiformis (photographed by G. Martinelli, Dietikon, Switzerland). c C. comatus (photographed
by G. Martinelli, Dietikon, Switzerland). d G. applanatum (photographed by G. Martinelli,
Dietikon, Switzerland).
of 16 kD [50]. A number of the Calvatia allergens were cross-reactive with
allergens from 4 other species of Calvatia [51].
Coprinus spp. (Inky Cap) (fig. 2c)
The allergenic activity of different Coprinus tissues was demonstrated by
RAST inhibition using separate extracts of spores, caps and stalks [43]. Mycelial
extracts of C. comatus and C. quadrifidus had allergenic activity both by skin
test and by RAST [52, 53]. IgE-binding bands from 22 to 100 kD were
detected by immunoblots in C. comatus; the most intense were at 29, 33 and
55 kD [22]. Recently, C. comatus allergens have been isolated using a molecular
phage display approach [54]. Since several Coprinus spp. are common, and at
times abundant, in lawns and gardens, these more ‘domestic’ rather than strictly
woodland species obviously cause greater exposure in humans.
Helbling/Brander/Horner/Lehrer
36
Ganoderma spp. (fig. 2d)
Ganoderma spores are morphologically very distinct. Consequently, these
spores are easily identified and more frequently reported in spore trap studies
than many other basidiospores. Spore and cap extracts of Ganoderma applanatum are skin-test reactive, and spore counts have been generally correlated
with patient symptoms [55, 56]. Using 6 sera from skin-test and RAST-positive
subjects, SDS-PAGE immunoblots of Ganoderma meredithae spore and cap
extracts revealed 10 IgE-binding proteins from 14 to 66 kD [45]. Seven of the
10 allergens occurred in both spore and cap extracts; the remaining allergens of
28, 50 and 66 kD were unique to the cap. IEF immunoprints revealed a total
of 16 proteins (pI 3.5–6.6) of which 8 were found in both the cap and the
spore [45]. In G. applanatum spore extracts, 14 allergens were detected by
CRIE and additional immunoblots revealed several IgE-binding bands between
18 and 82 kD [57]. Immunoblot inhibition studies with Ganoderma and other
basidiomycetes indicated that Ganoderma had little cross-reactivity with other
basidiomycetes [58]. Despite the fact that Ganoderma spp. have been studied
by more laboratories than any other basidiomycetes, no allergens have yet been
isolated or characterized.
Pleurotus spp. (Oyster Mushroom) (fig. 3a)
Since Pleurotus ostreatus is a cultivated mushroom to which significant
occupational exposures occur, the allergenicity of this mushroom and inhalation
of its spores resulting in hypersensitivity pneumonitis were recognized early
[26]. In an interesting contrast, there is significant occupational exposure to
spores of Pleurotus mushrooms but essentially none to the commonly cultivated button mushroom (Agaricus bitorquis). Pleurotus releases spores as soon
as the cap begins to expand, and prodigious quantities are released before the
caps grow to merchantable size. These exposures can lead to hypersensitivity
pneumonitis. In contrast, caps of the ordinary button mushrooms are generally
harvested before the caps open and release spores, which minimizes exposure.
Button mushrooms are grown in compost, which, however, is heavily colonized
by actinomycetes, and these spores can induce hypersensitivity pneumonitis in
workers.
Oyster mushroom spores contain potent allergens capable of inducing
rhinitis and bronchoconstriction [59, 60]. At least 5 allergens have been shown
by CRIE in spore extracts of Pleurotus [44]. Immunoblots revealed IgE-binding
proteins of 10, 29, 30 and 33 kD, as well as some larger IgE-binding molecular components. Using mycelial extracts, at least 13 allergens have been
Allergy to Basidiomycetes
37
a
b
Fig. 3. a Pleurotus ostreatus (photographed by G. Martinelli, Dietikon, Switzerland).
b Psilocybe cubensis (photographed by A. Helbling).
identified [61]. In a timed test of allergen release from intact spores, allergens
were released rapidly from Pleurotus spores [47]. This was in marked contrast
to other spore types and may increase the clinical relevance of Pleurotus and
other species with thin-walled spores.
Psilocybe spp. (fig. 3b)
Although exposure of most people to Psilocybe spp. is undoubtedly minimal, skin test reactivity to P. cubensis spore extracts is the most significant
of all basidiomycetes tested in Europe and the USA [20, 21]. This reactivity
probably results from exposure to related species. P. cubensis occurs naturally
on the northeastern coast of South America, the Caribbean and the southeastern coast of North America. Although related species occur throughout the
temperate northern hemisphere, the distribution of these mushrooms is probably less important clinically than the cross-reactivity patterns of its allergens.
Several species in genera closely related to Psilocybe are common lawn
Helbling/Brander/Horner/Lehrer
38
mushrooms, including several Coprinus spp., which are also known allergens
that cross-react.
Spore and mycelial extracts of P. cubensis (magic mushroom) have been
thoroughly characterized; 13 and 11 allergens, respectively, were identified
by SDS-PAGE immunoblot analysis [46, 62]. RAST inhibition demonstrated
common IgE-binding components, although the spore extract consistently had
greater inhibitory potency than the mycelial extract [46]. Using a panel of 11
individual sera (from SPT- and RAST-positive subjects), the allergens that
bound IgE from most sera were the 16-kD (82% reactive), the 35-kD (100%)
and the 76-kD (91%) proteins. IEF immunoprints indicated that the most
reactive proteins were at pI 5.0 (80%), 5.6 (87%), 8.7 (80%), and 9.3 (100%).
P. cubensis extracts showed the greatest inhibitory activity of all fungal species
tested by RAST inhibition in an evaluation of cross-reactivity [61]. Hence
Psilocybe allergens are potent as well as cross-reactive. Monoclonal antibodies
were prepared against the 48-kD allergen and may allow future structural analysis [63]. A cDNA clone, the first of a recombinant allergen from a basidiomycete, was isolated that codes for the 16-kD allergen [64]. The cDNA
sequence and deduced amino acid sequence indicate homology with
cyclophilin which is an abundant and widespread peptidyl prolyl-isomerase of
the lumen of the endoplasmic reticulum. In contrast to human cyclophilin,
which is not allergenic, fungal cyclophilin seems to be a major allergen in
P. cubensis.
Cross-Reactivity
Only three well-documented studies have addressed cross-reactivity
among or involving basidiomycetes [46, 58, 61]. These studies all used allergen
extracts to inhibit IgE-specific immunochemical assays. RAST inhibition was
first used to detect the cross-reactivity of various basidiospore allergen extracts
[61]. The second study included spores from six different mushrooms, and the
results were concordant with the taxonomic classification of basidiomycetes.
Four species from related orders (C. quadrifidus, C. cyathiformis, P. ostreatus,
P. cubensis) were significantly cross-reactive; species from two unrelated
orders (Ganoderma meredithae, Pleurotus tinctorinus) showed only minimal
cross-reactivity with one another or the other four species [58].
Another RAST inhibition study assessed allergenic relationships between
basidiomycete and conidial fungi [65]. In this study, there was minimal although
detectable cross-reactivity between the two groups. Far stronger reactions were
consistently observed though among species within each group. These results
indicate that relatively broad cross-reactivity occurs among basidiomycetes;
Allergy to Basidiomycetes
39
this generally follows accepted taxonomic groupings, but far more than 6
species should be tested in order to properly support this conclusion. These
observations may be important in future development of useful diagnostic as
well as therapeutic basidiomycete reagents. They also could serve as a basis for
monitoring basidiospore levels in the atmosphere.
Molecular Biological Approaches to Basidiomycete Allergens
The molecular analysis of basidiomycete allergens has lagged behind that
of other aeroallergens in general and other fungi in particular. This is mainly due
to a lack of sufficient source material and allergic sera and the fact that basidiomycetes have only recently been recognized as important fungal allergens
compared with other fungi. Nevertheless, progress has been made with allergen
characterization. Several basidiomycete allergens have been purified and/or
produced as recombinant allergens as listed in table 3. The first DNA sequence
identified for a basidiomycete allergen was for a 16-kD allergen of
P. cubensis [64]. Only limited progress was made with P. cubensis allergens,
but the 16-kD allergen was determined by cDNA sequence analysis to be homologous with cyclophilin, an abundant, widespread peptidyl prolyl isomerase.
However, the mushroom C. comatus, (extracts from which showed SPT reactivity in 58% basidiomycete-sensitive individuals [22]) has recently been used
successfully as a model system. Using the pJuFo filamentous phage display
system of Crameri et al. [66], 7 cDNAs were sequenced and 2 C. comatus allergens (Cop c 1 and Cop c 2) were characterized.
Screening of C. comatus Phage Display Library – 7 Putative Allergens
The synthesis of a pJuFo library [66] of C. comatus with sera of
basidiomycete-allergic individuals allowed the isolation of 7 putative allergens
[30, 54]. Homology searches on various databases showed that generally no other
homologies could be found except for Cop c 2, which encodes a C. comatus
thioredoxin (TRX). Cop c 1 may represent a putative transcription factor because
of the presence of two leucine zipper motifs. Cop c 1 and Cop c 2 were characterized in detail (table 4) [30, 54]. Altogether, 5 of the 7 different cDNAs were
cloned into different bacterial expression systems. This substantiates the assertion
that basidiomycete proteins are an important new spectrum of allergens in addition to the variety of known allergens from established plant, other mold and
animal sources.
Helbling/Brander/Horner/Lehrer
40
Table 3. Cloned basidiomycete allergens
Species
Allergen designation
P. cubensis
Psi c 1
Psi c 2
Cop c 1
C. comatus
1
Function
cyclophilin
leucine
zipper
thioredoxin
?
?
?
?
?
Cop c 2
Cop c 3
Cop c 4
Cop c 5
Cop c 6
Cop c 7
2
Homology
Reference
isomerase
transcription
factor
redox
?
?
?
?
?
Helbling1
Horner [64]
Brander [54]
Brander2
Helbling1
Helbling1
Helbling1
Helbling1
Helbling1
Unpublished.
Manuscript in preparation.
Table 4. Characteristics of Coprinus cDNA coding for allergens
Coprinus
designation
Accession No.
c1
AJ 132235
c2
c3
c4
c5
c6
c7
AJ 242791
AJ 242792
not submitted
AJ 242793
not submitted
AJ 242794
cDNA insert
size, bp
Open reading
frame, aa
463
81
463
985
1500
756
250
614
115
342
227
68
49
140
Homology
putative transcription
factor
TRX
?
?
?
?
?
Cop c 1, the First C. comatus Allergen (fig. 4)
Since Cop c 1 cDNA amplified most frequently during the screening
process, Brander et al. [54] cloned this cDNA into the pQE-System (Qiagen)
and produced recombinant protein in Escherichia coli (85 mg/l culture). The
expressed N-terminal His-tagged fusion protein was purified over a Ni-matrix
and dialyzed stepwise against decreasing concentrations of urea. The last
step was dialysis against phosphate-buffered saline. Pure, recombinant Cop c 1
Allergy to Basidiomycetes
41
a
b
c
Fig. 4. a cDNA sequence of Cop c 1 and deduced amino acid sequence. b Deduced
amino acid sequence of rCop c 1 fusion protein. Underlining N-terminal His-tag; italics
amino acid sequence that derives from multicloning site of -ZAPII phagemid SK. bold
sequence encoded by Cop c 1. c The same deduced amino acid sequence is shown twice.
Upper sequence: two leucine zipper motifs termed N- and C-terminal; lower sequence: two
different repeats, 6 times L-4-L and 7 times P-5-L.
Helbling/Brander/Horner/Lehrer
42
a
b
Fig. 5. SPT series with (a) rCop c 1 or (b) rCop c 2 (photographed by F. Schweizer,
Dermatology, Bern).
allergen (rCop c 1) was tested by SDS-PAGE, immunoblotting, ELISA, ELISAinhibitions, proliferation assays and SPTs [54]. rCop c 1 is recognized by 25%
of 92 tested sera from basidiomycete-sensitized subjects (fig. 5). Positive
immunoblots and proliferative responses also support the conclusion that rCop
c 1 is an IgE-binding component of C. comatus. As few as 11 fmol rCop c 1/l
induce a clearly positive wheal-and-flare reaction in SPTs.
Allergy to Basidiomycetes
43
Cop c 2
Cop c 2 most likely represents a basidiomycete (C. comatus) TRX [30].
TRX participates in various redox reactions through reversible oxidation of its
active center dithiol to a disulfide and catalyzes dithiol-disulfide exchange
reactions. TRX of different sources is highly conserved in the amino acid
sequence with typical conserved regions. IgE-ELISA data indicate that this
ubiquitous enzyme also possesses allergenic potential, since it bound IgE from
20% of sera from basidiomycete-sensitive subjects. Positive ELISA inhibitions
and proliferation assays, as well as strong skin reactions upon testing with 14
ng/ml of recombinant protein (fig. 5) confirm the allergenicity of this protein.
Conclusions
Basidiomycetes are important sources of aeroallergens and sensitization to
various species is by far more frequent than previously accepted. Basidiospores
have been demonstrated to cause respiratory allergy and are also suggested to
trigger inflammatory skin eruptions in a subgroup of patients with atopic
eczema. The clinical findings are supported by various in vitro methods demonstrating IgE-binding proteins. Through molecular biology techniques, several
recombinant allergens have been produced and their biologic activity has
been demonstrated in vivo by skin tests. To date, two recombinant C. comatus
proteins have been shown to meet all criteria of allergens. Six of the 7 isolated
C. comatus allergens are either coded in unknown genes, or genes not yet
identified as coding for allergens [unpubl. data]. Recently, robot-based highthroughput screening of an enriched C. comatus phage surface display library
has revealed the presence of at least 37 allergen-encoding cDNAs [67, 68].
Plants, other molds and other allergen sources all are known to have many
cross-reactive allergens. Hence, it is reasonable to assume that many basidiomycete allergens would also be cross-reactive, and indeed the limited data
available support this. This suggests that basidiomycetes may harbor an
unknown array of new allergens. Clearly, isolation and characterization of
basidiomycete allergens should be a major goal for future research.
References
1
2
3
Blackley C: Experimental researches on the causes and nature of catarrhus aestivus (hay fever or
hay asthma). London, Baillière, Tindall & Cox, 1873.
Van Leeuwen WS: Bronchial asthma in relation to climate. Proc R Soc Med 1924;17:19–26.
Bernton HS: Asthma due to a mold – Aspergillus fumigatus. JAMA 1930;95:189–191.
Helbling/Brander/Horner/Lehrer
44
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
Southworth D: Introduction to the biology of airborne fungal spores. Ann Allergy 1974;32:1–22.
Salvaggio J, Aukrust L: Mold-induced asthma. J Allergy Clin Immunol 1981;68:327–346.
D’Amato G, Spieksma FTHM: Aerobiologic and clinical aspects of mould allergy in Europe
(position paper). Allergy 1995;50:870–877.
Horner WE, Helbling A, Salvaggio JE, Lehrer SB: Fungal allergens. Clin Microbiol Rev 1995;8:
161–179.
Horner WE, Helbling A, Lehrer SB: Basidiomycete allergens. Allergy 1998;53:1114–1121.
Hawksworth DL, Kirk PM, Sutton BC, Pegler DN: Ainsworth & Bisby’s Dictionary of the Fungi,
ed 8. Cambridge, CAB International, 1995.
Adams KF, Hyde HA, Williams DA: Woodlands as a source of allergens with special reference to
basodiospores. Acta Allergol 1968;23:265.
Chatterjee J, Hargreave FE: Atmospheric pollen and fungal spores in Hamilton in 1972 estimated
by the Hirst automatic volumetric spore trap. Can Med Assoc J 1974;110:659–663.
Wilken-Jensen K, Graevesen S: Atlas of moulds in Europe causing respiratory allergy. Copenhagen,
ASK Publishing, 1984.
Ellis MH, Gallup J: Aeroallergens of southern California. Immunol Allergy Clin North Am 1989;
9:365–380.
Levetin E: Studies on airborne basidiospores. Aerobiologia 1990;6:177–180.
Li D-W, Kendrick B: A year-round study on functional relationships of airborne fungi with
meteorological factors. Int J Biometeorol 1995;39:74–80.
Decco ML, Wendland BI, O’Conell E: Volumetric assessment of airborne pollen and spore levels
in Rochester, Minn. 1992 through 1995. Mayo Clin Proc 1998;73:225–229.
Clot B: Suisse Meteorological Institute, Neuchâtel; personal communication, 1998.
Horner WE, Reese G, Lehrer SB: Field assessment of an immunochemical assay for basidiomycete fungal antigens in indoor dust; in Engineering Solutions to Indoor Air Quality Problems.
Proc Conf Air and Waste Management Assoc, Pittsburg, July 1995.
O’Rourke MK, Gunyan M, Bourdour A, Van der Water PK: The prevalence of basidiomycetes in
homes, Tucson, Arizona. 21st Conf Agricultural Aerobiology, San Diego, March 1994. Boston,
American Meteorological Society, 1994.
Lehrer SB, Lopez M, Butcher BT, Olson J, Reed M, Salvaggio JE: Basidiomycete mycelia
and spore-allergen extracts: Skin test reactivity in adults with symptoms of respiratory allergy.
J Allergy Clin Immunol 1986;78:478–485.
Lehrer SB, Hughes JM, Altman LC, Bousquet J, Davies RJ, Gell L, et al: Prevalence of basidiomycete allergy in the USA and Europe and its relationship to allergic respiratory symptoms.
Allergy 1994;49:460–465.
Helbling A, Gayer F, Pichler WJ, Brander KA: Mushroom (Basidiomycete) Allergy: Diagnosis
established by skin test and nasal challenge. J Allergy Clin Immunol 1998;102:853–858.
Bruce CA, Norman PS, Rosenthal RR, Lichtenstein LM: The role of ragweed pollen in autumnal
asthma. J Allergy Clin Immunol 1977;59:449–459.
Malling HJ: Diagnosis and immunotherapy of mould allergy. IV. Relation between asthma
symptoms, spore counts and diagnostic tests. Allergy 1986;41:342–350.
Epton JE, Martin IR, Graham P, Healy PE, Smith H, Balasurbramaniam R, Harvey IC, Fountain
DW, Hedley J, Town GI: Climate and aeroallergen levels in asthma: A 12-month prospective study.
Thorax 1997;52:528–534.
Norter V, Hansen BM, Felton G, Schulz KH: Mushroom workers’ lung caused by inhalation of spores of the edible mushroom Pleurotus florida. Dtsch Med Wochenschr 1976;101:
1241–1245.
O’Brien IM, Bull J, Creamer B, Sepulveda R, Harries M, Burge PS, Pepys J: Asthma and extrinsic allergic alveolitis due to Merulius lacrymans. Clin Allergy 1978;8:535–542.
Sastre J, Ibañez MD, Lopez M, Lehrer SB: Respiratory and immunological reactions among
shiitake (Lentinus edodes) mushroom workers. Clin Exp Allergy 1990;20:13–19.
Lopez M, Voigtlander JR, Lehrer SB, Salvaggio JE: Bronchoprovocation studies in basidiosporesensitive allergic subjects with asthma. J Allergy Clin Immunol 1989;84:242–246.
Helbling A, Brander KA, Gayer F, Fischer B, Borbély P, Bonadies N, Pichler WJ: IgE-vermittelte
Allergie auf Ständerpilze (Basidiomyzeten). Allergol J 2000;9:271–275.
Allergy to Basidiomycetes
45
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
de Bruin-Weller MS, Bruijnzeel-Koomen CAFM: Atopy patch testing – a diagnostic tool. Allergy
1999;54:784–791.
Fischer B, Yawalkar N, Brander KA, Pichler WJ, Helbling A: Coprinus comatus (shaggy cap) is a
potential source of aeroallergen that may provoke atopic dermatitis. J Allergy Clin Immunol 1999;
104:836–841.
Bruhn JN, Soderberg MD: Allergic contact dermatitis caused by mushrooms. Mycopathologia
1991;115:191–195.
Ueda A, Obama K, Aoyama K: Allergic contact dermatitis in shiitake (Lentinus edodes [Berk]
Sing) growers. Contact Dermatitis 1992;26:228–233.
Rosina P, Chieregato C, Schena D: Allergic contact dermatitis from Pleurotus mushroom. Contact
Dermatitis 1995;33:277–278.
Torricelli R, Johansson SGO, Wüthrich B: Inhalative and ingestive allergy to the mushroom
Boletus edulis. Allergy 1997;52:747–751.
Roncarolo D, Minale P, Mistrello G, Voltolini S, Falagiani P: Food allergy to Boletus edulis.
J Allergy Clin Immunol 1998;101:850–851.
Levy AM, Kita H, Philips F, Schkade PA, Dyer PD, Gleich GJ, Dubravec VA: Eosinophilia and
gastrointestinal symptoms after ingestion of shiitake mushrooms. J Allergy Clin Immunol
1998;101:613–620.
Lacaz CdS, Heins-Vaccari EM, Takahashi de Melo N, Hernandez-Arriagada GL: Basidiomycosis:
A review of the literature. Rev Int Med Trop São Paulo 996;38:379–390.
Nenoff P, Friedrich T, Schwenke H, Mierzwa M, Horn L-C, Haustein U-F: Rare and fatal simultaneous mould infection of the lung caused by Aspergillus flavus and the basidiomycete Coprinus
sp. in a leukemic patient. J Med Vet Mycol 1997;35:65–69.
Verweij PE, van Kasteren M, van de Nes J, de Hoog GS, de Pauw BE, Meis JFGM: Fatal
pulmonary infection caused by the basidiomycete Hormographiella aspergillata. J Clin Microbiol
1997;35:2675–2678.
de Hoog GS, van den Ende AHGG: Molecular diagnostics of clinical strains to filamentous
Basidiomycetes. Mycoses 1998;41:183–189.
Davis WE, Horner WE, Salvaggio JE, Lehrer SB: Basidiospore allergens: Analysis of Coprinus
quadrifidus spore, cap and stalk extracts. Clin Allergy 1988;18:261–267.
Weissman DN, Halmepura L, Slavaggio JE, Lehrer SB: Antigenic/allergenic analysis of basidiomycete cap, mycelia and spore extracts. Int Arch Allergy Appl Immunol 1987;84:56–61.
Horner WE, Helbling A, Lehrer SB: Basidiomycete allergens: Comparison of three Ganoderma
species. Allergy 1993;48:110–116.
Helbling A, Horner WE, Lehrer SB: Identification of Psilocybe cubensis spore allergens by
immunoprinting. Int Arch Allergy Immunol 1993;100:263–267.
Horner WE, Levetin E, Lehrer SB: Basidiospore allergen release: Elution from intact spores.
J Allergy Clin Immunol 1993;92:306–312.
Horner WE, Ibanez MD, Lehrer SB: Immunoprint analysis of Calvatia cyathiformis allergens.
I. Reactivity with individual sera. J Allergy Clin Immunol 1989;87:784–792.
Horner WE, Ibanez MD, Lehrer SB: Stability studies of Calvatia cyathiformis allergens. Int Arch
Allergy Appl Immunol 1989;90:174–181.
Horner WE, Lopez M, Salvaggio JE, Lehrer SB: Basidiomycete allergy: Identification and
characterization of an important allergen from Calvatia cyathiformis. Int Arch Allergy Appl
Immunol 1991;94:359–361.
Levetin E, Horner WE, Lehrer SB: Morphology and allergenic properties of basidiospores from
Calvatia species. Mycologia 1992;84:759–767.
Lehrer SB, Lopez M, Butcher BT, Olson J, Reed M, Salvaggio JE: Basidiomycete mycelia
and spore-allergen extracts: Skin test reactivity in adults with symptoms of respiratory allergy.
J Allergy Clin Immunol 1986;78:478–485.
Butcher BT, O’Neil CE, Reed MA, Altman LC, Lopez M, Lehrer SB: Basidiomycete allergy:
Measurement of spore-specific IgE-antibodies. J Allergy Clin Immunol 1987;80:803–809.
Brander KA, Borbely P, Crameri R, Pichler WJ, Helbling A: IgE-binding, proliferative responses
and skin test reactivity to Cop c 1, the first recombinant allergen from the basidiomycete Coprinus
comatus. J Allergy Clin Immunol 1999;104:630–636.
Helbling/Brander/Horner/Lehrer
46
55
56
57
58
59
60
61
62
63
64
65
66
67
68
Hasnain SM, Wilson JD, Newhook FJ: Allergy to basidiospores: Immunologic studies. N Z Med
J 1985;98:342–346.
Singh AB, Gupta SK, Pereira BMJ, Prakash D: Sensitization to Ganoderma lucidum in patients
with respiratory allergy in India. Clin Exp Allergy 1995;25:440–447.
Vijay HM, Comtois P, Sharma R, Lemieux R: Allergenic components of Ganoderma applanatum.
Grana 1991;30:167–170.
De Zubiria A, Horner WE, Lehrer SB: Evidence for cross-reactive allergens among basidiomycetes: Immunoprint-inhibition studies. J Allergy Clin Immunol 1990;86:26–33.
Helbling A, Gayer F, Brander KA: Respiratory allergy to mushroom spores: Not well recognized
but relevant. Ann Allergy Asthma Immunol 1999;83:17–19.
Horner WE, Ibañez MD, Liengswangwong V, Salvaggio JE, Lehrer SB: Characterization of
allergens from spores of the oyster mushroom, Pleurotus ostreatus. J Allergy Clin Immunol 1988;
82:978–986.
O’Neil CE, Hughes JM, Butcher BT, Salvaggio JE: Basidiospore extracts: Evidence for common
antigenic/allergenic determinants. Int Arch Allergy Appl Immunol 1988;85:161–166.
Helbling A, Horner WE, Lehrer SB: Identification of Psilocybe cubensis spore allergens by
immunoprinting. Int Arch Allergy Immunol 1993;100:263–267.
Reese G, Horner WE, Lehrer SB: Basidiomycete allergens. Characterization of epitopes with
monoclonal antibodies raised against Psilocybe cubensis spore extract; in Kraft D, Sehon A (eds):
Molecular Biology and Immunology of Allergens. Boca Raton, CRC Press, 1993, pp 275–277.
Horner WE, Reese G, Lehrer SB: Identification of the allergen Psi c 2 from the basidiomycete
Psilocybe cubensis as a fungal cyclophilin. Int Arch Allergy Immunol 1995;107:298–300.
O’Neil C, Horner WE, Reed M, Lopez M, Lehrer SB: Evaluation of Basidiomycete and
Deuteromycete (fungi imperfecti) extracts for shared allergenic determinants. Clin Exp Allergy
1990;20:533–538.
Crameri R, Suter M: Display of biologically active proteins on the surface of filamentous phage;
A cDNA cloning system for selection of functional gene products linked to the genetic information responsible for their production. Gene 1993;137:69–75.
Crameri R, Kodrius R, Konthur Z, Leehracl H, Blaser K, Walter G: Tapping allergen repertoires
by advanced cloning technologies. Int Arch Allergy Immunol 2001;124:43–47.
Crameri R: Recombinant Aspergillus fumigatus allergens: From the nucleotide sequence to
clinical applications. Int Arch Allergy Immunol 1998;115:99–114.
Prof. Samuel B. Lehrer, PhD, School of Medicine,
Department of Medicine SL57, Section of Clinical Immunology and Allergy,
1700 Perdido Street, New Orleans, LA 70112 (USA)
Tel. 1 504 588 5578, Fax 1 504 584 3686, E-Mail sblehrer@tulane.edu
Allergy to Basidiomycetes
47
Breitenbach M, Crameri R, Lehrer SB (eds): Fungal Allergy and Pathogenicity.
Chem Immunol. Basel, Karger, 2002, vol 81, pp 48–72
The Allergens of Cladosporium
herbarum and Alternaria alternata
Michael Breitenbach, Birgit Simon-Nobbe
Department of Genetics and General Biology, University of Salzburg,
Salzburg, Austria
The two mold species Cladosporium herbarum and Alternaria alternata
are important causes of allergies. Before 1990 little was known about the relevant allergens of these two worldwide-occurring fungi.
Both molds are important sources of allergens for mold-allergic patients
(perhaps after Aspergillus fumigatus [1]) and are not pathogens except, as
reported in recent years, for a minority of immuno-compromised patients [2–4].
However, A. alternata can be cultured from hypersensitivity pneumonitis [5].
A question that is largely unanswered even today is: What is the most important
way of sensitization of the patients? Several possibilities come to mind, among
them inhalation of dried mycelia in house dust or inhalation of spores from outdoor or indoor sources. It is well known that the spores of the two molds are
common in indoor and outdoor air [6]. The molds grow in soil, on decaying plant
material and as plant pathogens. Recently it was found that both C. herbarum and
A. alternata are also common plant endophytes. They grow within the extracellular space of plants without causing disease [H.J. Prillinger, pers. commun.].
Most of the allergens identified so far from the two molds are intracellular
housekeeping proteins. Notable exceptions usually are the major allergens, which
in the case of A. alternata and A. fumigatus are secreted proteins. It is an open
question how the intracellular proteins are presented to the immune system. The
spores of the two molds discussed here are too large to reach the alveoli of the
lung, but they may still be important inhalant allergens. However, none of the
allergens identified so far are spore specific.
What is further missing are longitudinal studies on children allergic to
A. alternata and C. herbarum, epidemiologic studies and a sufficient number of
well-documented case histories to draw conclusions about the ‘typical’ symptoms and time course of the mold-allergic patient.
Composting and mushroom-growing facilities have been suspected widely to
be dangerous to the workers exposed to the emanating mold spores and ‘organic
dust’. There seems to be a severe health risk of developing [7–10] allergic bronchopulmonary aspergillosis (ABPA) and other forms of pulmonary complications
only in asthmatic and atopic patients, but these are mainly due to thermophilic
actinomycetes and A. fumigatus. C. herbarum and A. alternata do occur in compost, however, in much smaller numbers than the species just mentioned.
The incidence of C. herbarum and A. alternata sensitization varies in the
different climatic zones of the world. In a European multicenter study [11] it
was demonstrated that 3–20% of all allergic patients tested showed positive
responses to A. alternata and/or C. herbarum in skin prick tests (SPT). A survey
done in Israel [12] revealed that 3.3 and 12% of the allergic patients had positive SPT responses to C. herbarum and A. alternata, respectively. In Austria the
number of patients with positive SPT responses to the two molds can be estimated as follows: in a large outpatient clinic, from where all sera used for the
investigations of the authors [13–16] of the present review were obtained, about
15,000 new patients are diagnosed as allergics every year. About 3% of those
patients usually show positive SPT and serum radioallergosorbent test (RAST)
responses to a ‘mold mix’. Considering the low quality of the commercial mold
extracts, the number may be underestimated, as assumed for all fungal allergies
[1]. About 30% of these mold allergics proved to be sensitized to A. alternata
and/or C. herbarum, as shown by ‘patient blots’ (IgE-specific immunoblots of
mold extract) performed in our laboratory. Our own mold extracts usually produced many more bands and contained more undegraded allergens than commercially available mold extracts. The patients were rarely sensitized to just one
mold species but in most cases to several mold species. One reason for this
might be the presence of cross-reactive phylogenetically conserved allergens
(discussed later in this chapter).
Problems with Reproducibility of Mold Extracts and
the Study of Mold Allergens
We will now discuss some of the reasons why mold allergens are more difficult to standardize than other aeroallergens such as pollen allergens and why
some of the commercially available extracts are very poor when analyzed for
the presence of major allergens. These commercial extracts were also inferior
to pure recombinant allergens when compared in clinical tests. Methods developed in our laboratory for optimal production and extraction of mold allergens
will be described.
C. herbarum and A. alternata Allergens
49
It is difficult to unequivocally identify closely related species and strains
by classical morphological criteria. Little is known about genetic variation
below the species level in these organisms. No sexual forms (ascospores) of
these molds are known, therefore identification on purely morphological criteria is difficult. The strains used by us were well-characterized strains from a
major European strain collection (Institut für Gärungsgewerbe, Berlin,
Germany). Identification was based on morphology and molecular criteria
(rDNA sequences; see Prillinger et al. [17, this volume]). It is unknown at the
moment how large intra species genetic variation is in C. herbarum and
A. alternata. The Alt a 1 cDNA sequences isolated from cDNA libraries which
originated from European and North American strains showed minor sequence
differences at the nucleotide level but no sequence differences at the amino acid
level [18] (also see ‘The major allergen of A. alternata, Alt a 1’ below).
The total number of relevant allergens is larger in molds than in pollens
and foodstuffs. The total number of IgE-reactive allergens from A. fumigatus
may be as high as 100 [19, this volume]; in C. herbarum so far at least 36 allergens have been cloned (also see ‘Other Allergens’ below). This large number of
partly comigrating proteins in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) makes it nearly impossible to unequivocally identify
a protein band in a so-called patient blot, particularly in the range of 30–60 kD.
This difficulty can only be overcome using pure recombinant or highly purified
natural allergens. However, based on data to be presented later it is likely that a
small number of purified recombinant allergens of the two molds are sufficient
to correctly diagnose patients sensitized to the two molds with a sensitivity and
specificity superior to those obtained with commercial extracts.
Growth conditions: The presence of specific allergens (including the major
allergens!) depends very much on the growth conditions. For instance, we
showed that when growing the molds on petri dishes containing complex
medium, allergen content was optimal on day 5, at the beginning of extensive
formation of conidiospores as tested by immunoblots. Complex medium contained 1% yeast extract, 2% peptone and 2% glucose solidified with 2% agar
and supported vigorous growth of C. herbarum and A. alternata. Growing the
molds in liquid medium with shaking or stirring resulted in much lower yields
of allergens. It is well known that for industrial purposes and for the production
of commercially available mold extracts, the fungi are batch grown for different periods of time in liquid tanks. This might partly explain the poor properties of these extracts.
Protein extraction methods are critical. In previous studies various extraction procedures have been tested. Paris et al. [20] could show that after breaking A. alternata cells, the use of carbonate buffer supplemented with protease
inhibitors (phenylmethylsulfonyl fluoride) and phenol-binding components
Breitenbach/Simon-Nobbe
50
(polyvinylpyrrolidone) resulted in better extracts than with the use of Coca’s
solution containing sodium carbonate and phenol. Portnoy et al. [21] tested four
different buffers and various extraction times in order to evaluate their effects
on the protein composition and content of the respective A. alternata extracts.
They could not determine an optimal extraction time valid for every allergen.
With respect to the different extraction buffers they could not detect major differences between the buffers tested, except that low pH resulted in a lower yield.
In our laboratory the following method for allergen extraction was developed. The fungal ‘mat’ was carefully removed from the surface of the petri dish
after 5 days of growth and immediately put into a mortar filled with liquid
nitrogen. The frozen material was ground under liquid nitrogen and the fine
powder was extracted with extraction buffer containing a mixture of protease
inhibitors and centrifuged at 4 °C [13]. Aliquots of the clear supernatant were
stored at ⫺70 °C. The use of these extraction methods allowed to generate
extracts showing the maximum number and intensity of IgE-binding bands in
immunoblots (fig. 1, 2).
Experience with Specific Immunotherapy in the
Treatment of Mold Allergies
Specific immunotherapy is defined as the administration of increasing
doses of an allergen extract to an allergic patient suffering from IgE-mediated
hypersensitivity. As a prerequisite the allergy has to be determined by an SPT
or an in vitro test like the RAST. Several questions arise in connection with
immunotherapy of mold allergy:
Which in vivo or in vitro tests yield the most reliable results in the diagnosis of fungal allergy?
How can the problem of variability of fungal extracts be overcome?
Is it possible and/or necessary to reduce the frequency of systemic side
effects after specific immunotherapy in order to reduce the patients’ symptoms?
In a placebo-controlled double-blind study [22] it was demonstrated that a
negative SPT response to C. herbarum was always correlated with the absence of
an allergy. In contrast a positive RAST always indicated clinical allergy; it was
thus concluded that a combination of these two tests would result in 100% agreement with a positive clinical diagnosis. In this high-dose immunotherapy study
81% of the group hyposensitized with C. herbarum extract showed an improvement of their clinical symptoms, whereas 19% of the treated patients exhibited
deterioration. In the untreated control group 73% of the patients showed aggravation of the symptoms whereas 27% improved. In a double-blind placebocontrolled study [23] 30 Cladosporium-allergic children suffering from asthma or
C. herbarum and A. alternata Allergens
51
Days of growth
kD
M
5
7
9
11
13
15
18
20
22
97 —
67 —
46 —
30 —
21 —
14 —
Days of growth
kD
5
7
9
11 13 15 18
20
22
97 —
67 —
46 —
30 —
21 —
14 —
Fig. 1. Time course of allergen expression in C. herbarum. a Extracts were prepared
on the days indicated after growth at 28 °C on solid YPD. Fungal proteins were separated by
SDS-PAGE and Coomassie stained. Equal amounts of fungal material were used for every
experiment. Protein bands between 14 and 21 kD, but also Cla h 1 at 30 kD were most
strongly expressed at day 5. b Aliquots of the same samples that were used in a were
separated by SDS-PAGE under the same conditions, blotted onto Immobilon-P membrane
and decorated with a serum pool of C. herbarum-allergic patients. Again it is shown that
the region corresponding to Cla h 1 is most strongly stained on day 5 extracts.
rhinoconjunctivitis and showing a positive SPT and RAST were studied. The
authors observed an increase in tolerance to conjunctival or bronchial challenge
in the 16 treated patients. Cantani et al. [24] reported the effects of Alternaria
immunotherapy on asthma and rhinitis in 79 children in a 3-year study. They
could show that immunotherapy was successful in 80% of the children sensitized
Breitenbach/Simon-Nobbe
52
Extraction time (h)
kD
M
1
6
12
24
36
48
97 —
67 —
46 —
30 —
21 —
14 —
Fig. 2. Time course of protein extraction from C. herbarum cells. C. herbarum was
grown as described in figure 1, harvested on day 5, treated with extraction buffer for varying times and the protein extracts were separated by SDS-PAGE. Routine extraction was
done overnight.
to Alternaria with doses above 80,000 protein nitrogen units (PNU). In another
double-blind placebo-controlled study [25] a ‘rush’ protocol was carried out with
a standardized Alternaria extract. The patients actively treated (13 out of 24) and
exclusively sensitized to Alternaria benefited from specific immunotherapy. The
efficacy was determined by patients’ self-evaluation, global symptom-medication
scores and nasal challenge tests. At the antibody level an unchanged specific IgE
level and an increased specific IgG level were detected.
Immunotherapy with fungal extracts has often been associated with a high
frequency of systemic side effects. It was shown that high doses of Cladosporium
extract (top doses of 100,000 biological units) resulted in an improved therapeutic effect [22]. According to preliminary data from Norman and Lichtenstein [26],
the elicitation of mild side effects was even intended and considered to indicate
that this would result in increased clinical efficacy. Tuchinda and Chai [27]
reported similar observations for Alternaria.
Although several studies about mold immunotherapy have been performed
and have shown clinical efficiency [22–25], the number of patients included in
the respective studies was rather small emphasizing the need for further studies.
One major problem in mold immunotherapy is the lack of reliable fungal
extracts [28–30]. In the last years, molecular biology has considerably contributed
to the solution of this problem. Several allergens of C. herbarum and A. alternata
C. herbarum and A. alternata Allergens
53
have been identified by cloning techniques [13, 14, 31, 32]. Using recombinant
allergens for skin tests Unger et al. [33] demonstrated that recombinant Alt a 1 and
Alt a 5 (enolase) achieved a higher specificity and sensitivity than commercial
extracts. Comparing specific allergen levels in mold extracts, Vailes et al. [33]
used recombinant Alt a 1 (rAlt a 1) (Biomay, Vienna, Austria) as internal standard for the determination of the Alt a 1 content in the different commercial
extracts. As a prerequisite Vailes et al. developed an Alt a 1-specific enzymelinked immunosorbent assay (ELISA) with a monoclonal antibody directed to
natural Alt a 1. ELISA dose response curves showed immunological equivalence
of recombinant (rAlt a 1) and natural Alt a 1 (nAlt a 1). This result suggests that
the biological properties of rAlt a 1 and nAlt a 1 are comparable. The rAlt a 1
used in this study may be suitable for immunotherapy. The use of recombinant
allergens for immunotherapy instead of natural mold extracts is desirable as it
was shown by Birkner et al. [35] that de novo sensitization after an immunotherapy with crude pollen extract can occur. An improvement could be achieved by
injection of only those allergens against which the patient is sensitized. Therefore
the use of recombinant proteins for the diagnosis and therapy of mold allergy
seems desirable.
Importance of Molecular Cloning Techniques
As the importance of molecular cloning techniques for research on mold
allergens has been reviewed several times [36–38], we will not deal with it
extensively here. However, it is clear from the facts just mentioned that the
importance of cloning methods can hardly be overestimated, especially in the
field of mold allergy. Speaking about diagnostics, the allergogram of a patient
often becomes clear only after pure recombinant allergens have been used.
Immunotherapy with mold extracts has not been widely used in the past
because of inherent problems and dangers related to commercially available
mold extracts, but could be done with the well-characterized pure recombinant
mold allergens that are available now.
Cloning, Analysis, Production and Clinical Testing of the
Allergens of Cladosporium and Alternaria
Cloning Methods
In our first attempts to clone the most important allergens of the two molds,
cDNA libraries were prepared in phage vectors and standard immunological
Breitenbach/Simon-Nobbe
54
screening methods were employed. A technical detail that seems to be important for the preparation of cDNA libraries from fungal materials is the presence
of a large excess of carbohydrate polymers in the RNA preparation from fungal
sources. Additionally, unidentified phenolic compounds that tend to bind to the
RNA have to be removed prior to subsequent in vitro enzymatic steps. Special
methods were developed to overcome these problems [13].
Growth conditions of the two molds were chosen exactly as described
above for protein extraction, i.e. the molds were cultured on solid YPD for
5 days. The reasoning was that the material containing all of the allergens should
also contain the corresponding mRNAs, which are the starting material for
cDNA synthesis. Care was taken to ensure that the starting material contained
vegetative hyphae as well as conidiospores, as we did not know beforehand
whether any one of the important allergens was spore specific. In reality it was
found that only a small minority of the allergens were actually spore specific
while the majority of the allergens were so-called housekeeping proteins.
PolyA⫹mRNA was purified from the mold material, and double-stranded
cDNA was prepared [13]. The cDNA was cloned directionally (EcoRI/XhoI)
into the cloning site of the phage vector, -ZAP II (Stratagene, La Jolla, Calif.,
USA) following the manufacturer’s instructions. Using -ZAP II instead of the
more conventional -gt11 has three major advantages: (1) directional cloning
improves the chance to create a correct in frame fusion; (2) the vector contains
a much smaller fusion part (the ␣-fragment of Escherichia coli -galactosidase)
of the recombinant protein as compared to -gt11, enabling much easier
purification and immunological testing of the recombinant fusion protein, and
(3) -ZAP II offers the possibility of ‘in vivo excision’ after obtaining a positive
clone, thus greatly speeding up the cloning procedure. The number of plaqueforming units of the primary libraries was always greater than 1 million. After
amplification the libraries were used for immunological screening with appropriate patient sera. It is our experience that in principle the majority of the
important allergens from one mold source can be cloned by this method.
However, there are exceptions that will be discussed. In all cases, the primary
immunopositive phage clones were plaque purified to homogeneity. In practice,
this required up to 4 rounds of plaque purification. After in vivo excision and
plasmid DNA preparation the cDNA clones were sequenced [39] and analyzed
for the presence of a complete open reading frame. If an N-terminal deletion
was found, the library was rescreened by plaque hybridzation with a 32P-labeled
probe prepared from the primary clone. Complete cDNA clones could be found
in every case.
The complete open reading frame of the allergen in question was then
recloned in pMW172 [40–42] resulting in a protein sequence identical to the
natural one. In case of secreted allergens (for instance, Alt a 1) the recombinant
C. herbarum and A. alternata Allergens
55
non-fusion (rnf) protein was expressed without the hydrophobic targeting
sequence. Methods were then developed to optimize expression in E. coli and
to purify the rnf protein for laboratory immunological tests and for clinical use.
In two cases (Alt a 1 and Alt a 5) these methods will be described in detail. For all
other allergens, analogous methods were used. We deliberately decided to produce
only rnf proteins (instead of tagged or fusion proteins) to be able to produce
proteins that are structurally and immunologically identical to the natural proteins
thus avoiding a concern for clinical use.
Although the methods described above were clearly successful, we envisaged a number of problems with immunological screening of phage cDNA
libraries for the cloning of mold allergens.
The number of clones corresponding to a given allergen depends on the
relative abundance of the mRNA in the given population of mRNAs which in
turn depends on the physiological status of the cell from which these mRNAs
were derived. In addition, it depends on a number of minor factors like the
possible loss of specific sequences during library amplification. The clone representing a given allergen might therefore be rare in the library and its identification by screening might become difficult.
If the allergen to be cloned is a secreted protein, which is the case for some
major allergens of fungi, like Alt a 1 [17] and Asp f 1 [43], the natural form of
the allergen may be different from the primary translation product. Posttranslational modification, such as proteolytic processing, oxidation to form disulfide bridges and glycosylation, might occur in the natural product. Postsynthetic
modifications of proteins pose a special problem for allergen research. To
test whether postsynthetic modifications were present on the highly purified
natural proteins, sugar analysis [44] as well as physicochemical measurements,
in particular mass spectrometry (MALDI-TOF-mass spectrometry [45]) of
the highly purified natural and recombinant proteins were employed. Some of the
postsynthetic modifications may have a strong influence on IgE binding. While
it is relatively easy to mimic the proteolytic trimming and oxidation in vitro,
glycosylation remains a problem. These items will be discussed, considering, as
an example, Alt a 1. The clinical importance of glycosylation for allergenicity
and for IgE binding is presently an open question.
Finally, the reducing milieu in the E. coli cell together with the absence of
postsynthetic modifications can in some cases be an obstacle for the correct
folding of a protein, thus preventing the isolation of the correct clone by
immunological screening with patient sera.
At least in principle, many of the problems just described can be overcome
by using the phage display technique [46], a method described in detail elsewhere in this volume [19]. Only a very short summary of the advantages of the
method is given here: The system produces filamentous phage particles which
Breitenbach/Simon-Nobbe
56
Fig. 3. Alignment of N-terminal sequences of Alt a 1. N-terminal peptide sequences of
Alt a 1 [48, 50, 51] were compared with the complete open reading frame of Alt a 1 [16, 18].
Identical amino acids are marked in grey. The numbering of the amino acids is according to
the complete open reading frame. Curran et al. [48] as well as Aden [50] identified 2 different variants with minor differences in the respective N-terminal sequences. Dashes represent
gaps in the sequence. Dots indicate that this part of the protein was not sequenced. The first
5 sequences were determined by solid-phase Edman degradation of nAlt a 1. The last two
sequences are deduced from cDNA sequences.
carry both the genetic information for a clone in their genome and the protein
encoded by the insert on their surface. This makes possible repeated rounds of
enrichment by bio-panning. Rare clones (say, 1 positive clone in 1 million
clones) can be isolated using this system [47]. In principle, the assembly of the
phage in the oxidizing milieu of the periplasmic space of E. coli should enable
disulfide bridges to be formed and should improve the folding of the recombinant protein and, therefore, its immunological reactivity. The results obtained so
far show that relatively large numbers of new allergen-encoding cDNAs of
C. herbarum can be cloned and that all of the clones tested so far code for
IgE-binding proteins.
The Major Allergen of A. alternata, Alt a 1
A 30-kD protein was recognized on nonreducing SDS-PAGE that reacted
with IgE of the majority of A. alternata-sensitized patients. Attempts to purify
this protein in several laboratories resulted in N-terminal amino acid sequences
determined by Edman degradation [17, 48–51]. A listing of these sequences
(fig. 3) shows rather large variability, which is, however, not due to genetic variability of the protein, but, rather, to inaccurate sequencing. It will become clear
from the following discussion why we can make such a statement. All attempts
to isolate a cDNA clone coding for Alt a 1 by screening standard gt11 or -ZAP
II expression libraries with patient sera failed [Vijay, pers. commun. and own
experience]. Finally, a cDNA clone was isolated in Vijay’s laboratory by screening a cDNA library in gt11 prepared from mRNA of a North American isolate
of A. alternata with a rabbit polyclonal antiserum raised against highly purified
C. herbarum and A. alternata Allergens
57
natural Alt a 1 [18]. In our own laboratory, a cDNA clone coding for Alt a 1 was
isolated by PCR cloning from a cDNA library prepared from mRNA of a
European isolate of A. alternata [16, 33]. The cDNA sequences were remarkably similar, with only 3 silent nucleotide differences [16] in the coding region
leading to identical deduced amino acid sequences. A comparison of the cDNA
sequence with the published N-terminal amino acid sequences showed that the
mature natural protein starts at aspartic acid 26. Computer analysis of the
deduced amino acid sequence gave clear evidence for an N-terminal hydrophobic leader sequence typical for ER targeting and also correctly predicted the
mature N-terminal amino acid. Thus the molecular weight of the protein is predicted to be 14.6 kD, which is observed under reducing conditions (SDS-PAGE
in the presence of -mercaptoethanol). However, under nonreducing conditions,
the protein migrates with an apparent molecular weight of 29–30 kD. The conclusion is that the natural allergen is a secreted protein, which presumably
resides in the periplasmic space of A. alternata cells, and must be either a
homodimer or a heterodimer consisting of two similar subunits. There is now
strong evidence that the allergen is, in fact, a homodimer: Firstly, the 14.6-kD
subunit cloned by us and De Vouge et al. [18] spontaneously dimerized in air,
and the resulting 29-kD protein shows full immunological reactivity with a
large number of patient sera. Cross-inhibition experiments showed that preincubation of serum with the recombinant homodimer completely inhibited the
appearance of the Alt a 1 band in the patient blots performed with A. alternata
extract [16]. Secondly and more importantly, this homodimer was used to raise
monoclonal antibodies and to create a quantitative immunological test (ELISA).
The ELISA dose-response curves for highly purified natural and recombinant Alt
a 1 were shown to be superimposable, thus showing that these two proteins
exhibit identical affinity and avidity for the antibody used [34]. Thirdly, it was
claimed that the second subunit present in the presumed heterodimer Alt a 1
should have a very similar but not identical amino acid sequence to Alt a 1 based
on the different N-terminal amino acid sequences obtained (fig. 3). We used the
cDNA clone coding for the 14.6-kD Alt a 1 subunit for hybridization screening
of the library, resulting in 12 new cDNA clones, all of identical sequence. This
renders the existence of a closely related but not identical second subunit improbable and also shows that different isoforms of Alt a 1 probably do not exist.
PCR methods were used to obtain a genomic clone of Alt a 1 (GenBank
AF288160) which displayed a short intron of 60 nucleotides after bp 344, as
shown by DNA sequence determination. In the same experiment, we showed by
genomic Southern blots that just one gene coding for the Alt a 1 subunit is
present on the haploid genome of A. alternata [16]. It is remarkable that extensive homology searches up to now revealed no Alt a 1 homolog from other
species and thus no clue as to the biological function of this major allergen.
Breitenbach/Simon-Nobbe
58
The Major Allergen, Cla h 1
Cla h 1, is the most important allergen of C. herbarum in terms of frequency of sensitization. Performing IgE blots of C. herbarum extract with
about 100 C. herbarum-sensitized individuals revealed that 61% of the patients
tested reacted specifically with a single, 30-kD protein band [13]. We termed
this protein Cla h 1.
In a first attempt it was not possible to clone the Cla h 1-encoding cDNA
by screening of a C. herbarum cDNA expression library with patient sera.
There are various possible explanations for this result: on the one hand it is conceivable that the mRNA coding for this allergen was short-lived or for other reasons underrepresented in the library. On the other hand it might be that the
overall three-dimensional structure of the protein expressed in E. coli is different from the native structure and, therefore, not recognized by human IgE antibodies. This could be due to posttranslational modifications of the protein or
simply to the reducing milieu in the E. coli cell and/or denaturing conditions
during plaque lifting hampering correct folding of the protein. We met similar
problems during our research on Alt a 1, the major allergen of A. alternata (see
the previous section of this chapter). This allergen is not recognized by IgE after
plaque lifting and the disulfide-bridged homodimeric protein could therefore
not be cloned using patient sera.
In two-dimensional IgE immunoblots, Cla h 1 was detected as a welldefined major spot with a minor spot probably corresponding to an isoform of
the allergen [15]. We have now purified the protein to homogeneity by preparative SDS-PAGE and raised a polyclonal rabbit antiserum against Cla h 1. This
will be used as described for Alt a 1 to clone the cDNA coding for Cla h 1 by
immunoscreening of the C. herbarum library.
As a complementary strategy, we will reclone the inserts of our new
-ZAP II library into the modified pJuFo vector pGA [46 and Achatz et al.,
unpubl. obs.] and select for IgE-binding phagemids as well as for phagemids
binding our polyclonal rabbit serum.
Enolase, an Important Allergen in C. herbarum and A. alternata,
Exhibits Cross-Reactive Properties
The glycolytic enzyme enolase (EC 4.2.1.11) catalyzes the interconversion
of 2-phospho-D-glycerate and phosphoenolpyruvate. Due to its essential role
in glycolysis, enolase is a rather highly conserved enzyme, which has been
identified and characterized from diverse sources ranging from bacteria to
C. herbarum and A. alternata Allergens
59
higher vertebrates. The active form of the enzyme is a dimer either forming a
heterodimer (e.g. Saccharomyces cerevisiae) [52] or a homodimer (e.g. Candida
albicans) [53].
Enolase was first identified as an allergen in S. cerevisiae [54–56] and
C. albicans [57]. Subsequently, we cloned the enolases of C. herbarum (Cla
h 6) [13] and A. alternata (Alt a 5) [14]. Testing the IgE-binding properties of
Cla h 6 and Alt a 5 revealed that 22% of the C. herbarum- and/or A. alternataallergic patients specifically reacted with the respective enolase. Investigation
of the protein sequence homologies between enolases from C. herbarum,
A. alternata, S. cerevisiae and C. albicans revealed identities ranging from 73
to 89%, the latter being observed between Cla h 6 and Alt a 5. The sequence
data together with observations of immunological cross-reactivity [54, 58]
made it very probable that enolase represents a fungal pan-allergen. Therefore
we performed a rather detailed investigation of the IgE-based cross-reactivity
between the enolases from C. herbarum, A. alternata, S. cerevisiae, C. albicans
and A. fumigatus. Analysis of IgE cross-reactivity was performed by crossinhibition tests as described [14]. All cross-inhibition experiments were carried
out with several patient sera stressing that the results obtained are not only true
for a single patient, but are of general relevance. Taken together, our results
clearly show extensive IgE cross-reactivity between fungal enolases.
Comparison of the IgE reactivities between Cla h 6 and Alt a 5 revealed
that preincubation of patient sera with rnfCla h 6 completely abolished
specific IgE reactivities to rnfAlt a 6. On the contrary, the sera depleted with
rnfAlt a 5 exhibited residual IgE binding to rnfCla h 6. Based on these experiments we conclude that Cla h 6 contains additional IgE-reactive epitopes as
compared with Alt a 5. A similar result was obtained when the cross-reactivity
between the enolases of C. herbarum and A. fumigatus was investigated.
Performing cross-inhibition experiments between Cla h 6 and the enolase of
S. cerevisiae we could show that both enolases seem to have the same IgEreactive epitopes, as no residual IgE reactivity was observed when the enolase
of S. cerevisiae was incubated with rnfCla h 6-depleted sera and vice versa.
Investigation of IgE cross-reactivities between Cla h 6 and the enolase of
C. albicans showed that there exist cross-reactive epitopes but, moreover, the
enolase of C. albicans also contains additional non-cross-reactive IgE-binding
epitopes.
Recently, enolase has also been shown to be an allergen in Hevea brasiliensis latex [58, 59]. Comparison of the protein sequences of the enolase of
H. brasiliensis with Cla h 6 and Alt a 5, revealed sequence identities between 62
and 60%. Although these identities are less close than those observed among
fungal enolases they are high enough to generate cross-reactivity between these
allergens [60].
Breitenbach/Simon-Nobbe
60
Consequently, enolase can be called a highly cross-reactive allergen, which
is not only a fungal pan-allergen, but also exhibits cross-reactivity with latex
enolase.
Epitope Mapping of C. herbarum Enolase
For a more detailed investigation of C. herbarum enolase, epitope mapping
was performed. Among the various methods available, we chose an approach
based on the polymerase chain reaction. Eight different oligonucleotide primers
equally distributed along the entire molecule were designed, whereby four of
them were ‘forward’ and the other four were ‘reverse’ oriented. These primers
were used to amplify 10 different, partially overlapping peptides of Cla h 6, all
of which were fused to the non-IgE-binding glutathione-S-transferase (GST) of
E. coli. Investigation of the IgE reactivities of these ten fusion proteins was performed with sera of 10 patients sensitized to C. herbarum. Analysis of the
respective immunoblots revealed that 6 of 10 peptides specifically reacted with
the sera of those patients, which also reacted with rnfCla h 6. Two peptides only
displayed IgE reactivity with few patients and another 2 peptides did not exhibit
any IgE reactivity at all. Control experiments showed that GST alone did not
bind patient IgE and that none of the peptides unspecifically reacted with the
sera of nonatopics.
One of the 6 IgE-reactive peptides, namely GST-rCla h 6 (120–189),
represents the overlapping region of the other 5 larger IgE-reactive fusion
peptides, indicating that this peptide harbors at least one immunodominant IgE
epitope of C. herbarum enolase. In order to investigate the topology of this peptide in the context of the entire enolase, modeling of the C. herbarum enolase
structure was performed [14] based on the crystal structure of S. cerevisiae
enolase-1 [61]. Modeling seemed possible as the enolase sequences from
C. herbarum and S. cerevisiae are 74% identical. The three-dimensional structure of Cla h 6 modeled in this way is shown in figure 4. The peptide rCla h 6
(120–189) is highlighted in the context of the entire protein. Based on the
modeling, it is apparent that rCla h 6 (120–189) exhibits an extended structure
spanning the body of the globular protein twice and reaching the surface three
times. The marked amino acids depict possible IgE-binding sites (fig. 4).
The 69 amino acids long peptide rCla h 6 (120–189) was not only
expressed as GST fusion protein but also subcloned as a nonfusion peptide.
Comparison of the IgE reactivity of the GST fusion and the nonfusion peptide
revealed that the GST fusion part seems to support a native-like threedimensional structure as the nonfusion peptide itself did not bind patient IgE.
For clarifying this observation, we analyzed the solution structure of rCla
C. herbarum and A. alternata Allergens
61
P177
K141
R127
R163
Fig. 4. Modeled three-dimensional structure of C. herbarum enolase. C. herbarum
enolase was modeled on the basis of the three-dimensional structure of S. cerevisiae enolase
[60, 62]. The C. herbarum enolase monomer is shown in magenta, whereas the peptide rCla
h 6 (120–189) is displayed in blue. Those residues which were predicted to interact with
patient IgE are marked. For further explanations, see text.
h 6 (120–189) fused to a 6xHis tag by circular dichroism spectroscopy (fig. 5).
The results indicate that the nonfusion peptide may adopt a new structure in
solution, which results in a loss of IgE reactivity.
Other Allergens
Since the 1980s several groups have undertaken the task of identifying the
allergens of A. alternata and C. herbarum by means of SDS-PAGE, Western
Blot, crossed radioimmunoelectrophoresis, crossed immunoelectrophoresis,
isoelectric focusing, RAST and RAST inhibition assays [63–69]. In the 1990s
the knowledge about fungal allergens has increased dramatically by the use of
molecular biological techniques.
Before giving an overview about our own work, we will briefly summarize
the cloning results together with data obtained by protein purification and
Breitenbach/Simon-Nobbe
62
0
mdeg
⫺5
⫺10
⫺15
185
195
205
215
225
235
245
255
Wavelength (nm)
Fig. 5. Circular dichroism spectroscopy of peptide (120–189) fused to a 6xHis-tag.
The circular dichroism spectrum of 6xHis-rCla h 6 (120–189) (protein concentration was
27 M in salt-free water) was recorded on a Jasco spectropolarimeter (J-810) at 185–260 nm.
Computer-assisted analysis of the data showed that the peptide is folded with 11% helix,
35% -sheet, 27% loop and 26% random structural elements.
characterization by various groups:
A. alternata
By immunological screening of an A. alternata gt11 cDNA library, an
IgE-binding fragment was identified, showing sequence homology to hsp70
(70-kD heat shock protein) from C. herbarum. This allergen, termed Alt a 3, is
recognized by 5% of the allergic subjects [70].
Alt a 2, another allergen, was also identified by immunological screening of
a cDNA library. Analysis of the cDNA did not reveal any homology to a known
allergen but to a transposable region and a mouse RNA-dependent eukaryote
initiation factor-2 a-kinase. The recombinant protein is recognized by 61% of the
A. alternata-allergic patients, rendering Alt a 2 a major allergen [32].
C. herbarum
The allergen Ag-54 (Cla h II) was isolated by protein-chemical methods
and represents a 25-kD protein with an isoelectric point of 5.0 [29, 71, 72]. This
allergen contains a large carbohydrate moiety accounting for 80% of its mass.
C. herbarum and A. alternata Allergens
63
Table 1. List of C. herbarum and A. alternata allergens
Allergen source
Systematic and
original names
A. alternata
Alt-1
Alt a 1
Alt a 29K
Alt a 11563
Alt a 1
Alt a 1
Alt a 2
Alt a 3
heat shock protein 70
Alt a 4
Alt a 5
Alt a 6
protein disulfide isomerase
enolase
acidic ribosomal protein P2
57
45
11
Alt a 7
Alt a 10
Alt a 12
YCP4 homolog protein
aldehyde dehydrogenase
acidic ribosomal protein P1
22
53
11
C. herbarum
Biological function
MW, kD Accession No.
References
31
28
29
31
Yunginger et al. [28]
Matthiesen et al. [51]
Curran et al. [48]
Paris et al. [66]
Unger et al. [16]
De Vouge et al. [18, 75]
Bush et al. [32]
De Vouge et al. [70]
25
Cla h 1
13
Cla h 2
23
Cla h 3
Cla h 4
aldehyde dehydrogenase
acidic ribosomal protein P2
53
11
Cla h 5
Cla h 6
Cla h 12
YCP4 homolog protein
enolase
acidic ribosomal protein P1
hsp70
22
46
11
70
U82633
U86752
U62442
U87807
U878078
X84217
U82437
X78222, P42037
U87806
X78225, P42058
X78227, P42041
X84216, P49148
X78228, P40108
X78223, P42039
X77253, P42038
X78224, P42059
X78226, P42040
X85180, P50344
X81860
Simon-Nobbe et al. [14]
Achatz et al. [13]
Achatz et al. [13]
Achatz et al. [13]
Aukrust et al. [71]
Sward-Nordmo et al. [73]
Aukrust et al. [71]
Sward-Nordmo et al. [73]
Achatz et al. [13]
Achatz et al. [13]
Zhang et al. [31]
Achatz et al. [13]
Achatz et al. [13]
Zhang et al. [31]
Information is taken from the ‘Official list of allergens’ of the IUIS Allergen Nomenclature Subcommittee (ftp://biobase.dk/
pub/who-iuis/allergen.list) and supplemented by additional relevant publications.
On the basis of RAST inhibition experiments and deglycosylation of the allergen it was shown that the protein moiety is responsible for the IgE-binding
capacity of Ag-54. This result was stressed by the fact that the carbohydrate
moiety alone did not specifically bind patient IgE [73].
Zhang et al. [31] isolated a cDNA clone (Ch3.1) showing high sequence
homology to hsp70. This allergen is recognized by 38% of the patients allergic
to C. herbarum.
Our group has cloned 11 allergens of C. herbarum and A. alternata by
immunological screening of -ZAP II expression libraries [13–16]. A complete
list of the C. herbarum and A. alternata allergens identified so far including
results from other groups [31, 32, 70, 74, 75] is given in table 1.
Breitenbach/Simon-Nobbe
64
However, the 9 allergens cloned from A. alternata and the 6 allergens
cloned from C. herbarum are not sufficient to explain the complex reactivity
patterns obtained in immunoblots using the corresponding mold extracts
developed with IgE from sera of sensitized patients. Therefore, we have to
assume that the major part of the allergenic structures produced by the molds
are still unknown. In an effort to clone the whole allergen repertoire of
C. herbarum, the premade -Zap II cDNA expression library was mass excised
in vivo [76] and phagemid DNA containing the inserts was isolated from
infected E. coli cells. Agarose-gel-purified cDNA inserts isolated from the
phagemids after EcoRI/XhoI restriction were used to construct a unidirectional C. herbarum phage surface display library in a modified pJuFo vector
[77], as described elsewhere in this volume [19]. The library kept in liquid
phase was affinity enriched for phage-displaying allergenic proteins using
solid-phase-immobilized IgE of a serum pool from C. herbarum-sensitized
individuals [78]. After four rounds of affinity selection, a large population of
phagemids displaying IgE-binding molecules potentially covering the whole
allergen repertoire present in the subcloned library was obtained. Obviously
enrichment of phagemids depends on both the frequency of primary clones
present in the original library and the abundance of allergen-specific IgE present in the serum pool used for screening [47]. A manual screening of a limited number of clones by restriction analysis is likely to detect only the most
efficiently enriched clones present in the large population. To be able to detect
all different clones enriched, more powerful screening formats are required
[79]. We have adapted robot technology to screen enriched cDNA libraries displayed on phage surface by high-density array hybridization [47, 79, 80]. 5,376
single clones from the enriched C. herbarum library were robot-picked and
arrayed onto 384-well culture plates to obtain working copies [47]. After highthroughput PCR amplification all inserts were high-density spotted onto nitrocellulose membranes and iteratively hybridized with labeled probes derived
from the known allergens. These experiments showed that 5 cDNAs previously
identified as coding for C. herbarum allergens were present with different
frequencies among the arrayed colonies. For further hybridization rounds, 10
nonhybridizing clones were chosen and sequenced, and inserts used to prepare
further hybridization probes by PCR [79]. After 3 rounds of hybridization,
more than 80% of the clones were identified, and representative inserts of each
hybridization class were sequenced. This work revealed 28 new sequences
potentially encoding IgE-binding molecules, among these nuclear transport
factor 2, also isolated from A. alternata, thioredoxin and two inserts without
any sequence homology to known proteins already shown to bind IgE from
C. herbarum-sensitized individuals [M. Weichel, pers. commun.]. The Davos
group is now in the process of subcloning all new sequences to produce
C. herbarum and A. alternata Allergens
65
proteins to be tested for their allergenicity and to identify the sequences of the
remaining 20% of the arrayed clones. They hope to be able to reconstruct the
almost complete allergenic repertoire of C. herbarum in the near future.
Production of Highly Purified Alt a 1 and A. alternata Enolase for
Clinical Use
Alt a 1
The cDNA sequence coding for the short (mature) form of the Alt a 1 subunit starting with asp-26 was cloned into the E. coli expression vector
pMW172 [41, 42]. The cloning site is devised so that the (partial) cDNA followed in frame just after the vector-encoded start codon. Purification of the
expressed protein was performed by inclusion body preparation followed by
ammonium sulfate precipitation and DEAE-chromatography, and yielded 99%
purity as estimated by SDS-PAGE and IgE immunoblots. In a nonreducing
PAGE this material displayed an apparent molecular weight of 29–30 kD indicating a homodimer. In a reducing SDS-PAGE Alt a 1 migrated at 14.6 kD.
Patient blots showed that this band strongly reacted with A. alternata-specific
serum IgE.
Samples of the same batch were tested for the absence of any cytotoxic
contaminations in a highly sensitive in vitro test [81]. The recombinant
proteins were added to EBV-transformed cytotoxic T cells in a concentration
range from 0.001 to 10 g/ml. No change in viability or tritium incorporation
was seen in the treated cells. In contrast, cells treated under the same conditions with the toxic allergen Asp f 1 were effectively killed. We conclude that
our preparation of Alt a 1 is nontoxic. Based on this and on earlier observations with other allergens purified using the same method, Alt a 1 and Alternaria
enolase (see below) were approved by the local ethics committee for clinical
testing.
A. alternata Enolase
The coding region of A. alternata enolase cDNA was also cloned into
pMW172 and transformed in E. coli. Purification of expressed Alt a 5 was done
by inclusion body preparation followed by isopropanol and ammonium sulfate
precipitations. Finally Sephacryl chromatography was performed ending up
with a material which appeared as a single band with essentially no contamination on reducing SDS-PAGE.
The immunological reactivity of the protein was determined by IgE
immunoblots and did not differ from that of the native enzyme. Furthermore, the
enzymatic activity of the protein was determined as described [82]. Although there
Breitenbach/Simon-Nobbe
66
are no literature data on the specific activity of A. alternata enolase, we conclude
that based on the values published for yeast enolase-1, a substantial part of the pure
recombinant A. alternata enolase isolated from E. coli is enzymatically active.
Finally, the preparation was tested for toxicity and approved for clinical testing as described above.
A third A. alternata allergen, shown to be a homolog of the yeast protein
YCP4, was also purified as an rnf protein using similar methods as above, tested
for toxicity and approved for clinical use.
Clinical Study with Recombinant A. alternata Allergens
We performed a clinical study [33] at a local hospital during 1997 in order
to assess the diagnostic value of the above-described pure recombinant allergens
of A. alternata in comparison with two commercial A. alternata extracts. Seven
patients with a positive clinical history for A. alternata sensitization, 5 normal
healthy subjects and 5 atopics not sensitized to any mold allergen according to
clinical history and previous SPT, were tested. The study was approved by the
local ethics committee.
All patients and control subjects were first routinely tested as follows: SPT
was performed with a standard panel of aeroallergens (including 5 molds) and
blood samples were taken for total and specific IgE determination. RAST for IgE
antibodies to A. alternata (Pharmacia, Uppsala, Sweden) was performed by using
the allergen-specific CAP system with results converted into RAST classes from
0 to 4 according to the manufacturer’s instructions. No antihistamine medication
was used by any subject. Six of the 7 patients (but no control subjects) were
RAST positive with a RAST class greater than 2. One patient was negative,
but this person still showed a positive skin reaction to rnf A. alternata enolase
(see below).
Both SPT (concentration range 0.1–100 g/ml) and intradermal tests (concentration range 1 ng/ml–1 g/ml) were performed. Positive and negative control
solutions were the same as in the routine SPT (physiological saline and 0.1% histamine, respectively). After 15 min the weal size was measured and photographs
were taken. The two commercial A. alternata extracts used in the study were
Pangramin from ALK (Horsholm, Denmark) and an A. alternata extract from
Stallergènes (Fresnes, France).
Six of the 7 patients reacted positively with rnfAlt a 1 and 2 of 7 patients
reacted positively with rnfAlt a 5 (enolase). The two recombinant allergens
together detected all of the 7 patients. None of the patients reacted with the
minor allergen, A. alternata YCP4. Very importantly, none of the 10 control
subjects showed any unspecific skin reaction with the rnf allergens used,
C. herbarum and A. alternata Allergens
67
showing that these solutions are safe and free of pyrogenic contaminations. The
performance of the commercial extracts was less convincing as 2 out of 10
healthy control subjects reacted positively in intradermal tests with Pangramin,
indicating that this extract contains unspecific pyrogenic compounds which is
not only a problem because of its diagnostic value but also because of safety
considerations. The SPT with the atopic patients revealed that the extract
from Stallergènes detected 7/7 patients whereas Pangramin detected only 5/7
patients.
Therefore we can say that SPT and intradermal testing showed that the two
recombinant allergens, Alt a 1 and A. alternata enolase were superior to the
commercial A. alternata extracts used with respect to positive and negative
predictability in the diagnosis of A. alternata allergy.
Discussion and Outlook
Taken together, all of the facts reported above about the allergens of
A. alternata and C. herbarum show that in spite of considerable problems with
commercially available extracts of the two molds, we are now in a position to
improve the situation through the use of pure rnf allergens of these two molds.
It is to be expected that not only diagnosis (as described in the preceding
section) but also therapy can be substantially improved. This applies not only to
conventional immunotherapy, but also to new forms of therapy (to be discussed
elsewhere) which are all based on knowledge of the complete allergen repertoire and on production and exploration of the immunological properties of rnf
allergens.
Acknowledgments
We are grateful to Hansjörg Prillinger for his help in identifying the molds and for
supplying well-characterized strains of C. herbarum and A. alternata from the strain collection of the Institut für Gärungsgewerbe, Berlin, Germany, and for his help and advice with
strain identifications. We are extremely grateful to Reto Crameri for his support and advice
over many years and for toxicity testing. Both Reto Crameri and Gernot Achatz helped us
with the improved phage display system. We are very much obliged to Markus Susani for
supply of purified nonfusion allergens of A. alternata and C. herbarum.
We are thankful to all present and previous members of our laboratory and to all
our collaborators for their excellent contributions to the research on mold allergens.
These are: Hannes Oberkofler, Andrea Unger, Erich Lechenauer, Gerald Probst, Barbara
Kessler, Ursula Denk, Fatima Ferreira, Peter Briza, Andreas Kungl, Andrey Kajava and Josef
Thalhamer.
This work was supported financially by the Austrian Science Fund, grant S8812-MED.
Breitenbach/Simon-Nobbe
68
References
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
Horner WE, Helbling A, Salvaggio JE, Lehrer SB: Fungal allergens. Clin Microbiol Rev 1995;8:
161–179.
Romano C, Valenti L, Miracco C, Alessandrini C, Paccagnini E, Faggi E, Difonzo EM: Two cases of
cutaneous phaeohyphomycosis by Alternaria alternata and Alternaria tenuissima. Mycopathologia
1997;137:65–74.
Acland KM, Hay RJ, Groves R: Cutaneous infection with Alternaria alternata complicating immunosuppression: Successful treatment with itraconazole. Br J Dermatol 1998;138:
354–356.
Palacio Ad, Gomez-Hernando C, Revenga F, Carabias E, Gonzales A, Cuetara MS, Johnson EM:
Cutaneous Alternaria alternata successfully treated with itraconazole. Clin Exp Derm 1996;21:
241–243.
Fink JN: Hypersensitivity pneumonitis; in Middleton E, Reed CE, Ellis EF, Adkinson NF,
Yunginger JW (eds.): Allergy, principles and practice, 3rd ed., vol. 2. St. Louis, Mosby, 1988, pp
1237–1252.
Horner WE, Lehrer SB, Salvaggio JE: Indoor air pollution. Fungi. Immunol Allergy Clin N Am
1994;14:551–566.
Slavin RG, Winzenburger P: Epidemiologic aspects of allergic aspergillosis. Ann Allergy 1977;
38:215–218.
Kramer MN, Kurup VP, Fink JN: Allergic bronchopulmonary aspergillosis from a contaminated
dump site. Am Rev Respir Dis 1989;140:1086–1088.
Giubileo L, Sarti AM, Bianchi LA, Calcaterra E, Colombi A: Review of risks of biological agents
and preventive measures to safeguard the health of compost production workers. Med Lav 1998;
89:301–315.
Marth E, Reinthaler FF, Haas D, Eibel U, Feierl G, Wendelin I, Jelovcan S, Barth S: Waste management – health: A longitudinal study (in German). Schriftenr Ver Wasser Boden Lufthyg 1999;
104:569–583.
D’Amato G, Chatzigeorgiou G, Corsico R, Gioulekas D, Jager L, Jager S, Kontou-Fili K,
Kouridakis S, Liccardi G, Meriggi A, Palma-Carlos A, Palma-Carlos ML, Pagan Aleman A,
Parmiani S, Puccinelli P, Russo M, Spieksma FT, Torricelli R, Wuthrich B: Evaluation of the
prevalence of skin prick test positivity to Alternaria and Cladosporium in patients with suspected
respiratory allergy. A European multicenter study promoted by the Subcommittee on Aerobiology
and Environmental Aspects of Inhalant Allergens of the European Academy of Allergology and
Clinical Immunology. Allergy 1997;52:711–716.
Katz Y, Verleger H, Barr J, Rachmiel M, Kiviti S, Kuttin ES: Indoor survey of moulds and prevalence of mould atopy in Israel. Clin Exp Allergy 1999;29:186–192.
Achatz G, Oberkofler H, Lechenauer E, Simon B, Unger A, Kandler D, Ebner C, Prillinger H,
Kraft D, Breitenbach M: Molecular cloning of major and minor allergens of Alternaria alternata
and Cladosporium herbarum. Mol Immunol 1995;32:213–227.
Simon-Nobbe B, Probst G, Kajava AV, Oberkofler H, Susani M, Crameri R, Ferreira F, Ebner C,
Breitenbach M: IgE-binding epitopes of enolases, a class of highly conserved fungal allergens.
J Allergy Clin Immunol 2000;106:887–895.
Simon B: Molekulargenetische und immunologische Charakterisierung von Cladosporium
herbarum-Allergenen. Kartierung der IgE-bindenden Epitope der Enolase von Cladosporium
herbarum; PhD-Dissertation, Salzburg, 1996.
Unger A: Molekulargenetische Untersuchung von Alt a 1, dem Hauptallergen und weiteren
Allergenen des Schimmelpilzes Alternaria alternata; PhD-Diss, Salzburg, 1996.
Prillinger HJ, Lopandic K, Schweigkofler W, Deak R, Aarts HJM, Bauer R, Maraz A: Molecular
phylogeny and systematics of the fungi with special reference to the Ascomycota and
Basidiomycota. Chem Immunol. Basel, Karger, 2002, vol 81, pp 207–294.
De Vouge MW, Thaker AJ, Curran IH, Zhang L, Muradia G, Rode H, Vijay HM: Isolation and
expression of a cDNA clone encoding an Alternaria alternata Alt a 1 subunit. Int Arch Allergy
Immunol 1996;111:385–395.
C. herbarum and A. alternata Allergens
69
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
Crameri R: Molecular cloning of Aspergillus fumigatus allergens and their role in allergic
bronchopulmonary aspergillosis. Chem Immunol. Basel, Karger, 2002, vol 81, pp 94–113.
Paris S, Fitting C, Ramirez E, Latge JP, David B: Comparison of different extraction methods of
Alternaria allergens. J Allergy Clin Immunol 1990;85:941–948.
Portnoy J, Pacheco F, Ballam Y, Barnes C: The effect of time and extraction buffers on residual
protein and allergen content of extracts derived from four strains of Alternaria. J Allergy Clin
Immunol 1993;91:930–938.
Malling HJ: Diagnosis and immunotherapy of mould allergy. With special reference to
Cladosporium herbarum. Dan Med Bull 1990;37:12–22.
Dreborg S, Agrell B, Foucard T, Kjellman NI, Koivikko A, Nilsson S: A double-blind, multicenter immunotherapy trial in children, using a purified and standardized Cladosporium herbarum
preparation. I. Clinical results. Allergy 1986;41:131–140.
Cantani A, Businco E, Maglio A: Alternaria allergy: A three-year controlled study in children
treated with immunotherapy. Allergol Immunopathol (Madr) 1988;16:1–4.
Horst M, Hejjaoui A, Horst V, Michel FB, Bousquet J: Double-blind, placebo-controlled
rush immunotherapy with a standardized Alternaria extract. J Allergy Clin Immunol 1990;85:
460–472.
Norman PS, Lichtenstein LM: The great debate: Immunotherapy and asthma. Clin Allergy 1986;
16:269–271.
Tuchinda M, Chai H: Effect of immunotherapy in chronic asthmatic children. J Allergy Clin
Immunol 1973;51:131–138.
Yunginger JW, Jones RT, Gleich GJ: Studies on Alternaria allergens. II. Measurement of the
relative potency of commercial Alternaria extracts by the direct RAST and by RAST inhibition.
J Allergy Clin Immunol 1976;58:405–413.
Aukrust L: Crossed radioimmunoelectrophoretic studies of distinct allergens in two extracts of
Cladosporium herbarum. Int Arch Allergy Appl Immunol 1979;58:375–390.
Aas K, Leegaard J, Aukrust L, Grimmer O: Immediate type hypersensitivity to common moulds.
Comparison of different diagnostic materials. Allergy 1980;35:443–451.
Zhang L, Muradia G, De Vouge MW, Rode H, Vijay HM: An allergenic polypeptide representing
a variable region of hsp 70 cloned from a cDNA library of Cladosporium herbarum. Clin Exp
Allergy 1996;26:88–95.
Bush RK, Sanchez H, Geisler D: Molecular cloning of a major Alternaria alternata allergen, rAlt
a 2. J Allergy Clin Immunol 1999;104:665–671.
Unger A, Stoger P, Simon-Nobbe B, Susani M, Crameri R, Ebner C, Hintner H, Breitenbach M:
Clinical testing of recombinant allergens of the mold Alternaria alternata. Int Arch Allergy
Immunol 1999;118:220–221.
Vailes L, Sridhara S, Cromwell O, Weber B, Breitenbach M, Chapman M: Quantitation of the
major fungal allergens, Alt a 1 and Asp f 1, in commercial allergenic products. J Allerg Clin
Immunol 2001;107:641–646.
Birkner T, Rumpold H, Jarolim E, Ebner H, Breitenbach M, Skvaril F, Scheiner O, Kraft D:
Evaluation of immunotherapy-induced changes in specific IgE, IgG and IgG subclasses in birch
pollen allergic patients by means of immunoblotting. Correlation with clinical response. Allergy
1990;45:418–426.
Scheiner O, Kraft D: Basic and practical aspects of recombinant allergens. Allergy 1995;50:384–391.
Valenta R, Vrtala S, Laffer S, Spitzauer S, Kraft D: Recombinant allergens. Allergy 1998;53:
552–561.
Kraft D, Ferreira F, Vrtala S, Breiteneder H, Ebner C, Valenta R, Susani M, Breitenbach M,
Scheiner O: The importance of recombinant allergens for diagnosis and therapy of IgE-mediated
allergies. Int Arch Allergy Immunol 1999;118:171–176.
Sanger F, Nicklen S, Coulson AR: DNA sequencing with chain-terminating inhibitors. Proc Natl
Acad Sci USA 1977;74:5463–5467.
Studier FW, Rosenberg AH, Dunn JJ, Dubendorff JW: Use of T7 RNA polymerase to direct
expression of cloned genes. Methods Enzymol 1990;185:60–89.
Way M, Pope B, Gooch J, Hawkins M, Weeds AG: Identification of a region in segment 1 of
gelsolin critical for actin binding. EMBO J 1990;9:4103–4109.
Breitenbach/Simon-Nobbe
70
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
Susani M, Jertschin P, Dolecek C, Sperr WR, Valent P, Ebner C, Kraft D, Valenta R, Scheiner O:
High level expression of birch pollen profilin (Bet v 2) in Escherichia coli: Purification and
characterization of the recombinant allergen. Biochem Biophys Res Commun 1995;215:250–263.
Moser M, Crameri R, Menz G, Schneider T, Dudler T, Virchow C, Gmachl M, Blaser K, Suter M:
Cloning and expression of recombinant Aspergillus fumigatus allergen I/a (rAsp f I/a) with IgE
binding and type I skin test activity. J Immunol 1992;149:454–460.
Kolarich D, Altmann F: N-glycan analysis by matrix assisted laser desorption/ionisation mass
spectrometry of electrophoretically separated non-mammalian proteins. Application to peanut
allergen Ara h 1 and olive pollen allergen Ole e 1. Anal Biochem 2000;285:64–75.
Engel E, Richter K, Obermeyer G, Briza P, Kungl AJ, Simon B, Auer M, Ebner C, Rheinberger HJ,
Breitenbach M, Ferreira F: Immunological and biological properties of Bet v 4, a novel birch
pollen allergen with two EF-hand calcium-binding domains. J Biol Chem 1997;272:28630–28637.
Crameri R, Suter M: Display of biologically active proteins on the surface of filamentous phages:
A cDNA cloning system for selection of functional gene products linked to the genetic information responsible for their production. Gene 1993;137:69–75.
Crameri R, Walter G: Selective enrichment and high-throughput screening of phage surfacedisplayed cDNA libraries from complex allergenic systems. Comb Chem High Throughput Screen
1999;2:63–72.
Curran IH, Young NM, Burton M, Vijay HM: Purification and characterization of Alt a-29 from
Alternaria alternata. Int Arch Allergy Immunol 1993;102:267–275.
Kleine-Tebbe J, Worm M, Jeep S, Matthiesen F, Lowenstein H, Kunkel G: Predominance
of the major allergen (Alt a I) in Alternaria sensitized patients. Clin Exp Allergy 1993;23:
211–218.
Aden E: Protein- und immunbiochemische Charakterisierung eines Alternaria tenuis (NEES) cf.
alternata Allergen-Extraktes sowie Isolierung und Quantifizierung des Hauptallergens Alt a I;
PhD-Diss, Hamburg, 1994.
Matthiesen F, Olsen M, Lowenstein H: Purification and partial sequenzation of the major allergen
of Alternaria alternata. J Allergy Clin Immunol 1992;89:241.
Holland MJ, Holland JP, Thill GP, Jackson KA: The primary structures of two yeast enolase genes.
Homology between the 5⬘ noncoding flanking regions of yeast enolase and glyceraldehyde-3phosphate dehydrogenase genes. J Biol Chem 1981;256:1385–1395.
Mason AB, Buckley HR, Gorman JA: Molecular cloning and characterization of the Candida albicans enolase gene. J Bacteriol 1993;175:2632–2639.
Baldo BA, Baker RS: Inhalant allergies to fungi: Reactions to bakers’ yeast (Saccharomyces
cerevisiae) and identification of bakers’ yeast enolase as an important allergen. Int Arch Allergy
Appl Immunol 1988;86:201–208.
Kortekangas-Savolainen O, Lammintausta K, Kalimo K: Skin prick test reactions to brewer’s yeast
(Saccharomyces cerevisiae) in adult atopic dermatitis patients. Allergy 1993;48:147–150.
Ito K, Ishiguro A, Kanbe T, Tanaka K, Torii S: Detection of IgE antibody against Candida albicans enolase and its cross-reactivity to Saccharomyces cerevisiae enolase. Clin Exp Allergy
1995;25:522–528.
Ishiguro A, Homma M, Torii S, Tanaka K: Identification of Candida albicans antigens reactive
with immunoglobulin E antibody of human sera. Infect Immun 1992;60:1550–1557.
Posch A, Chen Z, Wheeler C, Dunn MJ, Raulf-Heimsoth M, Baur X: Characterization and identification of latex allergens by two-dimensional electrophoresis and protein microsequencing.
J Allergy Clin Immunol 1997;99:385–395.
Breiteneder H, Scheiner O: Molecular and immunological characteristics of latex allergens. Int
Arch Allergy Immunol 1998;116:83–92.
Wagner S, Breiteneder H, Simon-Nobbe B, Susani M, Krebitz M, Niggemann B, Brehler R,
Scheiner O, Hoffmann-Sommergruber K: Hev b 9, an enolase and a new cross-reactive allergen
from Hevea latex and molds. Purification, characterization, cloning and expression. Eur J Biochem
2000;267:7006–7014.
Lebioda L, Stec B, Brewer JM: The structure of yeast enolase at 2.25-Å resolution. An 8-fold beta
⫹ alpha-barrel with a novel beta beta alpha alpha (beta alpha)6 topology. J Biol Chem 1989;264:
3685–3693.
C. herbarum and A. alternata Allergens
71
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
Stec B, Lebioda L: Refined structure of yeast apo-enolase at 2.25 A resolution. J Mol Biol
1990;211:235–248.
Kroutil LA, Bush RK: Detection of Alternaria allergens by Western blotting. J Allergy Clin
Immunol 1987;80:170–176.
Steringer I, Aukrust L, Einarsson R: Variability of antigenicity/allergenicity in different strains of
Alternaria alternata. Int Arch Allergy Appl Immunol 1987;84:190–197.
Vijay HM, Burton M, Young NM, Corlett M, Bernstein IL: Comparative studies of allergens from
mycelia and culture media of four new strains of Alternaria tenuis. Grana 1989;28:53–61.
Paris S, Fitting C, Latge JP, Herman D, Guinnepain MT, David B: Comparison of conidial and
mycelial allergens of Alternaria alternata. Int Arch Allergy Appl Immunol 1990;92:1–8.
Vijay HM, Burton M, Young NM, Copeland DF, Corlett M: Allergenic components of isolates of
Cladosporium herbarum. Grana 1991;30:161–165.
Lowenstein H, Aukrust L, Gravesen S: Cladosporium herbarum extract characterized by means of
quantitative immunoelectrophoretic methods with special attention to immediate type allergy.
Int Arch Allergy Appl Immunol 1977;55:1–12.
Sward-Nordmo M, Almeland TL, Aukrust L: Variability in different strains of Cladosporium
herbarum with special attention to carbohydrates and contents of two important allergens (Ag-32
and Ag-54). Allergy 1984;39:387–394.
De Vouge MW, Thaker AJ, Zhang L, Muradia G, Rode H, Vijay HM: Molecular cloning of IgEbinding fragments of Alternaria alternata allergens. Int Arch Allergy Immunol 1998;116: 261–268.
Aukrust L, Borch SM: Partial purification and characterization of two Cladosporium herbarum
allergens. Int Arch Allergy Appl Immunol 1979;60:68–79.
Sward-Nordmo M, Wold JK, Paulsen BS, Aukrust L: Purification and partial characterization of
the allergen Ag-54 from Cladosporium herbarum. Int Arch Allergy Appl Immunol 1985;78:
249–255.
Sward-Nordmo M, Smestad Paulsen B, Wold JK: Immunological studies of the glycoprotein allergen Ag-54 (Cla h II) in Cladosporium herbarum with special attention to the carbohydrate and
protein moieties. Int Arch Allergy Appl Immunol 1989;90:155–161.
Zhang L, Muradia G, Curran IH, Rode H, Vijay HM: A cDNA clone coding for a novel allergen,
Cla h III, of Cladosporium herbarum identified as a ribosomal P2 protein. J Immunol 1995;154:
710–717.
De Vouge MW, Thaker AJ, Zhang L, Curran IH, Muradia G, Rode H, Vijay HM: Isolation of a
cDNA clone encoding a putative Alternaria alternata Alt a I subunit. Adv Exp Med Biol 1996;
409:205–212.
Short JM, Fernandez JM, Sorge JA, Huse WD: Lambda ZAP: A bacteriophage lambda expression
vector with in vivo excision properties. Nucleic Acids Res 1988;16:7583–7600.
Crameri R, Achatz G, Weichel M, Rhyner C: Direct selection of cDNAs by phage display.
Methods Mol Biol, in press.
Crameri R, Jaussi R, Menz G, Blaser K: Display of expression products of cDNA libraries on
phage surfaces. A versatile screening system for selective isolation of genes by specific geneproduct/ligand interaction. Eur J Biochem 1994;226:53–58.
Crameri R, Kodzius R: The powerful combination of phage surface display of cDNA libraries and
high throughput screening. Comb Chem High Throughput Screen 2001;4:145–155.
Crameri R, Kodzius R, Konthur Z, Lehrach H, Blaser K, Walter G: Tapping allergen repertoires
by advanced cloning technologies. Int Arch Allergy Immunol 2001;124:43–47.
Hemmann S, Ismail C, Blaser K, Menz G, Crameri R: Skin-test reactivity and isotype-specific
immune responses to recombinant Asp f 3, a major allergen of Aspergillus fumigatus. Clin Exp
Allergy 1998;28:860–867.
Westhead EW: Enolase from yeast and rabbit muscle. Methods Enzymol 1966;9:670–679.
M. Breitenbach
University of Salzburg, Department of Genetics and General Biology,
Hellbrunnerstrasse 34, A–5020 Salzburg (Austria)
Tel. ⫹43 662 8044 5786, Fax ⫹43 662 8044 144, E-Mail michael.breitenbach@sbg.ac.at
Breitenbach/Simon-Nobbe
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Breitenbach M, Crameri R, Lehrer SB (eds): Fungal Allergy and Pathogenicity.
Chem Immunol. Basel, Karger, 2002, vol 81, pp 73–93
Molecular Cloning of Aspergillus
fumigatus Allergens and Their Role
in Allergic Bronchopulmonary
Aspergillosis
Reto Crameri
Swiss Institute of Allergy and Asthma Research, Davos, Switzerland
Allergic reactions to moulds, in particular to Aspergillus fumigatus, are
difficult to diagnose for various reasons. Most important, standardized A. fumigatus allergen preparations are not available. Commercial fungal extracts are
subject to large batch-to-batch variations and are therefore unsatisfactory for
diagnostic applications. Furthermore, moulds of the genus Aspergillus are associated with a wide variety of pulmonary complications, ranging from benign
colonization of the lung to life-threatening diseases, such as invasive aspergillosis or allergic bronchopulmonary aspergillosis (ABPA). A. fumigatus is able to
produce a wide number of more than 40 IgE-binding molecules. Expression of
many of these allergens depends on the growth phase of the fungus making
standardization of extracts a difficult task. Moreover, the molecular nature of
the IgE-binding molecules is largely unknown and the pathophysiologic role
played by each molecule in the clinically distinct pulmonary complications
remains to be elucidated. ABPA is a potentially fatal disease resulting from
IgE-mediated responses to A. fumigatus grown through noninvasive colonization
of the lung in conjunction with deterioration of the lung function. However, since
most of the criteria used to diagnose ABPA are not specific for the disease,
serologic findings should strongly contribute to confirm or exclude a suspected
ABPA based on clinical signs. The reliability of skin tests and serologic methods used to diagnose ABPA mainly depends on the composition of the antigen
preparations used. The large differences reported in the incidence of both,
sensitization to A. fumigatus and ABPA might partly be explained by the lack
of reliable A. fumigatus extracts. The aim of our research is to molecularly clone,
produce and evaluate the whole repertoire of IgE-binding molecules produced
by A. fumigatus. Large-scale serologic and skin test evaluations of some of the
cloned A. fumigatus allergens revealed the existence of disease-specific allergens, opening new perspectives for diagnosis and management of ABPA.
Host Defense Mechanisms and A. fumigatus-Related Diseases
A. fumigatus is a member of the genus Aspergillus, which, according
to Raper and Fennell [1] includes 132 species, subdivided into 18 groups.
Aspergilli are ubiquitously distributed in our environment and among the most
important opportunistic fungal pathogens for humans [2]. Only a few members
of the genus are recognized opportunistic pathogens and among these A. fumigatus is the etiological agent found in 80% of the Aspergillus infections in
humans [3]. The reasons for the pathogenicity of A. fumigatus, which mainly
affects patients suffering from asthma, cystic fibrosis or other forms of immunodeficiency [4, 5], are not fully understood. However, the immune status of the
individuals seems to be more important for the development of aspergillosis
than the virulence of the mould itself [6]. In fact, patients receiving chemotherapy, undergoing total-body irradiation, organ transplantation or prolonged highdose corticosteroid treatment are particularly prone to developing Aspergillus
infections [7, 8]. As an opportunistic pathogen that only causes disease accidentally when host defenses are impaired, A. fumigatus has not developed
sophisticated survival strategies, as have better-developed pathogens such as
Listeria or Yersinia. Therefore, it is not surprising that, in spite of considerable
efforts, no distinct A. fumigatus virulence factors have as yet been identified
unequivocally [9, 10]. Studies involving mutants obtained by targeted gene disruption showed that the potential virulence factors catalase, alkaline protease,
metalloprotease, hyphal growth factor chsE, and fungal ribotoxin restrictocin do
not influence pathogenicity of the fungus in mouse models [11–14]. Several
studies showed interactions between A. fumigatus conidia which, due to their
small size of 2–3 m, can reach the terminal respiratory tract and matrix proteins, such as fibrinogen, laminin, fibronectin and collagen, suggesting a role of
these physical interactions in host tissue adherence [15–18]. Adherence of a fungus to the host epithelial surfaces constitutes a primary and crucial step in the
onset of colonization [19, 20]. Several host defense mechanisms have developed
in the context of innate immunity and play a predominant role in the clearance
of A. fumigatus from the respiratory tract. The first line of defense against fungal colonization is mucociliary clearance, which can be modulated by different
secondary metabolites secreted by Aspergillus and other microorganisms. It has
been shown that substances secreted by the fungus are able to induce slowing
and disorganization of ciliary beating frequency resulting in enhancement of the
Crameri
74
retention time of the conidiae in the airways [21]. Since remaining spores are
ingested and killed by monocytes/macrophages [22], clearing the lung from
inhaled conidia, an important property for the onset of aspergillosis is the capacity of Aspergillus to interfere with both the phagocytosis and killing capacity of
phagocytic cells allowing A. fumigatus spores to survive ingestion by alveolar
macrophages [23]. Additionally, neutrophil granulocytes attack hyphae that are
too large for ingestion and kill them by oxidative and nonoxidative mechanisms
[24]. Each line of defense by itself is able to protect the host against a large
conidial inoculum over long periods of time: only if all defense lines are overcome can Aspergillus cause invasive disease [25].
A. fumigatus-Related Diseases
The different diseases related to A. fumigatus which usually remain mutually exclusive can be broadly classified according to their clinical conditions into
systemic mycoses, saprophytic colonization and allergic diseases [26]. Systemic
(invasive) aspergillosis is considered as an infection affecting immunocompromised subjects suffering from prolonged granulocytopenia or other hematological malignancies [4, 27, 28]. The rate of successful treatment in established
invasive aspergillosis is associated with a fatality rate of almost 100%, mainly
due to the lack of adequate diagnostic techniques for early detection of the fungal pathogen [4]. Saprophytic bronchopulmonary colonization refers to fungal
infestation of the airways without evidence for direct tissue damage [26]. As a
special case aspergilloma, also termed mycetoma or fungus ball, is a saprophytic
manifestation of an Aspergillus growing in preformed lung cavities of patients
with bronchogenic carcinoma, tuberculosis, sarcoidosis or, rarely, ABPA [29].
A. fumigatus-related allergic disorders include allergic rhinitis, allergic sinusitis,
allergic asthma and ABPA [26, 30]. A common feature of these atopic diseases
is a Th2-type immunologic response, which dictates the production of IgE antibodies against A. fumigatus allergens followed by an increased sensitization of
mast cells. A corresponding positive skin test to A. fumigatus extracts is therefore regularly found in subjects suffering from one of these diseases [26, 30].
The symptoms of A. fumigatus-induced allergic rhinitis are indistinguishable
from those caused by other allergens [26], and the diagnosis is based on case history, skin reactivity and positive RAST to the appropriate allergens [31]. Allergic
fungal sinusitis is a chronic disease that affects multiple sinuses without tissue
invasion [32]. Several fungi including A. fumigatus have been implicated in
allergic sinusitis. The patients may show skin reactivity, precipitating antibodies
and elevated allergen-specific IgG and IgE levels to A. fumigatus extracts [26,
32], immunologic characteristics also found in ABPA [33, and see below].
Aspergillus fumigatus Allergens
75
The symptoms related to mould-associated asthma can be distinguished
from those attributable to other allergen-induced asthma by a history of
exposure, skin test reactivity or in vitro demonstration of fungus-specific IgE
in patients’ sera [34, 35]. The immunologic and inflammatory response in
A. fumigatus-related asthma, responsible for the fungus-induced early and late
asthmatic reactions, is similar to the responses induced by inhalant allergens
derived from different allergenic sources such as the house dust mite [36].
However, for therapeutic reasons, A. fumigatus-related asthma must be clearly
distinguished from ABPA which requires steroid therapy to control ongoing
lung destruction by allergic inflammation and tissue reaction [37]. The diagnosis of ABPA is complicated by a number of characteristics shared by ABPA and
A. fumigatus-related asthma [37] and even more complicated in A. fumigatussensitized patients suffering from cystic fibrosis [30].
Diagnosis and Epidemiology of Allergic Bronchopulmonary
Aspergillosis
Transient colonization of the airways by A. fumigatus can induce an
abnormal host response to the fungus characterized by the presence of large
numbers of eosinophils and lymphocytes in the airway walls, increased levels
of serum IgE, precipitating antibodies to Aspergillus antigens [38] in conjunction with deterioration in lung function [39]. This clinical entity termed allergic
bronchopulmonary aspergillosis (ABPA) was first described by Hinson et al.
[40] in 1952 and considered a rarity [41], but is currently diagnosed with much
greater frequency [42] as a result of the improvement in serologic and radiological methods of diagnosis [43]. Greenberger et al. [37] and Patterson et al.
[44, 45] continued to refine the diagnostic criteria of this complex clinical
syndrome. The currently accepted diagnostic criteria for ABPA are reported
in table 1. The assessment of peripheral blood eosinophilia, chest roentgenographic infiltrates, central bronchiectasis and elevated total serum IgE poses
few problems to the experienced clinician. Unfortunately, these criteria are not
sufficient to diagnose the disease [47]. The remaining criteria, immediate skin
reactivity to Aspergillus, serum precipitins and elevated serum IgE and IgG to
A. fumigatus, although depending on the composition of the extract used [48,
49], should, however, strongly contribute to confirm or exclude ABPA suspected on clinical signs [50]. The lack of standardized A. fumigatus extracts
[51] may account partly for the discrepancy in the incidence of ABPA reported
to range from 7 to 22% among asthmatic patients sensitized to A. fumigatus [52,
53] and from 0.1 to 12% in patients with cystic fibrosis [54, 55]. The diagnosis
of ABPA in patients with cystic fibrosis is further complicated by a number of
Crameri
76
Table 1. Diagnostic criteria of ABPAa
1
2
3
4
5
6
7
8
Asthma
Current or previous pulmonary infiltrate
Central bronchiectasis
Peripheral blood eosinophilia (1,000/mm3)
Immediate skin reactivity to Aspergillusb
Serum precipitins to Aspergillusb
Elevated specific serum IgE and serum IgG to A. fumigatusb
Elevated total serum IgE (ⱖ1,000 ng/l)
a
According to Greenberger and Patterson [37].
Outcome strongly depending from the quality of the extract used. Not all of these criteria may be present in a patient at a given time because they vary with the activity and stage
of the disease [44, 46]. A serologic diagnosis of ABPA can be considered as established if
criteria 6, 7 and 8 are positive [45]. If skin tests are negative, ABPA can be excluded [37].
b
clinical findings shared by the two diseases, including pulmonary infiltrates
and centrally located bronchiectasis [54, 55].
The true prevalence of ABPA remains highly speculative: ABPA is not
recognized in the international classification of diseases and the diagnostic criteria lack clinical uniformity and standardized tests [56]. This intriguing situation
is best described by the prevalence of immediate skin sensitivity to crude
A. fumigatus extracts, a criterion absolutely required for diagnosing ABPA [37, 44,
45]. Depending on the study, prevalences varying between 2 and 38% for asthmatic patients and from 35 to 81% for patients suffering from cystic fibrosis
were found [56]. These studies highlight the difficulty in determining the prevalence of a condition in which diagnostic criteria are not established. The best
estimate of the prevalence of ABPA among asthmatic patients and patients with
cystic fibrosis calculated by Novey [56] from the available surveys suggests
that ABPA could be present in between 0.25 and 0.8% of all asthma patients in
the USA. An incidence rate of ABPA of no more than 11% and closer to 7% in
patients suffering from cystic fibrosis is suggested by the same author [56]
based on the limited data available. This incidence is high enough to consider
ABPA as an important disease entity, which should be excluded in every patient
with asthma or cystic fibrosis [37, 45].
Cloning and Molecular Characteristics of A. fumigatus Allergens
Several attempts have been made to characterize and purify relevant A. fumigatus allergens using conventional biochemical methods such as gel filtration
Aspergillus fumigatus Allergens
77
[57], preparative isoelectric focusing [58], ion exchange [59] and affinity chromatography [60] or use of monoclonal antibodies [61]. However, none of these
methods provided sufficient quantities of purified allergens for diagnostic applications [26]. Although biochemical purification of Asp f 1, a major allergen of the
fungus, allowed determination of the amino acid sequences of tryptic peptides
resulting in identification of the protein as a ribotoxin [62], it was also clearly
shown that such preparations can be contaminated with other allergens with
similar immunochemical properties [63], illustrating the difficulties in obtaining
pure native allergens. The sequence information obtained at protein level for this
ribotoxin was used to clone the gene coding for Asp f 1 [63], the first fungal allergen cloned. Asp f 1 revealed sequence identity to restrictocin, an 18-kD ribotoxin
produced by Aspergillus restrictus and never described as allergen [64] that is
>99% identical to the Asp f 1 sequences derived from different clinical isolates of
A. fumigatus [65, 66]. All these proteins are related to ribotoxins such as ␣-sarcin
[67] and mitogillin [68], ribonucleolytic enzymes strictly restricted to the genus
Aspergillus [69]. Therefore, Asp f 1 can be considered a species-specific allergen
in contrast to other IgE-binding proteins of A. fumigatus, which are highly crossreactive with related proteins from phylogenetically distant species [70, 71].
Together with honey bee venom phospholipase A2 [72] and the birch pollen allergen Bet v 1 [73], Asp f 1 is one of the best-investigated allergens at biochemical
and clinical level [30] and still the only fungal allergen for which a crystal structure is available [74].
Although screening of cDNA libraries constructed in bacteriophage with
sera of sensitized patients yielded some additional A. fumigatus allergens [75, 76]
(table 2), the breakthrough in cloning A. fumigatus allergens [30, 87, 88], and IgEbinding proteins from other complex allergenic sources [89–91], was achieved by
screening of cDNA libraries displayed on the surface of filamentous phage. The
basic concept of linking phenotype, expressed as gene product displayed on the
phage surface, to its genetic information integrated into the phage genome (fig. 1)
allows the screening of large libraries for the presence of specific clones using the
discriminative power of affinity purification. The selection procedure involves the
enrichment of phage binding to an immobilized target [92–94]. Identification of
allergens is facilitated by the common property of the molecules to interact with
IgE [95] achieved by interaction between gene product displayed on phage surface
and solid-phase-bound IgE during consecutive rounds of phage selection and
amplification [30]. The procedure allows selective isolation of all allergenexpressing clones present in a cDNA library consisting of millions of single
phages (fig. 2). As a consequence of the physical linkage between genotype and
phenotype, sequencing the DNA of the integrated section of the phage genome can
readily elucidate the amino acid sequence of the displayed allergen. This elegant
method of cloning has been used to identify an array of novel genes from cDNA
Crameri
78
Table 2. Characteristics of the cloned A. fumigatus allergens
Allergen Function
aa
CDSa MW IgE-binding Skin test Accession
kD
in vitro, %b reactivityc no.
References
Asp f 1
Asp f 2
Asp f 3
Asp f 4
Asp f 5
Asp f 6
Asp f 7
Asp f 8
Asp f 9
Asp f 10
Asp f 11
Asp f 12
Asp f 13
Asp f 15
Asp f 16
Asp f 17
Asp f 18
ribotoxin
fibrinogen binding protein?
peroxisomal protein
unknown
metalloprotease
MnOD
unknown
P2 ribosomal protein
unknown
aspartic protease
cyclophylin
heat shock protein 90
alkaline serine protease
serine protease?
149
268
168
286
388
207
112
111
302
325
171
?
282
152
C
C
C
P
C
C
P
C
P
C
C
P
C
C
63, 77
76
78, 79
47, 80, 81
30, 82
70, 80, 83
30, 82
71
30, 82
30, 82
84
75
85, 86
84
unknown
vacuolar serine proteinase
197 P
C
16.9
37
18.5
30
42.1
23
11.6
11.1
32.3
34.4
18.8
65
34
19.5
43
19.4
34
66
ND
88
42
85
35
39
10
66
18
50
ND
ND
29
ND
43
ND
pos.
NT
pos.
pos.
pos.
pos.
pos.
pos.
pos.
pos.
pos.
NT
NT
pos.
NT
pos.
NT
S889330
U56938
U58050
AJ001732
Z30424
U53561
AJ223315
AJ224333
AJ223327
X85092
U92465
Z11580
AJ002026
G3643813
AJ224865 84
Y13338
86
a
Coding sequences: C = complete, P = partial.
100 sera from patients with or without ABPA were tested. ND = not determined.
c
NT = Not tested, pos. = positive.
b
libraries displayed on phage surface [94, 96], among these also a wide variety of
A. fumigatus allergens [30, 88].
Physicochemical and biochemical characteristics of the A. fumigatus
allergens cloned thus far are listed in table 2. Many of these allergens do occur
as homologous allergens in more than one fungal species and could account for
cross-reactivity among moulds [82]. This is clearly the case for Asp f 3, a
peroxisomal protein demonstrated to be cross-reactive with two homologous
proteins of Candida boidiini [78] and recently isolated as IgE-binding protein
also from the yeast Malassezia furfur [90]. Asp f 6 (manganese superoxide
dismutase), Asp f 8 (P2 acidic ribosomal protein) and Asp f 11 (cyclophilin)
belong to the class of phylogenetically highly conserved proteins [70, 71, 82].
Interestingly, these proteins share a high degree of sequence identity of more
than 50% with the corresponding homologous human proteins [71, 83, 97]. The
human and A. fumigatus proteins expressed as recombinant proteins in E. coli
are recognized by IgE antibodies from individuals sensitized to the homologous
A. fumigatus allergens. Moreover, both the fungal as well as the human proteins
Aspergillus fumigatus Allergens
79
c
pr DN
od A
uc
t
Fos
Jun
g3p (~5/43 kD)
~900 nm
g6p (~5/12 kD)
ssDNA (~6,500 nt)
DNA Inserts
g8p (~2,800/5 kD
g7p (~5/3.5 kD)
g9p (~5/3.5 kD)
~6 nm
Fig. 1. Physical linkage between genetic information and gene product. The cDNAs
are cloned after the Fos leucine zipper and the protein products are captured and immobilized on the phage surface through interaction and covalent linkage with the modified gIII
gene product of filamentous phages fused to the Jun leucine zipper [92–96].
induced proliferation in peripheral blood mononuclear cells and positive skin
reactions in A. fumigatus-allergic patients showing specific IgE responses to the
recombinant A. fumigatus proteins [71, 83]. These observations clearly demonstrate that autoreactivity to self-antigens may occur in chronic allergic diseases
and suggest that the human proteins can act as autoallergens in vivo [71, 83].
The mechanisms responsible for these autoimmune reactions remain to be
elucidated; however, the responses against self-antigens in individuals sensitized
to the corresponding A. fumigatus allergens indicate an autoantigenic T-cellmediated pathogenesis in chronic inflammatory responses to the fungus [82].
Recent investigations in patients suffering from severe forms of atopic dermatitis showed frequent IgE reactivity against autoantigens [98], supporting our
hypothesis that autoimmune reactivity in atopy is not a rare event [97].
An array of allergenic proteins like Asp f 2, Asp f 4, Asp f 7, Asp f 9 and
other recently cloned allergens (table 2) fail to show any homology to known
protein sequences and therefore the biochemical function of these gene products remains unknown. However, these allergens can substantially contribute to
Crameri
80
Fig. 2. Selective enrichment of phage displaying allergens. Human serum IgE from
sensitized individuals coated to a solid phase surface is used to capture phages displaying
IgE-binding molecules. Phages that bind specifically to IgE can be selectively enriched
during consecutive rounds of phage growth and adsorption [92–96].
improve the diagnosis of sensitization to A. fumigatus [30]. Many other allergens, including Asp f 1, Asp f 5, Asp f 10, Asp f 13, Asp f 15 and Asp f 18,
correspond to enzymes showing a high degree of sequence homology to proteins
cloned from different fungal species [82]. In spite of the fact that many IgEbinding proteins cloned from different allergenic sources exhibit enzymatic
activity [99], the role of the biochemical function of a protein in the induction of
allergic diseases has to be questioned. In fact, engineered enzymatically inactive
forms of PLA2 from bee venom [100] and of Phl p 5b from timothy grass [99]
did not lose any histamine-releasing activity from basophils of sensitized individuals. In contrast, there is increasing evidence that the three-dimensional
structure of the allergens is important for the differential regulation of IgE and
IgG4 responses independently of the enzymatic activity of the involved molecules [101, 102]. This evidence is corroborated by the demonstration that structural proteins without enzymatic activity cloned using high-throughput
screening of an enriched A. fumigatus cDNA library displayed on phage surface
[95, see below] can act as allergens. We assume that the allergens reported in
Aspergillus fumigatus Allergens
81
table 2, together with the new allergens derived from robotic screening [103]
should cover the whole allergenic repertoire of A. fumigatus which represents
one of the most complex allergenic sources [104–106].
Clinical and Diagnostic Evaluation of Recombinant
A. fumigatus Allergens
Although in vitro methods, including enzyme linked immunosorbent
assay (ELISA) and Western blot analysis, provide evidence for the IgE-binding ability of a protein, the final demonstration of the clinical relevance of a
recombinant allergen is the ability of the preparation to elicit type I skin reactions in allergic individuals [83]. This is especially important for recombinant
proteins produced in heterologous expression systems, which may not be correctly folded and therefore unable to interact with IgE [107]. A major concern
in using recombinant allergens for the diagnosis of allergic diseases is
whether the results obtained in vitro with ELISA or ImmunoCAP® determinations correlate with the ability of the protein to induce skin reactions. The
recombinant allergens Asp f 1, Asp f 3, Asp f 4 and Asp f 6 have been tested
in large-scale skin studies in patients suffering from asthma or cystic fibrosis
and coexisting A. fumigatus-sensitization, and have been demonstrated to be
reliable diagnostic reagents [47, 49, 66, 79]. The overall results of these
studies that compare skin test reactivity with serum IgE levels in groups of
patients suffering from different A. fumigatus-related complications are
summarized in table 3. The fact that only individuals with detectable allergenspecific IgE levels in serum reacted against the corresponding allergen in
skin challenges is important for the reliability of in vitro tests (fig. 3).
Furthermore, the levels of rAsp f 1, 3, 4 and 6-specific IgE in serum significantly correlated with the magnitude of the wheal surfaces in all cases [47,
66, 79], a result confirmed by quantitative skin tests performed with recombinant phospholipase A2 [107, 110]. By using allergen extracts [111] and, in
particular, mould extracts [112], discrepancies between results of skin tests
and allergen-specific IgE determinations have been reported. Accordingly,
based on our studies, the specificity of in vivo and in vitro tests can be
substantially improved using recombinant allergens, whereas the sensitivity
depends on the frequency of the sera recognizing the single component. All
four allergens are suitable for the development of fully automated reagents for
a quantitative diagnosis of sensitization. rAsp f 1 [108], rAsp f 3 [79], rAsp
f 4 and rAsp f 6 [47] immobilized in the Pharmacia ImmunoCAP® System
yielded a highly significant correlation between specific IgE determined
by ELISA and the quantitative ImmunoCAP determinations. The excellent
Crameri
82
Table 3. Correlation between intradermal skin test reactivity and absolute levels of
allergen-specific IgE in serum of A. fumigatus-sensitized individuals and healthy controls
Patient group
Allergen-specific IgE (kUA/l) againsta
rAsp f 1
rAsp f 3
rAsp f 4b
rAsp f 6b
10.79
0.34
14.10
0.34
11.30
0.39
3.91
0.74
A. fumigatus-sensitized with
Positive skin test
Negative skin test
4.97
0.34
4.86
0.34
0.34
0.36
0.34
0.35
Healthy control individuals
All negative in skin test
0.34
0.34
0.36
0.34
ABPA with
Positive skin test
Negative skin test
a
Determined with the Pharmacia CAP system.
These allergens are highly specific for ABPA and all A. fumigatus-sensitized individuals
without ABPA score negative in skin challenges [47]. All individuals with allergen-specific
serum IgE levels below approximately 2 g/l are negative to intradermal allergen challenges,
demonstrating the specificity of the in vitro IgE determinations [47, 79, 108, 109].
b
correlations between wheal surface and allergen-specific IgE in serum
obtained for each single allergen [47, 79, 109] allow an approximate quantification of the amount of allergen-specific IgE needed for a positive skin
reaction. The cut-off values for a positive skin test response were found to be
approximately 0.35, 0.35, 0.9 and 1.2 kUA/l for rAsp f 1, 3, 4 and 6, respectively, corresponding to an absolute allergen-specific IgE serum concentration in the range of 2 g/l. The statistically significant correlation between
serologic data and skin test results suggests the possibility, after adequate calibration of the in vitro systems [108], of relying on serologic data obtained
with recombinant allergens to diagnose sensitization.
The Role of A. fumigatus Allergens in Allergic
Bronchopulmonary Aspergillosis
The availability of highly pure recombinant allergens from this complex
fungal allergenic source allows determination of patient-specific reactivity
patterns and therefore evaluation of the involvement of each single molecule
in the pathogenesis of the different A. fumigatus-related diseases. Large serologic studies involving patients sensitized to A. fumigatus suffering from
Aspergillus fumigatus Allergens
83
rAsp f 1
rAsp f 3
10,000
Sensitized/
asthma
ABPA
Sensitized/
asthma
ABPA
1,000
100
100
10
10
1
Specific serum IgE (EU/ml)
Specific serum IgE (EU/ml)
1,000
1
⫺
⫹
⫹
⫺
⫺
⫹
⫹
⫺
Intradermal skin test
Fig. 3. Intradermal skin test with rAsp f 1 and rAsp f 3 and allergen-specific IgE
values. rAsp f 1 or rAsp f 3 allergen solutions were injected into the skin of A. fumigatussensitized asthma patients with or without ABPA. Results were grouped according to negative (⫺) or positive (⫹) intradermal skin tests and plotted against the allergen-specific IgE
values determined by ELISA. The hatched area represents the cut-off value indicating the
level of allergen-specific IgE in serum required for a positive skin test.
asthma or cystic fibrosis with or without ABPA revealed the existence of
disease-specific allergens [47, 80]. Although it was known that serum IgE of
patients suffering from ABPA do recognize protein bands in Western blots
performed with fungal extracts not recognized by sensitized individuals without
ABPA [34, 46], the biochemical nature of these components remained undefined. Molecular cloning of A. fumigatus allergens allowed the biochemical
characterization of some of these components as cytoplasmic proteins with a
defined function, such as manganese superoxide dismutase (Asp f 6) [83] or
P2 acidic ribosomal protein (Asp f 8) [71] or, as in the case of Asp f 2 [76] and
Asp f 4 [30, 80, 81], as allergens with unknown biological functions. These
allergens provide a highly reliable, specific and sensitive serologic diagnosis
of ABPA (table 4) [80, 81, 113] contributing to the solution of an old diagnostic problem. The rAsp f 4- and rAsp f 6-based serological diagnosis of
ABPA has a specificity of 100% and reaches a sensitivity of 90% in asthmatic
patients sensitized to A. fumigatus [80], whereas the serological discrimination
between A. fumigatus sensitization and ABPA in patients suffering from cystic
fibrosis reached 100% [81]. Although sensitization to secreted A. fumigatus
allergens, like Asp f 1 or Asp f 5 [30], can easily be explained as a consequence
of fungal exposure in the respiratory tract, the specific IgE responses to
cytoplasmic allergens in patients with ABPA require further explanation.
Crameri
84
Table 4. Discrimination between ABPA and A. fumigatus sensitization by serology
with recombinant A. fumigatus allergens
Patient group
ABPA with
Allergic asthma (n ⫽ 60)
Cystic fibrosis (n ⫽ 20)
A. fumigatus-sensitized with
Allergic asthma (n ⫽ 40)
Cystic fibrosis (n ⫽ 20)
Control individuals
Healthy (n ⫽ 20)
CF-controlsb (n ⫽ 10)
rAsp f 1, %
rAsp f 3, %
rAsp f 4a, %
rAsp f 6a, %
83
100
88
100
80
80
55
70
45
60
53
90
0
0
0
0
0
0
0
0
0
0
0
0
a
These allergens are highly specific for ABPA and allow serologic discrimination between
ABPA and A. fumigatus allergy with a specificity of 100% and a sensitivity of 90% in
A. fumigatus-sensitized allergic asthmatics [30, 80]. In A. fumigatus-sensitized patients with
cystic fibrosis specificity and sensitivity reach 100% [81].
b
Cystic fibrosis patients not sensitized to A. fumigatus [30, 81].
Specific sensitization to nonsecreted proteins in ABPA suggests substantial
differences in the pathway of exposure to, and immunologic recognition of, the
pathogenic fungus between sensitized patients with and without ABPA.
Intracellular allergens are unlikely to be present as free aeroallergens in contrast to secreted allergens which might be present in the environment [114].
This could partly explain the lack of specific IgE raised against these proteins
in A. fumigatus-sensitized individuals [80]. Allergic individuals are assumed to
become sensitized to proteins secreted shortly after spore germination during
the time between deposition in the airway and clearance [77]. In contrast,
patients suffering from ABPA have or had the fungus growing in the lung [37,
38] and, as a result of fungal damage related to cellular defense mechanisms,
become exposed more strongly to secreted and nonsecreted proteins than
individuals suffering from simple allergy.
Although it is known that infection by pathogens or parasites can induce
differential immune responses in infected individuals [115], it was not yet
observed that a single pathogen can elicit differential IgE responses in clinically
related diseases. These observations open new perspectives for a serologic discrimination between clinically distinct IgE-mediated allergic diseases related to
A. fumigatus. The availability of the pure substances will aid development of
highly specific reagents such as monoclonal antibodies suitable for the in vivo
Aspergillus fumigatus Allergens
85
localization of the substances during the time course of infection and thus
contributing to further elucidate the pathophysiologic mechanisms involved in
the development of ABPA.
Utilizing the Allergenic Repertoire of A. fumigatus Identified
with Advanced Technologies
Allergen cloning by phage surface display technology has substantially
reduced the time required for the isolation of cDNAs encoding IgE-binding proteins to a short period of a few weeks [30, 88]. This approach has been successfully applied to cDNA libraries constructed using mRNAs derived from different
complex allergenic sources [88–93]. The subsequent cloning of IgE-binding molecules from these sources resulted in the production of whole panels of fungal
allergens [103–106]. However, single clones enriched from phage surface display
libraries derived from complex allergenic systems are expected to be multiply
represented in the large phagemid population selected, as a direct consequence of
amplification and enrichment of IgE-binding molecules occurring during each
round of phage selection and amplification [94]. Therefore, identification of
clones encoding products derived from rare mRNA species among billions of
phages obtained after several rounds of selection and amplification remains a
major problem. To enable fast and cost-effective characterization of all clones of
an enriched cDNA library, high-throughput screening technology is required. The
combination of selective enrichment and automated robot technology for picking
and high-density spotting of clones onto filter membranes has proven to be very
efficient for the isolation of virtually all allergen-specific cDNA clones present in
enriched libraries [103]. Thereby, all clones representing a specific gene present in
a library are identified by hybridization with labeled cDNA probes derived from
inserts encoding genes of interest. Negative clones can be regrouped on new filters
and used for further rounds of hybridization with a different set of DNA probes.
This technology permits a fast identification of all different clones present in the
enriched library and drastically reduces the manual work involved. Using three
rounds of hybridization, we were able to identify 71 different cDNAs encoding
allergens of an A. fumigatus cDNA library displayed on the phage surface,
enriched by selection with serum IgE of individuals sensitized to the mould
[Kodzius et al., in preparation]. This allergen panel is likely to represent the whole
IgE-binding repertoire of the pathogenic fungus and will allow to further investigate the immune responses occurring in the clinically distinct A. fumigatus-related
allergic diseases at the molecular level. Moreover, the consequent application of
functional enrichment and robotic-based screening will allow to rapidly generate
information and reagents to facilitate progress in the whole field of life science.
Crameri
86
Conclusions
The development of phage surface display technology for cDNA cloning
allowed rapid progress in the identification and characterization of allergenic
molecules produced by A. fumigatus. This technique, corroborated by roboticbased high-throughput screening technology, allowed identification at the
molecular level of basically the whole repertoire of IgE-binding molecules
produced by this fungal pathogen. It must be pointed out, that A. fumigatus
represents one of the most complicated inhalative sources of allergens. The
availability of the cDNAs encoding more than 70 A. fumigatus allergens will
contribute to generate rapid progress in understanding the pathophysiologic
role played by each single molecule in the clinically distinct allergic diseases
related to A. fumigatus. All recombinant allergens tested so far are able to elicit
strong type I skin reactions in sensitized individuals and, moreover, are applicable to fully automated diagnostic systems suitable for routine diagnosis. The
results show that recombinant allergens are clearly superior to fungal extracts
for diagnostic purposes as deduced from the significant correlations obtained
between skin test outcome and serology. Large-scale serological studies showed
that specific IgE responses to at least 4 allergens (Asp f 2, Asp f 4, Asp f 6 and
Asp f 8) are highly specific for sera of patients suffering from ABPA. These
disease-specific allergens allow a serological discrimination between ABPA
and A. fumigatus-sensitization with 100% specificity and 90% sensitivity in
asthmatic patients, whereas sensitivity and specificity in patients suffering from
cystic fibrosis can practically reach 100%. Such discrimination is not possible
using conventional fungal extracts, highlighting the potential of recombinant
allergens for diagnostic uses. Production, characterization and evaluation of the
whole IgE-binding repertoire of A. fumigatus obtained by phage display and
high-throughput screening technology are likely to allow further improvement
of the differential diagnosis of A. fumigatus-related allergic diseases. The wide
application of these advanced technologies will have implications far beyond
allergen cloning and characterization.
Acknowledgments
I am grateful to Prof. Dr. K. Blaser and Prof. Dr. H. Lehrach for continuous support
and encouragement. I am particularly indebted to the clinics in Davos for the intensive clinical support and to all my coworkers and collaborators for practical support which made
progress in this field of research possible at all. This work was supported by the Swiss
National Science Foundation (grants 31.50515.97 and 31.063381.00) and by the Ciba-Geigy
Jubiläumsstiftung.
Aspergillus fumigatus Allergens
87
References
1 Repar KG, Fennell DI: The Genus Aspergillus. Baltimore, Williams & Wilkins, 1965, pp 1–686.
2 Bardana EJ: The clinical spectrum of aspergillosis. 2. Classification and description of saprophytic allergic and invasive variants of human disease. CRC Crit Rev Clin Lab Sci 1980;
13:85–159.
3 Al-Doory Y, Wagner GE: Aspergillosis. Springfield, Thomas, 1985.
4 Saral R: Candida and Aspergillus infections in immunocompromised patients: An overview. Rev
Infect Dis 1991;13:487–492.
5 Levitz SM: Overview of host defense in fungal infections. Clin Infect Dis 1992;14:37–42.
6 Romani L, Howard DH: Mechanisms of resistance to fungal infections. Curr Opin Immunol
1995;7:517–523.
7 Palmer LB, Greenberger HE, Schiff MJ: Corticosteroid treatment as a risk factor for invasive
aspergillosis in patients with lung disease. Thorax 1991;46:15–20.
8 Murphy JW, Friedman H, Bendinelli M: Fungal infections and immune responses. New York,
Plenum Press, 1993.
9 Monod M, Fatih A, Joton-Ogay K, Paris S, Latgé JP: The secreted proteases of pathogenic species
of Aspergillus and their possible role in virulence. Can J Bot 1995;73:1081–1086.
10 Bouchara JP, Tronchin G, Larcher G, Chabasse D: The search for virulence determinants in
Aspergillus fumigatus. Trends Microbiol 1995;3:327–330.
11 Calera JA, Paris S, Monod M, Hamilton AJ, Debeaupuis JP, Diaquin M, Lopez-Medrano R, Leal F,
Latgé JP: Cloning and disruption of the antigenic catalase gene of Aspergillus fumigatus. Infect
Immun 1997;65:2717–2724.
12 Smith JM, Tang CM, Van Noorden S, Holden DW: Virulence of Aspergillus fumigatus double
mutants lacking restrictocin and an alkaline protease in a low-dose model of invasive pulmonary
aspergillosis. Infect Immun 1994;62:5247–5254.
13 Aufauver-Brown A, Mellado E, Gow NAR, Holden DW: Aspergillus fumigatus chsE: A gene
related to chs3 of Saccharomyces cerevisiae and important for hyphal growth and conidiospore
development but not pathogenicity. Fungal Genet Biol 1997;21:141–152.
14 Jaton-Ogay K, Paris S, Huerre M, Quadroni M, Falchetto R, Togni G, Latgé JP, Monod M:
Cloning and disruption of the gene encoding an extracellular metalloprotease of Aspergillus fumigatus. Mol Microbiol 1994;14:917–928.
15 Annaix V, Bouchara JP, Larcher G, Chabasse D, Tronchin G: Specific binding of human fibrinogen fragment D to Aspergillus fumigatus conidiae. Infect Immun 1992;60:1747–1755.
16 Tronchin G, Bouchara JP, Larcher G, Lissitzky JC, Chabasse D: Interaction between Aspergillus
fumigatus and basement membrane laminin: Binding and substrate degradation. Biol Cell 1993;
77:201–208.
17 Coulot P, Bouchara JP, Reiner G, Annaix V, Planchenault C, Tronchin G, Chabasse D: Specific
interaction of Aspergillus fumigatus with fibrinogen and its role in cell adhesion. Infect Immun
1994;62:2169–2177.
18 Gill ML, Penalver MC, Lopez-Ribot JK, O’Conner JE, Martinez JP: Binding of extracellular
matrix proteins to Aspergillus fumigatus conidia. Infect Immun 1996;64:5239–5247.
19 Ollert MW, Söhnchen R, Korting HC, Ollert U, Bräutigam S, Bräutigam W: Mechanisms of adherence of Candida albicans to cultured human epidermal keratinocytes. Infect Immun 1993;61:
4560–4568.
20 Bromley IMJ, Donaldson K: Binding of Aspergillus fumigatus spores to the lung epithelial cells
and basement membrane proteins: Relevance to the asthmatic lung. Thorax 1996;51:1203–1209.
21 Amitani R, Taylor G, Elezin EN, Lewellyn-Jones C, Mitchell J, Kuze F, Cole PJ, Wilson R:
Purification and characterization of factors produced by Aspergillus fumigatus which affect
human ciliated respiratory epithelium. Infect Immun 1995;63:3266–3271.
22 Schaffner A, Douglas H, Braude A: Selective protection against conidia by mononuclear and
against mycelia by polymorphonuclear phagocytes in resistance to Aspergillus. J Clin Invest
1982;69:617–631.
23 Kurup VP: Interaction of Aspergillus fumigatus spores and pulmonary alveolar macrophages of
rabbits. Immunobiology 1984;166:53–61.
Crameri
88
24 Schaffner A, Davis CE, Schaffner T, Markert M, Douglas H, Braude AI: In vitro susceptibility of
fungi to killing by neutrophil granulocytes discriminates between primary pathogenicity and
opportunism. J Clin Invest 1986;78:511–524.
25 Schneemann M, Schaffner A: Host defense mechanisms in Aspergillus fumigatus infections; in
Brakhage AA, Jahn B, Schmidt A (eds): Aspergillus fumigatus. Contrib Microbiol. Basel, Karger,
1999, vol 2, pp 57–68.
26 Kurup VP, Kumar A: Immunodiagnosis of aspergillosis. Clin Microbiol Rev 1991;4:439–456.
27 Cohen MS, Isturiz RE, Malech HL, Root RK, Wifert CM, Gutman L, Buckley RH: Fungal infection in chronic granulomatous disease: The importance of the phagocyte in defence against fungi.
Am J Med 1981;71:59–66.
28 Gerson SL, Tablot GH, Hurwitz S, Strom BL, Lusk EJ, Cassileth PA: Prolonged granulocytopenia:
The major risk factor for invasive pulmonary aspergillosis in patients with acute leukaemia. Ann
Intern Med 1984;100:345–351.
29 Shah A, Khan ZU, Chaturvedi S, Ramchandran S, Dandhawa HS, Jaggi OP: Allergic bronchopulmonary aspergillosis with coexistent aspergilloma: A long-term follow-up. J Asthma 1989;
21:109–115.
30 Crameri R: Recombinant Aspergillus fumigatus allergens: From the nucleotide sequences to clinical applications. Int Arch Allergy Immunol 1998;115:99–114.
31 Slavin RG: Rhinitis, sinusitis, otitis and oral lesions; in Lockey RF, Bukantz SC (eds):
Fundamentals of Immunology and Allergy. Philadelphia, Saunders, 1987, pp 27–44.
32 Gourley DS: Allergic fungal sinusitis. Insights Allergy 1989;4:1–7.
33 Greenberger PA: Allergic bronchopulmonary aspergillosis and fungoses. Clin Chest Med 1988;9:
599–609.
34 Borga A: Allergens of Aspergillus fumigatus; PhD thesis, Stockholm, 1990.
35 Tromplet J, Becker W, Schlaak M: Analysis of IgG and IgE responses in allergic disease caused by
Aspergillus fumigatus by immunolotting techniques. Int Arch Allergy Immunol 1994;104:390–398.
36 Kauffman HF, Tomee JFC, Werf TS, de Monchy JGR, Koëter GK: Review of fungus-induced asthmatic reactions. Am J Respir Crit Care Med 1995;151:2109–2116.
37 Greenberger PA, Patterson R: Allergic bronchopulmonary aspergillosis and the evaluation of the
patient with asthma. J Allergy Clin Immunol 1988;91:646–650.
38 Patterson R, Rosenberg M, Roberts M: Evidence that Aspergillus fumigatus growing in the airway
of man can be a potent stimulus of specific and nonspecific IgE formation. Am J Med 1977;63:
257–262.
39 Nicholai T, Arleth S, Spaeth A, Bertle-Harms R, Harms HK: Correlation of IgE antibody titre to
Aspergillus fumigatus with decreased lung function in cystic fibrosis. Pediatr Pulmonol 1990;8:
12–15.
40 Hinson KFW, Moon AJ, Plummer NS: Bronchopulmonary aspergillosis. A review and report of
eight new cases. Thorax 1952;7:317–333.
41 Slavin RG, Stanczyk DJ, Lonigro AJ, Brown GS: Allergic bronchopulmonary aspergillosis – a
North American rarity. Am J Med 1969;47:306–313.
42 Hoehne JH, Reed CE, Dickie HA: Allergic bronchopulmonary aspergillosis is not rare. Chest
1973;63:177–181.
43 Riechson RB, Stander PE: Allergic bronchopulmonary aspergillosis an increasingly common disorder among asthmatic patients. Postgrad Med 1988;88:217–219.
44 Patterson R, Greenberger PA, Radin RC, Roberts M: Allergic bronchopulmonary aspergillosis:
Staging as an aid to management. Ann Allergy 1986;56:444–453.
45 Patterson R, Greenberger PA, Roberts M: The diagnosis of allergic bronchopulmonary aspergillosis; in Patterson R, Greenberger PA (eds): Allergic Bronchopulmonary Aspergillosis. Providence,
Oceanside Publications, 1995, pp 1–3.
46 Leser C, Kauffman HF, Virchow C, Menz G: Specific serum immunopatterns in clinical phases of
allergic bronchopulmonary aspergillosis. J Allergy Clin Immunol 1992;90:589–599.
47 Hemmann S, Menz G, Ismail C, Blaser K, Crameri R: Skin test reactivity to 2 recombinant
Aspergillus fumigatus allergens in A. fumigatus-sensitized asthmatic subjects allows diagnostic
separation of allergic bronchopulmonary aspergillosis from fungal sensitization. J Allergy Clin
Immunol 1999;104:601–607.
Aspergillus fumigatus Allergens
89
48 Slavin RG, Knutsen AP: Purified Aspergillus proteins: Going where no one has gone before. J Lab
Clin Med 1993;121:380–381.
49 Nikolaizik WH, Crameri R, Blaser K, Schöni MH: Skin test reactivity to recombinant Aspergillus
fumigatus allergen I/a in patients with cystic fibrosis. Int Arch Allergy Immunol 1996;11:
403–408.
50 Roberts M, Greenberger PA: Serologic analysis of allergic bronchopulmonary aspergillosis; in
Patterson R, Greenberger PA (eds): Allergic Bronchopulmonary Aspergillosis. Providence,
Oceanside Publications, 1995, pp 11–15.
51 American Academy of Allergy, Asthma and Immunology. Position statement. The use of standardized allergen extracts. J Allergy Clin Immunol 1997;99:583–586.
52 Basich JE, Graves TS, Baz MN: Allergic bronchopulmonary aspergillosis in corticoid-dependent
asthmatics. J Allergy Clin Immunol 1981;68:98–102.
53 Henderson AH, Englis MP, Veckt RJ: Pulmonary aspergillosis, a survey of its occurrence in
patients with chronic lung disease and a discussion of the significance of diagnostic tests. Thorax
1968;23:513–519.
54 Nelson LA, Callerame ML, Schwartz RH: Aspergillosis and atopy in cystic fibrosis. Am Rev
Respir Dis 1979;120:863–873.
55 Slavin RG: ABPA in CF: A devastating combination. Pediatr Pulmonol 1996;21:1–2.
56 Novey HS: Epidemiology of allergic bronchopulmonary aspergillosis. Immunol Allergy Clin
North Am 1998;18:641–653.
57 Schonheider H, Anderson P: Fractionation of Aspergillus fumigatus antigens by hydrophobic
interaction chromatography and gel filtration. Int Arch Allergy Appl Immunol 1984;73:231–236.
58 Piechura JE, Huang CJ, Cohen SH, Kidd JM, Kurup VP, Calvanico NJ: Antigens of Aspergillus
fumigatus. II. Electrophoretic and clinical studies. Immunol 1983;49:657–665.
59 Longbottom JL, Austick PKC: Antigens and allergens of Aspergillus fumigatus. I.
Characterization by quantitative immunoelectrophoretic techniques. J Allergy Clin Immunol
1986;79:9–17.
60 Samuelsen H, Karlsson-Borga A, Paulsen BS, Wold JK, Rolfsen W: Purification of a 20 kD allergen from Aspergillus fumigatus. Allergy 1991;46:115–124.
61 Arruda LK, Platts-Mills TAE, Longbottom JL, El-Dahr JM, Chapman M: Aspergillus fumigatus:
Identification of 16, 18 and 45 kDa antigens recognized by human IgG and IgE antibodies and
murine monoclonal antibodies. J Allergy Clin Immunol 1992;89:1166–1176.
62 Arruda LK, Platts-Mills TAE, Fox JW, Chapman MD: Aspergillus fumigatus allergen I, a major
IgE binding protein, is a member of the mitogillin family of cytotoxins. J Exp Med 1990;172:
1529–1532.
63 Moser M, Crameri R, Menz G, Schneider T, Dudler T, Virchow C, Gmachl M, Blaser K, Suter M:
Cloning and expression of recombinant Aspergillus fumigatus allergen I/a (rAsp f 1/a) with IgE
binding and type 1 skin test activity. J Immunol 1992;149:454–460.
64 Lamy B, Davies J: Isolation and nucleotide sequence of the Aspergillus restrictus gene coding for
the ribonucleolytic toxin restrictocin and its expression in Aspergillus nidulans: The leader
sequence protects producing strains from suicide. Nucleic Acids Res 1991;19:1001–1006.
65 Lamy B, Moutaouakil M, Latgé JP, Davies J: Secretion of a potential virulence factor, a fungal
ribonucleotoxin, during human aspergillosis infections. Mol Microbiol 1991;5:1811–1815.
66 Moser M, Crameri R, Menz G, Suter M: Recombinant Aspergillus fumigatus allergen I/a (rAsp f
I/a) in the diagnosis of Aspergillus related diseases. Agents Actions 1993;43:131–137.
67 Sacco G, Drickamer K, Wool IG: The primary structure of the cytotoxin ␣-sarcin. J Biol Chem
1983;258:5811–5818.
68 Fernandez-Luna JL, Lopez-Otin C, Soriano F, Méndez E: Complete amino acid sequence of the
Aspergillus cytotoxin mitogillin. Biochemistry 1985;24:861–867.
69 Kao R, Davies J: Molecular dissection of mitogillin reveals that the fungal ribotoxins are a family of natural genetically engineered ribonucleases. J Biol Chem 1999;274:12576–12582.
70 Mayer C, Hemmann S, Faith A, Blaser K, Crameri R: Cloning, production, characterization and
IgE cross-reactivity of different manganese superoxide dismutases in individuals sensitised to
Aspergillus fumigatus. Int Arch Allergy Immunol 1997;113:213–215.
Crameri
90
71 Mayer C, Appenzeller U, Seelbach H, Achatz G, Oberkofler H, Breitenbach M, Blaser K, Crameri
R: Humoral and cell-mediated autoimmune reactions to human acidic ribosomal P2 protein in
individuals sensitized to Aspergillus fumigatus P2 protein. J Exp Med 1999;189:1507–1512.
72 Blaser K, Carballido JM, Faith A, Crameri R, Akdis CA: Determinants and mechanisms of human
immune responses to bee venom phospholipase A2. Int Arch Allergy Immunol 1998;117:1–10.
73 Breiteneder H, Pettenburger K, Bito A, Valenta R, Kraft D, Rumpold H, Scheiner O, Breitenbach
M: The gene coding for the major birch pollen allergen Bet v 1, is highly homologous to a pea
disease resistance response gene. EMBO J 1989;8:1935–1938.
74 Yang X, Moffat K: Insights into specificity of cleavage and mechanism of cell entry from the crystal structure of the highly specific Aspergillus ribotoxin, restrictocin. Structure 1996;4:837–852.
75 Kumar A, Reddy LV, Sochanik A, Kurup VP: Isolation and characterization of a recombinant heat
shock protein of Aspergillus fumigatus. J Allergy Clin Immunol 1993;91:1024–1030.
76 Banerjee B, Kurup VP, Phadnis S, Greenberger PA, Fink JN: Molecular cloning and expression of
a recombinant Aspergillus fumigatus protein Asp f II with significant immunoglobulin E reactivity in allergic bronchopulmonary aspergillosis. J Lab Clin Med 1996;127:253–262.
77 Arruda KL, Mann JB, Chapman MD: Selective expression of a major allergen and cytotoxin, Asp
f 1, in Aspergillus fumigatus. Implications for the immunopathogenesis of Aspergillus-related disease. J Immunol 1992;149:3354–3359.
78 Hemmann S, Blaser K, Crameri R: Allergens of Aspergillus fumigatus and Candida boidinii share
IgE-binding epitopes. Am J Respir Crit Care Med 1997;165:1956–1962.
79 Hemmann S, Ismail C, Blaser K, Menz G, Crameri R: Skin-test reactivity and isotype-specific
immune responses to recombinant Asp f 3, a major allergen of Aspergillus fumigatus. Clin Exp
Allergy 1998;28:860–867.
80 Crameri R, Hemmann S, Ismail C, Menz G, Blaser K: Disease-specific recombinant allergens for
the diagnosis of allergic bronchopulmonary aspergillosis. Int Immunol 1998;10:1211–1216.
81 Hemmann S, Nikolaizik WH, Schöni MH, Blaser K, Crameri R: Differential IgE recognition of
recombinant Aspergillus fumigatus allergens by cystic fibrosis patients with allergic bronchopulmonary aspergillosis or Aspergillus allergy. Eur J Immunol 1998;28:1155–1160.
82 Crameri R: Epidemiology and molecular basis of the involvement of Aspergillus fumigatus antigens in allergic diseases; in Brakhage AA, Jahn B, Schmidt A (eds): Aspergillus fumigatus.
Contrib Microbiol, Basel, Karger, 1999, vol 2, pp 44–56.
83 Crameri R, Faith A, Hemmann S, Jaussi R, Ismail C, Menz G, Blaser K: Humoral and cellmediated autoimmunity in allergy to Aspergillus fumigatus. J Exp Med 1996;184:265–270.
84 Hemmann S: Cloning, characterization and clinical evaluation of recombinant Aspergillus fumigatus allergens; PhD thesis, Zürich, 1998.
85 Jaton-Ogay K, Suter M, Crameri R, Falchetto R, Fatih A, Monod M: Nucleotide sequence of a
genomic and a cDNA clone encoding an extracellular alkaline protease of Aspergillus fumigatus.
FEMS Microbiol Lett 1992;92:163–168.
86 Chou H, Lin WL, Tam MF, Wang SR, Han SH, Shen HD: Alkaline serine proteinase is a major
allergen of Aspergillus flavus, a prevalent airborne Aspergillus species in the Taipei Area. Int Arch
Allergy Immunol 1999;119:282–290.
87 Crameri R, Blaser K: Cloning allergens from Aspergillus fumigatus: The filamentous phage
approach. Int Arch Allergy Immunol 1995;107:460–461.
88 Crameri R, Blaser K: Cloning Aspergillus fumigatus allergens by the pJuFo filamentous phage
display system. Int Arch Allergy Immunol 1996;110:41–45.
89 Kleber-Janke T, Crameri R, Appenzeller U, Schlaak M, Becker WM: Selective cloning of peanut
allergens, including profilin and 2S albumins, by phage display technology. Int Arch Allergy
Immunol 1999;119:265–274.
90 Lindborg M, Magnusson CGM, Zagari A, Schmidt M, Scheynius A, Crameri R, Withley P:
Selective cloning of allergens from the skin colonizing yeast Malassezia furfur by phage surface
display technology. J Invest Dermatol 1999;113:156–161.
91 Brander KA, Borbély P, Crameri R, Pichler WJ, Helbling A: IgE-binding, proliferative responses
and skin test reactivity to Cop c 1, the first recombinant allergen from the basidiomycete Coprinus
comatus. J Allergy Clin Immunol 1999;104:630–636.
Aspergillus fumigatus Allergens
91
92 Crameri R, Suter M: Display of biologically active proteins on the surface of filamentous phages:
A cDNA cloning system for selection of functional gene products linked to the genetic information responsible for their production. Gene 1993;137:69–75.
93 Crameri R, Jaussi R, Menz G, Blaser K: Display of expression products of cDNA libraries on
phage surfaces. A versatile screening system for selective isolation of genes by specific geneproduct/ligand interaction. Eur J Biochem 1994;226:53–58.
94 Crameri R: pJuFo: A phage surface display system for cloning genes based on protein-ligand
interaction; in Schaefer B (ed): Gene Cloning and Analysis: Current Innovations. Wymondham,
Horizon Press, 1997, pp 29–42.
95 Crameri R, Hemmann S, Blaser K: pJuFo: A phagemid for display of cDNA libraries on phage surface suitable for selective isolation of clones expressing allergens. Adv Exp Med 1996;409:103–110.
96 Suter M, Foti M, Ackermann M, Crameri R: A cDNA cloning system based on filamentous phage:
Selection and enrichment of functional gene products by protein/ligand interactions made possible by linkage of recognition and replication functions; in Kay B, Winter KJ, McCafferty J (eds):
Phage Display of Peptides and Proteins. San Diego, Academic Press, 1996, pp 187–205.
97 Appenzeller U, Mayer C, Menz G, Blaser K, Crameri R: IgE-mediated reactions to autoantigens
in allergic diseases. Int Arch Allergy Immunol 1999;118:193–196.
98 Natter S, Seiberler S, Hufnagl P, Binder BR, Hirsch AM, Ring J, Abeck D, Schmidt T, Valent P,
Valenta R: Isolation of cDNA clones coding for IgE autoantigens with serum IgE from atopic
dermatitis patients. FASEB J 1998;12:1599–1569.
99 Bufe A: The biological function of allergens: relevant for the induction of allergic diseases? Int
Arch Allegy Immunol 1998;117:215–219.
100 Förster E, Dudler T, Gmachl M, Aberer W, Urbanek R, Suter M: Natural and recombinant enzymatically active or inactive bee venom phospholipase A2 has the same potency to release histamine from basophils in patients with hymenoptera allergy. J Allergy Clin Immunol 1995;95:
1229–1235.
101 Wymann D, Akdis CA, Blesken T, Akdis M, Crameri R, Blaser K: Enzymatic activity of soluble
phospholipase A2 does not affect specific IgE, IgG4 and cytokine responses in bee sting allergy.
Clin Exp Allergy 1998;28:839–849.
102 Akdis CA, Blesken T, Wymann D, Akdis M, Blaser K: Differential regulation of human T-cell
cytokine patterns and IgE and IgG4 responses by conformational antigen variants. Eur J Immunol
1998;28:914–925.
103 Crameri R, Walter G: Selective enrichment and high-throughput screening of phage surfacedisplayed cDNA libraries from complex allergenic systems. Comb Chem High Throughput Screen
1999;2:63–72.
104 Crameri R, Kodzius R, Konthur Z, Lehrach H, Blaser K, Walter G: Tapping allergen repertoires
by advanced cloning technologies. Int Arch Allergy Immunol 2001;124:43–47.
105 Crameri R, Kodzius R: The powerful combination of phage surface display of cDNA libraries and
high throughput screening. Comb Chem High Throughput Screen 2001;4:145–155.
106 Crameri R: High throughput screening: A rapid way to recombinant allergens. Allergy 2001;56
(suppl 67):30–34.
107 Müller UR, Dudler T, Schneider T, Crameri R, Fischer H, Skrbic D, Maibach R, Blaser K, Suter
M: Type I skin reactivity to native and recombinant phospholipase A2 from honeybee venom is
similar. J Allergy Clin Immunol 1995;96:395–402.
108 Crameri R, Lidholm J, Grönlund H, Stüber D, Blaser K, Menz G: Automated specific IgE assay
with recombinant allergens: evaluation of the recombinant Aspergillus fumigatus allergen I in the
Pharmacia CAP system. Clin Exp Allergy 1996:26;1411–1419.
109 Moser M, Crameri R, Brust E, Suter M, Menz G: Diagnostic value of recombinant Aspergillus
fumigatus allergen I/a for skin testing and serology. J Allergy Clin Immunol 1994;93:1–11.
110 Müller U, Fricker M, Wymann D, Blaser K, Crameri R: Increased specificity of diagnostic
tests with recombinant major bee venom allergen phospholipase A2. Clin Exp Allergy 1997;27:
915–920.
111 Van der Zee JS, de Groot H, van Swieten P, Jansen HM, Aalberse RC: Discrepancies between the
skin test and IgE antibody assays: Study of histamine release, complement activation in vitro, and
occurrence of allergen specific IgE. J Allergy Clin Immunol 1988;82:270–281.
Crameri
92
112 Vijay HM, Perlmutter L, Bernstein IL: Possible role of IgG4 in discordant correlations between
intracutaneous skin tests and RAST. Int Arch Allergy Appl Immunol 1978;56:517–522.
113 Kurup VP, Banerjee B, Hemmann S, Greenberger PA, Blaser K, Crameri R: Selected recombinant
Aspergillus fumigatus allergens bind specifically to IgE in ABPA. Clin Exp Allergy 2000;30:
988–993.
114 Swanson MC, Agarwal MK, Reed CD: An immunochemical approach to indoor aeroallergen
quantitation with a new volumetric air sampler: Studies with mite, roach, cat, mouse and guinea
pig antigens. J Allergy Clin Immunol 1985;76:724–729.
115 Reiner SL, Locksley RM: The regulation of immunity to Leishmania major. Annu Rev Immunol
1995;13:151–177.
Prof. Dr. Reto Crameri, PhD
Head Molecular Allergology, Swiss Institute of Allergy and Asthma Research,
Obere Strasse 22, CH–7270 Davos (Switzerland)
Tel. ⫹41 81 410 08 48, Fax ⫹41 81 410 08 40, E-Mail crameri@siaf.unizh.ch
Aspergillus fumigatus Allergens
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Breitenbach M, Crameri R, Lehrer SB (eds): Fungal Allergy and Pathogenicity.
Chem Immunol. Basel, Karger, 2002, vol 81, pp 94–113
Defense Mechanisms of the Airways
against Aspergillus fumigatus:
Role in Invasive Aspergillosis
H.F. Kauffman, J.F.C. Tomee
Laboratory for Allergology and Pulmonology, Department of Allergology,
University Hospital, Groningen, The Netherlands
Fungal infections, including those by Aspergillus fumigatus, have become
more prevalent in recent years. Intensified medical treatment causing iatrogenic
immunosuppression (e.g. in organ transplantation and cancer therapy) may
explain the increase. Also in patients with acquired immunosuppression, as
with the acquired immuno deficiency (AIDS) pandemic, fungal infections are
increasingly found.
When fungi interact with the human host two factors determine the
outcome. Characteristics of the fungus facilitating its invasiveness and survival
in the human host (collectively described as the ‘fungal virulence factors’) and
the ability of the host to cope with the fungal threat (the host defense strategies).
Especially in the case of pathogenic fungi such as A. fumigatus the fungus may
overwhelm the host defense system by causing immunosuppression in various
ways and damaging the local barrier function of the respiratory epithelium [1].
In doing so, the fungus may cause derailment of host defenses leading to a selfperpetuating cycle of inflammation which will further facilitate fungal invasion
[1, 2]. The pathogenicity of A. fumigatus in the animal and human host is
remarkable and several factors may explain its pathogenicity.
The human airways are continuously exposed to large numbers of fungal
spores that enter the lower airways. Nevertheless, human airways have a remarkable capacity to eliminate most of the inhaled fungal spores and only a small number of fungi are known to cause airway diseases, often under conditions of altered
immune activity (e.g. in atopy or immunosuppression). The general strategy of the
airways to resist fungal infection is aimed at elimination of the offending fungus
by ciliary clearing of the airways, defensive properties of the resident cells in the
airways and phagocytic cell activity.
Although the genus Aspergillus encompasses over 350 species, most
infections are caused by A. fumigatus, Aspergillus niger, Aspergillus flavus and
Aspergillus terreus. Especially, A. fumigatus is known as a cause of multiple
airway diseases ranging from harmless saprophytic colonization, local inflammatory disease in immunocompetent individuals to life-threatening invasive
disease in immunocompromised patients. In this review we will focus on airway
diseases caused by A. fumigatus in both immunocompetent and immunocompromised patients with emphasis on the different defense strategies of the airways
against this fungus.
Overview of Manifestations of Aspergillosis
An intriguing aspect of the biological behavior of A. fumigatus is its ability
to colonize the human bronchopulmonary system under various circumstances.
Aspergillus species are ubiquitous in the environment and are inevitably inhaled
into the airways. Following the inhalation of Aspergillus conidia or mycelial fragments, colonization of the airways may result. Colonization does not necessarily
result in disease, yet, contrary to the seemingly innocuous ‘nonpathogenic saprophytic colonization’ in healthy individuals, acute and often fulminant invasive
disease may occur in patients who are vulnerable to infection due to underlying
lung diseases or immunosuppression. In addition to its ability to colonize the
human respiratory tract, Aspergillus has a significant potential to act as a powerful allergenic source. Aspergillus conidia (and mycelial fragments) in the
bronchial tree may result in the development of an allergic state in individuals
with a predisposition to develop allergic sensitization (atopy), as in patients with
Aspergillus-related asthma and allergic bronchopulmonary aspergillosis (ABPA).
Over the past decades various different classifications of pulmonary aspergillosis have been proposed. Probably one of the best known and widely accepted
of all has been presented by Bardana [3]. In this system pulmonary aspergillosis
is classified into four groups which will be discussed here in more detail.
Nonpathogenic Saprophytic Colonization
Aspergillus species may cause saprophytic infestation of the human respiratory tract without causing manifest tissue damage. Mostly, this form of saprophytic
colonization goes without disease and therefore this condition is not an infection as
such. Generally, saprophytic colonization remains untreated; however, in patients
with enhanced susceptibility to fungal infection, notably after organ transplantation, leukemia, or cancer chemotherapy, antifungal treatment is often warranted.
Defense Mechanisms against Fungi
95
Saprophytic colonization is found with increased incidence in patients
with underlying pulmonary diseases, such as in advanced stages of chronic
obstructive pulmonary disease, chronic asthma requiring administration of
adrenal corticosteroids, primary ciliary dyskinesia syndrome and cystic fibrosis [4–7]. A. fumigatus is the predominant species cultured from the respiratory
tract although other Aspergillus species may also be found occasionally.
Aspergilloma
Aspergilloma (mycetoma or fungus ball) usually is regarded as a saprophytic
growth of a fungus in a preformed and mostly poorly drained lung space, typically
in the upper lung fields. Pulmonary aspergilloma is mostly caused by A. fumigatus
although other Aspergillus species may also be associated with aspergilloma
formation. Aspergilloma may be found in patients with a history of a disease,
mostly tuberculosis or sarcoidosis, where cavities or large bronchiectatic cysts have
formed. Formation of aspergilloma may follow or may precede other pulmonary
illnesses including ABPA. The average size of a pulmonary aspergilloma is 3–5 cm
in diameter and may be visualized by conventional roentgenography or (computed) tomography of the thorax as a typical air-crescent sign (Monod’s sign)
surrounding a dense structure (i.e., the fungus ball). Diagnosis of pulmonary
aspergilloma requires this typical chest picture together with either a positive
culture of a specimen obtained from the cavitary lesion or positive IgG antibody
titers against A. fumigatus measured by an enzyme-linked immunosorbent assay
(ELISA) or serum precipitin. The pulmonary aspergilloma is composed of
cellular debris, fibrin, tangled fungal hyphae, mucus and a few inflammatory cells.
The occurrence of pulmonary aspergilloma in the general population is unknown,
but has been estimated to be 0.01–0.02%.
The natural history of pulmonary aspergilloma is highly variable. Pulmonary
aspergilloma may exist for a long period without any adverse effects. Spontaneous
lysis has been reported in up to 10% of the cases. The most common symptom is
recurrent hemoptysis, which may cause fatal asphyxia. Treatment of active disease
usually consists of resection of the fungus ball or antifungal therapy.
Hypersensitivity-Induced Aspergillosis
Aspergillus Asthma
Exposure to Aspergillus allergens in patients with atopy may lead to allergic
reactions in the upper (rhinitis, sinusitis) or lower airways (Aspergillus asthma,
ABPA). In the lower airways Aspergillus asthma is caused by a type I allergic
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reaction upon exposure to Aspergillus conidia or hyphae. Sensitization to
Aspergillus is found mostly in combination with sensitization to other inhalant
allergens, such as house dust mite, tree pollen and grass pollen.
Allergic Bronchopulmonary Aspergillosis
ABPA is regarded as a complication of asthma, and occurs in approximately
1% of asthmatic patients. In addition to fever and malaise, the acute phase
presents as an acute, easily reversible asthmatic syndrome with dyspnea and
transient pulmonary infiltrations of eosinophils, neutrophils and lymphocytes,
which may be effectively treated with corticosteroids. This condition usually
progresses to a corticosteroid dependent and more intractable asthmatic state
with transient pulmonary infiltrates (pulmonary eosinophilia) and a high sensitization to Aspergillus. From there the usual progression of the disease is to
fibrosis and bronchiectasis. Most patients come to medical attention before the
age of 35 years. The disease is a common complication of cystic fibrosis.
Eight diagnostic criteria for ABPA have been proposed by Rosenberg et al.
[8] and Patterson et al. [9] and these include: (1) episodic bronchial obstruction
(asthma); (2) peripheral blood eosinophilia (⬎1,000/mm3); (3) elevated total IgE
level in serum; (4) specific IgE and IgG to Aspergillus antigen in serum; (5) immediate (type I) skin reactivity to Aspergillus antigen; (6) precipitating antibodies to
Aspergillus; (7) transient pulmonary infiltrates, and (8) central bronchiectasis.
Sputum often contains A. fumigatus and sometimes may grow Aspergillus.
Proximal bronchiectasis is a characteristic bronchographic finding of patients with
ABPA, and atelectasis and cavitations can sometimes be found [10, 11].
ABPA is immunologically characterized by type I, type III and type IV
hypersensitivity reactions. The type I immune reactions mediated by IgE antibody result in bronchospasm, eosinophilia and immediate skin reactivity, all of
which can be experimentally induced by cutaneous or bronchial provocation
with A. fumigatus extracts. The severe pulmonary eosinophilic infiltrate is possibly mediated by an extended or late-type reaction induced by factors from mast
cell degranulation and activation of epithelial cells [2]. The development of the
bronchiectatic lesions may be the consequence of both the excretion of proteolytic enzymes by A. fumigatus and a type III (Arthus’ or immune-complexmediated) reaction mediated by circulating or precipitating antibody complexes.
Strongly elevated titers of IgG and IgA antibodies have been described in the
peripheral blood and in bronchoalveolar fluid [12, 13]. Type IV delayed hypersensitivity reaction may be involved as evidenced by in vitro lymphocyte transformation responses to Aspergillus antigens [8], and may cause granulomas and
contribute to mononuclear infiltration.
Although the airway damage, resulting in proximal bronchiectasis, may be
mediated by various of the aforementioned immunologic mechanisms, at least
Defense Mechanisms against Fungi
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some of the airway destruction may be explained by microinvasive processes in
the airway wall. Studies have suggested that patients with ABPA display evidence for ‘limited fungal invasion’ of the lung parenchyma [14–17]. Riley et al.
[18] demonstrated viable fungi within the parenchyma of a patient with ABPA.
A lung biopsy specimen obtained from a child with cystic fibrosis and ABPA
indicated a marked disruption of elastin layers in bronchioles, whereas hyphae of
A. fumigatus were observed in the parenchyma [19]. Some have suggested that
this microinvasive activity may be mediated by the release of fungal products,
such as proteases, in the area of the airway wall [2, 15, 20–23].
Hypersensitivity Pneumonitis
Hypersensitivity pneumonitis (or extrinsic allergic alveolitis) is an inflammatory interstitial lung disease possibly resulting from hypersensitivity type III
and type IV reactions following persistent or intense exposure to antigens
of bacterial, animal, or fungal origin or to reactive chemical sources.
Hypersensitivity pneumonitis occurs primarily in nonatopic individuals and
more frequently in men than in women and children. The clinical symptoms
occur typically within 4–8 h following the exposure to the offending agent
with resultant systemic and respiratory manifestations often with fever.
Histologically, the disease is characterized by a diffuse mononuclear cell infiltration of alveolar wall, alveoli, terminal bronchioles, and neighbouring interstitium leading to radiological abnormalities. The physiological correlates are
restriction of lung volumes and impaired oxygenation. Damage to the peripheral
airways and surrounding parenchyma results in lung restriction; however, overt
lung obstruction, probably as a result of lesions located in the bronchioles, is present in some patients. Diagnosis is often made on the basis of a history of environmental or occupational exposure, the presence of elevated IgG antibodies
against the environmental antigen as determined by ELISA or precipitating antibodies to the suspected antigen, the presence of suppressor cytotoxic lymphocytosis in bronchoalveolar lavage fluid, and granulomatous alveolitis in lung
biopsy specimens may confirm the diagnosis. The most frequent and commonly
known is ‘farmer’s lung disease’ resulting from inhalation of large quantities of
thermophilic actinomycetes (Thermoactinomycetes vulgaris, Micropolyspora
faeni). Various reports have related hypersensitivity pneumonitis to intense
exposure to Aspergilli as well. Aspergillus clavatus, present in mouldy barley on
brewery floors, may cause ‘malt-worker’s lung’ [24]. Aspergillus umbrosus,
A. fumigatus and A. terreus, present in mouldy oats, corn or hay, may cause
‘farmer’s lung disease’ [25–27]. A. fumigatus has been identified as the causative
organism in ‘greenhouse lung’ [28] and ‘tobacco worker’s lung’ [29]. Over the
past decades the immunopathogenesis and cellular mechanisms of hypersensitivity pneumonitis have been ascribed to cell-mediated immune mechanisms,
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resulting in perpetual lung injury and chronic inflammation characterized by
fibrotic and granulomatous processes in the lung interstitium.
Invasive Pulmonary Aspergillosis
The most severe and life-threatening pulmonary disease caused by
Aspergillus is invasive aspergillosis. The term invasive aspergillosis generally
denotes histopathologically demonstrated fungal invasion into tissue, preferably
with confirmation by culturing the organism. The incidence of invasive fungal
infections has increased dramatically in the last decades due to the growing
number of immunocompromised patients who survive longer than in the past,
the widespread use of immunosuppressive drugs, a large aging population with
increased numbers of malignancies, and the AIDS pandemic. Generally, as holds
true for bacterial and viral infections, immunosuppression is the major condition
that increases the susceptibility to fungal infections, including aspergillosis.
Immunosuppression may result from various causes, including immunosuppressive and antibacterial therapy or chemotherapy. Underlying diseases leading to
T cell depletion as in patients infected with human immunodeficiency virus and
the use of invasive procedures, such as catheters and prosthetic devices, are recognized as major risk factors for developing invasive aspergillosis. Prolonged
neutropenia is the leading cause of invasive aspergillosis. The risk has been
calculated to increase from 1% per day after the first 3 weeks of neutropenia to
4.5% per day after 5 weeks [30]. The important role of neutrophils in the protection against Aspergillus is also illustrated by the increased incidence of
invasive aspergillosis observed in patients with a genetic defect in neutrophil
function as in chronic granulomatous disease.
Host defense against Aspergillus
The defense system of the airways includes both the innate, nonadaptive
defense at the level of the mucociliary system and the adaptive immune
response. Special attention will be given to the innate defense system as an
important first-line defense against inhaled microorganisms.
Innate Defense Strategies of Airways against Fungi
The innate nonadaptive mucosal defense system of the airways of
healthy individuals is a highly efficient primary defense for the elimination of
Defense Mechanisms against Fungi
99
microorganisms. Some parts of this primary defense are not influenced by
immunosuppressive therapy and function as the only barrier against invading
fungi under severe immunosuppressed conditions.
The defense against A. fumigatus by the mucosal airway system is diverse.
Cell-mediated defense plays a crucial role in the host defense against most fungal pathogens, including Aspergillus. Alveolar macrophages, polymorphonuclear cells and defense effector proteins produced by resident cells cooperate
in the control and elimination of the fungus in the airways [2]. Different functional levels may be distinguished (fig. 1).
By junctional contacts the epithelial cells of the airway mucosa function as
a physical barrier that prevents the penetration and passage of microorganisms.
In addition, excretory cells present in the epithelial cell layer produce glycoconjugates, defensive proteins and enzymes that together constitute a protective
environment. The mucus layer (or epithelial lining fluid) of the airways facilitates the elimination of inhaled particles, including fungal spores. Mucociliary
clearance is mediated by coordinated ciliary movement from specialized epithelial cells that transport the epithelial lining fluid together with impacted material, e.g. fungal spores, out of the airways into the pharynx (fig. 1). The clearance
rate of the epithelial lining fluid is dependent on the ciliary beating frequency
that can be modulated by multiple factors and mediators, e.g. prostaglandins,
that are released during infectious and asthmatic inflammatory responses.
Pseudomonas aeruginosa, Haemophilus influenzae and Aspergillus may release
factors that inhibit the beating frequency [31], facilitating the survival of these
microorganisms in the airways.
The mucosal airway defense is mediated by defense effector proteins that
have a direct antimicrobial activity which is not dependent on the cellular defense
system (fig. 1) [32, 33]. Antibacterial and antifungal proteins are constitutively
produced by airway epithelial cells. Although the regulation of these defensive proteins is largely unknown, the production was shown to be enhanced by bacterial
stimulation [32–35]. These defensive proteins mainly belong to the family of the
cationic defensins, most of them being salt-sensitive, and contribute to the innate
defense in vertebrate and invertebrate organisms. Both ␣-defensins derived from
neutrophils and the human -defensin-1 from epithelial cells, have strong bactericidal activity [32]. Other members of the -defensin family, tracheal antimicrobial
peptide and lingual antimicrobial peptide have been shown to be inducible proteins
in epithelial cells of the mucosal surface [36]. Antileukoprotease, also known
as secretory leukoprotease inhibitor, has been studied with respect to its capacity
to inhibit serine proteases, but aside from being an antiprotease, it was also
demonstrated to possess strong antifungal activity [37, 38]. Recombinant
antileukoprotease expressed pronounced fungicidal and fungistatic activity
towards metabolically active A. fumigatus conidia and C. albicans yeast cells,
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Phagocytic cells
Alveolar macrophage
Monocyte
Neutrophil
Antifungal/bacterial
Clearance
Opsonization
proteins
Transport
system
• Ciliary
beating
frequency
• Mucin
Lysozyme
SLPI/ALP
-Defensins
TAP
LAP
LL-37
Sp-A
Sp-D
MBP
Complement
sIgA
Phagocytosis
Oxygen radicals
Nitric oxide
␣-Defensins
Killing
IL-1, TNF-␣
Fungal
spores
Proteases
Cytokines
IL-8
IL-6
MCP-1
Neutrophils
Lymphocytes
-ELF
-Epithelial cell layer
-Basal membrane
Inflammation
Fig. 1. Innate defense of the airway mucosa in the immune competent host. The first
barrier of the airway mucosa is the epithelial cell layer. On top of the epithelial cells, a mucus
layer (epithelial lining fluid, ELF) contains various proteins that are produced by both columnar
and basal epithelial cells, and by specific excretory cells. The rate of transportation of the mucus
layer by coordinated cilia beating to the pharynx determines the rate of clearance of particles that
are deposited on the mucus layer (first block, left upper corner). Within the mucus layer several
antifungal proteins are present with killing potency for bacteria and fungi (second block, upper
left). These components include lysozyme, secretory leukoprotease inhibitor (SLPI) or
antileukoprotease (ALP), -defensins, tracheal antimicrobial peptide (TAP), lingual antimicrobial peptide (LAP), and LL-37 as antimicrobial peptides. Block three shows glycoproteins that
bind to microorganisms (opsonization) which will prevent binding to the epithelial cells and will
give help to phagocytic leukocytes. Some of these components (the family of C-type lectins and
collectins) are the surfactant proteins A and D (Sp-A, Sp-D), the mannan-binding protein/lectin
(MBP) and components of the complement system. Secretory IgA also belongs to the first barrier defense and is produced as an adaptive response in contrast to the nonadaptive innate recognition. The second part of the defense is the phagocytosis and killing by phagocytic cells that
can recognize the microorganism by innate receptors of the Toll-like receptor family. The phagocytic cells will kill the fungi with the help of antifungal proteins (defensins) and the production
of oxygen radicals and nitric oxide (block upper right). During the activation and killing process
phagocytes will produce IL-1 and tumor necrosis factor-␣ (TNF-␣). These proinflammatory
cytokines will stimulate the epithelial cells in enhancing the production of defense proteins (see
second and third block) and will stimulate the epithelial cells to produce various chemokines
(IL-8, RANTES, MCP-1, IL-6). These chemokines will attract neutrophils, monocytes and
lymphocytes to give additional help in the defense against the fungi (lower part of the figure).
Defense Mechanisms against Fungi
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whereas metabolically quiescent A. fumigatus conidia were totally resistant.
Antileukoprotease is composed of two highly homologous domains. The COOHterminal domain contains the proteinase inhibitor site that also blocks the serine
proteinase activity of A. fumigatus. Until recently, the function of the second NH2
terminal domain has been largely unknown. However, the fungicidal activity of
recombinant antileukoprotease molecule was found to be localized primarily in the
NH2 terminal domain. This may be explained by the more basic properties of the
NH2 terminal domain. On a molar base, the fungicidal activity of recombinant
antileukoprotease was comparable with the fungicidal activity of a mixture of
human neutrophil -defensins (HNP1-3) and also with that of lysozyme.
Currently, more of these proteins are detected in the epithelial lining fluid that may
play an important role in the defense against infectious agents such as bacteria,
fungi and viruses [32–36, 38]. The mechanisms by which these cationic proteins,
especially defensins, kill microorganisms are not fully understood. The bactericidal activity of defensins were found to be associated with pore formation in the
outer membrane of bacteria. Comparable mechanisms may also pertain to the
fungicidal activity. In fact, binding of cationic antimicrobial proteins to the fungal
cell wall chitin, a prerequisite for pore formation, has been demonstrated. In addition to the cationic defensive proteins, products such as serum-derived transferrin
and polymorphonuclear cell-derived lactoferrin inhibit growth of microorganisms
by strongly binding to iron.
C-type lectins, glycolipids and glycoproteins are involved in the innate
defense of the airways by the binding to surface structures of fungi (fig. 1;
opsonization) [39]. These glycoproteins will impede the attachment of microorganisms to the epithelial surface, preventing access to the host. Components
of this defense are the collectins, e.g. surfactants (Sp-A, Sp-D) that together
with complement and mannose-binding protein/lectin act through the recognition of ‘patterns of molecular structures’ present on the surface of large groups
of microorganisms [40, 41].
Complement components are deposited on the surface of microorganisms
in an inflammatory milieu. These microorganism-bound opsonins may then bind
to the cell surface of macrophages and enhance phagocytosis and killing. The
importance of complement, particularly the opsonic component C3, has been
documented in the defense against different fungi [42, 43]. Yet, the role of complement (and antibody) in the defense against Aspergillus is far less clear, and
contrasting data have been reported [44–48]. Kurup [48] found that prior
opsonization of A. fumigatus conidia with normal rabbit serum, rabbit antiA. fumigatus serum, complement, or lung lavage fluid, did not markedly enhance
attachment, internalization or killing of the conidia by rabbit alveolar
macrophages, suggesting that opsonization of Aspergillus is not required for
binding to rabbit macrophages. Terminal components of the complement system
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may also cause lysis of microbial cell membranes. Fungi, however, are resistant
to lysis by these terminal components, even in the presence of specific antibody,
presumably as a result of their thick cell wall. Therefore, as opposed to bacteria
and viruses, fungi are not killed following incubation in serum.
Although the role of complement in the human host defense against
Aspergillus is not clear, some studies indicate that activation of complement by
Aspergillus depends on the presence of complement receptors on the fungal
surface. C3 deposition via activation of the alternative complement pathway was
shown for A. fumigatus conidia, especially during germination of the spores.
Like resting conidia, hyphae are poor activators of complement [49] and are
damaged by neutrophils in vitro in the absence of complement [50]. In summary,
the activation of the alternative pathway seems to be the main mechanism of the
activation of the complement system by the conidia. However, its role in vivo in
the defense against A. fumigatus needs additional studies.
In contrast to the nonadaptive defense system discussed so far, secretory
IgA (sIgA) is produced as the result of a specific adaptive immune response.
Binding of sIgA to the surface of fungi will prevent the binding of the spores
to the epithelial surface. Furthermore, it has been shown that sIgA bound to
immobilized antigen is important for the activation of certain cell types, e.g.
eosinophilic leukocytes [51, 52]. sIgA against A. fumigatus in human bronchoalveolar lavage fluid has been shown to be locally produced and is enhanced in
bronchoalveolar fluid in patients with ABPA [13].
Phagocytosis and killing by phagocytic cells such as alveolar macrophages
and polymorphonuclear cells in the epithelial lining fluid constitute a crucial
role in the innate defense of the airways (fig. 1). Macrophages selectively protect against conidia and their importance in mediating the first line of host
defense against inhaled Aspergillus has been clearly demonstrated. In murine
models macrophages destroyed inhaled conidia and prevented lethal infection
in vivo even in neutropenic or athymic mice. Phagocytic cells have receptors that
recognize surface structures of microorganisms, the so-called ‘pattern recognition receptors’ [53]. For example, studies by Turner [41] have shown that fungal
mannan is recognized by the mannan-binding protein present on phagocytic
cells. The efficiency of phagocytosis and subsequent killing is improved by
opsonization with sIgA and the various glycoproteins (described above) that are
present in the epithelial lining fluid.
Those conidia that have escaped the first line of cellular defense may
germinate and grow as the hyphal form (fig. 1). Aspergillus hyphae have a long,
dichotomously branched, filamentous shape that cannot be readily phagocytosed
by a single cell. Protection against the hyphal form is mediated by polymorphonuclear cells and monocytes (fig. 1). Adhesion to hyphae and release of the
reactive oxygen intermediates and cationic peptides by these cells mediating the
Defense Mechanisms against Fungi
103
second line of the innate defense are considered key mechanisms in causing
hyphal damage. The strong clinical association of severe neutropenia and pathological dysfunction of polymorphonuclear cells with the high prevalence of
invasive aspergillosis illustrates the importance of these cells in host defense.
Following phagocytosis, macrophages are able to kill intracellular
microorganisms by several mechanisms. Normally, phagocytosis leads to the
production of reactive oxygen species (referred to as the ‘respiratory burst’),
such as nitric oxide, hydrogen peroxide, superoxide anion, and hydroxyl radical, that mediate the oxidative killing of microorganisms. In some pathological
situations such as in patients with chronic granulomatous disease, the
macrophages, monocytes and neutrophils fail to produce reactive oxygen
species in response to phagocytosis. Aspergillus infections are common in these
patients, suggesting that reactive oxygen species are required to kill the fungus
in vivo.
After binding and phagocytosis, alveolar macrophages and monocytes
release pro-inflammatory cytokines, e.g. IL-1, interferon-␥ and tumor necrosis
factor-␣ (fig. 1). These cytokines activate resident cells (epithelial cells, fibroblasts) to produce chemokines such as IL-8, granulocyte-monocyte-colonystimulating factor, RANTES (regulated on activation normal T cell expressed
and secreted) that will result in a second wave of cell recruitment (mononuclear
and polymorphonuclear cells).
In recent years the epithelial cell has been recognized as a central player for
regulating the natural and acquired immune system of the host at mucosal surfaces. In a study with epithelial cell lines from human airways it was shown that
fungal serine protease activity present in culture filtrates from A. fumigatus
induced the production of interleukin IL-8 and IL-6 and monocyte chemotactic
protein-1 and caused cell detachment in a dose-dependent fashion [54, 55].
Chymostatin, antipain, phenylmethylsulfonyl fluoride and heat treatment completely inhibited fungal protease activity, cytokine production and cell detachment, whereas antileukoprotease partially inhibited these activities [55]. These
observations have shown that by causing cell detachment, fungal proteases may
decrease the physical barrier function of the epithelium. However, by eliciting a
cytokine response, the epithelium may signal the mucosal inflammatory
response resulting in the recruitment of phagocytes against A. fumigatus. In a
more recent paper it was also shown that, in contrast to other fungi, proteases
from A. fumigatus at higher concentrations are able to inhibit chemokine production by epithelial cells. It was proposed that this silencing of epithelial cells
may play an additional role in the pathogenicity of A. fumigatus by preventing
detection by infiltrating phagocytes [56].
Furthermore, it was shown that spores of A. fumigatus can directly bind to
epithelial surface cultured in vitro [57]. The role of this binding to epithelial
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cells in the defense system is as yet unclear. In one study it was shown that
attachment of spores to the epithelial surface is followed by phagocytosis by the
epithelial cell, suggesting a defensive role [57], whereas in another study binding of spores to the epithelial surface was followed by germination and penetration by hyphae into the epithelial cell [58]. Until now the significance of the
binding of fungal spores to the epithelium for a defensive or invasive role is not
clear, and further studies are needed. However, it is beyond doubt that binding
of microorganisms to host tissues is a prerequisite for survival of the organism
in the human airways in protecting it from mucociliary clearing.
The innate defense strategies described above likely are the major defense
mechanism of the central airways. These mechanisms are highly efficient in
eliminating fungi from the airways and may at least in part explain why
immunocompromised patients are only in part affected by life-threatening invasive aspergillosis. The significance of the innate defense is also supported by
the observation that cultures of sputum samples of patients with aspergillosis,
e.g. ABPA, often remain negative even if fungal hyphae can be detected in the
sputum samples by cytology, indicating the efficient killing of fungi in the
airways.
The Adaptive Immunological Response
During growth of Aspergillus in lung tissue, antigen presentation by antigenpresenting cells may result from either phagocytosis of intact spores, fungal particles or soluble antigens that are released from the growing tip of the fungus.
Antigen presentation will activate B and T lymphocytes, resulting in both cellular and humoral immune responses, while highly specific killer T cell response
may develop. Depending on the immunologic status of the patient, a Th1- or Th2like response may predominate.
In immunocompetent patients the immunologic response to Aspergillus is
dependent on the immune status of the patient. In nonatopic patients the presentation of antigens of Aspergillus to the immune system will result in a Th1-type
response with strong IgG and IgA antibody formation and recruitment of polymorphonuclear cells. This is seen in patients with aspergilloma and patients with
cystic fibrosis with Aspergillus infections. In patients with cystic fibrosis, infections with A. fumigatus regularly will develop into ABPA, with concurrent more
severe loss of airway function. Although it is generally assumed that antibody
formation will facilitate the elimination of the fungus, the proteases liberated
from polymorphonuclear cells may cause severe damage of the airway tissue.
In atopic patients the Th2-type response is characterized by IL-4 and IL-5
cytokine production. IL-4 dictates B cells to produce IgE antibodies against
Defense Mechanisms against Fungi
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Aspergillus antigens, followed by an increased sensitization of mast cells.
Furthermore, IL-5 is responsible for the recruitment of eosinophils from the
bone marrow to the airways. As was described above, aspergillosis in atopic
patients may develop into Aspergillus-associated asthma and in the more complicated manifestation of ABPA. An important difference between the two manifestations is the binding and growth of Aspergillus hyphae on and between
epithelial cells in ABPA, which has never been demonstrated for Aspergillus
asthma. The continuous presence of the fungus at the epithelial surface will
result in abundant liberation of antigen, followed by strong antibody responses
(IgG, IgA and IgE) in the blood and at local sites. These antibodies can be
detected in the bronchoalveolar lavage fluid [12, 13]. Furthermore, extremely
high titers of total IgE may be found, which is characteristic for exacerbations
of ABPA [12, 59].
It was described above that proteases from A. fumigatus are able to cause
epithelial cell detachment and induce the production of chemokines. Destruction
of the epithelial cell barrier is rapidly followed by repair mechanisms, resulting in
the influx of serum proteins and extracellular matrix proteins to the lumen site of
the epithelium [60]. Both spores and mycelium of A. fumigatus have surface
structures that are able to interact with extracellular matrix molecules and serum
factors, such as fibrinogen, collagen I and IV, laminin and complement C3 [47,
61–65]. Therefore, damage and repair mechanisms of the airway mucosa may
facilitate the binding of Aspergillus to the damaged sites of the airways (fig. 2).
Binding of Aspergillus to the epithelial surface may be responsible for local
chemokine production by proteolytic attack, resulting in a rapid recruitment of
phagocytic leukocytes. It has been argued that in patients with asthma this local
inflammatory response of epithelial cells [2] may result in the development of
ABPA with massive infiltration of eosinophils [19]. These eosinophils may
cause additional damage to the epithelial cell layer by release of their toxic granular proteins. This will cause additional binding of Aspergillus, which will
further facilitate the survival of the fungus in the tissue. In this way Aspergillus
will modulate a defensive immunological response in atopic asthmatic patients
into a perpetuating cycle of damage to the airways, promoting its continuous
growth in the airways.
Although information is lacking on the further progress of damage of the
airways of patients with ABPA, the continuous findings of bronchiectatic lesions,
fibrosis and haemoptysis during exacerbations of the disease, together with the
presence of antibodies against proteases of A. fumigatus, suggest that next to toxic
components eventually released by Aspergillus also the proteolytic components
may play an important role in the inflammatory responses found in patients with
ABPA. In summary: it may be concluded that in immunocompetent patients
A. fumigatus generates a strong perpetuating cycle of inflammatory responses
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Clearance
Transport
system
• Ciliary
beating
frequency
• Mucin
Antifungal/bacterial
Opsonization
proteins
Lysozyme
SLPI/ALP
-Defensins
TAP
LAP
LL-37
?
Phagocytic cells
Phagocytosis
Sp-A
Sp-D
MBP
Complement
sIgA
Oxygen radicals
Nitric oxide
␣-Defensins
Killing
IL-1, TNF-␣
?
A. fumigatus
Proteases?
Binding to MMP
?
Signaling
by
cytokines
Migration
of fungus
-ELF
-Epithelial cell layer
-Basal membrane
Blood vessel
Transport to other organs
Fig. 2. Innate defense against fungi in the immunocompromised patient. Abbreviations
as in figure 1. In the immunocompromised patient the phagocytic activity and production of
the first wave of proinflammatory cytokines (IL-1, TNF-␣) is largely or totally diminished
(right upper corner). In contrast to the cellular response, the innate barrier functions against
fungi shown in the upper left corner (blocks 1–3) remains active and will prevent infections
of the airways against the majority of bacteria and fungi that are inhaled. A. fumigatus
has the capacity to impair this innate response at several points and will be able to cause
damage to the epithelial surface by means of different proteases that are excreted. Damage
will result in exposure of mucosal extracellular matrix proteins (MMP), a target of binding
for Aspergillus spores and mycelium. Binding to the mucosal site is the first and often fatal
event that may start invasive aspergillosis. Whether epithelial cells are still able to respond
to the challenge by Aspergillus proteases is very likely but unknown. However, the cells
that have to respond to these signals are largely absent (lower left part). The way by which
Aspergillus is able to penetrate the lung tissue and spread (via the blood circulation) to
various organs of the body is largely unknown as well.
that induces additional damage to the airways. This self-damaging mechanism
will facilitate the local invasion by the fungus.
Although proteases of A. fumigatus appear to play a role in different
manifestations of aspergillosis in immunocompetent patients, their role in
Defense Mechanisms against Fungi
107
immunocompromised patients is much less clear and still a matter of debate.
Arguments pro and contra have been reviewed recently [66, 67]. In patients with
strong immunosuppression and neutropenia, activation of epithelial cells by
proteases may still be possible, but this will not result in a self-damaging inflammatory response as has been described for ABPA or cystic fibrosis patients
with Aspergillus infections. Recently we were able to isolate two strains of
A. fumigatus from a patient who died from invasive growth of Aspergillus in the
lung tissue. Both strains were unable to grow on a collagen-containing medium,
suggesting that these two strains of A. fumigatus were not easily producing
the elastin- and collagen-degrading serine proteases [Kauffman, unpubl. obs.].
The production of these collagen-degrading proteases has been proposed as
virulence factor for invasive strains of A. fumigatus.
Effect of Immunosuppressing Agents on the Innate
and Adaptive Defense Mechanisms
During immunosuppressed conditions the cellular defensive role of T cells
is most vulnerable (fig. 2). Immunosuppressive therapy following organ transplantation is aimed at suppressing cellular systems that are involved in the
rejection of the transplanted organ. This will result in the loss of the cytotoxic
T cell function, which is most vulnerable to immunosuppression. Generally this
will result in a partial loss of the cellular defense, while the humoral response
to Aspergillus still may be functional. Strong antibody responses against
A. fumigatus are found under immune-suppressive therapy after lung transplantation [68]. Also, observation in bronchoalveolar fluid after lung transplantation
still shows normal numbers of alveolar macrophages and polymorphonuclear
cells [Kauffman, unpubl. obs.]. Tuned therapy regimens try to strike a balance
between acute rejection of the transplanted organ and the threat of viral, bacterial or fungal infections. However, after strong immunosuppression, as is used
after e.g. bone-marrow transplantation, both cellular and humoral responses
may be lost (fig. 2). As described before, these immunocompromised patients
are vulnerable to severe and life-threatening invasive aspergillosis. In contrast
to immunosuppression of the adaptive immune response, the effect of immunosuppressive agents on the innate defense responses is very limited. However,
the limited information available suggests that the effect of corticosteroids and
cyclosporin A may be different on resident cells of the airways compared with
the effects on immune cells. It has been shown that incubation of epithelial cells
with corticosteroids increases both the mRNA expression and the protein
release of the secretory leukocyte protease inhibitor [69]. As described above,
SLPI has bactericidal and fungicidal activities, suggesting that under certain
Kauffman/Tomee
108
immunosuppressive conditions some components of the innate defense may be
enhanced. In contrast, Agerberth et al. [33] found lower antibacterial levels in a
single patient that was treated with corticosteroids before bronchoalveolar
lavage fluid collection, suggesting that the production of antibacterial components by airway epithelial cells was inhibited at the corticosteroid concentration
used in this study. Studies with other immunosuppressants, e.g. cyclosporin A,
FK506, showed increased production of cytokines by resident tissue cells
(mesangial cells, epithelial cells), indicating that the effect of immunosuppressive agents on resident tissue cells may be different as compared with their
effect on immune cells [70–72]. Similarly, we have recently shown that the
immunosuppressive drug cyclosporin A induces an enhanced production of
cytokines (IL-6 and IL-8) by human airway-derived epithelial cells [73].
Furthermore, the effect of corticosteroids showed bell-shaped dose responses
for these cytokines, with a first phase of activation of the cytokine response at
concentrations that are used under in vivo conditions after lung transplantation,
followed by an inhibitory response at higher concentrations [73]. These observations suggest that the influence of immunosuppressive agents on the local
innate defensive mechanism may be different (stimulatory) from the effect
(inhibitory) on the adaptive immune response. Further studies are necessary in
order to elucidate the modulating role of the different agents used for immunosuppression on the nonadaptive primary defensive system and the adaptive
immunological response and the significance for fungal infections.
Concluding Remarks
The pathology of Aspergillus-related disease is broad and depends on
fungal-related factors (the fungal virulence factors) and host-related factors
(anatomical abnormalities, atopy, impaired local or systemic immunity).
Aspergillus species are ubiquitous in the environment and are inevitably inhaled
into the airways. In healthy individuals A. fumigatus has low pathogenicity and
inhalation of A. fumigatus conidia may cause innocuous colonization of the
airways. However, opportunistic infections may occur in susceptible patients
who have breaches in their defense systems.
Defense against A. fumigatus is diverse and includes cellular and noncellular components. Cell-mediated defense by alveolar macrophages, polymorphonuclear cells and monocytes plays a central role in the host defense
against Aspergillus both in the innate and acquired defense mechanisms.
Soluble antimicrobial and fungicidal proteins produced by epithelial cells
may cause direct killing of inhaled fungal spores. In addition, opsonizing soluble products may facilitate phagocytosis and killing of Aspergillus by alveolar
Defense Mechanisms against Fungi
109
macrophages and polymorphonuclear leukocytes and may contribute to its
elimination by inhibiting attachment of the fungus to host tissues. Epithelial
cells also play a crucial role in the defense against Aspergillus by the release of
proinflammatory cytokines, recruiting cells of the host defense to the site of
fungal penetration.
It has been argued that the strategies of A. fumigatus may depend on the
immunological status of the patient.
Acting as a powerful allergen source, exposure to A. fumigatus may result
in the development of an allergic state in individuals with a predisposition to
develop allergic sensitization (atopy). In these patients proteases may facilitate the
pathogenic presence of Aspergillus, by inducing an inflammatory (eosinophilic)
response causing damage to the epithelial cell layer. In immunocompromised
patients inflammatory self-damaging by proteases of fungal origin is absent. In
these patients the crucial cell-mediated defense is lost and the residual defense is
mainly composed of the innate defense factors derived from epithelial cells. It is
discussed that immunosuppression mainly acts on the crucial cellular defense of
the airways. However, the effect of the immunosuppressive agents on the innate
response capacities of resident cells, such as epithelial cells or mucus-producing
cells, is hardly known. Immunosuppressive agents may influence such cells
differently as compared with their influence on immune cells.
References
1
2
3
4
5
6
7
8
9
10
11
12
Tomee JF, Kauffman HF: Putative virulence factors of A. fumigatus. Clin Exp Allergy 2000;30:
476–484.
Kauffman HF, Tomee JF: Inflammatory cells and airway defense against A. fumigatus. Immunol
Allergy Clin North Am 1999;18:619–640.
Bardana EJ, Jr: Pulmonary aspergillosis; in Al-Doory Y, Wagner GE (eds): Aspergillosis.
Springfield, Thomas, 1985, pp 43–78.
Andriole VT: Infections with Aspergillus species. Clin Infect Dis 1993;17(suppl 2):S481–S486.
Chimelli L, Mahler-Araújo MB: Fungal infections. Brain Pathol 1997;7:613–627.
Basisch JE, Graves TS, Baz MN, Scanlon G, Hoffman R, Patterson R, et al: Allergic bronchopulmonary aspergillosis in corticoid-dependent asthmatics. J Allergy Clin Immunol 1981;68:98–102.
Nelson LA, Callerame ML, Schwartz RH: Aspergillosis and atopy in cystic fibrosis. Am Rev
Respir Dis 1979;120:863–873.
Rosenberg M, Patterson R, Mintzer R, Cooper BJ, Roberts M, Harris KE: Clinical and immunological criteria for the diagnosis of allergic bronchopulmonary aspergillosis. Ann Intern Med
1977;86:405–414.
Patterson R, Greenberger PA, Robert RC, Roberts M: Allergic bronchopulmonary aspergillosis:
Staging as an aid to management. Ann Intern Med 1982;96:286–291.
Scadding JG: The bronchi in allergic aspergillosis. Scand J Respir Dis 1967;48:372–377.
Gefler WB, Epstein DM, Miller WT: Allergic bronchopulmonary aspergillosis: Less common
patterns. Radiology 1981;140:307.
Kauffman HF, Beaumont F: Serological diagnosis of Aspergillus infections. Mykosen 1988;31
(suppl 2):21–26.
Kauffman/Tomee
110
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
Kauffman HF, Koëter GH, van der Heide S, de Monchy JGR, Kloprogge E, de Vries K: Cellular
and humoral observations in a patient with allergic bronchopulmonary aspergillosis during a
nonasthmatic exacerbation. J Allergy Clin Immunol 1989;83:829–838.
Anderson CJ, Craig S, Bardana EJ, Jr: Allergic bronchopulmonary aspergillosis and bilateral
fungal balls terminating in disseminated aspergillosis. J Allergy Clin Immunol 1980;65:140–144.
Henderson AH: Allergic aspergillosis: Review of 32 cases. Thorax 1968;23:501–512.
Stevens EAM, Hilvering C, Orie NGM: Inhalation experiments with extracts of A. fumigatus on
patients with allergic aspergillosis and aspergilloma. Thorax 1970;25:11–18.
Wahner HW: Pulmonary aspergillosis. Ann Intern Med 1963;58:472.
Riley DJ, Mackenzie JW, Uhlman WE, Edelman NH: Allergic bronchopulmonary aspergillosis:
Evidence of limited tissue invasion. Am Rev Respir Dis 1975;111:232–236.
Slavin RG, Bedrossian CW, Hutcheson PS, Pittman S, Salinas-Madrigal L, Tsai CC et al: A pathologic study of allergic bronchopulmonary aspergillosis. J Allergy Clin Immunol 1988;81:
718–725.
Kurup VP, Resnick A, Scribner GH, Gunasekaran M, Fink JN: Enzyme profile and immunochemical characterization of A. fumigatus antigens. J Allergy Clin Immunol 1986;78:1166–1173.
Kothary MH, Chase TJ, Macmillan JD: Correlation of elastase production by some strains of
A. fumigatus with ability to cause pulmonary invasive aspergillosis in mice. Infect Immun 1984;
43:320–325.
Kauffman HF, Tomee JF, van der Werf TS, de Monchy JGR, Koeter GH: Review of fungusinduced asthmatic reactions. Am J Respir Crit Care Med 1995;151:2109–2116.
Tomee JF, Kauffman HF, Klimp AH, de Monchy JGR, Koëter GH, Dubois AEJ: Immunologic
significance of a collagen-derived culture filtrate containing proteolytic activity in Aspergillusrelated diseases. J Allergy Clin Immunol 1994;93:768–778.
Blyth W, Grant IW, Blackadder ES, Greenberg M: Fungal antigens as a source of sensitization and
respiratory disease in Scottish maltworkers. Clin Allergy 1977;7:549–562.
Ojanen TH, Terho EO, Mantyjarvi RA: Comparison of A. fumigatus and Aspergillus umbrosus
antigens in serological tests of farmer’s lung. Allergy 1982;37:297–301.
Baur X, Dexheimer E: Hypersensitivity pneumonitis concomitant with acute airway obstruction
after exposure to hay dust. Respiration 1984;46:354–361.
Reijula K, Sutinen S, Tuuponen T, Lahti R, Karkola P: Pulmonary fibrosis, with sarcoid granulomas and angiitis, associated with handling of mouldy lichen. Eur J Respir Dis 1983;64:625–629.
Yoshida K, Ueda A, Yamasaki H, Sato K, Uchida K, Ando M: Hypersensitivity pneumonitis resulting from A. fumigatus in a greenhouse. Arch Environ Health 1993;48:260–262.
Huuskonen MS, Husman K, Jarvisalo J, Korhonen O, Kotimaa M, Kuusela T, et al: Extrinsic allergic alveolitis in the tobacco industry. Br J Ind Med 1984;41:77–83.
Gerson SL, Talbot GH: Prolonged granulocytopenia: The major risk factor for pulmonary invasive
aspergillosis in patients with acute leukaemia. Ann Intern Med 1984;100:345–351.
Wanner A, Salathe M, O’Riordan TG: Mucociliary clearance in the airways. Am J Respir Crit
Care Med 1996;154:1868–1902.
Goldman MJ, Stotzenberg ED, Karl UP, Zasloff M, Wilson JM: Human -defensin-1 is a saltsensitive antibiotic in lung that is inactivated in cystic fibrosis. Cell 1997;88:553–560.
Agerberth B, Grunewald J, Castaños-Velez E, Olsson B, Jörnvall H, Wigzell H et al: Antibacterial
components in bronchoalveolar lavage fluid from healthy individuals and sarcoidosis patients. Am
J Respir Crit Care Med 1999;160:283–290.
Schonwetter BS, Stolzenberg ED, Zasloff MA: Epithelial antibiotics induced at sites of inflammation. Science 1995;267:1645–1648.
Ganz T, Lehrer RI: Defensins. Pharmacol Ther 1995;66:191–206.
Russell JP, Diamond G, Tarver AP, Scanlin TB, Bevins CL: Coordinate induction of two antibiotic
genes in tracheal epithelial cells exposed to the inflammatory mediators lipopolysaccharide and
tumor necrosis factor alpha. Infect Immun 1996;64:1565–1568.
Tomee JFC, Hiemstra PS, Heinzel-Wieland R, Kauffman HF: Antileukoprotease: An endogenous
protein in the innate mucosal defense against fungi. J Infect Dis 1997;176:740–747.
Tomee JFC, Koëter GH, Hiemstra PS, Kauffman HF: Secretory leukoprotease inhibitor: A native
antimicrobial protein presenting a new therapeutic option? Thorax 1998;53:114–116.
Defense Mechanisms against Fungi
111
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
Pearson AM: Scavenger receptors in innate immunity. Curr Opin Immunol 1996;8:20–28.
Epstein J, Eichbaum Q, Sheriff S, Ezekowitz RA: The collectins in innate immunity. Curr Opin
Immunol 1996;8:29–35.
Turner MW: Mannose-binding lectin: The pluripotent molecule of the innate immune system.
Immunol Today 1996;17:532–540.
Calderone RA, Linehan L: The role of complement in host resistance to systemic fungal infection.
Immunol Ser 1989;47:225–242.
Murphy JW: Immunity to fungi. Curr Opin Immunol 1989;2:360–367.
Washburn RG, Hammer CH, Bennett JE: Inhibition of complement by culture supernatants of
A. fumigatus. J Infect Dis 1986;154:944–951.
Robertson MD, Kerr KM, Seaton A: Killing of A. fumigatus spores by human lung macrophages:
A paradoxical effect of heat-labile serum components. J Med Vet Mycol 1989;27:295–302.
Sturtevant JE, Latgé JP: Interactions between conidia of A. fumigatus and human complement
component C3. Infect Immun 1992;60:1913–1918.
Sturtevant JE, Latgé JP: Participation of complement in the phagocytosis of the conidia of
A. fumigatus by human polymorphonuclear cells. J Infect Dis 1992;166:580–586.
Kurup VP: Interaction of A. fumigatus spores and pulmonary alveolar macrophages of rabbits.
Immunobiology 1984;166:53–61.
Kozel TR, Wilson MA, Farrell TP, Levitz SM: Activation of C3 and binding to A. fumigatus conidia and hyphae. Infect Immun 1989;57:3412–3417.
Diamond RD, Krzesicki R, Epstein B, Jao W: Damage to hyphal forms of fungi by human leukocytes in vitro. A possible host defense mechanism in aspergillosis and mucormycosis. Am J Pathol
1978;91:313–328.
Diaz P, Gonzalez MC, Galleguillos FR, Ancic P, Cromwell O, Shepherd D, et al: Leukocytes and
mediators in bronchoalveolar lavage during allergen-induced late-phase asthmatic reactions. Am
Rev Respir Dis 1989;139:1383–1389.
Kita H, Abu-Ghazaleh RI, Sanderson CJ, Gleich GJ: Effect of steroids on immunoglobulininduced eosinophil degranulation. J Allergy Clin Immunol 1991;87:70–77.
Medzhitov R, Janeway CA: Innate immunity: Impact on the adaptive immune response. Curr Opin
Immunol 1997;9:4–9.
Robinson BW, Venaille TJ, Mendis AH, McAleer R: Allergens as proteases: An A. fumigatus
proteinase directly induces human epithelial cell detachment. J Allergy Clin Immunol 1990;86:
726–731.
Tomee JF, Wierenga ATJ, Hiemstra PS, Kauffman HF: Proteases from A. fumigatus induce release
of proinflammatory cytokines and cell detachment in airway epithelial cell lines. J Infect Dis
1997;176:300–303.
Kauffman HF, Tomee JF, van de Riet MA, Timmerman AJ, Borger P: Protease-dependent activation of epithelial cells by fungal allergens leads to morphologic changes and cytokine production.
J Allergy Clin Immunol 2000;105:1185–1193.
Paris S, Boisvieux UE, Crestani B, Houcine O, Taramelli D, Lombardi L, et al: Internalization of
A. fumigatus conidia by epithelial and endothelial cells. Infect Immun 1997;65:1510–1514.
DeHart DJ, Agwu DE, Julian NC, Washburn RG: Binding and germination of A. fumigatus conidia on cultured A549 pneumocytes. J Infect Dis 1997;175:146–150.
Greenberger PA, Patterson R: Allergic bronchopulmonary aspergillosis and the evaluation of the
patient with asthma. J Allergy Clin Immunol 1988;81:646–650.
Persson CG, Erjefalt JS, Erjefalt I, Korsgren MC, Nilsson MC, Sundler F: Epithelial shedding –
restitution as a causative process in airway inflammation. Clin Exp Allergy 1996;26:
746–755.
Annaix V, Bouchara JP, Larcher G, Chabasse D, Tronchin G: Specific binding of human fibrinogen fragment D to A. fumigatus conidia. Infect Immun 1992;60:1747–1755.
Bouchara JP, Bouali A, Tronchin G, Robert R, Chabasse D, Senet JM: Binding of fibrinogen to
the pathogenic Aspergillus species. J Med Vet Mycol 1988;26:327–334.
Bouchara JP, Sanchez M, Chevailler A, Marot LA, Lissitzky JC, Tronchin G, et al: Sialic aciddependent recognition of laminin and fibrinogen by A. fumigatus conidia. Infect Immun 1997;65:
2717–2724.
Kauffman/Tomee
112
64
65
66
67
68
69
70
71
72
73
Tronchin G, Bouchara JP, Ferron M, Larcher G, Chabasse D: Cell surface properties of A. fumigatus conidia: Correlation between adherence, agglutination, and rearrangements of the cell wall.
Can J Microbiol 1995;41:714–721.
Donaldson K, Bromley ILJ: Binding of Aspergillus spores to lung epithelial cells and basement
membrane proteins: Relevance to the asthmatic lung. Thorax 1996;51:1203–1209.
Monod M, Fatih A, Jaton-Ogay K, Paris S, Latge JP: The secreted proteases of pathogenic species
of Aspergillus and their possible role in virulence. Can J Bot 1995;73:S1081–S1086.
Tomee JF, Kauffman HF: Putative virulence factors of A. fumigatus. Clin Exp Allergy 2000;30:
476–484.
Tomee JF, Mannes GP, van der Bij W, van der Werf TS, de Boer WJ, Koeter GH et al:
Serodiagnosis and monitoring of Aspergillus infections after lung transplantation. Ann Intern Med
1996;125:197–201.
Abbinante-Nissen JM, Simpson LG, Leikauf GD: Corticosteroids increase secretory leukoprotease inhibitor transcript levels in airway epithelial cells. Am J Physiol 1995;265:L286–L292.
Muraoka K-I, Fujimoto K, Sun X, Yoshioka K, Shimuzu K-I, Yagi M, et al: Immunosuppressant
FK506 induces interleukine-6 production through the activation of transcription factor nuclear
factor (NF)-B. J Clin Invest 1996;97:2433–2439.
Khanna A, Cairns V, Hosenpud JD: Tacrolimus induces increased expression of transforming
growth factor-b1 in mammalian lymphoid as well as non-lymphoid cells. Transplantation 1999;67:
614–619.
Hojo M, Morimoto T, Maluccio M, Asano T, Morimoto K, Lagman M, et al: Cyclosporin induces
cancer progression by a cell-autonomous mechanism. Nature 1999;397:530–534.
Borger P, Kauffman HF, Timmerman JA, Scholma J, van den Berg JW, Koeter GH: Cyclosporine,
FK506, mycophenolate mofetil, and prednisolone differentially modulate cytokine gene expression in human airway-derived epithelial cells. Transplantation 2000;69:1408–1413.
H.F. Kauffman, PhD., Assoc. Prof.
University Hospital Groningen, Clinic for Internal Medicine,
Department of Allergology, Laboratory of Allergology and Pulmonology,
Hanzeplein 1, NL–9713 GZ Groningen (The Netherlands)
Tel. ⫹31 50 3613703, Fax ⫹31 50 3121576, E-Mail h.f.kauffman@path.azg.nl
Defense Mechanisms against Fungi
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Breitenbach M, Crameri R, Lehrer SB (eds): Fungal Allergy and Pathogenicity.
Chem Immunol. Basel, Karger, 2002, vol 81, pp 114–128
Secreted Proteinases and
Other Virulence Mechanisms of
Candida albicans
Michel Monod a, Margarete Borg-von Zepelin b
a
Service de Dermatologie (DHURDV), Centre Hospitalier Universitaire Vaudois,
Lausanne, Switzerland, and
b
Department of Bacteriology, University Clinics, University of Göttingen, Germany
Candida albicans is a major source of different types of fungal infections
in immunocompromised patients [1]. Chronic superficial C. albicans infections
of the skin or the mucosae are commonly seen in patients with cell-mediated
immunological disorders, e.g. AIDS patients, whereas disseminated infections
due to C. albicans and related Candida spp. predominantly occur in patients
with severe defects of their phagocytic system, e.g. neutropenic patients, in
immunosuppressed patients following organ transplants or in cancer patients
undergoing aggressive cytostatic therapies (for a review, see Lortholary and
Dupont [2]). Furthermore, the widespread use of broad-spectrum antibiotics
has caused an increase in mucosal Candida infections, such as thrush, infections of the intestinal tract and vaginitis in normal hosts. In fact, Candida spp.
are ubiquitous human commensals, mainly residing in the gastrointestinal tract
without causing any harm. Since they are not highly virulent microorganisms,
Candida infections only occur under favorable conditions in the host, which
allow the fungus to do more than just colonize mucosae. Candida infections
have emerged as a significant medical problem at the end of the twentieth century concomitantly with AIDS and immunosuppressive therapies.
C. albicans and related Candida spp. are prototrophic, which contributes
to establish virulence. Uracyl or adenine auxotrophy diminishes the virulence
of C. albicans [3, 4]. Efficient iron assimilation is also essential for C. albicans
virulence [5]. Since prototrophy is also found in many other yeasts which are
not necessarily pathogenic, it is conceivable that C. albicans must possess traits
which make this species more adapted than others to overcome the mucosal
barriers protecting the host from infections and deep mycosis. The aim of this
chapter is to briefly review different traits of virulence of C. albicans and to
discuss the role of the secreted aspartic proteinases (Sap) in more detail.
Site-Directed Mutagenesis and Genomics to
Investigate Virulence Factors
Numerous putative virulence factors have been investigated using genetic
tools and animal models by observing the specific effects of gene disruption
and growth of the organism in vitro and in vivo. Many knockouts of genes
result in strains that show lower lethality for mice than their parent strains without affecting growth in vitro in a rich medium. The construction of mutants of
C. albicans in an isogenic background is technically difficult due to the permanent diploid status of this asexual yeast. The ‘ura-blaster’ system [6], which
allows the sequential disruption of targeted alleles, has been applied in many
investigations using a C. albicans with a ura3/ura3 background [7]. Strains
constructed by the ura-blaster technique have the URA3 gene of C. albicans
inserted into the targeted gene of interest. It is of critical importance that the
orotidine 5-decarboxylase enzyme, encoded by the URA3 gene inserted into the
targeted gene retains sufficient activity, since both uracyl auxotrophy and mutations in the pyrimidine biosynthetic pathway have previously been shown to
diminish the virulence of C. albicans. However, retrospectively, the URA3
activity within the disruption cassette was shown (1) to vary and (2) to be lower
than in the parental strain [8]. Therefore, the results of some virulence studies
in animal models has to be interpreted with precaution. It is possible now, to
replace URA3 with a dominant selection marker conferring resistance to
mycophenolic acid that does not influence virulence [9, 10]. This marker in
combination with the FLP recombinase allows sequential disruption of both
alleles of a gene to generate site-targeted mutants from wild-type strains [11].
The relevance of putative virulence attributes can be based on comparisons
of C. albicans with apathogenic yeasts like Saccharomyces cerevisiae and other
less pathogenic species. Shotgun sequencing of the C. albicans genome was
completed with the sequencing of 10.4 haploid genome equivalents [12].
Roughly 80% of the genes found are closely homologous to the genes of
S. cerevisiae. Interestingly, many genes shown to be involved in the virulence
of C. albicans belong to the remaining 20% of the genes which have no equivalents in the S. cerevisiae genome. Furthermore, virulence factors also appear
to be organized in large protein families specific for C. albicans. This applies
especially to the families of secreted aspartic proteinases and cell surface
agglutinin-like proteins. As discussed below, numerous genes necessary to
C. albicans Virulence Factors
115
C. albicans for establishing infection were shown to be involved in dimorphism
and adherence, which have to be considered as essential virulence traits, or
encode secreted hydrolases. However, disruption of genes involved in other
functions (e.g. nutritional factors and efflux pumps) for which a homolog exists
in many yeasts can also render C. albicans less virulent [3–5, 13].
Dimorphism
In response to certain environmental conditions, C. albicans can switch
from a yeast-like organism to pseudohyphal or hyphal forms. Hyphae grow by
apical tip extension, and division with true septa occurs in a fission-like manner. In contrast, pseudohyphae arise by budding and are chains of distinct cells
where the mother and daughter cells fail to separate after division [14]. The
ability to switch between yeast cells and long hyphae is considered an essential
virulence trait of C. albicans [15]. During mucosal and deep infections, budding yeasts transform to filaments (fig. 1). Evidence that hyphae are essential
for tissue invasion was demonstrated using mutants unable to switch to the filamentous form in serum and were avirulent in a mouse model [16]. The yeast
and filamentous forms of C. albicans (fig. 1) are interconvertible and differ in
many adhesion and hydrolytic factors themselves associated with virulence.
The formation of hyphae is induced by many signals, including N-starvation
and/or C-starvation, pH and embedded/microaerophilic conditions [for a
review, see Ernst 17].
Switching System of C. albicans
Also linked with virulence is a second form of cellular transformation in
which cells alternate at high frequency (10–2–10–3) between different phenotypes, each expressing a unique combination of characteristics [18–20].
Switching involves the coordinated regulation of many unlinked genes and can
affect many characters such as colony morphology, cell shape, cell size, cell
wall morphology, aspartic proteinase secretion, adherence, virulence in animal
models and susceptibility to antifungal drugs. The different phenotypes are
strain specific. Switching in C. albicans is ordinarily observed through changes
in colony morphology. The particular strain WO-1, that switches from a white
to an opaque form at frequencies of 10–2 –10–4 per cell cycle, has been the object
of intensive research [19, 20]. Recently, phenotypic switching events in this
strain have been shown to be regulated by the transcriptional factor Efg1p [21].
Monod/Zepelin
116
a
b
c
d
Fig. 1. Yeast and filamentous forms of C. albicans. a C. albicans grown in Sabouraud
liquid medium. b Direct mycogical examination of a vaginitis sample. c C. albicans colonizing human nonkeratinized buccal epithelium. A tissue sample (diameter 2 mm) was
infected with 2 ⫻ 105 blastospores of C. albicans CBS 2730. After 4 h of incubation, the
epithelial surface was invaded by germ tubes. d C. albicans invading endothelial cells.
Cultured endothelial cells from dog vessels were infected with C. albicans CBS 2730 blastoconidia (105 cells/ml). After 4 h of incubation, target cells were invaded by C. albicans
filaments. a, b Fluorescence micrographs of C. albicans stained with blankophor. c, d
Scanning electron micrographs.
Opaque cells produce the secreted aspartic proteinases Sap1p, Sap2p and
Sap3p in vitro, while the white form of the same strain cultured under identical
conditions produces Sap2p only [22–24]. In an intravenous infection model, the
white phenotype was more virulent than the opaque phenotype. In contrast, in
a murine cutaneous candidiasis model, the opaque phenotype was more infectious [25]. In addition to yeast-hyphal dimorphism, phenotypic switching can
provide a means for rapid adaptation of C. albicans to a changing environment
in different anatomical locations.
C. albicans Virulence Factors
117
Adherence
Infection with C. albicans generally involves adherence and colonization
of superficial tissues. Cell wall mannoproteins of C. albicans are pivotal for
these functions [for a review, see Calderone and Braun 26]. Glycosylation
occurs through either N-linkage to asparagine or O-linkage to serine or threonine residues via mannosyltransferases. Blocking the adhesion of C. albicans
to epithelial cells in vitro could be achieved by either mannose supplementation, pretreating yeasts with the mannose-specific lectin concanavalin A [27],
exposing C. albicans to tunicamycin [28], a drug which inhibits mannoprotein
synthesis, or by the addition of an anti-Candida antibody with specificity for
mannan [29]. Moreover, cell surface defects of C. albicans strains involving
mannoproteins are correlated with a reduction in adherence [30], and null
mutants of MNT1, which encodes for a mannosyltransferase involved in
O-glycosylation, showed reduced adherence to buccal epithelial cells [31]. The
virulence of these mutants was strongly attenuated in both systemic and vaginal
animal models.
The well-characterized agglutinin-like sequence (Als) proteins are highly
glycosylated cell surface proteins with both N- and O-linked carbohydrates
[32–34]. They are encoded by the ALS gene family, which contains at least 9
members [35] with similarities to ␣-agglutinin (Ag␣1p) of S. cerevisiae, a cell
surface glycoprotein involved in cell-to-cell interactions during mating [36]. The
C-terminus of each predicted Als protein encodes a consensus sequence for the
addition of a glycosylphosphatidylinositol (GPI) anchor. Heterologous expression of ALS genes in the nonadherent S. cerevisiae confers the ability to adhere
to extracellular matrix glycoproteins [37] and to cultured human cells [38]. Als
proteins, like other cell wall proteins, such as Hpw1p and Saps (see below), are
activated specifically during hyphal development. Immunohistological staining
demonstrates the production of Als proteins on the cell wall of C. albicans in
infected tissues of mice with disseminated candidiasis [39].
C. albicans possesses specific molecules with limited homology to the
vertebrate integrins able to bind to extracellular matrix proteins, such as collagen, fibronectin and laminin, but also ligands of host cells and iC3b of the complement system [for reviews, see Calderone and Braun 26 and Hostetter 40]. In
particular, a gene encoding a protein similar to mammalian leukocyte integrins,
INT1, has been cloned from C. albicans [41]. The INT1 gene product shows
homology to the ␣M chain of the human iC3b receptor CR3, and is not present
in other yeast species. INT1 expression in S. cerevisiae was sufficient to mediate adherence of this yeast which is normally nonadherent to epithelial cells.
Furthermore, disruption of INT1 in C. albicans suppressed hyphal growth,
adhesion to epithelial cells and virulence in mice.
Monod/Zepelin
118
Lectin activity is expressed by C. albicans [42] but so far, only one gene,
EPA1 (for epithelial adhesin 1), encoding a specific lectin of the related species
Candida glabrata was cloned [43]. From its predicted amino acid sequence,
this lectin protein appears to be GPI anchored. Deletion of this gene reduced
adherence of C. glabrata to epithelial cells in vitro by 95%. Heterologous
expression of the EPA1 gene in the nonadherent S. cerevisiae also confers the
ability to adhere to cultured cells.
Stable attachments between germ tubes and mammalian cells were found
with C. albicans [44]. A surface protein in the fungus with similarities to mammalian small prolin-rich protein, encoded by the gene HWP1 (hyphal wall protein) serves as a substrate for epithelial transglutaminase enzymes that can
covalently cross-link Hpw1p to epithelial proteins. This binding mechanism
involving covalent cross-linkage of the fungus to host cell surface proteins
appears to be unique for the adherence of microorganisms.
In summary, C. albicans possesses a broad panel of molecules involved in
adherence with different binding mechanisms which can function independently. Many of these molecules, such as members of the Als family, Int1p,
Hwp1p, appear to be specific for C. albicans and are not found in other yeasts.
Secreted aspartic proteinases, which are also a particularity of C. albicans are
additionally involved in adherence. Their multiple roles in virulence will be
discussed below.
Secreted Hydrolases
Many pathogenic microbes possess constitutive and inducible hydrolytic
enzymes that help them invading host tissues by destroying, altering, or damaging membrane integrity leading to dysfunction or disruption of host structures. C. albicans produces a variety of secreted hydrolases which are
implicated in pathogenesis.
Phospholipases
Extracellular phospholipases have been implicated as virulence factors in
many microorganisms, such as Listeria monocytogenes [45], Toxoplasma
gondii [46, 47], and Entamoeba histolytica [48]. The secretion of phospholipases by C. albicans was first detected by growing the yeast on solid media
containing egg yolk and CaCl2 and visualizing a halo of precipitation of phospholipid digestion products with calcium around the colonies [49].
Phospholipase activity was shown to be correlated with adherence to buccal
C. albicans Virulence Factors
119
epithelial cells and pathogenicity in mice [50]. C. albicans genes PLB1 and
PLB2, encoding phospholipases, were cloned [51, 52]. Results obtained from
PLB1-deficient mutants constructed using the ura-blaster methods were shown
to contribute to lethality of mice infected by C. albicans. Phospholipase Plb1p
is not involved in adherence, but contributes to virulence by facilitating
increased penetration and damage to host cells; this enzyme may also enhance
the ability of C. albicans to cross the gastrointestinal barrier and to enter blood
vessels.
Secreted Aspartic Proteinases
Among various potential virulence factors, Saps have been intensively
investigated. High titers of anti-Sap antibodies are observed in sera of patients
with candidiasis [53] and the presence of Sap antigens has been demonstrated
on the surface of fungal elements colonizing mucosa, penetrating tissues during disseminated infection and evading macrophages after phagocytosis of
Candida cells [54–56]. Evidence for the role of Sap in virulence has also been
derived from experiments with the specific inhibitor pepstatin A that is able to
block adherence and invasion of C. albicans [57–60].
Ten genes encoding Saps have been cloned from C. albicans to date [61,
62, our own unpublished results]. Sap1p, Sap2p and Sap3p as well as Sap4p,
Sap5p and Sap6p form two subgroups each containing closely related enzymes,
whereas Sap7p and Sap8p form distinct branches in clustering trees [61, 62].
The SAP9 and SAP10 deduced amino acid sequences show a putative GPI
anchor at the C-terminus of the translation products. The Candida Saps, are
synthesized as precursors in a preproprotein form. The prepeptide, or signal
peptide, of 16–18 amino acid (aa) residues is necessary for entering the secretory pathway by transporting the protein across the membrane of the endoplasmic reticulum. With the exception of Sap7p, which has a putative 195 aa
propeptide, the Candida Saps have a relatively short propeptide (32–58 aa)
which contains one to four Lys-Arg sequences, one of which is immediately
before the N-terminus of the mature form of the enzyme.
The fact that C. albicans inhabits diverse host niches leads to the question
whether different Saps are expressed by C. albicans in reaction to specific environmental conditions. This question has been addressed by a number of laboratories (1) by Northern blotting analysis [63], (2) by using RT-PCR performed
on in vitro and in vivo samples [64–66], (3) by using a gene expression reporter
system [67], (4) by constructing mutants bearing mutations in either individual
or multiple SAP genes [65, 68, 69] and (5) by determining which of these proteinases are involved during yeast adherence to mucosae and tissue invasion by
Monod/Zepelin
120
using immunological methods [70]. The availability of pure recombinant Sap1p
to Sap6p produced in Pichia pastoris helped to determine the specificity of
monoclonal and polyclonal antibodies raised against peptides or active individual Sap.
Aspartic Proteinases in the Adherence Process
Using RT-PCR techniques, the expression of different SAP genes was
detected in samples of human oral candidiasis [63, 65]. In an in vitro model of
oral candidiasis using reconstituted human epithelium, the different proteinase
genes were sequentially expressed [64]. SAP1 and SAP3, then SAP6 and finally
SAP2 and SAP8 expression was observed at the time when increasing damage
to reconstituted human epithelium occurred [64], and the lesions caused by
⌬sap1,3 double mutants were shown to be strongly attenuated [65]. In contrast,
no attenuation of the lesions in reconstituted human epithelium was observed
using a ⌬sap4,5,6 mutant [64]. In an experimental rat vaginitis model, the
expression of SAP1 and SAP2 was shown by Northern blot analysis [63]. In particular Sap2p seemed to have a potential importance in vaginal infection as
deduced from infection assays using proteinase-deficient mutant strains in a rat
vaginitis model [71]. Altogether these results show the importance of the SAP1SAP3 gene subfamily in mucosal adherence.
Since the introduction of highly active antiretroviral therapy using HIV
aspartic proteinase inhibitors and nucleoside analogs, oropharyngeal candidiasis, the most common fungal disease in patients suffering from HIV infection,
is less often observed even in the presence of viral resistance to treatment
[72–74]. The availability of purified recombinant Sap produced in P. pastoris
allowed testing the effects of four different HIV aspartic proteinase inhibitors,
ritonavir, saquinavir, indinavir and nelfinavir on C. albicans Sap activity.
Interestingly, Sap1p, Sap2p and Sap3p, apparently involved in Candida adherence, were those inhibited by HIV proteinase inhibitors [75]. The influence of
these HIV proteinase inhibitors on the adherence of various Candida strains to
epithelial cells was correlated to the direct effect of the HIV proteinase
inhibitors on the different Saps [75, 76] (fig. 2), leading to the hypothesis that
the frequency of oropharyngeal candidiasis might be decreased still more by
inhibition of Saps involved in C. albicans adherence. In another study, indinavir
and ritonavir were shown to have a good anti-C. albicans effect in a rat vaginitis
model [77]. Globally, these results suggest that the dramatically lower rates of
oropharyngeal candidiasis due to C. albicans in individuals receiving HIV
aspartic proteinase inhibitors reflect not only an improvement in the immune
system as measured by an increase in CD4 counts but that at least some of these
C. albicans Virulence Factors
121
a
b
Fig. 2. Fluorescence micrographs of C. albicans SC5314 adhering on epithelial Vero
cells in the absence a and in the presence b of the HIV proteinase inhibitor ritonavir at a
concentration of 200 m.
HIV proteinase inhibitors may display a specific anti-Sap activity leading to a
reduced number of C. albicans yeasts on epithelial cells. Therefore, development of specific aspartic proteinase inhibitors might be useful in the treatment
of mucosal candidiasis. The precise function of Candida Saps in the adherence
process is not known, but two hypotheses can be advanced: (1) the Candida
Saps could act as ligands to surface proteins of the specific host cells, which do
Monod/Zepelin
122
not necessarily require their enzymic activity. (2) The Candida cells use Saps
as active enzymes to affect target structures of their host cells leading to conformational changes of surface proteins or ligands of epithelial cells allowing a
better adherence of the yeasts.
Aspartic Proteinases in Deep-Seated Candidiasis
Deep-seated, or systemic candidiasis is a secondary disease of patients
suffering from an impairment of their immunological defense system due to
unrelated underlying diseases, such as hematological disorders or different
forms of cancer as well as organ transplantation, which often involve the cellular immune system. Specifically, a lack of granulocytes appears to be important
in the pathogenesis of candidiasis. Deep-seated candidiasis is considered an
endogenous infection. After colonizing the mucosal surfaces, Candida yeasts
penetrate the mucosal barrier, and to the blood system. The second defense
line formed by macrophages and granulocytes constitutes a major obstacle for
the establishment of systemic Candida infections. Engulfed Candida have the
ability to resist phagocytes [78] similarly to L. monocytogenes [79]. After ingestion of yeast cells by isolated peritoneal macrophages, Sap4p, Sap5p and Sap6p
have been shown to be expressed by C. albicans [69]. However, the three
closely related isoenzymes Sap4p, Sap5p and Sap6p could never be recovered
up to now from C. albicans in vitro cultures although SAP4, SAP5 and SAP6
mRNAs were detected during hyphal cell growth stimulated by serum at neutral
pH [80, 81]. SAP4, SAP5 and SAP6 appear to be expressed by one or several
yet unknown macrophage intracellular factors via the transcription factor
CaTec1p which specifically recognizes repeated CATTC(A/C) sequences in the
promoters [82]. Results of the viability tests of wild-type strain C. albicans
SC5314 and of the ⌬sap4,5,6 mutant DSY459 in macrophages suggest that
Sap4p–Sap6p have a role as pathogenicity factors [70]. Other results support
the importance of Sap4p–Sap6p in deep-seated candidiasis. (1) The members or
at least one individual member of the Sap4p–Sap6p subfamily were shown to
contribute to virulence in mice peritonitis [83]. (2) Mice and guinea pigs
infected with DSY459 survived significantly longer than animals infected with
the parental nonmutated strain Sc5314 [69].
Sap4p–Sap6p isoenzymes, which are optimally active at pH 5.0, could act
as cytolysins, as described for Trypanosoma cruzi, Listeria and Shigella [84].
Filamentous growth C. albicans has a destructive action on macrophages [16].
However, as previously reported [54], Sap4p–Sap6p could additionally be
involved in the direct destruction of intracellular components of the
macrophages by affecting some key enzymes of the macrophage oxidative
C. albicans Virulence Factors
123
metabolism which is important for optimal microbicidal activity [for a review,
see Miller and Britigan 85].
Immunohistological staining also allowed to demonstrate the presence of
Sap proteins on the cell wall of C. albicans in infected tissues of patients and
mice with disseminated candidiasis [56]. Therefore, Saps could also play an
important role in tissue invasion by the fungus. Using a gene expression
reporter system, SAP2 expression appears to be strongly induced after dissemination into deep organs [67].
In conclusion, the ability of C. albicans to adhere to mucosae in the oral
and vaginal tracts, to invade deep organs and to resist to phagocytic cells apparently requires the use of combinations of different specific proteinases suitable
to each particular condition during the infection. Expansions of genes to form
a gene family could reflect selection during evolution to allow a better adaptation of the organisms to different environmental conditions.
Acknowledgment
We thank Dr. Mary Holdom for a critical review of the manuscript and assistance with
the English.
References
1
2
3
4
5
6
7
8
9
10
Odds FC: Candida and candidosis. London, Saunders, 1988.
Lortholary O, Dupont B: Antifungal prophylaxis during neutropenia and immunodeficiency. Clin
Microbiol Rev 1997;10:477–504.
Kirsch DR, Withney RR: Pathogenicity of Candida albicans auxotrophic mutants in experimental infections. Infect Immun 1991;59:3297–3300.
Donovan M, Schumuke JJ, Fonzi WA, Bonar SL, Gheesling-Mullis K, Jakob GS, Davisson VJ,
Dotson SB: Virulence of a phosphoribosylaminoimidazole carboxylase-deficient Candida
albicans strain in an immunosuppressed murine model of systemic candidiasis. Infect Immun
2001;69:2542–2548.
Ramanan N, Wang Y: A high-affinity iron permease essential for Candida albicans virulence.
Science 2000;288:1062–1064.
Alani E, Cao L, Kleckner N: A method for gene disruption that allows for repeated use of URA3
selection in the construction of multiply disrupted yeast strains. Genetics 1987;116:541–545.
Fonzi WA, Irwin M: Isogenic strain construction and gene mapping in Candida albicans. Genetics
1993;134:717–728.
Lay J, Henry LK, Clifford J, Koltin Y, Bulawa CE, Becker JM: Altered expression of selectable
marker URA3 in gene-disrupted Candida albicans strains complicates interpretation of virulence
studies. Infect Immun 1998;66:5301–5306.
Kohler GA, White T, Agabian N: Overexpression of a cloned IMP dehydrogenase gene of Candida
albicans confers resistance to the specific inhibitor mycophenolic acid. J Bacteriol 1997;179:
2331–2338.
Beckerman J, Chibana H, Turner J, Magee PT: Single-copy IMH3 allele is sufficient to confer
resistance to mycophenolic acid in Candida albicans and to mediate transformation of clinical
Candida species. Infect Immun 2001;69:108–114.
Monod/Zepelin
124
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
Morschhäuser J, Michel S, Staib P: Sequential gene disruption in Candida albicans by FLPmediated site-specific recombination. Mol Microbiol 1999;32:547–556.
Tzung K-W, Williams RM, Scherer S, Federspiel N, Jones T, Hansen N, Bivolarevic V, Huizar L,
Komp C, Surzycki R, Tamse R, Davis RW, Agabian N: Genomic evidence for a complex sexual
cycle in Candida albicans. Proc Natl Acad Sci USA 2001;98:3249–3253.
Becker JM, Henry LK, Jiang W, Koltin Y: Reduced virulence of Candida albicans mutants
affected in multidrug resistance. Infect Immun 1995;63:4515–4518.
Shepherd MG, Yin CY, Ram SP, Sullivan PA: Germ tube induction in Candida albicans. Can J
Microbiol 1980;26:21–26.
Cutler JE: Putative virulence factors of Candida albicans. Annu Rev Microbiol 1991;45:187–218.
Lo HJ, Köhler JR, DiDomenico B, Loebenberg D, Cacciapuoti A, Fink GR: Nonfilamentous
C. albicans mutants are avirulent. Cell 1997;90:939–949.
Ernst JF: Transcription factors in Candida albicans-environmental control of morphogenesis.
Microbiology 2000;146:1763–1774.
Soll DR, Galask R, Isley S, Rao TVG, Stone D, Hicks J, Schmid J, Mac K, Hanna C: Switching
of Candida albicans during successive episodes of recurrent vaginitis. J Clin Microbiol 1989;27:
681–690.
Soll DR, Morrow B, Srikantha T: High-frequency phenotypic switching in Candida albicans.
Trends Genet 1993;9:61–65.
Soll DR: The emerging molecular biology of switching in Candida albicans. ASM News 1996;62:
415–420.
Sonneborn A, Tebarth B, Ernst JF: Control of white-opaque phenotypic switching in Candida
albicans by the Efg1p morphogenetic regulator. Infect Immun 1999;67:4655–4660.
Ray TL, Payne CD, Soll DR: Variable expression of Candida acid proteinase by switch phenotypes
of individual Candida albicans strains. Clin Res 1988;36:687.
Morrow B, Srikantha T, Soll DR: Transcription of the gene for a pepsinogen, PEP1, is regulated
by white-opaque switching in Candida albicans. Mol Cell Biol 1992;12:2997–3005.
White TC, Miyasaki SH, Agabian N: Three distinct secreted aspartyl proteinases in Candida
albicans. J Bacteriol 1993;175:6126–6133.
Rüchel R, de Bernardis F, Ray TL, Sullivan PA, Cole GT: Candida acid proteinases. J Med Vet
Mycol 1992;30:123–132.
Calderone RA, Braun PC: Adherence and receptor relationships of Candida albicans. Microbiol
Rev 1991;55:1–20.
Sandin RL, Rogers AL, Patterson RJ, Beneke ES: Evidence of mannose-related adherence of
Candida albicans to human buccal epithelial cells in vitro. Infect Immun 1982;35:79–85.
Douglas LJ, McCourtie J: Effect of tunicamycin treatment on the adherence of Candida albicans
to human buccal epithelial cells. FEMS Microbiol Lett 1983;16:199–202.
Rotrosen D, Calderone RA, Edwards JE: Adherence of Candida species to host tissues and plastic.
Rev Infect Dis 1986;8:73–85.
Calderone RA, Wadsworth E: Characterization of mannoproteins from a virulent Candida albicans strain and its derived, avirulent strain. Rev Infect Dis 1988;10:S423–S427.
Buurman ET, Westwater C, Hube B, Brown AJ, Odds FC, Gow NA: Molecular analysis of
CaMnt1p, a mannosyl transferase important for adhesion and virulence of Candida albicans. Proc
Natl Acad Sci USA 1998;95:7670–7675.
Hoyer LL, Scherer S, Shatzman AR, Livi GP: Candida albicans ALS1: domains related to a
Saccharomyces cerevisiae sexual agglutinin separated by a repeating motif. Mol Microbiol 1995;
15:39–54.
Hoyer LL, Payne TL, Bell M, Myers AM, Scherer S: Candida albicans ALS3 and insights into the
nature of the ALS gene family. Curr Genet 1998;33:451–459.
Hoyer LL, Payne TL, Hecht JE: The ALS2 and ALS4 genes of Candida albicans and localization
of Als proteins to the fungal cell surface. J Bacteriol 1998;180:5334–5343.
Hoyer LL, Hecht JE: The ALS6 and ALS7 genes of Candida albicans. Yeast 2000;16:847–855.
Lipke PN, Wojciechowicz D, Kurjan J: ␣Gal is the structural gene for the Saccharomyces cerevisiae ␣-agglutinin, a cell surface glycoprotein involved in cell-cell interactions during mating.
Mol Cell Biol 1989;9:3155–3165.
C. albicans Virulence Factors
125
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Gaur NK, Klotz SA: Expression, cloning and characterization of a Candida albicans gene, ALA1,
that confers adherence properties upon Saccharomyces cerevisiae for extracellular matrix proteins. Infect Immun 1997;65:5289–5294.
Fu Y, Rieg G, Fonzi WA, Belanger PH, Edwards JE Jr, Filler SG: Expression of the Candida albicans gene ALS1 in Saccharomyces cerevisiae induces adherence to endothelial and epithelial cells.
Infect Immun 1998;66:1783–1786.
Hoyer LL, Clevenger J, Hecht JE, Ehrhart EJ, Poulet FM: Detection of Als proteins on the cell wall
of Candida albicans in murine tissues. Infect Immun 1999;67:4251–4255.
Hostetter MK: Integrin-like proteins in Candida spp. and other microorganisms. Fungal genetics
and Biology 1999;28:135–145.
Gale CA, Bendel CM, McClellan M, Hauser M, Becker JM, Berman J, Hostetter MK: Linkage of
adhesion, filamentous growth, and virulence in Candida albicans to a single gene, INT1. Science
1998;279:1355–1359.
Calderone RA: Molecular interactions at the interface of Candida albicans and host cells. Arch.
Med. Res. 1993;24:275–279.
Cormak BP, Ghori N, Falkow S: An adhesin of the yeast pathogen Candida glabrata mediating
adherence to human epithelial cells. Science 1999;285:578–582.
Staab JF, Bradway SD, Fidel PL, Sundström P: Adhesive and mammalian transglutaminase substrate properties of Candida albicans Hwp1. Science 1999;283:1535–1538.
Smith GA, Marquis H, Jones S, Johnston NC, Portnoy DA, Goldfine H: The two distinct phospholipases C of Listeria monocytogenes have overlapping roles in escape from a vacuole and cellto-cell spread. Infect Immun 1995;63:4231–4237.
Saffer LD, Long Krug SA, Schwartzman JD: The role of phospholipase in host cell penetration by
Toxoplasma gondii. Am J Trop Med Hyg 1989;40:145–149.
Saffer LD, Schwartzman JD: A soluble phospholipase of Toxoplasma gondii associated with host
cell penetration. J Protozool 1991;38:454–458.
Ravdin JI, Murphy CF, Guerrant RL, Long-Krug SA: Effect of antagonists of calcium and
phospholipase A on the cytopathogenicity of Entamoeba histolytica. J Infect Dis 1985;152:542–549.
Price MF, Wilkinson ID, Gentry LO: Plate method for detection of phospholipase activity in
Candida albicans. Sabouraudia 1982;20:7–14.
Barret-Bee K, Hayes Y, Wilson RG, Ryley JF: A comparison of phospholipase activity, cellular
adherence and pathogenicity of yeasts. J Gen Microbiol 1985;131:1217–1221.
Leidich SD, Ibrahim AS, Fu Y, Koul A, Jessup C, Vitullo J, Fonzi W, Mirbod F, Nakashima S,
Nozawa Y, Ghannoum MA: Cloning and disruption of a caPLB1 a phospholipase B gene involved
in the pathogenicity of Candida albicans. J Biol Chem 1998;273:26078–26086.
Sugiyama Y, Nakashima S, Mirbod F, Kanoh H, Kitajima Y, Ghannoum MA, Nozawa Y:
Molecular cloning of a second phospholipase B gene, caPLB2 from Candida albicans. Med
Mycol 1999;37:61–67.
Rüchel R, Böning-Stutzer B, Mari A: A synoptical approach to the diagnosis of candidosis, relying on serological antigen and antibody tests, on culture, and on evaluation of clinical data.
Mycoses 1988;31:87–106.
Borg M, Rüchel R: Expression of extracellular acid proteinase by proteolytic Candida spp. during experimental infection of oral mucosa. Infect Immun 1988;56:626–631.
Borg M, Rüchel R: Demonstration of fungal proteinase during phagocytosis of Candida albicans
and Candida tropicalis. J Med Vet Mycol 1990;28:3–14.
Rüchel R, Zimmermann F, Böning-Stutzer B, Helmchen U: Candidiasis visualised by proteinasedirected immunofluorescence. Virch Arch Pathol Anat 1991;419:199–202.
Ollert MW, Söhnchen R, Korting C, Ollert U, Bräutigam S, Bräutigam W: Mechanisms of adherence of to cultured human epidermal keratinocytes. Infect Immun 1993;61:4560–4568.
Ray TL, Payne CD: Scanning electron microscopy of epidermal adherence and cavitation in
murine candidiasis: A role for Candida acid proteinase. Infect Immun 1988;56: 1942–1949.
Rüchel R, Ritter B, Schaffrinski M: Modulation of experimental systemic murine candidosis by
intravenous pepstatin. Zentralbl Bakteriol Mikrobiol Hyg 1990;273:391–403.
Fallon K, Bausch K, Noonan J, Huguenel E, Tamburini P: Role of aspartic proteases in disseminated Candida albicans infection in mice. Infect Immun 1997;65:551–556.
Monod/Zepelin
126
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
Monod M, Togni G, Hube B, Sanglard D: Multiplicity of genes encoding secreted aspartic proteinases in Candida species. Mol Microbiol 1994;13:357–368.
Monod M, Hube B, Hess D, Sanglard D: Differential regulation of SAP8 and SAP9 which encode
two new members of the secreted aspartic proteinase family in Candida albicans. Microbiology
1998;144:2731–2737.
De Bernardis F, Cassone A, Sturtervant J, Calderone R: Expression of Candida albicans SAP1 and
SAP2 in experimental vaginitis. Infect Immun 1995;63:1887–1892.
Schaller M, Schäfer W, Korting HC, Hube B: Differential expression of secreted aspartyl proteinases in a model of human oral candidosis and in patient samples from the oral cavity. Mol
Microbiol 1998;29:605–615.
Schaller M, Korting HC, Schäfer W, Bastert J, Wen Chieh C, Hube B: Secreted aspartic proteinase
(Sap) activity contributes to tissue damage in a model of human oral candidosis. Mol Microbiol
1999;34:169–180.
Naglik JR, Newport G, White TC, Fernandes-Naglik LL, Greenspan JS, Greenspan D, Sweet SP,
Challacombe SJ, Agabian N: In vivo analysis of secreted aspartyl proteinase expression in human
oral Candidiasis. Infect Immun 1999;67:2482–2490.
Staib P, Kretschmar M, Nichterlein T, Köhler G, Michel S, Hof H, Hacker J, Morschhäuser J: Host
induced, stage specific virulence gene activation in Candida albicans during infection. Mol
Microbiol 1999;533–546.
Hube B, Sanglard D, Odds FC, Hess D, Monod M, Schäfer W, Brown AJP, Gow NAR: Disruption
of each of the aspartyl proteinase genes SAP1, SAP2, and SAP3 of Candida albicans attenuates
virulence. Infect Immun 1997;65:3529–3538.
Sanglard D, Hube B, Monod M, Odds FC, Gow NAR: A triple deletion of the aspartyl proteinase
genes SAP4, SAP5, and SAP6 of Candida albicans causes attenuated virulence. Infect Immun
1997;65:3539–3546.
Borg-von Zepelin M, Beggah S, Boggian K, Sanglard D, Monod M: The expression of the
secreted aspartyl proteinases Sap4 to Sap6 from Candida albicans in murine macrophages. Mol
Microbiol 1998;28:543–554.
De Bernardis F, Arancia S, Morelli L, Hube B, Sanglard D, Schäfer W, Cassone A: Evidence that
members of the secretory aspartyl proteinase gene family, in particular SAP2, are virulence factors for Candida vaginitis. J Infect Dis 1999;179:201–208.
Egger M, Hirschel B, Francioli P, Sudre P, Wirz M, Flepp M, Rickenbach M, Malinverni R,
Vernazza P, Battegay M: Impact of new retroviral combination therapies in HIV
infected patients in Switzerland – Prospective multicentre study. Br Med J 1997;315:
1194–1199.
Palella FJ, Delaney KM, Moorman AC, loveless MO, Fuhrer J, Satten GA, Aschman DJ,
Holmberg SD: Declining morbidity and mortality among patients with advanced human immunodeficiency virus infection. N Engl J Med 1998;338:853–860.
Ledergerber B, Egger M, Opravil M, Telenti A, Hirschel B, Battegay M, Vernazza P, Sudre P,
Flepp M, Furrer H, Francioli P, Weber R, for the Swiss HIV Cohort Study: Highly active
antiretroviral therapy: Low rates of clinical disease progression despite high rates of virological
failure. Lancet 1999;353:863–868.
Borg-von Zepelin M, Meyer I, Thomssen R, Würzner R, Sanglard D, Telenti A, Monod M: HIVprotease inhibitors reduce cell adherence of Candida albicans strains by inhibition of yeast
secreted aspartic proteases. J Invest Dermatol 1999;113:747–751.
Korting HC, Schaller M, Eder G, Hamm G, Böhmer U, Hube B: Effects of the human immunodeficiency virus (HIV) proteinase inhibitors Saquinavir and Indinavir on in vitro activities of
secreted aspartyl proteinases of Candida albicans isolates from HIV-infected patients. Antimicrob
Agents Chemother 1999;43:2038–2042.
Cassone A, De Bernardis F, Torosantucci A, Tacconelli E, Tumbarello M, Cauda R: In vitro and in
vivo anticandidal activity of human immunodeficiency virus protease inhibitors. J. Infect Dis
1999;180:448–453.
Marquis G, Garzon S, Montplaisir S, Strykowski H, Benhamou N: Histochemical and immunochemical study of the fate of Candida albicans inside human neutrophil phagolysosomes.
J Leukocyte Biol 1991;50:587–599.
C. albicans Virulence Factors
127
79
80
81
82
83
84
85
De Chastellier C, Berche P: Fate of Listeria monocytogenes in murine macrophages: Evidence for
simultaneous killing and survival of intracellular bacteria. Infect Immun 1994;62:543–553.
Hube B, Monod M, Schofield A, Brown AJP, Gow NAR: Expression of seven members of the
gene family encoding secretory aspartyl proteinases in Candida albicans. Mol Microbiol 1994;14:
87–99.
White TC, Agabian N: Candida albicans secreted aspartyl proteinases: Isoenzyme pattern is
determined by cell type, and levels are determined by environmental factors. J Bacteriol 1995;177:
5215–5221.
Schweitzer A, Rupp S, Taylor BN, Röllinghoff, M, Schröppel K: The TEA/ATTS transcription
factor CaTec1p regulates hyphal development and virulence in Candida albicans. Mol Microbiol
2000;38:435–445.
Kretschmar M, Hube B, Bertsch T, Sanglard D, Merker R, Schröder M, Hof H, Nichterlein T:
Germ tubes and proteinases are virulence factors of Candida albicans in murine peritonitis. Infect
Immun 1999;67:6637–6642.
Andrews NW, Portnoy DA: Cytolysins from intracellular pathogens. Trends Microbiol 1994;2:
261–263.
Miller RA, Britigan BE: Role of oxidants in microbial pathophysiology. Clin Microbiol Rev 1997;
10:1–18.
Dr. Michel Monod, Service de Dermatologie,
Laboratoire de Mycologie, BT422, Centre Hospitalier Universitaire Vaudois,
1011 Lausanne (Switzerland)
Tel. ⫹41 21 314 0376, Fax ⫹41 21 314 0378, E-Mail Michel.Monod@chuv.hospvd.ch
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Breitenbach M, Crameri R, Lehrer SB (eds): Fungal Allergy and Pathogenicity.
Chem Immunol. Basel, Karger, 2002, vol 81, pp 129–166
Cutaneous Mycology
Thomas Hawranek
Department of Dermatology, Landeskliniken Salzburg, Salzburg, Austria
In 1839 it was shown for the first time that fungi can cause disease.
Schoenlein and Gruby discovered what was later called Trichophyton schoenleinii, the causative agent of scalp infection or favus [1]. But for many years,
fungi somehow lived in the shadow of bacterial diseases: their importance grew
during the last decades as a consequence of worldwide travel as well as the
increased use of immunosuppressive drugs and the appearance of AIDS.
Opportunistic infections caused by unknown fungi or fungi initially thought to
be nonpathogenic were increasingly recognized. Dermatology is involved not
only because the majority of fungal infections affect the skin or the subcutaneous tissues, but also because systemic infections may present with cutaneous
lesions and therefore lead to the diagnosis of systemic disease. Many of the
subcutaneous and systemic mycoses were first described by dermatologists.
Excellent reference books [1–7], comprehensive reviews [8–12] and web
sites are available on the theme. A good starting point for excursions into the
world of mycology online is, for instance http://fungusweb.utmb.edu/mycology/.
This short excursus on cutaneous mycology cannot and will not give an extensive overview on this vast field, but tries to expound some clinical aspects of
fungal-induced dermatological diseases. Whether they display pleasant activities, like the production of cheese or the fermentation of alcohol, or do harm by
destroying crops or by attacking man, fungi are closely linked to our lives.
Fungi may affect man in three ways: by causing allergy (cutaneous, respiratory and other forms), by their toxins (whether by ingestion of poisonous
fungi or by production of mycotoxins) or by invasion.
The molecular phylogeny and systematics of the fungi will be discussed
elsewhere in this book [13]. A simple division of fungi with respect to clinical
aspects is that into molds and yeasts, the former characterized by the formation of
septate and nonseptate hyphae, the latter by an unicellular life cycle with reproduction mainly by budding. The so-called dimorphic fungi, such as Histoplasma
or Coccidioides, may switch forms depending on environmental conditions, such
as temperature, grow as molds at room temperature and as yeasts at 37 °C or in
human tissue.
The immense number of names given not only to the whole diversity of up
to 250,000 species, but often also to one and the same of the approximately 200
pathogens, has led to some confusion, partly brought into order by an internationally accepted nomenclature. Confusion also arose from giving different
names to one and the same disease, depending on which of the many different
pathogenic agents was demonstrated in a special case.
Identification of a particular fungal pathogen is essential in many respects;
it will have an important role in therapeutic decision-making as in otomycosis,
and with the arrival of the new antifungals it has become important to differentiate between yeast and dermatophyte infection as well as between dermatophytes themselves, as they may not respond similarly. Identification of the
pathogen may also lead to the source of an infection and therefore enable
initiation of measures against spread.
A useful classification of fungal infections groups them according to the
site of the infection combined with the immune reaction of the host (table 1).
Pathogenesis of Cutaneous Fungal Infection
As the mammalian physiological barriers protect against microbial invasion,
fungal diseases in man are rather uncommon considering the wide distribution of
these microorganisms. In addition, nonimmunological and immunological
defenses hinder the virulence of fungi [14]. All humans have contact with dermatophytes during their lifetime, but only a few develop clinical symptoms, fungal
infection therefore usually depends on a breakdown of one of these defense lines.
Dermatophyte growth is restricted to the stratum corneum, the keratinized
hair shaft and the keratinized parts of the nail. From there, the disease is transmitted by direct contact or via fomites in the form of parasitic arthroconidia, the
infective agents of the fungi. These are extremely resistant to environmental
rigors surviving up to 20 months in scales or hair, which therefore are the usual
source of infection. As there is not much space between the layers of the stratum corneum, arthroconidia, which are formed by fragmentation of hyphae, are
the ideal form of growth. This fragmentation is promoted by high carbon dioxide tension, humidity, a temperature of ideally 37 °C, a physiological pH, certain metabolites and still unidentified cutaneous factors. Transversal growth
between the cell layers is facilitated by keratinases produced by fungi as well as
hydrolytic enzymes (e.g. lipases, proteases, phosphatases) and finds its clinical
expression in the peripheral expansion of the skin lesion.
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Table 1. Clinical classification of the most common fungal infections and their
pathogens
Superficial mycoses
Characterization: cosmetically apparent infections without invasion of living tissue or
immune response of the host
1. Pityriasis versicolor (M. furfur)
2. White piedra (T. ovoides, T. asakii, T. asteroides and others)
3. Black piedra (P. hortai)
4. Tinea nigra (H. werneckii)
Cutaneous mycoses
Characterization: superficial infection of skin, hair and nails; usually no invasion of living
tissue, defense reaction of the host
1. Dermatophytosis (ringworm, tinea) (multiple dermatophytes)
2. Candidiasis (C. albicans and others)
Subcutaneous mycoses
Characterization: chronic infection of skin and subcutaneous tissue after traumatic
implantation of the geophilic agent.
1. Sporotrichosis (S. schenckii)
2. Chromoblastomycosis (Fonsecaea pedrosoi, Cladophialophora carrionii and others)
3. Mycetoma (Madura foot) (Madurella mycetomi, Pseudoallescheria boydii, Acremonium,
Exophiala and others)
4. Subcutaneous zygomycosis (entomophthoromycosis and mucormycosis)
5. Lobomycosis (L. loboi)
6. Rhinosporidiosis (Rhinosporidium seeberi)
Systemic (deep) mycoses by pathogenic fungi
Characterization: the immunocompetent host is infected by the dimorphic fungi as they
change their morphological form; patients usually get infected by inhalation of conidia;
restricted to certain geographic areas
1. Coccidioidomycosis (C. immitis)
2. Paracoccidioidomycosis (P. brasiliensis)
3. Histoplasmosis (H. capsulatum)
4. Blastomycosis (B. dermatitidis)
Systemic (deep) mycoses by opportunistic fungi
Characterization: the immunocompromised host is overcome by fungi of actually low
virulence
1. Candidiasis (C. albicans and others)
2. Cryptococcosis (C. neoformans)
3. Systemic zygomycosis (mucormycosis) (Rhizopus arrhizus, Mucor, Absidia and others)
4. Aspergillosis (Aspergillus spp.)
5. Pseudallescheriasis (P. boydii)
Rare mycoses
1. Pheohyphomycosis (E. jeanselmei, Cladophialophora, Exophiala and others)
2. Hyalohyphomycosis (Acremonium spp., Penicillium spp., Beauveria spp. and many others)
3. Protothecosis (Prototheca spp.)
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Essential steps in dermatophyte infection are an initial close contact between
arthroconidia and corneocytes of the stratum corneum, adherence by physical or
chemical binding, rapid germination (usually within 4–6 h) to evade elimination
by continuous epithelial desquamation and finally penetration of the stratum
corneum. Defense mechanisms of the host include elimination by epidermal
turnover (decreased infection in psoriasis, increased in keratoderma), unsaturated
fatty acids of the sebum (explaining relative resistance of the adult scalp against
tinea capitis as well as clearing of this disease during puberty), vitamin-K-like
substances in sweat, unsaturated transferrin in human serum (binding of the iron
needed for fungal growth) and a body temperature of about 37 °C. Cell-mediated
immunity often leads to spontaneous resolution of infection, whereas a decrease
in T-helper cells and an increase in T-suppressor cells tend to lead to chronicity.
Antibodies seem to have no effect on the course of the infection. Neutrophils and
monocytes play an important role in the early phases of invasion. Other factors
associated with fungal infections are very young and very old age, endocrine disorders like diabetes, Cushing’s disease, sex, genetic and racial factors, hot and
humid climates, malnutrition as well as lack of knowledge about the prevalence
and carrier status and absence of control measures [15].
Clinical Mycology
Superficial Mycoses
The so-called superficial mycoses are characterized by invasion restricted
to the stratum corneum and thus are usually not associated with a remarkable
inflammatory response of the host.
Pityriasis versicolor
This cosmetically disturbing condition, first described in 1846, is caused
by the lipophilic yeast Malassezia furfur, the hypha-producing, invasive form
of Pityrosporum ovale (mainly on the scalp) sive orbiculare (mainly trunk),
physiological saprophytes of the human skin from where they may be cultured
in up to 100% of the cases. Whether or not these two latter forms represent different species or two forms of the same organism is still discussed. Certain predisposing factors (humidity, heat, oily skin, hyperhidrosis as well as hereditary
disposition and immunodeficiency) lead to the distinct clinical lesions presenting as asymptomatic, rarely itching macules of hypopigmentation (in summer
affected areas fail to tan) and hyperpigmentation (winter) on the upper trunk,
neck and shoulders, slightly scaling when scraped. The disease is uncommon in
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childhood. Recently reported entities are systemic disease by Malassezia –
fungemia described mostly in neonates receiving parenteral lipids through
central catheters as well as a form of onychomycosis in AIDS patients.
Usually, the clinical picture in combination with the detection of short
curved hyphae and ovoid budding yeast cells (the so-called ‘spaghetti and meatballs’) by light microscopy is sufficient for diagnosis. Often inspection under
Wood’s light (a lamp emitting UV light at a wavelength of above 365 nm) may
demonstrate weak yellow fluorescence, which however disappears after bathing
[16]. Occasionally, however, it may be necessary to culture the fungi on agar
with sterile olive oil. In most cases, topical treatment with selenium sulfide or a
number of other agents including imidazoles and terbinafine extended also to
areas not yet visually affected will be successful, repigmentation taking up to
several months. A single dose (400 mg) of ketoconazole or fluconazole as well
as itraconazole 200 mg daily for 5–7 days has been equally effective. Before
treatment, it is advisable to point out to the patient that relapse after successful
therapy is the rule, but may be avoided by prophylactic measures such as topical
therapy weekly (low compliance [17]) or ketoconazole 400 mg once a month.
Pityrosporum folliculitis, a distinct clinical picture, is characterized by scattered itching acneiform, small follicular papules, sometimes pustules, on the
back and shoulders of young patients. In contrast to acne, it is often aggravated
by the use of local antibiotics and sun exposure. Hyphae are usually absent by
light microscopic examination of pustular material. Pityrosporum yeasts take
part in the pathogenesis of seborrheic dermatitis and have also been associated
with certain facial eczemas in young atopic women.
White Piedra
Trichosporon asakii, Trichosporon inkin, and Trichosporon mucoides are
regularly isolated from clinical specimens. In addition, Trichosporon ovoides
(Trichosporon beigelii) was isolated as causative agent of capital white piedra.
These inhabitants of soil, lakes and plants in subtropical and temperate climates
including Europe, North America and Japan produce small and soft white to
cream-colored nodules on hair shafts of a usually restricted area which may be
easily stripped off. Breakage or splitting of hair is common. The condition is
not contagious. Cultures should be grown without the use of cycloheximide.
Treatment includes shaving of all affected hair and/or topical clotrimazole, oral
ketoconazole being an alternative possibility. In immunocompromised patients
T. asakii may lead to a serious systemic infection called trichosporonosis.
Black Piedra
This infection by Piedraia hortai presents with tightly adherent dark nodules on hair shafts causing breakage. Light microscopy and culture consolidate
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the diagnosis. Shaving is the treatment of choice; oral terbinafine 250 mg daily
for several weeks may be an alternative.
Tinea nigra
This harmless condition, caused by Hortaea (Phaeoannelomyces) werneckii, is acquired by direct inoculation from various sources via minor trauma
and most commonly involves one hand. This dematiaceous fungus is endemic in
maritime regions of tropical and subtropical climates and occasionally affects
travelers on vacation, producing mostly asymptomatic, sometimes itchy, slowly
extending brown to black macules after incubation periods of supposedly weeks
to decades. Misinterpretation as melanoma may lead to unnecessary excision.
After consolidation of diagnosis by light microscopy and culture, treatment with
topical keratolytic agents, 10% thiabendazole or topical imidazoles has been
effective.
Cutaneous Mycoses
Dermatophytosis (Ringworm, Tinea)
The dermatophytes, a group of filamentous fungi invading the epidermal
stratum corneum and keratinized skin appendages as hair and nails in up to
20% of the population [18], may be divided according to their natural habitats:
anthropophilic species are spread from human to human, zoophilic species parasitize animals, and geophilic species live on soil as saprophytes. The latter two
are also capable of causing human disease. Spread of zoophilic and geophilic
species from human to human is uncommon. Zoophilic species usually cause a
more severe clinical variant producing suppurative lesions. Trichophyton
rubrum and Trichophyton mentagrophytes var. interdigitale are the two most
commonly isolated species in Europe. Dermatophytes are discriminated by
macroscopical and microscopical features (table 2) described in the section on
laboratory diagnosis.
Special Clinical Patterns
Tinea corporis
This dermatophyte infection confined to the trunk and extremities takes a
subacute to chronic course (weeks to years) and produces the typical and rather
well-known lesion called ringworm (fig. 1), a usually round, often irregular
scaly lesion with a significantly more inflamed, raised border containing a
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Table 2. Characteristics of some dermatophytes
Species
Geography
Anthropophilic dermatophytes
T. rubrum
cosmopolitan
Remarks
Culture
Microscopy
most common and widely distributed
dermatophyte of man; mostly feet and
nails infected; Wood’s light negative
downy or fluffy white ‘snowball’
appearance; reverse: dark red,
sometimes melanoid; granular
and dysgonic varieties
Macroconidia long, cylindrical, thin
walled, usually absent; microconidia
rare also, clavate to pyriform,
situated along the sides of hyphae
T. mentagrophytes
var. interdigitale
cosmopolitan
mostly tinea pedis and corporis; no tinea
capitis, but positive hair penetration
test; positive urease test.
flat, downy, white in the beginning,
then becomes powdery and creamcolored, sometimes velvety
single-standing pear-shaped conidia
along the hyphae, typical spiral hyphae
occasionally, sometimes macroconidia;
chlamydospores
M. audouinii
cosmopolitan
used to be the most common cause of
epidemics of tinea capitis in Europe
and N. America, Ectothrix; Wood’s
light positive
downy white surface to grayishwhite ‘mouse-fur’, reverse salmon
to peach-pink, sometimes rusty;
slow growth
pectinate (‘comb-like’) and racquet
(rackett) hyphae, thick-walled
chlamydospores; rare micro- and
(bizarre) macroconidia
M. ferrugineum
E. Europe,
Asia, C. Afr.,
S. America
epidemics of tinea capitis in children,
ectothrix invasion of hair; Wood’s
light positive
waxy, convoluted, slowly growing
thallus, color cream to yellow
to red
no macro- or microconidia, but hyphae
of irregular shape with prominent septae
(‘bamboo hyphae’)
T. schoenleinii
Eurasia,
Africa
causes favus, a chronic and scarring
form of tinea capitis with typical
scutulae, Wood’s light positive
gray to gray-brown, cream-colored,
orange-brown; waxy and folded, then
becoming flat; grows well at 37 °C
no macro- nor microconidia, but hyphae
tend to build antler-like branches at the
end, sometimes resembling chandeliers
T. violaceum
cosmopolitan
finely scaling lesions, typical ‘black dot’
tinea capitis; endothrix hair invasion
violet, seldom white, glabrous or
waxy, heaped and folded, slowly
growing colonies; growth stimulated
by thiamine; reverse deep purple
branched, distorted, thick hyphae;
no macro- or microconidia
T. soudanense
N. C. Africa
endothrix tinea capitis; Wood’s light
negative
slowly growing, yellow-orange
(apricot) cultures with flat or folding,
velvety surface; often broad fringed
margin (‘eyelashes’); reverse apricot
typical is the right angled, reflexive
branching of hyphae; rarely
pear-shaped microconidia,
no macroconidia; chlamydospores
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Table 2. Continued
Species
Geography
Remarks
Culture
Microscopy
T. tonsurans
cosmopolitan
fine scaling lesions mainly on the scalp;
endothrix invasion; Wood’s light
negative
cream to buff or yellow-brown
powdery or velvety, sometimes
folded colonies; reverse yellowbrown or red-brown
irregular, branched (‘tree-like’)
hyphae with numerous
microconidia of different
morphology (‘balloon-like’,
‘matchstick-like’ etc.);
macroconidia rare
T. megninii
Europe,
Africa
onychomycosis and tinea corporis
mostly in the upper exposed parts
of the body of older men for unknown
reasons
white suede-like colonies turning to
pink or violet with time; wide radial
grooves; reverse light wine-red
clavate microconidia and long
pencil-shaped macroconidia
Epidermophyton
floccosum
cosmopolitan
common cause for skin lesions as well
as onychomycosis; epidemics among
persons using the same shower
and facilities
typical olive-green to khaki color,
slowly growing colonies with
pleomorphic tufts in older cultures;
reverse yellowish-brown
macroconidia borne directly from
hyphae, often in clusters, of broadly
clavate, ‘beaver tail’ appearance with
0–4 cells; no microconidia
Zoophilic dermatophytes
M. canis
cosmopolitan
cats (!) and dogs; tinea frequently
seen in children; ectothrix; Wood’s
light positive
white wooly surface, reverse shows
the typical deep yellow, rarely
orange or brownish
numerous spindle-shaped, rough and
thick-walled macroconidia, few
microconidia
T. verrucosum
cosmopolitan
cattle; highly inflammatory infections
of exposed areas, especially the
beard and scalp; ectothrix, but
Wood’s light negative; grow better
at 37 °C (!) and with thiamine
very slow growth (⫽ small
colonies) of white or buff, flat or
raised and folded colonies;
reverse white or buff
macro- and microconidia rarely seen;
on thiamine sometimes few, but typical
‘rat tail’ or ‘string bean’ shaped
macroconidia; chlamydospores in
chains (‘string of pearls’); terminal
vesicles with yeast extract
T. mentagrophytes
var. erinacei
UK, NZ
hedgehogs and their mites; in man
mostly exposed areas of the skin;
endothrix invasion in tinea capitis;
Wood’s light negative
white, flat, powdery colonies, the
reverse shows a typical bright
yellow
many large clavate microconidia on the
sides of the hyphae, macroconidia
smooth, thin-walled and of different size
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T. mentagrophytes
var. quinckeanum
cosmopolitan
‘mouse favus’; in man rarely scutulae;
seldom endo- or ectothrix invasion;
Wood’s light negative
white and downy first, then
becomes powdery and heaped and
folded; reverse yellow-brown
slender microconidia along the hyphae,
becoming broad and pear-shaped
with age; macroconidia rarely seen
M. equinum
Europe,
N. Am.,
Austr.
horses; rare; ectothrix; Wood’s light
positive
suede-like to fine powdery, white
to pale salmon, radial folds, reverse
buff to salmon
2 to 4-celled spindle-shaped, irregular
thick-walled macroconidia, rare
microconidia
M. nanum
cosmopolitan
pigs; hair invasion possible but Wood’s
light negative
white to cream-colored powdery,
reverse brown-orange
typically egg-shaped, rough-walled
macroconidia with 2 cells
M. persicolor
Eur., Austr.,
Africa, N. Am
voles and bats; rare cause of tinea in
man
white to pink powdery to downy
surface, reverse red-brown; rapid
growth
pear- to club-shaped microconidia,
single-standing or in clusters; rare
club-shaped macroconidia
T. gallinae
cosmopolitan
fowl; rare cause of tinea in man;
ectothrix hair invasion; Wood’s light
negative
white to pink, velvety to downy,
reverse orange-pink to
strawberry-red
multicellular, sometimes curved
thick-walled macroconidia with a
blunt tip, microconidia
T. equinum
cosmopolitan
horses; rare in man; ectothrix hair
invasion; Wood’s light negative
white to buff, velvety to downy
surface, often with yellow fringe;
reverse yellow, then red brown;
most require niacin for rapid growth
solitary pear-shaped to elongated
microconidia along the hyphae,
macroconidia rare and of clavate shape
of different size
Geophilic dermatophytes
M. gypseum
cosmopolitan
soil; solitary lesions of skin and scalp
(ectothrix but Wood’s light negative)
cinnamon-colored, powdery surface,
often with a fluffy white taft,
reverse yellow-brown
ellipsoidal cucumber-like, multicelled,
thin-walled macroconidia, clavate
microconidia
M. cookei
cosmopolitan
geophilic, but reported in dogs,
rodents, and man
powdery to granular, brownish,
reddish, yellowish, reverse deep
purple-red
ellipsoidal cucumber-like macroconidia,
but mostly thick-walled; numerous
microconidia
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137
majority of ‘active’ fungi (fig. 2) and often producing prominent hair follicles.
Multiple lesions are not uncommon. Facial involvement is characterized by
possible flares in sunlight.
Management: Besides measures as boiling or disinfecting clothes, topical
antimycotic therapy is the treatment of choice. Ointments should be applied
several centimeters beyond the border of the lesion and treatment should be
continued over at least 1 week after clearance of the lesions. However, these
recommendations are seldom followed, resulting in chronic or recurrent disease
(apart from the problem of resistance). In these cases, and when multilocular
lesions or an infection of extensive spread have to be treated, systemic antifungals usually for 1–2 weeks are necessary, terbinafine usually giving the best
results in dermatophytosis.
Tinea inguinalis (Tinea cruris)
This very contagious condition (usually spread via contaminated towels,
saunas) presents with bilateral and itchy scaling in the groin (fig. 2), usually
including the pubic region and the upper region of the thighs and sometimes the
perineum. The frequently occurring candidiasis of this region usually may be
easily differentiated by the typical peripheral satellite lesions, pustules and/or
papules outside the border of the main lesion. The bacterial erythrasma usually
is associated with fine wrinkling and has a typical red-brownish color; Wood’s
light may be helpful for diagnosis.
Management: Treatment rules are very similar to those of tinea corporis
(local therapy should be continued for at least another week after clearance of
the lesions), prophylactic measures may include boiling of underwear, usage of
different towels for the affected region, examination of the feet to see whether
tinea pedis may be the source of autoinoculation, avoidance of occlusive and
synthetic garments and possibly weight loss.
Tinea capitis
This dermatophyte infection of the scalp and hair of sometimes quite striking appearance is reported to have had tremendous influence on the lives of
people in old times: subjects with tinea capitis were allowed to keep their heads
covered in the presence of the monarch, and sometimes were not allowed to
emigrate to the United States [19]. Although such drastic measures are historical anecdotes, tinea capitis nevertheless may lead to some social discomfort
even today.
Usually not a disease of adults, this sporadic infection sometimes causes
epidemics in schools. Its clinical appearance varies from mildly scaling lesions
over alopecia (patchy hair loss) to highly inflamed, suppurative (kerion) variants, the latter usually caused by zoophilic species as the leading pathogen in
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Fig. 1. Ringworm. Typical round lesion
on the calf with inflamed, raised border.
Fig. 2. Tinea ‘cruris’. Note the typical
scaling on the ‘active’ border of the lesion
containing the majority of ‘active’ fungi.
Western Europe. Itching is no obligate symptom, hair loss usually is reversible
except in some cases of kerion. Lymphadenitis is a common complication even
in mild disease [20]. ‘Gray patch’ tinea capitis caused by Microsporum (canis
and audouinii) is differentiated from the other forms by some authors.
There are three forms of hair invasion: ectothrix (M. canis, M. audouinii,
Microsporum ferrugineum, Trichophyton verrucosum), endothrix (Trichophyton
tonsurans, Trichophyton violaceum, Trichophyton soudanense) and favic
(Trichophyton schoenleinii). Favus is a rarely seen condition in small endemic
areas of Northern Africa, the Middle East and the USA. It presents with typical
dish-like crusts (scutulae) and scarring alopecia and may also affect adults.
Wood’s light: This lamp emitting UV light at a wavelength of above 365 nm
is helpful in the diagnosis of tinea capitis (bright greenish yellow), erythrasma,
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a bacterial skin disease (brick red) and pityriasis versicolor (yellow) if observed
in a darkened room. Dermatophytes regularly inducing fluorescence are M. canis
and M. canis var. distortum, M. audouinii, M. ferrugineum and T. schoenleinii,
whereas Microsporum nanum and Microsporum gypseum do so only occasionally. Very recent infection may not yet show this quality, and fluorescence may
remain even after cessation of viability.
Management: tinea capitis is the typical example for the importance of an
accurate identification of the pathogen, as proceedings are different for anthropophilic dermatophytes transmitted from child to child and for zoophilic species
usually originating from one common source.
If more than one child is affected in the first case, the whole class as well
as the family members should be examined with brush samples (see Collection
of Material for Laboratory Diagnosis) done simultaneously. Active disease
should be treated systemically (see below); the carrier status (i.e. asymptomatic
patient ⫹ positive culture) demands only local therapy with ketoconazole shampoo (or selenium sulfide) for about 3 weeks. Initially, adjuvant measures may
include hair cut, protection (dressing or cap) and sterilization of important
fomites (e.g. combs, brushes, toys, etc.) even if the organisms may be found on
a great variety of locations and there are no uniform recommendations for the
method [21]. Cultures should be repeated after 1 month of treatment and again
before discontinuation of therapy. Whether or not infected children should be
kept away from kindergarden or school remains a moot point, the usual recommendation is to wait until light microscopy is negative [10], others [4] tend to
let children return after 1 week’s topical treatment with ketoconazole shampoo
(or selenium sulfide).
These precautions are not necessary with zoophilic infections, here the source
(housepets) as well as every infected person should be treated systemically.
Antifungal agents used in tinea capitis: griseofulvin 10–20 mg/kg body
weight for at least 6–8 weeks up to several months (still the ‘gold standard’)
or – if resistance to griseofulvin occurs – terbinafine 62.5 mg daily from 10 to
20 kg, 125 mg up to 40 kg and 250 mg above 40 kg body weight for 4 weeks or
itraconazole suspension 5 mg/kg daily for 2–4 weeks or fluconazole 2–8 mg/kg
daily for 4–6 weeks. Monitoring of hepatic, renal and hematopoietic function
may be indicated [22].
The role of griseofulvin as the gold standard of treatment may be questioned in the near future because of a growing risk/benefit ratio [23] compared
to the new drugs. Although valid studies concerning treatment with those new
antifungals are still lacking (e.g. terbinafine for T. tonsurans, itraconazole for
M. canis) and appropriate dosing and duration of therapy, it is very likely that
these drugs prove to be of excellent efficacy, offering pulse therapy and shortcourse therapy in addition [24].
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Fig. 3. Sycosis barbae. This infection
caused by T. verrucosum in a milker is an
impressive clinical example of the aggressive behavior of zoophilic dermatophytes
compared with anthropophilic species.
Bacterial superinfection (Staphylococcus aureus, gram-negative bacteria)
rarely demands antibiotic treatment; scarring may be prevented by prednisone
1 mg/kg daily for 1–2 weeks in severe cases.
Tinea barbae
Mainly adults handling animals (farm, zoo) or straw contaminated by
infected mice are affected by this highly inflammatory, usually pustular variant
(sycosis barbae, fig. 3), caused in most cases by T. verrucosum.
Tinea pedis (‘Athlete’s Foot’)
Up to 70% of the ‘western’ population is reported to suffer from this harmless, but stubborn disease unheard of in certain aboriginal tribes, a fact which
has been attributed to the avoidance of occlusive footwear (‘tinea pedis follows
in the footsteps of shoe-wearing nations’ [25]). So tinea pedis is rare in Southeast
Asia, the original endemic area of T. rubrum where the worldwide epidemic is
said to have originated from. Partly due to the lack of sebum, which contains
fungistatic lipids, even cultures of clinically normal toewebs give positive
results in up to 21% of the samples. Transmission usually occurs by walking
barefoot on contaminated floors where the fungi are able to survive in skin
scales for many months. The infection commonly starts with scaling in the third
or fourth interdigital space (interdigital form). The clinical picture may be mild
with erythematosquamous lesions sometimes covering the whole sole and
extending upwards (squamous form, ‘moccasin-type’) almost always caused by
T. rubrum or a more severe blistering disease (vesicobullous form) generally
attributable to T. mentagrophytes var. interdigitale. Especially in chronic courses
with interdigital maceration (‘dermatophytosis simplex’) concomitant bacterial
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infection (e.g. Pseudomonas aeruginosa leading to greenish discoloration,
Proteus, Staphylococcus aureus) is very common (‘dermatophytosis complex’),
most bacteria being resistant to penicillin and its derivates. In mild disease sensitivity of cultures has been reported to reach about 85%. The more symptoms,
bacterial superinfection and inflammation, the more difficult it is to isolate the
pathogenic fungus in cultures, lowering sensitivity by approximately 50%.
Management: Therapy is very similar to that of tinea corporis. Concomitant
onychomycosis bears the constant danger of reinfection, therefore a cure of this
disease should be aimed at. Chronic disease tends to require systemic therapy
(e.g. itraconazole or terbinafine for 2 weeks) with clinical recovery some time
after cessation of therapy. Additional measures include daily bathing of feet
with meticulous drying and daily change of absorbent socks (100% cotton)
which should be boiled or washed with special preparations. Nonocclusive
shoes should be preferred, changed and left to dry over 3 days, prepared daily
with antifungal footpowder inside. Relapses may be avoided by long-term
topical antifungals twice weekly at least [26]. Unfortunately, recurrences must
be regarded as a rule.
Tinea manuum
Infection of typically only one hand usually occurs simultaneously with
(bilateral) infection of the feet (or groins), often involving fingernails (in fact
onychomycosis may be a source of reinfection, thus suggesting treatment of
onychomycosis). Clinical appearance may be characterized by fine, nonsymptomatic, so-called ‘dyshidrotic’ scaling or by hyperkeratotic, chronic lesions.
Up to 90% of this disease is caused by T. rubrum.
Management: Treatment is very much the same as in tinea corporis, but
usually requires about 2–6 weeks of systemic medication; topical treatment
should be continued for several weeks after clearance of the lesions.
Onychomycosis
Fungal nail infections (fig. 4) are a very common disease with an incidence of approximately 5:1,000. The most common pathogens are T. rubrum
followed by T. mentagrophytes var. interdigitale, invading the nail from the
distal and lateral ends leading to onycholysis (i.e. separation of the nail from
the nailbed), discoloration, thickening and dystrophy. Isolation of the pathogen
by culture may prove difficult even in samples positive on light microscopy. An
extensive survey of onychomycosis is given in Hay et al. [27]. For onychomycosis caused by molds, see below.
Management: Because of the characteristic physical features of the nail,
pharmacokinetics play a major role in therapy [28]. Topical therapy (ciclopirox
and amorolfin lacquer), more successful with thin nails and if the lunula is
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Fig. 4. Onychomycosis. Another typical manifestation of dermatophyte infection,
starting from the distal border of the nail.
spared, may be tried if ⬍ 60% [29] of the nail is affected, another possibility is
atraumatic removal of the nail by bifonazole/urea followed by prolonged topical bifonazole. In extensive distal disease, proximal onychomycosis or failure
of local therapy, systemic therapy with itraconazole (400 mg/day for 1 week
every month for 3–4 months or terbinafine 250 mg daily for 3 and more months
or fluconazole) is indicated. In lateral nail mycosis, poor penetration even of the
new antifungals may lead to recurrences [30]. Treatment of concomitant tinea
pedis is essential. Long-term management with periodical use of topical therapy
for tinea pedis and nails should prevent relapses.
Skin and Nail Infections by Molds
Inflammation of the proximal nailfold (often misdiagnosed as bacterial
infection) strongly suggests onychomycosis by molds (except Acremonium),
especially when associated with the proximal subungual form of fungal nail
infection [31]. As most molds do not grow on agars containing cycloheximide,
this group of pathogens usually is missed on routine agars. This is why many of
these infections were first described only recently. The significance of finding
a mold in culture has to be seen with caution [32] as usually they represent a
contamination. Cases of onychomycosis caused by molds have been estimated
at about 5% [33], prevalence rates ranging from 1.45 to 17.6%.
Scytalidium Species. Scytalidium dimidiatum (formerly Hendersonula
toruloidea) and its nonpigmented variant, Scytalidium hyalinum, found in the
tropics, the USA and the Mediterranean (although a recently published article
[31] states that Scytalidium spp. have never been isolated in Italy) are very
common in endemic areas, but may be acquired by tourists as well. Clinically,
they lead to a scaly tinea pedis et manuum, sometimes including nails starting
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from the distal and lateral ends [34] without considerable thickening of the nail.
Scytalidium spp. were never found to cause tinea in areas of the skin with a thin
keratin layer. Diagnosis often is made by light microscopic demonstration of
irregular hyphae. Colonies mostly grow rapidly. If Whitfield’s ointment is not
effective, therapy presents a problem, even if there are anecdotal reports of
success with the newer antifungals.
Onychomycosis Caused by Scopulariopsis brevicaulis. S. brevicaulis is the
most common cause of onychomycosis by molds, presenting with a cinnamon
discoloration of the toenails. Direct examination reveals the distinctive, lemonshaped conidia taking up Parker’s stain quickly. On agar with cycloheximide the
partially resistant fungus grows slowly. Therapy is problematic, local therapy
may be tried after removal.
Onychomycosis Caused by Other Molds. Aspergillus spp., Fusarium spp.,
Acremonium spp. (all three mostly held responsible for ‘superficial white onychomycosis’), Onychocola canadiensis, Curvularia lunata and many others
have been described to cause secondary onychomycosis, often in elderly
patients with dystrophic nails due to ischemia. According to a recent study [31],
local factors do not play a significant role in fungal invasion. Therapeutic
efforts in these cases usually are fruitless, although the same study mentions
cure rates of 42.5% for S. brevicaulis, 20% for Acremonium and 29.4% for
Fusarium. The best results were obtained by the use of topical therapy (8%
ciclopirox nail laquer or topical terbinafine after nail avulsion) whereas systemic
therapy with itraconazole or terbinafine was often ineffective. Aspergillus infections were cured in 100% of cases with systemic or topical therapy.
Tinea incognito
This condition is characterized by an unusually aggressive behavior of
dermatophytes invading the upper dermis thus causing granulomatous folliculitis.
The main cause for this is seen in prolonged and inappropriate topical steroid
therapy for fungal disease, resulting in loss of itching, scaling and the typical
pronounced border of the lesion, producing prominent papules and pustules.
Other triggers that have been mentioned [35] are trauma (shaving), longstanding occlusion and the use of local steroid/antifungal/antiviral/antibacterial
combination therapy.
‘Superficial’ Candidiasis
The first step in the genesis of this common infection of mucous membranes
(of the mouth, gastrointestinal tract and vagina where they live as physiological
commensals) and the skin seems to be a change in host resistance, the origin
of which is detectable in most cases. Predisposing factors include age (very
young and very old), moisture, reduced general conditions (e.g. malignancies),
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hormonal influences (diabetes and other endocrine disorders, pregnancy),
immunosuppression (e.g. steroids, whole-body irradiation, immunosuppressive
drugs), destruction of the physiological bacterial flora by prolonged use of antibiotics and mechanical factors, whereas for vaginal candidiasis such factors have
yet to be found. Candida albicans, is by far the most important pathogen; other
pathogens include Candida krusei, Candida glabrata, Candida tropicalis. The
typical sign of the invasive phase of Candida is the production of hyphae.
Management: Three groups of antifungals are available: for the treatment
of candidiasis the polyenes (amphotericin, nystatin, natastatin), the imidazoles
(clotrimazole, miconazole, econazole, ketoconazole, fluconazole, itraconazole)
and flucytosine, the latter – which is used only for systemic candidiasis in combination with amphotericin B – being relatively susceptible to the development
of resistance. Amphotericin B in its recently developed lipid-associated form
(reduced renal toxicity) is used intravenously because of the minimal gastrointestinal uptake after oral administration. Of the imidazoles, besides their use as
topicals, mainly fluconazole (100–400 mg daily) and itraconazole (100–200 mg
daily, now in soluble form with better absorption) are used, as ketoconazole
was shown to be of considerable hepatotoxicity. With fluconazole, the problem
of primary (mainly C. glabrata, C. krusei and some strains of C. albicans) and
secondary (maintenance therapy in AIDS patients) resistance is emerging.
Specific antimycotic treatment should be combined with general measures
to modify predisposing factors, e.g. drying, hygienic procedures with dentures,
treatment of underlying diseases.
Oral Candidiasis (Thrush)
Management: Suspensions, tablets or lozenges of nystatin, amphotericin or
miconazole several times a day for 10–14 days are usually sufficient in acute
cases in children and immunocompetent adult patients. Chronic and unresponsive courses, AIDS patients and chronic mucocutaneous candidiasis usually
demand the use of systemic imidazoles, intermittently if possible, because
of the risk of development of resistance with continuous therapy [36]. Once
resistance has occurred, it can usually be overcome by increasing the dose of
fluconazole (stepwise up to 800 mg daily); however, success is seldom persisting as response to higher doses often is transitory. Alternative azoles (itraconazole, ketoconazole) show modest cross resistance, itraconazole (as a new liquid
formulation in cyclodextrin) being very effective for treatment of primary
disease at a dose of 200 mg daily. Unfortunately, this efficacy obviously cannot
be reached for fluconazole-resistant strains in the long term, so one is left with
toxic amphotericin B at 0.3–0.5 mg/kg intravenously per day initially (omission
once to three times per week). In angular cheilitis cure depends on treatment of
the oral disease and is usually accelerated by additional topical treatment.
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Fig. 5. Oral candidiasis (thrush). Note
the typical white, easily removable patches
of pseudomembranous candidiasis.
Pseudomembranous Candidiasis. This condition is characterized by white
to gray patches (fig. 5) which are easily removed leaving inflamed epithelium,
often accompanied by angular cheilitis (perlèche). In patients without known
predisposing factors, this disease, especially its chronic form, is highly suspicious of HIV infection, often extending to the pharyngeal and esophageal areas,
usually causing retrosternal pain on swallowing.
Erythematous (Atrophic) Candidiasis. No pseudomembranes; the mucosal
surface is inflamed, often associated with local discomfort. In its chronic
variant, bacteria probably play a pathogenic role too, so the use of antiseptics in
addition to antifungal treatment is essential.
Candida Leukoplakia (Chronic Plaque-Like or Hyperplastic Candidiasis).
Here the plaques, most commonly on the cheeks and on the tongue, are not
easily removable and may clear with prolonged antimycotic therapy. This
condition is difficult to differentiate from other types of leukoplakia.
Genital Candidiasis
Candida Balanitis. This infection preferably occurs in the uncircumcised
population. In its mild variant, papules develop to pustules or vesicles with
minimal inflammation and discomfort; in its severe form, these symptoms
aggravate and become persistent, often extending to the prepuce.
Vaginal Candidiasis. This condition, usually accompanied by itch and
discharge, may appear in its acute form or take a chronic relapsing course
presenting a burden for both patient and doctor. No satisfying explanation has
been found for chronic recurrent vaginal candidiasis; an interesting neuropsycho-endocrinological mechanism has been advanced [37], supported by
observations of an association with signs of depression [38] and atopy [39].
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Management: As for treatment, single-dose oral fluconazole 150 mg or
itraconazole 600 mg or single-dosed vaginal antimycotic suppositories (clotrimazole, econazole, isoconazole, nystatin) in combination with antimycotic
topical therapy is generally sufficient; sometimes prolonged therapy for up to
14 days may be necessary. If failure of therapy or relapse occurs, systemic treatment with fluconazole or itraconazole seems to be useful. As C. glabrata and
C. krusei usually are not eradicated by these drugs, high-dose treatment with
fluconazole for 3 weeks is recommended for the former, local therapy for the
latter. Treatment of the asymptomatic sexual partner is no longer regarded as
mandatory [29], whereas examination and appropriate treatment still seems
useful [40] in cases of continuing recurrences. The question of chronic relapsing vaginal candidiasis is left open in the recently established guidelines of the
European Dermatology Forum [29]; fluconazole 150 mg once a month after
menstruation or once a week as a possible maintenance therapy after initial
eradication [41] seems to be worth trying in certain cases, however leading to
relapse in half of the patients after stopping. Another approach is itraconazole
400 mg monthly [42] which is able to reduce the relapse rate but fails after
cessation of therapy.
Scrotal and Perianal Candidiasis. These manifestations usually accompany genital disease, but may also develop independently. On the scrotum
candidiasis often presents as erythema.
Candida Paronychia and Onychomycosis
Interdigital candidiasis and candida paronychia are connected with frequent
immersion of the hands in water leading to painful swelling of the nailfolds.
Discharge of pus is often associated with bacterial coinfection. The invasion
may also spread to the nails causing onycholysis, even if this seems to be a rare
occurrence in temperate climates [43]. Other causes of candida onychomycosis
are Cushing’s disease and Raynaud’s disease; it also appears in connection with
chronic mucocutaneous candidiasis. In the course of diagnostic procedures, a
dermatophyte isolated in culture is considered the cause of onychomycosis,
whereas C. albicans is envisaged as a possible pathogen. Other Candida spp.
may be contaminants requiring reculture [44].
Management: In paronychia, prolonged local therapy with polyene or imidazole lotions combined with drying and improvement of circulation are helpful,
chronic cases often demand the additional use of topical steroids. For onychomycosis, and perhaps also for paronychia, systemic imidazoles are promising.
Congenital Candidiasis
Generalized cutaneous candidiasis of newborns, mostly of mothers who
suffered from vaginal candidiasis prior to delivery, often is associated with
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prematurity or intrauterine contraceptive devices. It usually starts on the face
and chest and becomes generalized over the next days. Sometimes pulmonary
involvement has to be differentiated from Candida sepsis, which seldom
involves the skin.
Management: Topical antimycotic therapy is sufficient for skin infection,
whereas systemic infection demands systemic therapy with amphotericin B
and/or fluconazole.
Chronic Mucocutaneous Candidiasis
Rare syndrome of unknown origin (possibly based on the inability to
develop effective cell-mediated immune responses against Candida [45]) with
chronic oral (pseudomembranes), cutaneous (crusted plaques called Candida
granuloma) and nail (dystrophy) candidiasis, mostly in children (usually with
extensive immunologic defects), but also in adults. This condition should be
differentiated from severe candidiasis among plenty of other infections due
to a known underlying compromised immune system (it seems important not to
forget HIV infection in adults presenting with chronic candidiasis), whereas in
patients with chronic mucocutaneous candidiasis (CMC), the candidiasis of
skin and mucous membranes represents the dominant clinical feature. Despite
the existence of overlaps, differentiation into five clinical forms has proven
helpful: a mild autosomal recessive form, a more severe autosomal dominant
variant, which perhaps includes a third form (diffuse CMC) with the most
severe course (often bronchiectasis). The fourth variant is associated with
endocrinopathy, mostly familial polyendocrinopathy syndrome, candidiasis
sometimes preceding the endocrine disorder by 10 years, at the moment including a recently described form associated with hypothyroidism which may be
transmitted in an autosomal dominant way. The last subtype, late-onset CMC,
may be associated with thymoma. Association with vitiligo, pernicious anemia,
ovarian failure and lupus erythematosus has been reported. Patients usually
present other infections (e.g. warts). No underlying disease is found in most
cases, although investigations (endocrine screening tests, immune parameters,
chest X-ray to exclude thymoma) are recommended as well as follow-up investigations. Prognosis is variable, spontaneous remissions occur.
Management: Prolonged and repeated systemic therapy with fluconazole,
itraconazole or ketoconazole – always with the risk of development of resistance – is crucial, treatment of an underlying endocrine disorder has no effect
on candidiasis. Genetic counseling is indicated.
Other Forms of Candidiasis
Interdigital candidiasis mainly affects persons with continuous water
contact and is characterized by macerated skin with erosion in the toe or finger
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web spaces. Candida intertrigo (flexural candidiasis) is generally easily recognized by the satellite lesions, which are also typical for candida-induced nappy
(or napkin, diaper) candidiasis, usually a secondary infection of irritant
eczema. Gastrointestinal involvement should be considered.
Management: For intertrigo, local antimycotic therapy has to be combined
with drying of this region (e.g. linen strips, use of an antimycotic paste), local
therapy with potassium permanganate deals with this aspect and is effective
against concomitant bacteria as well. In the treatment of diaper candidiasis
these points of view are important as well (e.g. frequent changes of napkins);
steroids should be avoided if possible.
Subcutaneous Mycoses
This group of mycoses is characterized by traumatic inoculation of
geophilic fungi and is seen mainly in tropical and subtropical climates.
Sporotrichosis
Geography: Europe (rare), Americas, Australia, Japan, South Africa.
As the dimorphic fungus Sporothrix schenckii is a geophilic species found
in soil and on plants, persons handling those are predisposed to acquire this
chronic infection (‘drunken rose gardener syndrome’) which primarily affects
the cutaneous and subcutaneous tissues (‘fixed type’) as well as the adjacent
lymphatics (‘lymphangitic type’), forming nodule after nodule along the draining vessels. Prillinger et al. [46] have shown that S. schenckii isolates from soil
are genotypically different and belong to Sporothrix albicans. Sometimes, the
infection spreads to joints, bones and muscles as well as to the lungs, central
nervous system or genitourinary tract (‘disseminated type’). Diagnosis usually
is made by culture; histopathology (as a dimorphic fungus Sporothrix develops
the yeast form in tissue, often surrounded by eosinophilic asteroid bodies)
is less important because of the small number of fungal elements found in
biopsies [47]. Therapy with itraconazole 100 mg daily for weeks according to
clinical response is often successful; potassium iodide used to be the therapy of
choice. Heat therapy (42 °C for 30 min twice a day) may be tried in early and
solitary skin lesions, as S. schenckii does not grow above 38.5 °C [48].
Chromoblastomycosis
Geography: South-East Asia, Africa, Central and South America.
This chronic infection by dematiaceous fungi typically presents with
verrucous, slowly growing lesions on the lower legs and hands. Diagnosis is
made by light microscopy, culture and histopathological demonstration of
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thick-walled and pigmented ‘sclerotic bodies’ in scrapings or biopsies. Wide
excision of an early lesion is considered the simplest successful therapy [49].
Itraconazole 100 mg daily may be the therapy of choice, sometimes in combination with flucytosine. Other possibilities include amphotericin B and flucytosine, flucytosine alone, thiabendazole and local heat.
Mycetoma (Madura Foot)
Geography: Middle East, India, Africa, northern parts of South America.
This chronic subcutaneous infection (mycetoma after Greek mykes for fungus
and oma for tumor, meaning tumor produced by fungi [50]) may be caused by
fungi (eumycetoma) or by bacteria, namely Actinomycetales (Actinomycetoma)
which resemble fungi macro- and microscopically but have the characteristic
features of bacteria on a cellular level [51]. After much controversy about
nomenclature [52] these are the commonly used terms today. The typical
clinical picture is a swollen foot (rarely hand) with abscesses, which painfully
rupture and give birth to red, black and white grains. Black grains give a first
hint that the infection most likely is fungal. Immediate direct examination of a
grain with KOH is a reliable method to distinguish between mycotic (hyphae
visible) and bacterial mycetoma, a decision of some importance as actinomycetoma often heals under antibiotic therapy, whereas eumycetomas frequently
require surgery. Reasons for this common failure of antimycotic therapy probably are adaptive changes by the organisms, like cell-wall thickening or pigmentation [53]. Ketoconazole 200–400 mg daily, griseofulvin or itraconazole may be
tried. X-ray of the feet often reveals lytic bone lesions. Histopathology consolidates the diagnosis.
Subcutaneous Zygomycosis
Zygomycetes rarely lead to infections in tropical countries, causing
systemic (see systemic mucormycosis) or a localized, disfiguring disease.
Therapeutic efforts include surgery, potassium iodide and azoles.
Entomophthoromycosis
Basidiobolus haptosporus and Conidiobolus coronatus, an insect
pathogen, both lead to painless, wooden, slowly extending swellings, the first
mainly on the limbs, whereas the second is typically restricted to the nasal submucosa and the face, where the swellings usually assume bizarre dimensions.
Localized Mucormycosis
The same agents as in systemic mucormycosis may lead to a subcutaneous,
localized variant of mucormycosis, presenting with plaques, pustules, abscesses,
ulcerations and finally skin necrosis.
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Lobomycosis
Reports of this chronic granulomatous skin infection in man mostly come
from the Brazilian Amazon valley, where 21% of the cases reported in the
medical world literature occur in an Indian tribe obviously highly predisposed
to this disease [54]. The bottle-nosed dolphin is another host. Affected are
people living in tropical rain forests; the infection is sometimes attributed to
contact with dolphins. Attempts to culture the pathogenic fungus Loboa loboi
never succeeded. Clinically, keloidal or smooth skin nodules appear on legs and
trunk to a various extent. Histopathology reveals a dense infiltrate of ovoid or
lemon-shaped organisms. Excision may be successful.
Rhinosporidiosis
The majority of cases of this chronic granulomatous disease of the mucosal
membranes have been reported from the southern parts of India and from
Sri Lanka, others come from equatorial Africa, Europe and the Americas.
Clinical findings show inflamed masses with white structures in the nasal
cavities leading do nasal bleeding and obstruction. Other affected areas include
the sinuses, the (palpebral) conjunctiva, and the oral, penile and vaginal mucosa.
Rare skin lesions may stretch out from the conjunctiva; a disseminated cutaneous form has been reported [55]. Incubation and routes of infection remain
unknown, minor trauma has been incriminated as a trigger. Diagnosis is confirmed by the presence of spherules staining with mucicarmine in biopsies. By
continuing divisions, the spherules grow until about 2,000 sporangiospores are
contained in the sporangium, leading to rupture. Attempts to culture the fungus
have not been successful. Regarding therapy, electrosurgery, cryotherapy, ketoconazole and sulfones have been tried.
Systemic (Deep) Mycoses Caused by Pathogenic Fungi
Systemic mycoses are caused by dimorphic fungi (growing as molds at
26 °C and as yeasts at 37 °C, i.e. during invasion of the human body) and are
usually acquired by inhalation of infectious conidia, thus leading to subclinical
or flu-like infections or to primary acute illness [56]. Exposure is often demonstrated by positive skin testing only. Skin lesions may develop at a later stage of
dissemination and often lead to diagnosis of the systemic infection, as they are
particularly well suited for inspection and further microscopic examination,
which usually provides the diagnosis. Coincidence of both erythema nodosum
(as a marker of positive outcome) and/or erythema multiforme has been
reported in acute disease. Very rarely traumatic implantation of the pathogen
(e.g. laboratory accidents) causes local painless nodules or ulcers with regional
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lymphadenopathy (primary cutaneous disease), healing without treatment after
a few months.
The following short overview tries to present some clinical features of skin
involvement in these deep mycoses.
Coccidioidomycosis
The soil inhabitant Coccidioides immitis is endemic in semiarid areas of
the South-West of the USA (‘San Joaquin Valley fever’), Central and South
America. Its conidia are dispersed by earth-moving activities or simply the dust
of unpaved streets, causing pulmonary infection by inhalation, usually asymptomatic or appearing as a mild upper respiratory infection. A small percentage
of infected patients develop chronic pulmonary disease, and in about only 1%
dissemination produces rather nondistinct (papules, pustules, plaques, ulcers,
nodules, abcesses) as well as typical solitary large, warty skin lesions. Facial
involvement seemed to be connected to meningeal disease in a retrospective
study [57]. Serology may be helpful in the diagnosis, although there may be
negative results even in disseminated disease. Effective drugs include amphotericin B, ketoconazole, fluconazole and itraconazole.
Paracoccidioidomycosis
Habitat of Paracoccidioides brasiliensis is the soil of semitropical areas
along rivers and agricultural areas in Latin America. In children, acute dissemination causes severe illness, often associated with pustules or subcutaneous
abscesses. In adults (mainly males) – usually many years after an asymptomatic
primary pulmonary infection – chronic disseminated disease leads to mucocutaneous lesions (ulcerating papules and pustules, subcutaneous cold abscesses,
sometimes scrofuloderma-like manifestations), especially around nose or mouth
in about half of the patients. Itraconazole is the drug of choice.
Histoplasmosis
Two forms may be differentiated by pathogen, geographical distribution
and clinical features. The most common form of histoplasmosis is caused by
Histoplasma capsulatum, worldwide inhabitant of bird roosts and the guano of
bat caves (‘typical disease of speleologists’). Depending on the amount of
inhaled conidia and fitness of the immune system of the host, most infected
adults remain asymptomatic, children often presenting with a more severe
course. Cutaneous lesions appear in about 5% (up to 25% in immunocompromised patients) and again present as unspecific papules, pustules, cutaneous
and subcutaneous nodules, plaques, granulomas, nodules, exfoliative erythroderma, eczematous changes and lesions similar to molluscum contagiosum, the
most characteristic manifestation being painful oropharyngeal mucocutaneous
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ulcers. Again, amphotericin and azole compounds (especially itraconazole) are
used in treatment, which should be initiated as quickly as possible.
Histoplasma capsulatum var. duboisii causes African histoplasmosis, skin
manifestations being the prominent sign in this variant. Three patterns may be
differentiated clinically: superficial (ulcerating papules or nodules, infiltrated
plaques, ‘target phenomenon’), subcutaneous granulomas (first hot and tender,
then cold, rupture) and osteomyelitic lesions, the latter often demanding surgical procedures combined with amphotericin B and ketoconazole.
Blastomycosis
Blastomyces dermatitidis is highly endemic in river banks of most eastern
states of North America, and there are worldwide reports of this infection
including Africa, India and Europe, where its rare appearance is most probably
related to contact with fomites. Skin lesions are a very common sign of chronic
disease and appear as either pustules developing to ulcers or papules growing
to verrucous lesions over months. African blastomycosis is characterized by
bone infection and draining sinuses, traumatic implantation presenting as a
chancroid-like lesion. Treatment: amphotericin B is used for severe acute disease,
otherwise ketoconazole, fluconazole or itraconazole have been effective.
Systemic (Deep) Mycoses Caused by Opportunistic Fungi
Patients with solid organ transplants tend to suffer from oral candidiasis and
cryptococcosis, whereas neutropenic patients (chemotherapy because of cancer
or bone marrow transplantation) are susceptible to aspergillosis, systemic candidiasis or mucormycosis. AIDS patients – as a general sign of their defective
immune system – present with more severe courses of infectious diseases in
general, including mycoses like candidiasis, dermatophytosis or seborrhoic
dermatitis.
The opportunistic pathogens rarely disseminate to the skin, even if skin
manifestations of cryptococcosis are not that uncommon since the appearance
of the AIDS pandemic. Diagnosis may be consolidated by biopsy.
Candidiasis
Oral candidiasis – besides other predisposing factors – is a common disease
in the immunocompromised patient and should remind clinicians of the possibility of HIV infection. It is often associated with esophageal manifestations.
Relapsing or chronic persistent vulvovaginal candidiasis has been reported in up
to 50% of HIV-infected women, therefore some authors recommend HIV testing
in this condition too [58]. Resistance to antifungal drugs is a common problem
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with candidiasis and other fungal infections [36]. In contrast to these two conditions, systemic candidiasis is not a disease of AIDS but of neutropenic cancer
patients, intensive care patients and intravenous drug abusers [59]. Fever and
muscle tenderness may accompany the appearance of macules changing to
papules (with pale centers) and nodules, abscesses and purpuric lesions, follicular pustules being a typical sign of septicemia in heroin abusers. Janeway lesions
may be seen in candida endocarditis though rarely. Chronic disseminated
(hepatosplenic) disease is characterized by abdominal pain. If skin lesions occur,
which is the case in a minority of septicemic patients only, evidence of yeast
cells in skin biopsies allows a rapid diagnosis. As blood cultures are not very
sensitive, serodiagnosis in this setting offers some diagnostic help.
Cryptococcosis
The typical habitat of Cryptococcus neoformans (appearing in two
varieties) is soil with pigeon droppings. Despite this fact, pigeon breeders do
not show any occupational predisposition to the disease [60]. Infection usually
occurs via the respiratory tract, leading to systemic disease (mainly CNS
manifestations) in patients with severe underlying disease, systemic corticoid
therapy and is especially common in AIDS patients in whom the symptoms of
meningitis will often be muted (e.g. absence of a stiff neck because of minor
inflammatory reaction of the meninges). Skin lesions present as nodules, ulcers
or molluscum-contagiosum-like lesions and occur in up to 10% of the cases,
genital ulcerations may be found in up to 40% of the cases. Primary cutaneous
cryptococcosis is rare, but is regularly reported in HIV-positive and -negative
patients. Diagnosis may be made by light microscopy with Indian ink revealing
the encapsulated yeast and confirmed by growth of brown cultures on melaninprecursor-containing agars and by urease production. A rapid latex agglutination test and ELISA are available. Aggressive treatment with amphotericin B
and/or flucytosine may be followed by maintenance therapy with fluconazole or
itraconazole (which are sufficient in the primary cutaneous form). Studies on
triple antimycotic therapy in HIV patients have been published [61].
Systemic Zygomycosis
There is still some controversy on terminology depending on whether
precedence is given to mycological or clinical aspects. Systemic zygomycosis
is a very rare and potentially fatal disease of neutropenic, diabetic, nephropathic and burn patients, the latter being especially prone to skin manifestations
(i.e. necrosis). Pathogens include Rhizopus spp., Absidia corymbifera,
Rhizomucor pusillus, Mucor spp. and Cunninghamella bertholletiae. The most
common clinical manifestation is the rhinocerebral form (often presenting as an
erythematous induration with central purple-black discoloration), cutaneous
Hawranek
154
lesions may be primary (rapid change in morphology from papules and pustules
to necrosis) or secondary in the course of hematogenous dissemination (reddish
to purplish subcutaneous nodules). Vectors for transmission in primary cutaneous zygomycosis may include cloth tape securing endotracheal tubes [63] or
wooden tongue depressors [64]. Diagnosis by autopsy or biopsy, treatment with
lipid-associated amphotericin B.
Aspergillosis
Besides causing allergies and mycotoxicosis, Aspergillus (named by the
Italian priest Micheli in 1729 after a church instrument called aspergillum used
to sprinkle holy water during religious ceremonies [65]) species (Aspergillus
niger, Aspergillus flavus, Aspergillus fumigatus, Aspergillus terreus and others)
typically colonize preformed cavities (aspergilloma) or cause granulomatous
disease of inner organs (e.g. bronchopulmonary aspergillosis). On rare occasions, they initiate systemic and even fatal disease (invasive aspergillosis).
Infection occurs by inhalation or inoculation of spores from soil, water and
decaying plants. The most common manifestations are the pulmonary form and
sinusitis. Cutaneous embolization leads to erythematous, indurated plaques,
hemorrhagic bullae and ulcers; other lesions are described as abscesses and of
molluscum-like appearance [66]. Primary cutaneous aspergillosis has been
reported with the application of contaminated dressings to preexisting wounds,
presenting as ulcerating nodules sometimes leading to hematogenous dissemination. Rare cases have been reported in neonates [67] and in HIV patients [68].
Diagnosis is obtained by biopsy and culture; blood cultures are rarely positive.
The prognosis is poor in spite of therapy with amphotericin B and/or flucytosine. Itraconazole has been tried with good results. Prevention (laminar flow, air
filtration, amphotericin B nasal spray and aerosol) may be successful.
Pseudallescheriasis
Pseudallescheria boydii is a geophilic fungus causing mycetoma and may
be a rare cause of pulmonary infections (especially in immunocompromised
patients), infections of the CNS and inner organs as well as ear and eye infections.
There has been a recent report on an unusual case of cutaneous pseudallescheriasis with pustules and erythema resistant to azole treatment [69].
Rare Mycoses
Hyphomycosis
Many species of the vast group of ubiquitous hyphomycetes (i.e. mycelial
fungi) show the same tissue morphology and host response in the very rare
Cutaneous Mycology
155
human cases. Whereas pathogens with distinctive features are assigned to separate entities, many of these fungi are categorized into two main artificial
groups, the pheohyphomycetes (containing brown pigment) and the hyphomycetes (with colorless walls), each group containing examples of completely
unrelated fungi.
Pheohyphomycosis
According to definition, these so-called dematiaceous fungi contain
melanin in their cell walls which sometimes (e.g. S. schenckii) may be made
visible only by specific melanin stains [70]. Regarding the frequency of infections, the major pathogens of more than 100 species involved are Bipolaris
spicifera and Exophiala jeanselmei, others include Curvularia spp. Examples
of distinct clinical entities are mycetoma, sporotrichosis, chromoblastomycosis,
black piedra and tinea nigra, ranging from superficial to systemic disease.
Subcutaneous infection often presents with a solitary acral abscess attached to
the skin, but without connection to the underlying tissue. Fistulas as well as
dissemination may complicate the course in immunocompromised individuals,
initial lesions often presenting as firm subcutaneous, indurated lesions. Besides
surgical excision in subcutaneous disease, therapeutic attempts with mostly
disappointing results included amphotericin B, flucytosine, miconazole, ketoconazole and itraconazole, the latter with promising effectiveness.
Hyalohyphomycosis
Species included are Penicillium, Beauveria, Acremonium, Fusarium
and Scopulariopsis, with an everexpanding list of organisms involved in human
disease.
Protothecosis
The achloric algae Prototheca wickerhamii and Prototheca zopfi, although
not belonging to the fungi, produce yeast-like colonies on Sabouraud’s agar and
are often included with the fungi [54]. The usually verrucous lesions most
commonly are observed in immunocompromised patients as an opportunistic
infection, and extracutaneous manifestations of the disease are not rare. The
histopathologically typical structure is the so-called morula, daughter cells
within a theca resembling a soccer ball. Therapy with excision or with combinations of an oral tetracycline with topical amphotericin B may be successful.
Penicillium marneffei Infection
This dimorphic fungus, which does not fit into the definition of hyalohyphomycosis, causes disease in bamboo rats of Southeast Asia. Its relation to
human disseminated mycosis in immunocompromised as well as healthy persons
Hawranek
156
is not clear; most cases have been reported from Thailand where it is one of the
most common AIDS-associated diseases. The skin lesions, which occur in about
50% of the patients, present as papules, ulcers or resemble molluscum contagiosum, and thus have to be distinguished from cryoptococcosis and histoplasmosis by biopsy. Blood cultures are positive in about three fourths of the
patients. Because of the high mortality, treatment is necessary. Drugs of choice
are itraconazole or ketoconazole for mild to moderate cases and amphotericin B
for severe cases. Maintenance therapy may be considered with the azoles [66].
Laboratory Diagnosis
For reasons of limited space and the general focus on clinical aspects in
this review, not too much emphasis is laid on technical details, for these, see a
reference book [3] or review article [71].
Collection of Material
Before sampling, the lesions should be cleansed with 70% alcohol if
covered with ointment. For skin scales, specimens should be gathered with a
sterile blunt scalpel from the edge of a lesion (or the roof of a blister); the best
transport medium is folded black paper. Nail specimens should be collected
from under the edge of the nail with a raspatory (as proximal as possible),
debris in paronychia may be gained by the gentle use of a dental probe. Hair
(roots) can easily be epilated with an epilation forceps. If T. verrucosum is
suspected, it may be necessary to remove many hairs. For the diagnosis of tinea
capitis a brush is pressed on the agar after brushing the hair ten times at least,
a screening method most valuable in school epidemics. Specimens from mucous
membranes are sampled by swabs and should be examined immediately, as
yeasts do not survive for long outside a transport medium.
Direct Examination (Potassium Hydroxide Test)
The specimen is immersed in 10% potassium hydroxide dissolving the
keratin but leaving fungal elements. The microscopic picture may be enhanced
by staining with an equal amount of Parker’s ‘Quink’. Nail material usually
needs about 10 min (about 1 h has recently been recommended [29]) to get
soft enough to be flattened (the thinner the sample, the easier the detection
of fungus; gentle warming may help), hair starts to disintegrate after a few
Cutaneous Mycology
157
minutes. Fungal structures are first searched for at low magnification (⫻100),
then examined at higher magnification (⫻400). This test does not allow the
diagnosis of a specific dermatophyte; a negative result does not rule out mycosis. The superficial mycoses, especially pityriasis versicolor, and – to a lesser
degree – S. brevicaulis may be satisfactorily diagnosed by direct examination
alone.
The fluorochromes, Calcofluor White and Congo red bind to chitin and
cellulose, while acridine orange binds to nucleic acids of the fungi. These dyes
may be helpful when there is a lack of fungal elements (blastospores, hyphae
or pseudohyphae) in the specimen, as even few elements are visible under the
fluorescence microscope. By using specific antibodies conjugated with the
fluorochromes, it should be possible to identify specific fungi.
Culture and Additional Tests
The specimen is inoculated onto the culture medium, usually Sabouraud’s
dextrose agar without and with added antibiotics, e.g. actidione for elimination
of contaminating molds and chloramphenicol for suppression of bacterial
growth. If infection with the mold S. brevicaulis or yeasts is suspected, actidion
should not be added. One culture should be incubated at about 26–28 °C for the
propagation of dermatophytes, the other at 37 °C for yeasts. Most dermatophytes need approximately 1–2 weeks for growth; yeasts grow within 1–2 days
(slimy little colonies very similar to bacterial cultures). If there is no growth
after 4 weeks, cultures may be reported as negative.
The cultures are examined for macroscopic features such as color, surface
and growth rate (table 2). An easy and good method of preparation for microscopic study is to bring some material, peferably from the margin of the culture,
on a glass slide by touching the culture with a sticky tape and mounting it on
the slide after putting a drop of lactophenol cotton blue on it. The mount
may then be examined looking for unique morphologic features (table 2). As a
general rule, macroconidia of Microsporum spp. have rough, thick walls,
whereas those of Trichophyton and Epidemophyton spp. are smooth-walled and
clavate or broadly clavate. Microconidia of Microsporum spp. are typically
pear-shaped and single-standing along the hyphae, whereas the microconidia of
Trichophyton spp. are pear-shaped to spherical, numerous and tend to build
clusters. They are absent in Epidermophyton spp. Distinctive details of conidia
(fig. 6) are best searched for by performing slide cultures. A little piece of agar
is inoculated with the fungus on four sides and incubated at 25–30 °C in a moist
atmosphere between a mount and a cover slip until sufficient growth becomes
apparent, a method also used to set up permanent preparations.
Hawranek
158
Fig. 6. Slide culture. A definitive diagnosis is usually made by distinctive microscopic
features, such as the typical spiral hyphae of T. mentagrophytes var. interdigitale.
If sporulation is insufficient, the same procedure may be repeated with
cornmeal agar or potato agar, which is sometimes successful. If this fails too, a
number of additional tests exist for the identification of dermatophytes, including urea hydrolysis, in vitro hair perforation test, growth on polished rice
grains, temperature enhancement or tolerance tests as well as numerous tests
for the detection of special nutritional requirements, e.g. thiamine, niacin.
Histopathology, Serology, Identification of Yeasts and Molds
Histopathology (hematoxylin-eosin stain, periodic acid-Schiff stain,
Grocott’s methenamine-silver stain) may be helpful in the diagnosis of dermatophytosis and plays an important role in the diagnosis of subcutaneous mycoses.
Serology: The detection of specific antibodies does not play any role in the
diagnosis of superficial or cutaneous mycoses, but has its place in systemic
candidiasis, aspergillosis and especially cryptococcosis, histoplasmosis and
coccidioidomycosis.
Identification of yeasts: As dermatophytes, yeast colonies are examined for
macroscopic and microscopic criteria. C. albicans, the most prominent fungus
of this group, may be identified by the presence of terminal chlamydospores on
rice agar and of germ tubes after 2 h of incubation in serum at 37 °C (‘germ tube
test’). Besides classical tests for the specific identification of yeasts [3], several
kits working with biochemical reactions are commercially available.
Identification of molds: As macroscopic and microscopic examination of
colonies is the only way of identifying molds at the moment, special attention
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159
Table 3. Aspects of antifungal drug therapy
Substance
Appl.
Some characteristics
Some side effects
Some interactions
Imidazoles
Miconazole
top., i.v.
ind.: broad spectrum (excl. aspergillus)
antifungal agent; no therapy of choice
i.v.: tachycardia, seizures, anemia;
top.: irritation, contact dermatitis
i.v.: oral antidiabetics↑,
coumarin↑, phenytoin↑
Econazole
top.
ind.: most cutaneous mycoses
irritation, contact dermatitis
–
Ketoconazole
top., o.
ind.: most cutaneous mycoses, some
systemic mycoses; because of its
potential hepatotoxicity has been
replaced widely by the newer
antifungal agents
hepatotoxicity, inhibition of
steroidogenesis and other
endocrinologic side effects,
impotence
insulin-sparing effects,
cyclosporin↑, coumarin↑,
disulfiram-like reactions
with alcohol; cave
terfenadine and
astemizole (cardiac
arrhythmia)
Itraconazole
o.
ind.: most cutaneous, subcutaneous
and systemic infections; pulse regimen
possible; fungostatic, lipophilic drug
with extensive tissue distribution;
should be taken with meals
nausea; hepatotoxicity (rare);
hypokalemia, edema,
gynecomastia, impotence with
higher doses; embryotoxicity
may ↑ levels of oral
antidiabetics, coumarin,
digoxin, phenytoin,
cyclosporin
Fluconazole
o., i.v.
ind.: most cutaneous and systemic
(candidiasis, cryptococcosis) mycoses
gastrointestinal side effects
(common), hepatotoxicity (rare),
exfoliative dermatitis, thrombo-,
leukopenia
may ↑ levels of oral
antidiabetics, coumarin,
phenytoin, cyclosporin
top.
ind.: cutaneous dermatophyte and yeast
infections; fungicidal and
antiinflammatory
local irritation, contact dermatitis
–
Triazoles
Allylamines
Naftifine
Hawranek
160
Terbinafine
o., top.
ind.: very effective for dermatophyte
infections given orally, low effectivity
against yeasts (only fungistatic against
C. albicans) and molds; fungicidal (!),
few relapses
gastrointestinal side effects, loss
of taste, hepatitis, erectile
dysfunction, Stevens-Johnson
syndrome
no influence on clearance
of cytochrome
P450-dependent drugs, but
is affected by inhibitors
or inducers of this system
Amphotericin B
i.v., top.
ind.: Candida, Aspergillus and most
systemic mycoses, presumed fungal
infections in febrile neutropenic
patients; first systemic antifungal drug
(1960); less toxicity with new lipid
complex and liposomal formulations;
pre- and post-infusion hydration
advisable
i.v.: chills (50%), fever (33%),
renal toxicity (up to 20%),
hypokalemia, hypomagnesemia,
anemia
avoid other nephrotoxic
drugs (cyclosporine,
aminoglycosides etc.);
hypokalemia may be
deteriorated by steroids
Nystatin
o., top.
ind.: Candida
o.: occasional gastrointestinal side
effects with high doses
–
Natamycin
top.
ind.: Candida, dermatophytes
irritation
–
o.
ind.: dermatophytosis in children (and
adults), still considered as the ‘gold
standard’ for tinea capitis; not effective
against yeasts; good safety profile; to
be taken with fat-containing food,
improved absorption by micronization;
blood count and liver function tests
possibly unnecessary in healthy
children
gastrointestinal symptoms,
headaches, arthralgias,
photosensitivity, aggravation of
lupus erythematosus, allergic
reactions (up to 7%), side effects
of the nervous system
coumarin levels ↑,
cyclosporin ↓;
barbiturates lead to
griseofulvin ↓; possible
interference with
anticonceptive pills;
disulfiram-like reactions
with alcohol
Polyenes
Miscellaneous
Griseofulvin
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161
Table 3. Continued
Substance
Appl.
Some characteristics
Some side effects
Some interactions
Amorolfine
top.
ind.: most cutaneous mycoses; nail
lacquer for onychomycosis
local irritation (burning, itch etc.)
–
Tolnaftate
top.
ind.: dermatophytosis (not T. rubrum)
local irritation, contact dermatitis
–
Cyclopiroxolamine
top.
ind.: most cutaneous mycoses
local irritation
–
Flucytosin
i.v., o.
ind.: systemic mycoses, given in
combination with amphotericin B;
resistance when used as monotherapy
bone marrow toxicity,
nephrotoxicity, hepatotoxicity
avoid other nephrotoxic
drugs; cytarabin as
antagonist
Appl = Mode of application; i.v. = intravenously; o. = orally; top. = topically; ind.: indication.
Hawranek
162
has to be paid to the texture of the colony as well as to the type of conidia
and especially the way these conidia are borne on the conidiophores. This may
be achieved by a sticky tapemount, but the best method to preserve special
conidial arrangements without the conidia becoming detached is slide culture
(see above).
Principles of Therapy
Fungi in the stratum corneum may be attacked by keratolysis (⫽ elimination of the pathogen including the site of infection), e.g. by Whitfield’s ointment
or topicals containing salicylic acid, or by broad-spectrum antiseptics (mostly
dyes like gentian violet or sulfide-containing creams). The third approach
involves specific antifungal substances administered topically or systemically.
Table 3 gives some characteristic information on antifungal agents, for detailed
information see review articles [72–74]. Four groups may be differentiated, all
characterized by different modes of action: the allylamines and azoles blocking
ergosterol formation in the cell wall in different ways, the polyenes destroying
the cell membranes, griseofulvin blocking the intracellular microtubules and
flucytosine blocking DNA and RNA synthesis.
References1
1
2
3
4
5
6
7
8
9
10
11
Clayton Y, Midgley G: Pocket Picture Guide to Medical Mycology. London, Gower, 1985.
Crissey JT, Lang H, Parish LC: Manual of Medical Mycology. Oxford, Blackwell, 1995.
Evans EGV, Richardson MD: Medical Mycology – A Practical Approach. Oxford, Oxford
University Press, 1989.
Hay RJ: Fungi and Skin disease. London, Gower, 1993.
Kwon-Chung KJ, Bennett JE: Medical Mycology. Philadelphia, Lea & Febiger, 1992.
Rippon JW: Medical Mycology – The Pathogenic Fungi and the Pathogenic Actinomycetes, ed 3.
Philadelphia, Saunders, 1988.
Roberts SOB, Hay RJ, Mackenzie DWR: A Clinician’s Guide to Fungal Disease. New York,
Marcel Dekker, 1984.
Hay RJ, Moore M: Mycology; in Champion RH, Burton JL, Burns DA, Breathnach SM (eds):
Textbook of Dermatology, ed 6. Oxford, Blackwell, 1998, pp 1277–1376.
Martin AG, Kobayashi GS: Superficial fungal infection: Dermatophytosis, tinea nigra, piedra; in
Fitzpatrick TB, Eisen AZ, Wolff K, Freedberg IM, Austen KF (eds): Dermatology in General
Medicine, ed 4. New York, McGraw-Hill, 1993, pp 2421–2451.
Ginter G, Rieger E: State of the art in diagnosis and treatment of cutaneous mycoses. Acta
Dermatoven APA 1996;5:3–13.
Martin AG, Kobayashi GS: Yeast infections: Candidiasis, pityriasis (tinea) versicolor; in
Fitzpatrick TB, Eisen AZ, Wolff K, Freedberg IM, Austen KF (eds): Dermatology in General
Medicine, ed 4. New York, McGraw-Hill, 1993, pp 2421–2451.
Cutaneous Mycology
163
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
Shadomy HJ, Utz JP: Deep fungal infections; in Fitzpatrick TB, Eisen AZ, Wolff K, Freedberg IM,
Austen KF (eds): Dermatology in General Medicine, ed 4. New York, McGraw-Hill, 1993,
pp 2468–2497.
Prillinger HJ, Lopandic K, Schweigkofler W, Deak R, Aarts HJM, Bauer R, Maraz A: Molecular
phylogeny and systematics of the fungi with special reference to the Ascomycota and Basidiomycota. Chem Immunol. Basel, Karger, 2002, vol 81, pp 207–294.
Rashid A, Richardson MD: Pathogenesis of dermatophytosis; in Aly R, Beutner KR, Maibach H
(eds): Cutaneous infection and therapy. New York, Dekker, 1997, pp 127–137.
Figueroa JI, Hawranek T, Abraha A, Hay RJ: Tinea capitis in south-western Ethiopia: A study of
risk factors for infection and carriage. Int J Dermatol 1997;36:661–666.
Assaf RR, Weil ML: The superficial mycoses. Dermatol Clin 1996;14:57–67.
Faergemann J: Pityriasis versicolor: Current treatments; in Aly R, Beutner KR, Maibach H (eds):
Cutaneous infection and therapy. New York, Dekker, 1997, pp 211–215.
Drake LA, Dinehart SM, Farmer ER, Goltz RW, Graham GF, Hordinsky MK, Lewis CW,
Pariser DM, Skouge JW, Webster SB, Whitaker DC, Butler B, Lowery BJ: Guidelines of care for
superficial mycotic infections of the skin: Tinea corporis, tinea cruris, tinea faciei, tinea manuum,
and tinea pedis. J Am Acad Dermatol 1996;34:282–286.
Elgart ML: Cutaneous mycology – Preface. Dermatol Clin 1996;14(1):xiii.
Frieden IJ: Tinea capitis; in Aly R, Beutner KR, Maibach H (eds): Cutaneous Infection and
Therapy. New York, Dekker, 1997, pp 169–181.
Frieden IJ, Howard R: Tinea capitis: Epidemiology, diagnosis, treatment and control. J Am Acad
Dermatol 1994;31:S42–S46.
Drake LA, Dinehart SM, Farmer ER, Goltz RW, Graham GF, Hordinsky MK, Lewis CW,
Pariser DM, Skouge JW, Webster SB, Whitaker DC, Butler B, Lowery BJ: Guidelines of care for
superficial mycotic infections of the skin: Tinea capitis and tinea barbae. J Am Acad Dermatol
1996;35:290–294.
Roberts DT: The risk/benefit ratio of modern antifungal pharmacological agents; in Aly R,
Beutner KR, Maibach H (eds): Cutaneous infection and therapy. New York, Dekker, 1997,
pp 183–190.
Elewski BE: Tinea capitis: A current perspective. J Am Acad Dermatol 2000;42:1–20.
Aly R: Tinea pedis: Epidemiology, clinical manifestations, pathophysiology, and therapy; in Aly
R, Beutner KR, Maibach H (eds): Cutaneous infection and therapy. New York, Dekker, 1997,
pp 139–148.
Masri-Fridling GD: Dermatophytosis of the feet. Dermatol Clin 1996;14:33–40.
Hay RJ, Baran R, Haneke E: Fungal (onychomycosis) and other infections involving the nail
apparatus; in Baran R, Dawber RPR (eds): Diseases of the Nails and Their Management, ed 2.
Oxford, Blackwell, 1994, pp 97–134.
De Doncker P: Pharmacokinetics in onychomycosis; in Aly R, Beutner KR, Maibach H (eds):
Cutaneous Infection and Therapy. New York, Dekker, 1997, pp 157–168.
Korting HC, Seebacher C, Schaller M, Evans EGV, Ginter G, Tosti A, Dupont B, Nowicki R,
Monod M: Guidelines on mycoses of the skin and bordering mucosal surfaces. European
Dermatology Forum, January 2000 (pers. commun.).
Baran RL, Aly Raza: Diagnosis and new treatments in the management of onychomycosis; in
Aly R, Beutner KR, Maibach H (eds): Cutaneous Infection and Therapy. New York, Dekker, 1997,
pp 149–156.
Tosti A, Piraccini BM, Lorenzi S: Onychomycosis caused by nondermatophytic molds: Clinical
features and response to treatment of 59 cases. J Am Acad Dermatol 2000;42:217–224.
Midgley G, Moore MK, Cook J, Phan QG: Mycology of nail disorders. J Am Acad Dermatol
1994;31:S68–S74.
Denning DW, Evans EVG, Kibbler CC, Richardson MD, Roberts MM, Rogers TR,
Warnock DW, Warren RE: Fungal nail disease: A guide to good practice. Br Med J 1995;311:
1277–1281.
Hay RJ, Moore MK: Clinical features of superficial fungal infections caused by Hendersonula
toruloidea and Scytalidium hyalinum. Br J Dermatol 1984;110:677–683.
Elgart ML: Tinea incognito. An update on Majocchi granuloma. Dermatol Clin 1996;14:51–55.
Hawranek
164
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
Graybill JR: The clinical impact of antifungal drug resistance in patients with AIDS; in
Aly R, Beutner KR, Maibach H (ed): Cutaneous infection and therapy. New York, Dekker, 1997,
pp 233–243.
Hawranek U: Vaginalmykosen – Eine rein somatische Erkrankung?, PhD thesis, Salzburg, 1998.
Irving G, Miller D, Robinson A, Reynolds S, Copas AJ: Psychological factors associated with
recurrent vaginal candidiasis: A preliminary study. Sex Transm Infect 1998;74:334–338.
Moraes PS: Recurrent vaginal candidiasis and allergic rhinitis: A common association. Ann
Allergy Asthma Immunol 1998;81:165–169.
Drake LA, Dinehart SM, Farmer ER, Goltz RW, Graham GF, Hordinsky MK, Lewis CW, Pariser
DM, Skouge JW, Webster SB, Whitaker DC, Butler B, Lowery BJ: Guidelines of care for superficial mycotic infections of the skin: Mucocutaneous candidiasis. J Am Acad Dermatol 1996;
34:110–115.
Sobel JD: Controversial aspects in the management of vulvovaginal candidiasis. J Am Acad
Dermatol 1994;31:S10–S13.
Spinillo A, Colonna L, Piazzi G, Baltaro F, Monaco A, Ferrari A: Managing recurrent vulvovaginal candidiasis. Intermittent prevention with itraconazole. J Reprod Med 1997;42:83–87.
Midgley G, Moore MK: Nail infections. Dermatol Clin 1996;14:41–49.
Drake LA, Dinehart SM, Farmer ER, Goltz RW, Graham GF, Hordinsky MK, Lewis CW,
Pariser DM, Skouge JW, Webster SB, Whitaker DC, Butler B, Lowery BJ: Guidelines of care for
superficial mycotic infections of the skin: Onychomycosis. J Am Acad Dermatol 1996;34:
116–121.
Kirkpatrick CH: Chronic mucocutaneous candidiasis, J Am Acad Dermatol 1994;31:S14–S17.
Prillinger H, Messner R, König H, Bauer R, Lopandic K, Molnár O, Dangel P, Weigang F, Kirisits
T, Nakase T, Sigler L: Yeasts associated with termites: A phenotypic and genotypic characterization and use of coevolution for dating evolutionary radiations in Asco- and Basidiomycetes.
System Appl Microbiol 1996;19:265–283.
Davis BA: Sporotrichosis. Dermatol Clin 1996;14:69–76.
Belknap BS: Sporotrichosis. Dermatol Clin 1989;7:193–202.
Elgart GW: Chromoblastomycosis. Dermatol Clin 1996;14:77–83.
Magana M, Magana-García M: Mycetoma. Dermatol Clin 1989;7:203–217.
Warren NG: Actinomycosis, nocardiosis, and actinomycetoma. Dermatol Clin 1996;14:85–95.
McGinnis MR: Mycetoma. Dermatol Clin 1996;14:97–104.
Hay RJ: A thorn in the flesh – a study of the pathogenesis of subcutaneous infections. Clin Exp
Dermatol 1989;14:407–415.
Elgart ML: Unusual subcutaneous infections. Dermatol Clin 1996;14:105–111.
Thappa DM, Venkatesan S, Sirka CS, Jaisankar TJ, Gopalkrishnan, Ratnakar C: Disseminated
cutaneous rhinosporidiosis. J Dermatol 1998;25:527–532.
Body BA: Cutaneous manifestations of systemic mycoses. Dermatol Clin 1996;14:125–135.
Arsura EL, Kilgore WB, Caldwell JW, Freeman JC, Einstein HE, Johnson RH: Association
between facial cutaneous coccidioidomycosis and meningitis. West J Med 1998;169:13–16.
Conant MA: Fungal infections in immunocompromised individuals. Dermatol Clin 1996;14:
155–162.
Hay RJ: Yeast infections. Dermatol Clin 1996;14:113–124.
Hernandez AD: Cutaneous cryptococcosis. Dermatol Clin 1989;7:269–274.
Kappe R, Levitz S, Harrison TS, Ruhnke M, Ampel NM, Just-Nubling G: Recent advances in
cryptococcosis, candidiasis and coccidioidomycosis complicating HIV infection. Med Mycol
1988;36(suppl 1):207–215.
Elgart ML: Zygomycosis. Dermatol Clin 1996;14:141–146.
Dickinson M, Kalayanamit T, Yang CA, Pomper GJ, Franco-Webb C, Rodman D: Cutaneous
zygomycosis (mucormycosis) complicating endotracheal intubation: Diagnosis and successful
treatment. Chest 1998;114:340–342.
Mitchell SJ, Gray J, Morgan ME, Hocking MD, Durbin GM: Nosocomial infection with Rhizopus
microsporus in preterm infants: Association with wooden tongue depressors. Lancet 1996;348:
441–443.
Isaac M: Cutaneous aspergillosis. Dermatol Clin 1996;14:137–140.
Cutaneous Mycology
165
66
67
68
69
70
71
72
73
74
Myskowski PL, White MH, Ahkami R: Fungal disease in the immunocompromised host.
Dermatol Clin 1997;15:295–305.
Papouli M, Roilides E, Bibashi E, Andreou A: Primary cutaneous aspergillosis in neonates: Case
report and review. Clin Infect Dis 1996;22:1102–1104.
Arikan S, Uzun O, Cetinkaya Y, Kocagoz S, Akova M, Unal S: Primary cutaneous aspergillosis in
human immunodeficiency virus-infected patients: Two cases and review. Clin Infect Dis 1998;
27:641–643.
Ginter G, Petutschnig B, Pierer G, Soyer HP, Reischle S, Kern T, de Hoog S: Atypical cutaneous
pseudoallescheriosis refractory to antifungal agents. Mycoses 1999;42:507–511.
Rinaldi MG: Phaeohyphomycosis. Dermatol Clin 1996;14:147–153.
Weitzman I, Padhye A: Dermatophytes – gross and microscopic. Dermatol Clin 1996;14:9–22.
Gupta AK, Sauder DN, Shear NH: Antifungal agents: An overview. Part I. J Am Acad Dermatol
1994;30:677–698.
Gupta AK, Sauder DN, Shear NH: Antifungal agents: An overview. Part II. J Am Acad Dermatol
1994;30:911–933.
Lesher JL Jr: Recent developments in antifungal therapy. Dermatol Clin 1996;14:163–169.
Dr. Thomas Hawranek, MD, Landeskliniken Salzburg, Department of Dermatology,
Müllner Hauptstrasse 48, A–5020 Salzburg (Austria)
Tel. ⫹43 662 4482 3023, Fax ⫹43 662 4482 3003 E-Mail T.Hawranek@lks.at
1
References are mainly restricted to reviews.
Hawranek
166
Breitenbach M, Crameri R, Lehrer SB (eds): Fungal Allergy and Pathogenicity.
Chem Immunol. Basel, Karger, 2002, vol 81, pp 167–206
Toxins of Filamentous Fungi
Deepak Bhatnagar, Jiujiang Yu, Kenneth C. Ehrlich
US Department of Agriculture, Agricultural Research Service,
Southern Regional Research Center, New Orleans, La., USA
Natural toxins in food can be divided into five main categories: mycotoxins,
bacterial toxins, phycotoxins, plant toxins and zootoxins. The first three are
toxic compounds produced by living organisms, and are formed directly in
food or transferred through the food chain, whereas, the latter two are ‘inherent
components (of plants or animals) that are harmful to humans and animals’ [1].
Toxins are compounds that are toxic or poisonous to living things, phytotoxins are compounds toxic to plants, and zootoxins are compounds toxic
to animals [2]. By the same logic, mycotoxins should be compounds toxic to
fungi. However, these are toxic compounds produced by fungi.
The term mycotoxin is derived from the Greek words ‘’ (fungus)
and ‘’ (arrow-poisons). Most mycotoxins can, therefore, be defined
as natural products produced by fungi that evoke a toxic response in higher
vertebrates and other animals when fed at low concentrations. Mycotoxins
could also be toxic to plants or other microorganisms. However, these compounds are not classified with antibiotics of fungal origin. Biological conversion products of mycotoxins are also called mycotoxins. Mycotoxins have
recently been the subject of many reviews [3–5]. Toxic metabolites produced
by mushrooms and yeast are not included in this review and have been
reviewed by Chu [4]. In this chapter, a comprehensive review has been provided
of the most significant mycotoxins, their fungal origin and their toxic effects.
The reader will hopefully be able to appreciate the diversity of mycotoxins
that have been examined thus far, and will understand the need for research on
several of these toxins that exhibit the potential for harmful effects on animals
and humans.
After cessation of an active growth phase, fungi are known to produce numerous organic compounds called secondary metabolites, which are not required
for the growth of the producing fungus. Mycotoxins are low-molecular-weight,
nonproteinaceous compounds derived primarily from amino acids, shikimic
acid or malonyl CoA, and their effect in animals and humans can be significant.
Mycotoxins are generally produced in the mycelia of filamentous fungi, but
can accumulate in specialized structures of fungi such as conidia or sclerotia as
well as in the environment surrounding the organism. Fungal species that
produce mycotoxins are very diverse. However, some mycotoxins are only
produced by a single fungal species or even by specific strains of a fungal
species or a number of fungal species. The toxic effects of mycotoxins are as
diverse as the fungal species that make these toxins. Some mycotoxins have
acute toxic effects while others have toxic effects after long-term exposure
(chronic effects).
Mycotoxicosis
Mycotoxins can elicit acute toxic, mutagenic, teratogenic and carcinogenic effects, as well as estrogenic effects on animals. The toxic effects of
mycotoxins are termed ‘mycotoxicoses’, and the severity depends on the type
of mycotoxin, the extent of exposure (duration and dose), age, nutritional
status and health of the affected individual, and the synergistic effects of the
mycotoxin with other chemical exposure of the individual. Mycotoxicosis is
usually mediated by damage to cells of all the major organs, most commonly
liver, kidney, lungs and the nervous, endocrine and immune systems.
Mycotoxicosis differs from ‘mycosis’, a term that refers to diseases produced
by direct pathogenic invasion by fungi. Mycotoxicosis can be labeled
acute or chronic depending on the amount of toxin ingested and the length
of exposure, with low doses and long-term exposure leading to chronic
toxicity, and short-term exposure to extremely high doses leading to acute
toxicity.
Over 300 mycotoxins have been identified, but only those implicated in
mycotoxicoses involving humans have been studied in detail. Table 1 provides
a partial list of the best-studied mycotoxins, the fungal source and the reported
human mycotoxicosis associated with each toxin. Although most of these
mycotoxins have been implicated in human illnesses, a direct connection
between the mycotoxin and a corresponding mycotoxicosis has been demonstrated for very few. For example, ochratoxin A, and sterigmatocystin may be
carcinogenic to human beings, but there are insufficient or inconclusive data to
demonstrate a direct effect [6]. Only in the case of aflatoxins, ergot alkaloids,
ochratoxins and to some extent trichothecenes has a relationship between exposure to the toxin and acute and chronic human mycotoxicoses been reasonably
well established.
Bhatnagar/Yu/Ehrlich
168
Table 1. Selected mycotoxins and their human and animal mycotoxicoses
Mycotoxin
Producing fungi
Toxic effect
Mycotoxicosis
Alternaria (tenuazonic
acid, alternariol)
A. alternata
apoptosis
mutagen
onyalai disease
Aflatoxin
A. flavus
A. parasiticus
apoptosis
mutagen
hepatotoxin
carcinogen
teratogen
acute aflatoxicosis,
hepatocarcinogenesis,
childhood
cirrhosis,
Rye’s syndrome
Cyclopiazonic acid
A. flavus
P. aurantiogriseum
nephrotoxin
cardiovascular
lesion
Kodua poisoning
Ergot alkaloids
Claviceps sp.
A. fumigatus
P. chermesinum
neurotrophy
St. Anthony’s fire
ergotism
Moniliformin
F. verticillioides
neurotoxin
cardiovascular
lesion
onyalai disease
Ochratoxin
A. ochraceus
P. niger
A. alliaceus
A. terreus
P. viridicatum
apoptosis
nephrotoxin
teratogen
Balkan nephropathy,
renal tumors
Penicillic acid
P. aurantiogriseum
A. ochraceus
neurotoxin
mutagen
–
Roquefortine
P. roquefortii
neurotoxin
–
Rubratoxin
P. rubrum
P. purpurogenum
apoptosis
hepatotoxin
teratogen
–
Sterigmatocystin
A. versicolor
A. nidulans
A. parasiticus
A. flavus
hepatotoxin
carcinogen
hepatocarcinogenesis
Trichothecenes
(T-2, deoxynivalenol)
F. sporotrichioides
Microdochium nivale
S. atra
apoptosis
dermatoxin
neurotoxin
teratogen
alimentary toxic
aleukia,
Akakabi-byo disease,
stachybotryotoxicosis,
esophageal cancer,
red mold disease,
yellow rain
Zearalenone
F. graminearum
genitotoxin
estrogenic
effects
mutagen
cervical cancer,
premature menarche
Adapted from Bhatnagar et al. [5].
Toxins of Filamentous Fungi
169
Mycotoxicology
Mycotoxicology is the study of the mycotoxins and their corresponding
mycotoxicoses. It is a diverse discipline that encompasses basic sciences,
such as chemistry, biochemistry and molecular biology, and multidisciplinary
subjects, such as plant and animal pathology, toxicology, pharmacology and
immunology.
Natural Occurrence of Mycotoxins
Mycotoxins are found worldwide as contaminants of food. Their occurrence depends on geographic and seasonal factors, as well as cultivation,
harvesting, storage and transportation practices. Mycotoxin production is also
dependent on the factors affecting the susceptibility of the commodity to
contamination, e.g., the genetic, environmental and nutritional status of the
crop [7]. Physical factors such as time of exposure, temperature during exposure, humidity, and extent of insect or other damage to the commodity prior to
exposure determine mycotoxin contamination in the field or during storage.
Mycotoxin-producing or other coexisting fungal species, that may be plant
pathogens, act as biological factors. These fungi can colonize seeds before and
after harvest, thus affecting mycotoxin production based on interactions of
existing fungal populations. Chemical factors including the nutritional status of
the crops or chemicals (such as fungicides) used in crop management can affect
fungal populations, and consequently toxin production. Mycotoxin production
is often very variable from the effects of some or all of these factors.
The temperature and relative humidity optima for mycotoxin production
may differ from that supporting fungal growth. For example, in the field, the
temperature range for Aspergillus flavus and A. parasiticus growth varies from
12 to 48 °C (35–37 °C optimum), and water activity requirement could be as
low as 0.80 (0.95 optimum). However, aflatoxin production by these fungi
requires a narrower range of temperatures (28–33 °C; 31 °C optimum), and
water activity (0.85–0.97; optimum 0.90). In general, mycotoxins are produced
optimally at 24–28 °C, but some toxins such as T-2 toxin are produced maximally at 15 °C. Contamination during crop storage may be affected by changes
in temperature and water activity that allow ecological succession of different
fungi.
Filamentous fungi are ubiquitous in nature, and are found in soils from
temperate and tropical regions. While some of these fungi are pathogenic,
others are not. Mycotoxin contamination has been found in cereal plants,
oilseed crops and grasses [7–12]. Most commodities have been shown to be
Bhatnagar/Yu/Ehrlich
170
susceptible to mycotoxin contamination. A partial list of these commodities is
given in table 2; for an exhaustive list, see Malloy and Mars [6]. Often even
when fungal growth cannot be observed, a large amount of mycotoxins can be
produced.
History of Mycotoxins
Historical instances of mycotoxicoses can be surmised even though identification of a mycotoxin as a causative agent was unknown at the time of occurrence. For example, at least one of the 10 plagues in ancient Egypt recorded in
Exodus could have been associated with mycotoxin contamination of food
[13, 14]. The ‘Plague of Athens’ in approximately 430 BC could be attributed
to intake of fungal-contaminated food [15]. The festival ‘Robigalia’ in the seventh and eighth century BC, established to honor the god Robigus, was celebrated at the end of April probably to prevent grain and trees from being
affected by rust or mildew [16]. Pneumonic plague deaths during the Middle
Ages have also been attributed to food poisoning induced by various mycotoxins [17]. For almost 1,000 years, since the ninth century, ergotism from ergotinfected rye has afflicted large populations in Europe [18]. Ergotism was also
called ‘ignis sacer’ (sacred fire) or St. Anthony’s fire, because it was believed
that a pilgrimage to the shrine of St. Anthony would bring relief from the
intense burning sensation caused by the mycotoxin [19, 20]. In the 1850s, ergotism was finally found to be caused by alkaloids from Claviceps purpurea sclerotia. In the USA, the last major outbreak of ergotism was recorded in 1925; but
ergot-induced mycotoxicosis was still being reported in France thirty years later
[16, 21, 22].
Modern mycotoxicology began in 1960, after 10,000 turkeys died when
fed a mycotoxin-contaminated peanut meal (‘Turkey X’ disease). Although
major deaths of livestock were reported earlier from consumption of moldy
corn in feed of horses in Illinois (1933–1934 where 5,000 horses died) and
swine (1,500 lost in Southeastern United States) [23], it was the discovery of
aflatoxins, produced by Aspergillus flavus, as the causative agent in the peanut
meal causing the ‘Turkey X’ disease (as well as in moldy corn affecting the
earlier deaths of livestock) that provided the impetus to associate fungal toxins
with illness in animals [24]. In the last 40 years, many new mycotoxins have
been identified and characterized, and their biosynthetic origin in various fungi
elucidated [25].
It has been estimated that at least 25% of the world’s agricultural product
is contaminated with mycotoxins [26]. Modern epidemiology has allowed
certain diseases to be linked to ingestion of mycotoxins by documenting
Toxins of Filamentous Fungi
171
Table 2. Natural occurrence of selected mycotoxigenic fungi
Fungal species
Aspergillus
A. flavus
A. parasiticus
A. ochraceus
A. versicolor
A. nidulans
A. clavatus
Commodity contaminated
peanuts, corn, most treenuts
cottonseed
figs, cereals, cheese
cereals, beans, coffee
corn, grains, cheese
corn, grains, cheese
apple, apple juice, beans, wheat
Fusarium
F. verticillioides
F. crookwellense
F. culmorum
F. graminearum
F. poae
F. proliferatum
F. sporotrichioides
corn, corn products
cereals
cereals
cereals
cereals
corn, corn products
cereals
Penicillium
P. citrinum
P. aurantiogriseum (P. cyclopium)
P. islandicum
P. rugulosum
wheat, corn, rice
corn, sorghum, rice
corn, hay
corn, hay, peanuts, rice
P. viridicatum
P. verruculosum
P. griseofulvum
P. aurantiogriseum (P. puberulum)
P. rubrum
P. purpurogenum
P. palitans
P. roquefortii
barley, corn, rice, walnuts
peanut, corn, cheese
rice, sorghum
barley, beans, cereals, coffee
apple, apple juice, beans, wheat
barley, corn
corn
cheese, hay
Adapted from Scudmore [3], Bhatnagar et al. [5].
occurrence of the mycotoxin contaminations in various parts of the world
and correlating outbreaks of illnesses to enteric exposures to mycotoxins.
Indoor air pollution by toxigenic fungi may implicate mycotoxins as having
a more widespread role in chronic disease than was previously thought possible [27, 28].
Mycotoxins may not be effective tools in biological warfare because acute
toxic effects are not very pronounced for most of these toxins [29]. But there
Bhatnagar/Yu/Ehrlich
172
is strong evidence of use of these toxic compounds, such as aflatoxins, in
biological terrorism [30, 31].
Classification of Mycotoxins
Mycotoxins are low-molecular-weight organic compounds and have been
classified in different ways depending on whether or not the classification
was done by chemists, clinicians, mycologists or public health organizations.
Chemists have classified these compounds according to their chemical structures. But the number and variety of mycotoxins make this classification
challenging even for organic chemists. Mycotoxins have also been classified by
toxicologists and clinicians based on the illnesses associated with mycotoxins
or organs affected. Mycologists would prefer to classify these toxins based on
the fungal genera producing specific mycotoxins. However, since some fungi
produce a single mycotoxin and other genera may produce many mycotoxins,
and different genera may produce the same mycotoxin, this mode of classification could get confusing.
Carcinogenicity has been used by the World Health Organization as an
index for classifying mycotoxins [32]. However, the carcinogenic effects of
very few mycotoxins have been established or even directly correlated.
Therefore, this method of classifying mycotoxins may not be applicable at the
present time. But as the carcinogenic risk of more mycotoxins is established
based on ongoing research to identify the associations between human or
animal consumption of contaminated food and the incidence of associated
mycotoxicosis, this classification system may become very relevant.
Turner and Aldridge [33] have categorized fungal secondary metabolites
not by their properties, but according to their biosynthetic origins. Although
diverse in structure and activity, fungal metabolites are derived from a few
biosynthetic pathways as shown in table 3.
Economic Impact of Mycotoxins
The most obvious impact of mycotoxins is the inability to sell crops for
human or animal food due to contamination with even relatively low levels of
certain mycotoxins. In certain developing countries where there is less regulation or monitoring of human and animal exposure to toxins, there are risks of
higher human health costs and animal death. Additional losses can be from a
number of unseen problems such as reduction in birth rate in certain animals,
decline in milk production by dairy cattle and egg production in poultry, loss of
Toxins of Filamentous Fungi
173
Table 3. Representative mycotoxins grouped according to biosynthetic pathways
Biosynthetic origin
Mycotoxin
Producing fungal genera
Polyketide
aflatoxin
citrinin
citreoviridin
ochratoxin
penicillic acid
secalonic acid
sterigmatocystin
zearalenone
Aspergillus
Aspergillus, Penicillium
Penicillium
Aspergillus, Penicillium
Aspergillus, Penicillium
Claviceps, Penicillium
Aspergillus, Chaetomium, Bipolaris
Fusarium
Amino acids
brevianamide
cytochalasins
Penicillium
Aspergillus, Helminthosporium,
Metarhizium, Phoma, Zygosporium
Claviceps
ergot alkaloids
Terpenes
penitrem
trichothecenes
Penicillium
Fusarium, Myrothecium,
Stachybotris
Tricarboxylic acid
rubratoxin
tenuazonic acid
Penicillium
Alternaria, Aspergillus
Adapted from Kale and Bennett [222].
quality of animal products, increased susceptibility to infection, losses due to
reduced weight gain from toxin-contaminated animal feeds.
The perceived significance of mycotoxin contamination of foods for
human health has prompted international trade and health organizations and
governmental control authorities to regulate toxin levels permitted in commodities for commerce. Since 1977, the Food and Agricultural Organization
(FAO) of the United Nations has published over a dozen position papers on
mycotoxin regulation and control [34]. Over 50 countries of the world have
developed such guidelines. In the United States, only the Aspergillus mycotoxins, aflatoxins, have action levels under the Food, Drug and Cosmetic Act [35].
The US Food and Drug Administration prohibits interstate commerce of feed
grain containing more than 20 parts per billion (ppb) aflatoxin B1 and prohibits
the sale of milk and eggs containing more than 0.5 ppb aflatoxin M1. Levels
varying from zero tolerance to 50 ppb are accepted by at least 50 countries [36]
(table 4). And, as stated by Van Egmond and Speijers [37], ‘A recent development in this regard was the establishment in 1994 of the WHO Collaborating
Center for Mycotoxins in Food (WHO-CCMF), located in the Albert Ludwigs
University School of Medicine in Freiburg, Germany. A particular task of this
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174
Table 4. Limits for aflatoxins in foodstuffs
Countries
Maximum allowed
aflatoxins, ppb or g/kg
Austria
France
Germany
India
Japan
Malaysia
Mexico
Netherlands
United Kingdom
United States (food and feed)
(milk and eggs)
0.02–1
0.1–10
5
30
10
35
20
0.02–5
10
20
0.5
Adapted from Van Egmond [36].
Center will be to elucidate the role of mycotoxin-contaminated food in human
health and disease. In 1997, the National Institute of Public Health and the
Environment, Bilthoven, The Netherlands was appointed as a Community
Reference Laboratory (CRL) for mycotoxins in animal products.’
The regulatory issues are less well-defined for mycotoxins other than
aflatoxins. But with increasing data on specific toxicity of Fusarium mycotoxins such as fumonisins, regulatory guidelines are being considered for
these toxins as well. For example, in the USA, advisory limits exist for
deoxynivalenol (DON) at 5–10 mg/kg (5–10 parts per million; ppm) in animal
feeds [7].
Several countries strictly enforce laws to prevent sale of contaminated
commodities, whereas in others regulatory guidelines exist, but are not
enforced. Problems related to more precise methods for evaluating mycotoxin
contamination of food are still being developed. Errors can result from sampling,
subsampling, and analysis [38]. Such errors can lead to accidental exposure to
inappropriate levels of mycotoxins or in rejection of sound lots. Additionally,
costs of chemical analyses, quality control and regulatory programs, research
development, extension services, lawsuits, and the cost of treatment of human
illness must be considered in the overall economic burden that mycotoxins has
on the economy worldwide. Based on estimates of this burden, one can only
justify the need for a major research effort in mycotoxin identification and
characterization, prevention and control. The research efforts to date in many
Toxins of Filamentous Fungi
175
areas of the world have made significant progress toward understanding the
natural occurrence of several mycotoxins.
Detection and Screening of Mycotoxins
Efforts to minimize mycotoxins in foodstuffs include monitoring, managing and controlling the levels of mycotoxins in agricultural products from farm
to fork. Therefore, surveillance programs have been established to reduce the
risk of mycotoxin consumption by humans and animals. Analytical testing
methods for rapid analyses of a large number of samples of foodstuffs have
been developed for several mycotoxins [39]. A single method for analysis of all
mycotoxins is not feasible because of the varied chemical nature of mycotoxins,
their molecular weights and functional groups. Current analytical techniques
for accurate characterization and quantitation of mycotoxins include thin layer
chromotography (TLC), high pressure liquid chromatography (HPLC) and gas
chromatography (GC). However, radioimmunoassay (RIA) and enzyme-linked
immunosorbent assay (ELISA) tests are available, based on sera developed for
a majority of the mycotoxins [39–42], for rapid detection of mycotoxins in
foods at levels as low as one tenth of a nanogram per milliliter [6]. A dozen
commercial test kits are available for field testing of various agricultural commodities for toxins such as aflatoxins.
Selected Mycotoxins
Some of the most prevalent mycotoxins, the producing fungi, and their major
toxic effects in humans and animals are discussed below. Although specific
mycotoxicoses have been attributed to various mycotoxins, and the mechanisms
of toxicity have been established in vitro [43], there are many pitfalls in interpreting the results of in vitro studies and extrapolating them to in vivo effects, and
suggesting a natural disease associated with consumption of a mycotoxin. Riley
[44], in describing these difficulties, suggested, ‘… the factors that tend to confound in vitro mechanisms studied include: (1) failure to differentiate secondary
effects from the primary biochemical lesion; (2) failure to relate the effective
intracellular concentration in vitro to the tissue concentration of toxin which
causes disease in vivo; (3) choice of an in vitro model which is either deficient in
the biochemical target or unresponsive due to other inadequacies of the model
system; and (4) failure to adequately model the complexity of the in vivo exposure with regard to potential interactions with other toxins, drugs, environmental,
and/or nutritional factors’.
Bhatnagar/Yu/Ehrlich
176
Aflatoxins
Aflatoxins are by far the best-characterized class of mycotoxin. They are
a group of polyketide-derived bis-furan-containing dihydrofuranofuran and
tetrahydrofuran moieties (rings) fused with a substituted coumarin (fig. 1). At
least 16 structurally related toxins have been characterized [45]. These toxins
are primarily produced by Aspergillus flavus and A. parasiticus on agricultural
commodities, and infrequently by A. tamarii and A. nomius [46]. The four
major aflatoxins, B1, B2, G1 and G2, were originally isolated from A. flavus
hence the name A-fla-toxin. The B toxins fluoresce blue under UV light and the
G toxins fluoresce green. Other significant members of the aflatoxin family,
M1 and M2, are metabolites of aflatoxin B1 (AFB1) originally isolated from
bovine milk. Attempts to decipher the aflatoxin biosynthetic pathway began
with the discovery of the structure of these toxins [45]. However, the major biochemical steps and the corresponding genetics of the AFB1 biosynthesis have
been elucidated only in the last decade [47–53]. Starting with the polyketide
precursor, acetate, there are at least 23 enzymatic steps in the AFB1 biosynthetic
pathway. AFB2, G1, and G2 are synthesized from pathways that diverge from the
B1 pathway [54–56]. The genes for almost all the enzymes have been cloned
and a regulatory gene (aflR) coding for a DNA-binding, Gal4-type 47-kD protein has been shown to be required for transcriptional activation of all the structural genes [48, 50, 51, 53, 57–65]. It has also been shown by restriction
mapping of cosmid and phage DNA libraries of A. flavus and A. parasiticus that
all the AFB1 pathway genes are clustered within a 75-kb region of the fungal
genome [50, 51, 53, 64].
Since the discovery of the Turkey X disease in 1960, aflatoxins have been
established as mutagenic, teratogenic and hepatocarcinogenic in experimental
animals. AFB1 is the most toxic of this group of toxins and the order of
toxicity is B1⬎G1⬎B2⬎G2. Aflatoxin M1 is 10-fold less toxic than B1, but its
presence in milk is of concern in human health [66–68]. AFB1 is also one of
the most carcinogenic natural compounds known; therefore, extensive research
has been done on its synthesis, toxicity and biological effects [53, 69–72].
Liver is the target organ for aflatoxins, and aflatoxicosis leads to proliferation of the bile duct, centrilobular necrosis and fatty infiltration of the liver,
hepatomas and hepatic lesions. The susceptibility of animals to AFB1 varies
with species [reviewed in Eaton and Groopman, 72]. In addition to the liver,
AFB1 also affects other organs and tissues, such as the lungs and the entire
respiratory system [73–75].
In animal models, activation of AFB1 by microsomal cytochrome P-450 is
required for carcinogenicity [72, 76]. One of the cytochrome P-450 monooxygenases in the liver converts AFB1 to a variety of metabolites of increased
Toxins of Filamentous Fungi
177
a
b
R3
O
O
O
O
O
R1
R1
R
O
O
O
R2
O
H
4
c
H
OCH3
O
d
R1
H H
H H
O
H
R1
H H
H
O
O
H
O
H
O
Aflatoxins
Structure
R1
R2
R3
R4
B1
M1
P1
Q1
R0
R0H1
B2
B2a
M2
G1
G2
G2a
GM
a
a
a
a
a
a
ac
ad
ac
b
bc
bd
bc
H
OH
H
H
H
H
H
H
OH
H
H
H
OH
OCH3
OCH3
OH
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
–
–
–
–
⫽O
⫽O
⫽O
⫽O
OH
OH
⫽O
⫽O
⫽O
–
–
–
–
H
H
H
OH
H
OH
H
H
H
–
–
–
–
Fig. 1. Chemical structure of aflatoxins. (a) The B-type aflatoxins are characterized by a
cyclopentane E-ring. These compounds have a blue fluorescence under long-wavelength
ultraviolet light. (b) The G-type aflatoxins have a xanthone ring in place of the cyclopentane.
(c) Aflatoxins of the B2 and G2 type have a saturated bis-furanyl ring. Only the bis-furan is
shown. (d) Aflatoxins of the B1a and G1a type have a hydrated bis-furanyl structure.
Bhatnagar/Yu/Ehrlich
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polarity including AFB1-8, 9-epoxide, the ultimate carcinogen which binds covalently to N7 position of guanine in DNA, resulting in defective repair and DNA
damage, mutations and ultimately carcinomas in many animal species
[70, 77, 78]. The role of aflatoxins in carcinogenesis in humans is complicated
by hepatitis B virus infections in human beings which is also associated with
hepatocarcinoma [79–81]. An association of hepatocellular carcinoma and
dietary exposure to aflatoxins was established from patients living in high-risk
areas of the People’s Republic of China, Kenya, Mozambique, Phillippines,
Swaziland, Thailand, and the Transkei of South Africa [72, 78, 82]. The correlation was demonstrated by measuring guanine-aflatoxin adducts in urine samples.
The presence of the adducts is considered an indication of the modification of a
guanine residue at a mutation hot spot, the transversion of guanine to thymine at
the third base of codon 249 in the tumor suppressor gene p53. This gene (p53)
is a transcription factor involved in the regulation of the cell cycle which is
commonly mutated in human cancers [83]. Since multiple factors may be
involved in carcinogenesis [84], aflatoxins in combination with hepatitis B virus
may be significant dual etiological factors in hepatocellular carcinoma [85, 86].
Toxic Polyketides Other Than Aflatoxins
Ochratoxins
These mycotoxins are a group of dihydroisocoumarins (fig. 2) and are
produced by a number of Penicillium and Aspergillus species [87]; the largest
amounts of ochratoxins are made by A. ochraceus and P. verruculosum. The two
species A. ochraceus and P. verruculosum, occur frequently in nature, and were
first reported as ochratoxin producers [88–90]. Other fungi such as Petromyces
alliaceus (alternate name Aspergillus alliaceus) [91, 92] and A. niger have also
been reported to produce ochratoxins. Ochratoxins are produced primarily
in cereal grains (barley, oats, rye, corn, wheat) and mixed feed during storage
in temperate climatic conditions. However, this toxin has also been reported to
contaminate other commodities such as beans, coffee, nuts, olives, cheese, fish,
pork, milk powder, wine, beer and bread [1, 93]. Ochratoxins bind tightly to
serum albumin and are carried in animal tissues and body fluids. Consequently,
the occurrence of this toxin has been reported in pork sausage made from
contaminated organ meats, a product widely consumed in northern Europe and
the Balkans.
Ochratoxin A, the most toxic member of this group of mycotoxins, has
been found to be a potent nephrotoxin causing kidney damage in many animal
species as well as liver necrosis and enteritis [4, 89, 94–96]. This toxin also
acts as a immunosuppressor [96–98], and demonstrates teratogenic [99],
Toxins of Filamentous Fungi
179
O
R1
OH
O
C
O
H
CH3
R2
R3
Ochratoxins
R1
R2
R3
A
B
C
A methyl ester
B methyl ester
B ethyl ester
4-hydroxy ochratoxin A
␣

a
a
b
c
c
b
a
OH
OH
Cl
H
Cl
Cl
H
H
Cl
Cl
H
H
H
H
H
H
H
OH
H
H
a ⫽ C6H5CH2CH(COOH)NH-.
b ⫽ C6H5CH2CH(COOEt)NH-.
c ⫽ C6H5CH2CH(COOMe)NH-.
Fig. 2. Structure of the ochratoxins. These metabolites form different classes depending
on the nature of the amide group (a–c), and the presence or absence of a chlorine moiety at
R2 in the phenyl.
mutagenic [100], weak genotoxic [101] effects in test animals. Some studies
have shown that ochratoxins may be weakly carcinogenic, affecting both the
kidney and the liver [97, 102]. In vitro studies have shown that ochratoxin is an
inhibitor of aminoacyl transfer ribonucleic acid (tRNA) synthetase and protein
synthesis. Human exposure to ochratoxins can come from ingestion of contaminated sausage meats or cereals, cereal products, coffee, beer and even pulses
[4, 103].
Although the role of ochratoxins in human pathogenesis is still speculative,
the lesions of nephropathy in humans were reported to be similar to those observed
in porcine nephropathy [104]. Outbreaks of kidney disease (Balkan endemic
nephropathy) in rural populations in Bulgaria, Romania, Tunisia and the former
Yugoslavia were associated with ochratoxin A [104–106]. These correlations were
Bhatnagar/Yu/Ehrlich
180
CH3
O
N
H3C
H
CH3
H
H
OH
NH
O
Fig. 3. Chemical structure of cyclopiazonic acid.
based on detection of ochratoxin A in human serum, milk and kidneys in these
countries. Among 77 countries which have regulations for different mycotoxins, 8
have specific regulations for ochratoxin A, with limits ranging from 1 to 20 g/kg
in different foods [4]. Administration of phenylalanine can reverse some of the
toxic effects of ochratoxins. But other efforts to eliminate the presence of this toxin
in food have not been successful.
Cyclopiazonic Acid
This fungal metabolite is produced by several species of the genus
Penicillium, P. aurantiogriseum, P. crustosum, P. griseofulvum, P. camemberti,
as well as by A. flavus, A. tamarii, and A. versicolor, but not A. parasiticus
[46, 107]. Natural occurrence of cyclopiazonic acid (CPA, fig. 3) has been
reported in corn, peanuts and cheese, along with the presence of aflatoxins. The
association of CPA with aflatoxins together with a lack of well-developed analytical methods for its determination in foods has prevented an assessment of
the possible health effects of CPA. Cyclopiazonic acid apparently causes hyperesthesia and convulsions as well as liver, spleen, pancreas, kidney, salivary
gland and myocardial damage [71, 108]. Kodua poisoning resulting from ingestion in India of Kodo millet seeds contaminated with Aspergillus, has been
attributed to CPA.
Patulin
Patulin was originally considered desirable for its antibacterial properties.
However, its toxicity to mammals precluded its use as an antibiotic. Patulin
(fig. 4), a metabolite of many species of Penicillium and Aspergillus, has been
detected in damaged apples, apple juice and apple cider made from partially
decaying apples and in other fruit juices as well as in wheat. A heat-resistant
fungus Byssochlamys nivea, frequently found in foods also produces patulin
[109]. Due to highly reactive double bonds that readily react with sulfhydryl
Toxins of Filamentous Fungi
181
O
O
OH
H
Fig. 4. Structure of patulin showing
the closed-ring tautomeric form.
O
O
HO
O
CH2
C
C
C
3
O
C
C
C
HO
OH
CH3
O
CH3
O
C
CH2
Fig. 5. Chemical structure of penicillic acid showing both tautomeric forms. The
open-chain form is in equilibrium with the closed form shown on the right in the figure.
groups in foods, patulin is not very stable in foods containing these groups
[110]. Toxic effects include contact edema and hemorrhage upon ingestion. It
appears to be carcinogenic in experimental animals but no instances of human
cancers are known [71, 111]. At least 10 countries have regulatory guidelines
that limit the presence of patulin to 50 g/kg in various foods and juices.
Penicillic Acid
Penicillic acid (fig. 5), also a metabolite of many Penicillium and
Aspergillus species, is a contaminant of corn (‘blue eye corn’), beans and even
meat. Even though penicillic acid has been shown to be a liver toxin, no
instances of animal or human toxicity have been reported. In spite of this, the
potential for these toxins to act synergistically with other more potent toxins
is a concern since the species that produce these toxins usually produce other
toxins as well [71].
Citrinin
Frequently associated with the natural occurrence of ochratoxin A, citrinin is
produced by Penicillium citrinum and several other Penicillia and Aspergilli [112]
Bhatnagar/Yu/Ehrlich
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OH
O
H
O
O
O
OMe
H
Fig. 6. Structure of sterigmatocystin.
The bis-furanyl structure is similar to that
of the aflatoxins except that the E-ring is a
substituted phenol.
and Monascus ruber and M. purpureus [113]. As with patulin, citrinin was originally investigated for its antibacterial properties. But, this toxin has been reported
to be a nephrotoxin in chickens fed corn contaminated with Penicillium citrinum.
This golden-yellow compound is easily detected as a free compound, but upon
binding to proteins particularly during the drying of corn, the color is lost, while
the toxicity remains. Therefore, significant quantities of citrinin, while part of the
diet, could lead to chronic and hard to diagnose kidney disease in susceptible individuals and animals, particularly those that were fed products made from lowquality corn [71].
Sterigmatocystin
Sterigmatocystin (ST) is produced by various Aspergilli. But there are
reports which indicate that fungi of the genera Bipolaris and Chaetomium, and
Talaromyces luteus also produce ST [112]. Structurally related to aflatoxins
(fig. 6), and a precursor of AFB1 [71, 114, 115], ST is a hepatotoxic and
carcinogenic mycotoxin. But its carcinogenicity is several order of magnitudes
less than that of AFB1 in test animals [116, 117]. ST is a contaminant of cereal
grains (barley, rice and corn), coffee beans and cheese [118]. It has been
reported as a contaminant in foods from regions of the world with a high
incidence of cancer, e.g., esophageal cancer in Linxian province in the People’s
Republic of China [119] and liver cancer in Mozambique [117]. Although STproducing fungi have been isolated from patients with esophageal cancer, the
role of ST in human carcinogenesis appears to be indirect and inconclusive [4].
Zearalenone
This toxin is produced by several Fusarium spp., in particular by the scabby
wheat fungus F. graminearum (roseum) which also makes deoxynivalenol,
DON [71]. It has an unusual macrocyclic structure [6(10-hydroxy-6-oxotrans-1-undecenyl)-beta-resorcyclic acid--lactone] (fig. 7). Zearalenone (also
called F-2) is found in the gluten of contaminated wet-milled wheat, and also
Toxins of Filamentous Fungi
183
CH3
OH
H
O
O
HO
O
Fig. 7. Structure of zearalenone.
in corn and corn products. Zearalenone has estrogenic properties and can cause
premature onset of puberty in female animals, as well as hyperestrogenic
effects and reproductive problems in animals, especially swine. This toxin has
also been associated with an increased incidence of cervical cancer.
Zearalenone can be toxic to plants; it can inhibit seed germination and embryo
growth at low concentrations. Contamination of feed with zearalenone as well
as with DON may result in severe economic losses to the swine industry due to
both feed refusal and adverse estrogenic effects.
Fumonisins
Fumonisins are a group of toxins produced primarily by Fusarium verticillioides (formerly called F. moniliforme), F. proliferatum and other related species
which readily colonize corn all over the world [120–124]. Nine structurally
related fumonisins including B1, B2, B3, B4, A1 and A2, have been described
(fig. 8). Chemically, fumonisin B1 is a derivative (diester) of propane-1,2,3tricarboxylic acid of 2-amino-12,16-dimethyl-3,5,10,14,15-pentahydroxyicosane [121–130]. The other fumonisins lack the tricarballylic acid or other
ester groups [121, 131, 132]. Fumonisins are chemically similar in structure to
toxins (AAL) produced by Alternaria alternata [133]. Production of fumonisins
by Alternaria has also been reported [134, 135], and some fumonisin-producing
Fusaria have been known to produce AAL toxins [136].
Fumonisins B1, B2 and B3 are the major mycotoxin contaminants in corn
and corn-based foods and other grains such as sorghum and rice. The level of
fumonisins varies from negligible to greater than 100 ppm, but is generally
reported to be between 1 and 2 ppm. Corn frequently contains detectable but
low levels of fumonisins, even though there is no visible sign of fungal contamination. In laboratory cultures, highest levels of this toxin are produced on corn,
and less on rice; whereas peanuts and soybeans are very poor substrates for
fumonisin production [4].
Bhatnagar/Yu/Ehrlich
184
CH3
R
R1
CH3
R3
R4
R
R2
OH
O
O
OH
O
R⫽
⫹N
HO
O
OH
Tricarballylic acid
3-Hydroxypyridinium (3HP)
Fumonisins
R1
R2
R3
R4
B1
B2
B3
B4
A1
A2
C1
C4
P1
P2
P3
OH
H
OH
H
OH
H
OH
H
OH
H
OH
OH
OH
H
H
OH
OH
OH
H
OH
OH
H
NH2
NH2
NH2
NH2
NHCOCH3
NHCOCH3
H
H
3HP
3HP
3HP
CH3
CH3
CH3
CH3
CH3
CH3
H
H
CH3
CH3
CH3
Fig. 8. Chemical structures of different classes of fumonisins. The R residue is
3-hydroxypyridinium in fumonisins P1–P3 while the others are tricarballyl esters.
Like most other mycotoxins, fumonisins are quite stable and are not
removed from grains by changes in temperature, pH and salt concentration during food processing. Therefore, significant amounts of fumonisins are usually
found in foods and feeds. Stability of fumonisins is of concern because some
products, such as gluten from wet milling and distillers’ dried grains from fermentation of contaminated grains, contain higher levels of fumonisins than found
in the starting raw material. Acid hydrolysis of fumonisins causes the loss of the
tricarballylic acid moiety. Significant amounts of fumonisins are not transmitted
to milk, meat or eggs because animals rapidly excrete these toxins [137, 138].
Toxins of Filamentous Fungi
185
Fumonisin biosynthesis has been of significant research interest in the last
few years [reviewed in Proctor, 139]. A 20-carbon chain is the backbone of
fumonisins which structurally resembles fatty acids and linear polyketides
[140]. Studies with 13C- and 14C-labeled acetate have suggested that a polyketide synthase, alanine, methionine and other amino acids are involved in fumonisin synthesis. Four loci (designated fum 1, fum 2, fum 3 and fum 4) involved
in fumonisin biosynthesis have been identified by classical genetic analyses
utilizing Gibberella fujikuroi, the sexual stage of Fusarium verticillioides
[139]. These genes are clustered on chromosome 1 [140, 141].
Fumonisin B1, first isolated in South Africa, is regarded as the most
significant of the fumonisins. Fumonisins have been shown to have diverse
biological and toxicological effects as discussed below. The mechanism of
fumonisin toxicity is not well understood. Studies have shown that fumonisins
are inhibitors of ceramide synthase; their effect thus appears to be related to
interference with sphingolipid biosynthesis in multiple organs, such as brain,
lung, liver and kidney, of the susceptible animals. Fumonisin B1 has also been
shown to be a hepatotoxin and a carcinogen in rats resulting in liver cirrhosis
and hepatic nodules, adenofibrosis, hepatocellular carcinoma, ductular carcinoma and cholangio-carcinoma [125, 142–145]. Because of its potential role
in hepatocarcinoma formation, fumonisin B1 has been classified as a class II
carcinogen by the International Agency for Research on Cancer, 1993. In cell
culture systems, fumonisin B1 has been demonstrated to be mitogenic and cytotoxic, without genotoxic effects [144, 145]. Kidney cells have also been shown
to be targeted by these toxins. Porcine pulmonary edema caused by ingestion of
fumonisin-contaminated wheat and corn is one of the recorded toxic effects of
this group of mycotoxins. Reports have indicated a possible role of fumonisin
B1 in the etiology of human esophageal cancer in the regions of South Africa,
China and northeastern Italy where Fusarium species are common contaminants. Erythro-Leukoencephalomalacia (ELEM) in horses and other Equidae
(donkeys and ponies) is the best-characterized toxicological effect of fumonisins, which is a seasonal disease occurring in late fall and early spring. The disease is manifested by neurological changes in animals, including uncoordinated
movement and apparent blindness. Clinically, the disease is characterized by
edema in the cerebellum. The levels of fumonisins B1 and B2 in feeds associated with ELEM ranges from 1.3 to 27 ppm [4].
Current data suggest that fumonisins may have greater effect on the health of
farm animals than on humans, because studies on the impact of levels of fumonisins in human food have not yielded significant correlations [127, 146]. The
Mycotoxin Committee of the American Association of Veterinary Laboratory
Diagnostics recommended that permissible levels of fumonisins (B1) in feeds be
limited to 5 ppm for horses, 10 ppm for swine and 50 ppm for cattle and poultry.
Bhatnagar/Yu/Ehrlich
186
Trichothecenes
Several species of Fusarium infect corn, wheat, barley, and rice. Under
favorable conditions they elaborate a number of different types of tetracyclic
sesquiterpenoid mycotoxins that are composed of the epoxytrichothecene
skeleton and an olefinic bond with different side chain substitutions (fig. 9).
Based on the presence of a macrocyclic ester or ester-ether bridge between
C-4 and C-15, trichothecenes are generally classified as macrocyclic (type C)
or nonmacrocyclic (types A and B) (table 5). Other fungal genera producing
trichothecenes are Myrothecium, Trichoderma, Trichothecium, Acremonium,
Verticimonosporium and Stachybotrys. The term trichothecenes is derived from
trichothecin, the first compound isolated in this group [115, 147–153].
The biosynthesis of the trichothecenes involves the incorporation of three
molecules of mevalonate into the trichothecane nucleus of trichothecin. The
precursor farnesyl pyrophosphate can fold in different ways depending on the
position and stereochemistry of the three double bonds. The 6, 7-trans olefin
is favored to fold to give trichodiene, the first stable intermediate in the biosynthetic pathway, a process catalysed by the enzyme trichodiene synthase.
Trichodiene synthases have a high degree of similarity in different trichotheceneproducing fungi. Subsequent conversions of the trichothecane nucleus involve
a series of specific oxidations of the three rings, mediated by cytochrome P450
oxidases encoded by genes in a gene cluster as has been found for biosynthesis
of other fungal secondary metabolites. The most significant oxidation is
provided by a cytochrome P450 monooxygenase which introduces the C12, 13
epoxide. At least 12 genes (Tri1 to Tri12) are involved in the biosynthesis of
trichothecenes. These genes have been cloned, and are clustered on a 25-kb
region of the chromosome. Tri6 is a regulatory gene [139, 154–157].
The trichothecenes constitute a class of over 80 different members that
elicit different toxic effects in animals and humans. The trichothecenes causing
the most concern for food and feed consumption are nivalenol, deoxynivalenol
(DON), T-2 toxin, and the macrocyclic trichothecenes. The most potent trichothecenes are dermal toxins and cause severe blistering and necrosis of the
target tissue. Their cytotoxicity parallels their acute toxicity in animals, with T2 toxin being more potent than nivalenol. Nivalenol is much more potent as an
acute toxin than is deoxynivalenol. DON is of considerable importance to agricultural economies because it is more frequently found in corn and cereal
grains, and animals refuse to eat contaminated grain. Economic losses from
both rejected feed stocks and underfed animals are significant.
Unlike most mycotoxins, T-2 toxin is produced optimally at 15 °C instead
of 25–30 °C for other toxins [152, 158]. Contamination of cereal grains such as
barley, corn, oats, and wheat with T-2 toxin is less frequent than with DON.
Toxins of Filamentous Fungi
187
H
H
H
O
H3C
R1
O
H
R5
CH2
R4
R2
CH3
R
3
Trichothecenes
R1
R2
R3
R4
R5
Diacetoxyscirpenol
4-Monoacetoxyscirpenol
15-Monoacetoxyscirpenolb
Scirpentriol
Deoxynivalenol
Nivalenol
Fusarenon-x
T-2
HT-2
T-2 triol
3⬘-OH-T-2
T-2 tetraol
Neosolaniol
Roridin A,B,E,H,J
Roridin K
Verrucarin A,B,J,K
Verrucarin L
Satratoxin F,G,H
Verrucarol
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
H
H
H
H
H
H
OAca
OAc
OH
OH
H
OH
OAc
OAc
OH
OH
OAc
OH
OAc
MCa
MC
MC
MC
MC
OH
OAc
OH
OAc
OH
OH
OH
OH
OAc
OAc
OH
OAc
OH
OAc
MC
MC
MC
MC
MC
OH
H
H
H
H
OH
OH
OH
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
⫽O
⫽O
⫽O
ISVc
ISV
ISV
OH-ISVd
OH
OH
H
OH
H
OH
H
H
a
OAc ⫽ OCOCH3.
Macrocyclic.
c
ISV ⫽ OCOCH2CH(CH3)2.
d
OH-ISV ⫽ OCOCH2C(OH)(CH3)2.
b
Fig. 9. Chemical structures of different trichothecenes. MC ⫽ Macrocyclic;
ISV ⫽ isovalerate; OH-ISV ⫽ hydroxyisovalerate.
Bhatnagar/Yu/Ehrlich
188
Table 5. Classification of trichothecenes
Nonmacrocyclic
Macrocyclic
type A1
type B2
type C3
T-2 toxin
HT-2 toxin
Neosolaniol (NESO)
Diacetoxyscirpenol (DAS)
T-2 tetraol (T-4ol)
deoxynivalenol (DON)
nivalenol (NIV)
fusarenon-X
roridins
verrucarins
stachybotryotoxins
1
Contain a hydrogen- or ester-type side chain at the C-8 position.
Contain a ketone.
3
Contain a macrocyclic ring.
2
However, T-2 toxin is at least 20-fold more toxic to animals, and possibly to
humans, than DON [4].
The trichothecene mycotoxicoses are difficult to distinguish because they
affect many organs, including the gastrointestinal tract, hematopoietic, nervous,
immune, hepatobiliary, and cardiovascular systems [reviewed in Chu, 148].
Ingestion of trichothecene-contaminated food and feed causes many types of
mycotoxicoses in humans and animals, for example, moldy corn toxicosis,
scabby wheat toxicosis (Akakabi-byo disease), vomiting and feed refusal,
fusaritoxicosis, and hemorrhagic syndrome.
The trichothecenes have now been found to be responsible for the human
disease alimentary toxic aleukia in western Siberia prior to World War II, which
was then attributed to ingestion of moldy millet and wheat. This contaminated
grain contained certain macrocyclic epoxytrichothecenes and the severe blistering agent T-2 toxin. Feed refusal by pigs exposed to moldy corn containing
DON (also called vomitoxin) was a severe economic problem in the 1970s in
midwest United States. Head scab of wheat caused by F. graminearum was
also a serious problem for wheat and barley growers from 1991 to 1997 in the
eastern and midwestern US, and in the Canadian provinces of Ontario and
Manitoba [159–161]. The major mycotoxin associated with head scab is DON
[152, 162]. Red-mold disease caused by contamination of wheat and barley
with F. graminearum among other Fusaria was a sporadic problem in Japan and
Korea in the 1970s. The combined result of Fusarium mycotoxin contamination
is worldwide economic losses in the billions of dollars.
Another disease, termed stachybotryotoxicosis, caused the death of thousands of horses in the Soviet Union prior to World War II and was attributed
to contamination of straw and hay with the fungus Stachybotrys atra.
Toxins of Filamentous Fungi
189
More recently, spores from S. atra growing on moist wood or building materials in houses have been associated with pulmonary hemosiderosis in young
infants. Trichothecenes may be involved in this ‘sick building’ syndrome in
humans. Symptoms such as headaches, sore throats, hair loss, diarrhea, fatigue
and general malaise have been reported in humans living in water-damaged
houses [28, 149, 163].
The unusual 12,13-spiroepoxy ring of the trichothecenes has been shown
to be necessary for their toxicity [reviewed in Chu, 148]. Their toxicity is
mainly due to the inhibition of initiation of polypeptide synthesis or polypeptide chain elongation during protein synthesis. All types of trichothecenes interfere with the action of peptidyl transferase by binding to the 60S subunits of the
eukaryotic ribosome. The higher-molecular-weight trichothecenes inhibit initiation by binding to the P site of the ribosome and preventing translocation. The
lower-molecular-weight species inhibit both the initiation and elongation steps
of protein synthesis.
Alternaria Toxins
Alternaria has been known for centuries to cause various plant diseases
such as early blight of potato and various leaf spots and fruit rot. Species of
this fungus are widely distributed in soil and on aerial plant parts. Because
Alternaria requires high moisture levels (28–34%) for growth, infection of
seeds occurs when the seed moisture is high, either in early stages of development or after wetting of crops from rain.
More than 20 species of Alternaria are known to produce about 70 secondary metabolites belonging to a diverse chemical group including dibenzopyrones, tetramic acids, lactones, quinones, cyclic peptides. However, only
alternariol (fig. 10), tenuazonic acid, altertoxin-I, alternariol methyl ether and
altenuene are common natural toxic contaminants of consumable items like
fruits (apples), vegetables (tomato), cereals (sorghum, barley, oat) and other
plant parts (such as leaves) [118]. The most common species of Alternaria,
A. alternata produces all important Alternaria toxins including the five mentioned above and tentoxin, altenuisol, alteniric acid, altenusin, dehydroaltenusin
[164, 165]. As mentioned previously, A. alternata f. sp. lycopersici produces
a group of host-specific toxins named AAL toxins which are similar to
fumonisins.
Although most of the compounds produced by Alternaria are generally nontoxic, alternariol monomethyl ether has been shown to be mutagenic in bacteria
(Ames tests) [166]. Tenuazonic acid (TA) is a protein synthesis inhibitor and is
capable of chelating metal ions and forming nitrosamines. TA is also produced
Bhatnagar/Yu/Ehrlich
190
O
OH
O
HO
CH3
OH
Fig. 10. Structure of alternariol.
by Phoma sorghina and Pyricularia oryzae and may be related to ‘onyalai’, a
hematological disorder in humans living south of the Sahara in Africa.
Neurotropic Mycotoxins
Ergot Alkaloids and Related Toxins
Ergotism was one of the earliest documented incidences of a disease
caused by mycotoxin consumption. Contamination of rye and millet with the
‘ergot’, a name given to the hard, dark purple, sickle-shaped sclerotia from the
parasitic fungus Claviceps (C. purpurea and C. paspali) [167], led to accumulation of a number of toxic alkaloids that affect the central and peripheral nervous
systems and cause vasoconstrictive activity and hallucinations. Two types of
ergot epidemics occurred: gangrenous and convulsive. In the gangrenous type,
the victim first experienced violent burning pains termed ‘St. Anthony’s fire’
and then numbing and necrosis of the limbs, while the latter type caused severe
convulsions and death. Epidemics of convulsive ergotism occurred from the
Middle Ages in Europe until as late as 1928 in Russia. In one epidemic in
France in the 1700s, it was estimated that 8,000 people died from eating grain
containing as much as 25% ergot. Acute human ergotism [168–170] has essentially disappeared as a concern since the modern cleaning and milling processes
for grains remove most of the ergot sclerotia. However, ergot poisoning can still
be a problem for livestock feeding on wild grasses infected with Claviceps sp.
Additional alkaloid-producing fungi are also of concern. Rye grass staggers and fescue toxicoses are caused by ingestion of grasses infected with
endophytes Neotyphodium sp. and Epichloe sp. These fungi can produce ergot
alkaloids, indole diterpenes and indole terpenes. The indoleterpenes can cause
tremors in grazing livestock.
The ergot alkaloids are derivatives of the four-ring structure of ergoline.
These alkaloids can be divided into three groups: derivatives of lysergic acid
Toxins of Filamentous Fungi
191
(e.g. ergotamine and ergocristine); derivatives of isolysergic acid (e.g. ergotaminine); and derivatives dimethylergoline (clavines, e.g. agroclavine) [16].
The most biologically active forms are amide derivatives of D-lysergic acid, an
alkaloid whose diethylamide derivative, LSD, is famous in modern times for its
use as a recreational hallucinogen. This class of ergot alkaloids also includes
the cyclol-type peptide alkaloids in which a cyclic dipeptide forms the amide
linkage to lysergic acid. Another class of ergot alkaloids, the clavine alkaloids,
lack the carboxyl function at C-17. At least 37 different types of clavine alkaloids are known and are made by Claviceps as well as unrelated fungi, such as
Aspergillus fumigatus and Penicillium chermesinum. Ergot alkaloids have even
been found in a single family of higher plants, the Convolvulaceae, which
includes the common morning glory, whose seeds have been valued for their
hallucinogenic properties.
The source of the ergot strongly influences the type of alkaloids present,
as well as the clinical picture of ergotism [171]. The ergot alkaloids have three
types of physiological effects: they cause contraction of smooth muscle, they
block the action of serotonin and adrenaline, and they act on the hypothalamicpituitary system to inhibit the secretion of prolactin. These properties have led
to their being used to induce uterine contractions, to relieve migraine headaches, and to treat prolactin-dependent disorders.
The biosynthesis of the ergot alkaloids involves condensation of dimethylallyl pyrophosphate (derived from mevalonic acid) with tryptophan. Closure
of the C- and D-rings of the alkaloid involves specific hydroxylations by
cytochrome P450-dependent oxidases and rearrangements. Further modifications involve N-methylation in the presence of S-adenosyl methionine and/or
condensation with amino acids and peptides. Coupling of lysergyl CoA with
certain peptides forms the peptide alkaloids which are the most bioactive of the
ergot alkaloids.
Other Neurotropic Mycotoxins
A number of other mycotoxins exhibit neurotoxic activity. In many cases
these mycotoxins do not cause noticeable toxicity, but in some cases have
tremorgenic activity, as evidenced by uncontrollable shaking and disoriented
behavior. In some instances, animals will refuse food, and have lowered resistance to disease. Because the fungi that make some of these toxins, such as several strains of Aspergillus (A. fumigatus, A. flavus) and Penicillium (P. oxalicum)
are widespread and contaminate a variety of different grains, their importance
in causing mycological disease may be currently underestimated. Like the ergot
alkaloids, some of these are derived from tryptophan, for example the tremorgenic toxins fumitremorgin, verruculogen, and roquefortine produced by certain
strains of Aspergillus and Penicillium spp. These mycotoxins contain both the
Bhatnagar/Yu/Ehrlich
192
indole ring of tryptophan and a dioxopiperazine ring formed by condensation
of two amino acids. The fungus that makes roquefortine, P. roquefortii, is used
for the manufacture of roquefort cheese and other blue cheeses. The low levels
of toxin in these cheeses are not considered to be a problem for human health.
Aspergillus terreus, A. flavus and A. fumigatus have been found to produce
tremorgenic toxins territrem A, B, and C, and aflatrem [172]. A. flavus,
A. wentii, A. oryzae and Penicillium atrovenetum (as well as Arthrinium sp.) can
produce -nitropropionic acid which causes convulsions, congestion in lungs
and liver damage [173]. Citreoviridin and xanthomegnin, produced by
Penicillium spp. are also frequently found in foods. Toxins causing tremorgenic
effects in mice include penitrem (Penicillium crustosum and P. aurantiogriseum) and paspalitrem (also produced by Penicillium spp. and Claviceps
paspali) [174–179]. Fusarium verticilliodes also produces mycotoxins such as
fusarins [180–182], moniliformin [150, 183], fusaric acid, fusaproliferin and
beauvericin. Fusarin C appears to be a potent mutagen. Aspergillus terreus,
A. fumigatus and Trichoderma viride also produce gliotoxin that may have
immunosuppressive effects in animals [184].
Management of Mycotoxin Contamination
The economic implications of the mycotoxin problem and its potential
health threat to humans have clearly created a need to eliminate or at least
minimize mycotoxin contamination of food and feed. While an association
between mycotoxin contamination and inadequate storage conditions has
long been recognized, studies have revealed that seeds are contaminated with
mycotoxins prior to harvest [185]. Therefore, management of mycotoxin
contamination in commodities must include both pre- and postharvest control
measures.
Preharvest Control
Preharvest mycotoxin contamination can be reduced somewhat by appropriate cultural practices such as irrigation, control of insect pests [reviewed in
ref. 186–189]. In some cases, changes in cultural practices to minimize toxin
contamination are not feasible. For example, inclusion of extra irrigation
regimes in desert-grown cotton fields is not feasible because it would add
significant cost to the grower even if irrigation were available. However,
significant control of toxin contamination is expected to be dependent on a
Toxins of Filamentous Fungi
193
detailed understanding of the physiological and environmental factors that
affect the biosynthesis of the toxin, the biology and ecology of the fungus,
and the parameters of the host plant-fungal interactions. Efforts are under way
to study these parameters primarily for the most agriculturally significant
toxins, namely aflatoxins, fumonisins, and trichothecenes [reviewed in ref. 155,
189–191].
While fumonisins may not be essential for virulence, they may play a
significant role in fungal infection of corn [190]. And, since Fusarium verticilloides hyphae are found to be confined to the pedicel of the corn kernel, introduction by genetic engineering or plant breeding and expression of antifungal
proteins in the pedicel region of corn kernel could impart resistance in corn
against preharvest fumonisin contamination [192].
F. graminearum is the causative organism of head blight (scab) of wheat,
and trichothecene produced by this fungus appears to be involved in fungal
virulence [157]. This suggests that resistance in wheat against trichothecene
production may provide a means of controlling not only head blight but
preharvest contamination of crops with this mycotoxin as well [157].
Use of atoxigenic biocompetitive, native Aspergillus flavus strains to outcompete the toxigenic isolates has been effective in significantly reducing
aflatoxin contamination [193]. Breakthroughs in the understanding of the molecular biology of aflatoxin biosynthesis have enabled the selective designing of
effective biocompetitive agents whenever a native biocontrol strain is not available [189]. Biocompetition may be more feasible in crops such as cotton where
resistance against fungal infections is not available due to limited genetic diversity in the cotton germplasm. In the long term, however, significant control of the
aflatoxin problem will likely be linked to introduction of resistant germplasm,
which is resistant either to fungal invasion or toxin production. Naturally resistant germplasm is available, but the identification of specific biochemical factors
linked to resistance against A. flavus are assisting in enhancing the observed
resistance levels in the existing germplasm as well as in the identification of
novel germplasm that demonstrates the desired characteristics, i.e. the presence
of the biochemical factors [189, 194]. However, the aflatoxin contamination
process is so complex [187] that a combination of approaches will be required to
eliminate or even control the preharvest toxin contamination problem [48].
Postharvest Control
Postharvest mycotoxin contamination is prevalent in most tropical countries due to a hot wet climate coupled with sub-adequate methods of harvesting,
handling and storage practices, which often lead to severe fungal growth and
Bhatnagar/Yu/Ehrlich
194
mycotoxin contamination of food and feed [188, 195–197]. Sometimes contaminated food has been diverted to animal feed to prevent economic losses and
health concerns. However, this is not a solution to the contamination problem.
Irradiation has been suggested as a possible means of controlling insect and
microbial populations in stored food, and consequently, reducing the hazard of
mycotoxin production under these conditions [reviewed in Sharma, 198].
However, significant emphasis has been placed on detoxification methods to
eliminate the toxins from the contaminated lots or at least reduce the toxin
hazards by bringing down the mycotoxin levels under the acceptable limits.
Detoxification of aflatoxins has been investigated extensively, particularly in
corn and peanuts. Comparatively fewer reports are available on detoxification
of other mycotoxins, such as zearalenone, tricothecenes, citrinin and ochratoxin.
Detoxification of mycotoxins can be achieved by removal or elimination of
the contaminated commodities or by the inactivation of toxins present in the
commodities through various physical, chemical and biological means. For a
successful detoxification procedure, the following basic criteria have been
suggested [199]:
(1) It must be economical, so that the detoxified commodities may still be sold
at competitive prices.
(2) The process must be relatively simple, not too time-consuming, and
suitable for operation by unskilled labor.
(3) It must be capable of removing all traces of active toxin and the chemical
or biochemical residues must not constitute a health hazard.
(4) The nutritional quality must not be impaired.
Various detoxification processes can be categorized as follows:
Removal or elimination of mycotoxins. Since most of the mycotoxin
burden in contaminated commodities is localized to a relatively small number
or seeds or kernels [reviewed in Dickens, 200], removal of these contaminated
seeds/kernels is effective in detoxifying the commodity. Methods currently
used include (a) physical separation by identification and removal of damaged
seeds, mechanical or electronic sorting, flotation and density separation of
damaged or contaminated seeds; (b) removal by filtration and adsorption onto
filter pads, clays, activated charcoal; (c) removal of the toxin by milling
processes; and (d) removal of the mycotoxin by solvent extraction.
Inactivation of mycotoxins. When removal or elimination of mycotoxins is
not possible, mycotoxins can be inactivated by (a) physical methods such as
thermal inactivation, photochemical or gamma irradiation; (b) chemical methods
such as treatment of commodities with acids, alkalis, aldehydes, oxidizing
agents, and gases like chlorine, sulfur dioxide, ozone and ammonia [201]; and
(c) biological methods such as fermentations or enzymatic digestions that cause
the breakdown of mycotoxins [202].
Toxins of Filamentous Fungi
195
The commercial application of only some of these detoxifying mechanisms is feasible because, in a number of cases, the methods will be limited
by factors such as the toxicity of the detoxifying agent, nutritional or aesthetic
losses during treatment, and the cost of the sophistication of the treatment [188,
203]. Moreover, at this time there exist regulatory barriers against interstate
commerce of decontaminated commodities, and sale of any mycotoxincontaminated human food treated by detoxifying agents. Nevertheless, ammoniation process has been used by some countries and states of the USA for
detoxification of aflatoxins in corn and peanut meal for feed purpose.
Dietary Considerations
Modifications to diets, such as use of agents for absorption, distribution
and metabolism of mycotoxins, can affect the toxicity of mycotoxins. Dietary
additives including anticarcinogenic substances [204–206] and chemoprotective
agents [207–211] have been found to inhibit the carcinogenic effects of aflatoxins in test animals by preventing formation of aflatoxin-DNA adducts [212].
Antioxidants such as vitamin C and E reduced the toxic effects of ochratoxin A
[213, 214], whereas ascorbic acid provided protection against aflatoxins [215].
The toxic effects of ochratoxin have also been reduced by aspartame because it
competitively prevents the binding of ochratoxin to serum albumin [216].
The introduction of hydrated sodium calcium aluminosilicates in diets of
animals has reduced the toxicity of various mycotoxins (such as aflatoxins) to
animals because these and related compounds (hydrated sodium calcium
aluminosilicate or novasil) have a high affinity for mycotoxins [217–221] that
prevents the absorption of mycotoxins by animals. Other absorbants that have
been tested for mycotoxins such as T-2 toxin included zeolite, bentonite and
superactive charcoal.
Summary
Mycotoxins are low-molecular-weight secondary metabolites of fungi. The most
significant mycotoxins are contaminants of agricultural commodities, foods and feeds. Fungi
that produce these toxins do so both prior to harvest and during storage. Although contamination of commodities by toxigenic fungi occurs frequently in areas with a hot and humid
climate (i.e. conditions favorable for fungal growth), they can also be found in temperate
conditions. Production of mycotoxins is dependent upon the type of producing fungus and
environmental conditions such as the substrate, water activity (moisture and
relative humidity), duration of exposure to stress conditions and microbial, insect or other
animal interactions. Although outbreaks of mycotoxicoses in humans have been documented,
Bhatnagar/Yu/Ehrlich
196
several of these have not been well characterized, neither has a direct correlation between the
mycotoxin and resulting toxic effect been well established in vivo. Even though the specific
modes of action of most of the toxins are not well established, acute and chronic effects in
prokaryotic and eukaryotic systems, including humans have been reported. The toxicity of
the mycotoxins varies considerably with the toxin, the animal species exposed to it, and the
extent of exposure, age and nutritional status. Most of the toxic effects of mycotoxins are
limited to specific organs, but several mycotoxins affect many organs. Induction of cancer
by some mycotoxins is a major concern as a chronic effect of these toxins. It is nearly impossible to eliminate mycotoxins from the foods and feed in spite of the regulatory efforts at the
national and international levels to remove the contaminated commodities. This is because
mycotoxins are highly stable compounds, the producing fungi are ubiquitous, and food
contamination can occur both before and after harvest. Nevertheless, good farm management
practices and adequate storage facilities minimize the toxin contamination problems. Current
research is designed to develop natural biocontrol competitive fungi and to enhance host
resistance against fungal growth or toxin production. These efforts could prevent toxin
formation entirely. Rigorous programs for reducing the risk of human and animal exposure
to contaminated foods and feed also include economically feasible and safe detoxification
processes and dietary modifications. Although risk assessment has been made for some
mycotoxins, additional, systematic epidemological data for human exposure is needed for
establishing toxicological parameters for mycotoxins and the safe dose for humans. It is
unreasonable to expect complete elimination of the mycotoxin problem. But multiple
approaches will be needed to minimize the economic impact of the toxins on the entire
agriculture industry and their harmfulness to human and animal health.
References
1 Van Egmond HP, Speijers, GJA: Survey of data on the incidence and levels of ochratoxin A in food
and animal feed worldwide. Nat Toxins 1994;3:125–144.
2 Bennett JW: Mycotoxins, mycotoxicoses, mycotoxicology and mycopathologia. Mycopathologia
1987;100:3–5.
3 Scudmore KA: Mycotoxins: in Watson D (ed): Natural Toxicants in Food. Boca Raton, CRC Press
2000, pp 147–181.
4 Chu FS: Mycotoxins and mycotoxicosis: in Reimann D, Cliver DO (eds): Foodborne Infections
and Intoxications, ed 3. New York, Academic Press, in press.
5 Bhatnagar D, Ehrlich KC, Chang P-K: Mycotoxins in Agriculture. Encyclopedia of Life Sciences.
London, Nature Publishing Group, 2000;12:564–573.
6 Malloy CD, Marr JS: Mycotoxins and Public Health: A Review. J Publ Health Manage Pract
1997;3:61–69.
7 D’Mello JPF, Macdonald AMC: Mycotoxins. Animal Feed Sci Technol 1997;69:155–166.
8 Scott PM: The natural occurrence of trichothecenes; in Beasley VD (ed): Trichothecene Mycotoxicosis. Pathophysiologic Effects. Boca Raton, CRC Press, 1989, pp 1–26.
9 Strange RN: Natural Occurrence of mycotoxins in groundnuts, cottonseed, soys and cassava:
in Smith JE, Henderson RS (eds): Mycotoxins and Animal Foods. Boca Raton, CRC Press, 1991,
pp 341–362.
10 Shotwell OL: Natural occurrence of mycotoxins in corn; in Smith JE, Henderson RS (eds):
Mycotoxins and Animal Foods. Boca Raton, CRC Press, 1991, pp 325–340.
11 Marasas WFO: Fumonisins: Their implications for human and animal health. Nat Toxins 1995;
3:193–198.
Toxins of Filamentous Fungi
197
12 Yoshizawa T: Natural occurrence of mycotoxins is small grain cereals (wheat, barley, rye, oats,
sorghum, millet, rice): in Smith JE, Henderson RS (eds): Mycotoxins and Animal Foods. Boca
Raton, CRC Press, 1991, pp 301–324.
13 Schoental R: Mycotoxins and the Bible. Perspect Biol Med 1984;28:117–120.
14 Marr JS, Malloy CD: An epidemiologic analysis of the ten plagues of Egypt. Caduceus 1996;
12:7–24.
15 Schoental R: Mycotoxins in food and the plague of Athens. J Nutr Med 1994;4:83–85.
16 Peraica M, Radic B, Lucic A, Pavlovic M: Toxic effects of mycotoxins in humans. Bull World
Health Organ 1999;77:754–766.
17 Matossian MK: Poisons of the past. Molds, Epidemics, and History. New Haven, Yale University
Press, 1989.
18 Castiglioni A: A History of Medicine. New York, Knopf, 1947.
19 Cartwright, FF: Disease and History. New York, Thomas, Crowell, 1975.
20 Van Dongen PWJ, DeGroot ANJA: History of ergot alkaloids from ergotism to ergometrine.
Eur J Obstet Gynec Reprod Biol 1995;60:109–116.
21 Maxcy KF (ed): Rosenau Preventive Medicine and Public Health. New York, AppletonCentury-Crofts, 1956.
22 Ackerknocht EH: History and Geography of the Most Important Diseases. New York, Hafner, 1965.
23 Christiansen CM, Kauffman HH: Grain storage: The role of fungi in quality loss. Minneapolis,
University of Minneapolis Press, 1969.
24 Bradburn N, Coker RD, Blunden G: The aetiology of Turkey X disease. Phytochemistry 1994;
35:817.
25 Sharma RP, Salunkhe DK: Animal and plant toxins; in Gupta PK, Salunkhe DK (eds): Modern
Toxicology. New Delhi, Metropolitan Book Co, 1985, vol 1, pp 252–316.
26 Mannon J, Johnson E: Fungi down on the farm. New Sci 1985;1445:12–16.
27 Gordon KE, Masotti RE, Waddell WR: Tremorgenic encephalopathy: A role of mycotoxins in
production of CNS disease in humans. Can J Neurol Sci 1993;20:237–239.
28 Hendry KW, Cole EC: A review of mycotoxins in indoor air. J Toxicol Environ Health 1993;
38:183–198.
29 Torrey L: Yellow rain: Is it really a weapon. New Sci, August 4, 1983, p 350.
30 Dayton L: Iranian scientist fails in attempt to obtain toxic fungi. New Sci, September 18, 1989, p 22.
31 Hanchette J, Brewer N: UN intelligence reports show Iraq could have spread deadly aflatoxin.
Gannett News Service, December 7, 1996.
32 International Agency for Research on Cancer: Some naturally occurring substances: Food items,
and constituents, heterocyclic aromatic amines and mycotoxins: IARC Monogr on the Evaluation
of Carcinogenic Risks to Humans. Lyon, World Health Organization, 1993, vol 56.
33 Turner WB, Aldridge DC: Fungal Metabolites II. Academic Press, London, 1983.
34 Boutrif E: FAO programmes for prevention, regulation, and control of mycotoxins in food. Nat
Toxins 1995;3:322–326.
35 Sharma RP, Salunkhe DK: Mycotoxin and Phytoalexins in Human and Animal Health. Boca Raton,
CRC Press, 1991.
36 Van Egmond HP: Rationale for regulatory programmes for mycotoxins in human foods and
animal feeds. Food Addit Contam 1993;10:29–36.
37 Van Egmond HP, Speijers GJA: Natural Toxins. I. Mycotoxins: in Vander Heijden K, Younes M,
Fishbein L, Miller S (eds): International Food Safety Handbook: Science International Regulation
and Control. New York, Dekker, 1999, pp 341–355.
38 Coker RD: Design of sampling plans for determination of mycotoxin in food and feed; in Sinha KK,
Bhatnagar D (eds): Mycotoxin in Agriculture and Food Safety. New York, Dekker, 1998, pp 109–134.
39 Wilson DM, Sydenham EW, Lombaert GA, Trucksess MW, Abramson D, Bennett GA: Mycotoxin
analytical techniques; in Sinha KK, Bhatnagar D (eds): Mycotoxin in Agriculture and Food Safety.
New York, Dekker, 1998, pp 135–182.
40 Chu FS: Development and use of immunoassays in detection of the ecologically important
mycotoxins; in Bhatnagar D, Lillehoj EB, Arora DK (eds): Handbook of Applied Mycology.
Mycotoxins in Ecological Systems. New York, Dekker, 1991, pp 87–136.
Bhatnagar/Yu/Ehrlich
198
41 Chu FS: Mycotoxin analysis; in Jeon IJ, Ikins WG (eds): Analyzing Food for Nutrition Labeling
and Hazardous Contaminants. New York, Dekker, 1995, pp 283–332.
42 Chu FS: Recent studies on immunoassays for mycotoxins; in Beier RC, Stanker LH (eds):
Immunoassays for Residue Analysis. Food Safety ACS Symposium Series Book, Washington,
American Chemical Society, 1996, No 621, pp 294–313.
43 Riley RT, Norred WP: Mechanism of mycotoxicity: in DH Howard, JD Miller (eds): The Mycota.
Berlin, Springer, 1996, vol 6, pp 193–211.
44 Riley RT: Mechanistic interactions of mycotoxins. Theoretical considerations; in Sinha KK,
Bhatnagar D (eds): Mycotoxins in Agriculture and Food Safety. New York, Dekker, 1998,
pp 266–254.
45 Goldblatt LA: Aflatoxin – Scientific Background, Control and Implications. New York, Academic
Press, 1969.
46 Goto T, Wicklow DT, Ito Y: Aflatoxin and cyclopiazonic acid production by a sclerotiumproducing Aspergillus tamarii strain. Appl Environ Microbiol 1996;62:4036–4038.
47 Bhatnagar D, Lillehoj EB, Arora DK (eds): Mycotoxins in Ecological Systems. Handbook of
Applied Mycology. New York, Dekker, 1992.
48 Bhatnagar D, Payne G, Linz JE, Cleveland TE: Molecular biology to eliminate aflatoxin. Inte
News Fats, Oils Relat Mater 1995;6:262–271.
49 Dutton MF: Enzymes and aflatoxin biosynthesis. Microbiol Rev 1988;52:274–295.
50 Townsend CA: Progress towards a biosynthetic rationale of the aflatoxin pathway. Pure Appl
Chem 1997;58:227–238.
51 Trail F, Mahanti N, Linz J: Molecular biology of aflatoxin biosynthesis. Microbiology 1995;
141:755–765.
52 Payne GA, Brown MP: Genetics and physiology of aflatoxin biosynthesis. Annu Rev Phytopathol
1998;36:329–362.
53 Cary JW, Bhatnagar D, Linz JE: Aflatoxin: Biological significance and regulation of biosynthesis:
in Cary JW, Linz JE, Bhatnagar D (eds): Microbial Foodborne Diseases. Mechanism of pathogenesis and toxin synthesis. Lancaster, Technomic Publishing, 2000, pp 317–361.
54 Bhatnagar D, Cleveland TE, Kingston DGI: Enzymological evidence for separate pathways for
aflatoxin B1 and B2 biosynthesis. Biochemistry 1991;30:4343–4350.
55 Yu J, Chang P-K, Ehrlich KC, Cary JW, Montalbano B, Dyer JM, Bhatnagar D, Cleveland TE:
Characterization of the critical amino acids of an Aspergillus parasiticus cytochrome P450
mono-oxygenase encoded by ordA that is involved in the biosynthesis of aflatoxins B1, G1, B2 and
G2. Appl Environ Microbiol 1998;64:4834–4841.
56 Yabe K, Nakamura M, Hamasaki T: Enzymatic formation of G-group aflatoxin and biosynthetic
relationship between G and B group aflatoxins. Appl Environ Microbiol 1999;65:3867–3872.
57 Cary JW, Wright M, Bhatnagar D, Lee R, Chu FS: Molecular characterization of an Aspergillus
parasiticus dehydrogenase gene, norA, located on the aflatoxin biosynthesis gene cluster. Appl
Environ Microbiol 1996;62:360–366.
58 Chang P-K, Cary JW, Yu J, Bhatnagar D, Cleveland TE: The Aspergillus parasiticus polyketide
synthase gene pksA, a homolog of Aspergillus nidulans wA, is required for aflatoxin B1 biosynthesis. Mol Gen Genetics 1995;248:270–277.
59 Chang P-K, Ehrlich KC, Yu J, Bhatnagar D, Cleveland TE: Increased expression of Aspergillus
parasiticus aflR, encoding a sequence-specific DNA-binding protein, relieves nitrate inhibition of
aflatoxin biosynthesis. Appl Environ Microbiol 1995;61:2372–2377.
60 Chang PK, Ehrlich KC, Linz JE, Bhatnagar D, Cleveland TE, Bennett JW: Characterization of the
Aspergillus parasiticus niaD and niiA gene cluster. Curr Genet 1996;30:68–75.
61 Payne GA, Nystrom GJ, Bhatnagar D, Cleveland TE, Woloshuk CP: Cloning of the afl-2 gene
involved in aflatoxin biosynthesis for Aspergillus flavus. Appl Environ Microbiol 1993;59:
156–162.
62 Prieto R, Woloshuk CP: ord1, an oxidoreductase gene responsible for conversion of O-methylsterigmatocystin to aflatoxin in Aspergillus flavus. Appl Environ Microbiol 1997;63:1661–1666.
63 Woloshuk CP, Yousibova GL, Rollins JA, Bhatnagar D, Payne GA: Molecular characterization of
the afl-1 locus in Aspergillus flavus. Appl Environ Microbiol 1995;61:3019–3023.
Toxins of Filamentous Fungi
199
64 Yu J, Chang PK, Cary JW, Wright M, Bhatnagar D, Cleveland TE, Payne GA, Linz JE: Comparative
mapping of aflatoxin pathway gene cluster in Aspergillus parasiticus and Aspergillus flavus. Appl
Environ Microbiol 1995;61:2365–2371.
65 Yu JH, Butchko RAE, Fernandes M, Keller NP, Leonard TJ, Adams TH: Conservation of structure
and function of the aflatoxin regulatory gene aflR from Aspergillus nidulans and A. flavus. Curr
Genet 1996;29:549–555.
66 Cullen JM, Ruebrier BH, Hsieh LS, Hyde DM, Hsieh DP: Carcinogenicity of dietary aflatoxin M1
in male Fischer rats compared to aflatoxin B1. Cancer Res 1987;47:1913–1917.
67 Galvano F, Galofaro V, Galvano G: Occurrence and stability of aflatoxin M1 in milk and milk
products: A worldwide review. J Food Protect 1996;59:1079–1090.
68 Van Egmond HP: Current situation on regulations for mycotoxins: Overview of tolerances and
status of standard methods of sampling and analysis. Food Addit Contam 1989;6:134–188.
69 Busby WF Jr, Wogan GN: Food-borne mycotoxins and alimentary mycotoxicoses: in Rieman H,
Byran FL (ed): Food-borne Infections and Intoxications. New York, Academic Press, 1979.
70 Busby WF Jr, Wogan GN: Aflatoxins: in Shank RC (ed.): Mycotoxins and N-nitrosocompounds:
Environmental Risks. Boca Raton, CRC Press, 1981, vol 2, pp 3–45.
71 CAST: Mycotoxins, Economics and Health Risks. Council of Agricultural Science and
Technology (CAST), Task force report No. 116. Ames, CAST, 1989, p 92.
72 Eaton DL, Groopman JD (eds): The toxicology of aflatoxins-Human health, Veterinary and
Agricultural significance. New York, Academic Press, 1994.
73 Massey TE: The 1995 Pharmacological Society of Canada Merck Frosst award – Cellular and
molecular targets, in pulmonary chemical carcinogenesis – studies with aflatoxin B1. Can J Physiol
Pharmacol 1996;74:621–628.
74 Heinonen JT, Fisher R, Brendel K, Eaton DL: Determination of aflatoxin B1 biotransformation
and binding to hepatic macromolecules in human precision liver slices. Toxicol Appl Pharmacol
1996;136:1–7.
75 Kelly JD, Eaton DL, Guengerich FP, Coulombe RA: Aflatoxin B1 activation in human lung.
Toxicol Appl Pharmocol 1997;144:88–95.
76 Newberne PM, Rogers AE: Animal toxicity of major environmental mycotoxins: in Shank RC
(ed): Mycotoxins and Nitroso Compounds, Environmental Risks. Boca Raton, CRC Press, 1981,
vol 1, pp 51–106.
77 Wild CP, Kleihues P: Etiology of cancer in human and animals. Exp Toxic Pathol 1996;48:95–100.
78 Wogan GN: Aflatoxins as risk factors for hepatocellular carcinoma in humans. Cancer Res
1992;52(suppl):2114s–2118s.
79 Hsieh DPH: Potential human health hazards of mycotoxins: in Natori S, Hashimoto K, Ueno Y
(eds): Mycotoxins and Phycotoxins 88. Amsterdam, Elsevier, 1989, pp 69–80.
80 Peers F, Bosch X, Kaldor J, Linsell A, Pluijiman M: Aflatoxin exposure, hepatitis B virus infection and liver cancer in Swaziland. Int J Cancer 1987;39:545–553.
81 Wild CP, Shresma SM, Anwar WA, Montesano R: Field studies of aflatoxin exposure, metabolism
and induction of genetic alterations in relation to HBV infection and hepatocellular carcinoma in
The Gambia and Thailand. Toxicol Lett 1992;64/65:455–461.
82 Zhu JQ, Zhang LS, Hu X, Xiao Y, Chen JS, Xu YC, Fremy J, Chu FS: Correlation of dietary aflatoxin B1 levels with excretion of aflatoxin M1 in human urine. Cancer Res 1987;47:1848–1852.
83 Groopman JD, Wogan GN, Roebuck BD, Kensler TW: Molecular biomarkers for aflatoxins and
their application to human cancer prevention. Cancer Res 1994;54:1907S–1911S.
84 Harris CC, Sun TT: Interactive effects of chemical carcinogens and hepatitis B virus in the
pathogenesis of hepatocellular carcinoma. Cancer Surv 1986;5:765–780.
85 Chen CJ, Wang LY, Lu SN, Wu MH, You SL, Zhang YL, Wang LW, Santella RM: Elevated
aflatoxin exposure and increased risk of hepatocellular carcinoma. Hepatology 1996;24:38–42.
86 Chen CJ, Yu MW, Liaw YF, Wang LW, Chiamprasert S, Matin F, Hirvonen A, Bell DA, Santella
RM: Chronic hepatitis B carriers with null genotypes of glutathione S-transferase M1 and T1
polymorphisms who are exposed to aflatoxin are at increased risk of hepatocellular carcinoma.
Am J Human Genet 1996;59:128–134.
87 Chu FS: Mycotoxins: in Cliver DO (ed): Foodborne Diseases, ed 2. New York, Academic Press,
in press.
Bhatnagar/Yu/Ehrlich
200
88 Chu FS: Studies on ochratoxins. Crit Rev Toxicol 1974;2:499–524.
89 Kuiper-Goodman T, Scott PM: Risk assessment of the mycotoxin ochratoxin A. Biomed Environ
Sci 1989;2:179–248.
90 Pohland AE, Nesheirn S, Friedman L: Ochratoxin A: A review. Pure Appl Chem 1992;64:
1029–1046.
91 Abarca MK, Bragulat MR, Castella G, Cobanes FJ: Ochratoxin A production by strains of
Aspergillus niger var. niger. Appl Environ Microbiol 1994;60:2650–2652.
92 Ono H, Kataoka A, Koakutsu M, Tanaka K, Kawasugi S, Wakazawa M, Ueno Y, Manabe M:
Ochratoxin producibility by strain of Aspergillus niger group stored in IFO culture collection.
Mycotoxins 1995;41:47–51.
93 Studer-Rohr I, Dietrich DR, Schlatter J, Schlatter C: The occurrence of ochratoxin A in coffee.
Food Chem Toxicol 1995;33:341–355.
94 Chu FS: Studies on ochratoxins. Crit Rev Toxicol 1974;2:499–524.
95 Fink-Gremmels J, Jahn A, Blom MJ: Toxicity and metabolism of ochratoxin A. Nat Toxins
1995;3:214–220.
96 Simon P: Ochratoxin and kidney disease in the human. J Toxicol Toxin Rev 1996;15:239–249.
97 Boorman G: NTP technical report on the toxicology and carcinogenesis studies of ochratoxin A
(CAS No. 303-47-9) in F344/N rats (gavage studies). NIH publication No 89-2813. US Dept of
Health and Human Services, NIH, Bethesda, 1988.
98 Boorman GA, McDonald MR, Imoto S, Persing R: Renal lesions induced by ochratoxin A
exposure in the F344 rat. Toxicol Pathol 1992;20:236–245.
99 Hayes AW: Mycotoxin Teratogenicity and Mutagenicity. Boca Raton, CRC Press, 1981.
100 deGroene EM, Jahn A, Horbach GJ, FinkGremmels J: Mutagenicity and genotoxicity of the
mycotoxin ochratoxin A. Environ Toxicol Pharmacol 1996;1:21–26.
101 Dirheimer G: Mechanistic approaches to ochratoxin toxicity. Food Addit Contam 1996;13(suppl):
45S–48S.
102 Schlatter C, Studerrohr J, Rasonyi T: Carcinogenicity and kinetic aspects of ochratoxin A. Food
Addit Contam 1996;13(suppl):43S–44S.
103 Stegen GVD, Jorissen U, Pittet A, Saccon M, Steiner W, Vincenzi M, Winkler M, Zapp J, Schlatter
C: Screening of European coffee final products for occurrence of ochratoxin A (OTA). Food Addit
Contam 1997;14:211–216.
104 Krogh P: Epidemiology of mycotoxic porcine nephropathy. Nord Vet Med 1976;28:452–458.
105 Maaroufi K, Achour A, Zakharna A, Ellouz F, el May M, Creppy EE, Bacha H: Human
nephropathy related to ochratoxin A in Tunisia. J Toxicol Toxin Rev 1996;15:223–237.
106 Petkova-Bocharova T, Castegnaro M: Ochratoxin A contamination of cereals in an area of high
incidence of Balkan endemic nephropathy in Bulgaria. Food Addit Contam 1985;2:267–270.
107 Huang X, Dorner JW, Chu FS: Production of aflatoxin and cyclopiazonic acid by various
aspergilli: An ELISA analysis. Mycotoxin Res 1994;10:101–106.
108 Riley RT, Goeger DE: Cyclopiazonic acid: Speculation on its function in fungi; in Bhatnagar D,
Lillehoj EB, Arora DK (eds): Handbook of Applied Mycology. Mycotoxins in Ecological
Systems. New York, Dekker, 1991, vol 5, pp 385–402.
109 Tournas V: Heat-resistant fungi of importance to the food and beverage industry. Crit Rev
Microbiol 1994;20:243–263.
110 Scott PM: Patulin: in Purchase IFH (ed): Mycotoxins. New York, Elsevier, 1975, pp 383–403.
111 Wilson BJ, Hayes AW: Microbial toxins: in Toxicants Occurring Naturally in Foods. Washington,
NRC, US Natl Acad Sci, 1973, pp 372–423.
112 Cole RJ, Cox EH: Handbook of Toxic Fungal Metabolites. New York, Academic Press, 1981.
113 Pastrana L, Loret MO, Blanc PJ, Goma G: Production of citrinin by Monascus ruber submerged
culture in chemical defined media. Acta Biotech Environ Microbiol 1996;16:315–319.
114 Betina V: Mycotoxins: Chemical Biological and Environmental Aspects. Amsterdam, Elsevier, 1989.
115 Chu FS: Mycotoxins: Food contamination, mechanism, carcinogenic potential and preventive
measures. Mutat Res 1991;259:291–306.
116 Mori H, Kawai K: Genotoxicity in rodent hepatocytes and carcinogenicity of mycotoxins and
related chemicals: in Natori S, Hashimoto K, Ueno Y (eds): Mycotoxins and Phycotoxins 88.
Amsterdam, Elsevier, 1989, pp 81–90.
Toxins of Filamentous Fungi
201
117 Van der Watt JJ: Sterigmatocystin: in Purchase IFH (ed): Mycotoxins. New York, Elsevier, 1977,
pp 368–382.
118 Jelinek CF, Pohland AE, Wood GE: Worldwide occurrence of mycotoxins in foods and feeds-an
update. J Assoc Off Anal Chem 1989;72:223–230.
119 Zhang RF, Chen CS, Yu L, Sun HL, Fu CG, Xu HD: Aspergillus versicolor and sterigmatocystin
might be related to genesis of gastric cancer. Abstract 7th Int Union of Pure and Applied
Chemistry (IUPAC), International Symposium on Mycotoxins and Phytotoxins, Tokyo 1988.
120 Dutton MF: Fumonisins, mycotoxins of increasing importance: Their nature and their effects.
Pharmacol Ther 1996;70:137–161.
121 Jackson L, DeVries JW, Bullerman LB (eds): Fumonisins in Food. Plenum Press, New York, 1996.
122 Riley RT, Richard JL (eds): Fumonisins: A current perspective and view to the future.
Mycopathologia 1992;117:1–124.
123 Riley RT, Norred WP, Bacon CW: Fungal toxins in foods: Recent concerns. Annu Rev Nutr 1993;
13:167–189.
124 Scott PM: Fumonisins. Int J Food Microbiol 1993;18:257–270.
125 Gelderblom WCA, Jaskiewicz K, Marasas WFO, Thiel PG, Horak RM, Vleggar R, Krek NPJ:
Fumonisins-novel mycotoxins with cancer-promoting activity produced by Fusarium moniliforme. Appl Environ Microbiol 1988;54:1806–1811.
126 Gelderblom WC, Marasas WFO, Vleggaar R, Thiel PG, Cawood ME: Fumonisins: Isolation,
chemical characterization and biological effects. Mycopathologia 1992;117:11–16.
127 Marasas WFO, Nelson PE, Toussoun TA: Toxigenic Fusarium Species: Identity and mycotoxicology. University Park, Pennsylvania State University Press, 1984.
128 Nelson PE, Desjardins AE, Plattner RD: Fumonisins: Mycotoxins produced by Fusarium species:
Biology, chemistry, and significance. Annu Rev Phytopathol 1993;31:233–252.
129 Norred WP: Fumonisins – Mycotoxins produced by Fusarium moniliforme. J Toxicol Environ
Health 1993;38:309–328.
130 Shier WT: Sphingosine analogs: An emerging new class of toxins that includes the fumonisins.
J Toxicol Toxin Rev 1992;11:241–257.
131 Seo JA, Kim JC, Lee YW: Isolation and characterization of two new type C fumonisins produced
by Fusarium oxysporum. J Nat Prod 1996;59:1003–1005.
132 Musser SM, Gay ML, Mazzola EP, Plattner RD: Identification of a new series of fumonisins
containing 3-hydroxypyridine. J Nat Prod 1996;59:970–972.
133 Abbas HK, Tanaka T, Shier WT: Biological activities of synthetic analogues of Alternaria
alternata toxin (AAL) and fumonisin in plant and mammalian cells. Phytochemistry 1995;40:
1681–1689.
134 Chen J, Mirocha CJ, Xie W, Hogge L, Olson D: Production of the mycotoxin fumonisin B1 by
Alternaria alternata f. sp. lycopersici. Appl Environ Microbiol 1992;58:3928–3931.
135 Abbas HK, Riley RT: The presence and phytotoxicity of fumonisins and AAL-toxin in Alternaria
alternata. Toxicon 1996;34:133–136.
136 Mirocha CJ, Gilchrist DG, Shier WT, Abbas HK, Wen Y, Vesonder RF: AAL toxins, fumonisins
(biology and chemistry) and host-specificity concepts. Mycopathologia 1992;117:47–56.
137 Hammer P, Bluthgen A, Walte HG: Carry-over of fumonisin B1 into milk of lactating cows.
Milchwissenschaft 1996;51:691–695.
138 Richard JL, Meerdink G, Maragos CM, Tumbleson M, Bordson G, Rice LG, Ross PF: Absence of
detectable fumonisins in the milk of cows fed Fusarium proliferatum (Matsushima) Nirenberg
culture material. Mycopathologia 1996;133:123–126.
139 Proctor RH: Fusarium toxins: Trichothecenes and Fumonisins; in Cary JW, Linz JE, Bhatnagar D
(eds): Microbial Foodborne Diseases: Mechanism of pathogenesis and toxin synthesis. Lancaster,
Technomic Publishing 2000, pp 363–381.
140 Desjardins AE, Plattner RD, Proctor RH: Linkage among genes responsible for fumonisin biosynthesis in Gibberella fujikuroi mating population A. Appl Environ Microbiol 1996;62:2571–2576.
141 Xu JR, Leslie JF: A genetic map of Gibberella fujikuroi mating population A (Fusarium moniliforme). Genetics 1996;143:175–189.
142 Gelderblom WCA, Krick NPJ, Marasas WFO, Thiel PG: Toxicity and carcinogenicity of the
Fusarium moniliforme metabolite, fumonisin B1 in rats. Carcinogenesis 1991;12:1247–1251.
Bhatnagar/Yu/Ehrlich
202
143 Gelderblom WC, Semple AE, Marasas WFO, Farber E: The cancer-initiating potential of the
fumonisin B mycotoxins. Carcinogenesis 1992;13:433–437.
144 Gelderblom WCA, Cawood ME, Snyman SD, Marasas WFO: Structure-activity relationships of
fumonisins in short-term carcinogenesis and cytotoxicity assays. Food Chem Toxicol 1993;
31:407–414.
145 Gelderblom WCA, Cawood ME, Snyman SD, Marasas WFO: Fumonisin B1 dosimetry in relation
to cancer initiation in rat liver. Carcinogenesis 1994;15:209–214.
146 Norred WP, Voss KA: Toxicity and role of fumonisins in animal diseases and human esophageal
cancer. J Food Prot 1994;57:522–527.
147 Beasley VR: Trichothecene Mycotoxicosis, Pathophysiologic Effects. Boca Raton, CRC Press,
1989, vol 1, p 175, vol 2, p 198.
148 Chu FS: Trichothecene mycotoxicosis: in Dulbecco R (ed): Encyclopedia of Human Biology, ed 2.
New York, Academic Press, 1997, vol 8, pp 511–522.
149 Jarvis BB, Salenime J, Morais A: Stachybotrys toxins. 1. Nat Toxins 1995;3:10–16.
150 Marasas WFO, Nelson PE, Toussoun TA: Toxigenic Fusarium species: Identity and Mycotoxicology. University Park, Pennsylvania State University Press, 1986.
151 Miller JD, Trenholm HL: Mycotoxins in grain: Compounds other than aflatoxin. St Paul, Eagan
Press, 1994.
152 Ueno Y: Trichothecenes: Chemical, Biological, and Toxicological aspects. Amsterdam, Elsevier, 1983.
153 Vesonder RF, Golinski P: in Chelkowski J (ed): Fusarium Mycotoxins, Taxonomy, and Pathogenicity.
New York, Elsevier, 1989, pp 1–39.
154 Desjardins AE, Hohn TM, McCormick SP: Trichothecene biosynthesis in Fusarium species:
Chemistry, genetics and significance. Microbiol Rev 1993;57:595–604.
155 Hohn TM, McCormick SP, Desjardins AE: Evidence for a gene cluster involving trichothecenepathway biosynthetic gene in Fusarium sporotrichioides. Curr Genet 1993;24:291–295.
156 Keller NP, Hohn TM: Metabolic pathway gene clusters in filamentous fungi. Fungal Genet Biol
1997;21:17–29.
157 Proctor RH, Desjardins AE, McCormick SP, Hohn TM: Trichothecene toxins and wheat head scab.
Proceedings of the USDA-ARS Fusarium/Fumonisin Workshop, Beltsville 1995, p 30.
158 Park J, Smalley EB, Chu FS: Natural occurrence of Fusarium mycotoxins of the 1992 corn crop
in the field. Appl Environ Microbiol 1996;62:1642–1648.
159 Anonymous: Protection against trichothecenes. Reported by Committee on Protection Against
Mycotoxins, National Research Council, National Academy of Science, National Academy Press,
Washington. 1983, pp 17–166.
160 Rotter BA, Prelusky DB, Pestka JJ: Toxicology of deoxynivalenol (vomitoxin). J Toxicol Environ
Health 1996;48:1–34.
161 Trenholm HL, Prelusky DB, Young JC, Miller JD: Reducing mycotoxins in animal feeds,
Agriculture Canada Publication 1827E. Agriculture Canada, Ottawa, Canada, 1988.
162 Luo XY: Fusarium toxin contamination of cereals in China. Proc Jpn Assoc Mycotoxicol
1988;Spec issue 1:97–98.
163 Nikulin M, Pasanen AL, Berg S, Hintikka EL: Stachybotrys atra growth and toxin production in
some building materials and fodder under different relative humidities. Appl Environ Microbiol
1994;60:3421–3424.
164 Chelkowski J (ed): Alternaria – metabolites, Biology and Plant diseases. Amsterdam, Elsevier,
1992.
165 Bottalico A, Logrieco A: Toxigenic Alternaria species of economic importance: in Sinha KK,
Bhatnagar D (eds): Mycotoxin in Agriculture and Food Safety. New York, Dekker, 1998, pp
65–108.
166 Woody MA, Chu FS: Toxicology of Alternaria mycotoxins: in Chelkowski J (ed): AlternariaMetabolites, Biology and Plant diseases. Amsterdam, Elsevier, 1992, pp 409–434.
167 Flieger M, Wurst M, Shelby R: Ergot alkaloids – sources, structures and analytical methods. Folia
Microbiol 1997;42:3–30.
168 King B: Outbreak of ergotism in Wollo, Ethiopia. Lancet 1979;i:1411.
169 Krishnamachari KAVR, Bhat RV: Poisoning of ergoty bajra (pearl millet) in man. Indian J Med
Res 1976;64:1624–1628.
Toxins of Filamentous Fungi
203
170 Tulpule PG, Bhat RV: Food toxins and their implication in human health. Indian J Med Res
1978;68(suppl):99–108.
171 Burfening PJ: Ergotism. J Am Vet Med Assoc 1973;163:1288–1290.
172 Ling KH: Territrems, tremorgenic mycotoxins isolated from Aspergillus terreus. J Toxicol Toxin
Rev 1995;13:243–252.
173 Liu XJ, Luo XY, Hu WJ: Arthrinium spp. and the etiology of deteriorated sugarcane poisoning: in
Natori S, Hashimoto K, Ueno Y (eds): Mycotoxins and Phycotoxins 88. Amsterdam, Elsevier,
1988, pp 109–118.
174 Kurata H, Ueno Y (eds): Toxigenic fungi – their toxins and health hazard. Kodansha, Tokyo/
Elsevier, New York, 1984.
175 Plumlee KH, Galey FD: Neurotoxic mycotoxins: A review of fungal toxins that cause neurological disease in large animals. J Vet Intern Med 1994;8:49–54.
176 Steyn PS: Mycotoxins, general view, chemistry and structure. Toxicol Lett 1995;82:843–851.
177 Yamaguchi T, Nozawa K, Hosoc T, Nakajima S, Kawai KL: Indoloditerpenes related to tremorgenic mycotoxin penitrems, from Penicillium crustosum. Phytochemistry 1993;32:1177–1181.
178 Penn J, Swift R, Wigley LJ, Mantle PG, Bilton JN, Sheppard RN: Janthitrems B and C, two principal indole-diterpenoids produced by Penicillium janthinellum. Phytochemistry 1993;32:
1431–1434.
179 Wilkins AL, Miles CO, Ede RM, Gallagher RT, Munday SC: Structure elucidation of janthitrem
B, a tremorgenic metabolite of Penicillium janthinellum, and relative configuration of the A and
B rings of janthitrems B, E, and F. J Agric Food Chem 1992;40:1307–1309.
180 Gelderblom WCA, Marasas WFO, Steyn PS, Thiel PG, Van der Merwe KJ, Rooyen HP, Vleggaar
R, Wessels PL: Structure elucidation of fusarin C, a mutagen produced by Fusarium moniliforme.
J Chem Soc Chem Commun 1984;1984:122–124.
181 Lu FX, Jeffrey AM: Isolation, structural identification, and characterization of a mutagen from
Fusarium moniliforme. Chem Res Toxicol 1993;6:91–96.
182 Wiebe LA, Bjeldanes LF: Fusarin C, a mutagen from Fusarium moniliforme grown on corn.
J Food Sci 1981;46:1424–1426.
183 Burmeister HR, Ciegler A, Vesonder RF: Moniliformin, a metabolite of Fusarium moniliforme
NRRL 6322: Purification and toxicity. Appl Environ Microbiol 1979;37:11–13.
184 Waring P, Beaver J: Gliotoxin and related epipolythiodioxopiperazines. Gen Pharmacol 1996;
27:1311–1314.
185 Lisker N, Lillehoj EB: Prevention of mycotoxin contamination (principally aflatoxins and
Fusarium toxins) at the preharvest stage; in Smith JE, Henderson RS (eds): Mycotoxins and
Animal Foods. Boca Raton, CRC Press, 1991, pp 689–719.
186 Sinha KK, Bhatnagar D (eds): Mycotoxin in Agriculture and Food Safety. New York, Dekker, 1988.
187 Payne GA: Process of contamination by aflatoxin-producing fungi and their impact on crops; in
Sinha KK, Bhatnagar D (eds): Mycotoxins in Agriculture and Food Safety. New York, Dekker,
1998, pp 278–306.
188 Sinha KK: Detoxification of mycotoxins and food safety; in Sinha KK, Bhatnagar D (eds):
Mycotoxin in Agriculture and Food Safety. New York, Dekker, 1998, pp 381–406.
189 Brown RL, Bhatnagar D, Cleveland TE, Cary JW: Recent advances in preharvest prevention of
mycotoxin contamination; in Sinha KK, Bhatnagar D (eds): Mycotoxins in Agriculture and Food
Safety. New York, Dekker, 1998, pp 351–380.
190 Desjardins AE, Plattner RD, Nelson TC, Leslie J: Genetic analysis of fumonisin production and
virulence of Gibberella fujikuroi mating population A (Fusarium moniliforme) on maize (Zea
mays) seedlings. Appl Environ Microbiol 1995;61:79–86.
191 Hohn TM, Desjardins AE, McCormick SP, Proctor RH: Biosynthesis of trichothecenes, genetic
and molecular aspects; in Eklund M, Richard JL, Mise K (eds): Molecular Approaches to Food
Safety: Issues Involving Toxic Microorganisms. Ft. Collins, Alaken, 1995, pp 239–248.
192 Muhitch MJ: A genetic engineering approach to lowering fumonisin levels in maize kernels.
Proceedings of the USDA-ARS Fusarium/Fumonisin Workshop, Beltsville 1995, p 27.
193 Cotty PJ, Bhatnagar D: Variability among atoxigenic Aspergillus flavus strains in ability to
prevent aflatoxin contamination and production of aflatoxin biosynthetic pathway enzymes.
Appl Environ Microbiol 1994;60:2248–2251.
Bhatnagar/Yu/Ehrlich
204
194 Chen Z-Y, Cleveland TE, Brown RL, Bhatnagar D, Cary JW, Rajasekaran K: Corn as a source of
antifungal genes for genetic engineering of crops for resistance to aflatoxin contamination.
American Chemical Society Publication, in press.
195 FAO: Prevention of mycotoxins. FAO Food and Nutrition, paper No 10, 1979, p 71.
196 Phillips TD, Clement BA, Park DL: Approaches to reduction of aflatoxin in foods and feeds; in
Eaton DL, Groopman JD (eds): The Toxicology of Aflatoxins: Human Health, Veterinary and
Agricultural Significance. San Diego, Academic Press, 1994, pp 383–406.
197 Haumann F: Eradicating mycotoxins in food and feeds. Inform 1995;6:248–257.
198 Sharma A: Mycotoxins – Risk evaluation and management in radiation-processed food; in Sinha
KK, Bhatnagar D (eds): Mycotoxin in Agriculture and Food Safety. New York, Dekker, 1998, pp
435–458.
199 Heathcote JG, Hibbert JR: Biochemical effects, structure activity relationships: in Goldblatt LA
(ed): Aflatoxin: Chemical and Biological Aspects. Amsterdam, Elsevier, 1978, pp 112–130.
200 Dickens JW: Aflatoxin control programme for peanuts. J Am Oil Chem Soc 1977;54:225A–228A.
201 Goldblatt LA, Dollear FG: Review of prevention, elimination and detoxification of aflatoxins.
Pure Appl Chem 1977;49:1759–1764.
202 Bhatnagar D, Lillehoj EB, Bennett JW: Biological detoxification of mycotoxin: in Smith JE,
Henderson RS (eds): Mycotoxins and Animal Foods. Boca Raton, CRC Press, 1991, pp 815–826.
203 Lopez-Garcia R, Park DL: Effectiveness of post-harvest procedures in management of mycotoxin
hazards: in Sinha KK, Bhatnagar D (eds): Mycotoxin in Agriculture and Food Safety. New York,
Dekker, 1998, pp 407–434.
204 Newberne PM: Interaction of nutrients and other factors with mycotoxins; in Krogh P (ed):
Mycotoxins in Food. New York, Academic Press, 1987, pp 177–216.
205 Dashwood RH, Arbogast A, Fong T, Perieira C, Hendricks JD, Bailey GS: Quantitative interrelationships between aflatoxin B1 carcinogen dose, indole-3-carbinol anti-carcinogen dose, target
organ DNA adduction and final tumor response. Carcinogenesis 1989;10:175–181.
206 Whitty JP, Bjeldanes LF: The effects of dietary cabbage on xenobiotic-metabolizing enzymes and
the binding of aflatoxin B1 to hepatic DNA in rats. Food Chem Toxicol 1987;25:581–587.
207 Wattenberg LW: Protective effects of 2(3)-tert-butyl 4-hydroxanisole on chemical carcinogenesis.
Food Chem Toxicol 1986;24:1099–1102.
208 Williams GM, Tanaka T, Maeura Y: Dose-related inhibition of aflatoxin B1 induced hepatocarcinogenesis by the phenolic antioxidants, butylated hydroxyanisole and butylated hydroxtoluene.
Carcinogenesis 1986;7:1043–1050.
209 Cabral JRP, Neal GE: The inhibitory effects of ethoxyquin on the carcinogenic action of aflatoxin
B1 in rats. Cancer Lett 1983;19:125–132.
210 Buetler TM, Bammler TK, Hayes JD, Eaton DL: Oltipraz-mediated changes in aflatoxin B1
biotransformation in rat liver: implication for human chemointervention. Cancer Res 1996;56:
2306–2313.
211 Qin G, Gopalan-Kirczky P, Su J, Ning Y, Lotlikar PD: Inhibition of aflatoxin B1-induced initiation
of hepatocarcinogenesis in the rat by green tea. Cancer Lett 1997;112:149–154.
212 Jhee EC, Ho LL, Tsuji K, Gopalan P, Lotlikar PD: Effect of butylated hydroxyanisole pretreatment
on aflatoxin B1-DNA binding and aflatoxin B1-glutathione conjugation in isolated hepatocytes
from rats. Cancer Res 1989;49:1357–1360.
213 Bose S, Sinha SP: Modulation of ochratoxin produced genotoxicity in mice by vitamin C. Food
Chem Toxicol 1994;32:533–537.
214 Hoehler D, Marquardt RR: Influence of vitamins E and C on the toxic effects of ochratoxin A and
T-2 toxin in chicks. Poultry Sci 1996;75:1508–1515.
215 Netke SP, Roomi MW, Tsao C, Niedzwiecki A: Ascorbic acid protects guinea pigs from acute
aflatoxin toxicity. Toxicol Appl Pharmacol 1997;143:429–435.
216 Creppy EE, Baudrimont I, Belmadani A, Betbeder AM: Aspartame as a preventive agent of
chronic toxic effects of ochratoxin A in experimental animals. J Toxicol Toxin Rev 1996;15:
207–221.
217 Beaver RW, Wilson DM, James MA, Haydon KD, Colvin BM, Sangster LT, Pikul AH, Groopman
JD: Distribution of aflatoxin in tissues of growing pigs fed an aflatoxin contaminated diet
amended with a high affinity aluminosilicate sorbent. Vet Hum Toxicol 1990;32:16–18.
Toxins of Filamentous Fungi
205
218 Colvin BM, Sangster LT, Haydon KD, Beaver RW, Wilson DM: Effect of high affinity aluminosilicate sorbent on prevention of aflatoxicosis in growing pigs. Vet Hum Toxicol 1989;31:
46–48.
219 Harvey RB, Kubena LF, Philips TD, Huff WE, Corrier DE: Prevention of aflatoxicosis by addition
of hydrated sodium calcium aluminosilicate to the diets of growing barrows. Am J Vet Res 1989;
50:416–420.
220 Kubena LF, Harvey RB, Huff WE, Elissalde MH, Yersin AG, Philips TD: Efficacy of a hydrated
sodium calcium aluminosilicate to reduce the toxicity of aflatoxin and diacetoxyscirpenol. Poultry
Sci 1993;72:51–59.
221 Smith EE, Phillips TD, Ellis JA, Harvey RB, Kubena LF, Thompson J, Newton G: Dietary
hydrated sodium calcium aluminosilicate reduction of aflatoxin M1 residue in dairy goat milk and
effects on milk production and components. J Anim Sci 1994;72:677–682.
222 Kale SP, Bennett JW: Strain instability in filamentous fungi; in Handbook of Applied Mycology;
in Bhatnagar D, Lillehoj EB, Arora DK (eds): Mycotoxins in Ecological Systems. New York,
Dekker, 1992, pp 311–332.
Deepak Bhatnagar, USDA/ARS/SRRC,
1100 Robert E. Lee Boulevard, New Orleans, LA 70124 (USA)
Tel. ⫹1 504 286 4388, Fax ⫹1 504 286 4419, E-Mail dbhatnag@srrc.ars.usda.gov
Bhatnagar/Yu/Ehrlich
206
Breitenbach M, Crameri R, Lehrer SB (eds): Fungal Allergy and Pathogenicity.
Chem Immunol. Basel, Karger, 2002, vol 81, pp 207–295
Phylogeny and Systematics of the
Fungi with Special Reference to the
Ascomycota and Basidiomycota
Hansjörg Prillingera, Ksenija Lopandica, Wolfgang Schweigkoflera,
Robert Deakb, Henk J. M. Aartsc, Robert Bauerd, Katja Sterflingera,
Günther F. Krausa, Anna Marazb
a
Universität für Bodenkultur, Arbeitsgruppe Mykologie und Bodenmikrobiologie,
Wien, Austria; bSzent Istvan University, Department of Microbiology and
Biotechnology, Budapest, Hungary; cState Institute for Quality Control of
Agricultural Products, RIKILT, Wageningen-UR, The Netherlands, and dUniversität
Tübingen, Lehrstuhl spezielle Botanik und Mykologie, Tübingen, Germany
In 1965, Zuckerkandl and Pauling [1] argued that sequence comparison of
informational macromolecules permits the evaluation of evolutionary relatedness, thereby fomenting a phylogenetic revolution, especially in prokaryotic
organisms and protists [2, 3]. Protista were once considered as a distinct third
kingdom besides animals and plants by Haeckel [4]. Kimura’s neutral theory
of molecular evolution also had an impact on studies of the phylogeny and evolution, especially of microorganisms with an inadequate fossil record [5–7].
Meanwhile, molecular systematics has revolutionized our understanding of the
microbial world. Currently, phylogenies of the Eukarya depend principally on
small [3, 8, 9–17] or large [18–24] ribosomal RNA (rRNA) subunits, although
5S rRNA [25, 26] and a number of protein sequences [27, 28] also influence
phylogenetic interpretations. Based on 18S rDNA sequencing, the Ascomycota
and Basidiomycota form monophyletic clades within the kingdom Mycobionta
or chitinous Fungi (fig. 1) [14, 29–31]. Defined by a membrane-bounded
nucleus, the kingdom Mycobionta is one of several kingdoms within the crown
groups of the Eukarya or eukaryotes (fig. 1).
Dedicated to Dr. C.P. Kurtzman on the occasion of his 60th birthday and for his valuable help in establishing the VIAM culture collection.
1
Prillinger/Lopandic/Schweigkofler/Deak/Aarts/Bauer/Sterflinger/Kraus/Maraz
208
The Eukarya constitute one of the three principal domains of life [32].
According to Knoll [33], the Eukarya are an ancient group, as old as the prokaryotic Bacteria and Archea, or nearly so [2, 8]. Paleontological and biogeochemical
data suggest that Eukarya were significant organisms of ecosystems at least as
early as 1,700–1,900 million years ago [33]. Sequence analyses of proteinencoding genes that duplicated before the divergence of the domains now suggest
that the general tree of life should be rooted between the Bacteria and Archea, with
the Eukarya bearing a specific phylogenetic relationship to the Archea [34, 35].
Within the Eukarya, the earliest diverging organisms (not shown in fig. 1)
are aerotolerant anaerobes, most of which live parasitically or symbiontically
within animal hosts (microsporidia, diplomonads, oxymonads, hypermastigids,
parabasalia and some others) [3, 8, 36]. Microsporidia contain many promising species for biological control of harmful insects. It is remarkable that
microsporidia cluster within the kingdom Fungi or Mycobionta (fig. 1) if
gene sequences encoding the largest subunit of the RNA polymerase II or the
elongation factors EF-1 and EF-2 are used [37]. Doolittle [28] stresses the
importance of lateral gene transfers in prokaryotic and eukaryotic evolution.
This, however, may complicate phylogenetic interpretations. The diplomonad
Giardia infects the human intestine and can cause diarrhea, a disease known as
giardiasis, or ‘hiker’s diarrhea’. These organisms have a well-defined nucleus
and flagellum apparatus, but no mitochondria or chloroplasts and are included
in the kingdom Archezoa [38]. Representatives of the Archezoa have relatively
simple cytoskeletons and exhibit a number of ultrastructural (e.g. extranuclear
pleuromitosis) [39–41] and biochemical characters more similar to those of
prokaryotes than to other eukaryotes [38].
Protists occupying the middle branches of the phylogenetic tree [33] of the
Eukarya commonly contain mitochondria, but no chloroplasts. Euglenids are the
exception; about one third of them are photosynthetic. Euglenid chloroplasts
may be derived from symbiotic green algae [13], implying a relative late acquisition of photosynthesis within this group. The amoebaflagellate Naegleria
(fig. 1) is considered to be one of the earliest diverging protists with mitochondria. Nuclear rRNA phylogenies support this view. Heterolobosea emerge at
Fig. 1. Phylogenetic tree of eukaryotic organisms based on the primary structure of the
18S rRNA gene. Complete sequences of the 18S rRNA gene were aligned by means of the
CLUSTALX program [44]. Software package PHYLIP [45] was used for phylogenetic inferences. Distance matrix was constructed in the DNADIST program (Kimura 2 parameter
model) and the FITCH program was used for calculating phylogeny. The phylogenetic tree
was displayed in TREEVIEW [46]. Branch lengths are proportional to nucleotide differences
and the numbers given on branches represent the percentage of frequencies with which a
given branch appeared in 100 bootstrap replications. The sequences were retrieved from the
nucleotide sequence libraries (EMBL, GenBank and DDBJ).
Systematics of the Ascomycota and Basidiomycota
209
the base of mitochondria-bearing eukaryotes, together with trypanosomids and
euglenoids. All three taxa feature extraordinarily long branch lengths [3] (fig. 1).
Although predominantly aerobic, organisms in this part of the phylogenetic tree
commonly thrive under relatively oxygen-poor conditions [42].
Most eukaryotic diversity is nested within the densely branched crown of the
phylogenetic tree (fig. 1) [3, 13, 33, 43]. Major clades that branch near a common
point include the kingdoms (fig. 1) Zoobionta or Animalia (Metazoa unicellular relatives), Chlorobionta (green algae and terrestrial plants), Mycobionta (chitinous or true fungi), Heterokontobionta (Stramenopila or chromophyta: golden
brown algae, diatoms, brown algae, oomycetes, slimenets), Rhodobionta (red
algae) and Alveolobionta (Alveolates: ciliates, dinoflagellates, apicomplexans; not
shown in fig. 1) [3]. Because of rapid diversification, branching order within the
crown group of Eukarya remains uncertain.
Mitochondria and chloroplast genomes have molecular sequences that ally
them to the Bacteria (proteobacteria and cyanobacteria) [47, 48]. The sequence
data are congruent with ultrastructural and biochemical evidence supporting the
endocytobiotic theory for the origins of these organelles [49, 50]. Molecular data
are in agreement with a multiple origin of plastids, with some plastids originating from prokaryotic (simple plastids: chloroplasts, cyanelles, rhodoplasts) and
others from eukaryotic (complex plastids: cryptophytes, haptophytes, heterokontophytes, euglenophytes, chlorarachniophytes, dinoflagellates) algae [13, 43].
The Kingdom Mycobionta (Eumycota) or True Fungi
Among earlier phylogenetic speculations extensively discussed in Jahrmann
and Prillinger [51] and Barr [52], the concept of Cavalier-Smith [53, 54] and
Prillinger [41, 51, 55] is noteworthy. Based on a framework of data on cell wall
chemistry, biosynthetic pathway of lysine, storage carbohydrates, ultrastructure of
mitochondrial cristae, type of motile cells and ploidy of vegetative hyphae,
Cavalier-Smith and Prillinger only considered the chytridiomycetes,
zygomycetes, ascomycetes and basidiomycetes as true or chitinous fungi and
included them in the kingdom fungi or Eumycota. These four fungal groups are
characterized by chitinous cell walls [56, 57], the -aminoadipic acid lysine
biosynthetic pathway [58, 59], glycogen as storage carbohydrate [54], nondiscoid
plate-like mitochondrial cristae [54], the absence of heterocont flagella, and the
absence of diploid vegetative hyphal compartments in the higher Ascomycota and
Basidiomycota (exceptions: forced heterokaryons: e.g. Aspergillus, Penicillium;
solopathogenicity in the smuts: Ustilago; Hymenomycetes: Armillaria [41, 60];
see Oomycota [61]). The primarily heterotrophic origin of this group is extensively discussed by Jahrmann and Prillinger [51]. Within the Eumycota the
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210
chytridiomycetes are considered basal, the Entomophthorales (zygomycetes)
evolved from a chytridiomycete by loss of the flagella [54]. Based on a single posterior flagellum (opisthokont), flattened, nondiscoid mitochondrial cristae, a chitinous exoskeleton, storage of glycogen instead of starch, lack of chloroplasts, and
the code UGA for tryptophan, not chain termination, in their mitochondria,
Cavalier-Smith [54] suggests a common origin of the true fungi with animalia and
choanoflagellate protozoa (fig. 1).
The oomycetes, hyphochytrids, labyrinthuloids, and thraustochytrids are
included in the kingdom Heterokontobionta or pseudofungi [54] based on the
presence of cellulose in their cell walls, a tubular mitochondrial crista, heterokont flagella, one decorated with tripartite hairs, and the , -diaminopimelic
acid lysine biosynthetic pathway. The slime molds were classified into the kingdom Protozoa [38].
Evidence from complete 18S rDNA sequence divergence (fig. 1) [29, 62] put
an end to the discussion on the kingdom Eumycota or true fungi and
corroborated the existence of four naturally related phyla or divisions: the
Chytridiomycota, the Zygomycota, the Ascomycota and the Basidiomycota within
the kingdom Fungi or Mycobionta (fig. 1) [63, 64] or Eumycota [41, 52]. Specific
acyclic polyols [65], an exclusively absorptive or lysotroph nutrition [66] and a distinct ultrastructure of the flagellar apparatus of the Chytridiomycota [52] are additional characteristics which support the kingdom Mycobionta.
Based on the complete sequence of the 18S rRNA gene and the amino
acid sequence of the elongation factor, the Animalia or Zoobionta appeared as
a sister group of the Mycobionta or true fungi (fig. 1) [3, 67–72]. Nikoh et al.
[73] come to a similar conclusion from a phylogenetic analysis of 23 different
proteins. A closer phylogenetic relationship of Zoobionta and Chlorobionta,
however, becomes apparent from homologous comparisons of ribosomal proteins [74]. The protozoal Choanoflagellida are phylogenetically closely related
to the Zoobionta and Mycobionta (fig. 1) [52].
Prototheca is a ubiquitous achlorophyllous green alga (fig. 1) that lives on
decaying organic matter and exhibits a yeast-like growth pattern. Human infection usually involves the skin and underlying tissues. P. wickerhamii (fig. 1) is
recovered most often from human specimens, while P. zopfii usually is associated with infections in animals [75].
In the phylogenetic trees of Bruns et al. [29] and Sugiyama [14], the
phagotrophic plasmodial slime molds (Myxomycota) and cellular slime molds
(Dictyosteliomycota) diverged prior to the terminal radiation of eukaryotes
(fig. 1). Presently no data are available on the cellular Acrasiomycota. In contrast, parsimony analysis of amino acid sequences of EF-1, a protein involved
in the translation of messenger RNA, strongly supports a monophyletic origin
of the Dictyosteliomycota and Myxomycota and the amoeboflagellate protostelid
Systematics of the Ascomycota and Basidiomycota
211
Planoprotostelium (kingdom Mycetozoa). Among the multicellular eukaryotes, the Mycetozoa appear closer to Animalia and true fungi than to green
plants [27]. The use of EF-1 emphasizes the importance of developing multiple sequence data sets. As a conclusion, the phylogeny of the Acrasiomycota,
Dictyosteliomycota and Myxomycota remains uncertain at the moment, and
additional sequence data are urgently needed. Based on 18S ribosomal DNA
sequencing, the plant parasitic slime mold Plasmodiophora brassicae
(Plasmodiophoromycota), a severe pathogen of crucifers, may be more closely
related to the Alveolobionta than to any of the fungi [76].
The Oomycota (fig. 1, Achlya, Lagenidium, Leptolegnia, Phytophthora,
Pythium, Saprolegnia), Hyphochytridiomycota (fig. 1, Hyphochytrium) and net
slime molds or Labyrinthulomycota (fig. 1, Labyrinthula, Thraustochytrium,
Ulkenia) form a clade with brown algae (Phaeophyceae), diatoms (Bacillariophyceae), Chrysophyceae, Xanthophyceae, and Chloromonadophyceae. These
organisms have heterokont flagella, one decorated with tripartite hairs;
autotrophic species contain chlorophylls a and c, and are classified within the
kingdom Chromista [38], Heterokontobionta [64] or Stramenopila [63]. The
Oomycota lack acylic polyols [65] and differ in sterol biosynthesis from the
true fungi [77]. Based on the biosynthesis of sterols, Berg and Patterson [77]
suggest a heterotrophic origin of the Oomycota. The labyrinthuloids appear to
be basal to other heterokont algae, Oomycota and Hyphochytridiomycota
within the Heterokontobionta (fig. 1) [78, 79].
The division Oomycota mainly consists of two orders. The order
Saprolegniales comprises aquatic species, some of which are pathogenic to fish.
Representatives of the order Peronosporales mostly occur in soil or as parasites of
plants [63]. The latter order comprises one species of clinical significance,
Pythium insidiosum [80]. Two members of the order Peronosporales, Phytophthora
infestans and Plasmopara viticola, have been implicated as allergenic fungi [81].
Morphological Differentiation within the Kingdom Mycobionta
Figure 2 shows a phylogenetic and ontogenetic scheme accounting for the
range of morphological organization in the kingdom Mycobionta. Figure 2 is
based on a scheme proposed by Pascher [82] for algae, but, unlike the latter,
envisages evolution from polykaryotic, via oligokaryotic to mono- and dikaryotic systems [41]. Different types of morphological organization are extensively
discussed in Jahrmann and Prillinger [51] and Prillinger [41, 83]. The basal
position of the flagellate and rhizopodial types was further corroborated by a
compilation of ultrastructural data [41] and sequencing of the ribosomal RNA
genes [3, 84]. In figure 2 we used the term flagellate instead of monadal
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os
is
an
as
to
m
fv
eg
et
at
ive
no
olu
ti o
Plectenchyma
Ev
Terrestrial
habitat
Pseudoparenchyma
Hyphal aggregates
Trichal
Forcibly discharged
secondary spore
Hyphal aggregates
Siphonal
Phylogeny
Pseudotrichal
tio
n
Colonies
ic
ev
olu
Coccal
Po
lyp
hy
let
Colonies
Rhizopodial
Colonies
Flagellate
Aquatic
habitat
Ontogeny
Fig. 2. Evolutionary scheme for morphological differentiation within the kingdom
Mycobionta. Modified from Prillinger [83].
because the first Eucarya was most probably already a chimera of two prokaryotic organisms [49]. Presently, it is not clear whether the Amoebidiales with
free-living rhizopodial or amoeboid stages belong to the Zygomycota. The
yeast form, denoted by the term ‘coccal’ (i.e. a unicellular organism having a
rigid cell wall outside its plasma membrane), occupies a basal position among
the Zygomycota, Ascomycota, and Basidiomycota [Oberwinkler, pers. obs.],
but seems to be derived in the Chytridiomycota (e.g. Basidiobolus) [83, 85, 86].
Systematics of the Ascomycota and Basidiomycota
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3
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A yeast/hypha dimorphism is common in all classes of the Ascomycota (fig. 3:
Hemiascomycetes, Protomycetes, and Euascomycetes) and Basidiomycota (fig.
4: Urediniomycetes, Ustilaginomycetes, Hymenomycetes), especially in primitive representatives. It seems to be fundamental for a rapid evolution of the
fungi. Similarly, the pseudotrichal pattern is common in many dimorphic
Zygomycota, Ascomycota, and Basidiomycota and often reverts to the unicellular condition [51, 83]. The polykaryotic siphonal (coenocytic) type is characteristic for many eukarpic Chytridiomycota and the aflagellate Zygomycota.
The forcibly discharged secondary spore (ballistospore in the Basidiomycota)
is a specific adaptation of unicellular morphological differentiation to terrestrial life [87]. Its polyphyletic origin in the Entomophthorales and its early existance in the Basidiomycota is discussed by Tucker [88] and Prillinger [83].
Forcibly discharged secondary spores stimulate a faster spreading on new solid
habitats and may help to escape or establish parasitic interactions. Septate
hyphae, or the trichal type of morphological organization, are well known in
advanced groups of the Zygomycota (Dimargaritales, Kickxellales) and filamentous Ascomycota and Basidiomycota, the hyphal compartments may be
poly-, oligo- and monokaryotic in the Ascomycota or commonly dikaryotic in
the Basidiomycota [41]. The plectenchyma with vegetative anastomoses are
characteristic for the fruiting bodies of the higher Ascomycota (except dikaryotic ascogenous hyphae) and Basidiomycota. Pseudoparenchyma, with approximately isodiametric cells and synchronous cell and nuclear divisions, differ
from true parenchyma of plants by the absence of a phragmoplast and a meristematic tissue. They are common in the Laboulbeniales and some other meristematic Euascomycetes [89], aecidia of rust fungi, and fruiting structures of
diverse Ascomycota and Basidiomycota.
Sexual Differentiation within the Kingdom Mycobionta
Two markedly different concepts of sexuality in Mycobionta have arisen,
depending on the primary event(s) or mechanism believed to be involved.
According to the view favored here, the primary event is ‘sexual differentiation’
[41, 55, 83]; this being the process which leads to karyogamy and meiosis either
within the same strain (homothallism) or after the crossing of two different mating
Fig. 3. Phylogenetic tree of Ascomycota based on the primary structure of the 18S rRNA
gene. Alignment, distance matrix and calculation of phylogenetic distances were made by means
of different programs as described in the legend of figure 1. Carbohydrate cell wall composition
[129] is assigned as follows: 䊉 Glucose-mannose; 䉱 glucose-mannose-galactose; 䊏 glucose, mannose, galactose, rhamnose. Human pathogenic genera are indicated by arrows.
T Teleomorphic species; A anamorphic species; Y yeasts or yeast stages.
Systematics of the Ascomycota and Basidiomycota
215
4
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216
types (heterothallism). According to the other view, the primary mechanism is the
polarity resulting from sexual incompatibility [90], a phenomenon which suppresses karyogamy and meiosis in monoecious organisms. Fundamental differences between both concepts are extensively discussed in Prillinger [83].
To explain the evolution of sexuality via sexual differentiation in the
Mycobionta, two distinct steps seem to be worth discussing.
A. A polyphyletic evolution from mitotic to meiotic life cycles within
a polykaryotic and coenocytic homothallic organism (primary or primitive
homothallism [41, 83] (fig. 5).
B. A polyphyletic origin of heterothallism in different groups of fungi
[55, 83]. Mycoparasitic interactions which evolve to sexual symbiosis and a lateral gene transfer are considered to be of fundamental importance for the evolution of heterothallism [28, 83]. Burgeff [91, 92] was the first to detect a close
relationship between fungal sexuality and parasitism. In contrast to Prillinger
[83], however, he considered parasitism as a degenerated form of sexuality. A
polyphyletic evolution from heterothallism back to homothallism (secondary or
derived homothallism) appears to be very common in fungi [41, 55, 83, 93, 94]
(fig. 5). Yun et al. [95] analyzed mating type gene organization, together with a
phylogeny from ITS/glyceraldehyde-3-phosphate dehydrogenase gene sequences
to show that homothallism in Cochliobolus (C. luttrellii, C. cymbopogonis,
C. kusanoi, C. homomorphus; fig. 5) arose independently from heterothallic
ancestors. In the Basidiomycota there is in addition some evidence that unifactorial (bipolar) heterothallism as a mating system evolved polyphyletically [90]:
(1) primarily via mycoparasitic interactions, as suggested for the Ascomycota
(fig. 5) [55, 83]; (2) secondarily from bifactorial (tetrapolar) heterothallism
by close linkage of A and B loci, as demonstrated in Ustilago hordei [96]; (3)
secondarily from bifactorial mating systems by ‘self-compatible’ mutations in
either the A or B factors (or homothallism if both the A and B factors are
affected), as has been demonstrated in Coprinus [97].
During recent years, a lot of new information has accumulated in favor of
the concept of sexual differentiation:
(1) Hijri et al. [98], Hosny et al. [99] and Sanders [100] demonstrated that
polykaryotic, coenocytic and highly heterokaryotic homothallic strains without
recombination, a prerequiste for the evolution of sexuality according to the
Fig. 4. Phylogenetic tree of Basidiomycota based on the primary structure of the
18S rRNA gene. Alignment, distance matrix and calculation of phylogenetic distances were
made by means of different programs as described in the legend of figure 1. Human pathogenic genera are indicated by arrows. T Teleomorphic species; A anamorphic species;
Y yeasts or yeast stages.
Systematics of the Ascomycota and Basidiomycota
217
Heterothallism
Complex hetero-bifactorial (according to Kües, pers.com.)
o
ev
ev
ic
yle
t
tic
Unifactorial
tio
lu
IC, Two idiomorphs (RP)
Neurospora crassa, Podospora pauciseta
Saccharomyces cerevisiae
EC, Two idiomorphs?
Sexual symbionts
Asterophora yeasts (Asterotremella)
Nonhaustorial parasites
Carcinomyces
Parasitella, Chaetocladium
Haustorial parasites
Tremella, Christiansenia
Syncephalis, Piptocephalis
Primary homothallism
Benjaminiella multispora
n
Po
lyp
h
e
yl
A : EC, two idiomorphs (SP)
B : IC, multiple alleles (RP)
Ustilago maydis
h
yp
olu
Simple hetero-bifactorial
l
Po
tio
n
A : IC, two subunits, multiple highly divergent alleles
B : IC? two subunits, multiple highly divergent alleles
Schizophyllum commun, Coprinus cinereus
Secondary homothallism
Schizosaccharomyces pombe
Saccharomyces cerevisiae
Gelasinospora reticulospora
Anixiella sublineata
Neurospora terricola
Neurospora dodgei
Neurospora tetraspora
Podospora pauciseta
Cochliobolus luttrellii
Agaricus bisporus
Coprinus bilanatus
Haasiella venustissima
Mycena galericulata
Fig. 5. Hypothetical scheme for the evolution of heterothallism and secondary
homothallism in Mycobionta. For synchronous nuclear division in Benjaminiella multispora,
Cokeromyces and Mucor species with respect to primary homothallism, compare Forst and
Prillinger [159]. For molecular details, see Hiscock and Kües [90 and the literature cited
therein]. For secondary homothallsim, see Glass et al. [160] and Yun et al. [95].
IC Intracellular function; EC extracellular function; SP structural proteins which are
involved in pheromone binding; RP regulatory proteins which are involved in n-DNA
binding and regulation of transcription.
concept of sexual differentiation, indeed exist in the Mycobionta within the
arbuscular mycorrhiza forming order Glomales of the Zygomycota. The authors
detected nuclei in the polykaryotic spores (Scutellospora castanea: approximately 800 nuclei/spore) which were genetically different at the genus level.
Since vegetative anastomoses occur in the Glomales [101], it is not clear
whether the homothallism of S. castanea is primitive (primary homothallism;
fig. 5) or derived (secondary homothallism) [41, 55, 83, 94]. The Glomales are
truly ancient, having remained largely morphologically unchanged since plants
first colonized the land around 400 million years ago [102]. No sexual stage has
been observed in the Glomales life cycle.
(2) Four yeast strains were isolated from different species of higher
Basidiomycota (Polyporus, Marasmiellus, Ganoderma). In Polyporus, the yeast
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218
appeared on a cross of isogenized ramarioid inbreeding strains, which had lost
their ability to produce fertile hymenia (fig. 6 a, b) [83, 103, 104]. The yeast
isolate from Marasmiellus originates from aseptically grown cultures of young
fruiting body trama [104]. In Ganoderma two yeast strains were isolated during germination experiments from basidiospores harvested in nature. A spermatia–trichogyne-like recognition reaction (fig. 6 c, d) and a specific homing
reaction (fig. 6 e) [104–106] as well as undistinguishable diglobular interphase
spindle pole bodies of nuclei (fig. 6 f, g) and closely similar mol% GC values
suggested conspecificity between the yeasts and the corresponding mycelial
fungi. Using genotypic identification methods, we were able to identify all the
yeast isolates. All yeast strains belong to Ustilago maydis [106]. Our results
were corroborated by breeding experiments with plant pathogenic haploid
mating type strains [106]. From our data we conclude that the spermatiatrichogyne fertilization reaction can be traced back to mycoparasitic interactions and has evolved polyphyletically in the Ascomycota and rust fungi (fig. 7
[107]). This is in agreement with a phylogenetic tree of complete sequences of
the 18S rRNA gene, where the Uredinales appear as a derived clade which can
be traced back to a dimorphic Mixia-like phylogenetic ancestor without morphologically developed sexual organs within the Urediniomycetes (fig. 4).
(3) In auxotrophic mutants of Absidia glauca, a specific gene transfer
from the parasite Parasitella parasitica to the host A. glauca was detected by
Kellner et al. [108] and Wöstemeyer et al. [109].
(4) Prillinger [55] considered at least four different gene types to be
involved in sexual differentiation (mating-type genes, homothallism genes,
incompatibility genes and sterility genes). A more recent molecular characterization of mating-type genes based on DNA sequence comparisons [90 and literature cited therein] corroborates the interpretation of Prillinger [55] that
mating-type genes biochemically differ in function from the incompatibility
genes [110–120]. In addition, a similarity of the MAT-1 gene of Cochliobolus heterostrophus with Neurospora crassa mt A-1 protein, the Podospora pauciseta
(anserina) mat protein and the known DNA-binding region of Saccharomyces
cerevisiae MAT 1 protein was detected. On the other hand, the MAT-2 gene
of C. heterostrophus exhibits similarities to the N. crassa mt a-1 protein, the
P. pauciseta mat protein and the known DNA-binding protein of
Schizosaccharomyces pombe mat-Mc [121–123]. In Saccharomyces cerevisiae
the cell-type-specific gene regulation in a and -cells clearly corroborates our
concept of sexual differentiation [90 and literature cited therein]. There is no
evidence for an incompatibility function of the mating type loci in S. cerevisiae.
An evolution from extracellular to intracellular functions of mating type genes
as was postulated by Prillinger [55] was detected by Bölker et al. [124] and
Kämper et al. [125] in Ustilago maydis. Molecular data on mating type genes of
Systematics of the Ascomycota and Basidiomycota
219
a
c
b
d
e
f
g
0.1m
0.1m
Fig. 6. Ustilago maydis isolated from Polyporus ciliatus (a–e) and Ganoderma adspersum (f, g). a Isogenised ramarioid inbreeding strains with nonsporulating fruiting bodies;
bottom: haploid parents, top: dikaryotic cross. See Prillinger and Six [103]. b A yeast colony
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Saccharomyces cerevisiae, Candida albicans, Schizosaccharomyces pombe,
Neurospora crassa, Ustilago maydis, Coprinus cinereus and Schizophyllum commune [90, 125–128] point to a polyphyletic evolution of heterothallism in different groups of fungi, as suggested by Prillinger [55, 83, 94]. The complexity of
mating type genes and their respective number of base pairs nicely correlate with
an evolution from the Hemiascomycetes to the Protomycetes and from the
Protomycetes on the one hand to the Euascomycetes (fig. 3) and the other hand
to the Urediniomycetes, Ustilaginomycetes and Hymenomycetes (fig. 4).
(5) Similarly to a polyphyletic evolution of heterothallism, a polyphyletic
loss of meiosis becomes obvious in several genera of the Eurotiales, Hypocreales
and Onygenales. Using rDNA sequencing, meiotic and strictly mitotic taxa were
often recovered clustered together, indicating that multiple independent losses
of teleomorphs had occurred: Aspergillus and related teleomorphs [130–132],
Penicillium, Geosmithia and their related Talaromyces and Eupenicillium
teleomorphs [133, 134], Blastomyces, Histoplasma, Coccidioides, Emmonsia,
Trichophyton, Oidiodendron species pathogenic in humans and related
Ajellomyces and Myxotrichum teleomorphs [135–137], Fusarium and related
Gibberella and Nectria teleomorphs [138–140], Gliocladium, Trichoderma and
their Nectria and Hypocrea teleomorphs [141, 142], Acremonium and the phylogenetically different teleomorphs [143], Uredinales [144], and asco- [23] and
basidiomycetous yeasts [24].
The molecular phylogenies do not support the existence of the
Deuteromycetes or Deuteromycota as a distinct higher taxon within the Mycobionta. Molecular characters offer the potential for combining the dual classification into one natural classification [145]. Anamorphic genus and species
names of the Deuteromycetes, however, may also be necessary in the future for
purposes of identification. As shown in figures 3 and 4, mitotic genera could
be placed into meiotic orders and families [145].
(arrow) which appeared in the mycelium of the sterile dikaryotic cross. c, d Trichogyne-like
approach of a hypha (large arrow) from the Polyporus mycelial culture towards the yeast
cells (small arrow) produced on the pseudomycelium by the Ustilago maydis strain. Open
star site of inoculum of Polyporus ciliatus; closed star site of the Ustilago maydis inoculum; triangle empty hyphae of the pseudomycelium. e Recognition reaction between
pseudohyphae from U. maydis (coming from the left) and true hyphae from P. ciliatus (coming from the right), hyphal annealing (small arrow), possible vegetative anastomosis? (large
arrow). For hyphal enveloping and lethal reaction, see Prillinger [104]. f, g Spindle pole body
of the nucleus in the hypha of G. adspersum and yeast of U. maydis; the nuclear envelope is
arrowed. From Prillinger, H.: Yeasts and anastomoses: Their occurrence and implications for
the phylogeny of Eumycota, figure 24.5; in Rayner et al. (eds): Evolutionary Biology of the
Fungi. London, Cambridge University Press, 1987. With permission from Cambridge
University Press.
Systematics of the Ascomycota and Basidiomycota
221
Zygomycota
Ascomycota
Piptocephalis Dispira Parasitella Phycomyces
Chaetocladium
Penicillium
Podospora
t
ascog
t
s sp
sp
t
f
a
a
e
a
b
c
d
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1
1
3
3
c
2
b
o
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ap
v
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Trichal
u
t
i
Non-haustorial parasites
E
h
Siphonal
a
Haustorial parasites
7
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222
A problem concerning DNA as the type specimen has been considered by
Reynolds and Taylor [146] and Haines and Cooper [147]. Prillinger et al. [148]
found that phenotypically identified strains of Sporothrix schenckii may be
heterogeneous and stressed the importance of culture collections in modern
genotypic identification.
(6) Different yeasts were isolated when culturing the pileitrama of young
fruit bodies of the two agarics Asterophora lycoperdoides and A. parasitica.
Both agarics occur as mycoparasites on Russula nigricans in nature. In A.
lycoperdoides, yeasts also appeared endophytically during the germination of
chlamydospores [51, 54, 104]. In subcultures of the original yeast isolates
from both Asterophora species, agaricoid fruit bodies developed after 4–6
weeks in artificial culture [51, 94, and Prillinger unpubl. obs.]. All attempts to
repeat these experiments from single yeast cells or different crossing experiments, however, failed to produce fruit bodies, although some structures which
resemble holobasidia were observed [104]. These data suggest that the yeast
isolates from pileitrama were contaminated with Asterophora chlamydospores
and exclude parasitic interactions. Physiological patterns and the qualitative
and quantitative yeast cell wall carbohydrate spectrum of the yeast isolates
from the two Asterophora species closely resemble some mycoparasitic
Tremella species (e.g. T. encephala) [104, 149]. Using a positive selection vector to clone fungal nuclear DNA in Escherichia coli together with random
fragment hybridization analysis, the conspecificity of the Asterophora yeasts
and hyphae was excluded [150]. Based on mt-DNA RLFP [83] and
nDNA/nDNA hybridization as well as ribosomal DNA restriction fragment
analysis [455] the yeast isolates from A. lycoperdoides and A. parasitica can
be considered as distinct species. Based on complete sequences of the 18S
rDNA (fig. 4) and partial sequences of the 26S rDNA [24], the genus
Cryptococcus appeared heterogeneous with respect to the type species C. neoformans (fig. 4). We therefore have included C. humicola and the two
genotypically distinct yeast isolates from the agarics A. lycoperdoides and
Fig. 7. Evolution of heterothallism in the kingdom Mycobionta. a Haustorial parasites
(after Jeffries and Young [153]); ap appressorium; h haustorium; arrow shows cell wall
of fungal host. b, c Nonhaustorial parasites (after Burgeff [91, 92]); hatched nuclei and
cytoplasm of the host; stippled nuclei and cytoplasm of the parasite; hatched and stippled
heterokaryosis; the arrow indicates septum formation in the parasite. d Early successive
stages in the gametangiogamy of Phycomyces nitens (Orban [154], Burgeff [92]). e Successive
stages of gametangiogamy (arrows) in Talaromyces (Penicillium) stipitatus (after Emmons
[155]); note the budding asci (a). f Spermatia-trichogyne fertilization in Podospora fimbriata
(after Zickler [156, 157]: P. fimbriata (Bombardia lunata) after Mirza and Cain [158]); s
spermatia; insert shows a spermatogonium (sp) fusing with a trichogyne (t).
Systematics of the Ascomycota and Basidiomycota
223
A. parasitica in the new genus Asterotremella as As. humicola, As. lycoperdoides and As. parasitica [Prillinger et al., in preparation]. We interpret As.
lycoperdoides and As. parasitica together with the agarics from which they
were isolated as sexual symbionts and missing links in the polyphyletic evolution from mycoparasitism to heterothallism (fig. 5, 7). Haustorial mycoparasites are common in the closely related genus Tremella.
As a conclusion, the trinity system Russula, Asterophora, and Asterotremella
is remarkable, and may be fundamental in the evolution of sexuality, especially
primary homothallism and subsequently heterothallism in fungi.
(7) Based on 18S ribosomal DNA sequencing, we have traced back the uninucleate ascomycetous yeasts (Kluyveromyces, Saccharomyces) to a polykaryotic
(Eremothecium) ancestor [152].
Our arguments in favor of a sexual differentiation corroborate the idea that
genetic engineering was not discovered by molecular biologists of the 20th century: it is common in nature and fundamental in the evolution of heterothallism
in the Mycobionta.
Phylogenetic Relationships among the Chytridiomycota and
Zygomycota
In the view of many mycologists, it is believed that the Chytridiomycota
are the most primitive fungi within the Mycobionta, because they are zoosporic
(fig. 2), and sexual reproduction has been reported to be accomplished by a
variety of different methods (isogamy, anisogamy, oogamy, gametangiogamy,
and somatogamy [63]. This view has been corroborated recently by the detection of the new order Neocallimasticales [161] which consists of species of
obligately anaerobic chytrids that inhabit the rumen of herbivorous animals.
While some species have typical uniflagellate zoospores, others are exceptional
within the chytrids because they are polyflagellate with more than 10 flagella
observed. In contrast to the other orders of the Chytridiomycota, the zoospores
of the Neocallimasticales lack the microbody-lipid globule complex and mitochondria [63, 162]. The order appears to be monophyletic based on preliminary
results from cladistic analysis of structural and molecular characters [161, 163,
164] and is, furthermore, ecologically distinct.
Our phylogenetic analysis of the Chytridiomycota and Zygomy cota
(fig. 1) corroborates the view of Nagahama et al. [86], Sugiyama [14], Tanabe
et al. [165] that both phyla are not monophyletic and instead suggest that
losses of flagella occurred in several lineages during the course of fungal
evolution. The Blastocladiales (Allomyces, Blastocladiella) form a clade distinct from the Monoblepharidales (Monoblepharis, Monoblepharella),
Prillinger/Lopandic/Schweigkofler/Deak/Aarts/Bauer/Sterflinger/Kraus/Maraz
224
Spizellomycetales (Spizellomyces), Neocallimasticales (Neocallimastix) and
Chytridiales (Chytridium) [165]. Basidiobolus ranarum an aflagellate entomophthoralean fungus, which has a yeast stage [85] must be included in the
Chytridiomycota (fig. 1) [86]. As McKerracher and Heath [166] already mentioned based on ultrastructural data, the morphology of the nucleus-associated
organelle of Basidiobolus is unusual since no other nonflagellated organism
contains microtubules as structural components of their nucleus-associated
organelle.
Within the Zygomycota three different clades appear. Mucor racemosus
(Mucorales: M. mucedo, Mycotypha microspora, Rhizopus oligosporus,
Syncephalstrum racemosum) [165] forms a distinct clade with three representatives of the Entomophthorales (Entomophthora muscae, Conidiobolus
coronatus and Zoophthora radicans; fig. 1) [86]. The insect-pathogenic
Entomophthorales (Conidiobolus, Entomophaga, Entomophthora, Eryniopsis,
Pandora, Strongwellsea, Zoophthora) form a monophyletic group except
Basidiobolus [165]. Based on Micromucor and Mortierella, the Mucorales
appear polyphyletic [165]. The arbuscular mycorrhizal fungi (Acaulospora,
Gigaspora, Glomus: Glomales) form a clade together with the ectomycorrhizal
fungus Endogone (Endogonales) and Geosiphon pyriforme, a fungus forming endocytobiosis with Nostoc (Cyanobacteria; fig. 1) [165, 167]. As already
indicated by similarities in septal pore ultrastructure, cell wall structure,
asexual reproductive apparatus, and serological affinity [168], the Harpellales
(Smittium, Furculomyces), an order of the Trichomycetes, have a close
relationship to the Kickxellales (Coemansia, Martensiomyces, Linderina,
Kickxella; fig. 1) [169]. Spiromyces can be excluded from the Kickxellales; it
forms a distinct clade related to the Harpellales and Kickxellales (fig. 1) [169].
The monophyly of the mycoparasitic Dimargaritales received strong bootstrap
support [165]. Also the mycoparasitic and zooparasitic Zoopagales in which
Syncephalis, Thamnocephalis, and Rhopalomyces form a sister group to
Piptocephalis and Kuzuhaea appear monophyletic [165].
Basidiobolus ranarum (fig. 1) as a representative of the Chytridiomycota
rarely causes subcutaneous infections in humans [170]. B. haptosporus and
B. meristosporus are additional species of clinical importance [75]. Conidiobolus coronatus (fig. 1; Entomophthorales, Zygomycota), also known as
Delacroixia coronata, is a pathogen causing nasal granuloma in man [80, 171,
172] and other higher mammals [173]. C. incongruus is an extremely rare agent
of systemic mycosis with a pulmonary portal of entry [80]. No molecular information is presently available for some other medically important genera of the
Zygomycota (e.g. Apophysomyces, Absidia, Cunninghamella, Rhizomucor and
Saksenaea; also see De Hoog and Guarro [80]. Allergen characterization has
been reported only for Rhizopus nigricans [81].
Systematics of the Ascomycota and Basidiomycota
225
Although Tanabe et al. [165] present a good overview of the phylogeny
of Chytridio- and Zygomycota, further studies of molecular systematics,
chemotaxonomy and ultrastructure will be necessary to establish a phylogenetic
system of the Chytridiomycota and Zygomycota.
Phylogenetic Relationships among the Ascomycota and
Their Anamorphs
About 70,500 species of true fungi or Mycobionta have been described;
however, some estimates of total numbers suggest that 1.5 million species
may exist [174, 175]. Most of them so far belong to the Ascomycota (32,300)
and the mitosporic fungi (14,100) which can generally be included in the
Ascomycota using sequencing of ribosomal DNA. There are numerous
hypotheses on the phylogeny and evolution of higher fungi [for references, see
51, 107, 176–178]. Among these, Savile’s [179] phylogenetic considerations of
higher fungi [107, 179] have attracted many mycologists. Savile suggested that
Taphrina was the closest survivor of a common ancestor of the Euascomycetes
and the Basidiomycota. He suggested that two major lineages evolved from
‘Prototaphrina’, a common ancestor. One major lineage led to the presentday Taphrina and the higher Ascomycota – today’s Euascomycetes – whereas
another major route led to the Basidiomycota (the Uredinales line and the
parasitic Auriculariaceae line) through a ‘Protobasidiomycete’.
Phylogenetic trees inferred from 18S rDNA sequence divergence indicate
the existence of two distinct phyla or divisions among the higher fungi (fig. 1),
the Ascomycota and the Basidiomycota, e.g. [12, 14, 16, 29, 30, 180–182].
Using Mucor racemosus as an outgroup, our FITCH tree inferred from approximately 1,600 alignable sites of the 18S rRNA gene sequence from about 200
selected species (fig. 3, 4) supports that both phyla appear to be monophyletic.
As already mentioned above, the use of the Deuteromycota or Deuteromycotina
as a formal taxon decreases [14, 16, 145, 175, 183, 184]. Molecular sequence
data clearly demonstrate that ascoma characters traditionally used to delimit
ascomycete orders or classes converge. Eriksson [185] focused attention on the
orders of ascomycetes and discouraged the use of supraordinal taxa.
Plectomycetes, Pyrenomycetes, Loculoascomycetes, Discomycetes and other
traditional class level categories are no longer used formally in the fungal
classification system [14, 175]. Similarly, the classical dipartite systems of the
Ascomycota (Hemiascomycetes, Euascomycetes) [177] and Basidiomycota
(Heterobasidiomycetes, Homobasidiomycetes) [186] are not corroborated by
molecular and biochemical data, instead tripartite systems appear in molecular
phylogenies. Based on complete or nearly complete 18S rDNA sequencing,
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226
the qualitative and quantitative monosaccharide pattern of purified cell walls
(fig. 3) [129], the ultrastructure of septal pores and urease activity [Prillinger
unpubl. obs., and [187], three classes seem promising among the Ascomycota
(fig. 3): the Hemiascomycetes, the Protomycetes and the Euascomycetes
[16, 188–190]. We cannot confirm the concept of the basal Ascomycetes or
Archiascomycetes suggested by Berbee and Taylor [30], Nishida and Sugiyama
[181], and Sugiyama [14]. Based on our polyphasic biochemical and molecular
approach, the Euascomycetes and Protomycetes appear as a sister group (fig. 3),
whereas the Hemiascomycetes occupy a basal position.
The following data of the literature are in favor of our interpretation [also
see Cai et al., 191]:
(1) the nuclear genome size of the Hemiascomycetes appears to be about
one-third the size of Aspergillus, Schizosaccharomyces and the basidiomycetous yeasts [192, 193]. More recent data from the whole genome sequencing
project, however, suggest that the genome of Saccharomyces cerevisiae and
Schizosaccharomyces pombe are similar in size (S. cerevisiae 13.4 Mb [194];
Sch. pombe about 14 Mb [195]). The 14 Mb are organized in 3 compact chromosomes in Sch. pombe, which resemble higher eukaryotes. In S. cerevisiae 16
rather primitive chromosomes (see 2 and 5) were found.
(2) S. cerevisiae shows a rather unique cell cycle with the doubling of
the spindle pole body and the formation of a short mitotic spindle already in the
S-phase.The G2 phase is missing. On the other hand, the cell cycle of Sch.
pombe resembles the higher eukaryotes with a characteristic G1, S, G2 and
mitose phase [196].
(3) S. cerevisiae has a very compact nuclear genome with very few introns
(223 introns) [197, 198]. The higher frequency of introns in the genes of Sch.
pombe resembles the situation in the higher eukaryotes.
(4) S. cerevisiae is one of the few eukaryotes that can live without functional mitochondria. On the other hand, Sch. pombe requires functional mitochondria for survival as do the higher eukaryotes.
(5) The centromers of S. cerevisiae are smaller and lack the repeated
sequences which are typical for higher eukaryotes. Sch. pombe has centromers
which resemble those in ‘higher’ ascomycetes and have a similar function [199].
(6) The length of the mating type loci: S. cerevisiae has the shortest known
idiomorphs for the mating type loci (a: 640 bp, : 750 bp). The respective lengths
for Sch. pombe are P: 1.1 kb, M: 1.1 kb; for Podospora pauciseta mat: 3.8 kb,
mat–: 4.7 kb, for Neurospora crassa A: 5.3 kb, a: 3.2 kb; for Ustilago maydis a1:
4.5 kb, a2: 8 kb; and for the hymenomycetous yeast Cryptococcus neoformans
mat 35–45 kb (the mat-a locus of C. neoformans is not yet determined) [200].
(7) The lack of fruit bodies among the Hemiascomycetes could be
interpreted as a primitive rather than as a reduced character. Among the
Systematics of the Ascomycota and Basidiomycota
227
Protomyces/Schizosaccharomyces group the Neolectales produce club-shaped
fruit bodies, which are up to 7 cm tall and differ from other ascomycetous fruit
bodies mainly by the absence of sterile hyphae (paraphyses) between the asci,
the lack of ascogenous hooks (crosiers) prior to ascus development, and the
unusual combination of inoperculate asci having amyloid ascus walls [201].
(8) The coenzyme Q of the Hemiascomycetes contain a variable number
of isoprene units ranging from Q-5 to Q-9 (Q-10 was found in Lipomyces
lipofer). On the other hand, most strains of the Protomyces/Schizosaccharomyces
clade analyzed so far contain coenzyme Q-10 (with the exception of Schizosaccharomyces octosporus, which has Q-9), resembling the Euascomycetes,
which, in most cases contain coenzyme Q-10 and Q-10 (H2). Coenzyme Q-9
was found only rarely in some euascomycetous strains (e.g. Capronia parasitica,
Symbiotaphrina spp.). No strain with less isoprene units was found within the
Euascomycetes so far. Basidiomycetous yeasts possess coenzyme Q systems
with Q-7, Q-8, Q-9, Q-10 and Q-10 (H2) [202 and references therein].
(9) The Hemiascomycetes include morphologically primitive fungi (within
the genus Eremothecium) with coenocytic ‘siphonal’ [41, 83, 152] ontogenetic
stages which resemble the Zygomycota and Chytridiomycota.
(10) The haplo-diplontic life cycle of S. cerevisiae also shows some
similarities with the Chytridiomycota, where in contrast to the Euascomycetes,
this type of life cycle and diploid stages, are common [63].
Hemiascomycetes
Within the Hemiascomycetes, Kurtzman and Fell [202] presently accept a
single order, Saccharomycetales (Endomycetales), only. Based on the qualitative and quantitative monosaccharide pattern of purified yeast cell walls and
complete 18S rDNA sequences (fig. 3) [203], we accept four different orders:
Saccharomycetales, Dipodascales, Lipomycetales and Stephanoascales.
Whereas the Saccharomycetales and Lipomycetales can be delimited by the cell
wall monosaccharide pattern and the presence or absence of extracellular amyloid compounds (Saccharomycetales: glucose, mannose, extracellular amyloid
compound ; Lipomycetales: glucose, mannose, galactose, extracellular amyloid compound ) it is not possible to separate the Dipodascales and
Stephanoascales based on the cell wall monosaccharide pattern. Within both
orders, glucose, mannose and galactose dominate; however, species with the
glucose mannose pattern appear intermingled [203]. Sequences of the complete
18S rRNA gene are important to decide, whether a species belongs to the
Dipodascales or the Stephanoascales. Extracellular amyloid compounds (starch
formation) are absent in the Dipodascales and Stephanoascales. Kurtzman and
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228
Fell [202] extensively discuss the problem of using Saccharomycetales instead
of the older order Endomycetales. The order Dipodascales was already introduced by Batra [204]. However, based on molecular characteristics, many genera suggested by Batra cannot be included into this order. Additional complete
18S rDNA sequences are necessary to corroborate the orders Dipodascales,
Lipomycetales and Stephanoascales [203].
Within the Dipodascales, four yeast species are of medical importance:
Dipodascus capitatus and its anamorph Geotrichum capitatum, Yarrowia
lipolytica (anamorph: Candida lipolytica), G. candidum and G. clavatum.
Komagatella (Pichia) pastoris is a biotechnologically interesting yeast which
exhibits the glucose mannose cell wall monosaccharide pattern and comes close
to the Dipodascales [203] and [Prillinger unpubl. obs.). K. pastoris became an
important host for the expression of recombinant DNA in recent years [202].
Its gene expression system has been developed to produce large amounts of
medically and industrially important proteins [205]. D. capitatus is associated
with human lung disorders. It is increasingly being found in blood of immunocompromised hosts, particularly in cases of leukemia [80, 202]. C. lipolytica
has been isolated from patients with fungemia [75]. The main human disorders
caused by G. candidum are bronchial or pulmonary infections, in humans as
well as in nonhuman mammals [206]. Smith et al. [207] and Prillinger et al.
[208] have shown that Galactomyces geotrichum and its anamorph G. candidum
are genotypically heterogeneous. G. candidum should be conserved for a beneficial species common in cheese and other dairy products. G. clavatum is
involved in human mycoses, particularly in connection with pulmonary disorders.
Within the Saccharomycetales, the following species are of clinical importance: Candida albicans, C. parapsilosis, C. tropicalis, C. viswanathii,
Debaryomyces fabryii (anamorph: C. famata var. flareri), Pichia guilliermondii
(anamorph: C. guilliermondii) are phylogenetically closely related and may be
included within the family Debaryomycetaceae (fig. 3) [23]. Pichia norvegensis
(anamorph: C. norvegensis) and Issatchenkia orientalis (anamorph: C. krusei),
Kluyveromyces marxianus (C. kefyr), S. cerevisiae, and C. glabrata, Clavispora
lusitaniae (C. lusitaniae) and C. haemulonii are additional groups of phylogenetically related species which can be included in the Saccharomycetales
(fig. 3) [23]. Last but not least Pichia anomala and its anamorph C. pelliculosa
are representatives of the Saccharomycetales.
As can be seen from figure 3, Kurtzman and Robnett [23] and Suzuki et al.
[203], within the Hemiascomycetes, the genera Candida and Pichia are still
heterogeneous. It was not possible to separate the genus Issatchenkia genotypically from the genus Pichia represented by its type species P. membranifaciens
(fig. 3) [23]. Genotypically, the genera Kluyveromyces, Saccharomyces,
Torulaspora and Zygosaccharomyces appear intermingled (fig. 3) [23, 191].
Systematics of the Ascomycota and Basidiomycota
229
K. delphensis is the teleomorphic species closest to Candida glabrata (fig. 3)
[23, 191]. C. dubliniensis was recently described as a new species [209] isolated
from 60 HIV-infected and 3 HIV-negative persons. Although C. dubliniensis
closely resembles C. albicans phenotypically, it could be distinguished genotypically [23, 209]. Rapid identification of C. dubliniensis with commercial
yeast identification systems was described recently [210]. However, Sullivan
et al. [209] did not compare their isolates with strains representing the many
synonyms of C. albicans, so it is possible that the species may be synonymous
with an earlier described species.
C. albicans commonly occurs in the digestive tract. Candidiasis is by
far the most important mycosis. Vaginal candidiasis is extremely frequent.
Mucocutaneous candidiasis occasionally leads to osteomyelitis [211]. Further
information on the pathogenicity of C. albicans can be found in De Hoog and
Guarro [80] and Murray et al. [75]. C. albicans and Saccharomyces cerevisiae
were also considered as allergenic yeasts [81]. S. cerevisiae has been isolated
from deep infections in debilitated patients and in patients with impaired immunity, both natural and acquired [212, 213]. De Hoog [213] presents an excellent
overview of risk assessment of fungi reported from humans and animals.
An interesting process of ‘budding meiosis’ was reported by van der Walt
and Johannsen [214] in Candida albicans and C. tropicalis. Diploidization of
the sexually active haplophase appeared to involve somatogamous autogamy or
autodiploidization. The site of reduction divisions was identified when it was
shown that the diplophase formed multinucleate cells on which the haplophase
was delimited externally as buds. ‘Budding meiosis’ in ascomycetous yeast
may be a phylogenetic precursor of the concept of ‘yeast basidia’ which was
introduced by Prillinger et al. [215–217] and seemed to be fundamental in the
evolution of basidia in the Basidiomycota. Although the presence of sexuality
in C. albicans was corroborated recently by the presence of a mating-type-like
locus [128], the existence of ‘budding meiosis’ in hemiascomycetous yeasts
needs further confirmation by cytological and ultrastructural data.
C. glabrata is often involved in urogenital infections [218]. It can also be
involved in deep infections (heart [219]; lungs, occasionally with sepsis [220];
osteomyelitis [221]. There is evidence that C. glabrata may emerge after antiC. albicans therapy [222].
Debaryomyces hansenii var. fabryii and its anamorph C. famata var. flareri
were reported to be pathogenic by Vazquez et al. [223] and Nicand
et al. [224]. Using random amplified polymorphic DNA analysis (RAPD-PCR),
Prillinger et al. [208] considered D. hansenii var. fabryii as a distinct species, i.e.
D. fabryii.
A compilation of the pathogenicity of C. haemulonii, C. parapsilosis,
C. tropicalis, C. viswanathii, Clavispora lusitaniae, Issatchenkia orientalis,
Prillinger/Lopandic/Schweigkofler/Deak/Aarts/Bauer/Sterflinger/Kraus/Maraz
230
Kluyveromyces marxianus, Pichia anomala, P. guilliermondii, and P. norvegensis
can be found in De Hoog and Guarro [80] and Murray et al. [75]. BlaschkeHellmessen [225] gives an overview of the habitats of pathogenic Candida species.
Based on ribosmal DNA sequencing, Messner et al. [21] and Prillinger et al.
[152] included the two filamentous and plant parasitic species Eremothecium ashbyi and E. gossypii as well as the two dimorphic plant parasites E. coryli and
E. sinecaudum in the family Saccharomycetaceae (fig. 3). These data clearly indicate that yeasts cannot be separated taxonomically from filamentous fungi.
Within the Stephanoascales Stephanoascus ciferrii and its anamorph
C. ciferrii are the only pathogenic species so far. The species is often associated
with animals and occasionally isolated from clinical specimens [226] as an agent
of human onychomycosis [227]. Some strains are strongly hyphal and produce
conidia from characteristically inflated, denticulate heads. This anamorph has
been described as Sporothrix catenata.
No species of the order Lipomycetales is known to be pathogenic in humans
so far. The order comprises typical soil yeasts (Babjevia, Lipomyces) and mycelial
species (Dipodascopsis).
Protomycetes
Figure 3 shows there is good bootstrap support for the new class of the
Protomycetes in our FITCH tree. A similar tree topology was obtained when the
programs neighbor-joining and maximum likelihood of the PHYLIP packages
were used for tree construction [16, 151]. Presently, four orders are accepted
within the Protomycetes. These are the Neolectales, the Pneumocystidales, the
Schizosaccharomycetales and the Taphrinales [228]. A fifth order, the
Protomycetales, suggested by Eriksson and Winka [228] and Kurtzman and Fell
[202], is not supported by biochemical and molecular data [182, 229, 230].
Prillinger et al. [229, 230] consider the Protomycetaceae as a family of the
Taphrinales.
Whereas the order Taphrinales was already introduced in 1928 by Gäumann
and Dodge [see Eriksson and Winka, [228], the Schizosaccharomycetales,
Neolectales, and Pneumocystidales were suggested only recently based on molecular characters. The order Schizosaccharomycetales was introduced by
Prillinger et al. [229]. It was accepted by Kurtzman [193], Eriksson et al. [231],
and Kurtzman and Fell [202]. The orders Neolectales and Pneumocystidales (fig.
3) were suggested by Landvik et al. [232] and Eriksson [233]. Based on the qualitative and quantitative monosaccharide pattern of purified cell walls, Prillinger
et al. [215, 216, 229, 234] consider the Protomycetes (Protomyces-Typ,
Schizosaccharomycetales) ancestral to the Euascomycetes and Basidiomycota,
Systematics of the Ascomycota and Basidiomycota
231
especially the Urediniomycetes sensu Swann and Taylor [10]. Morphological and
ultrastructural data of Mixia osmundae [235 and Bauer unpubl. obs.] and 5S
rRNA sequences from Protomyces inundatus [25] and Taphrina deformans [236]
are additional characteristics which give support to the concept that the
Protomycetes are ancestors of the Basidiomycota, as was originally suggested by
Savile [107]. Based on the presence of fucose in cell walls of T. vestergrenii [217,
229], we consider T. vestegrenii a missing link on the route from the
Protomycetes to the Urediniomycetes. Based on complete sequences of the
18S rDNA T. vestergrenii occupies an intermediate position between the genera
Protomyces and Taphrina. We have suggested the new genus Fucotaphrina for
this species.
Based on the qualitative and quantitative monosaccharide pattern of purified cell walls (glucose: 70, mannose: 23, galactose: 7) and rDNA sequencing
(fig. 3) [237], the anamorphic pigmented yeast Saitoella complicata unequivocally belongs to the Protomycetes and can be excluded from the Urediniomycetes where it was included originally. A two-layered cell wall, a negative
diazonium blue B test and positive urease activity are additional characteristics of yeasts and yeast stages which belong to the Protomycetes [238].
Enteroblastic budding of S. complicata [14], however, suggests affinities to the
Basidiomycota.
The Neolectales are so far the only group of the Protomycetes where morphologically distinct fruting bodies, apothecia similar to clavarioid basidiocarps,
are produced. Landvik et al. [232] and Landvik [239] excluded the apothecial
ascomycetes Neolecta vitellina and N. irregularis from the Euascomycetes
(fig. 3). Asci of N. vitellina are occasionally filled with numerous conidia and
the ascospores become conidiogenous by producing a single apical collarette
from which the phialoconidia emerge [201]. These features are similar to budding of ascospores within the ascus of Taphrina, and therefore do not conflict
with the proposed molecular phylogeny.
The Pneumocystidales is the only order of the Protomycetes which harbors
species pathogenic in humans. Pneumocystis carinii is a unicellular eukaryotic
organism with a tropism for growth on respiratory surfaces of mammals [75].
Epidemic pneumonia may occur in institutional housing, such as orphanages,
under conditions of overcrowding and malnutrition. P. carinii has emerged as
one of the most common pulmonary infections in AIDS patients in recent years;
the presence of Pneumocystis has become one of the first indications for the
disease [80]. The molecular phylogeny and systematics of P. carinii have been
controversial for a long time (fig. 3 [75, 240, 241]). P. carinii has a number of
features that are atypical for fungi (e.g. cholesterol instead of ergosterol) [75].
There is emerging molecular evidence that there are different varieties, and possibly different species of Pneumocystis.
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232
The Schizosaccharomycetales comprise three distinct fermentative species
which reproduce by fission; none of them are of medical importance [202].
Schizosaccharomyces pombe is known from tropical millet beer.
The Taphrinales (fig. 3) comprise the Protomycetaceae and the
Taphrinaceae [230]. All species are dimorphic fungi with the mycelial phase
parasitic on ferns and especially woody dicotyledons, and the yeast phase saprophytic [176]. The mycelia and meiosporangia of the Protomycetaceae are
polykaryotic, within the Taphrinaceae mycelia are commonly dikaryotic and
the young meiosporangia contain one nucleus only. Prillinger et al. [229] regard
the ascus of Taphrina as a ‘siphonal’ germination state of a chlamydospore. This
siphonal germ tube acts as a meiosporangium, where an evolution from an undetermined number of meiotic nuclei in the case of Protomyces to a single meiotic
nucleus represented by the Taphrina species becomes obvious.
Euascomycetes
The Euascomycetes with comparatively well-developed fruiting bodies or
ascomata comprise the plectomycetes, pyrenomycetes, loculoascomycetes,
laboulbeniomycetes, and discomycetes based on traditional morphological
classifications. In the monophyletic euascomycete lineage (100% bootstrap
support in our FITCH tree fig. 3) [14], two major lineages, the Plectomycetidae
with closed ascomata (cleistothecia) and the Pyrenomycetidae with flaskshaped ascomata (perithecia), appeared monophyletic, each receiving 100%
bootstrap support (fig. 3). The tree topology in figure 3 supports the monophyly
of the Plectomycetidae and Pyrenomycetidae as already detected by Berbee and
Taylor [242] and Nishida and Sugiyama [181]. Our phylogenetic tree (fig. 3)
does not support the concept of Gargas and Taylor [243] that the apothecial
Pezizales (Ascobolus, Peziza, Gyromitra, Inermis, Morchella, Plectania) are
ancestral to the cleistothecial and perithecial forms. Based on the apothecial
Neolectales, however, Nannfeldt’s [244] phylogenetic hypothesis of primitive
apothecial ascomata with subsequent evolution of cleistothecial and perithecial
forms cannot be excluded. The cleistothecial Erysiphales which come close
to the Leotiales, however, appear as an exception (fig. 3) [63]. Within the
Hypocreales there are in addition some cleistothecial taxa, such as
Heleococcum, Mycoarachis and Roumegueriella, which have to be excluded
from the Plectomycetidae [142]. Analyses of 18S rDNA support that neither the
loculoascomycetes (fissitunicate ascomycetes) nor the discomycetes (apothecial ascomycetes) are monophyletic (fig. 3) [16, 89, 243, 245–248]. Within the
fissitunicate ascomycetes, the loculoascomycete order Pleosporales (fig. 3)
appears as a monophyletic group including the families Pleosporaceae and
Systematics of the Ascomycota and Basidiomycota
233
Lophiostomataceae; similarly, the loculoascomycete order Dothideales (fig. 3)
may also constitute a monophyletic group, however, with weaker statistical
support [89, 247]. On the other hand, the fissitunicate Chaetothyriales appear
as a sister group of the Plectomycetidae or the lichen-forming Lecanorales
and Peltigerales (fig. 3) [16, 89, 246–249]. Within the Laboulbeniales, the
Pyxidiophora-Rickia lineage (obligate parasites of arthropods) lies outside the
other perithecial ascomycetes among loculoascomycetes and discomycetes
where taxon sampling is still incomplete [250]. At the moment, therefore,
previous hypotheses including Pyxidiophora in the Hypocreales, the
Ophiostomatales or the Hemiascomycetes are not supported [250].
For allergenic and human-pathogenic Euascomycetes, the following three
orders are of special importance: Chaetothyriales, Eurotiales and Onygenales
(fig. 3). Additional species can be found within the Dothideales, Hypocreales,
Leotiales, Microascales, Ophiostomatales, Phyllachorales, Pleosporales and
Sordariales. Extensive explanations of terms and types of mycosis used in
clinical pathology can be found in De Hoog and Guarro [80].
Chaetothyriales
There is molecular evidence that at least the families Chaetothyriaceae
and Herpotrichiellaceae belong to the Chaetothyriales [249]. Unexpectedly,
the Chaetothyriales were found to be remote from the remaining Loculoascomycetes such as Dothideales and Pleosporales and relatively close to the
Onygenales and Eurotiales (fig. 3) [246] or the lichen-forming Lecanorales and
Peltigerales [249]. The diversity of anamorphs in Chaetothyriales is remarkable
and it is often difficult to distinguish them from anamorphs of the Dothideales
[246]. Many species are dimorphic and are able to grow in yeast form (black
yeasts). Similarly to the fissitunicate ascomycetes, the black yeasts are polyphyletic as well, and occur within the Chaetothyriales, Dothideales and
Pleosporales. Calcium regulates in vitro dimorphism in chromoblastomycotic
fungi [251]. Basic morphological differences are associated with differences in
thallus structure and maturation [252], which explains why anamorphs of
Chaetothyriales are smaller and more homogeneously pigmented than those
of Dothideales [246]. Whereas Capronia species, a teleomorphic genus of
the Herpotrichiellaceae, were described from plants such as Ericaceae [253],
most anamorphs, however, have been associated with a wide spectrum of
human diseases. Chromoblastomycosis is a disease which is not found outside
the Herpotrichiellaceae [246]. Untereiner and Malloch [187] discussed the
patterns of substrate utilization within the Herpotrichiellaceae. Cladophialophora
(C. arxii, C. bantiana, C. boppii, C. carrionii, C. devriesii), Exophiala (E. bergeri, E. castellani, E. dermatitidis, E. jeanselmei, E. lecanii-corni, E. moniliae,
E. pisciphila, E. salmonis, E. spinifera), Fonsecaea (F. compacta, F. pedrosoi),
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234
Phaeoannellomyces (P. elegans), Phialophora (P. bubaki, P. macrospora,
P. repens, P. richardsiae, P. verrucosa), Ramichloridium (R. mackenziei,
R. schulzeri), Rhinocladiella (R. aquaspersa, R. atrovirens), and Sarcinomyces
(S. phaeomuriformis) are important human pathogenic genera and species
within the Herpotrichiellaceae [75, 80, 246]. It was not possible to differentiate
S. phaeomuriformis from E. dermatitidis using complete sequences of the 18S
rDNA. Within the Herpotrichiellaceae, however, none of these genera appeared
phylogenetically homogeneous based on complete 18S rDNA sequences [246].
The genus Sarcinomyces is polyphyletic, S. crustaceus and S. petricola have to be
excluded from the Herpotrichellaceae [89]. The genus Wangiella was not
accepted by Haase et al. [246]. It is considered as a synonym of Exophiala.
Cladophialophora modesta takes a somewhat external position with respect to the
Herpotrichiellaceae and comes close to the Chaetothyriaceae. This is remarkable,
since the species was isolated from mycosis in the brain of a human patient [254].
Eurotiales
Benny and Kimbrough [255] redefined the classical morphological class
of the Plectomycetes with emphasis on centrum development and mode of discharge of the asci, and recognized six orders (Ascosphaerales, Elaphomycetales,
Eurotiales, Microascales, Onygenales and Ophiostomatales). Molecular data,
however, suggest that only the Ascosphaerales, Elaphomycetales, Eurotiales
and Onygenales can be accepted within the subclass Plectomycetidae (fig. 3).
Presently, it is not clear whether the Ascosphaerales which lack ascocarps and the
hypogeous Elaphomycetales can be accepted as distinct orders or families within
the Eurotiales (fig. 3) [63, 228, 256]. Eriksson and Winka [228] suggest five families within the Eurotiales (Ascosphaeraceae, Elaphomycetaceae, Eremascaceae,
Monascaceae and Trichocomaceae) based on molecular characterization. The
plectomycete family Trichocomaceae within the Eurotiales includes cleistothecial
teleomorphic genera which are associated with economically and medically
important anamorphs, such as Penicillium, Geosmithia, Merimblia, Aspergillus,
Peacilomyces and related genera [257, 258]. The teleomorphic genera associated
with a Penicillium anamorph are Talaromyces, Hamigera, Eupenicillium,
Trichocoma, Penicilliopsis and Chromocleista [14, and literature cited therein].
Berbee et al. [259] and Sugiyama [14] gave a good overview of molecular
phylogenetic studies in the Trichocomaceae. According to these studies, the genus
Penicillium is not monophyletic; one group diverged first within the Trichocomaceae cluster and contains different Talaromyces species with the Penicilliumproducing Talaromyces flavus and the Geosmithia-producing T. bacillisporus. The
second group consists of the Penicillium-producing Eupenicillium javanicum, the
Aspergillus-producing Eurotium rubrum and Neosartorya fischeri as well as the
Basipetospora-producing Monascus purpureus.
Systematics of the Ascomycota and Basidiomycota
235
Emericella (Aspergillus) nidulans is a remarkable fungus which lacks synaptic meiosis. Prillinger [41, 83] considers this fungus besides S. pombe important for
a polyphyletic evolution of meiosis within the Eumycota or Mycobionta [260, 261].
Within the genus Aspergillus especially three species are of clinical importance A. flavus, A. fumigatus and A. terreus (fig. 3 ) [262]. A. flavus is one of the
main agents of human allergic bronchial aspergillosis. The species also occurs in
the external ear and may be involved in otitis [263]. It is a common agent of
mycotic sinusitis [80]. Systemic infections occur in leukemic patients [264].
Together with A. parasiticus, A. flavus is well known for the production of the
mycotoxin aflatoxin [63]. A. fumigatus is the main agent of aspergillosis in
immunocompromised patients. It causes a typical inhalation mycosis, whereby
colonization and invasion are commonly accompanied by allergic reactions. De
Hoog and Guarro [80] gave a good overview of the clinical importance of A. fumigatus. This species has a natural habitat in rotten plant material at higher temperatures. It is especially common in air during biological waste treatment and
compost formation [265]. A. terreus causes allergic or invasive bronchopulmonary
aspergillosis [80]. De Hoog and Guarro [80] discuss the clinical importance of 29
additional Aspergillus species (A. alliaceus, A. caesiellus, A. candidus, A. carneus,
A. clavato-nanicus, A. clavatus, A. conicus, A. deflectus, A. janus, A. japonicus, A.
niger, A. ochraceus, A. oryzae, A. restrictus, A. sclerotiorum, A. sydowii, A.
tamarii, A. ustus, A. versicolor). Some of them have teleomorphs in Eurotium (E.
amstelodami, E. chevalieri, E. herbariorum, E. repens), Emericella (E. nidulans,
E. quadrilineata, E. unguis), and Fennellia (F. flavipes, F. nivea). Neosartorya
spinosa is a rare opportunistic human pathogen with an Aspergillus anamorph.
Cases of pulmonary infection and endocarditis have been reported [80].
Within the genus Penicillium only P. marneffei, a member of the subgenus
Biverticillium, is a true and common pathogenic fungus. P. marneffei occurs
naturally in bamboo rats in Southeast Asia. It is the third most common cause
of disseminated opportunistic infection in patients with AIDS in parts of
Southeast Asia. The species is unique among the genus Penicillium in being
dimorphic and forming a unicellular yeast stage that reproduces by planate
division (fission) in tissues [75, 80]. The mycosis is acquired by inhalation and
is mostly fatal when untreated. Ten additional Penicillium (P. chrysogenum,
P. citrinum, P. commune, P. decumbens, P. expansum, P. griseofulvum, P. purpurogenum, P. rugulosum, P. spinulosum, P. verruculosum) species are known
as rare opportunistic pathogenic fungi [80].
Within the genus Paecilomyces seven species (P. crustaceus, P. fumosoroseus,
P. javanicus, P. lilacinus, P. marquandii, P. variotii, P. viridis) are rare opportunistic fungi pathogenic in humans [80]. The genus Paecilomyces has teleomorphs in
Byssochlamys, Talaromyces, and Thermoascus. Paecilomyces variotii (fig. 3)
causes pneumonia [266], sphenoid sinusitis [267], soft tissue infection of the
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236
heel [268] and cutaneous hyalohyphomycosis [269] in humans. Further human
infections are discussed by De Hoog and Guarro [80]. P. tenuipes, a parasitic fungus of moth larvae and pupae can be excluded from the genus Paecilomyces based
on 18S rDNA sequencing [270]. The data suggest P. tenuipes may be the anamorph
of an entomogenous fungus of the genus Cordyceps (Hypocreales).
Eremomyces langeronii with its anamorph Arthrographis kalrae is a cleistothecial species whose inclusion in the Eurotiales has to be confirmed by ribosomal DNA sequence information. The species causes an onychomycosis and
is mainly isolated from human skin and nails [80, 271].
Onygenales
Eriksson and Winka [228] suggest three families within the Onygenales
(fig. 8; Arthrodermataceae, Gymnoascaceae and Onygenaceae). The Arthrodermataceae and Onygenaceae harbor two important groups of human pathogenic
fungi: the dermatophytes and the dimorphic systemic fungi. Dermatophytes
which were traditionally classified within the Hyphomycetes produce thallic,
one-celled microconidia in addition to multicelled macroconidia. Teleomorphs of
dermatophytes belong to the genus Arthroderma (e.g. Arthroderma simii is the
teleomorph of Trichophyton simii; Arthrodermataceae) [75, 80]. They have spherical evanescent asci containing 8 ascospores; the ascoma wall is often a loose
network of hyphae with complicated branching and ornamentation [80, 272].
Dermatophytes are keratinophilic fungi which are capable of invading the keratinous tissues of living mammals. They are grouped into three categories on the
basis of host preference and natural habitat. Anthropophilic species almost exclusively infect humans, rarely animals. Zoophilic species are essentially pathogens
of nonhuman mammals or birds, although animal to human transmission is not
uncommon. Geophilic species are soil-associated organisms, and soil per se or
soil-borne keratinous debris is a source of infection for humans as well as other
animals. Epidermophyton floccosum, Microsporum audouinii, M. ferrugineum,
Trichophyton concentricum, T. gourrilii, T. kanei, T. megninii, T. metagrophytes,
T. raubitschekii, T. rubrum, T. schoenleinii, T. soudanense, T. tonsurans, T. violaceum and T. yaoundei are the most important anthropophilic species. M. canis,
M. equinum, M. gallinae, T. equinum, T. simii and T. verrucosum are zoophilic
species and M. nanum, M. persicolor, M. praecox, M. vanbreuseghemii and
T. terrestre are geophilic species. The pathogenicity of these species is extensively
discussed in De Hoog and Guarro [80].
The dimorphic systemic fungi are phylogenetically closely related to the
dermatophytes and can be included in the family Onygenaceae (fig. 3, 8) [135,
273, 274]. Five clearly different genera can be distinguished: Blastomyces,
Coccidioides, Emmonsia, Histoplasma and Paracoccidioides. Each comprises
only a very few species and all are pathogenic. Species of Blastomyces
Systematics of the Ascomycota and Basidiomycota
237
8
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238
and Histoplasma have a teleomorph in Ajellomyces; for the other genera no
teleomorph is known so far. The natural habitat of all species are warm-blooded
animals. Humans are infected by inhalation of dry propagules or by trauma. In
healthy persons, symptoms are mild and mostly heal spontaneously [80].
Blastomyces dermatitidis is the agent of a chronic, granulomatous blastomycosis of the skin, mostly originating as a pulmonary infection or possibly also
from trauma. Coccidioides immitis causes a coccidioidomycosis. Emmonsia
parva is the agent of a adiaspiromycosis. Adiaspores are liberated conidia,
which, after inhalation, enlarge in the alveoli of the host [80, 275]. Histoplasma
capsulatum causes histoplasmosis. The species is the agent of an intracellular
mycosis of the monocyte-macrophage system. Budding yeast cells are produced within phagocytosing cells. Paracoccidioides brasiliensis causes a paracoccidioidomycosis. The species is responsible for a systemic, chronic disease.
It may cause painful, erosive stomatitis with loss of teeth, frequently associated
with swollen lymph nodes. The fungus is abundantly present with yeast cells in
pus and tissues. All species show a temperature-dependent yeast-hypha dimorphism. In the environment, they all produce a filamentous mycelial form at
room temperature; at 37 °C they reproduce as yeasts in the tissues.
Chrysosporium zonatum was detected recently as the agent of a disseminated infection in a patient with chronic granulomatous disease. C. zonatum is
the anamorph of the heterothallic ascomycete Uncinocarpus orissi [276].
Although a dimorphism is absent, this species is phylogenetically closely
related to Coccidioides immitis [277]. It produces abundant arthroconidia and
degrades cellulose as well as keratin [277].
Aphanoascus fulvescens (Onygenaceae), Arachnomyces nodososetosus
(anamorph: Onychocola canadensis; Gymnoascaceae), Gymnoascus dankaliensis,
Gymnascella hyalinospora (Gymnoascaceae), Myxotrichum deflexum, and
Neoarachnotheca keratinophilum (anamorph: Myriodontium keratinophilum;
Onygenaceae) are five rare opportunistic clinical fungi which belong to the
Onygenales [80, 278].
Hypocreales
The Hypocreales are pyrenomycetous ascomycetes with unitunicate
asci produced within fleshy, lightly or brightly colored, typically ostiolate
perithecial ascocarps [142]. However, as already mentioned above, they include
Fig. 8. Phylogenetic tree of pathogenic yeasts and fungi based on the partial sequences
of the 18S rRNA gene. Alignment, distance matrix and calculation of phylogenetic distances
were made by means of different programs as described in the legend of figure 1.
Approximately 400 bp long DNA fragments were compared corresponding to the position
582–1,006 bp in Saccharomyces cerevisiae.
Systematics of the Ascomycota and Basidiomycota
239
some cleistothecial species within the Bionectriaceae too [142]. Eriksson
and Winka [228] and Rossman et al. [279] accept five families within the
Hypocreales (Bionectriaceae, Clavicipitaceae, Hypocreaceae, Nectriaceae,
Niessliaceae).
Fusarium solani and its teleomorph Nectria haematococca is a common
clinical species causing keratitis, endophthalmitis and disseminated and cutaneous infections (fig. 3) [80]. It is also known as an allergenic fungus [81].
Besides Cladosporium cladosporioides, C. sphaerospermum and Alternaria
alternata, species of Fusarium are the most common allergenic fungi in Canada
[280]. F. aquaeductuum, F. chlamydosporum, F. dimerum, F. incarnatum,
F. oxysporum, F. proliferatum, F. sacchari, F. tabacinum, and F. verticillioides
are rare opportunistic Fusarium species [80]. Based on ribosomal DNA
sequencing, F. dimerum makes the genus Fusarium polyphyletic [139].
Genotypic identification methods are necessary to identify Fusarium species
unequivocally [281]. Cylindrocarpon is an additional anamorph of Nectria
species which is phylogenetically closely related to Fusarium [139]. C. destructans (teleomorph: Nectria radicicola) and C. lichenicola are two rare opportunistic clinical fungi within the Hypocreales. No sequence data are available
for C. cyanescens recovered from a human mycetoma [80].
Species of the genus Trichoderma (e.g. T. viride) are potentially pathogenic, toxigenic, and implicated in allergy or hypersensitivity pneumonitis
[80, 282]. Teleomorphs are known in different Hypocrea species [80, 283].
Trichoderma viride, T. longibrachiatum, T. pseudokoningii, T. koningii and
T. harzianum were reported in recent years to occur in humans [262]. As in the
case of Fusarium genotypic identification methods are necessary to identify
these species unequivocally [284, 285, Kubicek, pers. commun.].
Based on ribosomal DNA sequencing, Acremonium was shown to be a
highly polyphyletic genus affiliated to at least three ascomycetous orders [143].
Teleomorphs of Acremonium are found in several genera of the Euascomycetes
(Emericellopsis, Hapsidospora, Nectria, Nectriella, Neocosmospora, Pronectria
and Thielavia). A larger number of species including the type species A. alternatum and the human pathogenic A. kiliense exhibit affinities to the
Hypocreales. A. kiliense has been described to cause ulcerative, nodulous hyalohyphomycosis, mycetomes and keratitis [80]. In A. strictum, a rare opportunistic
human pathogen, phylogenetically closely related to A. kiliense, we have recently
detected a yeast stage. A. alabamense is the known anamorph of Thielavia terrestris. It is a rare opportunistic fungus pathogenic in humans and phylogenetically related to the Sordariales [80, 143]. No molecular data are available for
some additional more or less rare opportunistic Acremonium species (A. blochii,
A. curvulum, A. falciforme, A. hyalinulum, A. potronii, A. recifei, A. roseogriseum) pathogenic in humans [80].
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240
Conidia germinates on host cuticle
Hypha penetrates host
New infection
Red colouring due
to oosporein production
Saprophytic growth
in the soil?
New sporulation on
mummified larva
Blastospores proliferation
white grub dies
Fig. 9. Life cycle of the entomopathogenic fungus Beauveria brongniartii.
Stachybotrys chartarum is an anamorphic soil and indoor air toxigenic
fungus which especially degrades cellulose. It has been associated with a number
of human and veterinary health problems. Most notable among these has been
a cluster of idiopathic pulmonary hemorrhage cases that were observed in the
Cleveland, Ohio (USA), area [286]. A teleomorph, Melanomma pomiformis, is
known only in S. albipes. It is included in the Niessliaceae of the Hypocreales
based on morphology.
Beauveria bassiana and Metarhizium anisopliae are anamorphic soil fungi
well known as insect pathogens. Both genera have attracted a great deal of
attention because of their biological control potential [63]. Beauveria and
Metarhizium both produce mycotoxins, and the destruxins, a group of secondary metabolites produced by M. anisopliae, are considered an important
new generation of insecticides [287]. Although the proteinaceous insect cuticle
is an effective barrier to many fungi, insect pathogens, including Beauveria and
Metarhizium, have a series of extracellular proteolytic enzymes that degrade
native insect cuticle [288]. Whereas B. bassiana and M. anisopliae are polyphage
and attack a wide host range, B. brongniartii acts specifically against the
cockchafer (Melolontha melolontha) and is used as a biological control agent
(fig. 9). After the fungus has penetrated the cuticle and reached the hemocoel,
yeast-like blastospores are produced, most probably to overcome the host
Systematics of the Ascomycota and Basidiomycota
241
defense system. After the death of the host the filamentous form will be
expressed again. This dimorphism is similar to that of some human pathogens
(e.g. Histoplasma capsulatum, Sporothrix schenckii). In addition, B. bassiana
and M. anisopliae cause rare infections in humans and have been identified
as agents of keratitis. Evidence that B. bassiana is an invasive human pathogen
is doubtful because in the only reported case, the isolated mould was described
as having greenish colonies and had microscopic features inconsistent with
those of B. bassiana [75]. rDNA analyses place these anamorphic fungi among
the Hypocreales [142, 289].
Verticillium is a further heterogeneous anamorph genus of many species
which are pathogenic in insects and plants. Although Messner et al. [290]
suggested a relationship between the common plant pathogen V. dahliae and
the Hypocreales, partial sequences of the 28S rDNA [142] and a more comprehensive phylogenetic tree of complete 18S rDNA sequences (fig. 3) exclude
V. dahliae from the Hypocreales. In contrast, the entomopathogenous V. lecanii
clusters within the Hypocreales [142].
Ophiostomatales
This order is usually characterized by perithecial ascocarps, but with cleistothecia in one genus (Europhium) [291, 292] and evanescent asci. A yeast
stage is known in many species [148, 293]. The qualitative and quantitative
monosaccharide patterns of purified yeast cell walls resemble those of Protomyces
and Taphrina species containing rhamnose [148]. Based on cell wall sugars,
sensitivity to cycloheximide, rDNA sequencing (fig. 3) and some additional
characteristics [63], species of Ophiostoma are phylogenetically distinct from
morphologically similar Ceratocystis (Microascales; fig. 3) species [294, 295].
Genotypic methods are important to distinguish species unequivocally [148,
293]. Many species are associated with scolytid and platypodid bark beetles in
woody tissues where they occur as saprophytic blue stain fungi. O. novo-ulmi is
a highly virulent plant pathogenic fungus causing Dutch elm disease [63].
Ophiostoma stenoceras causes onychomycoses in humans [296].
Sporothrix schenckii (fig. 3) is an anamorphic species which is the agent of
human sporotrichosis; in addition it is known as an allergenic fungus [297].
Characteristic lesions at regional lymph nodes or localized cutaneous infection
are common [80]. Species with a Sporothrix anamorph are also known within
the Hemiascomycetes (Stephanoascales: Stephanoascus ciferrii) and
Basidiomycota (Cerinostereus cyanescens).
Phyllachorales
The characteristics of the perithecial order Phyllachorales are not clear-cut
and await further molecular characterization and investigation of additional
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242
species. The order is accepted based on Alexopoulos et al. [63] and Eriksson
and Winka [228] for Glomerella, Phyllachora and Polystigma species.
Glomerella cingulata, more often encountered as the anamorph Colletotrichum
gloeosporioides, has been reported as a parasite of over 100 angiosperms.
Sutton [298] provided descriptions for almost 40 species of Colletotrichum;
however, morphological criteria are of little use when information on a plant
pathogenic isolate is needed. Colletotrichum coccodes, C. dematium and C.
gloeosporioides are known as rare opportunistic fungi causing keratitis in
humans [80].
Sordariales
The perithecial order Sordariales is less important from the economic
point of view; however, it harbors genera well known from experimental mycology (Neurospora, Podospora, Sordaria; fig. 3). Neurospora sitophila
(Sordariaceae), the red bread mould, is known to infest bakeries and cause considerable contamination. In culture, the fungus literally lifts the lid of petri
dishes, and contaminates by rapid growth and the production of enormous numbers of pinkish air-dispersed conidia. N. sitophila is also known as an allergenic
fungus [297].
Species of the Chaetomiaceae differ from Sordariaceae by their usually
globose or ovoid asci that lack an apical ring and deliquesce within the perithecium or cleistothecium. In addition, the best-known species have conspicuous hyphal appendages on the ascocarp surface. Chaetomium atrobrunneum,
C. funicola and C. globosum are known as rare opportunistic human pathogens
[80]. Members of the Chaetomiaceae are cellulolytic and occur naturally on
paper, tapestries, and cotton fabrics, sometimes causing considerable damage.
C. globosum is also known as an allergenic fungus [297].
Corynascus heterothallicus, with its anamorph Myceliophthora thermophila, is another species of the Chaetomiaceae. This fungus was recovered
from a disseminated infection in a leukemic patient [80].
The genus Phaeoacremonium was introduced by Crous et al. [299] to
distinguish Acremonium species with pigmented vegetative hyphae and conidiophores. P. parasiticum, originally described as Phialophora parasitica, is
known as the agent of phaeohyphomycoses or mycetomes [80]. Partial
sequences of the 26S rDNA of P. parasiticum corroborate the exclusion of the
genus Phialophora [300]. De Hoog [pers. commun.] suggests a relationship
with Sordariales.
Lecythophthora hoffmannii, L. mutabilis, Phialemonium curvatum and
P. obovatum are rare opportunistic clinical fungi for which morphological and
molecular data suggest an affinity to Sordariales (Coniochaetaceae) [80,
Prillinger and Lopandic unpubl. obs.].
Systematics of the Ascomycota and Basidiomycota
243
Microascales
Members of this order are characterized by a lack of stromata, perithecia
in most species, but some possess cleistothecia. Previously, Microascaceae
were placed among plectomycetes by some mycologists because of the mature
condition of evanescent, scattered asci [255]. More recently, Barr [301] placed
the family among the pyrenomycetes. The work of Berbee and Taylor [242,
302] and Spatafora and Blackwell [295] using DNA analysis has clearly placed
the group within the perithecial ascomycetes (Pyrenomycetidae, fig. 3). The
conidia of Microascaceae are blastic, and conidiogenous cell proliferation
is percurrent or sympodial. Several anamorphs include Scopulariopsis,
Scedosporium and Wardomyces. Pseudallescheria boydii is a cleistothecial
species which is frequently encountered as a saprophyte in soil, manure and
polluted water. The species is reported worldwide as the agent of white grain
mycetomes. In addition, the fungus causes systemic infections in immunocompromised hosts or occurs in the respiratory tract where it triggers allergic
reactions, sinusitis, pneumonia or systemic pseudallescheriasis [80]. Species
causing onychomycosis include Scopulariopsis brevicaulis, by far the most
important as both a pathogen and a regular contaminant, S. candida, Microascus
cirrosus (fig. 3) and M. cinereus [75, 80]. S. brumptii is increasingly found as
a pulmonary invader in patients with impaired cellular immunity [80]. S. acremonium, S. asperula, S. flava, S. fusca and S. koningii are known as rare opportunistic pathogenic fungi in humans [80]. Microascus manginii has been
reported in several cases of onychomycosis [80].
Scedosporium prolificans has been frequently isolated from subcutaneous
lesions; in addition, it was recovered from a fatal case of endocarditis [80].
Ceratocystis fimbriata (fig. 3) is an aggressive primary pathogen with a
worldwide distribution that causes diseases in a wide range of plants (sweet
potato, rubber, coffee, quaking aspen, prune, apricot) [303]. Its long-necked
perithecia are morphologically closely similar to those of Ophiostoma species
[63]. In contrast to Ophiostoma, no yeast stage is known for Ceratocystis species.
Dothideales
Nannfeldt [244] first segregated the classical Loculoascomycetes (which he
called ascoloculares) from the other filamentous ascomycetes. While
the other filamentous ascomycetes usually have thin-walled asci with a single
functional wall layer, the Loculoascomycetes have thick-walled asci with two separable wall layers (fissitunicate) [63]. Luttrell [304] established the subclass
Loculoascomycetes to grant formal taxonomic status to Nannfeldt’s group.
He placed all other filamentous ascomycetes among the Euascomycetes.
Barr [253] accepted the Loculoascomycetes as a class and presented a highly
structured, hierachical view of its taxonomic subdivisions. Based on rDNA
Prillinger/Lopandic/Schweigkofler/Deak/Aarts/Bauer/Sterflinger/Kraus/Maraz
244
sequencing, Berbee [247] and Sterflinger et al. [89] showed that the Loculoascomycetes are not monophyletic and can be separated into three distinct
orders: the Chaetothyriales, the Dothideales and the Pleosporales. Although the
jack-in-the-box-type ascus is a good marker for large, monophyletic loculoascomycete orders (fig. 3), it must have evolved at least twice or been lost at least
once [247].
Whereas species of the genus Cladosporium pathogenic in humans show a
phylogenetic relationship to the Chaetothyriales (Herpotrichellaceae) and were
therefore assigned to the genus Cladophialophora by Masclaux et al. [300], endophytic, mycoparasitic, plant pathogenic, and saprophytic species of Cladosporium
can be included in the Dothideales (fig. 8). Cladosporium herbarum was proven
to represent the anamorph of Mycosphaerella tassiana [300]. C. cladosporioides
and C. sphaerospermum belong to the most common allergenic fungi in Canada
[282]. Mycosphaerella tassiana, C. herbarum, C. macrocarpum and C. cladosporioides had the same partial 26S rRNA sequence. C. sphaerospermum was found
to have 14 base differences [300]. C. cladosporoides and C. herbarum were commonly found as endophytes of grapevine [305].
Hortaea werneckii is a dimorphic black yeast which exclusively causes
tinea nigra palmaris on one or both hands or on the sole. It is restricted to tropical, subtropical and mediterranean areas [80]. H. werneckii is halotolerant having its natural habitat in salty environments [306]. Complete sequences of the
18S rDNA (fig. 3) [89] as well as partial sequences of the 26S rDNA suggest a
relationship of H. werneckii with the Dothideales.
Aureobasidium pullulans, a common dimorphic endophyte of grapevine
[305] and saprophyte on plant leaves, occurs in addition as an allergenic
fungus [297, 307] and as a rare opportunistic pathogen in humans, where it
caused keratitis, pulmonary infection, systemic infections, cutaneous infection,
peritonitis, and invasive mycosis in an AIDS patient [80]. Complete 18S as well
as partial 26S rDNA sequences corroborate a relationship of Aureobasidium
species with Dothideales (fig. 3) [16, 89, 300].
Hormonema dematioides is a very similar dimorphic fungus, but can be
differentiated from A. pullulans by the absence of synchronous conidiation, by
different physiological profiles and genotypic approaches like RAPD-PCR
[151]. It is also occasionally pathogenic in humans [262].
Madurella grisea and M. mycetomi are the main agents of human black
grain eumycetoma [80]. Presently no molecular data are available which
corroborate an affinity to Dothideales.
Nattrassia mangiferae (synanamorph: Scytalidium dimidiatum) is known as
a plant pathogen but is also commonly reported from human superficial infections in subtropical and tropical countries. In humans it causes extensive hyperkeratosis with scaling of the skin of the extremities, as well as onychomycoses
Systematics of the Ascomycota and Basidiomycota
245
[308]. Scytalidium hyalinum probably comprises a hyaline mutant of S. dimidiatum causing similar clinical symptoms [80, 308].
Lasiodiplodia theobromae is a rare opportunistic human pathogen causing
keratitis, onycho- and phaeohyphomycosis [80]. Botryosphaeria rhodina is
known as a teleomorph of L. theobromae. B. rhodina clusters with B. ribis
(fig. 3.) in the Dothideales [247].
Neotestudina rosatii and Piedraia hortai are two human pathogenic
species where morphological data suggest a classification among the Dothideales. N. rosatii occurs in soil of tropical countries and causes mycetoma in
humans [80]. P. hortai is the agent of black piedra on scalp hair [80].
Cenococcum geophilum is a cosmopolitan fungus and is the most widespread ectomycorrhizal fungus. It has an extremely broad host range and
habitat. Parsimony and distance analyses positioned C. geophilum as a basal,
intermediate lineage between the two Loculoascomycete orders, the
Pleosporales and the Dothideales [309]. At least four independent lineages of
mycorrhizal fungi were identified among the Ascomycota examined (compare
Elaphomyces, Tuber; fig. 3).
Pleosporales
The Pleosporales form a monophyletic group with high bootstrap support
(fig. 3) [247]. In phylogenetic trees based on complete 18S rDNA sequences, they
commonly appear as a sister group of the Dothideales (fig. 3). This, however, may
change if partial sequences are used (fig. 8). Alternaria alternata is a saprophyte
on dead plant material and a common endophyte [305], but it may also cause skin
lesions in humans after a trauma. Rare cases of systemic infection, onychomycosis and endophthalmitis following eye surgery were reported [80]. Besides
Cladosporium and Fusarium species A. alternata is the most common allergenic
fungus in Canada [282]. Breitenbach et al. [310] reported nucleotide sequences
of three cDNA clones coding for 53-, 22-, and 11-kD allergens. All of these allergens are homologous to Cladosporium herbarum allergens and are abundant,
cytosolic housekeeping proteins. Teleomorphs of Alternaria species are known in
Pleospora and Lewia [311]; they can be included in the Pleosporales (fig. 3;
Pleosporaceae) [311] based on complete sequences of 18S rDNA (fig. 3).
Eriksson and Winka [228] accept four families within the Pleosporales
(Leptosphaeriaceae, Lophiostomataceae, Melanommataceae and Pleosporaceae).
A. chlamydospora, A. dianthicola, A. infectoria, and A. tenuissima are additional
Alternaria species of clinical importance [80]. Ulocladium morphologically
closely resembles Alternaria. Ulocladium chartarum was recovered from extensive infection of subcutaneous human tissues and is considered to be allergenic
[80]. Further molecular data are necessary to clarify the phylogenetic relationship
between Alternaria and Ulocladium.
Prillinger/Lopandic/Schweigkofler/Deak/Aarts/Bauer/Sterflinger/Kraus/Maraz
246
Species of Phoma are known to have teleomorphs in Leptosphaeria
(e.g. Phoma lingam) or chlamydospores with muriform septation, resembling
the conidia of Alternaria (e.g. Phoma glomerata). Partial sequences of the 18S
rDNA of Phoma species exhibit a relationship to the teleomorph Cucurbitaria
which can be included in the Pleosporales (Leptosphaeriaceae; fig. 3) [16].
Further sequence data are necessary to clarify whether the genus Phoma is
heterogeneous. Phoma cava, P. cruris-hominis, P. eupyrena, P. glomerata,
P. herbarum, P. minutella, P. minutispora, P. oculo-hominis and P. sorghina are
known as rare opportunistic pathogens in humans [80]. P. betae is considered to
be an allergenic fungus. Epicoccum nigrum is an allergenic fungus; molecular
data suggest that it is a synanamorph of Phoma epicoccina. It was isolated from
Vienna monument surfaces [Sterflinger and Prillinger, unpubl. obs.]. Lehrer
et al. [322] reported that E. nigrum is one of the most important sources of
spores isolated outdoors. It is a frequent sensitizing agent in the Scandinavian
population [307].
Leptosphaeria senegalensis and L. thompkinsii cause mycetoma in Africa
[80]. Coniothyrium fuckelii is the anamorph of L. coniothyrium. It is known as
a plant pathogen especially on Rosaceae and from human infections [80].
The genus Cochliobolus harbors many fungi pathogenic in plants and
humans [80, 311]. Based on ITS and glyceraldehyde-3-phosphate dehydrogenase
gene sequences, Berbee et al. [311] support a suggestion by Tsuda and Ueyama
[312] to separate the genus into two closely related genera: Cochliobolus and
Pseudocochliobolus. Additional molecular sequences, especially, of species pathogenic in humans, however, are necessary to corroborate this concept. Species of
Cochliobolus exhibit a Bipolaris anamorph. For species of Pseudocochliobolus,
two anamorphs (Bipolaris, Curvularia) are known [311]. Although De Hoog and
Guarro [80] used the anamorph genus Drechslera for different Cochliobolus
species (D. hawaiensis, D. spicifera and D. australiensis), Berbee et al. [311]
restricted Drechslera to Pyrenophora species, which again cluster within the
Pleosporales (fig. 3). There is some molecular evidence [311] that highly virulent
species pathogenic in plants are common within Cochliobolus (C. carbonum and
C. victoriae) and species pathogenic in humans cluster within Pseudocochliobolus
(P. australiensis, P. geniculatus, P. hawaiiensis, P. lunatus and P. verruculosus).
Presently, no molecular data are available for C. spiciferus (anamorph: Bipolaris
spicifera) an agent of human and animal sinusitis and cutaneous phaeohyphomycoses [80]. Bipolaris hawaiiensis is a common saprophyte on plant material.
Sinusitis and pulmonary and cerebral mycosis have been reported [80].
B. australiensis and B. papendorfii are rare opportunistic fungi pathogenic in
humans [80]. Curvularia geniculata was found after traumatic implantation in the
eye and as the agent of allergic sinusitis [80]. Curvularia lunata is a ubiquitous
saprophyte on plant material. It is known from allergic bronchopulmonary disease
Systematics of the Ascomycota and Basidiomycota
247
[313], sinusitis, keratitis, phaeohyphomycosis, onychomycosis or mycetomas
[80]. C. brachyspora, C. clavata, C. pallescens, C. senegalensis and C. verruculosa are considered as rare opportunistic human pathogenic fungi [80].
Exserohilum is an anamorphic genus with teleomorphs in Setosphaeria
[311]. The genus comprises plant pathogenic species, mainly occurring on
grasses. Human mycoses mostly concern cases of sinusitis, partially with
cerebral involvement. E. mcginnisii, E. longirostratum and E. rostratum are
known from human infections [80]. Helminthosporium halodes is the
anamorph of Setosphaeria rostrata. It causes allergic bronchopulmonary mycosis [314].
Stemphylium is the anamorph which belongs to the teleomorphic
Pleospora species (fig. 3) [311]. S. macrosporoideum is recovered from a
mixed infection in antromycosis [315]. Together with Alternaria species,
Stemphylium botryosum is considered as one of the most important mold allergens in the United States [307]. There is some molecular evidence that the
genus Pleospora (P. herbarum, P. rudis) [89] is heterogeneous.
Botryomyces caespitosus is a rare opportunistic fungus which causes a
chromoblastomycosis-like subcutaneous infection after trauma in humans [316].
Leotiales
The Leotiales are the largest of the orders of inoperculate discomycetes.
They are characterized by either cup- or disk-shaped apothecia and asci that have
more or less thickened apices. Although the apothecial discomycetes are not a
monophyletic group based on molecular characters [243], there is support that
the Leotiales, Lecanorales and Pezizales are monophyletic orders within the
apothecial Euascomycetes. Many members of the Leotiales live saprobically on
the soil, some are parasitic on plants and belong to the worst fungal pathogens.
Among these are Monilinia fructicola, the cause of brown rot of stone fruits and
Sclerotinia sclerotiorum, the cause of lettuce drop and other vegetable diseases
(fig. 3). Moserella radicicola produces hypogeous apothecia [317]. Botrytis
cinerea and its teleomorph Botryotinia fuckeliana is known as gray mould of lettuce and strawberries. The fungus is also known from dessert wines, grapes are
left in vineyards purposely to become infected with B. cinerea, the ‘noble rot’,
which enhances the sweetness of the grapes. In addition, B. cinerea is a common
allergenic fungus [297]. Based on partial sequences of the 18S rDNA, B. cinerea
can be assigned to the Sclerotiniaceae [318].
Ochroconis gallopava is a common agent of encephalitis in poultry. In addition, it is known to cause subcutaneous phaeohyphomycosis and endocarditis in
humans [80]. O. constricta, O. humicola and O. tshawytschae are known as rare
opportunistic pathogenic fungi [80]. Presently, no molecular data are available
which corroborate the inclusion of the genus Ochroconis in the Leotiales.
Prillinger/Lopandic/Schweigkofler/Deak/Aarts/Bauer/Sterflinger/Kraus/Maraz
248
Interestingly, the cleistothecial powdery mildew Blumeria graminis (fig. 3;
Erysiphales) clusters with Sclerotinia sclerotiorum [319]. Additional complete
18S rDNA sequences of powdery mildew fungi are necessary to clarify the
phylogenetic relationship between Erisyphales and Leotiales. It is, however,
remarkable that many representatives of the Leotiales have asci with thickwalled apices [Oberwinkler, pers. commun.].
Pezizales
Pezizales are a large monophyletic order that contains the species commonly called operculate discomycetes as well as derived hypogeous forms that
have evanescent asci with ascospores spread by mycophagy. O’Donnell et al.
[320] have recently investigated phylogenetic relationships among ascomycetous truffels and the true and false morels. The results indicate that the hypogeous ascomycetous truffle and truffel-like taxa studied represent at least five
independent lineages within the Pezizales. The data also suggest that several
epigeous and most hypogeous taxa have been misplaced taxonomically. There
is strong support for a Tuberaceae-Helvellaceae clade which is a monophyletic
sister group of a Morchellaceae-Discinaceae clade (fig. 3). Members of the
Morchellaceae, the common morel (Morchella esculenta) and Tuberaceae
(Tuber melanosporum) are well known as food. There are only few poisonous
species (e.g. Gyromitra esculenta) of clinical importance.
Recently, a very rare and interesting fungus, Calyptrozyma arxii, was
isolated from a case of esophagitis. This fungus is typically dimorphic, initially
developing as a yeast and then producing hyphae. Both sexual and vegetative
reproductive structures are produced in the same thallus. Sexual reproduction is
represented by naked asci, and both blastic and thallic conidia are produced
[321]. Recent 5.8S rRNA sequence analysis suggests a close relationship with
Pezizales [262].
Phylogenetic Relationships of the Basidiomycota and
Their Anamorphs
Within the Basidiomycota neither the dipartite classical system of
Patouillard [323], which was recently improved by Oberwinkler [186, 324, 325],
nor the tripatite systems, which are based on the morphology of the basidium
according to Lowy [326], Talbot [327] and Donk [328, 329], could be corroborated by biochemical and molecular data. In contrast to Lowy, Talbot and
Donk, Patouillard and Oberwinkler considered the mode of basidiospore
germination important for the definition of different classes of the Basidiomycota.
Systematics of the Ascomycota and Basidiomycota
249
a
b
Fig. 10. A yeast stage in the agarics Collybia cirrata and C. tuberosa. a C. tuberosa on
decaying agaric; arrow indicates purple sclerotium. b C. cirrata: yeasts develop from
basidiospores on an acidic (pH 4.5) malt extract medium. From Prillinger et al.: Expel Mycol
1993;17:26. With permission from Academic Press.
Dörfler [330] and Prillinger et al. [215–217, 234] detected three phylogenetically distinct cell wall sugar patterns which correlate perfectly with the complete 18S rDNA sequences of Swann and Taylor [10, 331], Schweigkofler and
Prillinger [16] or the partial 26S rDNA sequences of Begerow et al. [22], leading to three distinct classes: Urediniomycetes, Ustilaginomycetes and
Hymenomycetes. Yeasts or yeast stages are known in all three classes of the
Basidiomycota (fig. 4). In addition, a yeast stage could be genotypically
demonstrated in three species of the agaric Collybia (fig. 10) [150]. Meanwhile
yeast stages are also known from symbiontic agarics (Agaricales) of leaf-cutting ants (e.g. different Cyphomyrmex species) [332]. A yeast/hypha dimorphism is considered of major importance in the evolution of Zygomycota,
Ascomycota and Basidiomycota.
With respect to basidia, Prillinger et al. [216] introduced two different
types of holobasidia. Whereas simple holobasidia are known in all three classes
of the Basidiomycota, complex holobasidia are reported from the Hymenomycetes only [215, 216, 234]. Complex holobasidia can be traced back by
partially septate basidia to tremelloid basidia (e.g. Syzygospora) [216, 333].
Yeast cells were considered to be the most primitive basidia within the
Prillinger/Lopandic/Schweigkofler/Deak/Aarts/Bauer/Sterflinger/Kraus/Maraz
250
Basidiomycota [215–217]. Similarly, the forcibly discharged basidiospore was
already established in yeast cells of the ‘Sporobolomyces’ type. Ballistospores
stimulate a faster spreading of yeast colonies on solid habitats and may help
to escape or establish parasitic interactions. In Melanotaenium endogenum, all
stages of transition from a mitotic ballistospore to the meiosporangium of
smuts can be observed [334]. Similarly, in the Zygomycota the structures of the
mitosporangium were used to disperse meiospores [63].
The new concept of the Urediniomycetes, Ustilaginomycetes and
Hymenomycetes can also be corroborated by ultrastructural data on septa and
spindle pole body morphology [325, 335, 336 and references cited therein; also
see Hibbett and Thorn, 337].
In tables 1–3 we have compiled some recent data of our cell wall sugar
analyses [129]. The Urediniomycetes are characterized by dominant amounts of
mannose and commonly the presence of fucose (table 1). Rhamnose, which is
characteristic of the Protomyces type [239], may occur sporadically together
with fucose (table 1). The absence of fucose in the Nahoidea/Sakaguchia clade
(table 1, fig. 4) needs further corroboration. Rhodotorula yarrowii is so far the
only species among the 64 investigated strains where xylose was detected as
well (table 1) [338]. In Mixia osmundae we cannot corroborate the data of
Sjamsuridzal et al. [182], who detected rhamnose instead of fucose. Based
on dominant amounts of mannose and the presence of fucose (table 1),
M. osmundae unequivocally belongs to the Microbotryum-type [217, 234], but
its position is uncertain. Morphological as well as ultrastructural data [235]
suggest that M. osmundae is a rather primitive representative of the Urediniomycetes. Sadebeck [339] already observed a Mixia-like exogenization of spore
formation in Taphrina carpini.
The qualitative and quantitative monosaccharide patterns of purified yeast
cell walls of Sterigmatomyces halophilus closely resemble those of some
Saccharomyces species (table 1). They can be distinguished from those of
the Saccharomyces type by the absence of glucose fermentation and a positive
diazonium blue B and urease test (Microbotryum type) [217, 234]. The glucose
mannose cell wall sugar pattern also appears in extremely derived
Hymenomycetes (table 3), but is different from that of the Urediniomycetes, as
it exhibits very high amounts of glucose (e.g. symbiotic yeast isolates from the
leaf-cutting ants Cyphomyrmex). It seems that the glucose mannose pattern is
the alpha and omega in the evolution of the Basidiomycota. Glucose is low at
the beginning and high at the end of evolution. Among different representatives
of the Ustilagionomycetes, the qualitative and quantitative cell wall sugar
patterns are commonly very homogeneous (table 2) [215, 217, 340]. Glucose
dominates over mannose, and galactose is commonly present. Among the
Hymenomycetes we have included 10 filamentous species representing
Systematics of the Ascomycota and Basidiomycota
251
Table 1. Cell wall sugars of yeasts or dimorphic Basidiomycota which belong to the Urediniomycetes
Species
Strain
Cell wall sugars
GLC
MAN
GAL
XYL
FUC
RHA
Urediniomycetes
Nahoidea/Sakaguchia clade
Erythrobasidium hasegawianum Y
Occultifur externus Y
Rhodotorula minuta Y
HB 62T
HB 262T
HB 477T
28
23
25
70
74
71
2
3
4
–
–
–
–
–
–
–
–
–
Agaricostilbales
Bensingtonia yuccicola Y
Kurtzmanomyces nectairei Y
K. tardus Y
Sporobolomyces ruber Y
S. xanthus Y
Sterigmatomyces elviae Y
St. halophilus Y
HB 419T
HB 106T
HB 268T
HB 317T
HB 316T
HB 104T
HB 100T
27
12
17
14
12
19
39
70
87
81
75
83
80
61
1
0.4
2
6
2
1
–
–
–
–
–
–
–
–
1
–
–
5
3
–
–
–
–
–
–
–
–
–
Microbotryomycetidae
Bensingtonia intermedia Y
Microbotryum salviae Y
M. succisae Y
Rhodotorula auriculariae Y
R. glutinis var. glutinis Y
R. glutinis var. glutinis Y
HB 417T
HB 315
HB 313
HB 413T
HB 476T
HB 462
15
10
10
16
14
20
80
59
45
79
86
78
3
7
12
1
–
1
–
–
–
–
–
–
2
24
33
1
tr
1
–
–
–
3
–
–
Uncertain position
Rhodotorula yarrowii Y
Kriegeria eriophori Y
Mixia osmundae Y
M. osmundae Y
Platygloea disciformis Y
HB 705T
HB 263
HB 748
HB 749
HB 267
20
22
33
35
23
58
49
55
55
73
2
16
10
7
1
5
–
–
–
–
2
9
2
2
1
13
4
–
–
–
Y=Yeast stage.
the Agaricales (Asterophora, Clitocybe, Mycena, Pholiota), Hymenochaetales
(Phellinus), Polyporales (Fomes, Laetiporus, Phanerochaete, Polyporus) and
Schizophyllales (Schizophyllum; fig. 4, table 3). Additional species can be
found in O’Brien and Ralph [341], Prillinger et al. [216, 217] and Messner
et al. [340]. Dominant amounts of glucose and the presence of xylose are wellestablished characters of the Hymenomycetes. Presently, only Coniophora
puteana (Boletales) [341] deviates from this pattern, showing glucose, mannose
and galactose as cell wall monosaccharides.
Prillinger/Lopandic/Schweigkofler/Deak/Aarts/Bauer/Sterflinger/Kraus/Maraz
252
Table 2. Cell wall sugars of dimorphic Basidiomycota which belong to the Ustilaginomycetes
Species
Strain
Cell wall sugars
GLC
MAN
GAL
XYL
FUC
RHA
11
14
14
–
–
–
–
–
–
–
–
–
Ustilaginomycetes
Doassansiales
Nannfeldtiomyces sparganii Y
Rhamphospora nymphaeae Y
R. nymphaeae Y
HB 304
HB 405
HB 406
87
85
86
Exobasidiales
Kordyana cubensis Y
HB 16
69
19
12
–
–
–
Ustilaginales
Schizonella sp. nov. Y
S. cocconii Y
S. melanogramma Y
Sporisorium ophiuri Y
Sp. reilianum Y
Ustilago avenae Y
U. bullata Y
U. hordei Y
HB 3
HB 112
HB 195
HB 19
HB 303
HB 302
HB 296
HB 297
88
93
87
96
96
71
72
84
5
2
4
2
1
27
25
13
7
5
9
2
3
2
3
3
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
2
0.7
0.4
Y Yeast stage.
Urediniomycetes
The Urediniomycetes presently comprise four distinct clades: the
Agaricostilbales, the Microbotryales, the Uredinales and the Cystobasidiales.
The Septobasidiales appear as a distinct order within the Uredinales clade. All
four clades are well supported by bootstrap factors close to 100% (fig. 4).
Swann et al. [11] recently introduced the subclass of the Microbotryomycetidae
which includes Heterogastridium pycnidiodeum (fig. 4) and Kriegeria
eriophori (table 1). In contrast to Swann and Taylor [10] and in agreement with
Sjamsuridzal et al. [342], the Cystobasidiales (Nahoidea/Sakaguchia clade)
occupy a basal position in our phylogenetic tree (fig. 4).
Other characters that generally corroborate the Urediniomycetes are the 5S
rRNA secondary structure of type A [236], plate-like spindle pole bodies, the
cell wall monosaccharide pattern (table 1) and simple septa tapering towards
the pore or poreless septa [22, 217, 234, 335, 336, 340, 343–348]. The members of this class are predominantly dimorphic except the Uredinales
Systematics of the Ascomycota and Basidiomycota
253
Table 3. Cell wall sugars of dimorphic and filamentous Basidiomycota which belong to the
Hymenomycetes
Species
Strain
Cell wall sugars
GLC
MAN
GAL
XYL
FUC
RHA
–
–
–
0.5
0.5
–
Hymenomycetes
Hymenomycetidae
Polyporus ciliatus M
Phanerochaete chrysosporium M
Fomes fomentarius M
Laetiporus sulfureus M
Phellinus torulosus M
Schizophyllum commune M
Asterophora parasitica M
Clitocybe phyllophila M
Collybia tuberosa Y
C. cookei Y
Mycena gallopus M
Pholliota squarrosa M
MB 15
MB 57
MB 79
MB 80
MB 125
MB 148
MB 29
MB 95
Pr 1986/93
Pr 1987/146
MB 140
MB 111
83
73
77
89
87
91
95
88
97
94
91
88
12
13
13
6
8
9
4
6
2
3
4
6
–
4
2
–
1
–
–
2
–
–
–
2
5
8
8
3
3
–
1
2
1
2
3
4
1
2
–
0.7
0.5
–
–
1
–
1
2
–
1
–
–
–
–
Lepiotaceae
Y.i. Cyphomyrmex minutus
Y.i. C. salvini
HB 667
HB 666
98
97
2
3
–
–
–
–
–
–
–
–
Tremellomycetidae
Asterotremella lycoperdoides Y
A. parasitica Y
A. humicola Y
Atractogloea stillata Y
Captotrema sp. Y
Christiansenia pallida Y
Filobasidiella neoformans Y
HB 81T
HB 82T
CBS 571T
HB 260
HB 259
HB 91
HB 420T
83
85
84
91
57
62
82
10
10
10
7
13
22
14
6
4
3
3
25
12
3
–
–
–
–
–
4
–
–
–
–
–
1
–
–
–
1
–
–
4
–
0.5
Y.i. Yeast isolate; M mycelium; Y yeast.
[5,000–8,000 species; Oberwinkler, pers. commun.] and produce rhodotorulic
acid as siderochrome [349]. The formation of secondary spores (fig. 2) is
common in rust fungi [324]. Teliospores, thick-walled resting spores of the rust
and smut fungi in which karyogamy occurs, are present or absent. Based on
M. osmundae, the Urediniomycetes can be traced back to the Upper
Carboniferous by coevolution and a fossil records of the Osmundaceae
(Discopteris, Todeopteris and Kidstonia) [229].
Prillinger/Lopandic/Schweigkofler/Deak/Aarts/Bauer/Sterflinger/Kraus/Maraz
254
Cystobasidiales
Oberwinkler [pers. commun.] suggested to include basidiomycetes which
cluster within the Nahoidea/Sakaguchia or Erythrobasidium clade in the order
Cystobasidiales (fig. 4) [Bauer et al., in preparation, table 1]. Sampaio et al.
[350] call the teleomorphic nature of Erythrobasidium hasegawianum [351]
into question and consider Erythrobasidium as an anamorphic genus. They
interpret the proposed holobasidium as a conidiogenic structure. E. hasegawianum and Sporobolomyces elongatus are phylogenetically closely related and
so far the only basidiomycetous yeasts that possess a hydrogenated ubiquinone
Q-10 (H2) [24, 351]. This ubiquinone system is common to many filamentous
Euascomycetes [352]. Sexual cycles within the Cystobasidiales differ.
Sakaguchia (Rhodosporidium) dacryoideum produces teliospores that germinate
to a 2- to 4-celled phragmobasidium with repetitively budding basidiospores.
Occultifur externus is a non-teliospore-forming fungus with a yeast stage
that produces auricularioid basidia with ballistospores and physiologically
resembles the anamorphic yeast Rhodotrula minuta [350]. O. internus is an
interesting mycoparasite within fruiting bodies of the Dacrymycetales [353].
R. minuta is a species of clinical importance within the Cystobasidiales.
R. minuta was isolated from bronchoscopy specimens and from postoperative
endophthalmitis [80]. The genus Rhodotorula, however, is heterogeneous and
has representative species at least in three different clades of the Urediniomycetes
(Microbotryum, Sporidiobolus and Erythrobasidium clades) [24]. Genotypic
methods are necessary to identify a Rhodotorula species unequivocally [208].
Microbotryales
The order Microbotryales was proposed by Bauer et al. [335] as ‘phytoparasitic members of the Basidiomycota having transversely septate basidia
with multiple production of sessile basidiospores and only intercellular
hyphae’. The order especially comprises phragmobasidial smut fungi from
dicotyledonous host plants (Liroa, Microbotryum, Sphacelotheca and
Zundeliomyces) and phragmobasidial species from monocotyledonous
host plants (Aurantiosporium, Bauerago, Fulvisporium and Ustilentyloma)
[24, 217, 234, 335]. In contrast to the phragmobasidiate members of the
Ustilaginomycetes, they do not produce intracellular hyphae or haustoria [335].
Parasitic species occurring on dicots have poreless septa, whereas parasitic
species on monocots have simple septal pores [335]. Yeast stages from phytoparasitic smut fungi of the Microbotryales commonly have a narrower oxydative
degradation spectrum of carbon and nitrogen compounds than yeasts of smuts
from the Ustilaginales [149, 234]. Celerin et al. [354] noted a specific glycosylation pattern that is unique to fimbriae from the Microbotryales. Based on
partial sequences of the 26S rDNA, Colacogloea peniophorae and Kriegeria
Systematics of the Ascomycota and Basidiomycota
255
eriophori as well as Heterogastridium pycnidioideum, which were traditionally
placed among the Platygloeales and Heterogastridiales [355], respectively, and
the basidiomycetous yeasts Leucosporidium, Mastigobasidium, Rhodotorula,
Bensingtonia, Sporobolomyces and Reniforma are closely related to the Microbotryales [24]. Presently, it is not clear whether the red pigmented teliosporic
yeasts Rhodosporidium and Sporidiobolus and their related anamorphs in the
genera Rhodotorula and Sporobolomyces form a distinct clade as suggested by
partial sequences of the 26S rDNA (Sporidiobolus clade) [24] or whether they
have to be included in the Microbotryales, which is supported by complete
sequences of the 18S rRNA gene (fig. 4).
Reniforma strues is unique among the basidiomycetous yeasts due to
the presence of kidney-shaped vegetative cells and the presence of ubiquinone
Q-7 [24].
Rhodotorula glutinis is a common saprophyte on various substrates;
disseminated cases in patients with compromised innate immunity do occur.
Sepsis due to the use of indwelling catheters has repeatedly been reported.
The species is also implicated in cases of keratitis and dacryoadenitis [80].
R. mucilaginosa is an additional species of clinical importance [262]. Genotypic
methods are necessary to identify these species unequivocally [208].
Sporidiobolus johnsonii and its anamorph Sporobolomyces holsaticus are
implicated in dermatitis [356]. The species is homothallic and forms simple
holobasidia with diploid basidiospores. Reduction division occurs with the
formation of dikaryotic hyphae with clamp connections [202].
Uredinales
Fungi belonging to the order Uredinales (fig. 4) commonly are referred to
as rust fungi. Approximately 5,000 species belonging to about 140–150 different genera which occur on spikemosses (lycophytes), ferns, gymnosperms
and angiosperms are known. They are especially important from the economic
point of view. All are parasitic on plants, often causing great losses to many
cultivated crops [63]. Based on traditional morphological systematics, the
Uredinales were considered for a long time as primitive Basidiomycota [357
and also see ref. 51, 83 and the literature cited therein]. New molecular data,
however, suggest that the Uredinales include many modern and advanced
taxa without yeast/hypha dimorphism, probably arising from simple-septate
primitive auricularioid parasites on mosses and ferns (fig. 4) [342] within the
Urediniomycetes.
Deml et al. [358, 359] and Prillinger et al. [216] isolated many different
tremelloid yeasts specifically from spermogonia of different rust fungi.
To the best of our knowledge, there are no fungi of clinical importance
within the Uredinales.
Prillinger/Lopandic/Schweigkofler/Deak/Aarts/Bauer/Sterflinger/Kraus/Maraz
256
Ustilaginomycetes
Although this clade comprises the least species of the three major lineages
of the Basidiomycota based on complete sequences of the 18S rRNA gene
(fig. 4), sufficient data from 5S rRNA [236], cell wall sugars [215, 217, 340]
(table 2), ultrastructure [335, 336], 18S rRNA gene (fig. 4) and partial
sequences of the large subunit rDNA (D1/D2 domain; [22, 24, 360] corroborate
the class of the Ustilaginomycetes. The species are usually dimorphic. Besides
some species pathogenic in humans (Malasseziales), almost all members of the
Ustilaginomycetes are known as phytoparasites. They share type B of 5S rRNA
secondary structure with the Hymenomycetes [236]. In addition, there are
some similarities in spindle pole body morphology with the Hymenomycetes
(fig. 6e, f) [336]. Figure 6e shows a characteristic hemispherical spindle pole
body of U. maydis. The spindle pole bodies of the Ustilaginomycetes roughly
resemble in their form those of the Hymenomycetes, but they have in common
with those of the Urediniomycetes an internal layering. Based on ultrastructural
data and partial sequences of the 26S rDNA there are three major lineages
which can be considered as subclasses: Entorrhizomycetidae, Exobasidiomycetidae and Ustilaginomycetidae [22, 335]. Teliospores are present or
absent. Presently, the Entorrhizomycetidae comprise one order: Entorrhizales,
the Ustilaginomycetidae two orders: Urocystales and Ustilaginales, and
the Exobasidiomycetidae seven orders: Malasseziales, Georgefischerales,
Tilletiales, Entylomatales, Microstromatales, Doassansiales, and Exobasidiales
[22, 335, 361]. Complete 18S rDNA sequences are urgently needed to corroborate these orders further.
Except for five species, the host range of the Ustilaginomycetes is restricted
to angiosperms. The lycophytes with species of Selaginella represent the most
primitive host group of the Ustilaginomycetes. A new genus Melaniella with two
species M. oreophila and M. selaginellae was recently described [362]. The
origin of lycophytes can be dated back to the Lower Devonian, about 400 million
years ago [363]. According to Bauer et al. [362], there are two possibilities to
explain the occurrence of Ustilaginomycetes on lycophytes: either it is the result
of a jump, or the Ustilaginomycetes arose as parasites of at least early vascular
plants and the parasitic smut fungi of Selaginella species represent extant
representatives of this ancestral ustilaginomycetous group. Because the
systematically different hosts of the Doassansiales are all paludal or aquatic
plants, Bauer et al. [362] believe the Doassansiales represent a good example for
evolution bound to an ecosystem, but not to a specific host relationship and favor
the jump hypothesis. Exoteliospora (Ustilago) osmundae is another representative of the Ustilaginomycetes which occur on primitive leptosporangiate ferns
[364]. As already mentioned for Mixia osmundae (Urediniomycetes), the
Systematics of the Ascomycota and Basidiomycota
257
Osmundaceae can be traced back to the Upper Carboniferous. Earlier known
reports of leptosporangiate ferns are in the Lower Carboniferous. Subsequent
major filicalean radiations during the early Mesozoic resulted in several families
with extant representatives, but it was obviously not until the Upper Cretaceous
that much of the extant diversity has appeared [365]. Based on coevolution, the
Urediniomycetes and Ustilagionmycetes appeared as distinct lineages at least
since the Carboniferous.
Species allergenic and pathogenic in humans are known among four orders
within the Ustilaginomycetes; representatives of the Malasseziales are of
special importance.
Malasseziales
The order was introduced by Moore [366]. A separate position of the
different Malassezia species is in agreement with morphological, physiological,
ultrastructural, and molecular characteristics [24, 361]. The cell wall of the
Malassezia yeasts is thick, multilamellate and reveals a unique substructure
with a helicoidal band that corresponds to a helicoidal evagination of the
plasma membrane [367–369]. The lipophilic, dimorphic yeast genus Malassezia
is presently divided into seven different species (M. furfur, M. pachydermatis,
M. sympodialis, M. globosa, M. obtusa, M. restricta, M. slooffiae) [369–371].
Of these, six are strictly lipid dependent with a requirement for long-chain fatty
acid supplementation in the medium to ensure their growth, and one (M. pachydermatis) for which the lipids present in rich media such as Sabouraud glucose
agar are sufficient. Whereas M. pachydermatis is isolated only rarely from
humans, the six lipid-dependent species are commonly found on human skin.
The yeasts are members of the normal human cutaneous flora and can be
cultured from almost all body areas. Under the influence of predisposing factors they become pathogenic and are associated with several diseases such as
pityriasis versicolor, Malassezia folliculits, seborrheic dermatitis, some forms
of atopic dermatitis, some forms of confluent and reticulate papillomatosis, and
even systemic infection [80, 371]. M. pachydermatis has occasionally been
implicated in cases of systemic infection.
A rapid and inexpensive identification method has been established to
separate the seven species routinely on the basis of morphological and physiological differences [371]. The new taxonomy must be applied to epidemiological
surveys before one can conclude whether all seven Malassezia species have
clinical importance.
Georgefischerales
Species of Tilletiopsis are frequently found as epiphytes on leaves,
especially those infected with powdery mildew or rust fungi [372, 373].
Prillinger/Lopandic/Schweigkofler/Deak/Aarts/Bauer/Sterflinger/Kraus/Maraz
258
T. pallescens is a potential biological control agent of Spaerotheca fulginea,
a powdery mildew which is common on cucumber leaves [373]. Among
the Ustilaginomycetes only the Melanotaeniaceae of the Ustilaginomycetidae,
the Georgfischeriaceae and Tilletiariaceae of the Georgefischerales and the
Entylomatales form Tilletiopsis-like pseudohyphal anamorphs that produce
ballistoconidia. This indicates that the genus Tilletiopsis is highly polyphyletic
[24, 361, 374]. T. albescens and T. pallescens are members of the Exobasidiomycetidae, but they cannot be assigned to any known order.
T. minor is a member of the Georgefischerales which caused subcutaneous
infection in an immunosuppressed patient [361, 375]. Based on the qualitative
and quantitative monosaccharide pattern of purified cell walls, T. minor fits
well in the Ustilaginomycetes [215].
Microstromatales
The genus Cerinosterus was erected by Moore [376] for basidiomycetous
hyphomycetes previously classified in Sporothrix. This transfer was necessary
because the type species of Sporothrix, S. schenckii, had been shown to be a
member of the euascomycetes order Ophiostomatales [377]. Two species were
accepted in Cerinostereus, with C. luteoalba as the type of the genus [376].
This species is the anamorph of Ditiola pezizaeformis (Femsjonia luteoalba),
which belongs to the Dacrymycetales. The second species, C. cyanescens was
excluded from the Dacrymycetales; 25S rRNA sequencing suggests an affinity
to Microstromatales [378]. Cells which produce conidia sympodially on
denticles, singly or in short chains, therefore can be found at least in four unrelated orders of the Ascomycota and Basidiomycota: Stephanoascales
(Hemiascomycetes), Ophiostomatales (Euascomycetes), Microstromatales
(Ustilaginomycetes) and Dacrymycetales (Hymenomycetes). C. cyanescens
has occasionally been isolated from human skin and blood and was involved in
nosocomial infections in patients with pneumonia [80]. Experimental inoculation showed low virulence [379].
Based on partial ribosomal DNA sequencing, Rhodotorula bacarum,
R. hinnulea and R. phylloplana can be excluded from the genus Rhodotorula.
Together with Sympodiomycopsis paphiopedili (fig. 4), they can be included
in the Microstromatales [24]. Presently there are no morphological and ultrastructural data to circumscribe this order [Oberwinkler, pers. commun.].
Ustilaginales
The genus Ustilago is representative of this order. Ustilago species
are commonly dimorphic phragmobasidial smut fungi parasitic on seeds and
flowers of many cereals and grasses [380]. Smut spores may be inhaled and
therefore may be isolated from sputum specimens. Based on partial sequences
Systematics of the Ascomycota and Basidiomycota
259
of the LSU rDNA, the genus Ustilago is heterogeneous. U. maydis appears to
be distinct from U. hordei, the type species of the genus [22, 24]. Rhodotorula
acheniorum is a candidate for reclassification. It clusters with the Ustilaginales
based on partial sequences of the LSU rDNA [24]. The species of Pseudozyma
are anamorphs of the Ustilaginales [360, 361].
Ustilago tritici is an allergenic species of the Ustilaginales [297].
Hymenomycetes
Representatives of the Hymenomycetes have dolipore septa with various
types of pore caps (without, cupulate, continuous or perforate) [325, 336], their
spindle pole bodies have a true globular morphology lacking obvious internal
differentiation [336]. In addition, they have a type B secondary structure of the
5S rRNA (cluster 5) [236]. A cell wall sugar analysis commonly exhibits dominant amounts of glucose and the presence of xylose (table 3) [216, 217, 340].
Based on sequence analysis of the small subunit rDNA, Swann and Taylor [10]
recommended two subclasses among the Hymenomycetes: (1) the Tremellomycetidae, which are commonly dimorphic and often yeast-like, and (2) the
Hymenomycetidae, containing the non-yeast-like macrofungi including the
mushrooms and puffballs. Nuclear (nuc) rDNA studies all support or are consistent with the view that the classical Homobasidiomycetes plus Auriculariales
s. str., Tualsnellales, and Ceratobasidiales form a monophyletic group (fig. 4)
[337], and that the Tremellomycetidae and Dacrymycetales are at the base of
the hymenomycete lineage. Based on complete sequences of the 18S rDNA
[381], the Tremellomycetidae comprise two distinct orders: the Tremellales and
the Cystofilobasidiales (fig. 4). This is in contrast to partial sequences of the
26S rDNA where Fell et al. [24] accept the Tremellales, the Trichosporonales,
the Filobasidiales and the Cystofilobasidiales (fig. 4). Presently, however, no
ultrastructural data give support to the Filobasidiales and Trichosporonales
[Bauer, unpubl. results]. Takashima and Nakase [381] subdivide the Tremellales
into six different lineages: the Filobasidium lineage, the Trichosporon lineage,
the Cryptococcus luteolus lineage, the Fillobasidiella lineage, the Bulleromyces
lineage and the Sterigmatosporidium lineage. Additional complete 18S rDNA
sequences especially of Tremella species are necessary to clarify systematic
relationships within the Tremellales unequivocally.
The Hymenomycetidae include the Auriculariales as well as all groups
of the classical Homobasidiomycetes. Cantharellus tubaeformis, however,
causes some problems (fig. 4). In a phylogram which is based on combined
nuc-ssu and mt-ssu rDNA sequences, C. tubaeformis clusters together with
Hydnum repandum and Clavulina cristata (Cantharelloid clade) inside the
Prillinger/Lopandic/Schweigkofler/Deak/Aarts/Bauer/Sterflinger/Kraus/Maraz
260
Hymenomycetidae [337, 382, 383]. In a phylogenetic tree based on nuc-ssu
rDNA, only C. tubaeformis remains distinct (fig. 4). As discussed by Pine et al.
[383], the rate of sequence evolution of nuc-ssu rDNA appears to be greater in
the cantharelloid clade than in most other Homobasidiomycetes, which makes
it very likely that long branch attraction is responsible for the placement
of C. tubaeformis near the base of the tree. We have observed a similar phenomenon with complete 18S rDNA sequences from Tulasnella pruinosa and
T. violea. We therefore have excluded these sequences from our phylogram
presented in figure 4 Pine et al. [383] present cytological (stichic nuclear
division in basidia) [384] and molecular (combined data for mit-ssu rDNA and
nuc-ssu rDNA) evidence that the Cantharellales (Cantharellus, Craterellus,
Clavulina, Hydnum, Stichoclavaria) are a well-established clade within the
Hymenomycetidae.
Presently, it is not clear whether the Dacrymycetales should be included in
the Hymenomycetidae as suggested by Swann and Taylor [10] or not (fig. 4).
The classical Homobasidiomycetes were tentatively separated into eight major
clades by Hibbett et al. [382] and Hibbett and Thorn [337] based on molecular
data. In accordance with Oberwinkler [186], these clades commonly comprise
resupinate, bracket-like, club-shaped or coralloid, pileate and gasteroid
(secotioid gasteroid and hypogeous gasteroid) fruiting bodies with various
types of hymenia (e.g. corticoid, toothed, poroid, agaricoid, gleba chambers).
Similarly to Oberwinkler [186], we used distinct orders to circumscribe these
clades: Agaricales, Boletales, Cantharellales, Gomphales, Hymenochaetales,
Polyporales, Russulales, Thelephorales. Some of these orders have already been
accepted by Gäumann [385] (Agaricales, Cantharellales, Dacrymycetales,
Polyporales) and Kreisel [386] within his subclass Hymenomycetidae
(Agaricales, Boletales, Cantharellales, Dacrymycetales, Polyporales, Russulales)
but the genera included generally differ remarkably if molecular characters are
used in addition. In contrast to Hibbett et al. [382], the Schizophyllales appear
as an additional distinct group in our phylogram (fig. 4).
The oldest unambiguous homobasidiomycete fossils are from the midCretaceous, but indirect evidence, including molecular clock dating, suggests
that the higher Hymenomycetes may have existed by the late Triassic (ca. 200
million years) [30, 387].
Tremellales
Cryptococcus neoformans is a zoopathogenic basidiomycetous yeast
which has a teleomorph in Filobasidiella (F. neoformans) but is usually encountered in the imperfect state [388, 389]. Fell et al. [390] proposed to conserve
Cryptococcus with C. neoformans (Sanfelice) Vuillemin as the neotype species.
Cryptococcosis is an inhalation mycosis, occurring nearly exclusively in
Systematics of the Ascomycota and Basidiomycota
261
immunocompromised patients. Pleural effusion is a first indicator of AIDS.
Dissemination leads to chronic meningitis which is usually fatal when
untreated [80]. Based on genetic recombination in the F1 generation C. neoformans consists of the following two varieties according to the current classification: C. neoformans var. neoformans, with serotypes A, D and C. neoformans
var. gattii, with serotypes B, C. According to Boekhout et al. [389], the two varieties differ in karyotype, RAPD-PCR patterns, in a number of physiological
characteristics, morphology of basidiospores, and in sensitivity to killer toxins
of Cryptococcus laurentii. The two varieties also differ in geographic distribution and habitat. C. neoformans var. neoformans occurs worldwide and is frequently isolated from bird droppings. C. neoformans var. gattii is restricted
to the tropics and the southern hemisphere; it is usually associated with
Eucalyptus species [389]. Differentiation between the two varieties is usually
performed on L-canavanine-glycine-bromthymol blue medium [391, 392] or by
testing D-proline assimilation [393]. Boekhout et al. [389] considered the two
varieties as distinct species, Filobasidiella neoformans and F. bacillispora.
All species of Cryptococcus are nonfermentative aerobes exhibiting a
positive urease test, a positive diazonium blue B test, and the presence of extracellular amyloid compounds. Cryptococcus differs from Trichosporon by the
presence of capsules and the absence of arthroconidia. Genotypic methods are
necessary to identify the species unequivocally [208].
C. laurentii is an additional species of clinical importance (pulmonary
abscess) which belongs to the Tremellales [24, 80, 262, 381]. Based on partial
26S rDNA and complete 18S rDNA sequences, the genus Cryptococcus is
polyphyletic and occurs in at least five different clades of the Tremellales, and
within the Cystofilobasidiales [24, 381] (fig. 4).
Filobasidium (Cryptococcus) uniguttulatum, Cryptococcus albidus, and
C. ater are three species of clinical importance which belong to the Filobasidium
lineage of the Tremellales [24, 262, 381] (fig. 4). The Filobasidium lineage may
be considered as a distinct family (Filobasidiaceae) within the Tremellales.
Although F. uniguttulatum has been isolated from human diseased nails or other
clinical specimens; it has not been documented to cause invasive disease [394].
Cases of meningitis and pulmonary infections have been reported to be caused
by C. albidus [80]. The type strain of C. ater was isolated from multiple ulcers
on the leg of a young man [202].
Species of Trichosporon are characterized by the presence of arthroconidia, positive urease and diazonium blue B tests and the absence of extracellular
amyloid compounds. The septa have dolipores, which may or may not have
tubular/vesicular parenthesomes. Except for T. pullulans, which is a member of
the Cystofilobasidiales (fig. 4), all species form a coherent clade within the
Tremellales, which suggests a distinct family (Trichosporonaceae) [15, 24, 381,
Prillinger/Lopandic/Schweigkofler/Deak/Aarts/Bauer/Sterflinger/Kraus/Maraz
262
395, 396]. Genotypic methods are essential to identify a Trichosporon species
unequivocally [208]. There are six Trichosporon species of clinical importance
(T. asahii, T. asteroides, T. cutaneum, T. inkin, T. mucoides, T. ovoides).
T. beigelii is a synonym of T. ovoides [202]. Two strains of Fissuricella
filamenta isolated from human skin showed DNA homology values around
85% with T. asteroides strains [395]. The groups of strains are thus genetically
close, despite the fact that F. filamenta strains were entirely composed of
meristematic cells, while such cells were absent from the strains of T. asteroides
[202]. T. asahii, T. inkin and T. mucoides are regularly isolated from clinical
specimens (white piedra), whereas the remaining species cause only occasional
infections [80, 262].
Cryptococcus humicola was isolated from human skin [262]. It is related to
the genus Trichosporon [24, 381, 397]. Based on 26S rDNA sequencing of the
D1/D2 region, C. humicola differs only by 2 bp from two yeast isolates of the
agarics Asterophora lycoperdoides and A. parasitica [151]. Prillinger et al. [190]
considered these yeast isolates, together with the agarics from which they were
isolated, as sexual symbionts and missing links in the evolution from mycoparasitism to sexuality (primary homothallism and heterothallism; fig. 5, 7). Based
on nucleotide divergence of the complete 18S rDNA from C. neoformans, the
type species of the genus Cryptococcus, we have placed the two yeast isolates
from the agarics and C. humicola in the new genus Asterotremella (fig. 4)
[Prillinger et al., in preparation].
Cantharellales
The Cantharellales (fig. 4) include cantharelloid to agaricoid (Cantharellus,
Craterellus), hydnoid (Hydnum), clavarioid to coralloid (Clavulina), clavarioid
(Multiclavula), and corticioid (Botryobasidium) fungi [337]. A distinctive
feature of the Cantharellales is the possession of ‘stichic’ basidia [383, 384].
Analyses of mt-rDNA sequences suggest that the classical heterobasidiomycete
order Tulasnellales belongs in the cantharelloid clade [398]. Based on morphological and ultrastructural data, Oberwinkler [pers. commun.] has some doubts
on this suggestion. Our alignments of complete 18S rDNA sequences suggests
a higher evolution rate in two Tulasnella species (see above). Except for
Botryobasidium, the species of this group have dolipores with perforate parenthosomes [325, 399, 400]. These data suggest that Botryobasidium occupies a
systematic position at the base of this group. Most members of the Cantharellales are known or presumed to be mycorrhizal, but Multiclavula is a basidiolichen. Cantharellus cibarius and Craterellus cornucopioides are delicious
edible mushrooms.
Based on molecular data [383], there is no evidence for the ‘Clavaria
theory’ of Corner [401]. Corner [401] treats the cantharelloid and clavarioid
Systematics of the Ascomycota and Basidiomycota
263
fungi as a basal paraphyletic group from which all other Homobasidiomycetes
derived.
Presently, the Cantharellales comprise about 170 described species of
Hymenomycetes.
Gomphales
This order originally was introduced by Jülich [402] based on comparative
morphology and a dark green reaction of the fruiting body plectenchyma when
treated with an aqueous solution of ferric sulfate; however, Jülich does not
include gasteroid genera and families as suggested by Pine et al. [383]. The
Gomphales (fig. 4) [337, 383, 398] include club-shaped fungi (Clavariadelphus),
cantharelloid forms (Gomphus), coralloid fungi (Lentaria, Ramaria), hydnoid
resupinate fungi (Kavinia), gilled mushrooms (Gloeocantharellus), and some
Gasteromycetes (false truffels: Gauteria; earthstars: Geastrum; cannon-ball
fungus: Sphaerobolus; stinkhorns: Clathraceae, Phallaceae). The corticioid
fungus Ramaricium probably also belongs in this group [337].
The gomphoid-phalloid clade includes presently about 350 described
species.
Thelephorales
The thelephoroid clade (fig. 4) [337, 398] is a morphologically diverse
group that includes corticioid fungi (Tomentella), clavarioid forms (Thelephora),
and pileate fruiting bodies with poroid (Boletopsis), toothed (Hydnellum,
Sarcodon), smooth to wrinkled or tuberculate (Thelephora), or lamellate
hymenophores (Lenzitopsis). Oberwinkler [186] suggested that the agaricoid
fungus Verrucospora is related to the Thelephorales based on spore morphology.
Characters used by Donk [403] to support the Thelephoraceae include dark,
ornamented spores with an angular outline, pigmentation of the fruiting body,
and the presence of thelephoric acid [404]. Thelephoric acid, which is a
terphenylquinone product of the shikimate-chorismate pathway is found in
Bankera, Boletopsis, Hydnellum, Phellodon, Polyozellus, Pseudotomentella,
Sarcodon and Thelephora. This is consistent with molecular characters, which
strongly support monophyly of the Thelephorales. Nevertheless, thelephoric
acid also occurs in Suillus and Rhizopogon (Boletales), Omphalotus and
Lampteromyces (Agaricales) and Trametes (Polyporales) [337].
All members of this group are thought to be ectomycorrhizal or orchid
symbionts [398]. The phylogenetic relationship to the Bankeraceae (Phellodon)
awaits further molecular studies. So far only dolipores with perforate parenthesomes are known in the Thelephorales [399, 400].
The Thelephorales presently include about 240 described species of
Hymenomycetes.
Prillinger/Lopandic/Schweigkofler/Deak/Aarts/Bauer/Sterflinger/Kraus/Maraz
264
Polyporales
The polyporoid clade (fig. 4) [337, 382] is primarily composed of corticioid
fungi and polypores, but also includes the gilled mushrooms Lentinus, Panus
and Faerberia as well as the ‘cauliflower fungus’ Sparassis. The brown rot root
parasite Sparassis spathulata appears to be phylogenetically related to the brown
rot polypores Laetiporus sulphureus, Phaeolus schweinitzii and Antrodia carbonica [337]. The phylogenetic relationship of the brown rot genera
Gloeophyllum and Neolentinus (Lentinus lepideus) to this clade (fig. 4) [337], is
presently not clear, however. Although the polyporoid clade as a whole is weakly
supported, there are four groups within the polyporoid clade that are strongly
supported and have corroborating anatomical and physiological characters.
They may be candidates for a new family delimitation (Fomitopsis-Daedalea
Piptoporus: brown rot, unifactorial mating system; Fomes Polyporus-LentinusGanoderma: white rot, bifactorial mating system; Bjerkandera-PhanerochaeteCeriporia-Phlebia: white rot; Antrodia-Phaeolus-Laetiporus-Sparassis: brown
rot) [405]. Dolipores with perforate parenthosomes are common in the
Polyporales [406, 407]; Phanerochaete so far is the only genus having imperforate parenthesomes [400]. A perforate parenthesome, however, was found in
Phanerochaete cremea [Bauer, unpubl. obs.]. Dimitic or trimitic hyphal systems
and hyphal pegs, fascicles of sterile hyphae that emerge from the hymenium are
common anatomical characters in the Polyporales [186].
Haploid apomixis was considered to be an extreme type of homothallic
breeding systems [41, 55, 83, 94], and its occurrence in the Basidiomycota in
nature was reviewed by Prillinger [93]. Using Polyporus ciliatus, we were able,
after eight inbreeding generations, to obtain isogenic strains which develop
fertile apomictic pileate fruiting bodies which could be distinguished from the
dikaryotic fruiting bodies only by microscopical investigations [103]. After 2
years of apomictic propagation, a Phlebia-like resupinate mutant (corticioid
morphology) appeared on a petri dish inoculated with one of these haploidapomictic pileate strains (fig. 11a). The Phlebia-like mutant exhibited a fertile
and predominantly bisporic apomictic hymenium, but it had still not lost its
mating capacity. It was therefore easy to obtain the three additional mating types
of the resupinate mutant by crossing with a compatible pileate strain
(fig. 11a). P. ciliatus is a complex hetero-bifactorial white rot fungus (fig. 5).
Compatible crosses of two resupinate strains yielded a morphologically similar
Phlebia-like dikaryotic strain, which, in contrast to the haploid-apomictic
strains, microscopically exhibited a clamped mycelium and predominantly
four-spored basidia (fig. 11b) [94]. This result clearly confirmed the polygenic
control of fruit body formation suggested by Prillinger and Six [103] and
excluded the existence of a ‘fruiting initiation gene’ which was postulated by
Esser et al. [408]. A genetic analysis of 200 haploid single-spore cultures of a
Systematics of the Ascomycota and Basidiomycota
265
a
b
Fig. 11. A Phlebia-like resupinate monogenic mutant of Polyporus ciliatus. The
resupinate mutant appeared in a culture of a haploid pileate fruiter strain after 2 years of
apomictic propagation. a Different mating types of the resupinate mutant were obtained from
a cross between the resupinate haploid mutant and a pileate haploid fruiter strain (bottom);
from the dikaryon (top) it is obvious that the resupinate mutant behaves as a recessive gene.
b A dikaryotic resupinate strain was obtained (top) in a cross of two compatible haploid
resupinate strains. For additional information, see Prillinger [94] and text.
Table 4. A monogenic fertile resupinate mutant of Polyporus ciliatus
Dikaryotic cross
Spores
Resupinate haploid
fruiting bodies
number
A1B1 A2B2
A1B2 A2B1
A2B1 A1B2
A2B2 A1B1
Total
Haploid progeny
germinated
resupinate
haploid
fruiting bodies
50
50
50
50
49
45
48
47
21
22
28
24
28
21
20
23
200
189
95
92
cross of resupinate with pileate strains yielded a clear-cut 1:1 (95:92) segregation pattern (table 4), indicating a typically monogenic character. In view of this
monogenic difference between resupinate and pileate strains with a corticoid and
poroid hymenium, respectively, these resupinate fruiting bodies can be interpreted as atavistic only [94]. Our data are in favor of a concept of Oberwinkler
[186] which suggests that the different fruiting bodies of the classical
Homobasidiomycetes have evolved repeatedly from morphologically simple
resupinate ancestors. Different types of haploid fruiting bodies (resupinate,
Prillinger/Lopandic/Schweigkofler/Deak/Aarts/Bauer/Sterflinger/Kraus/Maraz
266
clavarioid, coralloid, pileate) detected in single-spore cultures of P. ciliatus are a
valuable tool to trace the phylogeny of the Polyporales (fig. 6a) [103].
Within Lentinus tigrinus (fig. 4) Rosinski and Robinson [409] detected
complete intercompatibility between monokaryotic isolates of the pileate
L. tigrinus and the gasteromycete Lentodium squamulosum. They accept
Le. squamulosum as a recessive mutant of L. tigrinus and recognized it as a
variety.
The polyporoid clade contains roughly 1,350 described species of the
Hymenomycetes, including about 90% of the morphologically described
Polyporaceae and 25% of the Corticiaceae, as well as all Ganodermataceae and
Sparassidaceae [337].
Ganoderma applanatum, G. lucidum and G. meredithae are known to
cause allergy in humans [81]. Anamorphs of polypores and corticoid fungi are
encountered with increasing frequency in clinical mycology. Identification to
species has been problematic, especially in the case of haploid monokaryons
[410]. Molecular techniques like ribosomal DNA sequencing may faciliate the
identification of filamentous haploid Hymenomycetes in the future.
Bjerkandera adusta is one of the most common Hymenomycetes isolated in
North America from sputum, skin, urine and air [410]. Other Polyporales
(Phlebia rufa, Phanerochaete chrysosporium, anamorph: Sporotrichum
pruinosum) are rarely isolated from human sputum [410].
Hymenochaetales
The order Hymenochaetales (fig. 4) was introduced by Oberwinkler
[186] and corroborated by Hibbett and Thorn [337] by molecular data. The
Hymenochaetales comprise the classical Hymenochaetaceae and some additional genera morphologically placed in the Corticiaceae (Basidioradulum,
Hyphodontia) and Polyporaceae (Trichaptum, Oxyporus, Schizopora). Thus, the
Hymenochaetales include resupinate and bracket-like poroid, toothed, and
corticoid forms. In addition, the clavarioid Clavariachaete belongs to the
Hymenochaetales, based on anatomical features [186]. Based on the following
characters the Hymenochaetaceae have been considered monophyletic:
clampless generative hyphae, fruiting bodies darkening in KOH, production of
white rot, presence of thick-walled tapering cystidia (setae). In addition, most
members of the Hymenochaetales have been shown to have imperforate parenthosomes (Basidioradulum, Hyphodontia, Inonotus, Phellinus, Schizopora,
Trichaptum) [325, 411–413]. Coltricia is a remarkable exception having
perforate parenthesomes and being the only ectomycorrhizal member of the
Hymenochaetales [406]. In our phylogram (fig. 4), Schizopora paradoxa
clusters outside the Hymenochaetales. In a phylogram of combined data for
mit-ssu rDNA and nuc-ssu rDNA, S. paradoxa is within the Hymenochaetales
Systematics of the Ascomycota and Basidiomycota
267
[337, 382]. Presently, there are no morphological-anatomical data to include
Basidioradulum, Hyphodontia, Oxyporus, Schizopora and Trichaptum in the
Hymenochaetales [Oberwinkler, pers. commun.].
The Hymenochaetales presently include about 630 described species of
Hymenomycetes.
Russulales
Based on comparative anatomy and morphology, Oberwinkler [186] redefined the Russulales and discussed older concepts of this order. The russuloid
clade has a remarkable diversity of fruiting body morphologies (fig. 4)
[337, 382]. There are resupinate (Stereum), coralloid (Clavicorona), and pileate
(Russula) forms with smooth (Stereum), toothed (Hericium), lamellate
(Lentinellus, Russula), or poroid hymenophores (Bondarzewia). Gasteroid forms
are also common (e.g. Martellia, Macowanites, Zelleromyces). The russuloid
clade is also ecologically variable, having ectomycorrhizal (Lactarius,
Russula), parasitic (Heterobasidion), saprophytic (Auriscalpium, Stereum) and
possibly lichenized (Pleurogala igapoensis) [414] species. Heterobasidion
annosum (fig. 4) is one of the most important root pathogens in European
spruce forests [415]. Species of Hericeum occur as parasites (H. erinaceus) and
saprophytes (H. clathroides) and cause a white rot. As discussed by Donk [403]
and Oberwinkler [186], many members of the Russulales have spores with amyloid ornamentations and gloeoplerous cystidia or hyphae, but these characters are
variable within the group. Hibbett and Thorn [337] estimate that the russuloid
clade contains approximately 1,000 described species of Hymenomycetes,
including roughly 20% of the morphologically described Corticiaceae.
Lactarius deliciosus and Russula cyanoxantha are well known delicious,
edible mushrooms.
Boletales
The history and concept of the Boletales is extensively discussed in Bresinsky
[64]. The delimitation of the order is based on morphological (spindle-shaped
basidiospores), chemical (pigments: shikimate-chorismate pathway derivatives),
wood decay (brown rot), specific mycoparasites (Sepedonium) and more recently
and better reliable on molecular data [382, 398, 416–421]. Based on a sequence
database of the mitochondrial large subunit rRNA gene, Bruns et al. [398] divided
the Boletales into six groups. Complete 18S nuclear ribosomal DNA sequences
and additional species may be useful to corroborate this groups at the family
level: group 1 (Boletaceae): Boletus, Chamonixia, Gastroboletus, Leccinum,
Strobilomyces, Tylopilus, Xerocomus; within group 1 the genera Boletus and
Xerocomus appeared to be heterogeneous; group 2 (Paxillaceae): Chalciporus,
Paxillus s. str., Paragyrodon; group 3 (Tapinellaceae): Hygrophoropsis, Serpula,
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268
Tapinella; group 4 (Coniophoraceae): Coniophora; group 5 (Gyrodonaceae):
Gyrodon, Gyroporus, Phaeogyroporus, Pisolithus; group 6 (Suillaceae): Alpova,
Brauniellula, Chroogomphus, Gomphidius, Hymenogaster, Melanogaster,
Rhizopogon.
The bolete clade contains resupinate fungi (Coniophora, Serpula), cantharelloid forms (Hygrophoropsis), gilled mushrooms (Gomphidius, Paxillus,
Tapinella), false truffels (Alpova, Hymenogaster, Melanogaster, Rhizopogon),
secotioid fungi (Brauniellula, Chamonixia, Gastroboletus, Gastrosuillus), and
puffballs (Pisolithus, Scleroderma), including the unusual stalked, gelatinous
puffball Calostoma [337].
The shikimate-chorismate pathway produces a number of compounds that
are characteristic of the Boletales (atrotomentin, pulvinic acid derivatives,
cyclopentanoids, polyprenylquinones). These compounds have been found in
diverse corticoid, resupinate, cantharelloid, lamellate, boletoid, and gasteroid taxa
including Boletus, Chamonixia, Chroogomphus, Coniophora, Gomphidius,
Gyrodon, Hygrophoropsis, Leucogyrophana, Paxillus, Phylloporus, Pisolithus,
Rhizopogon, Scleroderma, Serpula, Suillus, and some others [422]. Atrotomentin,
pulvinic acid derivatives, and cyclopentanoids have also been found in the lignicolous white rot agarics Omphalotus and Lampteromyces [423]. However, analyses of rDNA sequences suggest that Omphalotus and Lampteromyces belong to a
distinct family Omphalotaceae within the Agaricales [421]. In addition, atrotomentin and cyclopentanoids are found outside the Boletales in Albatrellus,
a heterogeneous genus within the Polyporales and Russulales [337, 398] and
Hydnellum (Thelephorales). These observations imply that the production of atrotomentin, pulvinic acid derivatives, and cyclopentanoids has evolved repeatedly.
The Boletales presently include about 840 described species of
Hymenomycetes. Many species of the Boletales are known as tasty (Boletus
edulis, Xerocomus badius) and a few as poisonous (B. satanas, Paxillus involutus).
P. involutus caused immunohemolytic anemia after repeated consumption of its
carpophores. Bresinsky and Besl [424] present an extensive documentation of
poisonous mushrooms and fungal intoxications. Jarosch and Bresinsky [425]
investigated the problem of speciation and cryptic species in P. involutus. Boletus
species and Serpula lacrymans are known as allergenic fungi [297, 426, this volume]. The brown-rot fungus S. lacrymans is primarily important in Europe, where
the dreaded dry rot causes tremendous damage to wooden structural elements and
floors in houses and other buildings.
Schizophyllales
Different from the combined analysis of nuc-ssu and mt-ssu rDNA of
Hibbett et al. [382] and Hibbett and Thorn [337] Fistulina hepatica and
Schizophyllum commune form a distinct clade with high bootstrap support,
Systematics of the Ascomycota and Basidiomycota
269
closely related to the Agaricales in our phylograms of complete 18S nuc-DNA
and partial sequences of the 26S rDNA (fig. 4) [Schweigkofler unpubl.].
We tentatively use the order Schizophyllales originally suggested by Nuss [427]
based on comparative morphology for this clade. Additional sequences,
especially from cyphelloid fungi may be useful to corroborate or reject this order.
S. commune is probably the best-known basidiomycetous agent of infection. Reports involving the lung include fungus ball of the lung [428], a case of
allergic bronchopulmonary mycosis in an otherwise healthy female [429] and
repeated isolation of S. commune from the sputum of a patient with chronic
lung disease [430]. Other reports of S. commune infections include cases of
meningitis [431], sinusitis [432–434], ulcerative lesions of the hard palate
[435], and possible onychomycosis [436] in both immunocompetent and
immunosuppressed hosts. The presence of clamp connections on dikaryotic
hyphae and the development of fruiting bodies in culture are primary characters which allow the identification of S. commune in human infections. The
diagnostic problems presented by a monokaryotic nonclamped, nonfruiting isolate from a dense mass in the right upper lobe of the lung in a female with a past
history of pulmonary tuberculosis and diabetes are described and discussed by
Sigler et al. [428]. Genotypic identification methods like partial sequencing of
ribosomal DNAs [16] are a promising tool to solve these problems in the future.
Agaricales
The Agaricales sensu Fries have long been recognized as an artificial
taxon, but the number of independent evolutionary lines of gilled mushrooms,
and their order relationships, have remained controversial [186, 402, 437, 438].
In a molecular analysis Hibbett et al. [382] suggest that agaricoid forms evolved
at least six times independently within the Hymenomycetes. Our present understanding of the phylogeny of the Hymenomycetes is only partially consistent
with Corner’s [401] ‘Clavaria theory’; agarics are polyphyletic, but there is
no indication that they have all derived from paraphyletic grades of club and
coral fungi. The molecular data of Hibbett et al. [382] and Hibbett and Thorn
[337] agree well with a phylogenetic concept of Oberwinkler [186] and genetic
experiments (table 4, fig. 11) of Prillinger [94] which suggest that the agarics
have evolved polyphyletically from morphologically simple corticioid or
resupinate ancestors.
The Agaricales contain the majority of the gilled mushrooms in the
Agaricales sensu Singer [438]. The largest groups of gilled mushrooms outside
the Agaricales are Lentinus (Polyporales), Lactarius, Russula (Russulales),
Gomphidius, Paxillus, Tapinella (Boletales). Although the pigments of
Lampteromyces, Omphalotus and Ripartites point to a Boletales affinity, these
species were included in the Agaricales by Binder et al. [421] based on ribosomal
Prillinger/Lopandic/Schweigkofler/Deak/Aarts/Bauer/Sterflinger/Kraus/Maraz
270
DNA sequencing. Pegler [439] restricted Lentinus to dimitic species, and therefore, transferred the monomitic shiitake fungus, traditionally known as Lentinus
edodes, into Lentinula which is close to Collybia within the Agaricales [440,
441]. In addition, ribosomal DNA sequence information suggests that species
of the clavarioid genera Clavaria, Clavulinopsis and Typhula belong to the
Agaricales too [382, 383]. Whereas Hibbett and Thorn [337] present evidence
to include at least some representatives of the Ceratobasidiales in the Agaricales,
data from Lee and Jung [442] and our investigations (Rhizoctonia solani;
fig. 4), however, are not in agreement with this suggestion and warrant further
investigations.
Classical Gasteromycetes that have been sampled in the Agaricales include
bird’s nest fungi, puffballs and many secotioid fungi. Lycoperdon, Calvatia, and
Tulostoma form a weakly supported monophyletic group within the Agaricales
which is phylogenetically closely related to Lepiota procera [382]. The exact
placement of the bird’s nest fungi (Nidulariaceae) is not resolved with
confidence [382]. Further gasteroid species or genera which have been studied
include Hydnangium (H. carneum, H. sublamellatum, H. microsporium),
Podohydnangium sp. which is closely related to Laccaria (L. bicolor, L. oblongospora, L. laccata, L. trullisata, L. gomezii, L. vinaceobrunnea, L. glabripes,
L. proximella), Montagnea arenaria and Podaxis pistillaris, which are closely
related to Coprinus comatus (Coprinus appeared to be heterogeneous) and
Leratia and Weraroa, which show affinities to Stropharia [422, 443, 444].
According to Hibbett and Thorn [337], the Agaricales contain approximately 7,400 species of gilled fungi and 1,025 species of the classical Aphyllophorales and Gasteromycetes. These species represent more than half of
all known classical Homobasidiomycetes, including approximately 87% of
all known gilled mushrooms [175]. Although symbionts of plants (mycorrhizae: Amanita, Cortinarius, Hebeloma, Inocybe) dominate, symbionts of animals (Amylostereum, Termitomyces), saprotrophs (Agaricus bisporus,
Pleurotus ostreatus, Lentinula edodes), pathogens (Armillaria mellea,
Crinipellis perniciosa, Mycena citricolor, Oudemansiella mucida, Pholiota
aurivella), and mycoparasites (Asterophora lycoperdoides) can be found as
well [337].
Agaricus bisporus, Calvatia cyathiformis, Coprinus comatus, C. quadrifidus, Lentinula edodes, Pleurotus ostreatus, Psilocybe cubensis are known
allergenic fungi [81, 426, this volume]. Psilocybe mexicana and other related
species have been referred to collectively as the ‘sacred or divine mushrooms’
or ‘teonanácatl’ used for centuries in certain religious rites of endemic peoples
of Mexico. The halucinogenic compounds present in these fungi were first isolated
and identified by Hofmann et al. [445] and named psilocin and psilocybin. The
hallucinogenic properties of both compounds are similar to d-lysergic acid
Systematics of the Ascomycota and Basidiomycota
271
diethylamide (LSD). They are extensively discussed in Bresinsky and Besl
[425] and Alexopoulos et al. [63].
Phylogenetic analyses of partial sequences from nuclear 26S rDNA
indicate monophyletic Pleurotaceae, consisting of the monophyletic genera
Pleurotus and Hohenbuehelia. The attack and consumption of nematodes
(nematophagy) support the monophyly of this family [441].
Agaricus campestris, Amanita caesarea, Coprinus comatus, Macrolepiota
procera, Marasmius scorodonius, Pholiota mutabilis and Rozites caperata are
well-known edible mushrooms. Bresinsky and Besl [424] present an overview
of many poisonous species (e.g. Amanita muscaria, A. phalloides, Cortinarius
orellanus, Inocybe patouillardi, Lampteromyces japonicus, Omphalotus
olearius) and different fungal intoxications.
Genotypic Identification
A number of fingerprinting methods based on the analysis of genomic
DNA polymorphism have been developed in the last decades for genotypic
species identification and delimitation. In our laboratory, we use two techniques successfully. RAPD-PCR is a simple and highly specific method. It was
introduced independently by Welsh and McClelland [446] and Williams et al.
[447]. We used RAPD-PCR to separate different species of yeasts like Mrakia
and Sterigmatomyces [340], Kluyveromyces [448], Metschnikowia [449],
Saccharomyces [450, 451], to identify known and new species of yeasts from
nature [148, 452], and to identify or describe new species of filamentous microfungi (Fusarium [281]; Ophiostoma [293]; Verticillium [290]; endophytic fungi
from grapevine [16, 305]). Rapid DNA extraction without digestion or removal
of RNA yields the template for RAPD analysis, which is routinely used to
determine the concentration of chromosomal DNA and the DNA to RNA ratio
on the basis of the intensities of ethidium-bromide-stained bands. Since the
amplification reaction for RAPD analysis cannot be completely standardized,
the absolute numbers and sizes of the fragments formed are by themselves not
significant markers for strain differentiation. Therefore, all of the strains that
are to be compared must be processed with one stock solution of premixed
reagents in one run of the thermocycler used and loaded onto one gel for maximum information output [340].
Amplified fragment length polymorphism (AFLP), developed by Vos et al.
[453], is also a new technique for DNA fingerprinting. This technique is based
on the selective amplification of restriction fragments (SARE) by PCR from a
digest of total genomic DNA.
Prillinger/Lopandic/Schweigkofler/Deak/Aarts/Bauer/Sterflinger/Kraus/Maraz
272
AFLP involves four steps: (a) restriction enzymes digestion; (b) ligation of
adapters to the restriction fragments; (c) PCR amplification of the restriction/
ligation fragments, and (d) detection and analysis of the amplified fragments.
The restriction fragments are generated by two restriction enzymes, one of
them is a rarely cutting one and the other is a frequently cutting one. Specific
adapters are ligated to the restriction fragments at the temperature at which
restriction enzymes are still active to avoid concatamer formation of the restriction fragments. Two different adapters are used, one specific for the rare cutting
enzyme and one specific for the frequent cutting enzyme. After ligation of the
adapters, the fragments have appropriate attachment sites for the selective PCR
primers. One of the two primers used is labeled (by isotopes or fluorescently).
Amplified fragments are detected according to the labeling technique.
Automatic laser fluorescence analysis (ALFA) [454] provides an automated
detection and analysis of fluorescently labeled samples.
The AFLP has the power of PCR and the solidity of RFLP analysis.
As the AFLP is highly reproducible and has a deep enough resolution due to
the number of estimable detected fragments; this technique allows us to analyze
the strains at the intraspecies level. With this technique we can carry out
epidemiological studies or can generate a database for routine identification.
We proved that its application is very useful in the determination of the route of
an infection or in the correct identification of pathogenic yeasts.
Figure 12 shows a digital electrophoresis image of ALFA-AFLP fragments
of Candida guilliermondii and C. glabrata strains, run on an automatic DNA
sequencer. A high degree of similarity can be clearly recognized among the
strains belonging to the same species. They share several (supposedly speciesspecific) bands in the same position, while differentiation of some of the isolates
can be performed according to the noncommon fragments. Completely identical
patterns can be seen, e.g. lanes 12 and 13, indicating that these isolates are probably in epidemiological relation. Proof of the identity, however, requires the
performance of AFLP by an additional adapter and primer set. Separation of the
isolates belonging to the two different species is clear by a simple visual evaluation of the patterns.
Recently we have shown that it is not possible to identify ascomycetous
and basidiomycetous yeasts unequivocally using the classical phenotypic
approach [208]. The score of correct identification using the phenotypic
approach is rather low especially in basidiomycetous yeasts. Partial sequences
of the 18S and 26S rDNA have become a reliable tool to identify yeast species
correctly [148, 452]. Meanwhile, partial sequences of the D1/D2 region of the
large subunit rDNA are available for all known ascomycetous [23] and basidiomycetous [24] yeasts in GenBank. This offers the possibility of a reliable
genotypic identification using the D1/D2 region. According to our experience
Systematics of the Ascomycota and Basidiomycota
273
1 2 3 4 5 6 7
12 13
16
25
34
Fig. 12. Digital electrophoresis image of AFLP fragments of Candida strains run on
an automatic DNA sequencer. Markers: lanes 1, 16, 25, 34; Candida guilliermondii: clinical
isolates, lanes 2–6; Candida glabrata: type strain CBS 138 lane 7, clinical isolates: others.
it seems worthwhile to corroborate the identification data obtained with a
RAPD-PCR analysis with the respective type strain.
For the identification of new yeast species from nature we use a new
polyphasic approach [452]: (1) Species delimitation using RAPD-PCR or
Prillinger/Lopandic/Schweigkofler/Deak/Aarts/Bauer/Sterflinger/Kraus/Maraz
274
AFLP; (2) Species identification using partial sequences of the 18S or 26S
rDNA and corroboration of the identification data using RAPD-PCR and the
respective type strain; (3) Ubiquinone system according to Messner et al. [340],
and (4) Qualitative and quantitative monosaccharide pattern of purified and
hydrolyzed cell walls [449].
Just as for the identification of yeasts, partial sequences of the 18S and 26S
rDNA are also useful for identifying filamentous microfungi. In contrast to
yeasts, however, reliable identification [16] is not always possible due to a lack
of sequences in Genbank.
Acknowledgments
We would like to thank Dr. D. Begerow, Dr. T. Boekhout, Prof. M. Breitenbach,
Dr. T.D. Bruns, Emeritus Prof. F. Ehrendorfer, Prof. O.E. Eriksson, Emeritus Prof. K. Esser,
Prof. J.W. Fell, Dr. M. Fischer, Emeritus Prof. W. Gams, Mag. G. Hagedorn, Dr. M.
Hamamoto, Dr. D.S. Hibbett, Dr. G. Himmler, Prof. G.S. de Hoog, Dr. P. Hoffmann, Dr. J.
Kämper, Prof. C. Kubicek, Dr. U. Kües, Dr. C.P. Kurtzman, Dr. S. Landvik, Dr. J. Loidl,
Prof. M. Melkonian, DI. Dr. R. Messner, Prof. U.G. Mueller, Dr. T. Nakase, Prof. L. Sigler,
Emeritus Prof. J. Sugiyama, Dr. M. Suzuki, Dr. E. Swann, Emeritus Prof. Y. Yamada for
sending interesting strains, valuable literature, critical comments or unpublished sequences.
My wife J. Prillinger and Ms. I. Mondl improved figures 2, 6 and 7. Ing. S. Huss prepared
a CD version of all figures. Prof. G.S. de Hoog informed the first author that his excellent
Atlas of Clinical Fungi is scheduled in a new, fully revised and greatly expanded edition for
early 2000.
Finally the first author is grateful to Prof. F. Oberwinkler of the University Tübingen
for his excellent introduction into mycology, sending interesting strains and many valuable
comments on the manuscript.
References
1 Zuckerkandl E, Pauling L: Molecules as documents of evolutionary history. J Theor Biol 1965;8:
357–366.
2 Woese CR: Bacterial evolution. Microbiol Rev 1987;51:221–271.
3 Sogin ML, Morrison HG, Hinkle G, Silberman JD: Ancestral relationships of the major eukaryotic
lineages. Microbiologia 1996;12:17–28.
4 Haeckel E: Systematische Phylogenie der Protisten und Pflanzen. Berlin, Reimer, 1894.
5 Kimura M: Evolutionary rate at the molecular level. Nature 1968;217:624–626.
6 Kimura M: A simple method for estimating evolutionary rates of base substitutions through
comparative studies of nucleotide sequences. J Mol Evol 1980;16:111–120.
7 Kimura M: The Neutral Theory of Molecular Evolution. Cambridge, Cambridge University Press,
1983.
8 Sogin ML, Gunderson JH, Elwood HJ, Alonso RA, Peattie DA: Phylogenetic significance of the
Kingdom concept: An unusual eukaryotic 16S-like ribosomal RNA from Giardia lamblia. Science
1989;243:75–77.
Systematics of the Ascomycota and Basidiomycota
275
9 Bhattacharya D, Elwood HJ, Goff LJ, Sogin ML: Phylogeny of Gracilaria lemaneiformis
(Rhodophyta) based on sequence analysis of its small subunit ribosomal RNA coding region.
J Phycol 1990;26:181–186.
10 Swann EC, Taylor JW: Phylogenetic diversity of yeast-producing basidiomycetes. Mycol Res
1995;99:1205–1210.
11 Swann EC, Frieders EM, McLaughlin DJ: Microbotryum, Kriegeria and the changing paradigm
in basidiomycete classification. Mycologia 1999;91:51–66.
12 Suh S-O, Nakase T: Phylogenetic analysis of the ballistosporous anamorphic genera Udenomyces
and Bullera, and related basidiomycetous yeasts, based on 18S rDNA sequence. Microbiology
1995;141:901–906.
13 Melkonian M: Phylogeny of photosynthetic protists and their plastids. Verh Dtsch Zool Ges
1996;89:71–96.
14 Sugiyama J: Relatedness, phylogeny, and evolution of the fungi. Mycoscience 1998;39:487–511.
15 Sugita T, Nakase T: Trichosporon japonicum sp. nov. isolated from the air. Int J Syst Bacteriol
1998;48:1425–1429.
16 Schweigkofler W, Prillinger H: Molekulare Identifizierung und phylogenetische Analyse von
endophytischen und latent pathogenen Pilzen der Weinrebe. Mitt Klosterneuburg 1999;49:65–78.
17 Yamada Y, Kawasaki H, Nagatsuka Y, Mikata K: The phylogeny of the cactophilic yeasts based on
the 18S ribosomal RNA gene sequences: The proposals of Phaffomyces antillensis and Starmera
caribaea, new combinations. Biosci Biotechnol Biochem 1999;63:827–832.
18 Perasso R, Baroin A, Qu LH, Bachellerie JP, Adoutte A: Origin of the algae. Nature 1989;339:
142–144.
19 Yamada Y, Nagahama T, Kawasaki H, Banno I: The phylogenetic relationships of the genera
Phaffia Miller, Yoneyama et Soneda and Cryptococcus Kützing emend. Phaff et Spencer
(Cryptococcaceae) based on the partial sequences of 18S and 26S ribosomal ribonucleic acids.
J Gen Appl Microbiol 1990;36:403–414.
20 Yamada Y, Higashi T, Mikata K: The phylogeny of species of the ascogenous telemorphic yeast
genera Ambrosiozyma, Hormoascus, Hyphopichia, Arthroascus, and Botryoascus based on the
partial sequences of 18S and 26S ribosomal RNAs. Bull Fac Agric Shizuoka Univ 1998;48:1–13.
21 Messner R, Prillinger H, Ibl M, Himmler G: Sequences of ribosomal genes and internal transcribed spacers move three plant parasitic fungi, Eremothecium ashbyi, Ashbya gossypii, and
Nematospora coryli, towards Saccharomyces cerevisiae. J Gen Appl Microbiol 1995;41:31–42.
22 Begerow D, Bauer R, Oberwinkler F: Phylogenetic studies on nuclear large subunit ribosomal
DNA sequences of smut fungi and related taxa. Can J Bot 1997;75:2045–2056.
23 Kurtzman CP, Robnett CJ: Identification and phylogeny of ascomycetous yeasts from analysis of
nuclear large subunit (26S) ribosomal DNA partial sequences. Antonie Van Leeuwenhoek 1998;
73:331–371.
24 Fell JW, Boekhout T, Fonseca A, Scorzetti G, Statzell-Tallman A: Biodiversity and systematics of
basidiomycetous yeasts as determined by large-subunit rDNA D1/D2 domain sequence analysis.
Int J Syst Evol Microbiol 2000;50:1351–1371.
25 Walker WF: 5S ribosomal RNA sequences from Ascomycetes and evolutionary implications.
Syst Appl Microbiol 1985;6:48–53.
26 Blanz PA, Unseld M: Ribosomal RNA as a taxonomic tool in mycology. Stud Mycol
1987;30:247–258.
27 Baldauf SL, Doolittle WF: Origin and evolution of the slime molds (Mycetozoa). Proc Natl Acad
Sci USA 1997;94:12007–12012.
28 Doolittle WF: Phylogenetic classification and the universal tree. Science 1999;284:2124–2128.
29 Bruns TD, Vilgalys R, Barns SM, Gonzalez D, Hibbett DS, Lane DJ, Simon L, Stickel S, Szaro
TM, Weisburg WG, Sogin ML: Evolutionary relationships within the fungi: Analyses of nuclear
small subunit rRNA sequences. Mol Phylogenet Evol 1992;1:231–241.
30 Berbee ML, Taylor JW: Dating the evolutionary radiations of the true fungi. Can J Bot 1993;71:
1114–1127.
31 Bresinsky A, Kadereit JW: Systematik-Poster Botanik. Stuttgart, Fischer, 1998.
32 Woese CR, Kandler O, Wheelis M: Towards a natural system of organisms: Proposal for the
domains Archaea, Bacteria, and Eucarya. Proc Natl Acad Sci USA 1990;87:4576–4579.
Prillinger/Lopandic/Schweigkofler/Deak/Aarts/Bauer/Sterflinger/Kraus/Maraz
276
33 Knoll AH: The early evolution of eukaryotes: A geological perspective. Science 1992;256:
622–627.
34 Gogarten JP, Kibak H, Dittrich P, Taiz L, Bowman EJ, Bowman BJ, Manolson MF, Poole RJ, Date
T, Oshima T, Konishi J, Denda K, Yoshida M: Evolution of the vacuolar H-ATPase: Implications
for the origin of eukaryotes. Proc Natl Acad Sci USA 1989;86:6661–6665.
35 Iwabe N, Kuma K-I, Hasegawa M, Osawa S, Miyata T: Evolutionary relationship of archaebacteria, eubacteria, and eukaryotes inferred from phylogenetic trees of duplicated genes. Proc Natl
Acad Sci USA 1989;86:9355–9359.
36 Vossbrinck CR, Maddox JV, Friedman S, Debrunner-Vossbrinck BA, Woese CR: Ribosomal RNA
sequence suggests microsporidia are extremely ancient eukaryotes. Nature 1987;326:411–414.
37 Hirt RP, Logsdon JM, Healy B, Dorey MW, Doolittle WF, Embley TM: Microsporidia are related
to fungi: Evidence from the largest subunit of RNA polymerase II and other proteins. Proc Natl
Acad Sci USA 1999;96:580–585.
38 Cavalier-Smith T: Kingdom Protozoa and its 18 phyla. Microbiol Rev 1993;57:953–994.
39 Heath B: Variant mitoses in lower eukaryotes: Indicators of the evolution of mitosis? Int Rev Cytol
1980;64:1–80.
40 Raikov IB: The Protozoan Nucleus Morphology and Evolution. Cell Biol Monogr. Vienna,
Springer, 1982, vol 9.
41 Prillinger H: Zur Evolution von Mitose, Meiose und Kernphasenwechsel bei Chitinpilzen.
Z Mykol 1984;50:267–352.
42 Margulis L, Corliss JO, Melkonian M, Chapman DI: Handbook of Protoctista. Boston, Jones &
Barlett, 1989.
43 Melkonian M: II. Systematics and evolution of the algae: Endocytobiosis and evolution of the
major algal lineages. Prog Bot 1996;57:281–311.
44 Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG: The ClustalX windows
interface: Flexible strategies for multiple sequence alignment aided by quality analysis tools.
Nucl Acids Res 1997;24:4876–4882.
45 Felsenstein J: Phylip-Phylogeny inference package (version 3.5c). Distributed by the author,
Department of Genetics, University of Washington, Seattle, 1993.
46 Page RDM: Treeview: An application to display phylogenetic trees on personal computers.
Comput Appl Biosci 1996;12:357–358.
47 Giovannoni SJ, Turner S, Olsen GJ, Barns S, Lane DJ, Pace NR: Evolutionary relationships among
the cyanobacteria and green chloroplasts. J Bacteriol 1988;170:3584–3592.
48 Yang D, Oyaizu Y, Oyaizu H, Olsen GJ, Woese CR: Mitochondrial origins. Proc Natl Acad Sci
USA 1985;82:4443–4447.
49 Margulis L: Symbiosis in Cell Evolution. San Francisco, Freeman, 1981.
50 Sitte P: Symbiogenetic evolution of complex cells and complex plastids. Eur J Protistol
1993;29:131–143.
51 Jahrmann HJ, Prillinger H: Das Vorkommen eines ‘Hefe’-Stadiums bei dem Homobasidiomyceten
Asterophora (Nyctalis) lycoperdoides (Bull.) Ditm. ex S.F. Gray und seine Bedeutung für die
Phylogenese der Basidiomyceten. Z Mykol 1983;49:195–235.
52 Barr DJS: Evolution and kingdoms of organisms from the perspective of a mycologist. Mycologia
1992;84:1–11.
53 Cavalier-Smith T: Eukaryote kingdoms: Seven or nine? Biosystems 1981;14:461–481.
54 Cavalier-Smith T: The origin of fungi and pseudofungi; in Rayner ADM, Brasier CM, Moore D (eds):
Evolutionary Biology of the Fungi. Cambridge, Cambridge University Press, 1987, pp 339–353.
55 Prillinger H: Zur genetischen Kontrolle und Evolution der sexuellen Fortpflanzung und
Heterothallie bei Chitinpilzen. Z Mykol 1982;48:297–324.
56 Bartnicki-Garcia S: Cell wall composition and other biochemical markers in fungal phylogeny; in
Harborne JB (ed): Phytochemical Phylogeny. London, Academic Press, 1970, pp 81–103.
57 Bartnicki-Garcia S: The cell wall: A crucial structure in fungal evolution; in Rayner ADM, Brasier
CM, Moore D (eds): Evolutionary Biology of the Fungi. Cambridge, Cambridge University Press,
1987, pp 389–403.
58 Vogel HJ: Distribution of lysine pathways among fungi: Evolutionary implication. Am Nat 1964;
98:435–446.
Systematics of the Ascomycota and Basidiomycota
277
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
Vogel HJ: Lysine biosynthesis and evolution: Fungi, gymnosperms and angiosperms; in
Bryson V, Vogel HJ (eds): Evolving Genes and Proteins. New York, Academic Press, 1965,pp 25–40.
Korhonen K: Breeding units in the forest pathogens Armillaria and Heterobasidion; in Rayner
ADM, Brasier CM, Moore D (eds): Evolutionary Biology of the Fungi. Cambridge, Cambridge
University Press, 1987, pp 301–310.
Sansome ER: Fungal chromosomes as observed with the light microscope; in Rayner ADM,
Brasier CM, Moore D (eds): Evolutionary Biology of the Fungi. Cambridge, Cambridge University
Press, 1987, pp 97–113.
Bruns TD, White TJ, Taylor JW: Fungal molecular systematics. Annu Rev Ecol Syst 1991;22:
525–564.
Alexopoulos CJ, Mims CW, Blackwell M: Introductory Mycology, ed 4, rev. New York, Wiley &
Sons, 1996.
Bresinsky A: Abstammung, Phylogenie und Verwandtschaft im Pilzreich. Z Mykol 1996;62:
147–168.
Rast DM, Pfyffer GE: Acyclic polyols and higher taxa of fungi. Bot J Linnean Soc 1989;99:39–57.
Zuck RK: Alternation of generations and the manner of nutrition. Drew Univ Stud 1953;6:1–19.
Baldauf SL, Palmer JD: Animals and fungi are each other’s closest relatives: Congruent evidence
from multiple proteins. Proc Natl Acad Sci USA 1993;90:11558–11562.
Hasegawa M, Iida Y, Yano T, Takaiwa F, Miyata T: Phylogenetic relationships among eukaryotic
kingdoms inferred from ribosomal RNA sequences. J Mol Evol 1985;22:32–38.
Hasegawa M, Hashimoto T, Adachi J, Iwabe N, Miyata T: Early branching in the evolution of
eukaryotes: Ancient divergence of Entamoeba that lacks mitochondria revealed by protein
sequence data. J Mol Evol 1993;36:380–388.
Hendriks L, de Baere R, van de Peer Y, Neefs J, Goris A, de Wachter R: The evolutionary position
of the rhodophyte Porphyra umbilicalis and the basidiomycete Leucosporidium scottii among
other eukaryotes as deduced from complete sequences of small ribosomal subunit RNA. J Mol
Evol 1991;32:167–177.
Kumar S, Rzhetsky A: Evolutionary relationships of eukaryotic kingdoms. J Mol Evol 1996;
42:183–193.
Wainright PO, Hinkle G, Sogin ML, Stickel SK: Monophyletic origins of the Metazoa: An evolutionary link with fungi. Science 1993;260:340–342.
Nikoh N, Hayase N, Iwabe N, Kuma K, Miyata T: Phylogenetic relationships of the kingdoms
Animalia, Plantae, and Fungi inferred from 23 different protein species. Mol Biol Evol 1994;
11:762–768.
Veuthey AL, Bittar G: Phylogenetic relationships of Fungi, Plantae, and Animalia inferred from
homologous comparison of ribosomal proteins. J Mol Evol 1998;47:81–92.
Murray PR, Baron EJ, Pfaller MA, Tenover FC, Yolken RH: Manual of Clinical Microbiology,
ed 7. Washington, ASM Press, 1999.
Castlebury LA, Domier LL: Small subunit ribosomal RNA gene phylogeny of Plasmodiophora
brassicae. Mycologia 1998;90:102–107.
Berg LR, Patterson GW: Phylogenetic implications of sterol biosynthesis in Oomycetes.
Exp Mycol 1986;10:175–183.
Leipe DD, Wainright PO, Gunderson JH, Porter D, Patterson DJ, Valois F, Himmerich S, Sogin
ML: The stramenopiles from a molecular perspective: 16S-like rRNA sequences from
Labyrinthuloides minuta and Cafeteria roenbergensis. Phycologia 1994;33:369–377.
van der Auwere G, de Baere R, van der Peer Y, de Rijk P, van den Broeck I, de Wachter R: The
phylogeny of the Hyphochytridiomycota as deduced from ribosomal RNA sequences of
Hyphochytrium catenoides. Mol Biol Evol 1995;12:671–678.
De Hoog GS, Guarro J: Atlas of Clinical Fungi. Baarn, Centraalbureau voor Schimmelcultures,
Universitat Rovira i Virgili, 1995.
Horner WE, Helbling A, Salvaggio JE, Lehrer SB: Fungal allergens. Clin Microbiol Rev 1995;
8:161–179.
Pascher A: Systematische Übersicht über die mit Flagellaten in Zusammenhang stehenden
Algenreihen und Versuch einer Einreihung dieser Algenstämme in die Stämme des
Pflanzenreiches. Beih Bot Centralbl II 1931;48:317–332.
Prillinger/Lopandic/Schweigkofler/Deak/Aarts/Bauer/Sterflinger/Kraus/Maraz
278
83 Prillinger H: Yeasts and anastomoses: Their occurrence and implications for the phylogeny of
Eumycota; in Rayner ADM, Brasier CM, Moore D (eds): Evolutionary Biology of the Fungi.
Cambridge, Cambridge University Press, 1987, pp 355–377.
84 Sogin ML: Early evolution and the origin of eukaryotes. Curr Opin Gen Dev 1991;1:457–463.
85 Benjamin RK: Zygomycetes and their spores; in Kendrick WB (ed): The Whole Fungus. Ottawa,
National Museums of Canada, 1979, pp 573–616.
86 Nagahama T, Sato H, Shimazu M, Sugiyama J: Phylogenetic divergence of the entomophthoralean
fungi: Evidence from nuclear 18S ribosomal RNA sequence. Mycologia 1995;87:203–209.
87 McLauglin DJ, Beckett A, Yoon KS: Ultrastructure and evolution of ballistosporic basidiospores.
Bot J Linnean Soc 1985;91:253–271.
88 Tucker BE: A review of the nonentomogenous Entomophthorales. Mycotaxon 1981;13:481–505.
89 Sterflinger K, de Hoog GS, Haase G: Phylogeny and ecology of meristematic ascomycetes. Stud
Mycol 1999;43:5–22.
90 Hiscock SJ, Kües U: Cellular and molecular mechanisms of sexual incompatibility in plants and
fungi. Int Rev Cytol 1999;193:165–295.
91 Burgeff H: Sexualität und Parasitismus bei den Mucorineen. Ber Dtsch Bot Ges 1920;38:
318–327.
92 Burgeff H: Untersuchungen über Sexualität und Parasitismus bei Mucorineen; in Goebel K (ed):
Botanische Abhandlungen. Jena, Fischer, 1924, pp 1–135.
93 Prillinger H: Untersuchungen zur Fruchtkörper- und Artbildung bei Basidiomyceten: Das
Vorkommen von haploider Apomixis und Amphithallie in der Natur. Z Mykol 1982;48:275–296.
94 Prillinger H: Morphologische Atavismen bei Homobasidiomyceten durch natürliche und
künstliche Inzucht und ihre Bedeutung für die Systematik. Ber Dtsch Bot Ges 1986;99:31–42.
95 Yun S-H, Berbee ML, Yoder OC, Turgeon BG: Evolution of the fungal self-fertile reproduction
life style from self-sterile ancestors. Proc Natl Acad Sci USA 1999;96:5592–5597.
96 Bakkeren G, Kronstad JW: Linkage of mating-type loci distinguishes bipolar from tetrapolar
mating in basidiomycetous smut fungi. Proc Natl Acad Sci USA 1994;91:7085–7089.
97 Casselton LA, Kües U: Mating-type genes in homobasidiomycetes; in Esser K, Lemke PA (eds):
The Mycota. I. Growth, differentiation and sexuality. Berlin, Springer, 1994, pp 307–321.
98 Hijri M, Hosny M, van Tuinen, Dulieu H: Intraspecific ITS polymorphism in Scutellospora
castanea (Glomales, Zygomycota) is structured within multinucleate spores. Fungal Genet Biol
1999;26:141–151.
99 Hosny M, Hijri M, Passerieux E, Dulieu H: rDNA units are highly polymorphic in Scutellospora
castanea (Glomales, Zygomycetes). Gene 1999;226:61–71.
100 Sanders IR: No sex please, we’re fungi. Nature 1999;399:737–739.
101 Mosse B: Some studies relating to ‘independent’ growth of vesicular arbuscular endophytes. Can
J Bot 1988;66:2533–2540.
102 Remy W, Taylor TN, Hass H, Kerp H: Four hundred-million-year-old vesicular arbuscular
mycorrhizae. Proc Natl Acad Sci USA 1994;91:11841–11843.
103 Prillinger H, Six W: Genetische Untersuchungen zur Fruchtkörper- und Artbildung bei
Basidiomyceten: Genetische Kontrolle der Fruchtkörperbildung bei Polyporus ciliatus. Pl Syst
Evol 1983;141:341–371.
104 Prillinger H: Are there yeasts in Homobasidiomycetes? in de Hoog GS, Smith MT, Weijman ACM
(eds): The Expanding Realm of Yeast-Like Fungi. Amsterdam, Elsevier, 1987, pp 33–59.
105 Kemp RFO: Breeding biology of Coprinus spp. in the section Lanatuli. Trans Br Mycol Soc
1975;65:375–388.
106 Prillinger H, Altenbuchner J, Schulz B, Dörfler C, Forst T, Laaser G, Stahl U: Ustilago maydis
isolated from Homobasidiomycetes; in Galling G (ed): Proceedings Applied Plant Molecular Biology
(Braunschweig Symposium, Nov 1988). Braunschweig, Technische Universität, 1989, pp 408–425.
107 Savile DBO: A phylogeny of the Basidiomycetes. Can J Bot 1955;33:60–104.
108 Kellner M, Burmester A, Wöstemeyer A, Wöstemeyer J: Transfer of genetic information from the
mycoparasite Parasitella parasitica to its host Absidia glauca. Curr Genet 1993;23:334–337.
109 Wöstemeyer J, Wöstemeyer A, Burmester A, Czempinski K: Relationship between sexual
processes and parasitic interactions in the host-pathogen system Absidia glauca-Parasitella
parasitica. Can J Bot 1995;73 (suppl 1):S243–S250.
Systematics of the Ascomycota and Basidiomycota
279
110 Philley ML, Staben C: Functional analyses of the Neurospora crassa MT a-1 mating type polypeptide. Genetics 1994;137:715–722.
111 Saupe SJ, Descamps C, Turcq B, Begueret J: Inactivation of the Podospora anserina vegetative
incompatibility locus het-c, whose product resembles a glycolipid transfer protein, drastically
impairs ascospore production. Proc Natl Acad Sci USA 1994;91:5927–5931.
112 Saupe SJ, Stenberg L, Shiu KT, Griffiths AJ, Glass NL: The molecular nature of mutations in the
mt-A-1 gene of the Neurospora crassa A idiomorph and their relation to mating-type function.
Mol Gen Genet 1996;250:115–122.
113 Saupe SJ, Kuldau GA, Smith ML, Glass NL: The product of the het-C heterokaryon incompatibility gene of Neurospora crassa has characteristics of a glycine-rich cell wall protein. Genetics
1996;143:1589–1600.
114 Saupe SJ, Glass NL: Allelic specificity at the het-c heterokaryon incompatibility locus of
Neurospora crassa is determined by a highly variable domain. Genetics 1997;146:1299–1309.
115 Coustou V, Deleu C, Saupe S, Bequeret J: The protein product of the het-s heterokaryon incompatibility gene of the fungus Podospora anserina behaves as a prion analog. Proc Natl Acad Sci
USA 1997;94:9773–9778.
116 Espagne E, Balhadere P, Begueret J, Turcq B: Reactivity in vegetative incompatibility of the HETE protein of the fungus Podospora anserina is dependent on GTP-binding activity and a WD40
repeated domain. Mol Gen Genet 1997;256:620–627.
117 Ferreira AV, An Z, Metzenberg RL, Glass NL: Characterization of mat A-2, mat A-3 and
deltamatA mating-type mutants of Neurospora crassa. Genetics 1998;148:1069–1079.
118 Wu J, Saupe SJ, Glass NL: Evidence for balancing selection operating at the het-c heterokaryon
incompatibility locus in a group of filamentous fungi. Proc Natl Acad Sci USA 1998;95:
12398–12403.
119 Shiu PK, Glass NL: Molecular characterization of tol, a mediator of mating-type-associated
vegetative incompatibility in Neurospora crassa. Genetics 1999;151:545–555.
120 Wickner RB, Taylor KL, Edskes HK, Maddelein ML, Moriyama H, Roberts BT: Prions in
Saccharomyces and Podospora spp.: Protein-based inheritance. Microbiol Mol Biol Rev 1999;63:
844–861.
121 Glass NL, Grotelueschen J, Metzenber RL: Neurospora crassa A mating-type region. Proc Natl
Acad Sci USA 1990;87:4912–4916.
122 Staben C, Yanofsky C: Neurospora crassa A mating-type region. Proc Natl Acad Sci USA 1990;
87:4917–4921.
123 Turgeon BG, Christiansen SK, Yoder OC: Mating type genes in Ascomycetes and their
imperfect relatives; in Reynolds DR, Taylor JW (eds): The Fungal Holomorph: Mitotic, Meiotic
and Pleomorphic Speciation in Fungal Systematics. Wallingford, CAB International, 1993, pp
199–215.
124 Bölker M, Urban M, Kahmann R: The a mating type locus of Ustilago maydis specifies cell
signalling components. Cell 1992;68:441–450.
125 Kämper J, Bölker M, Kahmann R: Mating-type genes in Heterobasidiomycetes; in Esser K,
Lemke PA (eds): The Mycota, Growth, Differentiation and Sexuality. Heidelberg, Springer, 1994,
vol I, pp 323–332.
126 Hicks JB, Klar JS: Transposable mating type genes in Saccharomyces cerevisiae. Nature
1979;282:478–483.
127 Schulz B, Banuett F, Dahl M, Schlesinger R, Schäfer W, Martin T, Herskowitz I, Kahmann R: The
b alleles of Ustilago maydis, whose combinations program pathogenic development, code for
polypeptides containing a homeodomain-related motif. Cell 1990;60:295–306.
128 Hull CM, Johnson AD: Identification of a mating type-like locus in the asexual pathogenic yeast
Candida albicans. Science 1999;285:1271–1275.
129 Lopandic K: Genotypic Identification and Comparative Analysis of Cell Wall Sugars from
Asco- and Basidiomycetous Yeasts; PhD thesis, University of Zagreb, 1998.
130 Geiser DM, Timberlake WE, Arnold ML: Loss of meiosis in Aspergillus. Mol Biol Evol 1996;
13:809–817.
131 Peterson SW: Phylogenetic analysis of Penicillium species based on ITS and lsu-rDNA nucleotide
sequences. Proc 3rd Int Workshop Penicillium Aspergillus, Baarn, May 1997.
Prillinger/Lopandic/Schweigkofler/Deak/Aarts/Bauer/Sterflinger/Kraus/Maraz
280
132 Tamura M, Kawahara K, Sugiyama J: Molecular phylogeny of Aspergillus and associated
teleomorphs in the Trichocomaceae (Eurotiales). Proc 3rd Int Workshop Penicillium Aspergillus,
Baarn, May 1997.
133 LoBuglio KF, Pitt JI, Taylor JW: Phylogenetic analysis of two ribosomal DNA regions indicates
multiple independent losses of a sexual Talaromyces state among asexual Penicillium species in
subgenus Biverticillium. Mycologia 1993;85:592–604.
134 Ogawa H, Yoshimura A, Sugiyama J: Polyphyletic origins of species of the anamorphic genus
Geosmithia and the relationships of the cleistothecial genera: Evidence from 18S, 5S and 28S
rDNA sequence analyses. Mycologia 1997;89:756–771.
135 Bowman BH, Taylor JW: Molecular phylogeny of pathogenic and non-pathogenic Onygenales; in
Reynolds DR, Taylor JW (eds): The Fungal Holomorph: Mitotic, Meiotic and Pleomorphic
Speciation in Fungal Systematics. Wallingford, CAB International, 1993, pp 169–178.
136 Bowman BH, White TJ, Taylor JW: Human pathogenic fungi and their close nonpathogenic
relatives. Mol Phylogenet Evol 1996;6:89–96.
137 Hambleton S, Egger KN, Currah RS: The genus Oidiodendron: Species delimitation and phylogenetic relationships based on nuclear ribosomal DNA analysis. Mycologia 1998;90:854–869.
138 Guadet J, Julian J, Lafay JF, Brygoo Y: Phylogeny of some Fusarium species, as determined by
large subunit rRNA sequence comparison. Mol Biol Evol 1989;6:227–242.
139 O’Donnell K: Fusarium and its near relatives; in Reynolds DR, Taylor JW (eds): The Fungal
Holomorph: Mitotic, Meiotic and Pleomorphic Speciation in Fungal Systematics. Wallingford,
CAB International, 1993, pp 225–233.
140 O’Donnell K, Cigelnik E, Nirenberg HI: Molecular systematics and phytogeography of the
Gibberella fujikuroi species complex. Mycologia 1998;90:465–493.
141 Rehner SA, Samuels GJ: Taxonomy and phylogeny of Gliocladium analysed from nuclear large
subunit DNA sequences. Mycol Res 1994;98:625–634.
142 Rehner SA, Samuels GJ: Molecular systematics of the Hypocreales: A teleomorph gene
phylogeny and the status of their anamorphs. Can J Bot 1995;73(suppl 1):S816–S823.
143 Glenn AE, Bacon CW, Price R, Hanlin RT: Molecular phylogeny of Acremonium and its
taxonomic implications. Mycologia 1996;88:369–383.
144 Vogler DR, Bruns TD: Use of molecular characters to identify holomorphs: An example from the rust
genus Cronartium; in Reynolds DR, Taylor JW (eds): The Fungal Holomorph: Mitotic, Meiotic and
Pleomorphic Speciation in Fungal Systematics. Wallingford, CAB International, 1993, pp 237–245.
145 Reynolds DR, Taylor JW: The Fungal Holomorph: Mitotic, Meiotic and Pleomorphic Speciation
in Fungal Systematics. Wallingford, CAB International, 1993.
146 Reynolds DR, Taylor JW: DNA specimens and the ‘international code of botanical nomenclature’.
Taxon 1991;40:311–315.
147 Haines JH, Cooper CR: DNA and mycological herbaria; in Reynolds DR, Taylor JW (eds): The
Fungal Holomorph: Mitotic, Meiotic and Pleomorphic Speciation in Fungal Systematics.
Wallingford, CAB International, 1993, pp 305–315.
148 Prillinger H, Messner R, König H, Bauer R, Lopandic K, Molnár O, Dangel P, Weigang F, Kirisits
T, Nakase T, Sigler L: Yeasts associated with termites: A phenotypic and genotypic characterization
and use of coevolution for dating evolutionary radiations in Asco- and Basidiomycetes. Syst Appl
Microbiol 1996;19:265–283.
149 Laaser G: Vergleichende systematische Studien an Basidiomycetenhefen unter besonderer
Berücksichtigung der Hefestadien. Bibl Mycol 1989;130:2–335.
150 Prillinger H, Altenbuchner J, Laaser G, Dörfler C: Yeasts isolated from Homobasidiomycetes
(Asterophora, Collybia): New aspects for sexuality, taxonomy, and speciation. Exp Mycol 1993;
17:24–45.
151 Schweigkofler W: Molekulare Identifizierung und Charakterisierung von endophytischen und
latent pathogenen Pilzen aus Weinreben (Vitis vinifera L.) in Österreich und Südtirol; Dissertation,
Universität für Bodenkultur, Wien, 1998, pp 1–179.
152 Prillinger H, Schweigkofler W, Breitenbach M, Briza P, Staudacher E, Lopandic K, Molnár O,
Weigang F, Ibl M, Ellinger A: Phytopathogenic filamentous (Ashbya, Eremothecium) and
dimorphic fungi (Holleya, Nematospora) with needle-shaped ascospores as new members within
the Saccharomycetaceae. Yeast 1997;13:945–960.
Systematics of the Ascomycota and Basidiomycota
281
153 Jeffries P, Young TWK: Ultrastructure of infection of Cokeromyces recurvatus by Piptocephalis
unispora (Mucorales). Arch Microbiol 1976;109:277–288.
154 Orban G: Untersuchungen über die Sexualität von Phycomyces nitens. Beih Bot Centralbl I
1919;36:1–59.
155 Emmons CW: The ascocarps in species of Penicillium. Mycologia 1935;27:128–150.
156 Zickler H: Die Spermatienbefruchtung bei Bombardia lunata. Ber Dtsch Bot Ges 1937;55:
114–119.
157 Zickler H: Zur Entwicklungsgeschichte des Askomyceten Bombardia lunata Zckl. Arch Protistenk
1952;98:1–70.
158 Mirza JH, Cain RF: Revision of the genus Podospora. Can J Bot 1969;47:1999–2048.
159 Forst TG, Prillinger H: Vergleichende karyologische Untersuchungen an dimorphen
Zygomyceten. Z Mykol 1988;54:139–154.
160 Glass NL, Metzenberg RL, Raju NB: Homothallic Sordariaceae from nature: The absence of
strains containing only the a mating type sequence. Exp Mycol 1990;14:274–289.
161 Li J, Heath IB: The phylogenetic relationships of the anaerobic chytridiomycetous gut fungi
(Neocallimasticaceae) and the Chytridiomycota. II. Cladistic analysis of structural data and
description of the Neocallimasticales ord. nov. Can J Bot 1993;71:393–407.
162 Barr DJS, Kudo H, Jakober KD, Cheng K-J: Morphology and development of rumen fungi:
Neocallimastix sp., Piromyces communis, and Orpinomyces bovis gen. nov., sp. nov. Can J Bot
1989;67:2815–2824.
163 Li J, Heath IB: The phylogenetic relationships of the anaerobic chytridiomycetous gut fungi
(Neocallimasticaceae) and the Chytridiomycota. I. Cladistic analysis of rRNA sequences. Can J
Bot 1992;70:1738–1746.
164 Bowman BH, Taylor JW, White TJ: Molecular evolution of the fungi: Human pathogens. Mol Biol
Evol 1992;9:893–904.
165 Tanabe Y, O’Donnell K, Saikawa M, Sugiyama J: Molecular phylogeny of parasitic Zygomycota
(Dimargaritales, Zoopagales) based on nuclear small subunit ribosomal DNA sequences. Mol
Phylogenet Evol 2000;16:253–262.
166 McKerracher LJ, Heath IB: The structure and cycle of the nucleus-associated organelle in two
species of Basidiobolus. Mycologia 1985;77:412–417.
167 Gehrig H, Schüssler A, Kluge M: Geosiphon pyriforme, a fungus forming endocytobiosis
with Nostoc (Cyanobacteria), is an ancestral member of the Glomales: Evidence by SSU rRNA
analysis. J Mol Evol 1996;43:71–81.
168 Moss ST, Young TWK: Phyletic considerations of the Harpellales and Asellariales
(Trichomycetes, Zygomycotina) and the Kickxellales (Zygomycetes, Zygomycotina). Mycologia
1978;70:944–963.
169 O’Donnell K, Cigelnik E, Benny GL: Phylogenetic relationships among the Harpellales and
Kickxellales. Mycologia 1998;90:624–639.
170 Davis SR, Ellis DH, Goldwater P, Dimitriou S, Byard R: First human culture-proven Australian
case of entomophthoromycosis caused by Basidiobolus ranarum. J Med Vet Mycol 1994;32:
225–230.
171 Ng KH, Chin CS, Jalleh RD, Siar CH, Ngui CH, Singaram SP: Nasofacial zygomycosis. Oral Surg
Oral Med Oral Pathol 1991;72:685–688.
172 Gugnani HC: Entomophthoromycosis due to Conidiobolus. Eur J Epidemiol 1992;8:391–396.
173 Moll HD, Schumacher J, Hoover TR: Entomophthoromycosis conidiobolae in a lama. J Am Vet
Med Assoc 1992;200:969–970.
174 Hawksworth DL: The fungal dimension of biodiversity: Magnitude, significance, and conservation. Mycol Res 1991;95:641–655.
175 Hawksworth DL, Kirk PM, Sutton BC, Pegler DN: Dictionary of the Fungi, ed 8, rev. Wallingford,
CAB International, 1995.
176 Kramer CL: The Taphrinales; in de Hoog GS, Smith MT, Weijman ACM (eds): The Expanding
Realm of Yeast-Like Fungi. Amsterdam, Elsevier, 1987, pp 151–166.
177 Müller E: Systemfragen bei Ascomyceten; in Frey W, Hurka H, Oberwinkler F (eds): Beiträge zur
Biologie der niederen Pflanzen. Stuttgart, Fischer, 1977, pp 43–57.
178 Tehler A: A cladistic outline of the Eumycota. Cladistics 1988;4:227–277.
Prillinger/Lopandic/Schweigkofler/Deak/Aarts/Bauer/Sterflinger/Kraus/Maraz
282
179 Savile DBO: Possible interrelationships between fungal groups; in Ainsworth GC, Susmann AS
(eds): The Fungi, an Advanced Treatise. New York, Academic Press, 1968, pp 649–675.
180 Gargas A, DePriest PT, Grube M, Tehler A: Multiple origins of lichen symbioses in fungi
suggested by ssu rDNA phylogeny. Science 1995;268:1492–1495.
181 Nishida H, Sugiyama J: Archiascomycetes: Detection of a major new lineage within the
Ascomycota. Mycoscience 1994;35:361–366.
182 Sjamsuridzal W, Tajiri Y, Nishida H, Thuan TB, Kawasaki H, Hirata A, Yokota A, Sugiyama J:
Evolutionary relationships of members of the genera Taphrina, Protomyces, Schizosaccharomyces,
and related taxa within the archiascomycetes: Integrated analysis of genotypic and phenotypic
characters. Mycoscience 1997;38:267–280.
183 Taylor JW: A contemporary view of the holomorph: Nucleic acid sequence and computer
databases are changing fungal classification; in Reynolds DR, Taylor JW (eds): The Fungal
Holomorph: Mitotic, Meiotic and Pleomorphic Speciation in Fungal Systematics. Wallingford,
CAB International, 1993, pp 3–13.
184 Taylor JW: Making the Deutereomycota redundant: A practical integration of mitosporic and
meiosporic fungi. Can J Bot 1995;73(suppl 1):S754–S759.
185 Eriksson OE: Origin and evolution of the Ascomycetes. Opera Bot 1981;60:175–209.
186 Oberwinkler F: Das neue System der Basidiomyceten; in Frey W, Hurka H, Oberwinkler F (eds):
Beiträge zur Biologie der niederen Pflanzen. Stuttgart, Fischer, 1977, pp 59–105.
187 Untereiner WA, Malloch D: Patterns of substrate utilization in species of Capronia and allied
black yeasts: Ecological and taxonomic implications. Mycologia 1999;92:417–427.
188 Prillinger H, Lopandic K, Schweigkofler W: Zellwandzucker als Hilfsmittel zur Klärung
phylogenetischer Beziehungen bei Hefen. Abstr Tagung Ges Mykol Lichenol, Regensburg, Okt
1997, p 35.
189 Prillinger H, Lopandic K, Schweigkofler W: Phylogeny of ascomycetous yeasts and yeast stages
from the Ascomycota based on cell wall sugars, 18S ribosomal DNA sequences, and coevolution.
Abstr 6th Int Mycol Congr, Jerusalem, Aug 1998, p 93.
190 Prillinger H, Schweigkofler W, Lopandic K, Bauer R, Mueller UG: Evolution of Asco- and
Basidiomycota based on cell wall sugars, 18S ribosomal DNA sequences and coevolution with
animals and plants. Yeast Newslett 1999;48:12.
191 Cai J, Roberts IN, Collins MD: Phylogenetic relationships among members of the ascomycetous
yeast genera Brettanomyces, Debaryomyces, Dekkera, and Kluyveromyces deduced by smallsubunit rRNA gene sequences. Int J Syst Bacteriol 1996;46:542–549.
192 Kurtzman CP: Molecular taxonomy of the fungi; in Benett JW, Lasure LL (eds): Gene
Manipulations in Fungi. Orlando, Academic Press, 1985, pp 35–63.
193 Kurtzman CP: Systematics of the ascomycetous yeasts assessed from ribosomal RNA sequence
divergence. Antonie Van Leeuwenhoek 1993;63:165–174.
194 Goffeau A, Barrell BG, Bussey H, Davis RW, Dujon B, Feldmann H, Galibert F, Hoheisel JD, Jacq
C, Johnston M, Louis EJ, Mewes HW, Murakami Y, Philippsen P, Tettelin H, Oliver SG: Life with
6,000 genes. Science 1996;274:546–567.
195 Hoheisel JD, Maier E, Mott R, McCarthy L, Gregoriev AV, Schalkwyk LC, Nizetic D, Francis F,
Lehrach H: High resolution cosmid and P1 maps spanning the 14 Mb genome of the fission yeast
S. pombe. Cell 1993;73:109–120.
196 Byers B, Goetsch L: Electronic microscopic observations on the meiotic karyotype of diploid and
tetraploid Saccharomyces cerevisiae. Proc Natl Acad Sci USA 1975;72:5056–5069.
197 Mewes HW, Albermann K, Bähr M, Frishman D, Gleissner A, Hani J, Heuman K, Kleine K,
Maierl A, Oliver SG, Pfeifer F, Zollner A: Overview of the yeast genome. Nature 1997;387:7–65.
198 Mewes HW, Albermann K, Heumann K, Liebl S, Pfeifer F: MIPS: A database for protein
sequences, homology data and yeast genome information. Nucleic Acid Res 1997;35:28–30.
199 Clarke L, Baum MP: Functional analysis of a centromere from fission yeast: A role for
centromere-specific repeated DNA sequences. Mol Cell Biol 1990;10:1863–1872.
200 Moore TDE, Edman JC: The a-mating type locus of Cryptococcus neoformans contains a peptide
pheromone gene. Mol Cell Biol 1993;13:1962–1970.
201 Redhead SA: The genus Neolecta (Neolectaceae fam. nov., Lecanorales, Ascomycetes) in Canada.
Can J Bot 1977;55:301–306.
Systematics of the Ascomycota and Basidiomycota
283
202 Kurtzman CP, Fell JW: The Yeasts, a Taxonomic Study, ed 4, rev. Amsterdam, Elsevier, 1998.
203 Suzuki M, Suh S-O, Sugita T, Nakase T: A phylogenetic study on galactose-containing Candida
species based on 18S ribosomal DNA sequences. J Gen Appl Microbiol 1999;45:229–238.
204 Batra LR: Taxonomy and systematics of the Hemiascomycetes (Hemiascomycetidae);
in Subramanian CV (ed): Taxonomy of Fungi. Madras, University of Madras, 1973, pp 187–214.
205 Cregg JM, Vedvick TS, Raschke WC: Recent advances in the expression of foreign genes in
Pichia pastoris. Biotechnology 1993;11:905–910.
206 Rhyan JC, Stackhouse LL, Davis EG: Disseminated geotrichosis in two dogs. J Am Vet Med
Assoc 1990;197:358–360.
207 Smith MT, de Cock AWAM, Poot GA, Steensma HY: Genome comparisons in the yeastlike
fungal genus Galactomyces Redhead et Malloch. Int J Syst Bacteriol 1995;45:826–831.
208 Prillinger H, Molnár O, Eliskases-Lechner F, Lopandic K: Phenotypic and genotypic identification of yeasts from cheese. Antonie Van Leeuwenhoek 1999;75:267–283.
209 Sullivan DJ, Westerneng TJ, Haynes KA, Bennett DE, Coleman DC: Candida dubliniensis sp. nov.:
Phenotypic and molecular characterization of a novel species associated with oral candidosis in
HIV-infected individuals. Microbiology 1995;141:1507–1521.
210 Pincus DH: Rapid identification of Candida dubliniensis with commercial yeast identification
systems. J Clin Microbiol 1999;37:3533–3539.
211 Mateev G, Kantjardjiev T, Vassileva S, Tsankov N: Chronic mucocutaneous candidosis with
osteomyelitis of the frontal bone. Int J Dermatol 1993;32:888–889.
212 Clemons KV, McCusker JH, Davis RW, Stevens DA: Comparative pathogenesis of clinical and
nonclinical isolates of Saccharomyces cerevisiae. J Infect Dis 1994;169:859–867.
213 De Hoog GS: Risk assessment of fungi reported from humans and animals. Mycoses 1996;39:
407–417.
214 van der Walt JP, Johannsen E: The dangeardian and its significance in the taxonomy of ascomycetous yeasts. Antonie Van Leeuwenhoek 1974;40:185–192.
215 Prillinger H, Dörfler C, Laaser G, Hauska G: Ein Beitrag zur Systematik und Entwicklungsbiologie
höherer Pilze: Hefe-Typen der Basidiomyceten. III. Ustilago-Typ. Z Mykol 1990;56:
251–278.
216 Prillinger H, Laaser G, Dörfler C, Ziegler K: Ein Beitrag zur Systematik und Entwicklungsbiologie
höherer Pilze: Hefe-Typen der Basidiomyceten. IV. Dacrymyces-Typ, Tremella-Typ. Sydowia
1991;53:170–218.
217 Prillinger H, Oberwinkler F, Umile C, Tlachac K, Bauer R, Dörfler C, Taufratzhofer E: Analysis
of cell wall carbohydrates (neutral sugars) from ascomycetous and basidiomycetous yeasts with
and without derivatization. J Gen Appl Microbiol 1993;39:1–34.
218 Redondo-Lopez V, Lynch M, Schmitt C, Cook R, Sobel JD: Torulopsis glabrata vaginitis: Clinical
aspects and susceptibility to antifungal agents. Obstet Gynecol 1990;76:651–654.
219 Nishida T, Mayumi H, Kawachi Y, Tokunaga S, Murayama A, Yasui H, Tokunaga K: The efficacy
of fluconazole in treating prosthetic valve endocarditis caused by Candida glabrata: Report of a
case. Surg Today Tokyo 1994;24:651–654.
220 Hickley WF, Sommerville LH, Schoen FJ: Disseminated Candida glabrata: Report of a unique
severe infection and a literature review. Am J Clin Pathol 1983;80:724–727.
221 Owen PG, Willis BK, Benzel EC: Torulopsis glabrata vertebral osteomyelitis. J Spinal Disord
1992;5:370–373.
222 Hoppe JE, Klingebiel T, Niethammer D: Selection of Candida glabrata in pediatric bone marrow
transplant recipients receiving fluconazole. Pediatr Hematol Oncol 1994;11:207–210.
223 Vazquez J, Lundstrom T, Dembry L, Perry MB, Zervos M: Disseminated Torulopsis candida
(Candida famata) infection: An unusual human pathogen. Abstr Gen Meet Am Soc Microbiol
1993;93:534.
224 Nicand E, Buisson Y, Auzanneau G, Improvisi L, Dupont B: Septicémie à Debaryomyces hansenii
(Candida famata), levure pathogène opportuniste. J Mycol Méd 1993;4:242–244.
225 Blaschke-Hellmessen R: Standorte für Candida aus medizinisch-hygienischer Sicht. Mycoses
1999;42(suppl 1):22–29.
226 Furman RM, Ahearn DG: Candida ciferrii and Candida chiropterorum isolated from clinical
specimens. J Clin Microbiol 1983;18:1252–1255.
Prillinger/Lopandic/Schweigkofler/Deak/Aarts/Bauer/Sterflinger/Kraus/Maraz
284
227 De Gentile L, Bouchara JP, Cimon B, Chabasse D: Candida ciferrii: Clinical and microbiological
features of an emerging pathogen. Mycoses 1991;34:125–128.
228 Eriksson OE, Winka K: Families and higher taxa of Ascomycota. Myconet 1998;1:17–24.
229 Prillinger H, Dörfler C, Laaser G, Eckerlein B, Lehle L: Ein Beitrag zur Systematik und
Entwicklungsbiologie höherer Pilze: Hefe-Typen der Basidiomyceten. I. Schizosaccharomycetales,
Protomyces-Typ. Z Mykol 1990;56:219–250.
230 Prillinger H, Bacigálová K, Lopandic K, Binder M: Taphrina padi (Jacz.) Mix in Bayern und der
Slowakei. Hoppea, 2000;61:275–294.
231 Eriksson OE, Svedskog A, Landvik S: Molecular evidence for the evolutionary hiatus between
Saccharomyces cerevisiae and Schizosaccharomyces pombe. Systema Ascomycetum 1993;11:
119–162.
232 Landvik S, Eriksson OE, Gargas A, Gustafsson P: Relationships of the genus Neolecta (Neolectales
ordo nov.) inferred from 18S rDNA sequences. Systema Ascomycetum 1993;11:107–118.
233 Eriksson OE: Pneumocystis carinii, a parasite in lungs of mammals, referred to a new family and
order (Pneumocystidaceae, Pneumocystidales, Ascomycota). Systema Ascomycetum 1995;13:
165–180.
234 Prillinger H, Deml G, Dörfler C, Laaser G, Lockau W: Ein Beitrag zur Systematik und
Entwicklungsbiologie höherer Pilze: Hefe-Typen der Basidiomyceten. II. Microbotryum-Typ. Bot
Acta 1991;104:5–17.
235 Nishida H, Ando K, Ando Y, Hirata A, Sugiyama J: Mixia osmundae: Transfer from the
Ascomycota to the Basidiomycota based on evidence from molecules and morphology. Can J Bot
1995;73(suppl 1):S660–S666.
236 Gottschalk M, Blanz PA: Untersuchungen an 5S ribosomalen Ribonukleinsäuren als Beitrag zur
Klärung von Systematik und Phylogenie der Basidiomyceten. Z Mykol 1985;51:205–243.
237 Sugiyama J, Nishida H, Suh S-O: The paradigm of fungal diagnoses and descriptions in the era of
molecular systematics: Saitoella complicata as an example; in Reynolds DR, Taylor JW (eds):
The Fungal Holomorph: Mitotic, Meiotic and Pleomorphic Speciation in Fungal Systematics.
Wallingford, CAB International, 1993, pp 261–269.
238 Ahearn DG, Sugiyama J, Simmons RB: Saitoella S. Goto, Sugiyama, Hamamoto & Komagata;
in Kurtzman CP, Fell JW (eds): The Yeasts, a Taxonomic Study. ed 4. Amsterdam, Elsevier, 1998,
pp 600–601.
239 Landvik S: Neolecta, a fruit-body-producing genus of the basal ascomycetes, as shown by SSU
and LSU rDNA sequences. Mycol Res 1996;100:199–202.
240 Taylor JW, Bowman BH: Pneumocystis carinii and the ustomycetous red yeast fungi. Mol
Microbiol 1993;8:425–426.
241 Taylor JW, Swann EC, Berbee ML: Molecular evolution of ascomycete fungi: Phylogeny and
conflict; in Hawksworth DL (ed): Ascomycete Systematics: Problems and Perspective in the
Nineties. New York, Plenum Press, 1994, pp 201–212.
242 Berbee ML, Taylor JW: Two Ascomycetes classes based on fruiting-body characters and
ribosomal DNA sequence. Mol Biol Evol 1992;9:278–284.
243 Gargas A, Taylor JW: Phylogeny of discomycetes and early radiations of the apothecial
Ascomycotina inferred from SSU rDNA sequence data. Exp Mycol 1995;19:7–15.
244 Nannfeldt JA: Studien über die Morphologie und Systematik der nicht-lichenisierten inoperculaten Discomyceten. Nova Acta Reg Soc Sci Upsaliensis 1932;8:1–368.
245 Haase G, Sonntag L, van der Peer Y, Uijthof JMJ, Podbielski A, Melzer Krick B: Phylogenetic
analysis of ten black yeast species using nuclear small subunit rRNA gene sequences. Antonie Van
Leeuwenhoek 1995;68:19–33.
246 Haase G, Sonntag L, Melzer-Krick B, de Hoog GS: Phylogenetic inference by SSU gene analysis
of members of Herpotrichiellaceae with special reference to human pathogenic species. Stud
Mycol 1999;43:80–97.
247 Berbee ML: Loculoascomycete origins and evolution of filamentous ascomycete morphology
based on 18S rRNA gene sequence data. Mol Biol Evol 1996;13:462–470.
248 Sterflinger K, de Baer R, de Hoog GS, de Wachter R, Krumbein WE, Haase G: Coniosporium
perforans and C. appollinis, two new rock inhabiting fungi isolated from marble in the Sanctuary
of Delos (Cyclades, Greece). Antonie Van Leeuwenhoek 1997;72:349–363.
Systematics of the Ascomycota and Basidiomycota
285
249 Winka K, Eriksson OE, Bang A: Molecular evidence for recognizing the Chaetothyriales.
Mycologia 1998;90:822–830.
250 Blackwell M: Minute morphological mysteries: The influence of arthropods on the lives of fungi.
Mycologia 1994;86:1–17.
251 Mendoza L, Karuppayil SM, Szaniszlo PJ: Calcium regulates in vitro dimorphism in chromoblastomycotic fungi. Mycoses 1993;36:157–164.
252 Takeo K, de Hoog GS: Karyology and hyphal characters as taxonomic criteria in ascomycetous
black yeasts and related fungi. Antonie Van Leeuwenhoek 1991;60:35–42.
253 Barr ME: Prodromus to Class Loculoascomycetes. Amherst, published by the author 1987.
254 McGinnis MR, Lemon SM, Walker D, de Hoog GS, Haase G: Fatal cerebritis caused by a new
species Cladophialophora. Stud Mycol 1999;43:166–171.
255 Benny GL, Kimbrough JW: A synopsis of the orders and families of Plectomycetes with keys to
genera. Mycotaxon 1980;12:1–91.
256 Landvik S, Shailer NFJ, Eriksson OE: SSU rDNA sequence support for a close relationship
between the Elaphomycetales and the Eurotiales and Onygenales. Mycoscience 1996;37:237–241.
257 Pitt JI: Phylogeny in the genus Penicillium: A morphologist’s perspective. Can J Bot 1995;
73(suppl 1):S768–S777.
258 Pitt JI, Samson RA: Species names in current use in the Trichocomaceae (Fungi, Eurotiales); in
Greuter W (ed): Names in Current Use in the Families Trichocomaceae, Cladoniaceae, Pinaceae,
and Lemnaceae. Königstein, Koeltz, 1993, pp 13–57.
259 Berbee ML, Yoshimura A, Sugiyama J, Taylor JW: Is Penicillium monophyletic? An evolution in
the family Trichocomaceae from 18S, 5.8S and ITS DNA sequence data. Mycologia 1995;
87:210–222.
260 Egel-Mitani M, Olson LW, Egel R: Meiosis in Aspergillus nidulans: Another example for
lacking synaptonemal complexes in the absence of crossover interference. Hereditas 1982;97:
179–187.
261 Bähler J, Wyler T, Loidl J, Kohli J: Unusual nuclear structures in meiotic prophase of fission yeast:
A cytological analysis. J Cell Biol 1993;121:241–256.
262 Guarro J, Gené J, Stchigel AM: Developments in fungal taxonomy. Clin Microbiol Rev 1999;
12:454–500.
263 Jesenska Z, Durkovsky J, Rosinski I, Polak M, Zamboova E, Baca B: Filamentous micromycetes
in otitis. Cesk Epidemiol Mikrobiol Imunol 1992;41:337–341.
264 Shitara T, Yugami S-I, Sotomatu M, Oshima Y, Ijima H, Kuroume T, Matsumoto T: Invasive
aspergillosis in leukemic children. Pediatr Hematol Oncol 1993;10:169–174.
265 Göttlich E: Untersuchungen der Pilzbelastung der Luft an Arbeitsplätzen in Betrieben zur
Abfallbehandlung; thesis, University of Stuttgart, 1995.
266 Byrd RP, Roy TM, Fields CL, Lynch JA: Paecilomyces variotii pneumonia in a patient with
diabetes mellitus. J Diabetes Complications 1992;6:150–153.
267 Thompson RF, Bode RB, Rhodes JC, Gluckman JL: Paecilomyces variotii: An unusual cause of
isolated sphenoid sinusitis. Arch Otolaryngol Head Neck Surg 1988;114:567–569.
268 Williamson PR, Kwon-Chung KJ, Gallin JI: Successful treatment of Paecilomyces variotii
infection in a patient with chronic granulomatous disease and review of Paecilomyces species
infections. Clin Infect Dis 1992;14:1023–1026.
269 Jaisree N, Singh SM: Hyalohyphomycosis caused by Paecilomyces variotii: A case report, animal
pathogenicity and in vitro sensitivity. Antonie Van Leeuwenhoek 1992;62:225–230.
270 Fukatsu T, Sato H, Kuriyama H: Isolation, inoculation to insect host, and molecular phylogeny of
an entomogenous fungus Paecilomyces tenuipes. J Invert Pathol 1997;70:203–208.
271 Cochet G: Sur un nouveau champignon arthrosporé (Arthrographis langeroni n. g., n. sp.), agent
pathogène d’une onychomycose humaine. Ann Parasitol Hum Comp 1939;17:97–102.
272 Scott JA, Malloch DW, Gloer JB: Polytolypa, an undescribed genus in the Onygenales. Mycologia
1993;85:503–508.
273 Leclerc MC, Philippe H, Guého E: Phylogeny of dermatophytes and dimorphic fungi based on
large subunit ribosomal RNA sequence comparisons. J Med Vet Mycol 1994;32:331–341.
274 Pan S, Sigler L, Cole GT: Evidence for a phylogenetic connection between Coccidioides immitis
and Uncinocarpus reesii (Onygenaceae). Microbiology 1994;140:1481–1494.
Prillinger/Lopandic/Schweigkofler/Deak/Aarts/Bauer/Sterflinger/Kraus/Maraz
286
275 Sigler L: Agents of adiaspiromycosis; in Ajello L, Hay R (eds): Topley and Wilson’s Microbiology
and Microbial Infections, 9 ed. London, Arnold, 1998, pp 571–583.
276 Roilides E, Sigler L, Bibashi E, Katsifa H, Flaris N, Panteliadis C: Disseminated infection due
to Chrysosporium zonatum in a patient with chronic granulomatous disease and review of
non-Aspergillus fungal infections in patients with this disease. J Clin Microbiol 1999;37:18–25.
277 Sigler L, Flis AL, Carmichael JW: The genus Uncinocarpus (Onygenaceae) and its synonym
Brunneospora: New concepts, combinations and connections to anamorphs in Chrysosporium,
and further evidence of relationship with Coccidioides immitis. Can J Bot 1998;76:1624–1636.
278 Iwen PC, Sigler L, Tarantolo S, Sutton DA, Rinaldi MG, Lackner RP, McCarthy DI, Hinrichs SH:
Pulmonary infection caused by Gymnascella hyalinospora in a patient with acute myelogenous
leukemia. J Clin Microbiol 2000;38:375–381.
279 Rossman AY, Samuels GJ, Rogerson CT, Lowen R: Genera of Bionectriaceae, Hypocreaceae and
Nectriaceae (Hypocreales, Ascomycetes). Studies Mycol 1999;42:1–248.
280 Tarlo SM, Fradkin A, Tobin RS: Skin testing with extracts of fungal species derived from the
homes of allergy clinic patients in Toronto, Canada. Clin Allergy 1988;18:45–52.
281 O’Donnell K, Gherbawy Y, Schweigkofler W, Adler A, Prillinger H: Phylogenetic analyses of
DNA sequence and RAPD data compared in Fusarium oxysporum and related species from maize.
J Phytopathol 1999;147:445–452.
282 Sigler L, Abbott SP, Gauvreau H: Assessment of worker exposure to airborne molds in honeybee
overwintering facilities. Am Indust Hyg Assoc J 1996;57:484–490.
283 Kuhls K, Lieckfeldt E, Samuels GJ, Kovacs W, Meyer W, Petrini O, Gams W, Börner T,
Kubicek CP: Molecular evidence that the asexual industrial fungus Trichoderma reesi is a clonal
derivative of the ascomycete Hypocrea jecorina. Proc Natl Acad Sci USA 1996;93:7755–7760.
284 Kuhls K, Lieckfeldt E, Samuels GJ, Meyer W, Kubicek CP, Börner T: Revision of Trichoderma
sect. Longibrachiatum including related teleomorphs based on analysis of ribosomal DNA
internal transcribed spacer sequences. Mycologia 1997;89:442–460.
285 Turner D, Kovacs W, Kuhls K, Lieckfeldt E, Peter B, Arisan-Atac I, Strauss J, Samuels GJ, Börner
T, Kubicek CP: Biogeography and phenotypic variation in Trichoderma sect. Longibrachiatum
and associated Hypocrea species. Mycol Res 1997;101:449–459.
286 Vesper SJ, Dearborn DG, Yike I, Sorenson WG, Haugland RA: Hemolysis, toxicity, and randomly
amplified polymorphic DNA analysis of Stachybotrys chartarum strains. Appl Environ Microbiol
1999;65:3175–3181.
287 Tanada Y, Kaya HK: Insect Pathology. New York, Academic Press, 1993.
288 Paterson IC, Charnley AK, Cooper RM, Clarkson JM: Partial characterization of specific
inducers of a cuticle-degrading protease from the insect pathogenic fungus Metarhizium anisopliae.
Microbiology 1994;140:3153–3159.
289 Ito Y, Hirano T: The determination of the partial 18S ribosomal DNA sequences of Cordyceps
species. Lett Appl Microbiol 1997;25:239–242.
290 Messner R, Schweigkofler W, Ibl M, Berg G, Prillinger H: Molecular characterization of the plant
pathogen Verticillium dahliae Kleb. using RAPD-PCR and sequencing of the 18S rRNA-gene.
J Phytopathol 1996;144:347–354.
291 Hausner G, Reid J, Klassen GR: Ceratocystiopsis: A reappraisal based on molecular criteria.
Mycol Res 1993;97:625–633.
292 Hausner G, Reid J, Klassen GR: On the subdivision of Ceratocystis s.l., based on partial ribosomal DNA sequences. Can J Bot 1993;71:52–63.
293 Halmschlager E, Messner R, Kowalski T, Prillinger H: Differentiation of Ophiostoma piceae and
Ophiostoma quercus by morphology and RAPD analysis. Syst Appl Microbiol 1994;17:554–562.
294 Spatafora JW, Blackwell M: Molecular systematics of unitunicate perithecial Ascomycetes:
The Clavicipitales-Hypocreales connection. Mycologia 1993;85:912–922.
295 Spatafora JW, Blackwell M: The polyphyletic origins of ophiostomatoid fungi. Mycol Res 1994;
98:1–9.
296 Summerbell RC, Kane J, Krajden S, Duke EE: Medically important species and related
ophiostomatoid fungi; in Wingfield MJ, Seifert KA, Webber JF (eds): Ceratocystis and
Ophiostoma: Taxonomy, Ecology, and Pathology. St Paul, American Phytopathological Society,
1993, pp 185–192.
Systematics of the Ascomycota and Basidiomycota
287
297 Rieth H: Pilz-Datei: Erläuterungen zu einigen allergologisch wichtigen Dermatophyten, Hefen
und Schimmelpilzen, ed 5. Reinbek b. Hamburg, Allergopharma Joachim Ganzer KG, 1983.
298 Sutton BC: The genus Glomerella and its anamorph Colletotrichum; in Bailey JA, Jeger MJ (eds):
Colletotrichum: Biology, Pathology and Control. Wallingford, CAB International, 1992, pp 1–26.
299 Crous PW, Gams W, Wingfield MJ, van Wyk PS: Phaeoacremonium gen. nov. associated with wilt
and decline diseases of woody hosts and human infections. Mycologia 1996;88:786–796.
300 Masclaux F, Guého E, de Hoog GS, Christen R: Phylogenetic relationships of human-pathogenic
Cladosporium (Xylohypha) species inferred from partial LS rRNA sequences. J Med Vet Mycol
1995;33:327–338.
301 Barr ME: Prodromus to nonlichenized, pyrenomycetous members of class Hymenoascomycetes.
Mycotaxon 1990;39:43–184.
302 Berbee ML, Taylor JW: Convergence in ascospore discharge mechanism among pyrenomycete
fungi based on 18S ribosomal RNA gene sequence. Mol Phylogenet Evol 1992;1:59–71.
303 Wingfield MJ, de Berr C, Visser C, Wingfield BD: A new Ceratocystis species defined using
morphological and ribosomal DNA sequence comparisons. Syst Appl Microbiol 1996;19:
191–202.
304 Luttrell ES: The ascostromatic Ascomycetes. Mycologia 1955;47:511–532.
305 Schweigkofler W, Prillinger H: Untersuchungen von endophytischen und latent pathogenen Pilzen
aus Rebholz in Österreich und Südtirol. Mitt Klosterneuburg 1997;47:149–158.
306 Zalar P, de Hoog GS, Gunde-Cimerman N: Ecology of halotolerant dothideaceous black yeasts.
Stud Mycol 1999;43:38–48.
307 Karlsson-Borga A, Jonsson P, Rolfsen W: Specific IgE antibodies to 16 widespread mold genera
in patients with suspected mold allergy. Ann Allergy 1989;63:521–526.
308 Roeijmans HJ, de Hoog GS, Tan CS, Figge MJ: Molecular taxonomy and GC/MS of metabolites
of Scytalidium hyalinum and Nattrassia mangiferae (Hendersonula toruloidea). J Med Vet Mycol
1997;35:181–188.
309 LoBuglio KF, Berbee ML, Taylor JW: Phylogenetic origins of the asexual mycorrhizal symbiont
Cenococcum geophilum Fr. and other mycorrhizal fungi among the Ascomycetes. Mol Phylogenet
Evol 1996;6:287–294.
310 Breitenbach M, Achatz G, Oberkofler H, Simon B, Unger A, Lechenauer E, Kandler D, Ebner C,
Kraft D: Molecular characterization of allergens of Cladosporium herbarum and Alternaria
alternata. Int Arch Allergy Immunol 1995;107:458–459.
311 Berbee ML, Pirseyedi M, Hubbard S: Cochliobolus phylogenetics and the origin of known, highly
virulent pathogens, inferred from ITS and glyceraldehyde-3-phosphate dehydrogenase gene
sequences. Mycologia 1999;91:954–977.
312 Tsuda M, Ueyama A: Pseudocochliobolus australiensis, the ascigerous state of Bipolaris
australiensis. Mycologia 1981;73:88–96.
313 McAleer R, Kroenert DB, Elder JL, Froudist JH: Allergic bronchopulmonary disease caused by
Curvularia lunata and Drechslera hawaiiensis. Thorax 1981;36:338–344.
314 Hendrick DJ, Ellithorpe DB, Lyon F, Hattier P, Salvaggio JE: Allergic bronchopulmonary
helminthosporiosis. Am Rev Respir Dis 1982;126:935–938.
315 Bassiouny A, Maher A, Bucci TJ, Moawad MK, Hendawy DS: Noninvasive antromycosis
(diagnosis and treatment). J Laryngol Otol 1982;96:215–228.
316 Benoldi D, Alinovi A, Polonelli L, Conti S, Gerloni M, Ajello L, Padhye AA, de Hoog GS:
Botryomyces caespitosus as an agent of cutaneous phaeohyphomycosis. J Med Vet Mycol 1991;
29:9–13.
317 Pöder R, Scheuer C: Moserella radicicola gen. et sp. nov., a new hypogeous species of Leotiales
on ectomycorrhizas of Picea abies. Mycol Res 1994;98:1334–1338.
318 Holst-Jensen A, Kohn LM, Schumacher T: Nuclear rDNA phylogeny of Sclerotiniaceae.
Mycologia 1997;89:885–899.
319 Saenz GS, Taylor JW, Gargas A: 18S rRNA gene sequences and supraordinal classification of the
Erysiphales. Mycologia 1994;86:212–216.
320 O’Donnell K, Cigelnik E, Weber NS, Trappe JM: Phylogenetic relationships among ascomycetous
truffles and the true and false morels inferred from 18S and 28S ribosomal DNA sequence
analysis. Mycologia 1997;89:48–65.
Prillinger/Lopandic/Schweigkofler/Deak/Aarts/Bauer/Sterflinger/Kraus/Maraz
288
321 Boekhout T, Roeijmans H, Spaay F: A new pleomorphic ascomycete, Calyptrozyma arxii gen. et
sp. nov., isolated from the human lower oesophagus. Mycol Res 1995;99:1239–1246.
322 Lehrer SB, Aukrust L, Salvaggio JE: Respiratory allergy induced by fungi. Clin Chest Med 1983;
4:23–41.
323 Patouillard NT: Essai taxonomique sur les familles et les genres des Hyménomycètes. Paris,
Lonsle-Saunier, 1900, pp 1–184.
324 Oberwinkler F: Was ist ein Basidiomycet? Z Mykol 1978;44:13–29.
325 Oberwinkler F: Anmerkungen zur Evolution und Systematik der Basidiomyceten. Bot Jahrb Syst
1985;107:541–580.
326 Lowy B: Taxonomic problems in the Heterobasidiomycetes. Taxon 1968;17:118–127.
327 Talbot PHB: Fossilized pre-Patouillardian taxonomy? Taxon 1968;17:620–628.
328 Donk MA: The heterobasidiomycetes: A reconnaissance. I. A restricted emendation. Proc Kon
Ned Akad Wetensch Ser C 1972;75:365–375.
329 Donk MA: The heterobasidiomycetes: A reconnaissance. Part IV. Proc Kon Ned Akad Wetensch
Ser C 1972;76:109–125.
330 Dörfler C: Vergleichende Untersuchungen zum biochemischen Aufbau der Zellwand an
Hefestadien von niederen und höheren Basidiomyceten. Bibl Mycol 1990;129:1–164.
331 Swann EC, Taylor JW: Higher taxa of Basidiomycetes: An 18S rRNA gene perspective.
Mycologia 1993;85:923–936.
332 Mueller UG, Rehner SA, Schultz TR: The evolution of agriculture in ants. Science 1998;281:
2034–2038.
333 Oberwinkler F, Bandoni R: Carcinomycetaceae: A new family in the Heterobasidiomycetes. Nord
J Bot 1982;2:501–516.
334 Ingold CT: Ballistospores in Melanotaenium endogenum. Trans Br Mycol Soc 1988;91:712–714.
335 Bauer R, Oberwinkler F, Vánky K: Ultrastructural markers in smut fungi and allied taxa. Can
J Bot 1997;75:1273–1314.
336 McLaughlin DJ, Frieders EM, Lü H: A microscopist’s view of heterobasidiomycete phylogeny.
Stud Mycol 1995;38:91–109.
337 Hibbett DS, Thorn RG: Basidiomycota: Homobasidiomycetes; in McLaughlin DJ, McLaughlin
EJ, Lemke PA (eds): The Mycota: Systematics and Evolution. Heidelberg, Springer, 2000, vol VII,
part B, pp 121–168.
338 Boekhout T, Fell JW, Fonseca A, Prillinger H, Lopandic K, Roeijmans H: The basidiomycetous
yeast Rhodotorula yarrowii comb. nov. Antonie Van Leeuwenhoek 2000;77:355–358.
339 Sadebeck R: Untersuchungen über die Pilzgattung Exoascus und die durch dieselbe um Hamburg
hervorgerufenen Baumkrankheiten. Jahrb Hamburg Wissensch Anst 1884;1:93–124.
340 Messner R, Prillinger H, Altmann F, Lopandic K, Wimmer K, Molnár O, Weigang F: Molecular
characterization and application of random amplified polymorphic DNA analysis of Mrakia and
Sterigmatomyces species. Int J Syst Bacteriol 1994;44:694–703.
341 O’Brien RW, Ralph BJ: The cell wall composition and taxonomy of some Basidiomycetes and
Ascomycetes. Ann Bot NS 1966;30:831–843.
342 Sjamsuridzal W, Nishida H, Ogawa H, Kakishima M, Sugiyama J: Phylogenetic positions of rust fungi
parasitic on ferns: Evidence from 18S rDNA sequence analysis. Mycoscience 1999;40: 21–27.
343 Khan SR, Kimbrough JW: A reevaluation of the Basidiomycetes based upon septal and basidial
structures. Mycotaxon 1982;15:103–120.
344 Oberwinkler F, Bauer R: The systematics of gasteroid, auricularioid Heterobasidiomycetes.
Sydowia 1989;41:224–256.
345 Boekhout T, Yamada Y, Weijman ACM, Roeymans HJ, Batenburg-van der Vegte WH: The
significance of Coenzyme Q, carbohydrate composition and septal ultrastructure for the taxonomy
of ballistoconidia-forming yeasts and fungi. Syst Appl Microbiol 1992;15:1–10.
346 Bauer R, Oberwinkler F: Meiosis, septal pore architecture, and systematic position of the
heterobasidiomycetous fern parasite Herpobasidium filicinum. Can J Bot 1994;72:1229–1242.
347 Suh S-O, Hirata A, Sugiyama J, Komagata K: Septal ultrastructure of basidiomycetous yeasts and
their taxonomic implications with observations on the ultrastructure of Erythrobasidium hasegawianum and Sympodiomycopsis paphiopedili. Mycologia 1993;85:30–37.
348 Wells K: Jelly fungi, then and now! Mycologia 1994;86:18–48.
Systematics of the Ascomycota and Basidiomycota
289
349 Deml G: Taxonomy of phragmobasidial smut fungi. Stud Mycol 1987;30:127–135.
350 Sampaio JP, Bauer R, Begerow D, Oberwinkler F: Occultifur externus sp. nov., a new species of
simple-pored auricularioid heterobasidiomycete from plant litter in Portugal. Mycologia 1999;
91:1094–1101.
351 Hamamoto M, Sugiyama J, Komagata K: Transfer of Rhodotorula hasegawae to a new
basidiomycetous genus Erythrobasidium as Erythrobasidium hasegawae comb. nov. J Gen Appl
Microbiol 1988;34:279–287.
352 Kuraishi H, Katayama-Fujimura Y, Sugiyama J, Yokoyama T: Ubiquinone systems in fungi. I.
Distribution of ubiquinones in the major families of ascomycetes, basidiomycetes, and
deuteromycetes, and their taxonomic implications. Trans Mycol Soc Jpn 1985;26:383–395.
353 Oberwinkler F: New genera of auricularioid heterobasidiomycetes. Rept Tottori Mycol Inst 1990;
28:113–127.
354 Celerin M, Day AW, Castle AJ, Laudenbach DE: A glycosylation pattern that is unique to fimbriae
from the taxon Microbotryales. Can J Microbiol 1995;41:452–460.
355 Bandoni RJ: Dimorphic Heterobasidiomycetes: Taxonomy and parasitism. Stud Mycol 1995;
39:13–28.
356 Bergman AG, Kauffman CA: Dermatitis due to Sporobolomyces infection. Arch Dermatol 1984;
120:1059–1060.
357 De Bary A: Untersuchungen über die Peronosporeen und Saprolegnieen und die Grundlagen eines
natürlichen Systems der Pilze. Abh Senckenb Naturf Ges 1881;12:225–370, Tafel 1–6.
358 Deml G, Bauer R, Oberwinkler F: Studies in Heterobasidiomycetes. 9. Axenic cultures of
Coleosporium tussilaginis (Uredinales). I. Isolation, identification and characterization of the
cultures. Phytopathol Z 1982;104:39–45.
359 Deml G, Bauer R, Oberwinkler F: Untersuchungen an Heterobasidiomyceten. 16. Axenische
Kultur von Coleosporium tussilaginis (Pers.) Lév. (Uredinales). II. Kreuzungsversuche mit
monokaryotischen Stämmen. Phytopathol Z 1982;103:149–155.
360 Boekhout T, Fell JW, O’Donnell K: Molecular systematics of some yeast-like anamorphs
belonging to the Ustilaginales and Tilletiales. Stud Mycol 1995;38:175–183.
361 Begerow D, Bauer R, Boekhout T: Phylogenetic placements of ustilaginomycetous anamorphs as
deduced from nuclear LSU rDNA sequences. Mycol Res 2000;104:53–60.
362 Bauer R, Vánky K, Begerow D, Oberwinkler F: Ustilaginomycetes on Selaginella. Mycologia
1999;91:475–484.
363 Schweizer HJ: Introduction to the plant bearing beds and the flora of the Lower Devonian of the
Rhineland. Bonner Paläobot Mitt 1987;13:1–94.
364 Bauer R, Oberwinkler F, Vánky K: Ustilaginomycetes on Osmunda. Mycologia 1999;91:
669–675.
365 Pryer KM, Smith AR, Skog JE: Phylogenetic relationships of extant ferns based on evidence from
morphology and rbcL sequences. Am Fern J 1995;85:205–282.
366 Moore RT: Taxonomic proposals for the classification of marine yeasts and other yeast-like fungi
including the smuts. Bot Mar 1980;23:361–373.
367 Takeo K, Nakai E: Mode of cell growth of Malassezia (Pityrosporium) as revealed by using
plasma membrane configurations as natural markers. Can J Microbiol 1986;32:389–394.
368 Guillot J, Guého E, Prévost MC: Ultrastructural features of the dimorphic yeast Malassezia
furfur. J Méd 1995;5:86–91.
369 Guého E, Midgley G, Guillot J: The genus Malassezia with description of four new species.
Antonie Van Leeuwenhoek 1996;69:337–355.
370 Boekhout T, Kamp M, Guého E: Molecular typing of Malassezia species with PFGE and RAPD.
Med Mycol 1998;36:365–372.
371 Guého E, Boekhout T, Ashbee HR, Guillot J, van Belkum A, Faergemann J: The role of
Malassezia species in the ecology of human skin and as pathogens. Med Mycol 1998;36(suppl 1):
220–229.
372 Boekhout T: A revision of ballistoconidia-forming yeasts and fungi. Stud Mycol 1991;33:1–194.
373 Urquhart EJ, Punja ZK: Epiphytic growth and survival of Tilletiopsis pallescens, a potential
biological control agent of Sphaerotheca fulginea on cucumber leaves. Can J Bot 1997;75:
892–901.
Prillinger/Lopandic/Schweigkofler/Deak/Aarts/Bauer/Sterflinger/Kraus/Maraz
290
374 Takashima M, Nakase T: A phylogenetic study of the genus Tilletiopsis, Tilletiaria anomala and
related taxa based on the small subunit ribosomal DNA sequences. J Gen Appl Microbiol 1996;
42:421–429.
375 Ramani R, Kahn RT, Chaturvedi V: Tilletiopsis minor: A new etiologic agent of human subcutaneous mycosis in an immunocompromised patient. J Clin Microbiol 1997;35:2992–2995.
376 Moore RT: Micromorphology of yeasts and yeast-like fungi and its taxonomic implications. Stud
Mycol 1987;30:203–226.
377 Weijman ACM, de Hoog GS: Carbohydrate patterns and taxonomy of Sporothrix and Blastobotrys.
Antonie Van Leeuwenhoek 1985;51:111–120.
378 Middelhoven WJ, Guého E, de Hoog GS: Phylogenetic position and physiology of Cerinosterus
cyanescens. Antonie van Leeuwenhoek 2000;77:313–320.
379 Sigler L, Harris JL, Dixon DM, Flis AL, Salkin IF, Kemna M, Duncan RA: Microbiology
and potential virulence of Sporothrix cyanescens, a fungus rarely isolated from blood and skin.
J Clin Microbiol 1990;28:1009–1015.
380 Vánky K: Illustrated genera of smut fungi; in Jülich W (ed): Cryptogamic Studies. Stuttgart,
Fischer, 1987, vol 1, pp 1–159.
381 Takashima M, Nakase T: Molecular phylogeny of the genus Cryptococcus and related species
based on the sequences of 18S rDNA and internal transcribed spacer regions. Microbiol Cult Coll
1999;15:35–47.
382 Hibbett DS, Pine EM, Langer E, Langer G, Donoghue MJ: Evolution of gilled mushrooms and
puffballs inferred from ribosomal DNA sequences. Proc Natl Acad Sci USA 1997;94:
12002–12006.
383 Pine EM, Hibbett DS, Donoghue MJ: Phylogenetic relationships of cantharelloid and clavariod
Homobasidiomycetes based on mitochondrial and nuclear rDNA sequences. Mycologia 1999;
91:944–963.
384 Maire R: Recherches cytologiques et taxonomiques sur les Basidiomycètes. Bull Soc Mycol Fr
1902;18:1–212.
385 Gäumann E: Vergleichende Morphologie der Pilze. Jena, Fischer, 1926.
386 Kreisel H: Grundzüge eines natürlichen Systems der Pilze. Lehre, Cramer, 1969.
387 Hibbett DS, Grimaldi D, Donoghue MJ: Fossil mushrooms from Miocene and Cretaceous ambers
and the evolution of homobasidiomycetes. Am J Bot 1997;84:981–991.
388 Kwon-Chung KJ: Filobasidiaceae – A taxonomic survey. Stud Mycol 1987;30:75–85.
389 Boekhout T, van Belkum A, Leenders ACAP, Verbrugh HA, Mukamurangwa P, Swinne D,
Scheffers WA: Molecular typing of Cryptococcus neoformans: Taxonomic and epidemiological
aspects. Int J Syst Bacteriol 1997;47:432–442.
390 Fell JW, Kurtzman CP, Kwon-Chung KJ: Proposal to conserve Cryptococcus (Fungi). Taxon 1989;
38:151–152.
391 Kwon-Chung KJ, Polacheck I, Bennett JE: Improved diagnostic medium for separation of
Cryptococcus neoformans var. neoformans (serotypes A and D) and Cryptococcus neoformans
var. gattii (serotypes B and C). J Clin Microbiol 1982;15:535–537.
392 Swinne D: Study of Cryptococcus neoformans varieties. Mykosen 1984;27:137–141.
393 Dufait R, Velho R, de Vroey C: Rapid identification of the two varieties of Cryptococcus
neoformans by D-proline assimilation. Mykosen 1987;30:483.
394 Kwon-Chung KJ: Filobasidium; in Kurtzman CP, Fell JW (eds): The Yeasts, a Taxonomic Study,
ed 4, rev. Amsterdam, Elsevier, 1998, pp 663–669.
395 Guého E, Smith MT, de Hoog GS, Billon-Grand G, Christen R, Batenburg-van der Vegte WH:
Contributions to a revision of the genus Trichosporon. Antonie Van Leeuwenhoek 1992;61:289–316.
396 Fell JW, Roeijmans H, Boekhout T: Cystofilobasidiales, a new order of basidiomycetous yeasts.
Int J Syst Bacteriol 1999;49:907–913.
397 Guého E, Improvisi L, Christen R, de Hoog GS: Phylogenetic relationships of Cryptococcus
neoformans and some related basidiomycetous yeasts determined from partial large subunit
rRNA. Antonie Van Leeuwenhoek 1993;63:175–189.
398 Bruns TD, Szaro TM, Gardes M, Cullings KW, Pan JJ, Taylor DL, Horton TR, Kretzer A,
Garbelotto M, Li Y: A sequence database for the identification of ectomycorrhizal basidiomycetes
by phylogenetic analysis. Mol Ecol 1998;7:257–272.
Systematics of the Ascomycota and Basidiomycota
291
399 Langer G: Die Gattung Botryobasidium Donk (Corticiaceae, Basidiomycetes). Bibl Mycol
1994;158:1–459.
400 Keller J: Atlas der Basidiomycetes. Neuchâtel, Union des Sociétés Suisses de Mycologie, 1997.
401 Corner EJH: A monograph of Clavaria and allied genera. Ann Bot Mém 1950;2:1–740.
402 Jülich W: Higher Taxa of Basidiomycetes. Vaduz, Cramer, 1981.
403 Donk MA: A conspectus of the families of the Aphyllophorales. Persoonia 1964;3:199–324.
404 Bresinsky A, Rennschmid A: Pigmentmerkmale, Organisationsstufen und systematische Gruppen
bei höheren Pilzen. Ber Dtsch Bot Ges 1971;84:313–329.
405 Hibbett DS, Donoghue MJ: Progress toward a phylogenetic classification of the Polyporaceae through
parsimony analyses of mitochondrial ribosomal DNA sequences. Can J Bot 1995; 73(suppl 1):
s853–s861.
406 Moore RT: Taxonomic significance of septal ultrastructure in the genus Onnia Karsten
(Polyporineae/Hymenochaetaceae). Bot Notiser 1980;133:169–175.
407 Mims CW, Seabury F: Ultrastructure of tube formation and basidiospore development in
Ganoderma lucidum. Mycologia 1989;81:754–764.
408 Esser K, Stahl U, Meinhardt F: Genetic aspects of differentiation in fungi; in Meyrath J,
Bullock JD (eds): Biotechnology and Fungal Differentiation. New York, Academic Press, 1977,
pp 67–75.
409 Rosinski MA, Robinson AD: Hybridization of Panus tigrinus and Lentodium squamulosum.
Am J Bot 1968;55:242–246.
410 Sigler L, Abbott SP: Characterizing and conserving diversity of filamentous Basidiomycetes from
human sources. Microbiol Cult Coll 1997;13:21–27.
411 Moore RT: The challenge of the dolipore septum; in Moore D, Casselton LA, Wood DA,
Frankland JC (eds): Developmental Biology of Higher Fungi. Cambridge, Cambridge University
Press, 1985, pp 175–212.
412 Langer E, Oberwinkler F: Corticoid basidiomycetes. I. Morphology and ultrastructure. Windahlia
1993;20:1–28.
413 Langer E: Die Gattung Hyphodontia John Eriksson. Bibl Mycol 1994;154:1–298.
414 Redhead SA, Norvell L: Notes on Bondarzewia, Heterobasidion and Pleurogala. Mycotaxon
1993;48:371–380.
415 Jahn H: Pilze, die an Holz wachsen. Herford, Bussesche Verlagshandlung, 1979.
416 Bresinsky A, Bachmann R: Bildung von Pulvinsäurederivaten durch Hygrophoropsis aurantiaca
(Paxillaceae-Boletales) in vitro. Z Naturforsch 1971;26b:1086–1087.
417 Nilsson T, Ginns J: Cellulolytic activity and the taxonomic position of selected brown-rot fungi.
Mycologia 1979;71:170–177.
418 Besl H, Bresinsky A, Kämmerer A: Chemosystematik der Coniophoraceae. Z Mykol 1986;52:
277–286.
419 Besl H, Dorsch R, Fischer M: Zur verwandtschaftlichen Stellung der Gattung Melanogaster
(Melanogastraceae, Basidiomycetes). Z Mykol 1996;62:195–199.
420 Bresinsky A, Jarosch M, Fischer M, Schönberger I, Wittmann-Bresinsky B: Phylogenetic
relationships within Paxillus s. l. (Basidiomycetes, Boletales): Separation of a Southern hemisphere genus. Plant Biol 1999;1:327–333.
421 Binder M, Besl H, Bresinsky A: Agaricales oder Boletales? Molekularbiologische Befunde
zur Zuordnung einiger umstrittener Taxa. Z Mykol 1997;63:189–196.
422 Gill M, Steglich W: Pigments of fungi (Macromycetes). Prog Chem Org Nat Prod 1987;51:1–317.
423 Kämmerer A, Besl H, Bresinsky A: Omphalotaceae fam. nov. und Paxillaceae, ein
chemotaxonomischer Vergleich zweier Pilzfamilien der Boletales. Plant Syst Evol 1985;150:
101–117.
424 Bresinsky A, Besl H: A Colour Atlas of Poisonous Fungi. London, Wolfe, 1990.
425 Jarosch M, Bresinsky A: Speciation and phylogenetic distances within Paxillus s. str.
(Basidiomycetes, Boletales). Plant Biol 1999;1:701–706.
426 Helbling A, Brander KA, Horner WE, Lehrer SB: Allergy to basidiomycetes. Chem Immunol.
Basel, Karger, 2002, vol 81, pp 28–47.
427 Nuss I: Untersuchungen zur systematischen Stellung der Gattung Polyporus. Hoppea 1980;
39:127–198.
Prillinger/Lopandic/Schweigkofler/Deak/Aarts/Bauer/Sterflinger/Kraus/Maraz
292
428 Sigler L, de la Maza LM, Tan G, Egger KN, Sherburne RK: Diagnostic difficulties caused by a
nonclamped Schizophyllum commune isolate in a case of fungus ball of the lung. J Clin Microbiol
1995;33:1979–1983.
429 Kamei K, Unno H, Nagao K, Kuriyama T, Nishimura K, Miyaji M: Allergic bronchopulmonary
mycosis caused by the basidiomycetous fungus Schizophyllum commune. Clin Infect Dis 1994;
18:305–309.
430 Ciferri R, Chavez Batista A, Campos S: Isolation of Schizophyllum commune from sputum.
Atti Inst Bot Lab Crittogam Univ Pavia 1956;14:118–120.
431 Chavez-Batista A, Maia JA, Singer R: Basidioneuromycosis of man. Anais Soc Biol Pernambuco
1955;13:52–60.
432 Catalano P, Lawson W, Bottone E, Lebenger J: Basidiomycetous (mushroom) infection of the
maxillary sinus. Otolaryngol Head Neck Surg 1990;102:183–185.
433 Rosenthal J, Katz R, DuBois DB, Morrissey A, Machicao A: Chronic maxillary sinusitis associated with the mushroom Schizophyllum commune in a patient with AIDS. Clin Infect Dis 1992;
14:46–48.
434 Sigler L, Bartley JR, Parr DH, Morris AJ: Maxillary sinusitis caused by medusoid form of
Schizophyllum commune. J Clin Microbiol 1999;37:3395–3398.
435 Restrepo A, Greer DL, Robledo M, Osorio O, Mondragon H: Ulceration of the palate caused by
a basidiomycete Schizophyllum commune. Sabouraudia 1973;9:201–204.
436 Kligman AM: A basidiomycete probably causing onychomycosis. J Invest Dermatol 1950;14:
67–70.
437 Kühner R: Les Hyménomycètes agaricoides (Agaricales, Tricholomatales, Pluteales, Russulales).
Numéro spécial du Bulletin de la Sociéte Linnéenne de Lyon, 1980.
438 Singer R: The Agaricales in Modern Taxonomy, ed 4, rev. Koenigstein, Koeltz Scientific
Books, 1986.
439 Pegler DN: The genus Lentinus, a world monograph. Kew Bull Add Ser 1983;10:1–281.
440 Hibbett DS, Vilgalys R: Phylogenetic relationships of Lentinus (Basidiomycotina) inferred from
molecular and morphological characters. Syst Bot 1993;18:409–433.
441 Thorn RG, Moncalvo J-M, Reddy CA, Vilgalys R: Phylogenetic analyses and the distribution of
nematophagy support a monophyletic Pleurotaceae within the polyphyletic pleurotoid-lentinoid
fungi. Mycologia 2000;92:241–252.
442 Lee S-S, Jung HS: Phylogenetic analysis of the Corticiaceae based on gene sequences of nuclear
18S ribosomal DNAs. J Microbiol 1997;35:253–258.
443 Mueller GM, Pine EM: DNA data provide evidence on the evolutionary relationships between
mushrooms and false truffles. McIlvainea 1994;11:61–74.
444 Hopple JS, Vilgalys R: Phylogenetic relationships among coprinoid taxa and allies based on data
from restriction site mapping of nuclear rDNA. Mycologia 1994;86:96–107.
445 Hofmann A, Heim R, Brack A, Kobel H, Frey A, Ott H, Petrzilka T, Troxler F: Psilocybin and
Psilocin. Helv Chim Acta 1959;42:1557–1572.
446 Welsh J, McClelland M: Fingerprint genomes using PCR with arbitrary primers. Nucleic Acids
Res 1990;18:7213–7218.
447 Williams JGK, Kubelik AR, Livak KJ, Rafalski JA, Tingey SV: DNA-polymorphisms amplified
by arbitrary primers are useful as genetic markers. Nucleic Acids Res 1990;18:6531–6535.
448 Molnár O, Prillinger H, Lopandic K, Weigang F, Staudacher E: Analysis of coenzyme Q systems,
monosaccharide patterns of purified cell walls, and RAPD-PCR patterns in the genus
Kluyveromyces. Antonie Van Leeuwenhoek 1996;70:67–78.
449 Lopandic K, Prillinger H, Molnár O, Giménez-Jurado G: Molecular characterization and
genotypic identification of Metschnikowia species. Syst Appl Microbiol 1996;19:393–402.
450 Molnár O, Messner R, Prillinger H, Stahl U, Slavikova E: Genotypic identification of
Saccharomyces species using random amplified polymorphic DNA analysis. Syst Appl Microbiol
1995;18:136–145.
451 Molnár O, Messner R, Prillinger H, Scheide K, Stahl U, Silberhumer H, Wunderer W:
Genotypische Identifizierung von Saccharomyces-Arten aus der Getränkeindustrie mit Hilfe der
Zufallsprimer-abhängigen Polymerase-Kettenreaktion (RAPD-PCR). Mitt Klosterneuburg
1995;45:113–122.
Systematics of the Ascomycota and Basidiomycota
293
452 Prillinger H, Kraepelin G, Lopandic K, Schweigkofler W, Molnár O, Weigang F, Dreyfuss MM:
New species of Fellomyces isolated from epiphytic lichen species. Syst Appl Microbiol 1997;
20:572–584.
453 Vos P, Hogers R, Bleeker M, Reijaus M, van de Lee T, Hornes M, Frijtens A, Pot J, Peleman J,
Kuiper M, Zabeau M: AFLP: A new technique for DNA fingerprinting. Nucleic Acids Res
1995;23:4407–4414.
454 Aarts H, Keijer J: Genomic Fingerprinting of Micro-Organisms by Automatic Laser Fluorescence
Analysis (ALFA) of Amplified Fragment Length Polymorphism (AFLP™); in Akkermans ADL,
van Elsas JD, de Bruijn FJ (eds): Molecular Microbial Ecology Manual 3.4.9. Dordrecht, Kluwer
Academic, 1999.
455 Laaser G, Möller E, Jahnke KD, Bahnweg G, Prillinger H, Prell HH: Ribosomal DNA restriction
fragment analysis as a taxonomic tool in separating physiologically similar Basidiomycetous
yeasts. Syst Appl Microbiol 1989;11:170–175.
Notes added in proof
The 2nd edition of the Atlas of Clinical Fungi [De Hoog et al., 2000] includes 65 additional clinical fungi. It often presents small subunit, large subunit or ITS restriction maps
for genotypic identification. According to the data of Binder and Hibbett [2002], the
Schizophyllales can be included among the euagarics. A publication by Sterflinger and
Prillinger [2001] presents molecular evidence that the genera Phoma and Epicoccum can
be included among the Pleosporales. According to Sugita et al. [2000], C. laurentii is a
genetically heterogeneous species; this must be taken into consideration when identifying
C. laurentii clinical isolates. Data by Sugita et al. [in press] suggest that the IGS region has a
powerful capacity of distinguishing between phylogenetically closely related strains and that
there may be a geographic substructure among T. ashai clinical isolates. O’Donnell et al.
[2001] and Voigt and Wöstemeyer [2001] present overviews on the phylogenetic relationship
among the Zygomycota. Sterflinger et al. [1999] describe the RFLP technique used in the
new edition of the Atlas of Clinical Fungi [Hoog et al., 2000]. Dr. C. Kurtzman kindly
showed us, with strongly deleted files, that the alignment in the Ascomycota becomes much
better. With these files the Archiascomycetes sensu Nishida and Sugiyama move into a basal
position. Sipiczki [2001] presents molecular data on the Archiascomycetes; however, he did
not find many similarities between the Archiascomycetes and the Basidiomycota (e.g. 5S rDNA
of Taphrina, enteroblastic budding, carotin pigments in Saitoella).
New notes on Ascomycota can be obtained from Myconet, vol. 6, 2001, edited by
O.E. Eriksson.
References
Binder M, Hibbett DS: Higher-level phylogenetic relationships of the Homobasidiomycetes (mushroomforming fungi) inferred from four rDNA regions. Mol Phylogenet Evol 2002;22:76–90.
De Hoog GS, Guarro J, Gené J, Figueras MJ (eds): Atlas of Clinical Fungi, ed 2. Centraalbureau voor
Schimmelcultures, Utrecht/Universitat Rovira i Virgili, Reus, 2000.
O’Donnell K, Lutzoni F, Ward TJ, Benny GL: Evolutionary relationships among mucoralean fungi
(Zygomycota): Evidence for family polyphyly on a large scale. Mycologia 2001;93:286–296.
Sipiczki M: Identification of Schizosaccharomyces pombe genes that encode putative homologues of
Saccharomyces cerevisiae mediator complex subunits. Acta Microbiol Immunol Hung
2001;48:519–531.
Prillinger/Lopandic/Schweigkofler/Deak/Aarts/Bauer/Sterflinger/Kraus/Maraz
294
Sterflinger K, de Hoog GS, Haase G: Phylogeny and ecology of meristematic ascomycetes. Stud Mycol
1999;43:5–22.
Sterflinger K, Prillinger H: Molecular taxonomy and biodiversity of rock fungal communities in an
urban environment (Vienna, Austria). Antonie van Leeuwenhoek 2001;80:275–286.
Sugita T, Nakajima M, Ikeda R, Matsushima T, Shinoda T: Molecular analysis of intergenic spacer 1
region of the rRNA gene of Trichosporon species. J Clin Microbiol, in press.
Sugita T, Takashima M, Ideka R, Nakase T, Shinoda T: Intraspecies diversity of Cryptococcus laurentii
as revealed by sequences of internal transcribed spacer regions and 28S rDNA gene and taxonomic positions of C. laurentii clinical isolates. J Clin Microbiol 2000;38:1468–1471.
Voigt K, Wöstemeyer J: Phylogeny and origin of 82 zygomycetes from all 54 genera of the Mucorales
and Mortierellales based on combined analysis of actin and translation elongation factor EF-1
genes. Gene 2001;270:113–120
Prof. DI. Dr. Hansjörg Prillinger, Universität für Bodenkultur,
Institut für Angewandte Mikrobiologie, Arbeitsgruppe Mykologie und Bodenmikrobiologie,
Muthgasse 18, A–1190 Wien (Austria)
Tel. 43 1 36006 6207, Fax 43 1 3697615, E-Mail H.Prillinger@iam.boku.ac.at
Systematics of the Ascomycota and Basidiomycota
295
Glossary
ABPA
allergic bronchopulmonary
aspergillosis
adiaspore
a spherical spore with a
rigid cell wall
(chlamydospore)
produced in the lungs by
the enlargement of an
inhaled conidium of
Emmonsia
aecidium
vegetative fruiting body
of the Uredinales at the
end of the haploid stage
containing dikaryotic
aeciospores
agaric
fungus belonging to the
Agaricales
anamorph
structure of the asexual
cycle and name of the
fungus within the
artificial
Deuteromycota
anisogamy
copulation of gametes
with unlike morphology
anthropophilic
fungus prefers to grow
in humans
apomictic propagation
propagation without
karyogamy and meiosis
apomixis
development of sexual
cells into spores without
being fertilized
apothecial
a cup- or saucer-like
ascoma in which the
hymenium is exposed at
maturity
arbuscular mycorrhiza
a symbiotic, nonpathogenic or feebly
pathogenic endoinfection
formed by zygomycetes
of the order Glomales.
The penetrating hyphae
produce finely branched
haustorial structures
(arbuscules) or coils and
commonly vesicles as
well
arthroconidia
segments developed by
breaking up at the septal
sites of a hypha;
infectious parasitic form
in tinea
Arthrospores
a spore derived from the
disarticulation of hypha
ascocarp
an ascus-containing
morphological structure
(ascoma)
ascoma
see ascocarp
ascospore
meiotic spore of the
Ascomycota
ascus
meiotic sac-like cell
within the ascoma of the
Ascomycota
aspergilloma
a ‘fungus ball’ of hyphae
of Aspergillus, found in
the upper lobe of the
lung
auricularioid
transversely septate
(commonly four)
cylindrical
meiosporangium of the
Basidiomycota
296
autodiploidization
self-inducing fusion of
nuclei
autogamy
self-fertilization; fusion
of nuclei without cell
fusion
bacteriophage
virus that infects
bacteria
ballistoconidium
forcibly discharged
vegetative spore
ballistospore
forcibly discharged
basidiospore
basidia (pl.)
meiosporangium of the
Basidiomycota
basidiocarp
meiotic fruiting body
(basidioma) of the
Basidiomycota
basidiolichen
symbiosis between an
alga and a basidiomycete
basidiospore
meiospore of the
Basidiomycota
basidium
meiosporangium of the
Basidiomycota, organ
diagnostic for the
Basidiomycota
Bioaerosol
biological airborne
particle(s), for instance
fungal spores, pollen etc.
blastospore
a conidium formed by
budding
body trama
hyphal layer within
fruiting bodies
budding
process of reproduction
by which the daughter
Glossary
cell separates from the
parent cell leaving a bud
scar
carpophore
(1) stalk of sporocarp;
(2) basidiocarp
chitinous fungi
fungi with chitin in their
cell walls
chlamydospore
thick-walled conidium,
often formed as a resting
form in unfavorable
conditions; depending
on the site on a hypha,
chlamydospores are
called laterales (on the
side), terminales (on
the tip), or intercalares
(centrally located)
chloroplast
photosynthetic organelle
with chlorophyll a and b
choanoflagellate
unicellular flagellates
with phylogenetic
relationships to animals
and fungi
clade
monophyletic group of
organisms
clavarioid
club- or coral-like
fruiting structure
cleistothecium
a closed fruiting body
having no predefined
opening within the
Ascomycota
coenocytic
non-septate
multinucleate mass of
protoplasm
collarette
a cup-shaped structure at
the apex of a
conidiogenous cell
conidiophore
specialized
conidiogenous hyphal
structure
conidiospore
an exogenous, nonmotile vegetative spore
conidium
asexual spore
corticioid
flat fruiting body of the
Basidiomycota which
develops directly on the
substrate with the
hymenium on the outer
side
crozier
the hook of an
ascogenous hypha before
ascus development
cystidium
a sterile cell, frequently
of distinctive shape,
between basidia of the
Basidiomycota
dematiaceous
fungi with brown or
black pigment in the cell
wall, thus appearing
brown or black microand macroscopically
denticle
a small tooth-like
projection especially one
on which a spore is
produced
dicotyledonous plants
plants with two seed
leaves
dikaryotic
a cell or hyphal
compartment having two
genetically distinct
haploid nuclei
dimitic
fruiting body of
Basidiomycota having
297
two types of hyphae
(generative hyphae
commonly with clamps,
skeletal hyphae
commonly thick-walled,
aseptate, and of limited
length)
dimorphic
having two forms; in
mycology often meaning
the yeast (often
saprophytic) and the
hyphal (often parasitic;
e.g. Ustilago maydis)
stage
dolipore septa
septa of higher
Basidiomycota with a
barrel-shaped structure
in the middle portion
ectomycorrhizal fungus
fungus with a hyphal
sheath on the surface of
the roots of trees.
Hyphae extend outward
into the soil and inward
between outer cortical
cells forming a ‘Hartig
net’
ectothrix
fungus growing on the
outside of a hair shaft,
destroying the cuticula
endocytobiotic theory
see endosymbiotic
theory
endosymbiotic theory
theory stating that
mitochondria and
chloroplasts have been
once free living bacteria
and became
symbiotically included
in the cytoplasm of the
host cell thus leading to
the origin of the
eukaryotes
Glossary
endophyte
an organism that lives
within a plant
endothrix
fungus growing inside
the hair shaft
eukaryotic
cells having a
membrane-bound true
nucleus
exoskeleton
outer skeleton found in
insects and other
arthropods
filamentous phage
virus infecting a
bacterial cell with a
variable length of the tail
fissitunicate
asci with two functional
wall layers (bitunicate),
splitting at discharge
flagellum
cylindrical extension of
an eukaryotic cell
responsible for active
movement, bound by a
plasma membrane
fungi imperfecti
fungi without known
sexual reproduction
(Deuteromycota)
fungus ball
ball-like hyphal
aggregates of
Aspergillus, found in the
upper lobe of the lung
gametangiogamy
fusion of sexually
differentiated hyphae
gastroid
basidia which do not
actively discharge their
basidiospores
geophilic fungus
fungus growing on or in
soil
germ tube
a hypha growing out of a
spore
gleba chambers
hymenial cavities within
a fruiting body where
gastroid basidia are
produced
gloeoplerous
hypha with hyaline or
yellowish and highly
refractile fluid
halotolerant
tolerant of higher salt
concentrations
haplophase
the part of the life cycle
where the cells are
haploid
haustorium
a special hyphal branch
which extends in the
living cell of the host,
for absorption of food
hemiascomycetous
fungi which belong to
the class of the
Hemiascomycetes
hetero-bifactorial
a mating system with two
different factors. One
codes for pheromones
and their receptors and
the other one for DNAbinding proteins
heterokont flagellae
two different types of
flagellae which differ in
length, type of motion
and external appendages
heterothallism
condition of sexual
reproduction in which
conjugation is possible
only through the
interaction of different
mating types
298
heterotrophic
organisms using organic
compounds as primary
sources of energy
holobasidium
a basidium which is
not divided by primary
septa
homothallism
a condition where sexual
reproduction occurs
without the interaction
of two different mating
types
hydnoid
producing basidia on
spines or tooth-like
projections
hymenium
meiospore-bearing layer
of a fruiting body
hypha
septate or nonseptate
vegetative filament
hypogeous
having subterranean
fruiting bodies
hyphomycete
mitosporic fungus
forming a mycelium
with or without pigment
isogamy
conjugation of
morphologically similar
gametes
isogenized ramarioid
inbreeding strains
haploid offspring which
becomes
morphologically similar
(Ramaria-like) after
crossing of
morphologically similar
parental strains
isoprene unit
chemical building block
containing 5 C-atoms
Glossary
karyogamy
fusion of genotypically
different nuclei
lysotroph
to obtain food by
extracellular enzymes
(absorbtive nutrition)
macroconidium
larger asexual
extracellular spore
meiospore
a spore produced in the
meiosis
meristem
tissue at the tip of a
growing plant structure,
where cell division is
most active, true
meristems are absent in
fungi
mesophilic fungi
fungi growing between
10 and 40°C
microconidium
smaller asexual spore
mitochondrial crista
structure of the inner
mitochondrial
membrane where the
respiratory chain
complexes are located
mitosporangium
a sporangium where
spore formation occurs
after mitosis
mitosporic
spore formation after
mitosis
monadal
primitive type of
morphological
organisation in algae or
unicellular flagellates
monocotylendonous
plants
plants with one seed
leaf
monoecious
male and female sex
organs on the same
mycelium
monokaryon
hyphal compartments
with a single haploid
nucleus, in
Basidiomycota often the
mycelium of a
basidiospore
monophyletic
a group composed of a
collection of organisms,
including the most
recent common ancestor
of all those organisms
mycelium
mass of hyphae
mycetoma
a fungal disease of the
foot or other parts of the
human body, especially
in the tropics
mycophagy
the use of fungi as food
oligokaryotic
hyphal compartment
with 3–10 nuclei
oogamy
heterogamy with a nonmotile female egg and a
motile male sperm
opportunistic pathogens
pathogens that convert
from a saprophytic to a
parasitic form in a
predisposed host
paraphysis
a sterile upward growing
hyphal element in the
hymenium of the
Ascomycota
parenthesome
a curved double
membrane (which may
be perforate, continuous
299
or vesiculate) on each
side of a dolipore
septum in the
Basidiomycota,
septal pore cap
perithecium
a round or flask-like
fruiting body of the
Ascomycota with an
opening at the top
phagemid
a bacterial plasmid that
can be propagated both
as a plasmid and a
bacteriophage
phialoconidium
a conidium produced on
a special conidiogenous
cell (phialide)
phragmobasidium
a basidium which is
divided by primary
septa, usually transverse
or cruciate
phragmoplast
cell division structure
occurring only in higher
plants
pileate
stalked fruiting body
with a pileus (hymeniumsupporting part of the
basidioma, cap)
pileitrama
hyphae within the cap of
a basidiomycete
plectenchyma
fruiting body of firmly
interwoven hyphae
looking like a
parenchyma of plants
polygenic
genetically controlled by
many genes
polykariotic
hyphal compartment
with more than ten nuclei
Glossary
polyphyletic origin
a collection of
organisms in which the
most recent common
ancestor of all the
included organisms is
not included
pseudohypha
hypha-like structure
formed by budding
yeasts, totally
separated by septa
without cytoplasmic
exchange
pseudoparenchyma
see plectenchyma
ramarioid strains
haploid or dikaryotic
fruiting bodies which
resemble a Ramaria
(coralloid basidiocarp)
resupinate
flat fruiting body of
Basidiomycota directly
on the substrate with the
hymenium on the outer
side
rhizopodial
type of morphological
organisation forming
amoeboid cells which
lack a rigid cell wall
saprobe
an organism using
dead organic material as
food, and commonly
causing its
decomposition
scolytid
fungus associated with
beetles (Scolytidae,
beetle family)
secotioid
the margin of the pileus
does not break free from
the stipe, gastroid
basidiospores
septate basidium
a basidium having
transverse or cruciate
septa
shikimate-chorismate
pathway
the most common
biosynthetic pathway
leading to aromatic
compounds
siphonal
hyphal compartments
without septa having
many nuclei
solopathogenicity
a pathogenic
monosporidial line
(e.g. Ustilago maydis)
somatogamous autogamy
karyogamy without
plasmogamy in
vegetative cells
somatogamy
fusion of somatic cells
or hyphae involving
plasmogamy but not
karyogamy
spermatium
non-motile male
gamete
spermogonium
a walled structure in
which spermatia are
produced
spindle pole body
organelle for the division
of nuclei in Zygo-,
Asco- and
Basidiomycota
spore
sexually or asexually
produced reproductive
unit
stichic
horizontal orientation of
the spindle of nuclei in
the basidium
300
symbiosis
association between
unlike organisms, which
is advantageous for both
organisms
sympodial
spore formation
characterized by
continued growth, after
the main axis has
produced a terminal
spore, by the
development of a
succession of apices
each of which originates
below and to one side of
the previous apex
synanamorph
two or more anamorphs
of the same teleomorph
teleomorph
the perfect (sexual) form
of an anamorph;
Glossary
morphological structure
of the sexual cycle
thallus
body of mold colony
consisting of vegetative
hyphae
tremelloid basidium
cruciate septate basidium
of the Tremellales
trichal form
septate filamentous
growth form
trichogyne
the receptive hypha of
the female sexual organ
trimitic
fruiting body having
three kinds of hyphae:
generative (often with
clamps), skeletal (often
thick-walled) and
binding hyphae (thinwalled)
unifactorial
heterothallism
a system in which the
sexual propagation to the
mycelia is controlled by
two different mating
types (e.g. ⫹ and ⫺ or a
and ␣)
vegetative hypha
fungal thread without
mitotic or meiotic
fruiting bodies
xerophilic fungi
favouring habitats in
which water is scarce
zoophilic fungus
fungus which prefers
animals for growth
zoospore
a motile spore having
one or more flagellae
301
Author Index
Aarts, H.J.M. 207
Bauer, R. 207
Bhatnagar, D. 167
Borg-von Zepelin, M. 114
Brander, K.A. 28
Breitenbach, M. 5, 48
Chiu, A.M. 1
Crameri, R. 5, 73
Deak, R. 207
Hawranek, T. 129
Helbling, A. 28
Horner, W.E. 10, 28
Schweigkofler, W. 207
Simon-Nobbe, B. 48
Sterflinger, K. 207
Kauffman, H.F. 94
Kraus, G.F. 207
Tomee, J.F.C. 94
Yu, J. 167
Lehrer, S.B. 5, 28
Levetin, E. 10
Lopandic, K. 207
Maraz, A. 207
Monod, M. 114
Ehrlich, K.C. 167
Prillinger, H. 207
Fink, J.N. 1
302
Subject Index
Aerobiology
biochemical assays 14, 15
culture analysis 13, 14
epidemiological studies 22–24
historical perspective 10, 11
indoor mold exposure 23, 24
measurement problems
counting methods for Burkard spore
traps 19–21
culture-related errors 21, 22
reporting lag 21
microscopic analysis 14
passive sampling of spores 11, 12
sampling equipment 10–13
variation patterns
altitude effects 18
ascospores 17
atmospheric variability 18, 19
basidospores 16, 17
diurnal variation 15, 16
rain effects 17
seasonal effects 17, 18
wind effects 17
Aflatoxins
carcinogenesis 177, 179
structures 177, 178
toxicity 177
types 177
Agaricales, phylogenetic relationships
270–272
Air sampling, see Aerobiology
Allergens, see also individual species
environmental factors in exposure 1
extract reproducibility problems with
molds 49–51
recombinant allergens for testing 3
Allergic bronchopulmonary aspergillosis
allergens and serology 83–87
clinical features 97
diagnosis 73, 74, 76, 77, 97
histopathology 106, 107
hypersensitivity reaction types 97
microinvasive processes 98
prevalence 76, 77, 97
Allergy
prevalence 1
respiratory, due to fungi 2
Alternaria alternata
allergens
Alt a 1
characterization 57, 58
purification 66
Alt a 2 63
Alt a 3 63
enolase
allergen activity 59, 60
cross-reactivity 60, 61
purification 66, 67
extract reproducibility problems with
molds 49–51
gene cloning and expression in
Escherichia coli 54–57
growth conditions and expression 50
mass spectrometry 56
303
Alternaria alternata (continued)
allergens (continued)
skin testing 67, 68
table of allergens 64
asthma patient sensitivity 22
exposure routes 48, 49
sensitization rates 49
specific immunotherapy 51–54
Alternaria toxins, types and features 190,
191
Amorolfine, cutaneous mycoses
management 162
Amphotericin B, cutaneous mycoses
management 161
Amplified fragment length polymorphism,
genotypic identification 272–275
Antileukoprotease, Aspergillus fumigatus
mucosal defense 100, 102
Ascomycota, see also individual species
ascospore variation patterns in air 17
phylogenetic relationships
Chaetothyriales 234, 235
class interpretation 227, 228
Dothideales 244–246
Euascomycetes 233, 234
Eurotiales 235–237
Hemiascomycetes 228–231
Hypocreales 239–242
Leotiales 248, 249
Microascales 244
Onygenales 237, 239
Ophiostomatales 242
Pezizales 249
Phyllachorales 242, 243
phylogenetic tree based on 18S rRNA
sequence 214, 226
Pleosporales 246–248
Protomycetes 231–233
Sordariales 243
Aspartate proteases, Candida albicans
adherence role 121–123
deep-seated candidiasis role 123, 124
secretion 120, 121
Aspergillosis
deep mycosis features and management
155
neurotropic mycotoxins 193
Subject Index
Aspergillus fumigatus
allergens
Asp f 1 78
characterization 78–80
cloning 78–81
enzymes 81
phage display and screening 78, 79,
82, 86, 87
purification 77, 78
skin challenge testing of recombinant
proteins 82, 83
table 79
diagnosis of allergy 2, 73
diseases, see also Allergic
bronchopulmonary aspergillosis
aspergilloma 96
atopic diseases and asthma 75, 76,
95–97
hypersensitivity pneumonitis 98, 99
invasive aspergillosis 75, 99
proteases in pathology 106–108
saprophytic colonization 75, 95, 96
exposure routes 48, 49
genome sequencing 5–8
host defense
adaptive immunity 105–108
hyphal forms 103, 104
immunosuppressive therapy effects
108, 109
innate defense of airway mucosa 99,
100, 102–105
overview 74, 75, 94
pathogenicity 74
species 74, 95
virulence factor identification 74
Asthma
air quality effects on symptoms 22, 23
Alternaria sensitivity 22
Aspergillus fumigatus 75, 76, 95–97
Athlete’s foot, see Tinea pedis
Basidomycetes, see also individual species
allergens
Boletus edulis 35
Calvatia 35, 36
Coprinus 36
Coprinus comatus
304
Cop c 1 41, 43
Cop c 2 44
phage display library screening 40
potential allergens 40, 44
cross-reactivity 39, 40
Ganoderma 37
Pleurotus 37, 38
Psilocybe 38, 39
atopic eczema 32
bronchial and nasal challenge response
32
contact dermatitis 33
food allergy 33
invasive mycosis 34
phylogenetic relationships
Agaricales 270–272
Boletales 268, 269
Cantharellales 263, 264
cell wall sugar analysis 251–254
class overview 249–252
Cystobasidales 255
Georgefischerales 258, 259
Gomphales 264
Hymenochaetales 267, 268
Hymenomycetes 260, 261
Malasseziales 258
Microbotryales 255, 256
Microstromatales 259
phylogenetic tree based on 18S rRNA
sequence 216
Polyporales 265–267
Russulales 268
Schizophyllales 269, 270
Thelephorales 264
Tremellales 261–263
Uredinales 256
Urediniomycetes 253, 254
Ustilaginales 259, 260
Ustilaginomycetes 257, 258
respiratory allergy 31, 32
sensitization rates
skin test reactivity prevalence 28, 29
species differences 30, 31
source materials for allergy testing 34, 35
spores
abundance in atmosphere 28–30
indoor spores 30
Subject Index
sampling 28–30
variation patterns in atmosphere 16,
17
taxonomic distribution 28, 29
Black piedra, features 133, 134
Blastomycosis, deep mycosis features and
management 153
Boletales, phylogenetic relationships 268,
269
Boletus edulis
allergens 35
food allergy 33
nasal challenge response 32
sensitization rates 31
Calvatia, allergens 35, 36
Candida albicans, see also Candidiasis
adherence and adhesion molecules
118, 119
dimorphism and switching system
116, 117
genome sequencing 5–8, 115
opportunistic diseases 114
prototrophy 114, 115
site-directed mutagenesis 115
virulence factors
aspartate proteases
adherence role 121–123
deep-seated candidiasis role
123, 124
secretion 120, 121
identification 115, 116
phospholipases 119, 120
Candida balantitis, features 146
Candidiasis
budding meiosis 230
chronic mucocutaneous 148
congenital 147, 148
deep mycosis features and management
153, 154
genital 146, 147
interdigital 148, 149
management of superficial disease 145
opportunistic infection 153, 154
oral 145, 146
paronychia and onychomycosis 147
predisposing factors 144, 145
305
Cantharellales, phylogenetic relationships
263, 264
Chaetothyriales, phylogenetic relationships
234, 235
Chromoblastomycosis, management 149,
150
Chytridiomycota, phylogenetic
relationships 224–226
Citrinin, toxin features 182, 183
Cladosporium herbarum
allergens
Cla h 1 characterization 59
Cla h 2 63, 64
enolase
allergen activity 59, 60
cross-reactivity 60, 61
epitope mapping 61, 62
extract reproducibility problems with
molds 49–51
gene cloning and expression in
Escherichia coli 54–57
growth conditions and expression
50
phage display and screening 65, 66
table 64
exposure routes 48, 49
sensitization rates 49
specific immunotherapy 51–54
Coccidioidomycosis, deep mycosis features
and management 152
Collectins, Aspergillus fumigatus mucosal
defense 102
Complement, Aspergillus fumigatus
mucosal defense 102, 103
Coprinus, allergens 36
Coprinus comatus
atopic eczema 32
Cop c 1 41, 43
Cop c 2 44
phage display library screening for
allergens 40
potential allergens 40, 44
sensitization rates 31
Cryptococcosis, opportunistic infection
154
Cryptococcus neoformans, genome
sequencing 5–8
Subject Index
Culture
air spore samples 13, 14, 21, 22
cutaneous mycology diagnostics 158,
159
Cutaneous mycology
candidiasis
chronic mucocutaneous candidiasis
148
congenital candidiasis 147, 148
genital candidiasis 146, 147
interdigital candidiasis 148, 149
management of superficial disease
145
oral candidiasis 145, 146
paronychia and onychomycosis 147
predisposing factors 144, 145
cutaneous mycoses
dermatophytes 134–137
onychomycosis 142–144
tinea barbae 141
tinea capitis 138–141
tinea corporis 134, 138
tinea incognito 144
tinea inguinalis 138
tinea manuum 142
tinea pedis 141, 142
deep mycosis
opportunistic fungi 153–155
pathogenic fungi diseases 151–153
host defense 132
laboratory diagnosis
culture 158, 159
dye-conjugated antibody assays 158
histopathology 159
mold identification 159, 163
potassium hydroxide test 157, 158
sample collection 157
serology 159
yeast identification 159
pathogenesis of cutaneous fungal
infection 130, 132
rare mycoses 155–157
subcutaneous mycoses
chromoblastomycosis 149, 150
entomophthoromycosis 150
lobomycosis 151
mycetoma 150
306
rhinosporidiosis 151
sporotrichosis 149
zygomycosis 150
superficial mycoses
black piedra 133, 134
pityriasis versicolor 123, 133
tinea nigra 134
white piedra 133
treatment agents and principles 160–163
Cyclopiazonic acid, toxin features 181
Cyclopiroxolamine, cutaneous mycoses
management 162
Cystobasidales, phylogenetic relationships
255
Defensins, Aspergillus fumigatus mucosal
defense 100, 102
Dermatophytes, types and characteristics
134–137
Dothideales, phylogenetic relationships
244–246
Econazole, cutaneous mycoses
management 160
Enolase
allergen activity in molds 60
cross-reactivity between Alternaria
alternata and Cladosporium herbarum
60, 61
epitope mapping of Cladosporium
herbarum protein 61, 62
function 59
purification of recombinant Alternaria
alternata protein 66, 67
Entomophthoromycosis, management 150
Epithelium, Aspergillus fumigatus mucosal
defense 104, 105, 110
Ergosterol, air sample analysis 14
Ergot alkaloids
biosynthesis 192
classification 191, 192
history of epidemics 191
physiological effects 192
Euascomycetes, phylogenetic relationships
233, 234
Eukarya
origins 209
Subject Index
phylogenetic tree of eukaryotic
organisms based on 18S rRNA
sequence 207–210
Eurotiales, phylogenetic relationships
235–237
Fluconazole, cutaneous mycoses
management 160
Flucytosin, cutaneous mycoses
management 162
Fumonisins
biosynthesis 186
food contamination 184, 185
fumonisin B1 186
types and structures 184, 185
Fungi
air sampling, see Aerobiology
clinical classificaton of infections 130,
131
extracts in allergy testing 3
kingdom, see Mycobionta
molds vs yeasts 129, 130
Ganoderma, allergens 37
Georgefischerales, phylogenetic
relationships 258, 259
-Glucans
air sample analysis 14
indoor respiratory complaint role 23
Gomphales, phylogenetic relationships 264
Griseofulvin
cutaneous mycoses management 161
tinea capitis management 140
Hemiascomycetes, phylogenetic
relationships 228–231
Histoplasmosis, deep mycosis features and
management 152, 153
Hortaea (Phaeoannelomyces) werneckii,
tinea nigra 134
Hyalohyphomycosis, features 156
Hymenochaetales, phylogenetic
relationships 267, 268
Hymenomycetes
cell wall sugar analysis 254
phylogenetic relationships 260, 261
Hyphomycosis, features 155, 156
307
Hypocreales, phylogenetic relationships
239–242
Immunoglobulin A, Aspergillus fumigatus
mucosal defense 103
Impactor samplers, types and features 12,
13
Itraconazole, cutaneous mycoses
management 160
Keratinases, cutaneous mycology 130
Ketoconazole, cutaneous mycoses
management 160
Lentinus edodes
food allergy 33
respiratory allergy 31
Leotiales, phylogenetic relationships 248,
249
Lobomycosis, management 151
Macrophage, Aspergillus fumigatus
mucosal defense 103, 104
Malasseziales, phylogenetic relationships
258
Merulius lacrymans, respiratory allergy 31
Miconazole, cutaneous mycoses
management 160
Microascales, phylogenetic relationships
244
Microbotryales, phylogenetic relationships
255, 256
Microstromatales, phylogenetic
relationships 259
Morphological differentiation, Mycobionta
212, 213, 215
Mycetoma, management 150
Mycobionta
features of kingdom 1, 2, 209
morphological differentiation 212, 213,
215
sexual differentiation 215, 217–219,
221, 223, 224
taxonomic overview 210–212
Mycotoxin, see also specific toxins
biosynthesis 167, 168, 174
Subject Index
classification 173
contamination management
preharvest control 193, 194
postharvest control 194–196
dietary considerations 196
removal 195
inactivation 195, 196
definition 167
detection and screening 176
economic impact and regulations
173–176
indoor respiratory complaint role 23, 24
mycotoxicology
definition 170
historical perspective 171–173
mycotoxicosis 168
natural occurrence 170, 171
neurotropic mycotoxins 191–193
table of examples 169
Naftifine, cutaneous mycoses management
160
Natamycin, cutaneous mycoses
management 161
Nystatin, cutaneous mycoses management
161
Ochratoxins
food contamination 179, 181
mechanism of action 180
structures 179, 180
Onychomycosis
fungal nail infections and management
142, 143
mold infections 143, 144
Onygenales, phylogenetic relationships
237, 239
Ophiostomatales, phylogenetic
relationships 242
Paracoccidioidomycosis, deep mycosis
features and management 152
Patulin, toxin features 181, 182
Penicillic acid, toxin features 182
Penicillium marneffei, infection 156,
157
Pezizales, phylogenetic relationships 249
308
Phage display
Aspergillus fumigatus allergen screening
78, 79, 82, 86, 87
Cladosporium herbarum allergen
screening 65, 66
Coprinus comatus allergen screening 40
Pheohyphomycosis, features 156
Phospholipases, Candida albicans 119,
120
Phyllachorales, phylogenetic relationships
242, 243
Phylogenetic tree, see Ribosomal RNA
genes
Piedraia hortai, black piedra 133
Pityriasis versicolor, features 132, 133
Pityrosporum folliculitis, features 133
Pleosporales, phylogenetic relationships
246–248
Pleurotus, allergens 37, 38
Pleurotus ostreatus, respiratory allergy 31
Pleurotus pulmonarius
atopic eczema 32
sensitization rates 31
Polymerase chain reaction
air sample analysis 14, 15
genotypic identification techniques
272–275
Polyporales, phylogenetic relationships
265–267
Potassium hydroxide, cutaneous mycology
testing 157, 158
Protomycetes, phylogenetic relationships
231–233
Protothecosis, features 156
Pseudallescheriasis, deep mycosis features
and management 155
Psilocybe, allergens 38, 39
RAPD-PCR, genotypic identification
272–275
Rhinosporidiosis, management 151
Ribosomal RNA genes
genotypic identification 272–275
phylogenetic trees based on 18S rRNA
sequences
Ascomycota 214, 226
Basidomycota 216
Subject Index
eukaryotic organisms 207–210
pathogenic yeasts and fungi 238
Russulales, phylogenetic relationships 268
Saccharomyces cerevisiae, genome features
5, 6, 8
Schizophyllales, phylogenetic relationships
269, 270
Scrotal candidiasis, features 147
Sexual differentiation, Mycobionta 215,
217–219, 221, 223, 224
Sick building syndrome, clinical features 2
Sordariales, phylogenetic relationships 243
Spores
air sampling, see Aerobiology
basidomycetes, see Basidomycetes
Sporotrichosis, management 149
Stachybotrys atra, pulmonary hemorrhage
and hemosiderosis 2
Sterigmatocystin, toxin features 183
T helper cell, Aspergillus fumigatus defense
105, 106
Terbinafine, cutaneous mycoses
management 161
Thelephorales, phylogenetic relationships
264
Thrush
erythematous candidiasis 146
leukoplakia 146
management 145
opportunistic infection 153, 154
pseudomembranous candidiasis 146
Tinea barbae, features 141
Tinea capitis
clinical presentation 138, 139
diagnosis 139, 140
management 140, 141
Tinea corporis, features 134, 138
Tinea incognito, features 144
Tinea inguinalis, features 138
Tinea manuum, features 142
Tinea nigra, features 134
Tinea pedis, features 141, 142
Tolnaftate, cutaneous mycoses management
162
Toxin, definition and classification 167
309
Tremellales, phylogenetic relationships
261–263
Trichosporon, white piedra 133
Trichothecenes
biosynthesis 187
diseases 189, 190
food contamination 187, 189
mechanism of action 190
structures 187, 188
types 187, 189
Ustilaginomycetes
cell wall sugar analysis 253
phylogenetic relationships 257, 258
Uredinales, phylogenetic relationships 256
Urediniomycetes
cell wall sugar analysis 252
phylogenetic relationships 253–254
Ustilaginales, phylogenetic relationships
259, 260
Zearalenone, toxin features 183, 184
Zygomycosis
superficial disease management 150
systemic disease 154, 155
Zygomycota, phylogenetic relationships
224–226
Subject Index
Vaginal candidiasis
clinical features 146
management 147
White piedra, features 133
310