Acetate Metabolism in The Fungal Pathogen Cryptococcus Neoformans

ACETATE METABOLISM IN THE FUNGAL
PATHOGEN CRYPTOCOCCUS NEOFORMANS
A Dissertation
Presented to
the Graduate School of
Clemson University
In Partial Fulfillment
of the Requirements for the Degree
Doctor of Philosophy
Genetics
by
Oly Ahmed
May 2023
Accepted by
Dr. Kerry Smith, Committee Chair
Dr. Cheryl Ingram-Smith
Dr. James Morris
Dr. Meredith Morris
ABSTRACT
Cryptococcus neoformans is an environmental basidiomycetous fungus with
a worldwide distribution and a wide range of habitats. Inhalation of the desiccated
yeasts or spores of C. neoformans often leads to opportunistic pulmonary infections
in immunocompromised individuals, and in severe cases causes lethal meningitis
following hematogenous dissemination. During infection, depending on the tissue
and disease state, the invading fungi experience a range of nutrient
microenvironments within the host body. As a result, rapid metabolic adaptations
geared towards efficient utilization of carbon sources alternative to glucose become
one of the prime determinants of survival and growth for the pathogen. Incidentally,
cryptococcal infection has a long-standing association with acetate metabolism as
underlined by previous cryptococcal transcriptomic studies in infection models that
revealed an increased activity of alternative carbon metabolism during infection, and
highlighted by the observation that cryptococcomas (infectious granulomas) are
enriched with the two-carbon metabolite acetate, a physiologically important
alternative carbon source. In this work I investigated the direct transcriptomic
impacts of acetate utilization compared to glucose utilization via RNA-sequencing,
and noted that cryptococcal transcriptome bears signatures of nutrient starvation
and metabolic adaptations in acetate-grown conditions, as well as a remarkable
upregulation of virulence-associated genes. Moreover, to investigate the importance
of acetate production in C. neoformans we biochemically and kinetically
characterized acetate kinase (Ack), an enzyme involved in acetate metabolism, and
found that kinetically Ack has a preference in the acetate forming direction
compared acetyl phosphate forming direction. This observation is consistent with
ii
the previously discovered biochemical role of another cryptococcal enzyme xylulose-
5-phosphate/ fructose-6-phosphate phosphoketolase (Xfp2), which produces acetyl
phosphate from phosphoketose sugars. Taken together, here I propose that Ack and
Xfp2 forms a pathway of acetate production in C. neoformans, and hence are likely
to be involved in acetate homeostasis. Possibility of the Xfp-Ack pathway of acetate
production in the fungal species C. neoformans prompted us to search for Ack
sequences in other eukaryotic genomes. Here I employed a hidden Markov model
based strategy to predict Ack sequences in publicly available and curated eukaryotic
genomes, and found that an overwhelming majority of the predicted Ack sequences
can be observed in dikaryotic fungi. Maximum likelihood based phylogeny of the
predicted eukaryotic Ack sequences suggest an evolutionary trajectory involving
divergence from a common ancestor and loss in most eukaryotic lineages. Finally, to
interpret the observation that acetate utilization appears to be connected with the
upregulation of virulence-associated genes in C. neoformans, I have proposed a new
theoretical framework to explain the emergence of virulence in fungal pathogens.
The hypothesis posits that molecular response to various environmental stresses and
virulence phenotypes are modular in nature and hence are often not connected in
most fungal species. Occasionally, species may co-opt virulence modules, and
hierarchically nest it with stress-response modules, thereby acquiring the ability of
opportunistic pathogenicity in mammalian hosts. Overall, this work furthers our
current understanding of the impacts of acetate metabolism in the human fungal
pathogen C. neoformans.
iii
TABLE OF CONTENTS
TITLE PAGE...........................................................................................................i
ABSTRACT ........................................................................................................... ii
LIST OF TABLES................................................................................................... ix
LIST OF FIGURES .................................................................................................. x
CHAPTER 1 ..........................................................................................................1
Cryptococcal virulence in the context of acetate metabolism .................... 1
1.1. Cryptococcus and cryptococcosis ........................................................ 2
1.1.1. The genus Cryptococcus ................................................................2
1.1.2. Distribution and morphology........................................................ 3
1.1.3. Cryptococcosis ..............................................................................3
1.1.4. Pathogenesis and host immune response ..................................... 4
1.1.5. Cryptococcal virulence factors ...................................................... 6
1.2. Carbon metabolism and virulence in C. neoformans ........................... 7
1.2.1. Glucose metabolism: Glycolysis and gluconeogenesis................... 8
1.2.2. Peroxisome and the glyoxylate-shunt ......................................... 10
1.2.3. Mitochondria: β-oxidation, respiration, and the electron

transport chain (ETC) ..................................................................................... 11
iv
1.2.4. The pentose phosphate pathway (PPP), intracellular NADPH and
trehalose metabolism .................................................................................... 13
1.3. Cryptococcal virulence: acetate and acetyl-CoA ................................ 16
1.3.1. Routes to acetyl-CoA .................................................................. 17
1.3.2. Routes to acetate ....................................................................... 18
1.3.3. Acetate kinase ............................................................................ 20
1.4. Exploring acetate metabolism in C. neoformans ............................... 21
TABLE ...................................................................................................... 23
FIGURES .................................................................................................. 24
REFERENCES ............................................................................................ 27
CHAPTER 2 ........................................................................................................ 34
Analysis of transcriptomic response in fungal pathogen Cryptococcus

neoformans grown on acetate and glucose as carbon sources .......................... 34
ABSTRACT ............................................................................................... 35
2.1. INTRODUCTION ................................................................................ 36
2.2. RESULT AND DISCUSSION ................................................................. 40
2.2.1. Chromosomal distribution of differentially expressed genes ...... 41
2.2.2. Differential expression of putative transcription factors ............. 41
2.2.3. Downregulation of genes in acetate-grown conditions ............... 42
v
2.2.4. Genes for acetate-metabolism, the glyoxylate cycle, and
peroxisomal activities were upregulated. ...................................................... 44
2.2.5. Upregulated genes bear the signature of nutrient-starvation. .... 46
2.2.6. Gluconeogenesis and certain anabolic pathways are also

upregulated. .................................................................................................. 49
2.2.7. Some upregulated genes are implicated in virulence and

pathogenesis. ................................................................................................ 51
2.3. CONCLUSION .................................................................................... 51
2.4. METHODS AND MATERIALS .............................................................. 55
2.4.1. RNA isolation and sequencing .................................................... 55
2.4.2. Transcriptomic analysis .............................................................. 56
TABLES .................................................................................................... 57
FIGURES .................................................................................................. 71
REFERENCES ............................................................................................ 73
CHAPTER 3 ........................................................................................................ 80
Biochemical characterization of acetate kinase from the fungal pathogen

Cryptococcus neoformans and its role in acetate production ............................ 80
ABSTRACT ............................................................................................... 81
3.1. INTRODUCTION ................................................................................ 82
3.2. METHODS AND MATERIALS .............................................................. 84
vi
3.2.1. Production and purification of CnAck ......................................... 84
3.2.2. Kinetic assays of CnACK .............................................................. 85
3.2.3. Identification of putative eukaryotic Ack .................................... 85
3.2.4. Construction of the phylogenetic tree ........................................ 86
3.3. RESULTS ........................................................................................... 87
3.3.1. Ack in C. neoformans is possibly unidirectional in its biological

role ................................................................................................................ 87
3.3.2. Majority of the detected eukaryotic Ack are of fungal origin ...... 89
3.3.3. Non-fungal Ack may be functionally distinct from fungal-Ack ..... 90
3.4. DISCUSSION ...................................................................................... 91
TABLES .................................................................................................... 96
FIGURES .................................................................................................. 98
REFERENCES .......................................................................................... 101
CHAPTER 4 ...................................................................................................... 104
Modularity hypothesis of fungal virulence ............................................ 104
4.1. Why we need a regulatory network-based understanding of the origin

of virulence in fungi ........................................................................................ 105
4.2. Fungal infections in mammals are rare, and virulence factors can have
niche-specific protective roles for fungi ........................................................... 107
vii
4.3. Stress response and virulence can be conceived as modules of a larger
conceptual network ........................................................................................ 109
4.4. The hypothesis and its implications ................................................ 111
TABLE .................................................................................................... 113
FIGURES ................................................................................................ 114
REFERENCES .......................................................................................... 116
CHAPTER 5 ...................................................................................................... 119
Screening modularity in transcriptional hierarchy requires undertaking

larger exploratory studies ............................................................................... 119
5.1. Transcriptional circuitry of acetate utilization ................................ 120
5.2. Possibility of an acetate switch....................................................... 121
5.3. Xfp-Ack pathway as a route of acetate production ......................... 122
5.4. The trio: Xfp-Pta-Ack ...................................................................... 122
5.5. Gene co-expression network in C. neoformans ............................... 124
REFERENCE ............................................................................................ 125
APPENDIX A .......................................................................................... 126
RNA-sequencing data of Cryptococcus neoformans ............................ 126
APPENDIX B .......................................................................................... 148
Putative acetate kinases of eukaryotic origin ..................................... 148
viii
LIST OF TABLES
Table 1.1. Ten species of Cryptococcus............................................................... 23
Table 2.1. GO term enrichment of upregulated genes ........................................ 57
Table 2.2. GO term enrichment of downregulated genes ................................... 58
Table 2.3. KEGG pathway enrichment of upregulated genes............................... 59
Table 2.4. KEGG pathway enrichment of downregulated genes .......................... 60
Table 2.5. List of upregulated genes................................................................... 61
Table 2.6. List of downregulated genes .............................................................. 68
Table 2.7. Upregulated cryptococcal genes with reported association with

pathogenicity .................................................................................................... 69
Table 3.1. Kinetics of CnAck for common divalent metal cofactors ..................... 96
Table 3.2. Specific activities of CnAck for common NTPs..................................... 96
Table 3.3. Kinectic parameters of CnAck ............................................................ 97
Table 4.1. Common fungal infections in mammals ........................................... 113
ix
LIST OF FIGURES
Figure 1.1. Pathways of central carbon metabolism of C. neoformans ................ 24
Figure 1.2. Routes to acetate and acetyl-CoA synthesis in C. neoformans ........... 26
Figure 2.1. Effects of either acetate or glucose as sole carbon source on

transcriptome of C. neoformans......................................................................... 71
Figure 2.2. Chromosomal distribution of the proportion of differentially expressed

genes in C. neoformans...................................................................................... 72
Figure 3.1. Maximum likelihood tree of 526 eukaryotic Ack sequences ............... 98
Figure 3.2. Phylogenetic of eukaryotic Ack ......................................................... 99
Figure 3.3. Proposed XFP2/ACK pathway for acetate production in C. neoformans

....................................................................................................................... 100
Figure 4.1. Stress-response and virulence traits in fungi can be thought of as

separate modules of the same regulatory networks ......................................... 114
Figure 4.2. Modularity in biological networks ................................................... 115
x
CHAPTER 1
Cryptococcal virulence in the context of acetate
metabolism
Oly Ahmed
Department of Genetics and Biochemistry; Eukaryotic Pathogen Innovation Center,
Clemson University, SC, USA
1
1.1. Cryptococcus and cryptococcosis
1.1.1. The genus Cryptococcus
The organism that is the focus of this research is Cryptococcus neoformans,
an opportunistic fungal pathogen of humans. The genus Cryptococcus has the
potential to generate profuse confusion in the literature for historical reasons. One
example is the scale insect Cryptococcus fagisuga. Feeding behavior of this insect
leads to beech bark disease in American beech (Fagus grandifolia) (Ćalić et al., 2017).
Or, consider for example many other basidiomycetes fungi that are classified as
Cryptococcus based on morphological data, although modern molecular phylogeny
shows their paraphyletic relationship with C. neoformans. These paraphyletic
‘cryptococci’ still retain the generic epithet, now called basionym, in the literature,
but are no longer incorporated into the genus Cryptococcus (Liu et al., 2015). To date,
ten species of Cryptococcus have been reported, seven of which have pathogenic
potential for humans (Table 1.1).
Another problematic relic of the older literature is the differential
nomenclature of teleomorphic (sexually reproductive) and anamorphic forms
(asexual form) of the same fungus; Cryptococcus (anamorph) is also known as
Filobasidiella (teleomorph) in the literature. Fortunately, this practice was officially
discontinued in 2013 (Hawksworth, 2011), and the anamorphic name Cryptococcus
has been adopted as the generic name. Other confounding basionyms have been
renamed based on new molecular data. However, entomologists carry on to date
calling a genus of insect Cryptococcus.
2
1.1.2. Distribution and morphology
C. neoformans is not an obligate pathogen, rather an environmental species
with worldwide distribution. This species has been isolated from niches as diverse as
decaying materials in the soil and tree-hollows, and bird guano. It has also been
observed to proliferate inside soil amoebae and nematodes (May et al., 2016).
For the majority of its life cycle, C. neoformans is found in yeast forms;
however, as a true dimorphic species, switching between the yeast and the hyphal
morphotypes occurs regularly. This morphotypic switching has clinical importance
as the hyphal form is more immunogenic than the yeast form in mammalian hosts,
and spores are the infectious propagules that arise from sexual reproduction (both
bisexual and unisexual modes of reproduction are seen in C. neoformans). An
extensive review of the sexual reproduction and morphotypic switching in C.
neoformans is provided by (Zhao et al., 2019).
1.1.3. Cryptococcosis
Cryptococcosis is a generic term for mycosis caused by the two known
pathogenic species of the genus Cryptococcus: C. neoformans and C. gattii (Maziarz
and Perfect, 2016). Like almost every other fungal infection of mammals,
cryptococcosis can be localized on the skin (primary cutaneous cryptococcosis);
however, unlike most fungal infections, skin can be a secondary site of disseminated
cryptococcosis (Noguchi et al., 2019). Other rare secondary organs of disseminated
cryptococcosis include the eye and the prostate (Maziarz and Perfect, 2016).
3
Disseminated cryptococcosis usually follows successful initial infection of the
lungs. Infectious propagules such as desiccated yeasts or spores enter the lungs
through inhalation where successful evasion of the surveillance by the host
pulmonary components of both the innate and the adaptive immune systems may
lead to hematogenous disseminations to secondary organs (Setianingrum et al.,
2019). However, direct dissemination is exceedingly rare in immunocompetent
people, and hence cryptococcosis is typically classified as an opportunistic fungal
infection.
A relatively more usual route of infection is the reactivation of the latent form
of cryptococcosis. Childhood infection may be controlled by intact immunity and go
into dormancy, which may later resurface and disseminate if the person becomes
immunocompromised (e.g. HIV infection, etc.) or immunosuppressed (e.g. bone-
marrow transplant, etc.) later in life (Alanio, 2020). The most lethal form of
cryptococcosis is the infection of the central nervous system (CNS) that invariably
results in subacute meningoencephalitis, dubbed cryptococcal meningitis
(Williamson et al., 2017).
1.1.4. Pathogenesis and host immune response
Establishment of infection is thought to be initiated by inhalation of
desiccated yeast and/or spore of C. neoformans, where the small size of these
infectious propagules (e.g. spores, 1-3 µm; yeast form, ~3 µm; growing yeast cell, 4-10
µm) facilitates the deposition in deep alveoli (Rathore et al., 2022). The initial
response to the alveolar deposition of infectious propagules is phagocytosis of
cryptococcal cells by alveolar macrophages (AM) (Giles et al., 2009; Wang et al., 2022)
4
and opsonization following the detection of spores through binding with specific
pattern-recognition receptors (PRRs) (Romani, 2004; Wang et al., 2022).
Encapsulated yeast cells can deflect recognition and opsonization owing to low
immunogenicity of cryptococcal capsules, a virulence factor (see below). Moreover,
phagocytosis of cryptococcal cells is not guaranteed to lead to clearance as the
fungal cells are capable of proliferating inside the host macrophage as a facultative
intracellular pathogen (Wang et al., 2022). For effective clearance of the cryptococcal
cells both the innate and adaptive components of immunity are important, as
defective cell-mediated immune response leads to failure of complete clearance
(Wang et al., 2022).
Successful evasion of host immune response by C. neoformans in the lungs
can lead to hematogenous dissemination. In the bloodstream, the complement
system functions as the early humoral response against the migrating cryptococcal
cells; antibodies and B and T cell-mediated responses emerge afterwards (an
extensive review is provided by Wang et al., 2022). Secondary infection of the CNS
requires the migrating cryptococcal cells to cross the blood-brain barrier (BBB), a
layer of endothelial cells with tight junctions and selective permeability around the
brain. C. neoformans has been reported to employ the following processes to cross
the BBB: (1) paracytosis, or going around the cells through the tight junctions, where
the secreted cryptococcal proteins Mpr1 (metalloprotease) and urease have been
implicated in the process (Vu et al., 2014; Shi et al., 2010); (2) transcytosis, or traversing
the BBB through the cytoplasm of the endothelial cells (Chang et al., 2004); (3) a
Trojan horse strategy, where the engulfed, but intact, cryptococcal cells are carried
across the BBB via phagocytes and released non-lytically (Charlier et al., 2009; May et
5
al., 2016); and (4) direct invasion of the BBB via the enzymatic action of various
secreted cryptococcal proteins such as phospholipases and ureases (Rathore et al.,
2022).
1.1.5. Cryptococcal virulence factors
Traditionally the term “virulence factor” has been used loosely to refer to any
(1) morphological characteristics, (2) phenotypes, or (3) genes and gene products,
that are found to be implicated in pathogenesis. As such a comprehensive, and
uncontroversial, list of virulence factors is difficult to produce. A well-rounded review
of our current knowledge of cryptococcal virulence factors can be found here
(Zaragoza, 2019).
The ability of C. neoformans strains to grow at physiologically relevant
temperature is presumably the most important virulence factor as the mammalian
host’s core temperature creates an effective physical barrier to fungal growth in vivo
(Casadevall, 2012). Other physiologically relevant characteristics implicated in
virulence involve growth adaptations to different pH environments, tolerance to
oxidative and nitrosative stresses, and acquisition of important metals such as iron
(Zaragoza, 2019).
In addition to the above mentioned phenotypic virulence factors, some
virulence factors are gene products that are secreted by the fungus in the
surrounding host-tissue environment for nutrient acquisition (e.g. proteases and
lipases) and immunomodulation (e.g. urease and phospholipase B; see Wang et al.,
2022).
6
The most studied virulence factors are the morphological characteristics
unique to C. neoformans: polysaccharide capsule, melanin formation, and anti-
phagocytic protein 1 (App1). The unique cryptococcal capsule helps prevent
phagocytosis, complement and antibody opsonization, and thus contributes to
persistence and immune-evasion; tissue-specific factors such as temperature, pH,
CO2 and iron concentrations, all influence the capsule synthesis within mammalian
hosts (Momin and Webb, 2021).
The virulence factor melanin is relatively typical of fungal pathogens; in C.
neoformans melanization is thought to be involved in protection against oxidative
and nitrosative challenges from phagocytic activities of immune cells. Additionally,
the unique secreted protein App1 is known to inhibit phagocytosis via complement-
mediated mechanisms (Momin and Webb, 2021).
1.2. Carbon metabolism and virulence in C. neoformans
Our current understanding of carbon metabolism in C. neoformans is based
on extensive studies done in model organism Saccharomyces cerevisiae. However,
S. cerevisiae differs from C. neoformans in two important aspects. First, it is an
ascomycete fungus unlike Cryptococcus spp., which are basidiomycete fungi.
Second, S. cerevisiae shows Crabtree effect, which is the ability to carry on
fermentation under aerobic conditions provided a critical threshold of glucose
concentration is met. Despite such qualifications, searching for analogous metabolic
pathways in C. neoformans proved useful in discovering homologous genes and
describing nutritional conditions that are implicated in cryptococcal virulence
(Figure 1.1).
7
Historically, the importance of carbon metabolism in C. neoformans during
the disease progression was inferred from many transcriptional studies done under
various in vivo and in vitro conditions using a wide range of techniques including
differential display RT-PCR (ddRT-PCR), serial analysis of gene expression (SAGE), and
microarray (Kronstad et al, 2012; Hu et al, 2008; Steen et al, 2003; Rude et al, 2002).
From such studies one pattern became apparent: C. neoformans employs metabolic
reprogramming at different stages of the disease progression depending on the
host-tissue microenvironments. (For an earlier review see Kronstad et al., 2012.) In
recent years techniques such as RNA-seq improved the resolution of such
transcriptomic studies. For example, comparing the transcriptomes of clinical
isolates from the cerebrospinal fluid (CSF) of 31 cryptococcal meningitis patients with
that of the fungi grown on rabbit CSF, artificial CSF, and carbon-rich in vitro
conditions highlighted the importance of glycolysis, the TCA cycle, oxidative
phosphorylation, and sugar transport in cryptococcal meningitis and the dynamic
adaptability of this infectious fungus in the central nervous system of human hosts
(Yu et al., 2021).
1.2.1. Glucose metabolism: Glycolysis and gluconeogenesis
The mechanism of glucose sensing in C. neoformans is not understood in its
totality to date. Hexose transporters Hxs1 and Hxs2 in C. neoformans are described to
be important for glucose uptake, and expression of HXS1 is reduced in high glucose
concentration and important for growth in low glucose conditions (Liu et al., 2013).
However, the expression of HXS2 is not regulated by glucose, suggesting the role of
Hxs1 in glucose sensing and regulation of glucose uptake in C. neoformans.
8
Moreover, HXS1 is shown to be required for virulence in murine inhalation models
(knock-out experiment), indicating the importance of regulation of glucose
metabolism during pulmonary infection (Liu et al., 2013).
The impact of glucose utilization, as reflected in response to glucose
starvation in virulence, is also highlighted by the positive association observed
between incremental glucose starvation in media and increased expression of the
gene for antiphagocytic protein 1 (APP1). App1 is a unique cryptococcal virulence
protein involved in resistance to phagocytosis by macrophages (Williams and Del
Poeta, 2011).
The impact of glycolysis on cryptococcal virulence has been studied utilizing
deletion mutants. Two C. neoformans mutants defective in glycolytic pathway either
at the first step (hxk1Δ/hxk2Δ; hexokinases) or the last step (pyk1Δ; pyruvate kinase)
both show attenuated virulence in mouse models; however, there remains a
distinction between the ATP-generation capabilities of these two strains in ex vivo
human CSF. ATP production was found to be unaffected in the hxk1Δ/hxk2Δ mutant,
but was severely reduced in pyk1Δ mutant, suggesting that different mechanisms of
virulence attenuations are at play depending on which phase in glycolysis was
blocked (Price et al., 2011).
In parallel to glycolysis, the impact of gluconeogenesis on virulence has been
studied in mutant strains lacking phosphoenolpyruvate carboxykinase (PCK1), one of
four dedicated gluconeogenic enzymes. The C. neoformans pck1Δ strain can grow on
glucose rich media; however, as expected, it has reduced growth on acetate and the
3-carbon source lactate (Panepinto et al., 2005). Moreover, this mutant is severely
attenuated in a mouse model (Panepinto et al., 2005). In a later study, although the
9
PCK1 gene was observed to be highly expressed in the low-glucose environment of
rabbit CSF, deletion of PCK1 did not seem to have any impact on the persistence of
the fungus in the rabbit CSF model (Price et al., 2011).
1.2.2. Peroxisome and the glyoxylate-shunt
Peroxisomes, a single membraned, virtually universal eukaryotic organelle, is
involved in diverse cellular functions, including, but not limited to, β-oxidation, the
glyoxylate-shunt, and reactive oxygen species (ROS) metabolism. Generation of
acetyl-CoA and carbohydrates for cell wall synthesis and maintenance of turgor
pressure via peroxisomal metabolic pathways, as well as synthesis of melanin and
certain mycotoxins, implicated this organelle in virulence in the context of plant-
fungal pathogenicity (Falter and Reumann, 2022).
Peroxisome biogenesis requires the involvement of proteins known as
peroxins (or, PEX proteins). Knock-out mutants for PEX1 and PEX6 in C. neoformans
were shown to have reduced growth on fatty acid diets; however, these mutants
can grow on acetate as sole carbon source, implying a heavy reliance on
peroxisomes for fatty acid degradation in C. neoformans (Idnurm et al., 2007). One
unusual feature of C. neoformans, in contrast to other known fungi, is its apparent
need of intact peroxisomal functions for growth on simple sugars such as glucose,
fructose, and mannose (Idnurm et al., 2007). Surprisingly, PEX1 mutants were found
to be as virulent as the wild-type when tested in mouse models (Idnurm et al., 2007).
As noted above, C. neoformans defective in peroxisomal functions can still
grow on acetate as sole carbon source, suggesting plausible extra-peroxisomal
mechanisms of acetate utilization. However, disrupting the glyoxylate-shunt via
10
deletion of isocitrate lyase (ICL1), a gene encoding one of the two unique enzymes,
renders the fungus unable to grow on oleic acid or acetate (Rude et al., 2002).
Additionally, ICL1 is also shown to be upregulated by acetate or ethanol (2-carbon
metabolites), and suppressed at high concentration of glucose (Rude et al., 2002).
Deletion of the gene encoding malate synthase (MLS1), the other glyoxylate-shunt
enzyme, also leads to growth defects on acetate, highlighting the essentiality of the
glyoxylate-shunt in acetate utilization (Idnurm et al., 2007). However, despite the
importance of glyoxylate-shunt in acetate utilization, defects in this pathway do not
seem to impact virulence (Idnurm et al., 2007).
1.2.3. Mitochondria: β-oxidation, respiration, and the electron transport
chain (ETC)
In addition to peroxisomes, mitochondria also contribute to β-oxidation; hence
attempts were made early on to distinguish between the differential roles of these
two organelles in lipid metabolism and virulence. The Kronstad laboratory
(Kretschmer et al., 2012) created C. neoformans deletion strains for the enzymes
Mfe2 (multifunctional enzyme 2: peroxisomal β-oxidation), and Had1 and Had2
(Hydroxyacyl-CoA dehydrogenases: mitochondrial β-oxidation) and observed that
although had1Δ is as virulent as wild-type, mfe2Δ and mfe2Δ::had1Δ are hypovirulent
in mouse inhalation models, suggesting that peroxisomal β-oxidation might be
slightly more important for virulence than the mitochondrial version.
C. neoformans has been considered an obligate aerobe since the observation
that aeration of the culture media through rotation stimulates growth of the clinical
isolates tested, compared to static growth conditions, irrespective of the
11
concentration of glucose in the media (Odds et al., 1995). More than a decade later,
experimentation in CoCl2-based hypoxia-mimetic conditions with randomly
mutagenized (T-DNA insertions) C. neoformans strains showed growth-defect in
low-oxygen conditions demonstrated the essential connection between the
integrity of mitochondrial structure and function in respiration and ROS deactivation
and the survival of C. neoformans in low-oxygen environments (Ingavale et al., 2008).
This link is deemed important as oxygen levels available to C. neoformans for
respiration differs drastically among diverse environmental habitats and various
host-tissues. Partial pressure of O2 at sea level is 159 mmHg, while it is 104-108 mmHg
in alveoli, and 30-48 mmHg in the brain (Ortiz-Prado et al., 2019).
The importance of mitochondria in cryptococcal virulence has been
highlighted by some recent studies. Inhibition of either the classical (involving
proton efflux) or the alternative oxidase pathway (reduces oxygen without creating
proton gradient) of the electron transport chain (ETC) is found to be sufficient for
inhibition of capsule growth, one of the most salient cryptococcal virulence traits
(Trevijano-Contador et al., 2017). Additionally, this study also found that capsule
growth in C. neoformans seems positively associated with increased respiratory
activity in mitochondria, as reflected by upregulation of COX1 (cytochrome c oxidase;
electron transport), an increase in mitochondrial membrane potential, and a
consequent increase in ROS level (Trevijano-Contador et al., 2017). However, this
discovery is more perplexing considering that capsule synthesis is thought to be a
form of stress response stimulated by nutrient-starvation, and yet seems to be
heavily reliant on energy generation via the ETC.
12
Corroborating evidence of the requirement of the ETC in C. neoformans
survival came from study of the chaperone protein Mrj1 (mitochondrial respiration J-
domain protein 1). Mrj1 functions in respiration through its interaction with Complex
III of the ETC in the mitochondrion. The mrj1Δ and non-functional mrj1 (single amino
acid alteration) strains show growth defects at physiological temperature and on
alternative carbon sources, and are deficient in capsule elaboration. Moreover, mrj1Δ
strain is also avirulent in the mouse model (Horianopoulos et al., 2020).
In addition to virulence, mitochondrial activities may also moderate drug
resistance in C. neoformans. For example, it has recently been reported that low
glucose increases fluconazole tolerance in C. neoformans (Bhattacharya et al., 2021).
The proposed mechanism of fluconazole resistance involves transcriptional
modulation induced by low-glucose condition that leads to increased activities of
efflux proteins, enzymes of β-oxidation, and SOD (superoxide dismutase; ROS
inactivation pathway). Increased β-oxidation and SOD activities modify
mitochondrial functions causing increase in intracellular ATP concentration, which
in turn accelerates fluconazole efflux via actions of efflux proteins (Bhattacharya et
al., 2021).
1.2.4. The pentose phosphate pathway (PPP), intracellular NADPH and
trehalose metabolism
The disaccharide trehalose, known for its role in stress response in fungi, is
intricately linked to glucose metabolism. Genes of the trehalose biosynthetic
pathway have been studied for their involvement in the metabolism, cell wall
homeostasis, stress response and virulence in few pathogenic fungal species
13
(Thammahong et al., 2017). Not surprisingly, mutants in trehalose 6-phosphate
synthase (TPS1) of both the model fungus S. cerevisiae and the rice-blast fungus
Magnaporthe grisea show growth inhibition on glucose. However, M. grisea tps1Δ
showed a variable growth phenotype on glucose based on what other nitrogen
sources are available. No growth defect was seen on ammonium, nitrite, and amino
acids (except cysteine), but growth was impaired on nitrate, suggesting a link
between nitrate utilization and glucose metabolism (Wilson et al., 2007).
Additionally, in tps1Δ, the nitrogen metabolite repressor 1 (NMR1) was upregulated,
whereas nitrite and nitrate reductases were downregulated when grown on nitrate
(N-source) with glucose (C-source). In parallel, tps1Δ mutants showed an increased
accumulation of glucose-6-phosphate, but reduction of glucose-6-phosphate in
hxk1Δ (i.e. defective in glycolysis) and hxk1Δ::tps1Δ mutants when grown on glucose.
These observations led the authors to propose that TPS1 is presumably connecting
the carbon and nitrogen metabolism via influencing the intracellular level of NADPH
generated through the action of G6Pdh (glucose 6-phosphate dehydrogenase) in
the oxidative phase of PPP (Wilson et al., 2007; Masi et al., 2021).
Traditionally, the role of trehalose metabolism in fungal pathogenesis has
been studied from the perspective of trehalose’s involvement in germination/
sporulation and as stress response/ protectant, as such studies involving C.
neoformans were no different. Defect in trehalose synthesis via deletion of TPS1 in C.
neoformans has been shown to cause virulence attenuation and faster clearance
(i.e., reduced persistence) when tested both for persistence in an
immunosuppressed rabbit model and survival in a mouse inhalation model (Petzold
et al., 2006). On the other hand, the impact of trehalose utilization on cryptococcal
14
virulence was tested on a mice tail-vein model using null mutants of neutral
trehalase (NTH) — nth1Δ, nth2Δ, and nth1Δ::nth2Δ — and was observed that although
nth1Δ showed little defect in virulence, both nth2Δ and nth1Δ/nth2Δ were
hypervirulent. Moreover, deletion of NTH1 and NTH2 led to a defect in sporulation as
well (Botts et al., 2014).
As the example above of Magnaporthe illustrates, a plausible link between
carbon and nitrogen metabolism through cellular NADPH has the potential to
confound studies aimed at elucidating the effect of trehalose synthesis on fungal
virulence. This possibility becomes more likely considering the studied effect of
NADPH generating pathways on virulence in C. neoformans. There are at least three
different pathways for NADPH generation in C. neoformans: (1) the oxidative phase
of the PPP involving G6Pdh and 6Pgd (6-phosphogluconate dehydrogenase), (2)
alternative oxidative PPP involving Gnk (gluconate kinase), and (3) Idp (NADP+-
dependent isocitrate dehydrogenase). An earlier study reported that deletion of
G6PDH (zwf1Δ) does not seem to alter the sensitivity of C. neoformans to
temperature or oxidative stress, and intracellular concentration of NADPH appears
to stay constant, implying other active routes of NADPH generation (Brown et al.,
2010). Later, a different study reported that deletion of GNK (alternative oxidative
PPP) severely impairs sporulation (Jezewski et al., 2022). Finally, deletion of IDP in C.
neoformans leads to sensitivity to high temperature, a virulence factor, and
nitrosative stress (Brown et al., 2010). Taken together, the role of NADPH in cellular
and reproductive fitness, and in carbon and nitrogen metabolism might be
combined conceptually to better guide our future studies of cryptococcal virulence.
15
1.3. Cryptococcal virulence: acetate and acetyl-CoA
An NMR-based study of a rat pulmonary cryptococcoma model, an induced
infection of C. neoformans, revealed a high abundance of trehalose, mannitol,
glycerophosphorylcholine, ethanol, and acetate in cryptococcomas compared to
healthy lung tissue (Himmelreich et al., 2003). This study sparked the interest of the
community to explore the connection between acetate and cryptococcosis. Later a
serial analysis of gene expression (SAGE) study of a murine pulmonary infection
model suggested the potential involvement of transcriptional activation of acetyl-
CoA metabolism in C. neoformans during the early stages of pulmonary
cryptococcosis (Hu et al., 2008). The same study also found that deletion of ACS1, the
enzyme responsible for conversion of acetate to acetyl-CoA, leads to growth
impairment on acetate, and a slight delay in causing disease in the murine
inhalation model, presumably due to compensatory actions of other acetyl-CoA
generating pathways active in C. neoformans (Hu et al., 2008).
These earlier studies raised further questions about the utility of
acetate/acetyl-CoA metabolism in C. neoformans virulence; is acetate production
(and subsequent secretion) and/or is acetate utilization important for pathogenicity?
Both of these possibilities seem plausible in the context of cryptococcosis. For
example, acetate production and secretion may explain the observed acidic pH of
cryptococcoma in vivo (Wright et al., 2002); however, this acidification is unlikely to
be an effective defense mechanism against the cryptococcoma-clearance
(analogous to tumor-clearance) by natural killer (NK) cells of the innate immune
system (Islam et al., 2013). On the other hand, acetate utilization may be one of the
major pathways necessary for survival in the glucose-limited microenvironments of
16
host-tissues. For context, pulmonary airway surface liquid (ASL) contains roughly 10-
fold lower glucose than serum (Philips et al., 2003; Ries et al., 2018), whereas this
amount is 60-70% of the serum-level in CSF (Seehusen et al., 2003; Ries et al., 2018).
However, acetate utilization vs. acetate production/secretion does not have to
be a false dichotomy as appears to be here in the preceding discussion. The stages
of disease progression, tissue microenvironments, and interaction with the
components of the immune system are likely to wildly influence the state of acetate
metabolism in C. neoformans during infection. A regulated switching between
production and utilization of acetate could be co-opted for adaptation to the
constantly changing nutrient pool within the host body during infection; equivalent
systems of “acetate switch” have been documented first in prokaryotes (e.g. see the
classic Wolfe, 2005) and recently in an archaeon (Kuprat et al., 2021).
1.3.1. Routes to acetyl-CoA
As mentioned above, ACS1 deletion has mild impact on cryptococcal virulence
in a murine inhalation model (Hu et al., 2008), and the authors suggested that
defects in acetyl-CoA synthesis via ACS1 may have been compensated by other
routes of acetyl-CoA synthesis. Subsequently, another route of acetyl-CoA synthesis
was investigated by Griffiths et al. (Griffiths et al., 2012). Deletion of ACL1, encoding
ATP-citrate lyase which synthesizes acetyl-CoA from cytoplasmic pool of citrate,
leads to decreased survival of C. neoformans within murine macrophages, increased
susceptibility to fluconazole, and virulence defects in a murine inhalation model
(Griffiths et al., 2012).
17
Recently, another direct route of acetyl-CoA synthesis from acetoacetate, an
end product of various amino acid degradation pathways, has been discovered.
KBC1 encodes 3-keto-butanoyl-CoA synthetase which converts acetoacetate to
acetoacetyl-CoA, which can be further converted to acetyl-CoA via the action of
acetoacetyl-CoA lyases (Alden et al., 2022). Although the KBC1 knock-out does not
show any discernible defect in virulence, mice infected with acs1Δ::kbc1Δ double
mutant were found to have reduced CNS burden compared to those infected with
either single mutant or wild-type strain (Alden et al., 2022).
1.3.2. Routes to acetate
To date, there are at least two pathways identified in C. neoformans that can
potentially contribute to the intracellular acetate-pool: (1) conversion of pyruvate to
acetate (analogous to that in S. cerevisiae; see Orlandi et al., 2014 for an introduction);
and (2) conversion of phosphoketose sugar to acetate (analogous to that in bacteria,
see Henard et al., 2015 for an introduction); (Figure 1.2). The first pathway involves
decarboxylation of pyruvate into acetaldehyde by pyruvate decarboxylase (Pdc),
followed by oxidation of acetaldehyde to acetate by aldehyde dehydrogenase (Ald).
The second pathway is relatively rare in eukaryotic organisms, and involves first the
breakdown of xylulose 5-phosphate (or fructose 6-phosphate) to glyceraldehyde 3-
phosphate (or erythrose 4-phosphate) and acetyl phosphate using inorganic
phosphate catalyzed by xylulose 5-phosphate/ fructose 6-phosphate
phosphoketolase (Xfp), followed by conversion of acetyl phosphate to acetate via the
action of acetate kinase (Ack).
18
The Pdc-Ald pathway is known as Pyruvate-Acetaldehyde-Acetate (PAA)
pathway, and has been described in many fungi as a route of fermentation. In S.
cerevisiae, PAA pathway can lead to increased intracellular acetate, which in turn
may reduce ethanol utilization under respiration condition, as a form of catabolite
repression (Simpson-Lavy and Kupiec, 2019). Similarly, in pathogenic fungus
Gibberella zeae, the causative agent of head blight disease in cereals, the PAA
pathway is thought to play an important role in lipid synthesis in the aerial mycelia
(Son et al., 2012). These examples highlight the diverse biological roles that PAA
pathway plays in fungi.
It has been observed that many fungal genomes contain genes for Xfp and
Ack, raising the possibility of a potential pathway connecting xylulose metabolism
with acetate production (Ingram-Smith et al., 2006). Little is known about the
biological significance of the Xfp-Ack pathway, dubbed the phosphoketolase
pathway, in fungi. Metabolic flux analysis in a phosphoketolase (XFP) overexpression
mutant of the filamentous fungus Aspergillus nidulans suggests that this pathway
might have evolved to provide added flexibility to the central carbon metabolism
(Panagiotou et al., 2008).
The relative importance of the above-mentioned routes to acetate synthesis
in C. neoformans cellular metabolism is not fully understood. However, a case can be
argued for a metabolic scheme that links the Xfp-Ack pathway to the oxidative
phase of the PPP. In non-dividing cells, metabolic needs such as fatty acid synthesis
(growth and membrane integrity) and glutathione metabolism (oxidative damage)
create high demands for NADPH regeneration without the excess supply of ribose 5-
phosphate through the canonical PPP. In these situations the Xfp-Ack route can act
19
as a siphoning mechanism by utilizing the sugar phosphate intermediates and
thereby essentially decoupling the NADPH regeneration from excess ribose 5-
phosphate build-up. Additionally, such siphoning mechanism also implies the
existence of a possible regulatory system between the PPP and acetate synthesis via
Xfp-Ack route.
1.3.3. Acetate kinase
From our discussion above it follows that acetate kinase, Ack, is likely to play
important metabolic roles in addition to acetate metabolism in C. neoformans, and
presumably in other eukaryotic species as well. In fact, recently it has been proposed
that Ack in the amoebic parasite Entamoeba histolytica, a eukaryote, might not be
as important in acetate production as it is in maintaining intracellular NAD+/NADH
ratio during ethanol production via glycolysis (Dang et al., 2022).
Compared to the Ack of bacterial and archaeal origins, our knowledge of
eukaryotic Ack is limited. Eukaryotic Ack of E. histolytica differs from Acks of well-
studied bacterial genera (e.g. Escherichia, Bacillus, Salmonella etc.) and archaeal
genus Methanosarcina (see Ferry, 2011) in that it requires inorganic pyrophosphate
(PPi), rather than ATP, as phosphoryl donor (Tielens et al., 2010). Furthermore, in free-
living alga Chlamydomonas reinhardtii, ACK is expressed in aerobic condition and
Ack is localized in mitochondria (Atteia et al., 2006). However, transferring this alga to
an anaerobic, heterotrophic culture condition seems to induce transcription of
another ACK predicted to be localized in chloroplast (designated as ACK1), and the
accumulation kinetics of this transcript appears to correspond with acetate
accumulation in this species (Dubini et al., 2009; Tielens et al., 2010). Apart from such
20
kinetic or expression studies, eukaryotic Ack has largely remained understudied. The
structure of Ack enzymes from E. histolytica and C. neoformans have been resolved,
and it was observed that substrate specificity difference between these eukaryotic
Ack (i.e. ATP versus PPi) may be attributed to subtle amino acid changes in the active
site without drastically altering the backbone structure of these proteins (Thaker et
al., 2013). These structures of eukaryotic Ack are also observed to be consistent for
the reaction mechanism of direct in-line transfer of phosphoryl group proposed for
prokaryotic Acks (Thaker et al., 2013). Here it is worth mentioning that in C.
reinhardtii phosphotransacetylase (Pta) is assumed to form a pathway of acetate
production or assimilation with Ack analogous to that found in many bacterial
species, whereas in E. histolytica traditional partner of Ack such as Pta ro Xfp of
acetate pathway are absent (Ingram-Smith et al., 2006).
1.4. Exploring acetate metabolism in C. neoformans
In this work, I set out to explore the acetate metabolism in the fungal
pathogen C. neoformans. I began by asking how acetate utilization as the sole
carbon source brings about changes in the transcriptional landscape of this species
compared to that during glucose utilization. C. neoformans KN99 strains were
grown in identical condition except for the addition of two different carbon sources
in the media. RNA-seq experiments were performed on the fungus following growth
in these conditions to measure the changes in the expression levels of thousands of
cryptococcal genes (Chapter 2).
In Chapter 3, I describe the in vitro kinetic characteristics of C. neoformans
Ack, isolated following heterologous expression in Escherichia coli, to better
21
understand the role of Ack in C. neoformans. This work was also extended to
incorporate in silico identification of ACK in currently available genome sequences of
eukaryotic organisms and phylogenetic reconstruction of these putative eukaryotic
ACK genes to glean the evolutionary history of this enzyme within the domain
Eukarya.
Chapter 4 includes a synthesis of evolutionary network biology with fungal
pathogenesis. Here I have proposed the modularity hypothesis to explain the
emergence of virulence traits in present day fungi that enables them to cause
accidental pathogenicity in mammalian hosts. This expository piece introduces the
modularity hypothesis for the first time as an explanatory framework to describe
evolution of fungal pathogenicity, as well as a pragmatic framework to guide
research programs aimed at efficient discovery of molecular pathways for antifungal
drug targeting.
Finally, in Chapter 5, I conclude this dissertation by summarizing my findings
and suggesting possible future avenues in our exploration of acetate metabolism in
the fungal pathogen Cryptococcus neoformans.
22
TABLE
Table 1.1. Ten species of Cryptococcus
Species complex1 Species Pathogenic to humans?
C. neoformans (sensu stricto) Yes

C. neoformans
C. deneoformans Yes
C. gattii (sensu stricto) Yes
C. bacillisporus Yes
C. gattii C. deuterogattii Yes
C. tetragattii Yes
C. decagattii Yes
ND C. depauperatus No
ND C. luteus No
ND C. amylolentus No
Note:
1. Modern phylogeny supports a ‘seven species’ view over ‘two species’ view of C.
neoformans/gattii complex (Hagen et al., 2015)
ND: not defined
23
FIGURES
Figure 1.1. Pathways of central carbon metabolism in C. neoformans. Schematic
showing prominent intermediates and gene products (enzymes and transporters) of
cryptococcal central carbon metabolism; genes studied to date and mentioned in
the text are shown in blue, italics. (caption continues into the next page)
24
(Continued from the last page) Abbreviations (metabolites): G6P: glucose-6-
phosphate; F6P: fructose-6-phosphate; Gly 3P: glyceraldehyde-3-phosphate; DHAP:
dihydroxyacetone phosphate; E4P: erythrose-6-phosphate; R5P: ribose-5-phosphate;
X5P: xylulose-5-phosphate; Ru5P: ribulose-5-phosphate; 6PG: 6-phosphogluconate;
6PG lactone: 6-phosphogluconolactone; PEP: phosphoenolpyruvate; Pyr: pyruvate;
OxAc: oxaloacetate. Abbreviations (gene products): HXS: hexose transporter; HXK:
hexokinase; G6PDH: glucose-6-phosphate dehydrogenase; 6PGD: 6-
phosphogluconate dehydrogenase; GNK: gluconate kinase; PYK: pyruvate kinase;
PCK: phosphoenolpyruvate carboxykinase; ACS: acetyl-CoA synthetase; ACL: ATP-
citrate lyase; ICL: Iso-citrate lyase; MLS: malate synthase; MFE2: multifunctional
enzyme 2; HAD: 3-hydroxyacyl-CoA dehydrogenase. Colors and shapes: Thick
magenta vertical line denotes separation between the outside (left) and the inside
(right) of the cell; rectangular box on the magenta line denotes glucose transporter;
solid arrows denote single step in the pathway; dashed arrows indicate multiple
intermediate steps in the pathway omitted from display for improved clarity; red
arrows: gluconeogenic steps; purple arrows: the TCA cycle steps; orange arrows:
steps of the glyoxylate shunt; green arrows: oxidative and alternative oxidative
phases of the pentose phosphate pathway.
25
Figure 1.2. Routes to acetate and acetyl-CoA synthesis in Cryptococcus
neoformans. Abbreviations: Ack: acetate kinase; Acs: acetyl-CoA synthase; Acl: ATP-
citrate lyase; Ald: aldehyde dehydrogenase; Kbc: 3-keto butanoyl-CoA synthetase;
Pdc: pyruvate decarboxylase; Pdh: pyruvate dehydrogenase; Xfp: xylulose 5-
phosphate/ fructose 6-phosphate phosphoketolase.
26
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33
CHAPTER 2
Analysis of transcriptomic response in fungal
pathogen Cryptococcus neoformans grown on
acetate and glucose as carbon sources
Oly Ahmed, Bridget Luckie, and Kerry Smith
Authors contribution:
KS: conceived and designed the experiment; BL: cultured the cells and harvested
RNA for sequencing; OA: analyzed the RNA-seq data and prepared the manuscript
34
ABSTRACT
The two-carbon short-chain fatty acid acetate is an important alternative
carbon source in fungal pathogens, as its abundance and the abundance of the
preferred carbon source glucose varies significantly depending on the host-tissue
microenvironments. This supports the importance of rapid metabolic adaptation in
the invading fungal pathogens for efficient utilization of alternative carbon sources
for survival and disease progression. Previous transcriptomic and proteomic studies
of pathogenic ascomycete fungi such as Aspergillus fumigatus, Paracoccidioides
spp., and Candida glabrata highlight the species-specific metabolic reprogramming
resulting in these fungi following acetate utilization compared to glucose utilization.
Here we report the impact of acetate utilization on the transcriptome of the
pathogenic basidiomycete Cryptococcus neoformans for the first time. Of the 6,967
cryptococcal genes quantified in this study, 282 and 74 genes were upregulated and
downregulated, respectively, in acetate-grown cells compared to glucose-grown
cells. Acetate utilization resulted in an induction of genes in the metabolic pathways
such as the TCA, the glyoxylate cycle, acetyl-CoA production, fatty acid and polyol
metabolism, gluconeogenesis, glycogen synthesis and glucose mobilization, amino
acid degradation, lysine biosynthesis, and many putative transporters involved in
nutrient acquisition. Acetate utilization also resulted in an induction of fifteen
transcription factors (TF); however, no TFs were found to be downregulated.
Additionally, many genes previously reported to be associated with cryptococcal
virulence were also found to be upregulated in acetate-grown cells. Taken together,
35
these observations suggest that acetate utilization in C. neoformans leads to
metabolic reprogramming for efficient growth and survival along with elaboration of
virulence traits as a response to nutritional stress.
2.1. INTRODUCTION
Acetate (C2H3O-), a short chain fatty acid, is known to be an important carbon
source for fueling the cellular metabolism in Bacteria, Archaea, and Eukarya (Pandey
et al., 2018). For fungal pathogens of humans, the ability to exploit carbon sources in
various host tissue-environments is more important for survival as the concentration
of the preferred carbon source (typically glucose; C6H12O6) varies from tissue to tissue
within the host body. For example, the glucose level in human bloodstream is
around 0.06% (3.3 mM)- 0.1% (5.5 mM) (Gow and Brown, 2018), in vaginal lumen the
level is around 0.5% (27.8 mM) (Owen and Katz, 1999), in airway surface liquid the
level is about 0.008% (0.4 mM) (Baker and Baines, 2018), and in cerebrospinal fluid
the level is approximately 0.06-0.07% (3.3 mM - 3.8 mM) (Seehusen et al., 2003).
Until quite recently, the impact of acetate utilization on the fitness and
pathogenicity of fungal pathogens has rarely been studied. Ries et al. (Ries et al.,
2021) have investigated the role of acetate utilization in shaping the virulence
phenotypes of the human fungal pathogen Aspergillus fumigatus, an ascomycete,
and observed that the transcription factor FacB is a master regulator of acetate
utilization, and acetate utilization is also under carbon catabolite repression in the
presence of glucose. The authors also examined secreted secondary metabolites in
A. fumigatus under low glucose (0.1% w/v) and acetate (0.1% w/v and 1% w/v)
conditions and the level of secretion of secondary metabolites, which might be
36
essential for growth and survival of the fungus, is presumably influenced by the
available carbon sources and carbon starvation in host environments (Ries et al.,
2021). Additionally, growth on acetate appears to modify the cell wall composition in
A. fumigatus by increasing the concentration of chitin and the structural
polysaccharide β-1-3-glucan, and decreasing the concentration of the cementing
polysaccharide α-1-3-glucan, which is supposed to have notable consequences for
the fungal survival owing to the importance of the cell wall composition in
conferring resistance to oxidative, antifungal, and neutrophil-induced stresses (Ries
et al., 2021).
In ascomycete genus Paracoccidioides, proteomic changes with respect to
acetate utilization have also been studied. The study included one isolate of
Paracoccidioides lutzii and three isolates of Paracoccidioides brasiliensis, and
observed that utilization of acetate as the sole carbon source results in metabolic
reprogramming through induction of proteins in the pathways such as
gluconeogenesis, the glyoxylate cycle, and degradation of amino acids (Baeza et al.,
2017). Moreover, depending on the isolates, induction of the tricarboxylic acid (TCA)
cycle, the electron transport chain (ETC), ethanol production, reassimilation of cell
wall components for gluconeogenesis, β-oxidation, and the methylcitrate cycle were
also observed (Baeza et al., 2017).
Similarly, gluconeogenesis and the glyoxylate cycle a were upregulated in
transcriptomic study of the acetate-grown ascomycete pathogen Candida glabrata.
Other pathways were downregulated, including the biosynthesis of certain amino
acids and the pentose phosphate pathway (PPP) (Chew et al., 2021). The same study
also performed proteomic experiments and reported upregulation of oxidative
37
phosphorylation related proteins in acetate-grown cells compared to glucose-grown
ones, suggesting the involvement of an active electron transport chain (ETC) for ATP
generation in the acetate-grown cells under glucose-deprived conditions (Chew et
al., 2021).
By contrast, the impact of acetate utilization on the expression and
elaboration of virulence phenotypes has not yet been studied in the fungal
pathogen C. neoformans. C. neoformans is an aerobic, basidiomycete yeast with a
worldwide distribution (Bahn et al., 2020). Cryptococcus spp. are found in diverse
ecological niches, with a particular abundance in bird guano (Springer et al., 2017).
The medical relevance of C. neoformans involves its opportunistic pathogenicity in
immunocompromised people; inhaled fungal propagules from the environment
might not be cleared by the immune system, and may lead to a systemic infection
following hematogenous dissemination (May et al., 2016). In addition to organs such
as skin, eyes, prostrate, and bones, major sites of infection by C. neoformans are
lungs and the central nervous system (CNS) (Maziarz and Perfect, 2016), where an
infection of the CNS leads to a lethal meningoencephalitis in humans.
At various stages of cryptococcal infection, yeast cells can act as both an
intracellular and an extracellular parasite (Feldmesser et al., 2000), which implies
that C. neoformans is highly capable of rapid metabolic adaptations within the host
body to cope with the changing nutrient landscape depending on the types of
tissue or cell under invasion. For example, previous gene expression studies of
cryptococcal cells that are phagocytosed revealed an indication of nutrient-limited
and stressful environments within the phagocytic cells (Kronstad et al., 2012).
Additionally, transcriptomic profiles of C. neoformans isolated from a murine
38
pulmonary infection model compared to those grown under standard in vitro
condition showed an elevated expression of genes involved in the glyoxylate
pathway, gluconeogenesis, β-oxidation, amino acid biosynthesis and acetyl-CoA
utilization (Hu et al., 2008), implying that utilization of the alternative carbon sources
such as lactate and acetate are likely to be important early in the establishment of
pulmonary infections (Price et al., 2011). Similarly, transcriptional profiles of C.
neoformans isolated from cerebrospinal fluid (CSF) of human patients showed
elevated expression of genes involved in cell wall metabolism, extracellular transport,
fatty acids and glucose metabolism, iron regulation, pH response, pentose
phosphate pathway, and translational machineries (Yu et al., 2021). These
transcriptional studies have drawn our attention to the possible connection between
a reliance on acetate utilization for survival in the course of disease progression by C.
neoformans and the subsequent and/or concurrent expression and elaboration of
virulence phenotypes in this pathogen.
Similar to the glucose concentrations mentioned above, concentration of
acetate in the human body varies from tissue to tissue. For example, the plasma
concentration of acetate in humans is estimated to be around 0.0003% - 0.0012%
(0.05 mM - 0.2 mM), which may rise above 0.003% (0.5 mM) following alcohol
ingestion (Bose et al., 2019); and in vaginal lumen the estimation is 0.02% - 0.05% (3.4
mM - 8.5 mM) (Owen and Katz, 1999). The aim of this study is to investigate the
effects of acetate utilization on the global transcription of C. neoformans genome.
Here, we report for the first time the changes in the transcriptome of C. neoformans
attributable to the differential utilization of either acetate or glucose as the sole
carbon source.
39
2.2. RESULT AND DISCUSSION
We grew C. neoformans in the presence of either glucose or acetate as the
sole carbon source to observe the effect of these carbon sources on the global gene
expression in this pathogenic fungus. We identified and quantified the expression of
a total of 6,967 genes, of which 282 were upregulated and 274 were downregulated
in acetate-grown cells compared to glucose-grown cells (Summary of the
differentially expressed genes, DEGs can be found in Figure 2.1 (A) and (B);
expression pattern of DEGs across all conditions and replicates is represented in
Figure 2.1 (C) as a heatmap). Owing to the incompleteness of the functional
annotation of C. neoformans genome (nearly 42% of the total protein coding genes
lacks functional annotation), we used a stringent cutoff value for GO enrichment
analysis (using the built-in tools in the FungiDB database for the “Biological Process”
terms): p-value < 0.01 and FDR < 0.05. Table 2.1 lists GO term enrichment of
upregulated genes, and Table 2.2 lists GO terms enrichment of downregulated
genes. At any rate, we are in favor of a conservative interpretation of GO enrichment
analysis for C. neoformans genome owing to its poor functional annotation status, as
statistical tests such as Fisher’s exact test employed in enrichment analysis loses
explanatory power for incompletely annotated genomes.
Moreover, we analyzed the differentially expressed gene (DEG) set for
enrichment in KEGG pathways (using built-in tools in the FungiDB database: p-value
< 0.01 and FDR < 0.05). Table 2.3 lists KEGG pathway enrichment of upregulated
genes, and Table 2.4 lists KEGG pathway enrichment of the downregulated genes.
40
2.2.1. Chromosomal distribution of differentially expressed genes
Histone acetylation is known to be sensitive to the intracellular availability of
acetyl-CoA (Pietrocola et al., 2015). It is plausible that acetate-grown cells likely
experience a global reshuffling of nuclear organization through histone acetylation,
thereby adding nuances to the transcriptional control of acetate utilization. A
cursory analysis of chromosomal distribution of differentially regulated genes
(normalized as proportion) suggests a possible association between a gene’s
regulation status (up-, down-, or non-regulated) and the chromosome it is located in
(Figure 2.2). However, a Chi-square test of independence (14 × 3 contingency, df = 26)
does not point to any significant association (p = 0.071, Cramer’s V = 0.052) between
regulation status and chromosome position. This might be due to the violation of a
tacit assumption of the contingency analysis (i.e., observations are independent of
one another) by the commonplace trans-activation of non-cis-chromosomal genes
during global transcription.
However, in this study, we did not attempt to confirm such epigenetic
regulation of differential gene expression. Moreover, one of the upregulated genes,
cytochrome c oxidase subunit III (CNAG_09004), is on the mitochondrial genome.
Interestingly, expression of no other mitochondrial gene was impacted by the choice
of nutrient source.
2.2.2. Differential expression of putative transcription factors
To investigate the role of transcription factors in differential regulation of
nutrient-utilization genes, we searched the C. neoformans genome for potential
transcription factors (TF). For this study, we used the deep neural network-based tool
41
DeepTFactor (Kim et al., 2021) which resulted in the prediction of 200 TFs (Table A2).
None of these predicted and known TFs were found to be present in the set of
downregulated genes; however, a total of 15 predicted and known TFs were
upregulated in acetate-grown cells, suggesting their potential involvement in
acetate-utilization, stress-response, and/or glucose-deprivation. The list of
upregulated TFs comprises 4 previously unknown TFs predicted in this study
(CNAG_02525, CNAG_04618, CNAG_01847, CNAG_05990), as well as 11 TFs previously
described elsewhere ( (Jung et al., 2015); (Lee et al., 2020)): CCD4 (CNAG_03279),
FZC15 (CNAG_06188), FZC31 (CNAG_03741), FZC34 (CNAG_00896), YRM103
(CNAG_04093), STB4 (CNAG_05785), LIV3 (CNAG_05835), BZP4 (CNAG_03346),
ARO8001 (CNAG_04345), MLN1 (CNAG_04837), GAT201 (CNAG_01551).
2.2.3. Downregulation of genes in acetate-grown conditions
Many downregulated genes are enriched in KEGG pathways such as
glycolysis/ gluconeogenesis; pentose phosphate pathway; glycine, serine and
threonine metabolism; and steroid biosynthesis; pentose and glucuronate
interconversions; metabolism of xenobiotics; tyrosine metabolism among many
others (Table 2.4). About 40% (30/74) downregulated genes have unknown
functions (‘hypothetical proteins’). Two prominent genes of central carbon
metabolism, fructose bis-phosphate aldolase 1 (FBA1; CNAG_06770) and pyruvate
decarboxylase (PDC1; CNAG_04659), were downregulated, whereas expression of
other glycolytic/ gluconeogenic genes were not significantly impacted by glucose
uptake and metabolism. Notably, FBA1 is an essential gene for survival in C.
42
neoformans; however, by contrast, targeted mutagenesis experiment shows that
PDC1 is not essential for its viability (Ianiri and Idnurm, 2015).
Pentose phosphate pathway genes such as 6-phosphogluconate
dehydrogenase (CNAG_07561) and a transketolase (CNAG_07445), were also
downregulated in acetate-grown cells. Other genes with putative roles in carbon
metabolism found in our set of downregulated genes include xylulose-5-
phsophate/fructose-6-phosphate phosphoketolase (XFP2; CNAG_06923),
phosphoglycerate dehydrogenase (CNAG_00774), mannitol dehydrogenase
(CNAG_00515), enoyl reductase (CNAG_04652), endo-1,3(4)-beta-glucanase
(CNAG_05458), cellulase (CNAG_00799), aldo-keto reductase (CNAG_01954), and an
NADP-alcohol dehydrogenase (CNAG_01896). However, no TCA cycle gene was
downregulated.
Putative transporters such as CNAG_01960 (efflux protein), CNAG_03438
(HXT1), CNAG_06377 (Solute carrier 25, mitochondrial phosphate transporter),
CNAG_00749 (alternative sulfate transporter) were upregulated in glucose-grown
cells (i.e. downregulated in acetate-grown cells), suggesting their involvement in
glucose uptake and/or secretion of metabolites.
Additionally, cells grown on acetate showed decreased expression of
ribosomal protein genes such as CNAG_06447 (L17), CNAG_02928 (L5e), CNAG_03577
(LP0), CNAG_04114 (S0), CNAG_02754 (S12e). Also downregulated is a chaperone
regulator (CNAG_05252) and conidiation-specific protein 6 (CNAG_03759), a homolog
of a conserved fungal gene involved in sporulation in Neurospora crassa (Wang et
al., 2012). One cyclophilin A homolog, CPA2 (CNAG_03621), which is known to be
43
important in normal growth but plays an ancillary role in virulence was
downregulated in acetate-grown cells (Wang et al., 2001).
Other noteworthy downregulated genes with known functions include a thiol
peroxidase (TSA1; CNAG_03482), known to be important for growth at physiological
temperature (Missall et al., 2004); aspartate kinase (CNAG_04347), an essential kinase
involved in threonine biosynthesis (Kingsbury and McCusker, 2008); and a putative
calcineurin substrate, HAD-like hydrolase (CNAG_01744), required for cell wall
integrity, virulence, and growth at physiological temperatures (Jung et al., 2018).
2.2.4. Genes for acetate-metabolism, the glyoxylate cycle, and
peroxisomal activities were upregulated.
Almost 35% (98/282) of the upregulated genes in acetate-grown cells are
functionally uncharacterized (‘hypothetical proteins’), while many other genes are
enriched in KEGG pathways such as valine, leucine and isoleucine degradation; fatty
acid degradation; propanoate and butanoate metabolism; β-alanine metabolism;
lysine degradation; fatty acid elongation; pyruvate metabolism; glyoxylate and
dicarboxylate metabolism among many others (Table 2.3).
Increased acetyl-CoA production and utilization in acetate-grown cells was
suggested by the higher expression of acetyl CoA synthetase (ACS; CNAG_00797),
and two acetyl CoA acyltransferases (CNAG_00490 and CNAG_00524). Three
carnitine O-acetyltransferases (CNAG_00537, CNAG_06551, and CNAG_05042) also
had increased expression in acetate-grown cells compared to glucose-grown ones;
however, information regarding their cellular localization has not been reported in
the literature. In connection to acetate assimilation, the TCA and the glyoxylate
44
cycles play an important role: two glyoxylate cycle enzymes, isocitrate lyase (ICL1,
CNAG_05303) and malate synthase A (MLS1, CNAG_05653), and a putative TCA cycle
enzyme malate dehydrogenase (MDH; CNAG_06374), were found to be upregulated
in acetate-grown cells. Interestingly, although C. neoformans is known to be a
Crabtree negative yeast (Ries et al., 2018), a homolog of fumarate reductase
(CNAG_07862), an enzyme essential for maintaining redox balance during anaerobic
growth in S. cerevisiae (Camarasa et al., 2007), was found in our upregulated gene-
set.
Peroxisome-related genes such as PEX1 (CNAG_07403), PEX2 (CNAG_00171),
PEX3 (CNAG_02939), PEX5 (CNAG_03729), PEX12 (CNAG_04937), PEX16
(CNAG_02096), one peroxisome targeting signal receptor (CNAG_01926), and two
peroxisomal long-chain fatty acid importers CNAG_00651 and CNAG_02764 (PXA2),
were found in the upregulated gene-set, indicating increased peroxisomal
biogenesis in acetate-grown cells. Interestingly, a gene for a putative NIPSNAP
family protein (CNAG_05310) is upregulated in acetate-grown cells. NIPSNAP
proteins are evolutionarily conserved and known to act as a mitophagy-inducing
signal following their accumulation on the mitochondrial surface (Kumar and
Reichert, 2021). Other interesting genes upregulated in the acetate-grown cells
include CNAG_03051 (polyamine transporter) and CNAG_04794 (spermine
transporter), which are involved in polyamine transport. This observation is
particularly interesting as polyamine metabolism has been linked to cell survival
during stress (Valdés-Santiago and Ruiz-Herrera, 2013).
45
2.2.5. Upregulated genes bear the signature of nutrient-starvation.
The transcriptome of acetate-grown cells in our study bore the signature of
nutrient-starvation as signaled by the upregulation of various catabolic genes. Table
2.5 lists a subset of the upregulated genes grouped by their involvement in various
biological pathways. A similar subset of downregulated genes is compiled in Table
2.6. Table A3 contains the comprehensive list of DEGs found in this study.
Upregulated genes involved in fatty acid catabolism included long chain acyl-
CoA synthetase (CNAG_03019), peroxisomal 2,4-dienoyl-CoA reductase
(CNAG_04238), three acyl-CoA dehydrogenases (CNAG_04688, CNAG_03666,
CNAG_02562), enoyl-CoA hydratase (CNAG_04531), multifunctional beta-oxidation
protein (MFE2; CNAG_05721,), enoyl-CoA hydratase/isomerase (CNAG_03010), 3-
hydroxyacyl-CoA dehydrogenase (HAD1; CNAG_04308), and 3-hydroxybutyryl-CoA
dehydrogenase (HAD2; CNAG_03134).
Genes involved in glucose mobilization and metabolism such as glucan 1,3-
beta-glucosidase (EXG104; CNAG_02225,), glycosyl hydrolase (CNAG_05991), beta-
glucosidase (CNAG_07600), glyceraldehyde-3-phosphate dehydrogenase type I
(CNAG_04523), glycosyl-hydrolase (CNAG_04291), UDP-glucose, sterol transferase
(ATG2602; CNAG_03133), alpha-glucosidase (CNAG_05913), and exo-beta-1,3-
glucanase (CNAG_05803) were also upregulated in acetate-grown cells, suggesting a
possible mechanism of reassimilation of cell wall components and other ancillary
storage molecules in response to glucose-deprived conditions.
A few genes involved in protein and amino acid degradation were also
upregulated in acetate-grown fungi: Gly-Xaa carboxypeptidase (CNAG_03960),
glycine cleavage system T protein (CNAG_02818), L-serine ammonia-lyase
46
(CNAG_06913), sarcosine oxidase (CNAG_05115), aryl-alcohol dehydrogenase
(CNAG_01952), and proline oxidase (CNAG_02049, PUT1). An unusually high number
of genes involved in branched-chain amino acids (BCAA) degradation was found to
be upregulated in acetate-grown cells; these genes include 2-oxoisovalerate
dehydrogenase E1 components (alpha subunit; CNAG_02284, and beta subunit;
CNAG_00397), 2-oxoisovalerate dehydrogenase E2 component (dihydrolipoyl
transacylase: CNAG_00484), isovaleryl-CoA dehydrogenase (CNAG_00452), 3-oxoacid
CoA-transferase (CNAG_05031), 3-methylcrotonyl-CoA carboxylase alpha subunit
(CNAG_01680), and two forms of methylmalonate-semialdehyde dehydrogenase
(acylating) (CNAG_01075 and CNAG_04351). Other upregulated genes involved in
nitrogen and sulfur metabolism were urea carboxylase (CNAG_07944), glutamate
dehydrogenase (CNAG_00879), cystathionine beta-synthase (CNAG_00637),
cystathionine gamma-lyase (CNAG_06448), and two isoforms of 2-nitropropane
dioxygenase (CNAG_05644 and CNAG_03243).
Polyols play an important role as carbohydrate reserves in fungi (Jennings,
1985). As such, a number of genes for transporters and enzymes involved in polyol
metabolism were found to be upregulated in acetate-grown cells. Enzymes of polyol
metabolism that were upregulated include sorbitol dehydrogenase (CNAG_00269),
and two isoforms of L-iditol 2-dehydrogenase (CNAG_05658 and CNAG_06977).
Upregulated transporters include the polyol transporter protein 1 (PTP1;
CNAG_05662), and three myo-inositol transporters: ITR2 (CNAG_00864), ITR3c
(CNAG_05381), and ITR6 (CNAG_03910).
Other enzymes of carbon metabolism that were found to be upregulated
include dihydroxyacetone kinase (DAK101, CNAG_00826), formate dehydrogenase
47
(CNAG_06672), aldehyde dehydrogenase (NAD) (CNAG_06628), acetoacetate-CoA
ligase (CNAG_02045), alcohol dehydrogenase (CNAG_02489), and D-glycerate 3-
kinase (CNAG_07779). Other notable catabolic genes that were also upregulated
include uracil phosphoribosyltransferase (CNAG_02344) from the pyrimidine salvage
pathway, and cytochrome C peroxidase (CNAG_02147), a cytosolic enzyme involved
in peroxide neutralization. The xenobiotic degradation enzyme alpha-ketoglutarate-
dependent 2,4- dichlorophenoxyacetate dioxygenase (CNAG_04417), a taurine
metabolic enzyme taurine catabolism dioxygenase (TAUD, CNAG_01542), and a
hydroxymethylglutaryl-CoA lyase (CNAG_03067) involved in fat and protein
catabolism were also upregulated.
Two enzymes of the pentose phosphate pathways (PPP), transketolase
(CNAG_06172) and ribose 5-phosphate isomerase (CNAG_00827), were also
upregulated in acetate-grown cells. Although the PPP has a major role in
biosynthetic processes and is less likely to be upregulated during nutrient-starvation
(glucose-deprived conditions in our case), it is not surprising to observe upregulation
of certain PPP genes in acetate-grown cells considering the special role PPP plays in
generation of reducing equivalents (Deacon and Deacon, 2006).
Moreover, many putative transporters involved in the uptake of various
nutrient sources were also upregulated in acetate-grown cells. Such transporters are
involved in sugar transport: CNAG_00048, CNAG_03772 (HXS1), CNAG_04931 (HXS2),
CNAG_04092, CNAG_02586, CNAG_05324, CNAG_05867 (L-fucose transporter),
CNAG_01936, CNAG_07874, CNAG_05387 (galactose transporter), CNAG_02733,
CNAG_07641; amino acid transport: CNAG_07367, CNAG_00597 (DIP5), CNAG_05685,
CNAG_07902; nicotinic acid transport: CNAG_00028, CNAG_00598, CNAG_03242,
48
CNAG_06942, CNAG_04536, CNAG_04795, CNAG_06204; organic acids and alcohols
transport: CNAG_04038 (quinate), CNAG_06817 (uric acid xanthine permease, UAP1),
CNAG_02254 (quinate, LPI12), CNAG_06561 (allantoate), CNAG_04142 (tartrate),
CNAG_04704 (JEN4; lactate), CNAG_05119 (gamma-aminobutyric acid); and
phosphate transport: CNAG_02777 (PHO84). Other upregulated genes with putative
transporter function include CNAG_00796 (MDR1), CNAG_05718, CNAG_00284,
CNAG_00499, CNAG_05300, CNAG_00904, CNAG_02288, CNAG_03242; as well as
alpha-glucoside: H+ symporters CNAG_05330, CNAG_06527, CNAG_05914,
CNAG_05929, CNAG_06259.
2.2.6. Gluconeogenesis and certain anabolic pathways are also
upregulated.
In addition to two glyoxylate cycle enzymes (see above), the gluconeogenesis-
defining enzyme, phosphoenolpyruvate carboxykinase (PCK1, CNAG_04217), was
upregulated in acetate-grown cells. Fungal cells grown on acetate as the sole carbon
source depend on gluconeogenesis for production of sugars that are eventually
utilized in synthesis of cell wall, nucleotides, storage molecules and many other
biosynthetic processes (Deacon and Deacon, 2006). Additionally, certain genes
involved in glycogen and fatty acid synthesis were also upregulated in acetate-
grown cells. Upregulated glycogen synthesis genes include 1,4-alpha-glucan-
branching enzyme (CNAG_00393), glycogen synthase (CNAG_04621), and glycogenin
glucosyltransferase (CNAG_05293). Other upregulated fatty acid synthesis genes
include acetyl/propionyl CoA carboxylase (CNAG_01671) and beta-ketoacyl reductase
(CNAG_01116).
49
Other noteworthy anabolic enzymes include the proline synthesis pathway
gene ornithine-oxo-acid transaminase (CNAG_05134), a putative tetrahydrobiopterin
synthesis pathway gene cyclohydrolase (CNAG_06009), and a ferroxidase (CFO1,
CNAG_06241) involved in iron uptake.
One particularly interesting observation was that two lysine biosynthesis
genes, saccharopine reductase (LYS9, CNAG_00247) and dihydrodipicolinate
synthase (CNAG_04346), were upregulated. Saccharopine reductase [saccharopine
dehydrogenase (NADP+, L-glutamate forming)] catalyzes the formation of the
penultimate intermediate of lysine biosynthesis through the α-aminoadipate (AAA)
pathway and was observed to be a part of chimera with spermidine synthase (SPE3)
in C. neoformans (Kingsbury et al., 2004). On the other hand, dihydrodipicolinate
synthase catalyzes the first committed step of lysine biosynthesis via the
diaminopimelate (DAP) pathway, which is found in bacteria, lower fungi, and plants
and is believed to be absent in ascomycetes and basidiomycetes fungi (Zabriskie
and Jackson, 2000). Presence of a dihydrodipicolinate synthase (DHDPS) gene in
many fungal genomes, including C. neoformans, appears to contradict the claim
that AAA, and not DAP, is the only route of lysine production in higher fungi.
However, recently, Desbois and colleagues (Desbois et al., 2018) have shown that
DHDPS from another pathogenic fungus Coccidioides immitis, does not possess the
said function, rather has the 2-keto-3-deoxygluconate aldolase (KDGA) and 4-
hydroxy-2-oxoglutarate aldolase (HOGA) activities, suggesting an instance of
functional misannotation.
50
2.2.7. Some upregulated genes are implicated in virulence and
pathogenesis.
Recent studies in cryptococcal virulence mechanisms and pathogenesis have
shed light on three primary processes: adaptation to host environment, evasion of
the immune system, and production of virulence factors (Zaragoza, 2019). While our
study was aimed at learning adaptive mechanisms in Cryptococcus at transcription
level in response to low-glucose and high-acetate environments, we have found that
a handful of genes with connection to virulence and pathogenesis were
upregulated. Table 2.7 lists the virulence-associated genes that were found to be
significantly upregulated in this study.
2.3. CONCLUSION
For a fungus like Cryptococcus neoformans, invasion of and mobilization to
different organ systems within a multicellular host offers a range of challenges in
addition to the ones offered by the immune system of the host. Nutrient challenge is
one of those challenges as different organs differ in nutrient profiles in terms of
availability of various nutritional resources. Prior to this study we assumed that such
a flexibility in the metabolic response to alternative carbon source is existent in
Cryptococcus spp. and should be reflected in the transcriptional profiles. Hence, we
set out to seek for such transcription-level modifications through capturing a
snapshot of global transcriptomic landscape of C. neoformans during the growth in
quasi-starvation (YNB w/o amino acids) environments with either glucose or acetate
as sole carbon source. To magnify any subtle transcription-level modifications, we
have deliberately chosen the media concentration of the carbon sources to be high
51
[compare the concentrations used in this study for glucose (2% w/v or 111 mM) and
acetate (2% w/v or 339 mM) with those of glucose (5.5 mM) and acetate (0.2 mM) in
human blood]. Additionally, under the assumption that, in glucose-limited
environments C. neoformans will prioritize the uptake and utilization of available
alternative carbon sources, acetate appears to be a straightforward candidate for
alternative carbon source to study owing to its abundance in various Cryptococcus-
infected tissues, as well as its direct assimilation in energy metabolism pathway
through conversion to acetyl-CoA that sits at the crossroad of anabolism and
catabolism: i.e. its overall physiological relevance. Our data suggests, as
hypothesized, C. neoformans does show variation in transcriptome in response to
variation of carbon source, and such differential regulation accounts for about 5% of
the genome.
Not surprisingly, the transcriptome of acetate-grown cells shows
characteristic signature of starvation via upregulation of genes involved in certain
catabolic pathways to readily supply the fuel, and certain other anabolic pathways
for synthesis of sugar and energy storage molecules. In addition to that, pathways
involved in the maintenance and construction of cellular structures appear to have
decelerated in acetate-only conditions. None of these seems unusual within the
context of growth in a glucose-limited environment. However, many genes, hitherto
reported to have implications in cryptococcal virulence and pathogenicity in various
host models, are also upregulated in acetate-grown cells. This suggests a possible
connection between acetate utilization and induction of pathogenicity-associated
phenotypes. Modulation of fungal phenotypes to shape the disease outcome of the
52
host, mediated by acetate utilization, has already been observed, quite recently, in
another human pathogenic fungus, Aspergillus fumigatus (Ries et al., 2021).
The mechanism through which acetate utilization can accentuate pathogenic
phenotypes of C. neoformans is not clear; however, we would like to propose
modularity as a plausible mechanism in this context. It is possible that the regulatory
circuitry evolved in C. neoformans to deal with acetate as a carbon source might
have also incorporated virulence-associated genes, and such regulatory circuitries
are modular in structure (e.g., modules as observed in various transcriptional
regulatory networks or TRNs).
This speculation is consistent with the idea that C. neoformans’ capacity for
virulence is accidental (Fu et al., 2021)— an exaptation (Naranjo-Ortiz and Gabaldón,
2019)— and has evolved under the selection pressure from amoebic predation in soil
(Casadevall et al., 2019). Since adaptation through natural selection can occur only in
response to ever-present or ever-recurring features of the surroundings (Garvey,
2007), this hypothesis can be extended to incorporate other naturally occurring
stresses imposed on the soil-dwelling fungi by their physical surroundings.
It is more likely that these physical stresses— including, but not limited to,
oxidative, osmotic, thermal, radiational, and nutritional stresses— come in a bundle
as a recurring geographic and seasonal feature of the soil-dwelling fungi’s
surroundings. Most of the pathogenicity-associated phenotypes are, probably not
surprisingly, found to be correlated. For example, growth at physiological
temperature (thermal stress), otherwise considered too high for environmental
fungi, is also associated with antioxidant response (oxidative stress), trehalose
accumulation (osmotic stress), and others (Zaragoza, 2019). Similarly, some virulence
53
factors, such as capsule and melanin production, can even provide the fungi with
actual physical protection in the wild (Naranjo-Ortiz and Gabaldón, 2019). This
implies that a more efficient way for adaptation to occur would be for C. neoformans
to evolve a blanket-defense against the recurring bundle of environmental stresses.
This may explain why most of those defense- (and, in the context of immunology,
‘virulence’)-related phenotypes are expressed simultaneously in C. neoformans.
In our study, we found transcriptomic hints of simultaneous activation of
various stress responses (e.g., starvation avoidance, maintenance of redox balance,
synthesis and uptake of compounds with osmotic potential), as well as changes in
expression of a few pathogenicity-associated genes with seemingly no involvement
in acetate utilization. Here, we propose to explain this observation by the partial
activation of blanket defense against environmental stressor-bundle (that we posit
to be extant in C. neoformans owing to natural selection) through pleiotropic effect
of the regulatory network involved in acetate utilization. While this hypothesis will
require further experimental validation, this framework holds a potential for
understanding cryptococcal pathogenicity in a different way. For example, if the
blanket defense mechanism is at work, then developing drugs that target only one
virulence factor at a time may soon lead to drug resistance.
54
2.4. METHODS AND MATERIALS
2.4.1. RNA isolation and sequencing
Cryptococcus neoformans var grubii KN99 cells were grown on Yeast
Nitrogen Base without amino acids (YNB w/o aa) with 2% (w/v) glucose, overnight, to
a density of ~1.5 x 108 cells/mL. These starting cultures were washed with the pre-
warmed media accordingly and were transferred to either YNB without amino acids
with 2% (w/v) acetate (acetate-grown) or YNB without amino acids with 2% (w/v)
glucose (glucose-grown) media for incubation on a rotor shaker for 5 hours.
Following incubation on experimental growth conditions, cells were harvested and
flash-frozen in liquid nitrogen. Total RNA was isolated (three biological replicates per
condition) using RiboPureTM RNA Purification Kit, yeast (InvitrogenTM, Thermo Fisher
Scientific, MA, USA) following manufacturer’s instruction. RNA-seq was performed on
an Illumina Hiseq 2500 sequencing platform using NEBNext® Ultra™ RNA Library
Prep Kit for Illumina (NEB, USA). Prior to sequencing, the index-coded samples were
clustered in a cBot Cluster Generation System using PE Cluster Kit cBot-HS
(Illumina). Library construction, quality control and sequencing were outsourced to
Novogene Co., Ltd, China. Sequencing generated an average of 57,360,137 raw and
54,766,198 clean reads (Table A1).
55
2.4.2. Transcriptomic analysis
Quality of the raw reads were checked using FastQC (v-0.11.7). Raw reads were
aligned against the C. neoformans KN99 genomes (fasta and GFF3) from FungiDB
48th release. STAR (v-2.7.5), Bowtie2 (v-2.3.5.1), and RSEM (v-1.3.3) were used for
alignment, mapping, and counting of the reads. Differential expression analysis was
conducted on the RSEM-generated transcript counts using R package DESeq2.
Figures for data visualization were prepared in RStudio (v-1.4). For ease and
consistency, final functional annotation of the KN99 strain’s transcripts were based
on the corresponding CNAG-IDs of the genome of C. neoformans H99 strain.
56
TABLES
Table 2.1. GO term enrichment of upregulated genes
Total
gene Upregulated Fold
GO term ID Biological process count gene count enrichment FDR
GO:0055085 transmembrane transport 350 57 3.88 9.7E-18
GO:0006810 transport 649 59 2.16 4.4E-07
GO:0051234 establishment of localization 654 59 2.15 4.4E-07
GO:0051179 localization 674 59 2.08 1.1E-06
GO:0007031 peroxisome organization 9 6 15.87 3.4E-05
GO:0008643 carbohydrate transport 23 8 8.28 1.7E-04
GO:0055114 obsolete oxidation-reduction process 335 33 2.35 1.7E-04
GO:0016054 organic acid catabolic process 24 8 7.94 1.7E-04
GO:0046395 carboxylic acid catabolic process 24 8 7.94 1.7E-04
GO:0044282 small molecule catabolic process 43 10 5.54 3.5E-04
GO:0009063 cellular amino acid catabolic process 19 5 6.27 3.6E-02
GO:0032787 monocarboxylic acid metabolic process 51 8 3.73 4.2E-02
GO:0072329 monocarboxylic acid catabolic process 6 3 11.9 4.3E-02
GO:0034440 lipid oxidation 2 2 23.81 4.3E-02
GO:0019395 fatty acid oxidation 2 2 23.81 4.3E-02
GO:0009062 fatty acid catabolic process 2 2 23.81 4.3E-02
GO:0006635 fatty acid beta-oxidation 2 2 23.81 4.3E-02
GO:0005978 glycogen biosynthetic process 2 2 23.81 4.3E-02
GO:0043436 oxoacid metabolic process 204 18 2.1 4.6E-02
GO:0019752 carboxylic acid metabolic process 204 18 2.1 4.6E-02
GO:0006631 fatty acid metabolic process 14 4 6.8 4.6E-02
GO:0006082 organic acid metabolic process 207 18 2.07 4.9E-02
57
Table 2.2. GO term enrichment of downregulated genes
Total
gene Downregulated Fold
GO term ID Biological process count gene count enrichment FDR
obsolete oxidation-reduction
GO:0055114 process 335 14 4.55 0.00026
GO:0019752 carboxylic acid metabolic process 204 8 4.27 0.03472
GO:0043436 oxoacid metabolic process 204 8 4.27 0.03472
GO:0006082 organic acid metabolic process 207 8 4.21 0.03472
GO:0008152 metabolic process 2044 30 1.6 0.03969
GO:0046394 carboxylic acid biosynthetic process 85 5 6.41 0.03969
GO:0044283 small molecule biosynthetic process 128 6 5.11 0.03969
GO:0016053 organic acid biosynthetic process 90 5 6.05 0.04442
GO:0005975 carbohydrate metabolic process 140 6 4.67 0.04916
58
Table 2.3. KEGG pathway enrichment of upregulated genes
Total gene Upregulated Fold

KEGG ID KEGG pathway count gene count enrichment FDR
Valine, leucine and isoleucine
ec00280 degradation 61 23 7.51 8.1E-14
ec00071 Fatty acid degradation 78 22 5.62 2.0E-10
ec00281 Geraniol degradation 30 14 9.3 7.8E-10
ec00640 Propanoate metabolism 109 24 4.39 2.9E-09
ec00650 Butanoate metabolism 110 24 4.35 2.9E-09
ec00410 beta-Alanine metabolism 49 15 6.1 8.7E-08
ec00930 Caprolactam degradation 52 12 4.6 9.2E-05

Carbon fixation pathways in
ec00720 prokaryotes 91 15 3.28 4.0E-04
ec00310 Lysine degradation 94 15 3.18 4.8E-04

alpha-Linolenic acid
ec00592 metabolism 94 15 3.18 4.8E-04
ec00062 Fatty acid elongation 23 7 6.06 9.7E-04
ec00620 Pyruvate metabolism 113 16 2.82 9.9E-04
ec00362 Benzoate degradation 81 13 3.2 1.2E-03

Glyoxylate and dicarboxylate
ec00630 metabolism 72 12 3.32 1.5E-03
59
Table 2.4. KEGG pathway enrichment of downregulated genes
Total gene Downregulated Fold

KEGG ID KEGG pathway count gene count enrichment FDR
ec00592 alpha-Linolenic acid metabolism 94 8 5.8 0.0032

Glycine, serine and threonine
ec00260 metabolism 109 8 5 0.0047
ec00930 Caprolactam degradation 52 5 6.56 0.0238
ec00010 Glycolysis / Gluconeogenesis 126 7 3.79 0.0327

Metabolism of xenobiotics by
ec00980 cytochrome P450 65 5 5.25 0.0327
Carbon fixation in photosynthetic
ec00710 organisms 40 4 6.82 0.0327
ec00830 Retinol metabolism 104 6 3.93 0.0327
ec00626 Naphthalene degradation 106 6 3.86 0.0327
ec00561 Glycerolipid metabolism 73 5 4.67 0.0327
ec00350 Tyrosine metabolism 187 8 2.92 0.0327
ec00902 Monoterpenoid biosynthesis 7 2 19.48 0.0327

Pentose and glucuronate
ec00040 interconversions 77 5 4.43 0.0327
ec00071 Fatty acid degradation 78 5 4.37 0.0327
ec00620 Pyruvate metabolism 113 6 3.62 0.0327
ec00030 Pentose phosphate pathway 80 5 4.26 0.0332

Drug metabolism - cytochrome
ec00982 P450 87 5 3.92 0.0446
ec00100 Steroid biosynthesis 30 3 6.82 0.0499
60
Table 2.5. List of upregulated genes in acetate-grown C. neoformans compared to
glucose-grown cells
Gene Name log2 Fold-

Gene ID Gene annotation or Symbol Chromosome Change FDR
Acetyl-CoA metabolism
CNAG_00490 acetyl-CoA acyltransferase N/A 1 4.150523898 0
CNAG_00524 acetyl-CoA acyltransferase 2 N/A 1 2.230319617 9.46E-135
CNAG_00537 Carnitine O-acetyltransferase N/A 1 5.18957041 0
CNAG_00797 acetyl-CoA synthetase N/A 1 2.593962989 1.06E-135
CNAG_05042 carnitine acetyltransferase N/A 4 2.339243859 9.32E-136
Branched-chain amino acid degradation pathway

2-oxoisovalerate dehydrogenase E1
CNAG_00397 component, beta subunit N/A 1 3.327788507 4.05E-204
CNAG_00452 Isovaleryl-CoA dehydrogenase N/A 1 4.381863876 3.22E-133

component (dihydrolipoyl
CNAG_00484 transacylase) N/A 1 2.659748528 8.88E-180
methylmalonate-semialdehyde
CNAG_01075 dehydrogenase (acylating) N/A 5 2.139409989 9.16E-54
3-methylcrotonyl-CoA carboxylase
CNAG_01680 alpha subunit N/A 11 4.653411548 0
CNAG_02284 component, alpha subunit N/A 6 2.059866905 8.43E-78
CNAG_05031 3-oxoacid CoA-transferase N/A 4 3.444662639 0
Cell cycle
CNAG_00442 cyclin N/A 1 2.465150614 1.41E-123
Fatty acid catabolism
CNAG_02562 acyl-CoA dehydrogenase N/A 3 3.63243594 1.89E-266
61
CNAG_03010 enoyl-CoA hydratase/isomerase N/A 3 4.251823055 0
CNAG_03019 long-chain acyl-CoA synthetase N/A 3 3.790271235 0
CNAG_03134 3-hydroxybutyryl-CoA dehydrogenase HAD2 8 2.006338715 1.23E-53
CNAG_03666 acyl-CoA dehydrogenase N/A 2 4.942071419 0

peroxisomal 2,4-dienoyl-CoA
CNAG_04238 reductase N/A 9 3.041104459 6.05E-230
CNAG_04308 3-hydroxyacyl-CoA dehydrogenase HAD1 9 2.103182554 9.28E-98
CNAG_04531 Enoyl-CoA hydratase N/A 9 2.432157388 2.46E-129

multifunctional beta-oxidation
CNAG_05721 protein MFE2 7 4.022808323 0
Fatty acid synthesis
CNAG_01116 beta-ketoacyl reductase N/A 5 2.767779821 4.50E-79
CNAG_01671 acetyl/propionyl CoA carboxylase N/A 11 3.563038208 0
Protein catabolism
CNAG_03067 hydroxymethylglutaryl-CoA lyase N/A 3 3.176153974 2.07E-274
Gluconeogenesis
phosphoenolpyruvate carboxykinase
CNAG_04217 (ATP) PCK1 9 3.26714125 6.60E-163
Glucose mobilization
CNAG_02225 glucan 1,3-beta-glucosidase EXG104 6 3.371935116 2.52E-249
CNAG_03133 UDP-glucose,sterol transferase ATG2602 8 2.111181869 4.92E-101
CNAG_04291 glycosyl-hydrolase N/A 9 2.44488372 2.31E-117

glyceraldehyde-3-phosphate
CNAG_04523 dehydrogenase, type I N/A 9 2.281267594 9.36E-153
CNAG_05803 exo-beta-1,3-glucanase N/A 7 2.501116431 3.85E-120
CNAG_05913 Alpha-glucosidase N/A 7 4.759365762 0
CNAG_05991 glycosyl hydrolase family 88 N/A 12 2.774307079 2.36E-176
CNAG_07600 Beta-glucosidase N/A 11 2.565018508 5.55E-137
Glycogen synthesis
CNAG_00393 1,4-alpha-glucan-branching enzyme N/A 1 2.535777799 2.86E-184
CNAG_04621 glycogen(starch) synthase, variant N/A 10 2.940035403 5.84E-184
CNAG_05293 glycogenin glucosyltransferase N/A 4 2.353347363 2.03E-55
62
Glyoxylate cycle
CNAG_05303 isocitrate lyase ICL1 4 6.900794065 0
CNAG_05653 malate synthase A MLS1 14 4.950928228 0
Iron uptake
CNAG_06241 acidic laccase CFO1 12 2.22727825 7.13E-83
Lysine synthesis
alpha-aminoadipic semialdehyde
CNAG_00247 synthase LYS9 1 4.647012781 0
CNAG_04346 dihydrodipicolinate synthase N/A 9 2.238276473 7.54E-60
Carbon metabolism
CNAG_00826 dihydroxyacetone kinase DAK101 1 3.981063756 0
CNAG_02045 acetoacetate-CoA ligase N/A 6 4.784163223 0

alcohol dehydrogenase, propanol-
CNAG_02489 preferring N/A 6 6.655716374 0
CNAG_06628 aldehyde dehydrogenase (NAD) N/A 7 3.554003489 2.07E-269
CNAG_06672 formate dehydrogenase N/A 7 2.019028288 5.82E-38
CNAG_07779 D-glycerate 3-kinase TDA10 9 2.205650117 5.10E-43
Mitophagy
CNAG_05310 nipsnap family protein N/A 4 5.003188347 1.07E-195
Nitrogen and Sulfur metabolism
CNAG_00637 Cystathionine beta-synthase N/A 1 3.059496886 5.75E-290
CNAG_00879 Glutamate dehydrogenase N/A 5 5.152721407 0
CNAG_03243 2-Nitropropane dioxygenase N/A 8 4.194067019 2.51E-143
CNAG_05644 2-Nitropropane dioxygenase N/A 14 3.729505188 0
CNAG_06448 Cystathionine gamma-lyase N/A 13 2.437034447 7.63E-64
CNAG_07944 Urea carboxylase N/A 13 3.394992027 0
Peroxide neutralization
CNAG_02147 cytochrome c peroxidase N/A 6 4.190300827 4.20E-208
Peroxisomal organization and transport
CNAG_00171 peroxin-2 N/A 1 2.002408397 2.54E-89

ATP-binding cassette, subfamily D
(ALD), peroxisomal long-chain fatty
CNAG_00651 acid import protein N/A 1 3.10426851 0
63
CNAG_01926 peroxisome targeting signal receptor PEX5 11 2.424142535 1.99E-146
CNAG_02096 peroxin-16 N/A 6 2.367732307 1.17E-77

ATP-binding cassette, subfamily D
(ALD), peroxisomal long-chain fatty
CNAG_02764 acid import protein PXA2 3 2.582242246 5.34E-143
CNAG_02939 peroxin-3 N/A 3 2.206201302 1.33E-121
CNAG_03729 peroxin-5 N/A 2 2.91316122 3.71E-263
CNAG_04937 peroxin-12 N/A 4 2.199480608 8.13E-88
CNAG_07403 Peroxisomal ATPase PEX1 PEX1 5 2.50683658 9.57E-158
Phagocytosis resistance
CNAG_06574 antiphagocytic protein 1 APP1 7 3.088293978 3.40E-122
Polyamine metabolism
CNAG_03051 polyamine transporter N/A 3 2.098521697 2.80E-74
CNAG_04794 spermine transporter N/A 10 2.584536287 1.19E-135
Polyol metabolism
CNAG_00269 Sorbitol dehydrogenase N/A 1 2.472085629 7.34E-54
CNAG_00864 myo-inositol transporter, putative ITR2 5 3.035563112 9.98E-182
CNAG_03910 myo-inositol transporter, putative ITR6 2 6.517998365 0
CNAG_05381 myo-inositol transporter, putative ITR3C 14 2.065739418 9.34E-37
CNAG_05658 L-iditol 2-dehydrogenase N/A 14 4.051104215 3.05E-223
CNAG_05662 Polyol transporter protein 1 PTP1 14 7.859393735 0
Pentose phosphate pathway
CNAG_06172 transketolase N/A 12 2.848885737 9.10E-75
CNAG_00827 ribose 5-phosphate isomerase N/A 1 4.99834915 1.33E-143
Proline synthesis
CNAG_05134 ornithine-oxo-acid transaminase N/A 4 2.241942301 7.64E-110
Protein and amino acid degradation
CNAG_01952 aryl-alcohol dehydrogenase N/A 11 2.509646461 5.96E-67
CNAG_02049 Proline dehydrogenase PUT1 6 3.09586931 7.34E-92
CNAG_02818 Glycine cleavage system T protein N/A 3 2.037473869 5.18E-181
CNAG_03960 Gly-Xaa carboxypeptidase N/A 2 2.174955689 1.79E-141
64
CNAG_05115 sarcosine oxidase N/A 4 2.406894282 9.41E-91
CNAG_06913 L-serine ammonia-lyase N/A 3 2.744533923 2.68E-146
Pyrimidine salvage pathway
CNAG_02344 Uracil phosphoribosyltransferase N/A 6 2.909303709 4.74E-132
Redox balance
CNAG_07862 fumarate reductase N/A 10 5.402443646 0
Taurine metabolism
CNAG_01542 taurine catabolism dioxygenase TauD N/A 11 4.087034641 1.74E-305
Tricarboxylic acid cycle

malate dehydrogenase (oxaloacetate-
CNAG_06374 decarboxylating)(NADP) N/A 13 2.564851405 8.46E-69
Tetrahydrobiopterin synthesis
CNAG_06009 cyclohydrolase N/A 12 2.962930691 1.30E-190
Transcription factors
CNAG_00896 transcription factor FZC34 5 2.628890692 9.44E-74
CNAG_01551 GATA family transcription factor GAT201 11 3.675353875 5.24E-221
CNAG_01847 hypothetical protein N/A 11 2.678662233 1.49E-183
CNAG_03279 hypothetical protein CCD4 8 2.071978705 6.91E-79
CNAG_03346 bZip transcription factor, putative BZP4 8 2.060783343 7.19E-57

0.0062721257
CNAG_03741 hypothetical protein FZC31 2 2.265537065 68
CNAG_04093 putative transcription factor YRM103 2 2.821424664 9.87E-138

specific RNA polymerase II
CNAG_04345 transcription factor ARO8001 9 4.057102724 0
CNAG_04837 bHLH family transcription factor MLN1 10 5.658104561 0
CNAG_05785 putative transcription factor STB4 7 4.142636148 2.09E-218
CNAG_05835 wor1/pac2 family transcription factor LIV3 7 2.020214829 1.01E-114
CNAG_06188 hypothetical protein FZC15 12 2.051555525 1.23E-43
Transport of metabolites
65
high-affinity nicotinic acid
CNAG_00028 transporter, variant N/A 1 2.833517394 1.41E-167
CNAG_00048 Sugar transporter N/A 1 2.165703801 2.68E-70
CNAG_00284 efflux protein EncT N/A 1 2.051495086 1.00E-40

solute carrier family 25 (mitochondrial
carnitine/acylcarnitine transporter),
CNAG_00499 member 20/29 N/A 1 3.69160675 0
CNAG_00597 amino acid transporter DIP5 1 4.518136614 0

nicotinamide mononucleotide
CNAG_00598 permease N/A 1 5.436810579 0
ATP-binding cassette, subfamily B
CNAG_00796 (MDR/TAP), member 1 MDR1 1 2.664776154 4.21E-53
CNAG_00904 aflatoxin efflux pump AFLT N/A 5 2.533320701 2.99E-10
CNAG_01936 Sugar transporter N/A 11 5.398218881 0
CNAG_02254 quinate permease LPI12 6 3.294651913 9.77E-295

CNAG_02288 citrate transporter), member 1 N/A 6 5.725525264 0
CNAG_02733 Monosaccharide transporter N/A 3 4.838391609 7.50E-302
CNAG_02777 phosphate:H symporter, variant PHO84 3 2.235545414 2.05E-09

solute carrier family 25 (peroxisomal
adenine nucleotide transporter),
CNAG_03242 member 17 N/A 8 2.249113409 2.69E-81
CNAG_03772 high-affinity glucose transporter HXS1 2 8.11764169 0
CNAG_04038 MFS quinate transporter QutD N/A 2 2.289231867 3.47E-79
CNAG_04142 tartrate transporter N/A 9 5.329645146 0

CNAG_04536 permease N/A 9 4.261264549 5.91E-218
MFS transporter, SHS family, lactate
CNAG_04704 transporter N/A 10 4.877479239 4.20E-260
CNAG_04795 adenine nucleotide transporter N/A 10 2.528076177 3.98E-97

0.0077011887
CNAG_04931 Putative hexose transporter HXS2 10 4.37816534 05
CNAG_05119 GABA permease N/A 4 2.758643737 1.60E-87
CNAG_05300 mitochondrial carrier protein N/A 4 2.730932228 1.93E-140
66
MFS transporter, SP family, general
CNAG_05330 alpha glucoside:H symporter N/A 4 5.501723558 1.13E-94
CNAG_05387 galactose transporter N/A 14 5.431201544 0
CNAG_05685 neutral amino acid transporter N/A 7 3.83528674 0
CNAG_05718 multidrug resistance protein fnx1 N/A 7 2.073586105 9.63E-64
CNAG_05867 L-fucose transporter N/A 7 7.624377564 0

CNAG_05914 alpha glucoside:H symporter N/A 7 7.804574725 0
CNAG_06204 high-affinity nicotinic acid transporter N/A 12 2.529064146 1.85E-150

alpha glucoside:H symporter, variant
CNAG_06527 2 N/A 7 3.840821525 4.20E-149
CNAG_06561 allantoate transporter N/A 7 2.745084933 6.78E-57
CNAG_06817 uric acid xanthine permease UAP1 5 2.413959934 1.46E-110

CNAG_07367 amino acid transporter, variant N/A 1 2.147164551 1.15E-33
CNAG_07902 AAT family amino acid transporter N/A 12 2.079429146 1.41E-74
Xenobiotic metabolism
alpha-ketoglutarate-dependent 2,4-
CNAG_04417 dichlorophenoxyacetate dioxygenase N/A 9 3.539262119 3.83E-88
67
Table 2.6. List of downregulated genes in acetate-grown C. neoformans compared to
glucose-grown one
Gene Name log2 Fold-
Gene ID Gene annotation or Symbol Chromosome Change FDR
Carbon metabolism
CNAG_00515 mannitol dehydrogenase N/A 1 -2.056718088 4.69E-46
CNAG_00774 phosphoglycerate dehydrogenase N/A 1 -2.320407504 3.88E-102
CNAG_00799 cellulase N/A 1 -2.169536781 3.35E-61
CNAG_01896 alcohol dehydrogenase (NADP) N/A 11 -2.146751373 1.19E-74
CNAG_01954 aldo-keto reductase N/A 11 -2.155508762 7.61E-60
CNAG_04652 enoyl reductase N/A 10 -2.326270665 1.76E-80
CNAG_04659 Pyruvate decarboxylase PDC1 10 -2.640566552 1.33E-184
CNAG_05458 endo-1,3(4)-beta-glucanase N/A 14 -2.647163092 1.60E-125
CNAG_06770 fructose-bisphosphate aldolase 1 FBA1 2 -2.061906962 7.67E-97

xylulose 5-phosphate/fructose 6-
CNAG_06923 phosphate phosphoketolase XFP2 3 -4.900496595 0
CNAG_02542 fructosamine kinase IRK2 6 -2.425279714 8.50E-56

ketol-acid reductoisomerase,
CNAG_05725 mitochondrial N/A 7 -2.89523887 1.73E-149
alcohol dehydrogenase, propanol-
CNAG_07745 preferring MPD1 8 -3.292011686 1.18E-193
Pentose phosphate pathway
CNAG_07445 transketolase N/A 5 -2.170823672 1.27E-54

6-phosphogluconate dehydrogenase,
CNAG_07561 decarboxylating 1 N/A 3 -2.431142436 1.95E-88
Ribosomal organization
CNAG_02754 small subunit ribosomal protein S12e N/A 3 -2.111585423 1.84E-118
CNAG_02928 large subunit ribosomal protein L5e N/A 3 -2.002523096 9.01E-82
CNAG_03577 large subunit ribosomal protein LP0 N/A 8 -2.155719974 5.55E-69
CNAG_04114 small subunit ribosomal protein S0 N/A 9 -2.199848081 2.32E-50

Large subunit ribosomal protein L17,
CNAG_06447 putative RPL17 13 -2.005085008 1.11E-127
Transport
CNAG_00749 alternative sulfate transporter N/A 1 -2.733300095 1.15E-66
68
CNAG_01960 efflux protein EncT N/A 11 -2.313456416 6.47E-57
CNAG_03438 hexose transporter HXT1 8 -2.501359805 9.23E-144

CNAG_06377 phosphate transporter), member 3 N/A 13 -2.152642318 3.17E-72
Table 2.7. Upregulated cryptococcal genes with reported association with

pathogenicity
Gene CNAG ID Function and Reference
APP1 CNAG_06574 Inhibition of phagocytosis (Luberto et al., 2003)
CFO1 CNAG_06241 Loss of CFO1 attenuates virulence (Jung et al., 2009);

Required for adapting to oxidative, osmotic, genotoxic, and
heavy metal stress (Lee et al., 2014);
Mutant shows susceptibility to fluconazole treatment (Kim et al.,
2012);
PCK1 CNAG_04217 Deletion mutant shows attenuated virulence in murine

inhalation model, but shows persistence in rabbit CSF model
(Price et al., 2011)
LIV3 CNAG_05835 Mutant is defective in lung infectivity (Liu et al., 2008);

TF implicated in quorum sensing (Homer et al., 2016)
MLN1 CNAG_04837 Increased expression in in vivo and ex vivo CSF conditions

compared to YPD media (Chen et al., 2014)
DAK101 CNAG_00826 Core-virulence kinase, and is required for lung and brain
infectivity (Lee et al., 2020)
DPP101 CNAG_06499 One of the phosphatases that were upregulated during host
infection; further characterization required (Jin et al., 2020)
PHO84 CNAG_02777 One of the deleted genes in the triple mutant phoΔΔΔ (pho84,
pho840, pho89) which fails to replicate within macrophage
(Kretschmer et al., 2014)
BZP4 CNAG_03346 TF involved in melanin synthesis; and is expressed at lower

levels in clinical isolates compared to environmental ones (Yu et
al., 2020); loss-of-function leads to reduced melanization
(Desjardins et al., 2017)
69
FZC31 CNAG_03741 Core-virulence TF required for lung and brain infectivity (Jung
et al., 2015)
FZC34 CNAG_00896 Upregulated (moderately, since FDR > 0.05) in ex vivo and in
vivo CSF conditions compared to YPD media (Chen et al., 2014)
GAT201 CNAG_01551 Regulation of anti-phagocytic mechanism (Santiago-Tirado et

al., 2015);
deletion leads to hypocapsular phenotype (Maier et al., 2015);
core -virulence TF for lung and brain infectivity (Lee et al., 2020)
RCV1 CNAG_01983 Deletion leads to impaired GXM production and secretion,

defective cytokinesis, reduced growth at 37°C, and loss of
virulence in murine model (Paes et al., 2018)
STB4 CNAG_05785 TF required for brain infectivity (Lee et al., 2020)
ICL1 CNAG_05303 Highly expressed during infection, however not required for
infection progression (Rude et al., 2002)
MFE2 CNAG_05721 loss -of-function leads to reduction in capsule production and

diminished ability to colonize brain (Kretschmer et al., 2012)
ISP6 CNAG_00456 Upregulated in in vivo and ex vivo CSF conditions compared to

YPD media, a putative target for Gat201 TF (Chen et al., 2014);
one of the proteins enriched in cryptococcal spore (Huang et al.,
2015)
MLS1 CNAG_05653 Mutant is unable to grow on acetate, however does not show
any reduction in virulence (Idnurm et al., 2007)
EXG104 CNAG_02225 Extracellular protein; downregulated in kcs1Δ mutants [kcs1Δ do

not elicit strong immune response and are not readily
phagocytosed] (Lev et al., 2015)
PXA2 CNAG_02764 Mutants showed survival defects in CSF model (Lee et al., 2010)
MEP1 CNAG_04735 Target of Gat201 [master virulence TF] (Brown and Madhani,
2012);
reduced expression in kcs1Δ mutants [kcs1Δ do not elicit strong
immune response and are not readily phagocytosed] (Lev et al.,
2015)
70
FIGURES
Figure 2.1. Effect of either acetate or glucose as sole carbon source on transcriptome
of Cryptococcus neoformans. (A) Venn diagram showing number of DEGs for
acetate-grown and glucose-grown cells. (B) Volcano plot depicting DEGs as
turquoise dots and non-DEGs as orange dots. (C) Heatmap showing expression
patterns of all 356 DEGs across six replicates under two different carbon conditions.
71
Figure 2.2. Chromosomal distribution of the proportion of Differentially expressed
genes (DEGs) in C. neoformans. Proportion of total genes that are either upregulated
(lime green bar) or downregulated (purple bar) in each of the 14 chromosomes
calculated as the percentage of the total genes on the corresponding chromosome.
Vertical lines represent global expected proportions (i.e. overall percentage of the
upregulated [green line] or downregulated [purple line] gene for all 14
chromosomes).
72
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CHAPTER 3
Biochemical characterization of acetate kinase
from the fungal pathogen Cryptococcus
neoformans and its role in acetate production
Oly Ahmed, Cheryl Ingram-Smith, Ann Guggisberg, Sheena

Henry, Ashley Mattison, Aigerim Bizhanova, and Kerry S. Smith
Authors contribution:
KS and CIS conceived and designed the experiments; CIS, AG, SH, AM, AB cloned the
cDNA, designed the protocol for production and isolation of Ack, and performed
kinetic assays in acetyl phosphate forming direction; OA performed the kinetic
assays in acetate forming direction, designed and performed the in silico
identification and phylogenetic analysis of the eukaryotic Acks, and prepared the
manuscript.
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ABSTRACT
Acetate kinase (EC 2.7.2.1; Ack) catalyzes the interconversion of acetyl
phosphate (AcP) and acetate where the phosphoryl group donor/acceptor for the
reaction is either ATP/ADP in genera of Bacteria and the genus Methanosarcina of
Archaea or inorganic pyrophosphate (PPi)/inorganic phosphate (Pi) in eukaryotic
amoebic pathogen Entamoeba histolytica. Excepting the example of E. histolytica
Ack, which is a PPi/Pi-dependant Ack, there is a dearth of information regarding the
kinetic properties of eukaryotic Ack (eAck). Ack from the human fungal pathogen
Cryptococcus neoformans (CnAck) is thought to play an important role in
acetate/acetyl-CoA metabolism, which has been implicated in cryptococcal
infections. Here we studied the kinetic behavior of CnAck and observed that
kinetically CnAck has a preference in the direction of acetate formation over that of
AcP formation. Based on the directionality of Ack catalyzed reaction we propose that
Ack forms a pathway with Xfp2 (xylulose-5-phosphate /fructose-6-phosphate
phosphoketolase; xylulose-5-phosphate / fructose-6-phosphate + Pi →
glyceraldehyde-3-phosphate / erythrose-4-phosphate + AcP) to form a pathway of
acetate production in C. neoformans. In addition, we have predicted 526 putative
eAck from a database constructed from 2,119 published proteomes/genomes, and
built a maximum likelihood tree. A majority of the predicted eAcks were of fungal
origin and monophyletic, with a comparable rate of evolution within the fungal
superclade suggesting a history of common ancestry. This is the first report of
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biochemical characterization of an ATP/ADP-dependent Ack of eukaryotic origin.
Moreover, this Ack’s possible involvement in an acetate production pathway in C.
neoformans may have interesting implications for cryptococcal virulence and hence
warrants further study.
3.1. INTRODUCTION
Acetate kinase (Ack; EC 2.7.2.1) catalyzes the reaction acetyl phosphate (AcP) +
ADP ↔ acetate + ATP, and is abundant in Bacteria and one methanogenic genus
from Archaea (Ferry, 2011). In Bacteria, Ack partners with phosphotransacetylase (Pta,
EC 2.3.1.8) to form a pathway of acetate formation during fermentative growth
(Zhang et al., 2021); whereas in the Archaea, such as Methanosarcina spp., Ack-Pta
pathway is utilized for methane formation (Ferry, 2011). Additionally, Ack has been
predicted in many eukaryotic genomes, notably in fungi (Ingram-Smith et al., 2006).
Outside the kingdom Fungi, the Ack from the eukaryotic amoebic pathogen
Entamoeba histolytica has been studied. The E. histolytica Ack differs from well-
studied prokaryotic Acks in its preference for inorganic pyrophosphate
(PPi)/inorganic phosphate (Pi) over ATP/ADP as phosphoryl group donor/acceptor
(Fowler et al., 2012). Surprisingly, Pta, the typical Ack partner, is not present in E.
histolytica (Ingram-Smith et al., 2006; Fowler et al., 2012). Instead, Ack is suggested to
play a role in maintaining intracellular NAD+/NADH ratio during growth on glucose
through an extended glycolytic pathway (Dang et al., 2022). Thus, variation in the
metabolic role of Ack appears to be niche and species-dependent, which opens up
the possibility of exploiting Ack’s functional relevance in other pathogens for
targeted intervention.
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The structure of Ack from the archaeon Methanosarcina thermophila was the
first one to be solved (Buss et al., 2001; Fowler et al., 2012). Later employment of
transition-state analog (Miles et al., 2002; Gorrell et al., 2005) and substrate analog
(Gorrell et al., 2005) to study the structural and kinetic properties of M. thermophila
Ack provided evidence for a direct in-line mechanism of phosphoryl group transfer.
Subsequently, the structures of the eukaryotic Ack from the amoebic species E.
histolytica and fungal species Cryptococcus neoformans were solved. The difference
between the fungal and amoebic Ack with respect to their specificity for ATP and
PPi, respectively, was explained by subtle amino acid substitutions within the
catalytic cleft (Thaker et al., 2013). The structures of the eukaryotic Ack strongly
resemble those of their prokaryotic counterparts and thus are considered to be
consistent with the proposed direct in-line mechanism of phosphoryl group transfer
(Thaker et al., 2013).
Human fungal pathogen Cryptococcus neoformans belongs to the phylum
Basidiomycota, and has a wider distribution both geographically and ecologically.
Spores and desiccated yeast of C. neoformans can become airborne and are inhaled
by its mammalian hosts, including humans. This mostly leads to initial pulmonary
infection in immunocompromised people, which can subsequently lead to fungal
meningitis through systemic dissemination. During pulmonary infection, C.
neoformans is exposed to a low glucose environment, and appears to metabolically
adapt to the pulmonary microenvironment through upregulation of pathways of
alternative carbon source metabolism (for an updated and extensive review see
(Berguson et al., 2022)). In the absence of simple sugars, C. neoformans relies on 2-C
compounds (e.g., acetate, ethanol) via upregulating the glyoxylate cycle; however
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this pathway seems to be dispensable for virulence (Berguson et al., 2022). Thus, it is
plausible that C. neoformans utilizes other non-canonical pathway(s) of acetate
metabolism both in the context of alternative carbon utilization and maintenance of
intracellular pool of acetate for growth (e.g. lipid synthesis) and epigenetic
regulations (e.g. histone acetylation). Presence of Ack in C. neoformans (FungiDB
gene ID: CNAG_06432) raises the possibility of a hitherto unknown acetate metabolic
pathway.
In this study, we investigated the kinetic characteristics of Ack from C.
neoformans (CnAck), which is the first report of biochemical characterization of a
fungal Ack. Moreover, we surveyed a curated, reference proteome database to
identify putative Ack across the domain Eukarya, and reconstructed a phylogeny to
better understand the possible evolutionary relationships among this large set of
identified eukaryotic acetate kinases (eAck).
3.2. METHODS AND MATERIALS
3.2.1. Production and purification of CnAck
Production and purification of CnAck were achieved following methods
described in (Thaker et al., 2013) with slight modifications. Briefly, the cDNA encoding
CnAck was cloned into the pET21b expression plasmid and transformed into E. coli
Rosetta2 (DE3) cells. Transformants were cultured in LB broth with appropriate
antibiotics at 37°C with shaking to a final OD600 of 0.75, followed by induction of the
ack gene expression through addition of IPTG to a final concentration of 1 mM. Cells
were harvested by centrifugation and resuspended in the purification buffer as
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described: 25 mM Tris, 150 mM NaCl, 20 mM imidazole, and 10% glycerol, pH 7.4
(Thaker et al., 2013). Harvested cells were lysed using French press (20,000 psi),
followed by clarification of the lysate through ultracentrifugation. Finally, CnAck was
purified to electrophoretic homogeneity on a 5 mL HisTrap Ni2+-affinity column using
the exact elution protocol described previously (Thaker et al., 2013).
3.2.2. Kinetic assays of CnACK
The reaction in the direction of AcP/ADP formation was measured using
hydroxamate assay exactly as previously described (Ingram-Smith et al., 2015). The
reaction in the direction of acetate/ATP formation was measured by coupling CnAck
reaction with hexokinase and glucose-6-phosphate dehydrogenase at 37°C in a
reaction buffer consisting of 100 mM Tris-HCl (pH 7.6), 10 mM MgCl2, 5.5 mM
dextrose, 0.2 mM DTT, 1 mM NADP+, with one substrate held constant at saturation
level and the other varied, and measuring the formation of NADPH at 340 nm.
Measurements for various metal cofactors and NTPs were performed using
hydroxamate assays in the AcP/ADP formation direction. Reactions were done in
triplicate; error was calculated in standard deviation; Prism 5 (GraphPad Software,
Dotmatics) was used for curve-fitting.
3.2.3. Identification of putative eukaryotic Ack
To reconstruct the phylogenetic relations among the eukaryotic Ack we
selected a total of 526 putative Ack sequences that were identified using a profile
hidden Markov model (HMM) and searched against a database of 34,059,850 protein
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sequences from 2,119 genomes (proteomes) (see below). The HMM profile and the
query database used in this study were based on the available latest releases from
PRIAM (Claudel‐Renard et al., 2003) and Uniprot (The UniProt Consortium, 2021),
respectively (PRIAM ID: PRI000886, 2018 release; Uniprot Reference Proteomes,
release 2022_03).
The search was conducted using HMMER version 3.3.2 (Eddy, 2011), and the
returned positive hits were further filtered by the reported E-values and bit scores.
Sequences with dissimilar E-values/ bit scores for ‘overall’ hit versus ‘best-domain’ hit
were considered spurious. Remaining sequences were aligned using KALIGN
(default parameters; Unipro UGENE v 43.0 (Okonechnikov et al., 2012)). The resulting
multiple sequence alignment (MSA) was manually edited to remove excess gaps
and mismatches. Shorter sequences (i.e. < 95% of the length of the longest sequence
in the MSA; modal length is 331 amino acids) were further removed from the final
alignment to avoid suspicious branching patterns due to the long-branch attraction
effect in the subsequent tree-construction. Finally, redundant genomes (i.e. multiple
sequenced genomes and/or strains of the same species) were discarded from the
analysis (only one Ack per species was used in the final tree). A complete list of eACK
used for construction of the phylogenetic tree, along with their corresponding
UniProt accession ID can be found in Table B1.
3.2.4. Construction of the phylogenetic tree
A maximum likelihood (ML) tree was constructed using IQ-TREE 2 (Minh et al.,
2020). Following evolutionary models were tested: Blosum 62, Dayhoff, JTT, LG,
Poisson, VT, and WAG. Based on the Bayesian Information Criterion (BIC), LG+I+I+R9
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(i.e, LG substitution model and, both invariant site and FreeRate with 9 categories)
were chosen for the subsequence ML tree construction. To reduce the
computational burden, 2,000 bootstrap replicates were constructed using UFBoot
(an ultrafast bootstrap approximation (Hoang et al., 2018)) (with option appended for
further optimization in case of severe model violation). Readers should be cautious
about the interpretation of the bootstrap values obtained using UFBoot as they
differ from those of standard nonparametric bootstrapping: the authors of IQ-TREE 2
recommend using 95% bootstrap as a reasonable cutoff. The tree was rooted using
Ack from the bacteria Salmonella typhimurium (PDB: 3SLC). The tree was visualized
using the web interface of the Interactive Tree of Life (https://itol.embl.de/).
3.3. RESULTS
3.3.1. Ack in C. neoformans is possibly unidirectional in its biological role
Acetate kinase (Ack) catalyzes the interconversion between ATP/acetate and
ADP/acetyl phosphate, and is reliant on divalent metal cofactors for the reaction
(Chittori et al., 2012). Previously, crystallographic structures of two eAck enzymes
have been reported: E. histolytica and C. neoformans (Thaker et al., 2013).
Catalytically, the major difference between these two eAcks is the preference for
either ATP/ADP or PPi/Pi as the phosphoryl group donor/acceptor in CnAck and
EhAck catalyzed reactions respectively (Thaker et al., 2013). Although kinetic
experiments were performed on EhAck previously (Fowler et al., 2011; Pineda et al.,
2016; Dang and Ingram-Smith, 2017), a biochemical characterization of an ATP-
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dependent eAck has not been reported yet. Our current study of CnAck kinetics is
thus the first report of kinetic properties of any ATP-dependent eAck.
In the current study we observed that the catalytic efficiency of CnAck is
higher for Mg2+ compared to Mn2+, Co2+, and Zn2+ (in the AcP forming direction; Table
3.1); hence in the subsequent kinetic assays, we used Mg2+ as the preferred cofactor
of CnAck. It should be noted that although the catalytic efficiencies (kcat/KM) of CnAck
with Zn2+ and Mg2+ are somewhat comparable, the KM is higher for Zn2+ than for Mg2+.
CnACK specific activity was determined with different NTPs to identify the
best phosphoryl donor. The specific activity of CnAck was found to be considerably
higher with ATP than CTP, GTP, TTP, UTP, or ITP (assays were performed in the AcP
forming direction; Table 3.2), indicating ATP is the preferred phosphoryl donor.
As Ack is classified as a member of the acetate/propionate kinase superfamily
(InterPro ID: IPR004372; Chittori et al., 2012), we examined CnAck’s ability to use
propionate as a substrate. and found the apparent KM for propionate to be 80.7 ± 8.3
mM, 2.8-fold higher than that for acetate, but the kcat was only 0.05 ± 0.01 sec-1, a
0.03-fold decrease compared to that for acetate. In the acetyl phosphate/ADP
forming direction, CnAck has a high apparent KM for the substrate acetate (KM = 28.8
± 1.1 mM) compared to ATP (KM = 1.5 ± 0.1 mM) and the substrates in the acetate
forming direction (AcP KM = 0.5 ± 0.1 mM; ADP KM = 2.3 ± 0.4 mM). The catalytic
efficiency (kcat/KM) of CnAck is also low for the substrates in the AcP forming direction
compared to acetate forming direction, and has a 19-fold higher Kcat for AcP versus
acetate forming direction (Table 3.3). Taken together, our results suggest a strong
preference of CnAck for the acetate/ATP forming direction in vitro.
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3.3.2. Majority of the detected eukaryotic Ack are of fungal origin
Historically, Ack was thought to be found only in genera of the Bacteria and
the single archaeon M. thermophila (Ingram-Smith et al., 2006). However, with the
increase in the number of sequenced genomes, it was soon discovered that many
eukaryotic organisms belonging to Ascomycota, Basidiomycota, Chytridiomycota,
Oomycota, Amoebozoa, and Chlorophyta have Ack genes (Ingram-Smith et al.,
2006). In this work, we also wanted to update that study as the modern genome
databases experienced an exponential increase in the number of genome
sequences deposited, and computation techniques and resources have improved
significantly ever since the previous study. Figure 3.1 summarizes the number of
species in various taxonomic groups with putative Ack genes identified in this study
along with the constructed maximum likelihood tree showing the phylogenetic
relation among putative eAcks.
Here we found that the majority of the Ack detected in this study form a
single monophyletic clade with comparable branch lengths (Figure 3.2 A); not
surprisingly these Ack are of fungal origin, belonging to phylum Ascomycota (63.5%)
and Basidiomycota (22.4%). Other fungal species (dubbed as ‘Enigmatic Fungi’ in
reference to the uncertainty around their placement in the tree of life) also form
sister clades to the abovementioned dikaryotic fungi groups, representing a small
fraction of the dataset (i.e. 2.9%; comprising of Mucoromycota, Chytridiomycota,
Olpidiomycota, and Zoopagomycota). The only non-fungal Ack that forms a tight
cluster with all the fungal Ack is detected in the dicot (also the only dicot found in
the dataset) plant Carpinus fangiana (Monkeytail hornbeam). It is not certain if this
dicot plant has acquired the gene for Ack through some parallel evolutionary
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mechanism (possibly via endophytic fungus); however, C. fangiana Ack shares 40.8%
identity with Ack of C. neoformans. With the exception of C. fangiana, all the
remaining non-fungal Ack (i.e. 11%) are paraphyletic to the said fungal super-clade
(Figure 3.2 B).
3.3.3. Non-fungal Ack may be functionally distinct from fungal-Ack
The second largest taxonomic group in our dataset is the SAR (Stramenopiles,
Alveolata, Rhizaria) supergroup, followed by Viridiplantae (green plants). As
mentioned above, except for the dicot C. fangiana, other members of the
Viridiplantae are paraphyletic to the fungal superclade. From the SAR group, only
stramenopiles and alveolates are represented in our dataset; however presence of
Ack in Rhizaria cannot be ruled out at this point since the taxonomic group of
Rhizaria is not well represented in the proteome database built on UniProt release
(proteomes of only three species of Rhizaria are present out of 2,119 reference
proteomes). This also raises the possibility of underrepresentation of certain
taxonomic groups, and hence our uncertainty regarding the presence of Ack in
many other minor eukaryotic groups yet to be sequenced.
Putative Ack sequences are also detected in metazoan species: two
arthropods (Tigriopus californicus and Hyalella azteca), a cnidarian (Clytia
hemisphaerica), and a nematode (Caenorhabditis remanei). Additionally, three
amoebic species also have Ack: Planoprotostelium fungivorum, Entamoeba
histolytica, and Entamoeba invadens. Unlike the CnAck reported here, Ack in E.
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histolytica utilizes inorganic pyrophosphate as sole phosphate donor (Fowler et al.,
2012); this functional distinction between fungal Ack and amoebic Ack leads us to
speculate about the potential existence of similar pyrophosphate-dependent Ack in
non-fungal eukaryotes.
3.4. DISCUSSION
Here we studied the kinetic parameters of CnAck heterologously produced in
E. coli and found that CnAck preferentially utilizes Mg2+ as the divalent metal
cofactor for interconversion between AcP and acetate with ATP/ADP as preferred
phosphoryl group donor/acceptor. Comparison of KM values and catalytic efficiency
for the substrates acetate and AcP suggests that Ack in C. neoformans may be
metabolically unidirectional with a strong preference in the acetate-forming
direction.
In many fermentative bacteria and the archaea Methanosarcina, Ack and Pta
form a pathway which is, in principle, bidirectional. Moreover, the relevance of the
Ack-Pta pathway is niche-specific. For example, Ack-Pta pathway is abundant in
fermentative bacteria, where it functions in the conversion of acetyl-CoA to acetate
and ATP production, or in archaea Methanosarcina spp. as the initial steps of
methanogenesis (Gorrell and Ferry, 2007). This pathway is also an important player
in the acetate switch, i.e., the cellular metabolic process involved in regulating either
acetate secretion or assimilation based on nutrient availability, in many bacteria
(Barnhart et al., 2015). Depending on the species and niche this pathway may
actually be very essential for survival as well. For example, in methanogenic
archaeon Methanosarcina acetivorans, the Ack-Pta pathway is required for growth
91
on acetate as a carbon and energy source for methane formation and on carbon
monoxide as the sole carbon source as this pathway is involved in acetogenesis from
carbon monoxide (Rother and Metcalf, 2004). Outside the bacteria and
Methanosarcina, the Ack-Pta pathway has only been investigated in the unicellular,
soil-dwelling, green alga Chlamydomonas reinhardtii, where both the enzymes have
mitochondrial and chloroplastic homologs. Ack1-Pat2 localizes in chloroplast and
plays a more prominent role in acetate production under dark, anoxic conditions
compared to Ack2-Pat1, the mitochondrial counterpart (note the abbreviation for Pta
in this species is Pat) (Yang et al., 2014). The other example of eukaryotic Ack studied
so far is from amoeba E. histolytica. However, in E. histolytica, Ack lacks the typical
Ack partners Pta and Xfp (Fowler et al., 2012), and it does not seem to play a major
role in acetate production, but rather helps maintain the NAD+/NADH balance
during ethanol synthesis in the extended glycolytic pathway (Dang et al., 2022).
The gene for Pta is missing from C. neoformans genome, hence Ack-Pta
pathway is not tenable in this organism. Incidentally, AcP, the substrate for Ack, can
also be synthesized from xylulose 5-phosphate (X5P) or fructose 6-phosphate (F6P)
and inorganic phosphate (Pi) via the action of the enzyme xylulose 5-phosphate/
fructose 6-phosphate phosphoketolase (Xfp) (Ingram-Smith et al., 2006); previously
an isozyme of Xfp from C. neoformans has been reported and characterized (Glenn
et al., 2014). Xfp can form a pathway with Ack for acetate and ATP production (e.g.
bifid shunt in bacteria Bifidobacterium spp. which is responsible for conversion of
hexose sugar into acetate), or Xfp can also form a pathway with Pta for acetyl-CoA
production from hexoses (e.g. as in lactic acid bacteria and bifidobacteria) (Henard et
al., 2015).
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The cryptococcal isozyme Xfp2 is allosterically inhibited by ATP (energy
pathway), phosphoenol pyruvate (glycolysis), and oxaloacetic acid (the TCA cycle);
and is activated by AMP (low energy condition) (Glenn et al., 2014). Thus Xfp2, in
combination with Ack, is likely to form a pathway that leads to the formation of ATP
and acetate from phosphoketose sugars and inorganic phosphate (Figure 3.3).
There is a dearth of information regarding the metabolism of AcP in eukaryotic cells;
however in recent times application of modern analytical techniques such as 2D-
NMR spectroscopy helped detect the transient nature of AcP formation in human
mitochondria, suggestive of its role as an intermediate of acetate formation in
mammalian cells, although this may not be the primary mechanism (Xu et al., 2018).
A plausible analogous mechanism of fast conversion of AcP to acetate in C.
neoformans becomes explicable through our observation that CnAck shows high
catalytic efficiency in acetate formation direction.
To understand the evolutionary history of Ack in Eukarya, we have used a
hidden Markov model (HMM)-based searching strategy against a curated database
of 2,119 sequenced eukaryotic genomes. Our conservative estimation is that 526
eukaryotic species appear to have putative Ack sequences in their genomes (note
that this estimate excludes extraneous multiple versions or strains of the same
species). The majority (~88%) of this list belongs to the group Fungi. Phylogenetic
analysis shows a tight clustering of the fungal Ack in a single super-clade with
comparable branch lengths. Such monophyly and nearly uniform rate of evolution
across the fungal Ack is strongly suggestive of a common ancestry. Similarly, most, if
not all, non-fungal Ack also show reasonable monophyletic relation within a given
taxa; however the branch lengths differ notably from one taxon to the next,
93
suggesting taxon-specific rates of divergence which may be reflective of the
organism’s niche and life-history.
At the moment nothing conclusive can be said from our dataset about the
distributions of Ack among fungi and non-fungal eukaryotes, since the
disproportionate distribution (i.e. 88.8% fungi vs. 11.2% non-fungi) is likely to have
arisen from a corresponding disproportionate representation of these two groups in
the total genomes sequenced to date. Despite such methodological bias it became
apparent from our phylogenetic tree that modern day protozoan, fungal, and algal
Acks have likely been derived from a common ancestral sequence. The diversity of
the ecological niches occupied and the sheer variations in the life-histories of the
species identified here make convergent evolution of this enzyme improbable;
however, overlap between species-taxon and Ack-clade, and similar branch-length
within the clades are consistent with both the scenarios of parallel and divergent
evolution. Widespread distribution of Ack in Bacteria, and in few species of Archaea,
points to the existence of a more ancient ancestral sequence and subsequent
divergence; in fact, from the consideration of structural conservation, a possibility of
an ancestral sequence has previously been suggested which might have led to the
evolution of both Ack and Acetyl-CoA synthetase (Acs), another crucial enzyme of
acetate metabolism, via duplication and divergence in all three domains of life
(Barnhart et al., 2015). Moreover, if AcP is indeed a primordial energy currency of the
ancient world that potentially coupled the energy with carbon flux as argued by
(Whicher et al., 2018), it will not be surprising to assume that any ancestral enzyme
that enhanced the rate of phosphorylation by AcP and thereby catalyzed the crucial
process of coupling carbon flux and energy generation, might have belonged to the
94
Last Universal Common Ancestor (LUCA)-protein set (Elias and Tawfik, 2012) and is at
the heart of the ancient cellular evolution.
However, we do not believe that all the eAck sequences in our dataset (and
many more that might be evident with future sequencing projects) have a neat
history of duplication and divergence. Some eukaryotes might have undergone
reticulate evolution and have likely come to possess Ack through horizontal gene
transfer (HGT). Consider, for example, that from the land-plant group (Streptophyta)
Ack is found in charophytic alga Chara braunii, liverwort Marchantia polymorpha,
moss Physcomitrium patens, dicot plant Carpinus fangiana. Although we believe
that our strategy for detecting eAck was limited by the size and the distribution of
various taxa in the genome database (e.g. Rhizaria: 0.1% representation vs. Metazoa:
41.0% representation), the situation cannot be argued to be the same for land plants
(Streptophyta: 9.0%; Chlorophyta: 1.2%; Amoebozoa: 0.4%, vs. Fungi: 41.2%
representation). The near-absence of Ack in Streptophyta is best explained by loss of
Ack in their last common ancestor, followed by reacquisition through HGT in a
handful of lineages. Similar reasoning seems more applicable for metazoan Ack
detected in our study.
95
TABLES
Table 3.1. Kinetics of CnAck for common divalent metal cofactors in the acetyl
phosphate forming direction
kcat KM kcat/KM
Cofactor
(s-1) (mM) (s-1 mM-1)
Mg2+ 1.42 ± 0.02 1.5 ± 0.1 0.95 ± 0.06
Zn2+ 2.39 ± 0.07 2.8 ± 0.2 0.85 ± 0.07
Mn2+ 2.36 ± 0.02 4.1 ± 0.1 0.58 ± 0.01
Co2+ 2.57 ± 0.07 4.5 ± 0.3 0.57 ± 0.04
Table 3.2. Specific activities of CnAck for common nucleotide triphosphates

(NTPs) in the acetyl phosphate forming direction
Specific activity Percentage activity

NTP
(µmol min-1 mg-1) (%)
ATP 0.93 ± 0.021 100.0
ITP 0.28 ± 0.001 30.2
CTP 0.26 ± 0.024 28.4
GTP 0.24 ± 0.010 25.9
UTP 0.22 ± 0.003 24.2
TTP 0.03 ± 0.004 3.1
96
Table 3.3. Kinetic parameters of CnAck
kcat KM kcat/KM
Substrate
(s-1) (mM) (s-1 mM-1)
Direction of acetyl phosphate formation

Acetate 1.54 ± 0.03 28.8 ± 1.1 0.05 ± 0.01
ATP 1.42 ± 0.02 1.5 ± 0.1 0.95 ± 0.06
Direction of acetate formation
Acetyl phosphate 30.22 ± 1.04 0.5 ± 0.1 60.44 ± 12.27
ADP 61.53 ± 4.08 2.3 ± 0.4 26.75 ± 4.98
97
FIGURES
Figure 3.1. Maximum Likelihood tree of 526 eukaryotic Ack sequences, rooted using
Ack of bacteria Salmonella typhimurium (red branch). Different taxonomic groups
are color coded (legend). For visual clarity of the tree topology, bootstrap values
(2000 replicates) and branch lengths are not shown. Legend also includes summary
(chart on the right) of the counts (column 3) and corresponding distribution of Ack in
various taxonomic supergroups (bold, left column) and subgroups (middle column).
98
Figure 3.2. (A) Ack of fungal origin (blue, orange, and yellow) and one dicot plant
(green) are highlighted as they form a monophyletic superclade (see text);
remaining paraphyletic non-fungal Ack are not color-coded. (B) the superclade in (A)
is collapsed (gray triangle) and non-fungal Ack are highlighted. In both (A) and (B)
branch lengths are shown. Outgroup branch is colored red.
99
Figure 3.3. Proposed XFP2/ACK pathway for acetate production in C. neoformans.
XFP2 catalyzes the conversion of xylulose-5-phosphate (X5P) or fructose-6-
phosphate (F6P) into glyceraldehyde-3-phosphate (G3P) or erythrose-4-phosphate
(E4P) and acetyl phosphate (AcP) using inorganic phosphate (Pi). Acetate kinase
(ACK) can then convert AcP to acetate by transferring the phosphate group to
adenosine diphosphate (ADP). Phosphoenol pyruvate (PEP), oxaloacetic acid (OAA),
and ATP (adenosine triphosphate), generated via metabolic processes such as
glycolysis, and the tricarboxylic acid (TCA) cycle negatively regulate activity of XFP2
(Xylulose-5-phosphate/ fructose-6-phosphate phosphoketolase 2), whereas
adenosine monophosphate (AMP) positively upregulates XPF2 (Glenn et al, 2014).
Abbreviation: PPP - pentose phosphate pathway.
100
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CHAPTER 4
Modularity hypothesis of fungal virulence
Oly Ahmed, and Kerry Smith

104
4.1. Why we need a regulatory network-based understanding
of the origin of virulence in fungi
To date fungal infection persists as a substantial problem in terms of human
health and healthcare cost (Warnock, 2017). An estimated one billion people
worldwide are affected by fungi, especially through superficial infections, resulting in
1.6 million infections annually (Siscar-Lewin et al., 2022). It is generally assumed that
throughout human history fungal infection was not a substantial threat; however, in
recent times we have noticed a significant rise in fungal infections (Dangarembizi et
al., 2022). This rise can at least be attributed to two anthropogenic sources: first,
climate change, which appears to be driving the emergence of new traits such as
thermotolerance in relatively harmless fungi, and thus evolution of pathogenic traits
in these species (Nnadi and Carter, 2021). Second, improved medical interventions,
for example immunosuppressant therapy, which renders the recipient of the
therapy ill-suited for fighting against opportunistic fungal infections (Dangarembizi
et al., 2022).
And yet, the unfortunate reality is that we are limited in our arsenal to fight
against fungal pathogens. The antifungal agents at our disposal suffer from
limitations with regards to activity, pharmacological properties, potential toxicity,
and cost of administration and management (Lewis, 2017). Additionally, secondary
resistance to antifungal drugs has also been reported (Johnson, 2017). Under such
circumstances, researchers are working incessantly to discover new antifungal
therapy via drug screening and drug repurposing (Donlin and Meyers, 2022).
Our understanding of the fungal virulence factors and mechanism (Brunke et
al., 2016) and the role of immunity in defense and disease progression (Casadevall,
105
2022) has advanced significantly. A noteworthy achievement of the community is
the proposal of a hypothesis that enables us to glean the evolutionary story of the
origin of virulence in fungi. The thesis posits that interactions in nature among
environmental fungi and their amoebic predators worked as a backdrop whereby
virulence factors imparted selective advantages to the fungi, which might have been
co-opted (as an exaptation) for pathogenicity in mammalian hosts (Casadevall et al.,
2019; Radosa and Hillmann, 2021). Although this hypothesis provides a theoretical
framework that is of tremendous use for understanding the evolutionary origin of
virulence traits in fungi, it is limited in expounding a framework useful for
understanding the emergence of such virulence traits from a cellular and molecular
biology perspective. Understanding the cell and molecular biology of fungal
virulence is paramount for developing research strategies that alleviates the
problem of blind screening for novel drugs.
Here we propose a hypothesis to explain the emergence of virulence in fungi
based on an underlying cellular regulatory network architecture that connects stress
perception and response traits modules with virulence traits modules in fungi. This
hypothesis, in our view, has a potential to predict connectedness between such
modules, and thus directs us to such links which, in principle, can be targeted to be
severed to remove or diminish pathogenicity in fungi.
106
4.2. Fungal infections in mammals are rare, and virulence
factors can have niche-specific protective roles for fungi
Fungal pathogenicity in mammals appears to be accidental as they do not
seem to suffer many fungal infections, and most cases are found in
immunocompromised individuals (for a recent review, see Gnat et al., 2021). Table 4.1
compiles a list of common fungal diseases reported in mammalian hosts.
It is worthwhile to ponder about the rarity of fungal pathogens in mammals.
First, out of an estimated 2.2 ~ 3.8 million fungal species, only a handful of the species
(presumably 0.01%; Köhler et al., 2017) cause any serious infection in plants and
animals (a more elaborate list of infectious fungi can be found in Sun et al., 2020).
This implies that not all fungi have pathogenic potential.
Second, not all fungal infections lead to disease progression. Disease, the
penultimate outcome of virulence, requires successful completion of multiple steps
involving different virulence factors and physiological adaptations on the invading
pathogen’s part within the invaded host body (Diard and Hardt, 2017). Thus, success
or failure at each of the pre-disease steps of infection is important in driving the
evolution and/or maintenance of virulence traits/factors in any pathogen (Diard and
Hardt, 2017). Mammalian body temperature and possession of a complex immune
system are thus two major challenges for fungal pathogens inside mammalian hosts
(Casadevall, 2022).
Attempts have been made to understand how a handful of fungal pathogens
might have developed (or perhaps, retained) the ability for pathogenicity in
mammalian hosts (dubbed as “The amoeboid predator - fungal animal virulence
hypothesis”, see the previous section and Casadevall et al., 2019). What all these
107
potential pathogenic fungi have in common — perhaps, in contrast to their non-
pathogenic counterparts — is a set of morphological and biochemical traits (dubbed
as virulence factors) that enables them to establish initial infection, evade the
patrolling immune system, and effectively disseminate systemically. However, what
is considered as a virulence factor is unique to the pathogenic species (an excellent
review of the examples of species-specific virulence factors can be found in Brunke
et al., 2016).
Our current understanding is that these virulence factors have tangible
utilities in terms of providing protection to the fungi from naturally occurring
predators and various environmental stresses. For example, morphological virulence
factors, such as dimorphic transition (Candida spp.), capsule formation
(Cryptococcus spp.), or growth at high temperatures (seen in all pathogenic fungi),
as well as biochemical factors, such as secretion of hydrolytic enzymes
(dermatophytes, Malassezia, Cryptococcus spp.), synthesis of reactive oxygen
species (ROS) and radiation protective compound melanin (Aspergillus,
Cryptococcus), etc. are likely to help fungi counter environmental changes such as
nutrient exhaustion, amoebic predation, rise in temperature, micro-nutrient
deprivation, change in pH and UV intensity, etc (Brunke et al., 2016). Thus, it is highly
likely that such ecologically relevant, niche-specific fungal phenotypes (known to us
as virulence factors) are also useful toolkits that can be co-opted for accidental
pathogenicity in an unfortunate mammalian host (Rokas, 2022).
108
4.3. Stress response and virulence can be conceived as
modules of a larger conceptual network
From gene knock-out experiments to -omics studies, investigations of
infectious agents (bacterial, fungal, protozoan) have pointed to numerous examples
illustrating the relatedness of virulence and stress response. Simply searching the
PubMed database on November 13, 2022 for “stress response and virulence” resulted
in 7,804 publications. The underlying assumption is that any infectious agent,
obligate or opportunistic, is constantly being exposed to various physical and
chemical stresses within the host; hence, a rapid adaptation to this host-specific
stresses are imperative for growth and survival of the infecting organism (Brown and
Goldman, 2016). Thus, it appears that a microbe’s ability of quick response and
adjustment to host-imposed stresses are necessary, if not sufficient, for
pathogenicity. On the flip side, non-pathogenic environmental species appear to
lack virulence traits, but they cannot possibly do without any niche-specific stress-
response mechanisms. These considerations point us to thinking of stress response
and virulence as separate modules (Figure 4.1).
Modularity can be roughly defined as a community of nodes in a given
network that shares a significantly high number of within-connections compared to
their connections with other such modules elsewhere in the network (Alcala-Corona
et al, 2021; Serban 2020) (Figure 4.2). Imagining stress response and virulence as
separate modules urges us to look for connections (or links, or edges) between
them. This is not inconceivable. For example, a single gene product such as YjbH
appears to tie oxidative stress response and virulence in opportunistic bacterial
pathogen Staphylococcus aureus (Paudel et al, 2021), whereas cAMP/PKA pathway is
109
speculated to be involved in nutrient sensing and adaptation, and virulence in
opportunistic fungal pathogen Cryptococcus neoformans (Caza and Kronstad, 2019).
In recent years, advancement in transcriptomic and proteomic technologies
and computational techniques has opened up an opportunity to take a systems-
level examination at our pathogens of interest. Quite recently an attempt was made
to reconstruct the Transcriptional Regulatory Network (TRN) of plant pathogenic
fungus Fusarium graminearum which revealed a modular architecture of important
traits such as virulence, sexual reproduction, and mycotoxin production (Guo et al,
2020). Similarly, Gene Co-expression Network (GCN) analysis has also been exploited
to discover gene regulatory modules that hint at previously unknown virulence
mechanisms in plant pathogenic bacteria Pseudomonas syringae (Nobori et al,
2020).
Although the examples above suggest a possibility of existence of virulence
and stress response as modules at gene regulatory levels, this need not be the case,
as modularity can be sought for across various scales in a given biological system
(Serban, 2020). Keeping aside any debate about the ontological status of such
modules, thinking in terms of a network and its (subnetwork) modules to
understand the regulatory connectivity of virulence traits and stress sensing and
response traits, we believe, does have an immense pragmatic importance.
110
4.4. The hypothesis and its implications
To summarize, stress sensing and response are connected to virulence in
many pathogenic organisms, whereas non-pathogenic organisms have functioning
mechanisms for stress sensing/response without any virulence traits, implying a
plausible existence of two separate modules. Moreover, previous TRN and GCN
analyses have been used to explore the modular nature of the underlying regulatory
network in connection to virulence in plant pathogens.
From such considerations, we hypothesized that various stress responses (and
associated sensing and signaling) and virulence traits in fungal pathogens of
mammals can be thought of as interconnected modules, where the connections
between them are likely to be regulatory in nature.
This picture of fungal virulence can also be used to explain the emergence of
pathogenicity, since modularity leads to distinctive functions with a possibility of
subsequent co-option, thereby facilitating evolutionary innovations (Sambamoorthy
et al, 2019). For example, in mycobacteria, the evolution of host-specific adaptations,
as well as the ability to emerge in new niches might have come about through
acquisition of new genetic materials, which needed functional repurposing following
integration. Such successful functional repurposing suggests an underlying modular
structure for evolution to work on (see Pepperell, 2022 for an updated review of the
evolution of tuberculosis pathogenesis).
Another major implication of the proposed hypothesis is that it predicts the
existence of interconnections between stress response and virulence modules. In
terms of gene regulatory networks, such interconnectedness may be exemplified by
the existence of one or few transcription factors or other regulatory elements. A
111
conception of fungal virulence in terms of network topology — a plausible
evolutionary design principle — is sure to lead to an understanding of connections
between stress response and virulence factors in fungal pathogens. Such discoveries
have the potential to usher in rational strategies for developing molecular and
pharmacological interventions for fungal infections in mammalian hosts.
112
TABLE
Table 4.1. Common fungal infections in mammals (Compiled and adapted from
Sayedmousavi et al, 2018)
Opportunistic infection (non-transmissible) Aspergillosis

Mucormycosis
Candidiasis
Cryptococcosis
Various black-fungi related
infections
Environmental sources (indirect Coccidioidomycosis

transmission) Histoplasmosis
Paracoccidioidomycosis
Blastomycosis
Other infections (direct transmission and White-nose syndrome

zoonosis) Microsporum infection
Sporothrix infection
Trichophyton infections
Encephalitozoon infections
Enterocytozoon infections
113
FIGURES
Figure 4.1. Stress-response and virulence-associated traits in fungi can be

thought of as separate modules of the same regulatory network. Fungi both with
and without pathogenic potential have all the necessary signaling and response
mechanisms for recurrent stresses experienced in their natural habitat, such as
nutrient starvation, pH, osmotic and temperature fluctuations, radiation, predators
etc. Moreover, pathogenic species possess virulence-associated phenotypes and
genetic factors which are absent in their non-pathogenic counterparts. Virulence
traits/factors are thought to play roles in stress-response in these pathogenic species
outside their host body, and are presumably co-opted for causing accidental
damages to the hosts. However, absence of virulence traits/factors in non-
pathogenic species does not diminish their ability to cope with the environmental
stresses. In pathogenic species, virulence traits/factors appear to be linked to the
stress response within the host body, and hence are presumably connected via
regulatory mechanism. Such consideration points to the possibility of stress-
response and virulence as two separate, but regulatorily interconnected modules in
pathogenic fungi.
114
Figure 4.2. Modularity in biological networks. Modularity is a widespread
phenomenon in any complex system such as ecological interactions, gene co-
expression and transcriptional regulatory networks in biology. Within a given
network (here boxes represent nodes of a hypothetical network, e.g. genes, and solid
connecting lines represent relationships among the nodes, e.g. co-expression or
transcriptional regulation), modules are clusters of nodes that are heavily
interconnected compared to other nodes elsewhere in the same network (here
nodes within a module share the same color). Two most common types of
modularity are depicted here: (A) simple modules, and (B) hierarchical modules. In
contrast to the simple modules, a module can be nested within a larger module
(depicted by the large red rectangle in B) giving rise to a hierarchical topology.
Virulence traits/ factors can be thought of as a module nested under stress-response
module in most pathogenic fungi.
115
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CHAPTER 5
Screening modularity in transcriptional hierarchy
requires undertaking larger exploratory studies
Oly Ahmed
119
In this work I set out to explore the mechanism(s) and effect(s) of acetate
metabolism in the human pathogenic fungus Cryptococcus neoformans owing to
the suggested relevance of acetate utilization in the survival and virulence
elaboration of this organism during the course of infection (Chapter 1). I have also
investigated biochemical properties of acetate kinase (Ack) from C. neoformans as
Ack forms a potential pathway for acetate production in this species (Chapter 3). In
this chapter I aim to suggest potential studies designed to answer important
questions raised in my work.
5.1. Transcriptional circuitry of acetate utilization
I initiated our exploration through the investigation of direct effects of acetate
utilization, in lieu of glucose as the sole carbon source, on the transcriptomic
landscape, and noticed that a subset of genes is regulated as to bear the signature
of nutrient stress, glucose deprivation, and global adaptation for efficient nutrient
acquisition (Chapter 2). Additionally, I have noted significant upregulation of a
number of genes previously described in the literature for their association with
virulence and fitness in C. neoformans (Chapter 2).
In this study (Chapter 2) I have computationally predicted a total of 200
putative transcription factors (TFs), of which a subset of 15 TFs were found to be
upregulated during acetate utilization, suggesting a possible role of these TFs in the
regulation of acetate metabolism. To date 11 out of 15 of these TFs have been
characterized previously, and the remaining 4 TFs are predicted for the first time in
the current study (Chapter 2). This opens up the possibility for studying the
120
individual and interactive roles of these putative TFs in acetate utilization. I suggest
that the first step towards that aim may be achieved through in silico detection of
the binding sites of these putative TFs. Under the assumption that these DNA-
binding sites (DBS) are likely to act as the initial cis-regulatory elements (CRE) for
proximal genes (for simplicity, the trans-acting regulatory mechanism should be
ignored to begin with), comparison among the proximal genes of the predicted DBS
with the differentially regulated gene-set from the transcriptomic study in Chapter 2
can be used, in principle, to predict the primary regulatory circuitry of acetate
utilization in C. neoformans.
Furthermore, the involvement of these 15 upregulated TFs need not be
restricted to acetate utilization. Expression of this subset of TFs can be monitored
using quantitative RT-PCR techniques under various growth conditions with carbon
sources such as lactate and trehalose, two other important physiologically relevant
alternative carbon sources (Chapter 1). These experiments will shed light on TFs
uniquely connected to acetate utilization by comparison.
5.2. Possibility of an acetate switch
In chapter 1 I discussed the possibility of the acetate switch (i.e., the ability of
either secreting out or assimilating in acetate from the surroundings depending on
the availability of the nutrients) in C. neoformans depending on the tissue of
infection and the disease state of the host as these parameters influence the
availability of acetate in various host microenvironments. An extension of this
hypothesis posits that C. neoformans thereby may possess the ability of acetate
switch. To investigate the possibility of an acetate-switch phenotype in cryptococcal
121
species, acetate concentrations in the spent media can be measured at equally
spaced time intervals (colorimetric assays, LC-MS/MS etc.), followed by time-series
and autocorrelation analyses. This may help us probe the presence of such
phenotype in unicellular fungal species, presumably, for the first time in this group
of eukaryotes.
5.3. Xfp-Ack pathway as a route of acetate production
The kinetic characterization of acetate kinase (Ack) reported here and
previous reports of kinetic properties of xylulose-5-phosphate/fructose-6-phosphate
phosphoketolase 2 (Xfp2) from C. neoformans consistently point to a pathway of
acetate synthesis from phosphoketose sugars (Chapter 3). Thus, a coupled reaction
involving both Xfp2 and Ack can be devised to characterize the properties of the
entire pathway, as a single metabolic entity, as the proof of principle.
Moreover, C. neoformans genome encodes for a paralog of Xfp, Xfp1, which is
yet to be characterized biochemically. The Xfp1 gene from C. neoformans thus can
be cloned, recombinantly produced, purified and kinetically characterized in
addition to examining its post-translational regulation. This will broaden our
understanding of the role of the Xfp paralogs in the Xfp-Ack pathway in C.
neoformans.
5.4. The trio: Xfp-Pta-Ack
In Chapter 3, I predicted Ack sequences from the published and curated
genomes of over two thousand species and constructed a phylogeny to investigate
the evolutionary history of this enzyme in Eukarya. The pathway of acetate
122
production (or assimilation) in prokaryotes and some eukaryotes involves Xfp and/or
phosphotransacetylase (Pta) (Chapter 3). Thus, a similar in silico study for predicting
and reconstructing the evolutionary history of Xfp and Pta, the two prominent
partners of Ack, can be compared across the domain Eukarya to further our
understanding of the evolution of the entire pathway in eukaryotes.
Moreover, special attention can be focused on the kingdom Fungi for their
biotechnological and medical relevance and potential. Strong functional ties
between Ack and its partner (say Xfp) are likely to lead to chromosomal proximity of
the genes and hence linkage disequilibrium (LD). Although it will be logistically
challenging to perform an LD experiment of this magnitude (i.e. across the kingdom
Fungi), available published genomes are easy to scan for in silico distance
measurement between Ack and partner genes.
In case of LD between Ack and partner genes, this new information will be
quite useful down the line. For example, Ack and the partner gene can be used to
check for synteny in closely related fungal species. Syntenic relationship combined
with LD may be suggestive of the selective sweep as a mechanism of evolution for
the entire pathway in eukaryotes. This implies that these genes have the
contributory potential along with other well-known near-universal single-copy
orthologs (e.g., OrthoDB, Zdobnov et al., 2021) for multi-loci phylogenomic studies in
Fungi for better phylogenomic resolution.
123
5.5. Gene co-expression network in C. neoformans
In chapter 4 I have proposed a new theoretical framework to explain the
possible mechanism of molecular evolution of various virulence traits in pathogenic
fungi. In chapter 2 we first saw a glimpse of the modular nature of virulence
phenotype and stress response phenotype in C. neoformans by noting the
association between the transcriptional regulation of the known stress response
genes with the known virulence genes in C. neoformans when grown with acetate
as the sole carbon source.
An extension of my work depicted in chapter 2, and a proof of concept of my
hypothesis introduced in chapter 4, can be systematically tested via gene co-
expression network (GCN) studies. Note that modularity at the transcriptional level is
not an absolute requirement for the emergence of fungal virulence, however,
logistically GCN is a good starting point. Hence, cryptococcal cells can be exposed to
various stress conditions, including but not limited to, heat stress, oxidative and/or
nitrosative stress, carbon and/or nitrogen starvation, radiation stress, and the
resultant transcriptomic profiles under each stress condition can be combined to
construct a GCN. Such analyses easily reveal the underlying modular structure of the
network; however, the challenge would be to tease out the recurrent patterns of
connectivity under various stress conditions between the genes of the
corresponding stress response and those of virulence and pathogenicity. An offshoot
result of GCN can be the classic guilt-by-association analysis where constant
expression of one or few virulence-associated genes under all or most stress
conditions tested may help us narrow down the most important virulence genes as
well.
124
REFERENCE
Zdobnov, E.M., Kuznetsov, D., Tegenfeldt, F., Manni, M., Berkeley, M., Kriventseva, E.V.,
2021. OrthoDB in 2020: evolutionary and functional annotations of orthologs.
Nucleic Acids Res. 49, D389–D393. https://doi.org/10.1093/nar/gkaa1009
125
APPENDIX A
RNA-sequencing data of Cryptococcus neoformans
Table A1. Preliminary stats of RNA-sequencing data
Clean read
Sample ID Raw read counts counts Clean bases GC content
Glucose-1 61,638,480 60,023,740 9.0 Gb 52.35 %
Glucose-2 55,954,158 53,621,164 8.0 Gb 52.49 %
Glucose-3 58,790,866 56,394,978 8.5 Gb 52.74 %
Acetate-1 53,419,220 51,696,148 7.8 Gb 51.57 %
Acetate-2 55,160,712 51,022,146 7.7 Gb 51.62 %
Acetate-3 59,197,388 55,839,016 8.4 Gb 51.6 %
Average 57,360,137.33 54,766,198.67 N/A 52.06 %
Median 57,372,512 54,730,090 N/A 51.99 %
Table A2. List of putative transcription factors identified in the study
CNAG_ID Chromosome Gene Name or Product Description

Symbol
CNAG_00014 1 N/A hypothetical protein
CNAG_00018 1 FZC6 hypothetical protein
CNAG_00027 1 N/A transcriptional activator
CNAG_00031 1 MLR1 hypothetical protein
CNAG_00039 1 ZFC6 hypothetical protein
CNAG_00043 1 N/A hypothetical protein, variant
CNAG_00068 1 N/A specific RNA polymerase II transcription factor
126
CNAG_00239 1 YAP1 hypothetical protein
CNAG_00460 1 LIV1 virulence related protein of unknown function
CNAG_00559 1 BZP3 bZip transcription factor, putative
CNAG_00732 1 CCD3 hypothetical protein
CNAG_00828 1 SIP401 pathway-specific nitrogen regulator
CNAG_00871 5 CLR3 hypothetical protein
CNAG_00883 5 ECM2201 Putative Zn2-Cys6 zinc-finger transcription factor
CNAG_01315 5 N/A protein transporter
CNAG_01454 5 STE12 alpha transcription factor STE12
CNAG_01551 11 GAT201 GATA family transcription factor
CNAG_01781 11 N/A hypothetical protein, variant 3
CNAG_01858 11 HOB2 hypothetical protein
CNAG_01948 11 FZC36 nuclear protein
127
CNAG_02022 6 N/A DNA-directed RNA polymerase II subunit RPB3
CNAG_02046 6 PBP1 poly(A)-binding protein binding protein
CNAG_02134 6 RSC8 rsc chromatin remodeling complex subunit
CNAG_02215 6 HAP3 transcriptional activator, variant
CNAG_02359 6 N/A small subunit ribosomal protein S25e
CNAG_02435 6 BWC2 white collar 2 protein
CNAG_02476 6 YRM101 putative transcription factor
CNAG_02516 6 HLH5 hypothetical protein
CNAG_02555 6 SIP402 Putative Zn2-Cys6 zinc-finger transcription factor
CNAG_02566 3 FKH2 hepatocyte nuclear factor
CNAG_02603 3 N/A early growth response protein 1
CNAG_02767 3 N/A transcription initiation factor TFIIE subunit alpha
CNAG_02774 3 MAL13 Fungal Zn(2)-Cys(6) binuclear cluster domain protein
CNAG_02986 3 YSA1 ADP-ribose pyrophosphatase
CNAG_03018 3 ASG101 Putative zinc finger transcription factor
128
CNAG_03116 8 HCM1 forkhead transcription factor 3
CNAG_03129 8 N/A transcription factor IIIA
CNAG_03155 8 N/A ENTH domain-containing protein
CNAG_03212 8 HCM101 forkhead domain-containing protein
CNAG_03229 8 YOX101 specific transcriptional repressor
CNAG_03279 8 CCD4 hypothetical protein
CNAG_03346 8 BZP4 bZip transcription factor, putative
CNAG_03366 8 ZNF2 C2H2 type zinc finger transcription factor
CNAG_03527 8 HEL2 cytoplasmic protein
CNAG_03636 2 N/A DNA-directed RNA polymerase I and III subunit RPAC2
CNAG_03710 2 ECM22 hypothetical protein
CNAG_03894 2 PDR802 Putative Zn2-Cys6 zinc-finger transcription factor
CNAG_03902 2 RDS2 Regulator of drug sensitivity 2, putative
CNAG_03998 2 RLM1 MADS-box transcription factor
CNAG_04090 2 ATF1 activating transcription factor
CNAG_04176 9 HSF2 hypothetical protein
129
CNAG_04263 9 BZP2 hypothetical protein
CNAG_04303 9 N/A E3 ubiquitin-protein ligase bre1
CNAG_04316 9 UTR1 NAD kinase
CNAG_04345 9 ARO8001 specific RNA polymerase II transcription factor
CNAG_04353 9 CLR1 hypothetical protein, variant
CNAG_04372 9 N/A DNA-directed RNA polymerase I, II, and III subunit RPABC2
CNAG_04398 9 ARO80 specific RNA polymerase II transcription factor
CNAG_04586 10 HOB7 LIM-homeobox protein
CNAG_04588 10 ERT1 hypothetical protein
CNAG_04630 10 YAP2 hypothetical protein
CNAG_04798 10 N/A regulatory protein Cys-3
CNAG_04836 10 FZC10 nuclear protein
CNAG_04837 10 MLN1 bHLH family transcription factor
CNAG_04841 10 FZC43 transcriptional regulatory protein, variant
CNAG_04864 10 CIR1 iron regulator 1
CNAG_04878 10 FZC1 hypothetical protein, variant
CNAG_04925 10 N/A nuclear protein
CNAG_04933 4 N/A hypothetical protein, variant 2
CNAG_05010 4 ZFC7 hypothetical protein
130
CNAG_05049 4 PIP201 hypothetical protein
CNAG_05067 4 CLR5 hypothetical protein
CNAG_05153 4 GAT5 hypothetical protein
CNAG_05170 4 PIP2 hypothetical protein
CNAG_05221 4 H2A-4 histone H2A.Z
CNAG_05222 4 NRG1 transcriptional regulator Nrg1
CNAG_05392 14 ZAP104 specific RNA polymerase II transcription factor
CNAG_05420 14 USV101 Nutrient and stress factor 1, putative
CNAG_05431 14 RIM101 pH-response transcription factor pacC/RIM101
CNAG_05442 14 N/A TATA-box-binding protein
CNAG_05479 14 N/A Fungal specific transcription factor, putative
CNAG_05761 7 N/A cyclin-dependent kinase regulatory subunit CKS1
CNAG_05785 7 STB4 putative transcription factor
CNAG_06005 12 N/A U3 small nucleolar RNA-associated protein 4
CNAG_06203 12 MAT2 HMG-box transcription factor, putative
131
CNAG_06276 13 CEP3 Centromere DNA-binding protein complex CBF3 subunit B,

putative
CNAG_06327 13 MIG1 DNA-binding protein creA
CNAG_06425 13 PPR1 fungal specific transcription factor
CNAG_06641 7 N/A DNA-directed RNA polymerase III subunit RPC6
CNAG_06734 2 N/A DNA-directed RNA polymerase I, II, and III subunit rpabc5
CNAG_06818 5 HAP1 hypothetical protein
CNAG_07435 5 HAP2 Transcriptional activator HAP2, putative
CNAG_07460 2 N/A heat shock transcription factor
CNAG_07494 2 N/A U3 small nucleolar RNA-associated protein MPP10
CNAG_07532 3 PHO23 hypothetical protein
CNAG_07545 3 N/A nuclear protein
CNAG_07590 11 N/A V-type H -transporting ATPase subunit C
CNAG_07680 7 HAP5 transcriptional activator hap5
132
CNAG_07922 13 FZC4 transcription factor
CNAG_07924 13 MCM1 Pheromone receptor transcription factor, putative
Table A3. List of differentially expressed genes (DEGs) in C. neoformans on

acetate-grown versus glucose-grown conditions
Gene log2 Fold-

Gene Annotation Symbol Chromosome Change FDR
xylulose 5-phosphate/fructose
CNAG_06923 6-phosphate phosphoketolase XFP2 3 -4.900496595 0
CNAG_05079 hypothetical protein N/A 4 -3.91100802 6.10E-15

Myo-inositol-1-phosphate
CNAG_01539 synthase N/A 11 -3.884280467 1.69E-261
CNAG_03759 conidiation-specific protein 6 N/A 2 -3.494376442 6.76E-16
alcohol dehydrogenase,
CNAG_07745 propanol-preferring MPD1 8 -3.292011686 1.18E-193
CNAG_04313 NADPH2 dehydrogenase N/A 9 -3.0553792 8.97E-163
CNAG_02692 nitroreductase N/A 3 -3.00643794 8.88E-162
CNAG_02418 asparagine-tRNA ligase N/A 6 -2.99493202 1.78E-299
133
ketol-acid reductoisomerase,
CNAG_05725 mitochondrial N/A 7 -2.89523887 1.73E-149
CNAG_01562 pr4/barwin domain protein BLP4 11 -2.849443331 7.93E-145
CNAG_03983 oxidoreductase N/A 2 -2.764136537 4.95E-219
glucose-methanol-choline
CNAG_05258 oxidoreductase SMG1 4 -2.753581817 1.05E-66
CNAG_00749 alternative sulfate transporter N/A 1 -2.733300095 1.15E-66
CNAG_05458 endo-1,3(4)-beta-glucanase N/A 14 -2.647163092 1.60E-125
CNAG_04659 Pyruvate decarboxylase PDC1 10 -2.640566552 1.33E-184
CNAG_03482 Thiol peroxidase TSA1 8 -2.539433406 5.94E-55
CNAG_03438 hexose transporter HXT1 8 -2.501359805 9.23E-144
transglycosylase SLT domain-

CNAG_04861 containing protein N/A 10 -2.449181211 2.91E-48
6-phosphogluconate
dehydrogenase,
CNAG_07561 decarboxylating 1 N/A 3 -2.431142436 1.95E-88
CNAG_02542 fructosamine kinase IRK2 6 -2.425279714 8.50E-56
CNAG_04652 enoyl reductase N/A 10 -2.326270665 1.76E-80

phosphoglycerate
CNAG_00774 dehydrogenase N/A 1 -2.320407504 3.88E-102
CNAG_01464 Flavohemoglobin FHB1 11 -2.317039085 1.70E-49
CNAG_01960 efflux protein EncT N/A 11 -2.313456416 6.47E-57
134
CNAG_01907 PLK/PLK1 protein kinase N/A 11 -2.244250572 1.69E-40
CNAG_04347 Aspartate kinase N/A 9 -2.241789382 1.03E-158
CNAG_05379 regucalcin N/A 14 -2.211205825 6.16E-75

small subunit ribosomal protein
CNAG_04114 S0 N/A 9 -2.199848081 2.32E-50
CNAG_07445 transketolase N/A 5 -2.170823672 1.27E-54
CNAG_00799 cellulase N/A 1 -2.169536781 3.35E-61
large subunit ribosomal protein

CNAG_03577 LP0 N/A 8 -2.155719974 5.55E-69
CNAG_01954 aldo-keto reductase N/A 11 -2.155508762 7.61E-60
solute carrier family 25

(mitochondrial phosphate
CNAG_06377 transporter), member 3 N/A 13 -2.152642318 3.17E-72
L-aminoadipate-semialdehyde
CNAG_03588 dehydrogenase LYS2 8 -2.152612104 5.88E-188
0.019946052
CNAG_04551 hypothetical protein N/A 10 -2.152320791 42
CNAG_01896 alcohol dehydrogenase (NADP) N/A 11 -2.146751373 1.19E-74

C-4 methyl sterol oxidase,
CNAG_01737 putative ERG25 11 -2.14256466 3.93E-103
CNAG_03621 Cyclophilin A CPA2 2 -2.127098841 1.83E-103
CNAG_01744 phosphatase HAD1 11 -2.126660815 4.36E-77
small subunit ribosomal protein

CNAG_02754 S12e N/A 3 -2.111585423 1.84E-118
CNAG_05623 chorismate synthase N/A 14 -2.062610548 1.17E-155
fructose-bisphosphate aldolase
CNAG_06770 1 FBA1 2 -2.061906962 7.67E-97
CNAG_00515 mannitol dehydrogenase N/A 1 -2.056718088 4.69E-46
135
CNAG_00519 lathosterol oxidase ERG3 1 -2.030366682 2.58E-76
Large subunit ribosomal

CNAG_06447 protein L17, putative RPL17 13 -2.005085008 1.11E-127
CNAG_05252 chaperone regulator N/A 4 -2.005078939 1.78E-79
large subunit ribosomal protein

CNAG_02928 L5e N/A 3 -2.002523096 9.01E-82
CNAG_05641 hypothetical protein, variant N/A 14 -2.000948487 2.24E-18
CNAG_00171 peroxin-2 N/A 1 2.002408397 2.54E-89

3-hydroxybutyryl-CoA
CNAG_03134 dehydrogenase HAD2 8 2.006338715 1.23E-53
CNAG_06672 formate dehydrogenase N/A 7 2.019028288 5.82E-38

wor1/pac2 family transcription
CNAG_05835 factor LIV3 7 2.020214829 1.01E-114
BNR/Asp-box repeat family
CNAG_02255 protein N/A 6 2.027277009 2.77E-61
CNAG_06000 glycoprotein N/A 12 2.029189017 9.97E-42

Glycine cleavage system T
CNAG_02818 protein N/A 3 2.037473869 5.18E-181
CNAG_06499 hypothetical protein DPP101 13 2.040968099 2.72E-111
CNAG_00284 efflux protein EncT N/A 1 2.051495086 1.00E-40
CNAG_06188 hypothetical protein FZC15 12 2.051555525 1.23E-43
CNAG_04892 hypothetical protein, variant 4 N/A 10 2.057446252 6.20E-59
2-oxoisovalerate
dehydrogenase E1 component,
CNAG_02284 alpha subunit N/A 6 2.059866905 8.43E-78
bZip transcription factor,
CNAG_03346 putative BZP4 8 2.060783343 7.19E-57
myo-inositol transporter,
CNAG_05381 putative ITR3C 14 2.065739418 9.34E-37
136
CNAG_03279 hypothetical protein CCD4 8 2.071978705 6.91E-79
multidrug resistance protein
CNAG_05718 fnx1 N/A 7 2.073586105 9.63E-64
AAT family amino acid
CNAG_03051 polyamine transporter N/A 3 2.098521697 2.80E-74

3-hydroxyacyl-CoA
CNAG_04308 dehydrogenase HAD1 9 2.103182554 9.28E-98
CNAG_03133 UDP-glucose,sterol transferase ATG2602 8 2.111181869 4.92E-101
CNAG_01601 lipase N/A 11 2.125510186 1.15E-94
CNAG_00488 hypothetical protein, variant N/A 1 2.126659399 2.94E-57
CNAG_06186 hypothetical protein, variant 2 N/A 12 2.139409259 3.82E-84
CNAG_07367 amino acid transporter, variant N/A 1 2.147164551 1.15E-33
CNAG_05266 membrane protein N/A 4 2.169742691 1.19E-36
CNAG_03960 Gly-Xaa carboxypeptidase N/A 2 2.174955689 1.79E-141
CNAG_04937 peroxin-12 N/A 4 2.199480608 8.13E-88
137
CNAG_07779 D-glycerate 3-kinase TDA10 9 2.205650117 5.10E-43
CNAG_02939 peroxin-3 N/A 3 2.206201302 1.33E-121

0.0211717349
CNAG_00446 hypothetical protein N/A 1 2.216615026 1
MFS transporter, SP family,

general alpha glucoside:H
CNAG_06259 symporter N/A 13 2.221697213 4.28E-52
CNAG_06241 acidic laccase CFO1 12 2.22727825 7.13E-83
CNAG_00524 acetyl-CoA acyltransferase 2 N/A 1 2.230319617 9.46E-135
CNAG_02777 phosphate:H symporter, variant PHO84 3 2.235545414 2.05E-09
CNAG_04346 dihydrodipicolinate synthase N/A 9 2.238276473 7.54E-60

ornithine-oxo-acid
CNAG_05134 transaminase N/A 4 2.241942301 7.64E-110
CNAG_06651 amidohydrolase N/A 7 2.244280371 5.90E-30

(peroxisomal adenine
nucleotide transporter),
CNAG_03242 member 17 N/A 8 2.249113409 2.69E-81
protein-L-isoaspartate O-
CNAG_03837 methyltransferase N/A 2 2.264403293 7.91E-60
0.006272125
CNAG_03741 hypothetical protein FZC31 2 2.265537065 768
glyceraldehyde-3-phosphate
CNAG_04523 dehydrogenase, type I N/A 9 2.281267594 9.36E-153
CNAG_04038 MFS quinate transporter QutD N/A 2 2.289231867 3.47E-79
138
CNAG_00456 Identified spore protein 6 ISP6 1 2.332279337 3.51E-110
CNAG_05042 carnitine acetyltransferase N/A 4 2.339243859 9.32E-136
CNAG_05293 glycogenin glucosyltransferase N/A 4 2.353347363 2.03E-55
CNAG_02096 peroxin-16 N/A 6 2.367732307 1.17E-77
CNAG_04096 racemase N/A 2 2.40182565 1.68E-126
CNAG_05115 sarcosine oxidase N/A 4 2.406894282 9.41E-91
CNAG_06817 uric acid xanthine permease UAP1 5 2.413959934 1.46E-110

peroxisome targeting signal
CNAG_01926 receptor PEX5 11 2.424142535 1.99E-146
CNAG_04531 Enoyl-CoA hydratase N/A 9 2.432157388 2.46E-129
CNAG_06568 RAN protein kinase SKS1 7 2.433420944 3.38E-88
CNAG_06448 Cystathionine gamma-lyase N/A 13 2.437034447 7.63E-64
CNAG_04291 glycosyl-hydrolase N/A 9 2.44488372 2.31E-117
CNAG_03697 hormone-sensitive lipase N/A 2 2.45694956 1.94E-194
CNAG_00442 cyclin N/A 1 2.465150614 1.41E-123
CNAG_04869 para-nitrobenzyl esterase PNB1 10 2.471842926 2.99E-117
CNAG_00269 Sorbitol dehydrogenase N/A 1 2.472085629 7.34E-54
139
CNAG_06776 membrane protein N/A 2 2.485269115 2.00E-59
CNAG_03199 FAD dependent oxidoreductase N/A 8 2.500247586 7.11E-96
CNAG_05803 exo-beta-1,3-glucanase N/A 7 2.501116431 3.85E-120
CNAG_07403 Peroxisomal ATPase PEX1 PEX1 5 2.50683658 9.57E-158
CNAG_01952 aryl-alcohol dehydrogenase N/A 11 2.509646461 5.96E-67
Protein required for cytokinesis

CNAG_01983 and virulence 1 RCV1 11 2.524657503 1.67E-115
cardiolipin-specific
CNAG_02505 phospholipase N/A 6 2.52513257 1.47E-50
CNAG_04795 adenine nucleotide transporter N/A 10 2.528076177 3.98E-97

CNAG_00904 aflatoxin efflux pump AFLT N/A 5 2.533320701 2.99E-10

1,4-alpha-glucan-branching
CNAG_00393 enzyme N/A 1 2.535777799 2.86E-184
malate dehydrogenase
(oxaloacetate-
CNAG_06374 decarboxylating)(NADP) N/A 13 2.564851405 8.46E-69
CNAG_07600 Beta-glucosidase N/A 11 2.565018508 5.55E-137
CNAG_06433 AMP-binding protein N/A 13 2.571617498 8.00E-197
ATP-binding cassette, subfamily

D (ALD), peroxisomal long-chain
CNAG_02764 fatty acid import protein PXA2 3 2.582242246 5.34E-143
CNAG_04794 spermine transporter N/A 10 2.584536287 1.19E-135
CNAG_00797 acetyl-CoA synthetase N/A 1 2.593962989 1.06E-135
CNAG_00896 transcription factor FZC34 5 2.628890692 9.44E-74
140
2-oxoisovalerate
dehydrogenase E2 component
CNAG_00484 (dihydrolipoyl transacylase) N/A 1 2.659748528 8.88E-180

CNAG_00796 B (MDR/TAP), member 1 MDR1 1 2.664776154 4.21E-53

0.000177492
CNAG_05300 mitochondrial carrier protein N/A 4 2.730932228 1.93E-140
CNAG_06913 L-serine ammonia-lyase N/A 3 2.744533923 2.68E-146
CNAG_06561 allantoate transporter N/A 7 2.745084933 6.78E-57
CNAG_05119 GABA permease N/A 4 2.758643737 1.60E-87
extracellular elastinolytic
CNAG_04735 metalloproteinase MEP1 10 2.759144137 5.75E-151
CNAG_01116 beta-ketoacyl reductase N/A 5 2.767779821 4.50E-79
CNAG_05991 glycosyl hydrolase family 88 N/A 12 2.774307079 2.36E-176
CNAG_02473 endoplasmic reticulum protein N/A 6 2.780784919 4.57E-184
CNAG_00047 pyruvate dehydrogenase kinase PKP1 1 2.818316495 1.03E-112
CNAG_04093 putative transcription factor YRM103 2 2.821424664 9.87E-138
141

CNAG_00028 transporter, variant N/A 1 2.833517394 1.41E-167
CNAG_06172 transketolase N/A 12 2.848885737 9.10E-75


Uracil
CNAG_02344 phosphoribosyltransferase N/A 6 2.909303709 4.74E-132
CNAG_03729 peroxin-5 N/A 2 2.91316122 3.71E-263
CNAG_05299 oxidoreductase N/A 4 2.914464089 8.17E-75

glycogen(starch) synthase,
CNAG_04621 variant N/A 10 2.940035403 5.84E-184
CNAG_06009 cyclohydrolase N/A 12 2.962930691 1.30E-190

CNAG_00864 putative ITR2 5 3.035563112 9.98E-182
CNAG_05639 hypothetical protein PPS1 14 3.038252281 0
peroxisomal 2,4-dienoyl-CoA
CNAG_04238 reductase N/A 9 3.041104459 6.05E-230
CNAG_00637 Cystathionine beta-synthase N/A 1 3.059496886 5.75E-290
CNAG_00141 monooxygenase N/A 1 3.082581858 4.71E-45
CNAG_06574 antiphagocytic protein 1 APP1 7 3.088293978 3.40E-122
CNAG_02049 Proline dehydrogenase PUT1 6 3.09586931 7.34E-92
142
D (ALD), peroxisomal long-chain
CNAG_00651 fatty acid import protein N/A 1 3.10426851 0

hydroxymethylglutaryl-CoA
CNAG_03067 lyase N/A 3 3.176153974 2.07E-274

NAD-dependent
CNAG_00094 epimerase/dehydratase N/A 1 3.227931305 1.14E-261

phosphoenolpyruvate
CNAG_04217 carboxykinase (ATP) PCK1 9 3.26714125 6.60E-163
CNAG_07747 acyl-CoA oxidase N/A 8 3.284455701 2.29E-250
CNAG_02254 quinate permease LPI12 6 3.294651913 9.77E-295
2-oxoisovalerate
dehydrogenase E1 component,
CNAG_00397 beta subunit N/A 1 3.327788507 4.05E-204
CNAG_02225 glucan 1,3-beta-glucosidase EXG104 6 3.371935116 2.52E-249
CNAG_07944 Urea carboxylase N/A 13 3.394992027 0
CNAG_05031 3-oxoacid CoA-transferase N/A 4 3.444662639 0
CNAG_02165 cytoplasmic protein N/A 6 3.468757517 1.26E-167
alpha-ketoglutarate-dependent
2,4- dichlorophenoxyacetate
CNAG_04417 dioxygenase N/A 9 3.539262119 3.83E-88
143
CNAG_06628 aldehyde dehydrogenase (NAD) N/A 7 3.554003489 2.07E-269
acetyl/propionyl CoA
CNAG_01671 carboxylase N/A 11 3.563038208 0
CNAG_04392 sterol-binding protein MFE2 9 3.64229792 9.91E-116
CNAG_01551 GATA family transcription factor GAT201 11 3.675353875 5.24E-221

(mitochondrial
carnitine/acylcarnitine
CNAG_00499 transporter), member 20/29 N/A 1 3.69160675 0
aldehyde dehydrogenase family

CNAG_00735 7 member A1 N/A 1 3.713063302 1.61E-259
CNAG_05644 2-Nitropropane dioxygenase N/A 14 3.729505188 0
phosphotransferase enzyme
CNAG_02295 family protein N/A 6 3.785156862 1.02E-301
CNAG_03019 long-chain acyl-CoA synthetase N/A 3 3.790271235 0
CNAG_05685 neutral amino acid transporter N/A 7 3.83528674 0
thiosulfate/3-mercaptopyruvate
CNAG_01252 sulfurtransferase N/A 5 3.839575537 1.09E-151

CNAG_06527 symporter, variant 2 N/A 7 3.840821525 4.20E-149
CNAG_00826 dihydroxyacetone kinase DAK101 1 3.981063756 0

multifunctional beta-oxidation
CNAG_05721 protein MFE2 7 4.022808323 0
specific RNA polymerase II

CNAG_04345 transcription factor ARO8001 9 4.057102724 0
taurine catabolism dioxygenase
CNAG_01542 TauD N/A 11 4.087034641 1.74E-305
144
0.04074920
CNAG_05229 Stomatin family protein N/A 4 4.133932227 1.17E-232
CNAG_05785 putative transcription factor STB4 7 4.142636148 2.09E-218
CNAG_00490 acetyl-CoA acyltransferase N/A 1 4.150523898 0
CNAG_02147 cytochrome c peroxidase N/A 6 4.190300827 4.20E-208
CNAG_03243 2-Nitropropane dioxygenase N/A 8 4.194067019 2.51E-143
CNAG_03010 enoyl-CoA hydratase/isomerase N/A 3 4.251823055 0

0.007701188
CNAG_04931 Putative hexose transporter HXS2 10 4.37816534 705
CNAG_00452 Isovaleryl-CoA dehydrogenase N/A 1 4.381863876 3.22E-133

0.045335569
CNAG_00597 amino acid transporter DIP5 1 4.518136614 0
alpha-aminoadipic
CNAG_00247 semialdehyde synthase LYS9 1 4.647012781 0
3-methylcrotonyl-CoA
CNAG_01680 carboxylase alpha subunit N/A 11 4.653411548 0
CNAG_05913 Alpha-glucosidase N/A 7 4.759365762 0

0.007461162
CNAG_02045 acetoacetate-CoA ligase N/A 6 4.784163223 0
145
MFS transporter, SHS family,

CNAG_04704 lactate transporter N/A 10 4.877479239 4.20E-260
CNAG_06431 acyl-CoA oxidase N/A 13 4.916807567 0
CNAG_03666 acyl-CoA dehydrogenase N/A 2 4.942071419 0
CNAG_05653 malate synthase A MLS1 14 4.950928228 0
CNAG_00827 ribose 5-phosphate isomerase N/A 1 4.99834915 1.33E-143
CNAG_05310 nipsnap family protein N/A 4 5.003188347 1.07E-195
CNAG_00879 Glutamate dehydrogenase N/A 5 5.152721407 0
CNAG_04142 tartrate transporter N/A 9 5.329645146 0

0.001460120
CNAG_07862 fumarate reductase N/A 10 5.402443646 0
CNAG_05387 galactose transporter N/A 14 5.431201544 0
CNAG_00598 permease N/A 1 5.436810579 0

CNAG_04837 bHLH family transcription factor MLN1 10 5.658104561 0

(mitochondrial citrate
CNAG_02288 transporter), member 1 N/A 6 5.725525264 0

CNAG_03910 putative ITR6 2 6.517998365 0
alcohol dehydrogenase,
CNAG_02489 propanol-preferring N/A 6 6.655716374 0
CNAG_05303 isocitrate lyase ICL1 4 6.900794065 0
146
CNAG_05867 L-fucose transporter N/A 7 7.624377564 0

CNAG_05914 symporter N/A 7 7.804574725 0
CNAG_05662 Polyol transporter protein 1 PTP1 14 7.859393735 0

high-affinity glucose
CNAG_03772 transporter HXS1 2 8.11764169 0
Notes:
1. List contains DEGs of the nuclear genome
2. The 5th column is color coded: yellow for downregulated genes and red for
upregulate genes
147
APPENDIX B
Putative acetate kinases of eukaryotic origin
Table B1. List of putative eukaryotic acetate kinases identified in the study
UniProt Length Species Taxon

accession (amino
acid)
A0A2P6P0X2 428 Planoprotostelium fungivorum Amoebozoa
A0A0A1U119 394 Entamoeba invadens IP1 Amoebozoa
C4M1C3 392 Entamoeba histolytica Amoebozoa
A0A0F4GWL1 420 Zymoseptoria brevis Ascomycota
A0A165G327 414 Xylona heveae (strain CBS 132557 / TC161) Ascomycota
A0A3M9Y9Y9 415 Verticillium nonalfalfae Ascomycota
A0A0D2A747 421 Verruconis gallopava Ascomycota
A0A370U2T7 457 Venustampulla echinocandica Ascomycota
A0A4Z1NHJ3 426 Venturia nashicola Ascomycota
A0A8H3U544 426 Venturia inaequalis (Apple scab fungus) Ascomycota
A0A517LHE8 426 Venturia effusa Ascomycota
A0A423W2V6 472 Valsa sordida Ascomycota
A0A423X684 460 Valsa malicola Ascomycota
A0A194UQZ8 447 Valsa mali var. pyri Ascomycota
A0A8E5MF26 420 Ustilaginoidea virens (Rice false smut fungus) Ascomycota

(Villosiclava virens)
148
C4JZC3 417 Uncinocarpus reesii (strain UAMH 1704) Ascomycota
A0A178EUJ9 410 Trichophyton rubrum (Athlete's foot fungus) Ascomycota

(Epidermophyton rubrum)
A0A059J4T7 401 Trichophyton interdigitale (strain MR816) Ascomycota
A0A2T4CH84 415 Trichoderma longibrachiatum ATCC 18648 Ascomycota
A0A0F9WTJ8 804 Trichoderma harzianum (Hypocrea lixii) Ascomycota
A0A2P4Z935 417 Trichoderma gamsii Ascomycota
A0A2T3ZQT0 417 Trichoderma asperellum CBS 433.97 Ascomycota
A0A395NJV2 417 Trichoderma arundinaceum Ascomycota
A0A0A1TI16 417 Torrubiella hemipterigena Ascomycota
A0A2S4L229 414 Tolypocladium paradoxum Ascomycota
A0A0L0N8L1 1157 Tolypocladium ophioglossoides (strain CBS 100239) Ascomycota

(Elaphocordyceps ophioglossoides)
A0A507BFA5 1602 Thyridium curvatum Ascomycota
A0A0F4Z849 414 Thielaviopsis punctulata Ascomycota
G2R1K7 436 Thermothielavioides terrestris (strain ATCC 38088 / Ascomycota

NRRL 8126) (Thielavia terrestris)
149
R4XA18 415 Taphrina deformans (strain PYCC 5710 / ATCC 11124 / Ascomycota
CBS 356.35 / IMI 108563 / JCM 9778 / NBRC 8474)
(Peach leaf curl fungus) (Lalaria deformans)
B8M7Z6 415 Talaromyces stipitatus (strain ATCC 10500 / CBS 375.48 Ascomycota
/ QM 6759 / NRRL 1006) (Penicillium stipitatum)
A0A7H8QRV8 420 Talaromyces rugulosus Ascomycota
B6Q5T9 452 Talaromyces marneffei (strain ATCC 18224 / CBS 334.59 Ascomycota
/ QM 7333) (Penicillium marneffei)
A0A0U1LTG9 421 Talaromyces islandicus (Penicillium islandicum) Ascomycota
A0A1Q5Q8B9 413 Talaromyces atroroseus Ascomycota
A0A364KLK3 415 Talaromyces amestolkiae Ascomycota
A0A364N0R6 419 Stemphylium lycopersici Ascomycota
A0A178AIA2 419 Stagonospora sp. SRC1lsM3a Ascomycota
A0A084QIB7 416 Stachybotrys chlorohalonata (strain IBT 40285) Ascomycota
U7Q038 443 Sporothrix schenckii (strain ATCC 58251 / de Perez Ascomycota

2211183) (Rose-picker's disease fungus)
150
A0A162I8M3 423 Sporothrix insectorum RCEF 264 Ascomycota
M3B0H8 421 Sphaerulina musiva (strain SO2202) (Poplar stem Ascomycota

canker fungus) (Septoria musiva)
F7W1G1 461 Sordaria macrospora (strain ATCC MYA-333 / DSM 997 / Ascomycota
K(L3346) / K-hell)
A0A3N2PRN0 415 Sodiomyces alkalinus F11 Ascomycota
R0ICZ8 419 Setosphaeria turcica (strain 28A) (Northern leaf blight Ascomycota
fungus) (Exserohilum turcicum)
A0A425BU85 396 Scytalidium sp. 3C Ascomycota
A0A8H2VYZ8 419 Sclerotinia trifoliorum Ascomycota
W9CR99 423 Sclerotinia borealis (strain F-4128) Ascomycota
A0A4U0U5S2 421 Salinomyces thailandica Ascomycota
A0A0E9N8B6 510 Saitoella complicata (strain BCRC 22490 / CBS 7301 / Ascomycota
JCM 7358 / NBRC 10748 / NRRL Y-17804)
A0A2S7PA73 428 Rutstroemia sp. NJR-2017a BVV2 Ascomycota
A0A1E1K2L5 417 Rhynchosporium commune Ascomycota
A0A0D2ITC7 412 Rhinocladiella mackenziei CBS 650.93 Ascomycota
A0A2D3VN54 424 Ramularia collo-cygni Ascomycota
151
B2WH19 412 Pyrenophora tritici-repentis (strain Pt-1C-BFP) (Wheat Ascomycota
tan spot fungus) (Drechslera tritici-repentis)
E3RRQ8 419 Pyrenophora teres f. teres (strain 0-1) (Barley net blotch Ascomycota
fungus) (Drechslera teres f. teres)
A0A178DWV0 419 Pyrenochaeta sp. DS3sAY3a Ascomycota
A0A179HNH1 414 Purpureocillium lilacinum (Paecilomyces lilacinus) Ascomycota
A0A1B8GHL8 419 Pseudogymnoascus verrucosus Ascomycota
L8FPJ8 419 Pseudogymnoascus destructans (strain ATCC MYA- Ascomycota

4855 / 20631-21) (Bat white-nose syndrome fungus)
(Geomyces destructans)
A0A8H6VPD4 419 Pseudocercospora fuligena Ascomycota
M3AJ32 419 Pseudocercospora fijiensis (strain CIRAD86) (Black leaf Ascomycota

streak disease fungus) (Mycosphaerella fijiensis)
A0A139GYJ5 419 Pseudocercospora eumusae Ascomycota
A0A1Y2FSE6 408 Protomyces lactucae-debilis Ascomycota
A0A2B7YE97 415 Polytolypa hystricis UAMH7299 Ascomycota
152
B2ALS8 437 Podospora anserina (strain S / ATCC MYA-4624 / DSM Ascomycota
980 / FGSC 10383) (Pleurage anserina)
A0A179FGW7 415 Pochonia chlamydosporia 170 Ascomycota
A0A0N0NP39 407 Phialophora attinorum Ascomycota
A0A0D2E2K0 418 Phialophora americana Ascomycota
A0A1L7X722 416 Phialocephala subalpina Ascomycota
Q0U2Y7 419 Phaeosphaeria nodorum (strain SN15 / ATCC MYA- Ascomycota

4574 / FGSC 10173) (Glume blotch fungus)
(Parastagonospora nodorum)
A0A0G2GIQ0 413 Phaeomoniella chlamydospora Ascomycota
R8BCA5 433 Phaeoacremonium minimum (strain UCR-PA7) (Esca Ascomycota

disease fungus) (Togninia minima)
A0A5N6FI44 409 Petromyces alliaceus (Aspergillus alliaceus) Ascomycota
W3XNS0 422 Pestalotiopsis fici (strain W106-1 / CGMCC3.15140) Ascomycota
A0A2V1D5Z2 419 Periconia macrospinosa Ascomycota
A0A1V6SBD3 403 Penicillium vulpinum Ascomycota
A0A1Q5TGI6 418 Penicillium subrubescens Ascomycota
A0A1V6T0M6 414 Penicillium steckii Ascomycota
153
A0A1V6RQP5 389 Penicillium solitum Ascomycota
B6HBY2 437 Penicillium rubens (strain ATCC 28089 / DSM 1075 / Ascomycota
NRRL 1951 / Wisconsin 54-1255) (Penicillium
chrysogenum)
A0A1V6P4I6 414 Penicillium polonicum Ascomycota
A0A135LIQ9 421 Penicillium patulum (Penicillium griseofulvum) Ascomycota
S7ZIS8 414 Penicillium oxalicum (strain 114-2 / CGMCC 5302) Ascomycota

(Penicillium decumbens)
A0A0M8NYD5 414 Penicillium nordicum Ascomycota
A0A1V6Z6R9 463 Penicillium nalgiovense Ascomycota
A0A0A2KAM9 395 Penicillium italicum (Blue mold) Ascomycota
A0A101MGX6 414 Penicillium freii Ascomycota
A0A1V6SPN5 431 Penicillium flavigenum Ascomycota
A0A0A2KEA4 459 Penicillium expansum (Blue mold rot fungus) Ascomycota
A0A1V6PMA8 414 Penicillium decumbens Ascomycota
A0A1V6V7D8 403 Penicillium coprophilum Ascomycota
A0A0F7TI52 414 Penicillium brasilianum Ascomycota
A0A1F5LCL1 423 Penicillium arizonense Ascomycota
A0A1V6QLJ1 407 Penicillium antarcticum Ascomycota
154
A0A1L9SX51 416 Penicilliopsis zonata CBS 506.65 Ascomycota
A0A6H0Y0H4 419 Peltaster fructicola Ascomycota
A0A177BWQ2 418 Paraphaeosphaeria sporulosa Ascomycota
C1H8T9 439 Paracoccidioides lutzii (strain ATCC MYA-826 / Pb01) Ascomycota

(Paracoccidioides brasiliensis)
S3CRH5 435 Ophiostoma piceae (strain UAMH 11346) (Sap stain Ascomycota
fungus)
A0A2C5XTH9 413 Ophiocordyceps australis Ascomycota
A0A0C3CSB4 417 Oidiodendron maius Zn Ascomycota
G4U9W7 469 Neurospora tetrasperma (strain FGSC 2509 / P0656) Ascomycota
Q7SH17 469 Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / Ascomycota

CBS 708.71 / DSM 1257 / FGSC 987)
A1D6Z2 425 Neosartorya fischeri (strain ATCC 1020 / DSM 3700 / Ascomycota
CBS 544.65 / FGSC A1164 / JCM 1740 / NRRL 181 / WB 181)
(Aspergillus fischerianus)
A0A0P7BCD8 842 Neonectria ditissima Ascomycota
A0A6A6Y5A7 417 Mytilinidion resinicola Ascomycota
155
G2Q2D7 447 Myceliophthora thermophila (strain ATCC 42464 / Ascomycota
BCRC 31852 / DSM 1799) (Sporotrichum thermophile)
A0A3N4L0W6 435 Morchella conica CCBAS932 Ascomycota
A0A4Q4T869 415 Monosporascus sp. MC13-8B Ascomycota
A0A4Q4TS32 415 Monosporascus ibericus Ascomycota
A0A5N6JU89 470 Monilinia laxa (Brown rot fungus) (Sclerotinia laxa) Ascomycota
A0A5M9JCQ9 501 Monilinia fructicola (Brown rot fungus) (Ciboria Ascomycota

fructicola)
A0A507QMX6 421 Monascus purpureus (Red mold) (Monascus anka) Ascomycota
A0A194XKF5 416 Mollisia scopiformis Ascomycota
A0A168B4M1 416 Moelleriella libera RCEF 2490 Ascomycota
A0A136IWQ0 434 Microdochium bolleyi Ascomycota
A0A162J5H1 415 Metarhizium rileyi (strain RCEF 4871) (Nomuraea rileyi) Ascomycota
A0A0D9PCA3 416 Metarhizium anisopliae BRIP 53293 Ascomycota
A0A0B2WYL4 416 Metarhizium album (strain ARSEF 1941) Ascomycota
E9EB84 416 Metarhizium acridum (strain CQMa 102) Ascomycota
A0A7C8HY93 440 Massariosphaeria phaeospora Ascomycota
156
A0A218Z4D0 553 Marssonina coronariae Ascomycota
K1X5B9 404 Marssonina brunnea f. sp. multigermtubi (strain Ascomycota

MB_m1) (Marssonina leaf spot fungus)
G4MPH1 432 Magnaporthe oryzae (strain 70-15 / ATCC MYA-4617 / Ascomycota

FGSC 8958) (Rice blast fungus) (Pyricularia oryzae)
A0A6P8B9W0 432 Magnaporthe grisea (Crabgrass-specific blast fungus) Ascomycota

(Pyricularia grisea)
A0A175W805 423 Madurella mycetomatis Ascomycota
K2RPL2 443 Macrophomina phaseolina (strain MS6) (Charcoal rot Ascomycota

fungus)
A0A1E3QE05 419 Lipomyces starkeyi NRRL Y-11557 Ascomycota
A0A8H6CKG4 412 Letharia lupina Ascomycota
E5A190 425 Leptosphaeria maculans (strain JN3 / isolate v23.1.3 / Ascomycota

race Av1-4-5-6-7-8) (Blackleg fungus) (Phoma lingam)
A0A8E2E485 421 Lepidopterella palustris CBS 459.81 Ascomycota
A0A5N5DLW6 422 Lasiodiplodia theobromae Ascomycota
A0A559M5D1 406 Lachnellula willkommii Ascomycota
A0A8H8RTH2 417 Lachnellula subtilissima Ascomycota
157
A0A8H8UK53 417 Lachnellula occidentalis Ascomycota
A0A8H8QT93 417 Lachnellula hyalina Ascomycota
A0A7D8UN39 427 Lachnellula cervina Ascomycota
A0A1Y2UG58 417 Hypoxylon sp. CO27-5 Ascomycota
G9N905 417 Hypocrea virens (strain Gv29-8 / FGSC 10586) Ascomycota

(Gliocladium virens) (Trichoderma virens)
G0RDG0 415 Hypocrea jecorina (strain QM6a) (Trichoderma reesei) Ascomycota
G9P656 417 Hypocrea atroviridis (strain ATCC 20476 / IMI 206040) Ascomycota
(Trichoderma atroviride)
A0A2J6SD10 416 Hyaloscypha variabilis F Ascomycota
A0A2J6PU90 417 Hyaloscypha hepaticicola Ascomycota
A0A2J6SYZ9 417 Hyaloscypha bicolor E Ascomycota
A0A1Z5SL83 505 Hortaea werneckii EXF-2000 Ascomycota
A0A8H7Z444 421 Histoplasma ohiense (nom. inval.) Ascomycota
A0A0F7ZSK2 415 Hirsutella minnesotensis 3608 Ascomycota
A0A2B7XQ83 417 Helicocarpus griseus UAMH5409 Ascomycota
158
F0XC80 655 Grosmannia clavigera (strain kw1407 / UAMH 11150) Ascomycota
(Blue stain fungus) (Graphiocladiella clavigera)
A0A8E2ETB1 417 Glonium stellatum Ascomycota
S3DEK6 416 Glarea lozoyensis (strain ATCC 20868 / MF5171) Ascomycota
A0A0E0RQ82 415 Gibberella zeae (strain ATCC MYA-4620 / CBS 123657 / Ascomycota
FGSC 9075 / NRRL 31084 / PH-1) (Wheat head blight
fungus) (Fusarium graminearum)
A0A8H5P1M9 835 Gibberella subglutinans (Fusarium subglutinans) Ascomycota
A0A2K0WDE8 415 Gibberella nygamai (Bean root rot disease fungus) Ascomycota
(Fusarium nygamai)
W7LRF8 415 Gibberella moniliformis (strain M3125 / FGSC 7600) Ascomycota

(Maize ear and stalk rot fungus) (Fusarium
verticillioides)
S0DI41 415 Gibberella fujikuroi (strain CBS 195.34 / IMI 58289 / Ascomycota
NRRL A-6831) (Bakanae and foot rot disease fungus)
(Fusarium fujikuroi)
159
J3NJ82 468 Gaeumannomyces tritici (strain R3-111a-1) (Wheat and Ascomycota
barley take-all root rot fungus) (Gaeumannomyces
graminis var. tritici)
A0A8H4U7Z2 414 Fusarium zealandicum Ascomycota
A0A2L2TNY0 415 Fusarium venenatum Ascomycota
C7YK38 414 Fusarium vanettenii (strain ATCC MYA-4622 / CBS Ascomycota

123669 / FGSC 9596 / NRRL 45880 / 77-13-4) (Fusarium
solani subsp. pisi)
A0A395SX31 419 Fusarium sporotrichioides Ascomycota
A0A8H4SWU7 414 Fusarium sarcochroum Ascomycota
A0A8H5L2W2 415 Fusarium pseudocircinatum Ascomycota
A0A1B8B3W3 415 Fusarium poae Ascomycota
A0A8H5IME6 908 Fusarium phyllophilum Ascomycota
A0A0D2X879 541 Fusarium oxysporum f. sp. lycopersici (strain 4287 / Ascomycota

CBS 123668 / FGSC 9935 / NRRL 34936) (Fusarium
vascular wilt of tomato)
A0A8H5IJJ4 403 Fusarium napiforme Ascomycota
A0A8H6D3E6 415 Fusarium mundagurra Ascomycota
A0A395RYV7 419 Fusarium longipes Ascomycota
A0A3M2S4Z2 414 Fusarium kuroshium Ascomycota
160
A0A8H5XXI0 415 Fusarium globosum Ascomycota
A0A8H4WR54 838 Fusarium gaditjirri Ascomycota
A0A395MIC5 415 Fusarium flagelliforme Ascomycota
A0A2T4HAR1 710 Fusarium culmorum Ascomycota
A0A8H4P5B2 415 Fusarium austroafricanum Ascomycota
A0A8H4L2G8 414 Fusarium albosuccineum Ascomycota
A0A8H4NFG0 415 Fusarium acutatum Ascomycota
A0A4V6WL79 1761 Friedmanniomyces simplex Ascomycota
A0A4U0VI43 430 Friedmanniomyces endolithicus Ascomycota
A0A0D2GMI4 436 Fonsecaea pedrosoi CBS 271.37 Ascomycota
A0A0D2JUH1 451 Fonsecaea multimorphosa CBS 102226 Ascomycota
A0A178ZNV0 417 Fonsecaea erecta Ascomycota
A0A0D2EQT4 421 Exophiala xenobiotica Ascomycota
A0A0D2C375 421 Exophiala spinifera Ascomycota
A0A0D1XF34 424 Exophiala sideris Ascomycota
A0A0D2B121 421 Exophiala oligosperma Ascomycota
A0A0D1ZH21 414 Exophiala mesophila (Black yeast) Ascomycota
H6BV64 423 Exophiala dermatitidis (strain ATCC 34100 / CBS 525.76 Ascomycota
/ NIH/UT8656) (Black yeast) (Wangiella dermatitidis)
161
A0A072PV08 423 Exophiala aquamarina CBS 119918 Ascomycota
M7T3L5 418 Eutypa lata (strain UCR-EL1) (Grapevine dieback Ascomycota

disease fungus) (Eutypa armeniacae)
A0A6G1G3F4 412 Eremomyces bilateralis CBS 781.70 Ascomycota
A0A1Y2LXG4 419 Epicoccum nigrum (Soil fungus) (Epicoccum Ascomycota

purpurascens)
A0A7S9PV96 421 Epichloe festucae (strain Fl1) Ascomycota
A0A2B7ZID5 433 Emmonsia crescens Ascomycota
Q5B3G6 420 Emericella nidulans (strain FGSC A4 / ATCC 38163 / CBS Ascomycota
112.46 / NRRL 194 / M139) (Aspergillus nidulans)
A0A1J9QSB5 426 Emergomyces pasteurianus Ep9510 Ascomycota
A0A1B7P5T7 527 Emergomyces africanus Ascomycota
A0A232LPM9 414 Elaphomyces granulatus Ascomycota
N1PRG8 423 Dothistroma septosporum (strain NZE10 / CBS 128990) Ascomycota

(Red band needle blight fungus) (Mycosphaerella pini)
A0A1S8BEI3 422 Diplodia seriata Ascomycota
A0A1J9RQK1 422 Diplodia corticola Ascomycota
162
A0A162WCX8 419 Didymella rabiei (Chickpea ascochyta blight fungus) Ascomycota
(Mycosphaerella rabiei)
A0A1Y2XC97 415 Daldinia sp. EC12 Ascomycota
A0A423XIR8 446 Cytospora leucostoma Ascomycota
W2RIW9 417 Cyphellophora europaea CBS 101466 Ascomycota
A0A4U0XC36 424 Cryomyces minteri Ascomycota
A0A179I9K0 412 Cordyceps confragosa (Lecanicillium lecanii) Ascomycota
R7Z350 432 Coniosporium apollinis (strain CBS 100218) (Rock- Ascomycota

inhabiting black yeast)
A0A420XXW1 416 Coniochaeta pulveracea Ascomycota
A0A1J7J5G2 781 Coniochaeta ligniaria NRRL 30616 Ascomycota
A0A2T3ABD5 462 Coniella lustricola Ascomycota
A0A4R8RIR5 414 Colletotrichum trifolii Ascomycota
A0A161YKA7 494 Colletotrichum tofieldiae Ascomycota
A0A4U6XVE9 416 Colletotrichum tanaceti Ascomycota
A0A066Y2I4 438 Colletotrichum sublineola (Sorghum anthracnose Ascomycota

fungus)
A0A5Q4C5U8 415 Colletotrichum shisoi Ascomycota
A0A135UQL4 417 Colletotrichum salicis Ascomycota
163
A0A8H6JX88 414 Colletotrichum plurivorum Ascomycota
A0A1G4APG6 417 Colletotrichum orchidophilum Ascomycota
N4V0K9 414 Colletotrichum orbiculare (strain 104-T / ATCC 96160 / Ascomycota

CBS 514.97 / LARS 414 / MAFF 240422) (Cucumber
anthracnose fungus) (Colletotrichum lagenarium)
A0A8H6U8R8 414 Colletotrichum musicola Ascomycota
A0A167B7J0 449 Colletotrichum incanum Ascomycota
A0A4T0VN07 414 Colletotrichum higginsianum Ascomycota
E3QCU7 415 Colletotrichum graminicola (strain M1.001 / M2 / FGSC Ascomycota

10212) (Maize anthracnose fungus) (Glomerella
graminicola)
A0A8H4FI56 692 Colletotrichum gloeosporioides (Anthracnose fungus) Ascomycota

(Glomerella cingulata)
L2FYU5 415 Colletotrichum fructicola (strain Nara gc5) Ascomycota

(Anthracnose fungus) (Colletotrichum gloeosporioides
(strain Nara gc5))
A0A010RVP2 805 Colletotrichum fioriniae PJ7 Ascomycota
A0A1Q8RZ87 414 Colletotrichum chlorophyti Ascomycota
164
A0A8H3W5T5 415 Colletotrichum asianum Ascomycota
A0A3D8SRF7 419 Coleophoma cylindrospora Ascomycota
A0A8H5ZG49 419 Cochliobolus sativus (Common root rot and spot Ascomycota
blotch fungus) (Bipolaris sorokiniana)
M2UZ24 419 Cochliobolus heterostrophus (strain C5 / ATCC 48332 / Ascomycota

race O) (Southern corn leaf blight fungus) (Bipolaris
maydis)
E9D099 415 Coccidioides posadasii (strain RMSCC 757 / Silveira) Ascomycota

(Valley fever fungus)
J3KC18 415 Coccidioides immitis (strain RS) (Valley fever fungus) Ascomycota
A0A1Y1ZKL3 419 Clohesyomyces aquaticus Ascomycota
M1VYV3 415 Claviceps purpurea (strain 20.1) (Ergot fungus) Ascomycota

(Sphacelia segetum)
W9W7X7 418 Cladophialophora yegresii CBS 114405 Ascomycota
W9XCJ0 416 Cladophialophora psammophila CBS 110553 Ascomycota
A0A0D2AW14 437 Cladophialophora immunda Ascomycota
A0A1C1CVD9 476 Cladophialophora carrionii Ascomycota
A0A3M7N4L1 400 Chaetothyriales sp. CBS 132003 Ascomycota
165
G0S0H4 431 Chaetomium thermophilum (strain DSM 1495 / CBS Ascomycota
144.50 / IMI 039719)
Q2HAJ4 459 Chaetomium globosum (strain ATCC 6205 / CBS 148.51 Ascomycota
/ DSM 1962 / NBRC 6347 / NRRL 1970) (Soil fungus)
A0A2I0RV10 420 Cercospora zeina Ascomycota
A0A2S6BZ10 420 Cercospora berteroae Ascomycota
A0A0F8B793 416 Ceratocystis fimbriata f. sp. platani Ascomycota
W9YRV4 423 Capronia epimyces CBS 606.96 Ascomycota
W9ZHA7 423 Capronia coronata CBS 617.96 Ascomycota
A0A8H7WGP5 416 Cadophora malorum Ascomycota
A0A443HQM9 415 Byssochlamys spectabilis (Paecilomyces variotii) Ascomycota
A0A4Z1KSA5 420 Botrytis porri Ascomycota
A0A8H6ECL4 415 Botrytis fragariae Ascomycota
A0A4Z1HMR2 419 Botryotinia narcissicola Ascomycota
A0A384JW64 419 Botryotinia fuckeliana (strain B05.10) (Noble rot Ascomycota

fungus) (Botrytis cinerea)
A0A4Y8CXK7 407 Botryotinia calthae Ascomycota
R1GMM0 424 Botryosphaeria parva (strain UCR-NP2) (Grapevine Ascomycota

canker fungus) (Neofusicoccum parvum)
166
A0A8H4IQ47 425 Botryosphaeria dothidea Ascomycota
A0A0H1BBD6 429 Blastomyces silverae Ascomycota
A0A1J9R0J2 433 Blastomyces percursus Ascomycota
A0A2B7WQB0 422 Blastomyces parvus Ascomycota
W6Y338 419 Bipolaris zeicola 26-R-13 Ascomycota
J4VTG6 427 Beauveria bassiana (strain ARSEF 2860) (White Ascomycota

muscardine disease fungus) (Tritirachium shiotae)
M2NIJ1 422 Baudoinia panamericana (strain UAMH 10762) (Angels' Ascomycota

share fungus) (Baudoinia compniacensis (strain UAMH
10762))
A0A074YGF3 421 Aureobasidium subglaciale (strain EXF-2481) Ascomycota

(Aureobasidium pullulans var. subglaciale)
A0A074Y071 421 Aureobasidium pullulans EXF-150 Ascomycota
A0A074WH41 421 Aureobasidium namibiae CBS 147.97 Ascomycota
A0A074W1K0 421 Aureobasidium melanogenum CBS 110374 Ascomycota
A0A1L9RW86 411 Aspergillus wentii DTO 134E9 Ascomycota
A0A3F3PUF1 417 Aspergillus welwitschiae Ascomycota
167
A0A2V5HGC9 409 Aspergillus violaceofuscus (strain CBS 115571) Ascomycota
A0A1L9PG63 419 Aspergillus versicolor CBS 583.65 Ascomycota
A0A319BZ17 421 Aspergillus uvarum CBS 121591 Ascomycota
A0A8E0QMC4 416 Aspergillus udagawae Ascomycota
A0A229Z1T7 416 Aspergillus turcosus Ascomycota
A0A397GCA0 415 Aspergillus thermomutatus Ascomycota
A0A5M3YT85 415 Aspergillus terreus Ascomycota
A0A5N6UBN6 433 Aspergillus tamarii Ascomycota
A0A1L9TNW8 422 Aspergillus sydowii CBS 593.65 Ascomycota
A0A2I2GJ85 421 Aspergillus steynii IBT 23096 Ascomycota
A0A317WE84 448 Aspergillus sclerotioniger CBS 115572 Ascomycota
A0A319FM33 421 Aspergillus sclerotiicarbonarius (strain CBS 121057 / IBT Ascomycota

28362)
A0A3A2ZKT8 415 Aspergillus sclerotialis Ascomycota
A0A318ZCW3 421 Aspergillus saccharolyticus JOP 1030-1 Ascomycota
A0A017SCU8 415 Aspergillus ruber CBS 135680 Ascomycota
A0A0F8XMD7 425 Aspergillus rambellii Ascomycota
A0A7R8AKY2 419 Aspergillus puulaauensis Ascomycota
168
A0A5N6SME9 430 Aspergillus pseudotamarii Ascomycota
A0A5N6HTP9 432 Aspergillus pseudonomiae Ascomycota
A0A0F0IGJ5 417 Aspergillus parasiticus (strain ATCC 56775 / NRRL 5862 Ascomycota
/ SRRC 143 / SU-1)
A0A1S9DMN7 420 Aspergillus oryzae (Yellow koji mold) Ascomycota
A0A5N6E964 433 Aspergillus novoparasiticus Ascomycota
A0A2I1C393 423 Aspergillus novofumigatus (strain IBT 16806) Ascomycota
A0A0L1J511 441 Aspergillus nomiae NRRL 13137 Ascomycota
A0A100I4C4 417 Aspergillus niger Ascomycota
A0A3D8SJ69 420 Aspergillus mulundensis Ascomycota
A0A5N6IJR6 433 Aspergillus minisclerotigenes Ascomycota
A0A5N5WUW1 433 Aspergillus leporis Ascomycota
A0A7R7WT65 417 Aspergillus kawachii (White koji mold) (Aspergillus Ascomycota

awamori var. kawachi)
A0A395H5T0 420 Aspergillus ibericus CBS 121593 Ascomycota
A0A395I5J3 421 Aspergillus homomorphus (strain CBS 101889) Ascomycota
A0A8H6Q8F1 416 Aspergillus hiratsukae Ascomycota
A0A317X1F7 424 Aspergillus heteromorphus CBS 117.55 Ascomycota
169
A0A1L9VNY8 415 Aspergillus glaucus CBS 516.65 Ascomycota
A0A8H4H1M9 416 Aspergillus fumigatiaffinis Ascomycota
A0A8H6QN79 416 Aspergillus felis Ascomycota
A0A319DPW6 416 Aspergillus ellipticus CBS 707.79 Ascomycota
A0A1E3BFQ1 1383 Aspergillus cristatus Ascomycota
A0A5N6YXL9 418 Aspergillus coremiiformis Ascomycota
A1CKH9 416 Aspergillus clavatus (strain ATCC 1007 / CBS 513.65 / Ascomycota
DSM 816 / NCTC 3887 / NRRL 1 / QM 1276 / 107)
A0A7R7ZLY7 414 Aspergillus chevalieri (Eurotium chevalieri) Ascomycota
A0A1R3RZ98 421 Aspergillus carbonarius (strain ITEM 5010) Ascomycota
A0A2I2F688 421 Aspergillus candidus Ascomycota
A0A0U5CDX5 415 Aspergillus calidoustus Ascomycota
A0A5N6ZP99 433 Aspergillus caelatus Ascomycota
A0A8H6ED23 420 Aspergillus burnettii Ascomycota
A0A1L9UX41 417 Aspergillus brasiliensis (strain CBS 101740 / IMI 381727 / Ascomycota
IBT 21946)
A0A1F7ZS77 420 Aspergillus bombycis Ascomycota
A0A5N7BLA0 417 Aspergillus bertholletiae Ascomycota
170
A0A401KGW0 417 Aspergillus awamori (Black koji mold) Ascomycota
A0A5N6TUU5 420 Aspergillus avenaceus Ascomycota
A0A2G7G6G2 433 Aspergillus arachidicola Ascomycota
A0A1L9X9V3 421 Aspergillus aculeatus (strain ATCC 16872 / CBS 172.66 / Ascomycota
WB 5094)
A0A168CDI7 1026 Ascosphaera apis ARSEF 7405 Ascomycota
A0A4S2N8C9 441 Ascodesmis nigricans Ascomycota
A0A8H7IVU4 419 Ascochyta lentis Ascomycota
A0A3N4I9P2 458 Ascobolus immersus RN42 Ascomycota
C5FE50 425 Arthroderma otae (strain ATCC MYA-4605 / CBS Ascomycota

113480) (Microsporum canis)
E5R3R6 421 Arthroderma gypseum (strain ATCC MYA-4604 / CBS Ascomycota

118893) (Microsporum gypseum)
D4AN76 428 Arthroderma benhamiae (strain ATCC MYA-4681 / CBS Ascomycota

112371) (Trichophyton mentagrophytes)
A0A436ZWC8 435 Arthrobotrys flagrans Ascomycota
A0A2T3B3F1 416 Amorphotheca resinae ATCC 22711 Ascomycota
A0A8H7B9U5 419 Alternaria burnsii Ascomycota
171
A0A177E2D3 419 Alternaria alternata (Alternaria rot fungus) (Torula Ascomycota
alternata)
A0A8H3II19 409 Alectoria fallacina Ascomycota
A0A168AV52 416 Akanthomyces lecanii RCEF 1005 Ascomycota
A0A086T8T6 428 Acremonium chrysogenum (strain ATCC 11550 / CBS Ascomycota

779.69 / DSM 880 / IAM 14645 / JCM 23072 / IMI 49137)
A0A2H3JKU9 438 Wolfiporia cocos (strain MD-104) (Brown rot fungus) Basidiomycota
A0A4T0SWP1 1142 Wallemia mellicola Basidiomycota
A0A4T0JHR1 2038 Wallemia ichthyophaga Basidiomycota
A0A4T0FS99 2528 Wallemia hederae Basidiomycota
A0A0D1DVK7 488 Ustilago maydis (strain 521 / FGSC 9021) (Corn smut Basidiomycota
fungus)
I2FWP7 493 Ustilago hordei (strain Uh4875-4) (Barley covered smut Basidiomycota
fungus)
A0A8H6HDF1 457 Tulosesus angulatus Basidiomycota
A0A0C3Q271 434 Tulasnella calospora MUT 4182 Basidiomycota
K1W6E2 446 Trichosporon asahii var. asahii (strain CBS 8904) Basidiomycota
(Yeast)
A0A4Q1BIA8 428 Tremella mesenterica (Jelly fungus) Basidiomycota
A0A1Y2J0G5 449 Trametes coccinea BRFM310 Basidiomycota
172
A0A066VW28 475 Tilletiaria anomala (strain ATCC 24038 / CBS 436.72 / Basidiomycota
UBC 951)
A0A177ULE8 608 Tilletia walkeri Basidiomycota
A0A0B7G051 446 Thanatephorus cucumeris (strain AG1-IB / isolate Basidiomycota

7/3/14) (Lettuce bottom rot fungus) (Rhizoctonia solani)
A0A0D0BEH4 431 Suillus luteus UH-Slu-Lm8-n1 Basidiomycota
A0A0C9UJU9 434 Sphaerobolus stellatus (strain SS14) Basidiomycota
A0A401H0V8 455 Sparassis crispa Basidiomycota
A0A166EG66 428 Sistotremastrum suecicum HHB10207 ss-3 Basidiomycota
F8PZ61 424 Serpula lacrymans var. lacrymans (strain S7.3) (Dry rot Basidiomycota
fungus)
A0A0C3AK39 403 Serendipita vermifera MAFF 305830 Basidiomycota
G4TD17 411 Serendipita indica (strain DSM 11827) (Root endophyte Basidiomycota
fungus) (Piriformospora indica)
A0A0C2ZWQ1 460 Scleroderma citrinum Foug A Basidiomycota
A0A0H2RWH9 442 Schizopora paradoxa Basidiomycota
D8Q9B6 411 Schizophyllum commune (strain H4-8 / FGSC 9210) Basidiomycota

(Split gill fungus)
A0A427YIM2 430 Saitozyma podzolica Basidiomycota
A0A4Y7PPQ7 435 Rickenella mellea Basidiomycota
173
A0A2S5BFW5 445 Rhodotorula taiwanensis Basidiomycota
A0A5C5FVE3 464 Rhodotorula diobovata Basidiomycota
A0A0K3C947 445 Rhodosporidium toruloides (Yeast) (Rhodotorula Basidiomycota

gracilis)
A0A1B7NE26 432 Rhizopogon vinicolor AM-OR11-026 Basidiomycota
A0A1J8R3R8 431 Rhizopogon vesiculosus Basidiomycota
A0A074S3V5 445 Rhizoctonia solani 123E Basidiomycota
A0A060SBW5 449 Pycnoporus cinnabarinus (Cinnabar-red polypore) Basidiomycota

(Trametes cinnabarina)
A0A5C3QLT6 511 Pterula gracilis Basidiomycota
A0A409WF51 469 Psilocybe cyanescens Basidiomycota
A0A8H7Y0P0 451 Psilocybe cubensis (Psychedelic mushroom) Basidiomycota

(Stropharia cubensis)
A0A5C3F5Y1 508 Pseudozyma flocculosa Basidiomycota
A0A1X6MN41 428 Postia placenta MAD-698-R-SB12 Basidiomycota
A0A371CPC3 451 Polyporus brumalis Basidiomycota
A0A5C3PIC0 448 Polyporus arcularius HHB13444 Basidiomycota
A0A067NK60 456 Pleurotus ostreatus PC15 Basidiomycota
A0A0C3PYC9 456 Pisolithus tinctorius Marx 270 Basidiomycota
174
A0A0D0A9Q1 448 Pisolithus microcarpus 441 Basidiomycota
A0A0C3BD59 441 Piloderma croceum (strain F 1598) Basidiomycota
A0A0C3RZX5 449 Phlebiopsis gigantea 11061_1 CR5-6 Basidiomycota
A0A4S4LAC0 427 Phellinidium pouzarii Basidiomycota
K5WFK2 453 Phanerochaete carnosa (strain HHB-10118-sp) (White- Basidiomycota

rot fungus) (Peniophora carnosa)
A0A165ZJB9 428 Peniophora sp. CONT Basidiomycota
A0A0D0EC35 436 Paxillus rubicundulus Ve08.2h10 Basidiomycota
A0A0C9U4C7 430 Paxillus involutus ATCC 200175 Basidiomycota
A0A409VE85 446 Panaeolus cyanescens Basidiomycota
A0A8E2J5T2 465 Obba rivulosa Basidiomycota
A0A8H3TY10 438 Naganishia liquefaciens Basidiomycota
A0A1Y2B794 430 Naematelia encephala Basidiomycota
A0A8H7CAS0 423 Mycena venus Basidiomycota
A0A8H6ZFB5 401 Mycena sanguinolenta Basidiomycota
A0A8H6U2C7 856 Mycena kentingensis (nom. inval.) Basidiomycota
A0A8H6WH39 877 Mycena indigotica Basidiomycota
A0A8H6WGQ6 434 Mycena chlorophos (Agaric fungus) (Agaricus Basidiomycota

chlorophos)
A0A0W0EXN9 454 Moniliophthora roreri Basidiomycota
175
A0A2X0NU47 454 Microbotryum silenes-dioicae Basidiomycota
A0A2X0LUS4 457 Microbotryum saponariae Basidiomycota
U5HFH2 454 Microbotryum lychnidis-dioicae (strain p1A1 Lamole / Basidiomycota

MvSl-1064) (Anther smut fungus)
A0A238FFF7 454 Microbotryum intermedium Basidiomycota
A0A1Y2G1A2 441 Leucosporidium creatinivorum Basidiomycota
A0A8H5LJK8 423 Leucoagaricus leucothites Basidiomycota
A0A5C2SCR8 449 Lentinus tigrinus ALCF2SS1-6 Basidiomycota
A0A5M6C8X4 426 Kwoniella shandongensis Basidiomycota
A0A1B9I896 432 Kwoniella pini CBS 10737 Basidiomycota
A0A1B9INM5 434 Kwoniella mangroviensis CBS 10435 Basidiomycota
A0A1B9GVS7 430 Kwoniella heveanensis BCC8398 Basidiomycota
A0A1A6A2N3 432 Kwoniella dejecticola CBS 10117 Basidiomycota
A0A1B9G008 434 Kwoniella bestiolae CBS 10118 Basidiomycota
A0A1Y1UJC5 430 Kockovaella imperatae Basidiomycota
A0A369JYC4 455 Hypsizygus marmoreus (White beech mushroom) Basidiomycota

(Agaricus marmoreus)
A0A0C9W6X7 432 Hydnomerulius pinastri MD-312 Basidiomycota
176
A0A2R6RQ14 456 Hermanssonia centrifuga Basidiomycota
A0A4Y9ZXK1 445 Hericium alpestre Basidiomycota
A0A5C3MMS3 439 Heliocybe sulcata Basidiomycota
A0A409XZM8 440 Gymnopilus dilepis Basidiomycota
S7RN70 438 Gloeophyllum trabeum (strain ATCC 11539 / FP-39264 / Basidiomycota

Madison 617) (Brown rot fungus)
A0A2G8S8E1 452 Ganoderma sinense ZZ0214-1 Basidiomycota
A0A067TGK8 446 Galerina marginata (strain CBS 339.88) Basidiomycota
A0A4Y9YDG2 452 Fomitopsis rosea Basidiomycota
S8E9U1 448 Fomitopsis pinicola (strain FP-58527) (Brown rot Basidiomycota

fungus)
J4I8V7 455 Fibroporia radiculosa Basidiomycota
A0A165JH73 433 Exidia glandulosa HHB12029 Basidiomycota
A0A4Q9NLQ0 437 Dichomitus squalens Basidiomycota
A0A4S8MBV0 448 Dendrothele bispora (strain CBS 962.96) Basidiomycota
M5G1H6 450 Dacryopinax primogenitus (strain DJM 731) (Brown rot Basidiomycota
fungus)
A0A0D7BPQ1 429 Cylindrobasidium torrendii FP15055 ss-10 Basidiomycota
A0A0J0XQ60 432 Cutaneotrichosporon oleaginosum Basidiomycota
177
P0CL98 430 Cryptococcus neoformans var. neoformans serotype D Basidiomycota
(strain JEC21 / ATCC MYA-565) (Filobasidiella
neoformans)
A0A0D0VDK0 430 Cryptococcus gattii CA1280 Basidiomycota
A0A1E3I9K5 430 Cryptococcus depauperatus CBS 7841 Basidiomycota
A0A1E3HDW9 431 Cryptococcus amylolentus CBS 6039 Basidiomycota
A0A5C3M3Y8 452 Crucibulum laeve Basidiomycota
A0A5C3L588 452 Coprinopsis marcescibilis Basidiomycota
A8NT51 470 Coprinopsis cinerea (strain Okayama-7 / 130 / ATCC Basidiomycota

MYA-4618 / FGSC 9003) (Inky cap fungus)
(Hormographiella aspergillata)
A0A4Y7STS6 455 Coprinellus micaceus Basidiomycota
M2R586 466 Ceriporiopsis subvermispora (strain B) (White-rot Basidiomycota

fungus) (Gelatoporia subvermispora)
A0A5N5QWH6 443 Ceratobasidium theobromae Basidiomycota
A0A8H8IUK0 439 Ceratobasidium sp. AG-Ba Basidiomycota
A0A4Q2DE89 442 Candolleomyces aberdarensis Basidiomycota
A0A165DAE4 450 Calocera cornea HHB12733 Basidiomycota
A0A067N0D0 449 Botryobasidium botryosum FD-172 SS1 Basidiomycota
178
A0A4S4LYZ9 440 Bondarzewia mesenterica Basidiomycota
A0A8I2YLL0 432 Boletus reticuloceps Basidiomycota
A0A550CYV4 472 Auriculariopsis ampla Basidiomycota
A0A8H7FRU8 425 Athelia sp. TMB Basidiomycota
A0A2H3BXR6 442 Armillaria solidipes Basidiomycota
A0A284R5K4 426 Armillaria ostoyae (Armillaria root rot fungus) Basidiomycota
A0A2H3E7I4 427 Armillaria gallica (Bulbous honey fungus) (Armillaria Basidiomycota

bulbosa)
A0A427YBS6 437 Apiotrichum porosum Basidiomycota
A0A4S4MQJ6 505 Antrodiella citrinella Basidiomycota
A0A2A9NSX0 423 Amanita thiersii Skay4041 Basidiomycota
A0A0C2T4V5 425 Amanita muscaria (strain Koide BX008) Basidiomycota
A0A8H4QNH9 449 Agrocybe pediades Basidiomycota
A0A8H7PVX7 399 Umbelopsis vinacea Mucor
A0A507BZE9 906 Synchytrium microbalum Chytrid
A0A507D9N5 454 Synchytrium endobioticum Chytrid
A0A0L0HQX9 430 Spizellomyces punctatus (strain DAOM BR117) Chytrid
A0A015K194 441 Rhizophagus irregularis (strain DAOM 197198w) Mucor

(Glomus intraradices)
A0A2Z6QKJ3 437 Rhizophagus clarus Mucor
179
A0A507DYC3 406 Powellomyces hirtus Chytrid
A0A8H7ZQU2 507 Olpidium bornovanus Olpidiomycota
A0A8H7U6M7 399 Mortierella isabellina (Filamentous fungus) Mucor

(Umbelopsis isabellina)
A0A068RX46 399 Lichtheimia corymbifera JMRC:FSU:9682 Mucor
A0A433D2I3 419 Jimgerdemannia flammicorona Mucor
A0A397TCR1 440 Glomus cerebriforme Mucor
A0A433PL30 420 Endogone sp. FLAS-F59071 Mucor
A0A261XTH6 415 Bifiguratus adelaidae Mucor
A0A1Y1XRM4 417 Basidiobolus meristosporus CBS 931.73 Zoopagomycota
A0A553PHG5 403 Tigriopus californicus (Marine copepod) Arthropod
A0A8B7NMF7 294 Hyalella azteca (Amphipod) Arthropod
A0A7M5WS78 431 Clytia hemisphaerica Cnidaria
E3NU68 417 Caenorhabditis remanei (Caenorhabditis vulgaris) Nematode
A0A0L0DCK8 1513 Thecamonas trahens ATCC 50062 Zooflagellate
A0A5J4Z201 483 Porphyridium purpureum (Red alga) (Porphyridium Rhodophyte

cruentum)
180
L1JQA4 435 Guillardia theta (strain CCMP2712) (Cryptophyte) Cryptophyte
R1DK22 404 Emiliania huxleyi (Coccolithophore) (Pontosphaera Coccolithophore

huxleyi)
A0A1V9ZFZ7 412 Thraustotheca clavata Stramenopiles
A0A812R312 959 Symbiodinium pilosum (Dinoflagellate) Alveolata
A0A812PTZ6 1053 Symbiodinium necroappetens Alveolata
A0A1Q9EJJ4 1092 Symbiodinium microadriaticum (Dinoflagellate) Alveolata

(Zooxanthella microadriatica)
A0A067CR29 414 Saprolegnia parasitica (strain CBS 223.65) Stramenopiles
T0PVD3 414 Saprolegnia diclina (strain VS20) Stramenopiles
A0A5D6Y223 433 Pythium brassicum Stramenopiles
A0A0P1AMX3 427 Plasmopara halstedii (Downy mildew of sunflower) Stramenopiles
G4ZDQ9 429 Phytophthora sojae (strain P6497) (Soybean stem and Stramenopiles
root rot agent) (Phytophthora megasperma f. sp.
glycines)
A0A6A3N222 429 Phytophthora rubi Stramenopiles
H3G8B6 431 Phytophthora ramorum (Sudden oak death agent) Stramenopiles
V9EEB5 428 Phytophthora parasitica P1569 Stramenopiles
A0A2P4WYF8 428 Phytophthora palmivora var. palmivora Stramenopiles
181
A0A0W8E017 428 Phytophthora nicotianae (Buckeye rot agent) Stramenopiles
A0A225V054 425 Phytophthora megakarya Stramenopiles
A0A3R7KB39 776 Phytophthora kernoviae Stramenopiles
A0A833TB11 428 Phytophthora infestans (Potato late blight agent) Stramenopiles

(Botrytis infestans)
A0A6A4E9J1 429 Phytophthora fragariae Stramenopiles
A0A329S542 428 Phytophthora cactorum Stramenopiles
B7G0U3 401 Phaeodactylum tricornutum (strain CCAP 1055/1) Stramenopiles
A0A3M6VFC5 428 Peronospora effusa Stramenopiles
A0A7J6NHW6 449 Perkinsus olseni (Perkinsus atlanticus) Alveolata
C5KJY4 436 Perkinsus marinus (strain ATCC 50983 / TXsc) Alveolata
A0A7J6MWY0 434 Perkinsus chesapeaki (Clam parasite) (Perkinsus Alveolata

andrewsi)
A0A662Y1L8 432 Nothophytophthora sp. Chile5 Stramenopiles
M4BZ04 434 Hyaloperonospora arabidopsidis (strain Emoy2) Stramenopiles

(Downy mildew agent) (Peronospora arabidopsidis)
K3WFJ6 435 Globisporangium ultimum (strain ATCC 200006 / CBS Stramenopiles

805.95 / DAOM BR144) (Pythium ultimum)
182
A0A484DVB3 434 Bremia lactucae (Lettuce downy mildew) Stramenopiles
A0A485LS90 500 Aphanomyces stellatus Stramenopiles
A0A024URZ4 406 Aphanomyces invadans Stramenopiles
A0A6G0XF49 407 Aphanomyces euteiches Stramenopiles
W4HBB2 406 Aphanomyces astaci Stramenopiles
A0A024GN63 2399 Albugo candida Stramenopiles
A0A1V9ZUK1 411 Achlya hypogyna Stramenopiles
D8TQ81 452 Volvox carteri f. nagariensis Chlorophyte
A0A383VQY2 414 Tetradesmus obliquus (Green alga) (Acutodesmus Chlorophyte

obliquus)
A0A7J7Q2V3 415 Scenedesmus sp. NREL 46B-D3 Chlorophyte
A0A2V0NTE8 416 Raphidocelis subcapitata Chlorophyte
A9TTS6 407 Physcomitrium patens (Spreading-leaved earth moss) Streptophyte

(Physcomitrella patens)
A0A2P6VED2 885 Micractinium conductrix Chlorophyte
A0A176VRW3 407 Marchantia polymorpha subsp. ruderalis Streptophyte
I0Z3H5 408 Coccomyxa subellipsoidea (strain C-169) (Green Chlorophyte

microalga)
A0A2P6TRH5 2189 Chlorella sorokiniana (Freshwater green alga) Chlorophyte
183
A0A2K3CPG8 414 Chlamydomonas reinhardtii (Chlamydomonas smithii) Chlorophyte
A0A835SSY5 431 Chlamydomonas incerta Chlorophyte
A0A388JX09 406 Chara braunii (Braun's stonewort) Streptophyte
A0A5N6KXJ9 421 Carpinus fangiana Dicot
A0A087SL08 406 Auxenochlorella protothecoides (Green microalga) Chlorophyte

(Chlorella protothecoides)
184

Acetate Metabolism in The Fungal Pathogen Cryptococcus Neoformans

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Acetate Metabolism in The Fungal Pathogen Cryptococcus Neoformans

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ACETATE METABOLISM IN THE FUNGAL

PATHOGEN CRYPTOCOCCUS NEOFORMANS

Cryptococcus neoformans is an environmental basidiomycetous fungus with

a worldwide distribution and a wide range of habitats. Inhalation of the desiccated

yeasts or spores of C. neoformans often leads to opportunistic pulmonary infections

in immunocompromised individuals, and in severe cases causes lethal meningitis

following hematogenous dissemination. During infection, depending on the tissue

and disease state, the invading fungi experience a range of nutrient

microenvironments within the host body. As a result, rapid metabolic adaptations

geared towards efficient utilization of carbon sources alternative to glucose become

cryptococcal infection has a long-standing association with acetate metabolism as

underlined by previous cryptococcal transcriptomic studies in infection models that

revealed an increased activity of alternative carbon metabolism during infection, and

highlighted by the observation that cryptococcomas (infectious granulomas) are

enriched with the two-carbon metabolite acetate, a physiologically important

alternative carbon source. In this work I investigated the direct transcriptomic

impacts of acetate utilization compared to glucose utilization via RNA-sequencing,

and noted that cryptococcal transcriptome bears signatures of nutrient starvation

and metabolic adaptations in acetate-grown conditions, as well as a remarkable

upregulation of virulence-associated genes. Moreover, to investigate the importance

of acetate production in C. neoformans we biochemically and kinetically

characterized acetate kinase (Ack), an enzyme involved in acetate metabolism, and

compared acetyl phosphate forming direction. This observation is consistent with

5-phosphate/ fructose-6-phosphate phosphoketolase (Xfp2), which produces acetyl

to be involved in acetate homeostasis. Possibility of the Xfp-Ack pathway of acetate

production in the fungal species C. neoformans prompted us to search for Ack

sequences in other eukaryotic genomes. Here I employed a hidden Markov model

can be observed in dikaryotic fungi. Maximum likelihood based phylogeny of the

predicted eukaryotic Ack sequences suggest an evolutionary trajectory involving

upregulation of virulence-associated genes in C. neoformans, I have proposed a new

theoretical framework to explain the emergence of virulence in fungal pathogens.

hierarchically nest it with stress-response modules, thereby acquiring the ability of

opportunistic pathogenicity in mammalian hosts. Overall, this work furthers our

current understanding of the impacts of acetate metabolism in the human fungal

LIST OF FIGURES .................................................................................................. x

Cryptococcal virulence in the context of acetate metabolism .................... 1

1.1. Cryptococcus and cryptococcosis ........................................................ 2

1.1.1. The genus Cryptococcus ................................................................2

1.1.2. Distribution and morphology........................................................ 3

1.1.3. Cryptococcosis ..............................................................................3

1.1.4. Pathogenesis and host immune response ..................................... 4

1.1.5. Cryptococcal virulence factors ...................................................... 6

1.2. Carbon metabolism and virulence in C. neoformans ........................... 7

1.2.1. Glucose metabolism: Glycolysis and gluconeogenesis................... 8

1.2.2. Peroxisome and the glyoxylate-shunt ......................................... 10

1.2.3. Mitochondria: β-oxidation, respiration, and the electron

1.3. Cryptococcal virulence: acetate and acetyl-CoA ................................ 16

1.3.1. Routes to acetyl-CoA .................................................................. 17

1.3.2. Routes to acetate ....................................................................... 18

1.3.3. Acetate kinase ............................................................................ 20

1.4. Exploring acetate metabolism in C. neoformans ............................... 21

Analysis of transcriptomic response in fungal pathogen Cryptococcus

2.1. INTRODUCTION ................................................................................ 36

2.2. RESULT AND DISCUSSION ................................................................. 40

2.2.1. Chromosomal distribution of differentially expressed genes ...... 41

2.2.2. Differential expression of putative transcription factors ............. 41

2.2.3. Downregulation of genes in acetate-grown conditions ............... 42

2.2.5. Upregulated genes bear the signature of nutrient-starvation. .... 46

2.2.6. Gluconeogenesis and certain anabolic pathways are also

2.2.7. Some upregulated genes are implicated in virulence and

2.3. CONCLUSION .................................................................................... 51

2.4. METHODS AND MATERIALS .............................................................. 55

2.4.1. RNA isolation and sequencing .................................................... 55