ARTICLE IN PRESS Toxicon 50 (2007) 600–611 www.elsevier.com/locate/toxicon Isolation and characterization of novel peptides from chilli pepper seeds: Antimicrobial activities against pathogenic yeasts Suzanna F.F. Ribeiroa, André O. Carvalhoa, Maura Da Cunhaa, Rosana Rodriguesb, Luana P. Cruza, Vânia M.M. Meloc, Ilka M. Vasconcelosd, Edesio J.T. Meloa, Valdirene M. Gomesa, a Universidade Estadual do Norte Fluminense, Centro de Biociências e Biotecnologia, Campos dos Goytacazes, 28015-602 RJ, Brazil b Universidade Estadual do Norte Fluminense, Centro de Ciências e Tecnologias Agropecuárias, UENF, Campos dos Goytacazes, 28015-602 RJ, Brazil c Universidade Federal do Ceará, Departamento de Biologia, 60451-970 Fortaleza, Ce, Brazil d Universidade Federal do Ceará, Departamento de Bioquı´mica, 60451-970 Fortaleza, Ce, Brazil Received 9 March 2007; received in revised form 8 May 2007; accepted 9 May 2007 Available online 23 May 2007 Abstract Different types of antimicrobial peptides have been identified in seeds from different plant species. The aim of this study was to isolate and characterize peptides present in chilli pepper seeds (Capsicum annuum L.) and evaluate their toxic activities against some yeast species. Initially, proteins from seed flour were extracted in phosphate buffer, pH 5.4, for 3 h at 4 1C and the pellet obtained at 90% saturation with ammonium sulfate was heated at 80 1C for 15 min. The resulting suspension was clarified by centrifugation and the supernatant was extensively dialyzed against water; the peptide-rich extract was then named F/0-90. Cation-exchange chromatography was performed to separate low molecular mass proteins. One of the resulting fractions, named F3, enriched with basic proteins of 6–16 kDa, was submitted to reversephase chromatography in a C2/C18 column by HPLC, resulting in four fractions denominated RP1, RP2, RP3 and RP4. When these fractions were submitted to N-terminal sequencing, the comparative analysis in databanks revealed homology for two of these peptides, isolated from fractions RP3 and RP4, with sequences of proteinase inhibitors and 2S albumins, respectively. The F3 fraction, rich in peptides, inhibited the growth of yeasts Saccharomyces cerevisiae, Candida albicans, Candida parapsilosis, Candida tropicalis, Pichia membranifaciens, Kluyveromyces marxiannus and Candida guilliermondii. The RP3 and RP4 fractions showed high inhibitory activity against the growth of the yeast S. cerevisiae. The F3 fraction was also able to inhibit glucose-stimulated acidification of the medium by yeast cells of S. cerevisiae and to cause several morphological changes in different yeasts, such as cell wall disorganization, bud formation as well as the formation of pseudohyphae. r 2007 Elsevier Ltd. All rights reserved. Keywords: Protease inhibitor; Napin; Capsicum annuum; Antimicrobial peptides; Yeast 1. Introduction Corresponding author. Fax: +55 22 2726 1520. E-mail address: valmg@uenf.br (V.M. Gomes). 0041-0101/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.toxicon.2007.05.005 Antimicrobial peptides (AMPs) are an important component of the natural defenses of most living ARTICLE IN PRESS 601 S.F.F. Ribeiro et al. / Toxicon 50 (2007) 600–611 organisms against invading pathogens. These are relatively small, cationic and amphipathic peptides of variable length, sequence and structure. During the past two decades, several AMPs have been isolated from a wide variety of animals, both vertebrates and invertebrates, and also plants (Broekaert et al., 1997; Garcia-Olmedo et al., 1998; Segura et al., 1999; Soares-Costa et al., 2002). These peptides serve as an ancient defense mechanism against a wide range of microorganisms including bacteria, protozoa, yeast, fungi and viruses that easily come into contact with the host through the environment. These molecules are considered as part of the innate immune system of all species (Sugiarto and Yu, 2004). Many of these peptides have been extensively studied in order to elucidate their antimicrobial mode of action. Different models for this action have been proposed: peptides might form pores (Brogden, 2005), or act by disorganizing the membrane (Heller et al., 2000) or by destabilizing the membrane bilayer (Gazit et al., 1995). The net result of these models is increased permeability of the membrane and lysis of the pathogen cell. Osmotin, for example, a tobacco pathogenesis-related protein of family 5 (PR-5), has antifungal activity in vitro and in vivo (Abad et al., 1996; Yun et al., 1997) and the mechanism of action of this osmotin is heightened by its structural and functional similarities with AMPs that are considered to be components of the related innate immune response of plants and animals (Taylor, 1998; Narasimhan et al., 2001). Various classes of AMPs have been implicated in the resistance mechanism of plants against pathogens; these include peptides isolated from seeds of Mirabilis jalapa (Cammue et al., 1992), Amaranthus caudatus (Broekaert et al., 1992) and Zea mays (Duvick et al., 1992), members of the thionin family (Garcia-Olmedo et al., 1989), members of the lipid transfer proteins (LTPs) family (Cammue et al., 1995) and members of the plant defensins family (Terra et al., 1992) (Table 1). During recent years, it has become increasingly clear that these peptides play an important role in protection against microbial infection (Egorov et al., 2005). Investigations on AMPs can significantly contribute to the development of resistance to different pathogens. In this study, we report the purification and characterization of two novel peptides from chilli pepper seeds (Capsicum annuum L.) and their inhibitory activity against different yeasts. Table 1 Plant antimicrobial peptides and their biological activities Name Inhibitory activity References Defensins G+ bacteria, fungi and insect aamylase G+ bacteria and fungi G+, G bacteria and fungi G+ bacteria and fungi G+, G bacteria and fungi Fungi Thomma et al. (2002), Bloch and Richardson (1991) Kader (1996), Carvalho and Gomes (2007) Broekaert et al. (1997) Lipid-transfer proteins Thionins Knotins Snakins Ac-AMP1, AcAMP2 and ArAMP1 Mj-AMP1 and Mj-AMP2 MBP-1 Broekaert et al. (1997) Segura et al. (1999) Broekaert et al. (1992), Lipkin et al. (2005) G+ bacteria and Cammue et al. (1992) fungi G+, G and fungi Duvick et al. (1992) G+, Gram positive; G, Gram negative. 2. Materials and methods 2.1. Plant material Seeds of C. annuum L. (accession UENF1381) were provided by the Laboratório de Melhoramento Genético Vegetal, Centro de Ciências e Tecnologias Agropecuárias, Universidade Estadual do Norte Fluminense, Brazil. This accession was previously described as being resistant to bacterial spot disease and it has been used in crosses aiming to breed pepper and chilli pepper for multiple disease resistance (Riva et al., 2004). 2.2. Yeasts The yeasts Candida parapsilosis (CE002), Candida guilliermondii (CE013), Pichia membranifaciens (CE015), Candida tropicalis (CE017), Candida albicans (CE022), Kluyveromyces marxiannus (CE025) and Saccharomyces cerevisiae (1038) were obtained from the Departamento de Biologia, Universidade Federal do Ceará, Fortaleza, Brazil. Yeasts were maintained on Sabouraud agar (1% peptone, 2% glucose and 1.7% agar-agar). 2.3. Purification of antifungal peptides The purification of antifungal peptides from chilli pepper seeds was performed basically as described ARTICLE IN PRESS 602 S.F.F. Ribeiro et al. / Toxicon 50 (2007) 600–611 by Diz et al. (2006), with some modifications. Proteins were extracted from these seeds’ flour (5 g) after 3 h incubation with 50 mL of extraction buffer (10 mM Na2HPO4, 15 mM NaH2PO4, 100 mM KCl, 1.5% EDTA), pH 5.4, at 4 1C. The precipitate obtained with 90% ammonium sulfate saturation was solubilized in distilled water and heated at 80 1C for 15 min. The resulting suspension was clarified by centrifugation (10,000g for 10 min) and the supernatant was extensively dialyzed against distilled water. The dialyzed solution was recovered by freeze drying and submitted to chromatographic methods. A cation-exchange CM-Sepharose column was employed for further separation of proteins from the peptide-rich extract. This column (1.5  50 cm) was equilibrated and initially eluted with 0.02 M phosphate buffer, pH 8.0. Elution of the bound fraction was carried out in a stepwise manner using NaCl from 0 to 1 M in the equilibration buffer. The F3 fraction, which demonstrated high inhibitory activity, was collected and diluted in a solution containing 0.065% (v/v) TFA plus 2% acetonitrile (v/v) and injected onto an HPLC C2/C18 ST 4.6/100 reverse-phase (RP) column (GE Healthcare). The chromatography was developed at a flow rate of 0.5 mL min1 with 100% solvent A (0.065% TFA plus 2% acetonitrile) for 10 min, 100% solvent B (80% acetonitrile containing 0.05% TFA) over 40 min and finally 100% solvent B over 5 min. Proteins were monitored by on-line measurement of the absorbance at 280 nm. 2.4. Gel electrophoresis and Western blotting SDS–tricine-gel electrophoresis was performed according to the method of Schägger and Von Jagow (1987). Western blotting was done by transferring proteins to nitrocellulose membranes after tricine polyacrylamide gel electrophoresis, according to the method described by Towbin et al. (1979). Antisera against LTP from C. annuum seeds were prepared by the immunization of white New Zealand rabbits with an approximately 10 kDa band protein that showed sequence homology to LTP (Diz et al., 2006). sequences of the peptides were determined by Edman degradation carried out in a Shimadzu PSQ-23A protein sequencer (Shimadzu, Kyoto, Japan). PTH-amino acids were detected at 269 nm after separation on an RP C18 column (0.46  25 cm) under isocratic conditions, according to the manufacturer’s instructions. Searches for sequence homology were performed with the BLASTp program (Altschul et al., 1990). 2.6. Preparation of yeast cells and effect of peptides on yeast growth For the preparation of yeast cell cultures, an inoculum from each stock was transferred to Petri dishes containing Sabouraud agar and allowed to grow at 28 1C for 2 days; after this period, cells were transferred to sterile 0.15 M NaCl solution (10 mL). Yeast cells were quantified in a Neubauer chamber for further calculation of appropriate dilutions. A quantitative assay for fungal growth inhibition was performed following the protocol developed by Broekaert et al. (1990), with some modifications. To assay the effect of different fractions on yeast growth, the cells (10,000 cells mL1 in 1 mL of saline solution) were incubated at 28 1C in 200 mL microplates in the presence of different concentrations of peptide solutions (2–64 mg mL1) of F3 fraction and isolate peptides. Optical readings at 620 nm were taken at zero hours and at every 6 h for the following 42 h. Cell growth controls without the addition of peptides were also determined. Experiments were performed in triplicate and the standard errors (coefficients of variation were less than 20%) were omitted for clarity. 2.7. Optical microscopy After a 42 h growth period, S. cerevisiae cells were separated from the growth medium by centrifugation, washed in 20 mM phosphate buffer, pH 8.0, and observed using an optical microscope at 1000  magnification (Axiovert 135). All the experiments were run in triplicate and the reading averages, standard errors and coefficients of variation were calculated. 2.8. Scanning electron microscopy 2.5. Amino acid sequence analysis Peptides from the F3 fraction were separated by HPLC C2/C18 chromatography. The obtained peaks were freeze dried and submitted to amino acid sequence analysis. N-terminal amino acid For scanning electron microscopy, yeast cells grown for 42 h in Sabouraud broth in the absence or presence of F3 fraction (16 mg mL1) were fixed for 30 min at room temperature in a solution containing 2.5% glutaraldehyde and, 4.0% formaldehyde in ARTICLE IN PRESS S.F.F. Ribeiro et al. / Toxicon 50 (2007) 600–611 0.1 M phosphate buffer, pH 7.3. Subsequently, the materials were rinsed three times with the above buffer, post-fixed for 30 min at room temperature with 1.0% osmium tetroxide, diluted in the same buffer and rinsed with distilled water. After this procedure, the yeast cells were dehydrated in acetone, critical point dried in CO2, covered with 20 nm gold and observed in a DSEM 962 Zeiss scanning electron microscope. The yeast cells grown without the addition of peptides were also determined. 2.9. Inhibition of glucose-stimulated acidification of the medium by S. cerevisiae yeast The Sabouraud broth (100 mL) containing the S. cerevisiae culture was shaken for 16 h at 30 1C with good aeration. The culture (A660 ¼ 0.2) was pelleted by centrifugation at 2000g (5 min, 4 1C), followed by two water-washing steps. The antifungal activity of the peptide-rich F3 fraction was determined by the incubation of S. cerevisiae cells (1  107) with 800 mL of 10 mM Tris–HCl, pH 6.0. Chilli pepper peptides were added to final concentrations of 2, 4, 8 and 16 mg mL1. After different incubation times (60, 30 and 10 min), a 0.5 M glucose solution was added to a final concentration of 0.15 M. pH measurements were taken at each subsequent minute for the next 30 min. Negative (BSA was added at 160 mg mL1 instead of peptide fraction) and positive (1000 U nistatin added) controls were run in order to evaluate the influence of peptides on H+ extrusion by yeast cells. The concentration of H+ extrusion was calculated as the difference between initial (T ¼ 0) and final (T ¼ 60 min) pH (rpH), the value of which was obtained from the equation pH ¼ log [H+]. The graphs were drawn based on the assumption that the values obtained using the negative control corresponded to 100% acidification. 3. Results and discussion 3.1. Purification of antimicrobial peptides from chilli pepper seeds AMPs such as LTPs and defensins were found in seeds of C. annuum (Do et al., 2004; Park et al., 2002; Oh et al., 1999; Lee et al., 2004). Do et al. (2004) and Park et al. (2002) characterized members of gene families encoding defensins and LTP, both derived from cDNA clones from C. annuum plants. More recently, Diz et al. (2006) purified an LTP from C. annuum seeds; however, information about 603 the antifungal activity, as well as action mechanisms in vitro for C. annuum peptides, was only analyzed in this work. Our working hypothesis is that C. annuum seeds contain different defense proteins as well as different AMPs. Thus, we herein investigated the presence and antifungal activity of novel AMPs in C. annuum seeds, accession UENF 1381. The resulting suspension after extraction was initially fractionated by cation-exchange chromatography. Three protein peaks, named F1, F2 and F3, were eluted with equilibrium buffer, 0.1 and 0.2 M NaCl, respectively (Fig. 1A). The adsorbed fraction (F3) from cation-exchange chromatography was further fractionated by RP chromatography in the C2/C18 ST 4.6/100 column. The F3 fraction was separated into four new fractions named RP1, RP2, RP3 and RP4 (Fig. 1B). The purification of two peptides was confirmed by rechromatographing the RP3 and RP4 peaks. The peaks were now named RP30 (Fig. 1C) and RP40 (Fig. 1D). After these steps the peptides were finally purified. 3.2. Characterization of antimicrobial peptides from chilli pepper seeds The analysis of the protein profile of the F1, F2 and F3 fractions obtained by cation-exchange chromatography demonstrated the presence of several peptides in SDS–tricine-gel electrophoresis under reducing conditions (Fig. 2A) (Diz et al., 2006). Because of its strongest antifungal activity, the F3 fraction was chosen for further characterization. The F3 fraction was mainly composed of four bands with molecular masses of between 6 and 16 kDa (Fig. 2A). The analysis of proteins from the RP1, RP2, RP3 and RP4 peaks, obtained by RP chromatography, is shown in Fig. 2B. RP2 and RP3 were composed of one unique peptide with molecular masses of between 6 and 8 kDa. RP4 was composed of one unique protein constituted of two chains with relative molecular masses of 4 and 8 kDa, respectively, as determined by SDS–tricinegel electrophoresis. The native protein can also be visualized with a molecular mass of 14 kDa (Fig. 2B). The immunoscreening of the F3 fraction with an antiserum against purified LTP from C. annum seeds (Diz et al., 2006) by Western blotting showed that none of the proteins cross-reacted with the antiserum, indicating no presence of an LTP in this fraction (data not shown). The Nterminal amino acid sequence of peptides from the RP3 and RP4 fractions showed homology, ARTICLE IN PRESS 604 S.F.F. Ribeiro et al. / Toxicon 50 (2007) 600–611 A 1.6 Absorbance, 280 nm 1.4 1.2 1 NaCl – 0.1 M 0.8 0.6 NaCl – 0.2 M NaCl – 1 M NaCl – 0.5 M 0.4 F2 F1 0.2 F3 0 10 0 20 30 40 50 60 70 80 Tubes B mAbs 1000 500 RP2 RP 4 RP 1 RP3 0 0 C D 200 200 mAbs mAbs 60 40 20 100 RP4 , 100 RP3’ 0 0 0 20 40 Time ,min 60 0 40 20 Time ,min 60 ARTICLE IN PRESS 605 S.F.F. Ribeiro et al. / Toxicon 50 (2007) 600–611 M A PRE F1 F2 RP2 RP3 respectively, to trypsin inhibitor and napin isolated from different plants (Tables 2 and 3). Our data show that the RP3 fraction peptide, now named CaTI, belongs to the proteinase inhibitor f amily, displaying some homology to other inhibitors of this family isolated from seeds of Nicotiana tabacum (Q40561), Erythrina variegate (P81366), E. latissima (P68171), C. annuum (Q9SDL4) and Psophocarpus tetragonolobus (P10822). The RP4 fraction, now named CaNap, belongs to the napin family (named as 2S albumins), displaying high homology to other proteins of this family isolated from seeds of Brassica napus (P01090), Brassica juncea (Q42413), Raphanus sativus (AAA32745), Passiflora edulis f. flavicarpa (Agizzio et al., 2003), Sesamum indicum (Dond and Dunstan, 1999) and Glycine max (Odani et al., 1897). Both proteinase inhibitor and the napin family play an important role in the protection of plants against different types of microorganisms. The analysis of the N-terminal amino acid sequence (WQCPPQEKLLECQG) of the peptide from the RP1 peak revealed sequence homology with vicilin-like AMP precursor isolated from plants of Gossypium hirsutum (P09801) and Macadamia integrifolia (Q9SPL3, Q9SPL4 and Q9SPL5). The analysis of the N-terminal amino acid sequence of the peptide from the RP2 peak revealed no sequence homology with any other known peptide, even those isolated from plant seeds. Further analyses to better characterize this peptide are under way. F3 16.950 14.400 10.600 8.160 6.200 M RP 1 RP4 B 16.950 14.400 10.600 8.160 6.200 Fig. 2. SDS–tricine-gel electrophoresis of C. annuum proteins during the purification process. (A) Fractions eluted from the cation exchange column: PRE, peptide-rich extract; F1, nonretained fraction; F2, retained fraction eluted with 0.1 – NaCl; F3, retained fraction eluted with 0.2 – NaCl. (B) Fractions eluted from the RP column: RP1, first peak; RP2, second peak; RP3, third peak; RP4, fourth peak. (C, D) Rechromatography in RP C2/C18 of RP3 and RP4 fractions, respectively. M, molecular mass markers (kDa). Table 2 Comparison of N-terminal sequence of C. annuum with those of related proteins Species (protein) C. annuum (fraction RP3) C. annum C. annum N. tabacum E. latissima E. variegate P. tetragonolobus 1 Sequences – 1 K – – – – – 1 S A 128 A 76 S 66 F 66 F 58 A E C C D I I K Accession number P P P P P P T R R R K D D G N N N N D D N E C C D D D E P D D P E K P T T T – V V C E D R D R R P I I I I I I L S A A A G G T Y Y Y Y F F V S M S S A A V V V K K Y Y R A C C C A A S P P P P P P P S S R R K K N V S S S C C V S19 G20 E146 E93 A84 A84 S77 46396269 Q9SDL4 Q40561 P68171 P81366 P10822 S and S19 indicate that the first N-terminal amino acid is S and the 19th N-terminal amino acid is S, respectively. Fig. 1. Purification of the antifungal peptide fraction from C. annuum seeds. (A) Cation-exchange CM-Sepharose chromatography. The column was previously equilibrated with 0.02 M phosphate buffer, pH 8.0. Elution was carried out in a stepwise manner using NaCl from 0 to 1 M in the equilibration buffer, at 50 mL h1; (B, C, D) RP-HPLC chromatography. The F3 fraction was applied to a C2/C18 ST 4.6/ 100 RP column and run in a Shimadzu apparatus. Elution was carried out as described in Section 2. The line represents the acetonitrile gradient and the dark line represents the protein elution profile at 220 nm. ARTICLE IN PRESS 606 S.F.F. Ribeiro et al. / Toxicon 50 (2007) 600–611 Table 3 Comparison of N-terminal sequence of C. annuum with those of related proteins Species (protein) Sequences C. annuum (fraction RP4) B. napus B. juncea R. sativus P. edulis f. flavicarpa S. indicum G. max 1 P P 1 P 1 P – – – 1 – F F – – – – Q R R R 1 P 1 Q 1 Q Accession number/reference C I I – S S Q P P P Q E Q D R K K R R Q S C C C C C C C S R R Q R R R Q K K K R Q K Q E E E Q Q Q F F F F M L L Q Q Q Q Q – Q Q Q Q Q G Q G A A A S Q G V K Q Q Q D R N Q H H H F Q L L L L L S15 F T R R R R – R P16 C – V – – S16 – S A – A – – – C C C C – – – Q Q Q Q – – – S22 Q22 Q22 R22 – – – P01090 Q42413 AAA32745 Agizzio et al. (2003) Tai et al. (1999) Odani et al. (1897) 1 P and S22 indicate that the first N-terminal amino acid is P and the 22nd N-terminal amino acid is S. 3.3. Effect of peptides on yeast growth Results obtained by Diz et al. (2006) indicated that the protein fractions of chilli pepper seeds display antifungal activities against different fungi. An inhibitory effect of F1, F2 and F3 fractions on the growth of all fungi tested was noticed at concentrations of 70 and 150 mg mL1. A notable inhibitory effect, mainly of the F3 fraction, was also observed on the growth of S. cerevisiae yeast at concentrations of 70 and 150 mg mL1, demonstrating 70% and 100% of inhibition, respectively. In this study, we tested the F3 fraction against the yeasts S. cerevisiae, C. guilliermondii, C. parapsilosis, K. marxiannus, P. membranifaciens, C. tropicalis and C. albicans. It was possible to see that in the presence of the F3 fraction the growth of the S. cerevisiae yeast was inhibited at all concentrations used. The IC50 value for C. albicans and C. guilliermondii, for example, can be observed at a concentration of o16 mg mL1. We also observed an accentuated reduction of growth from the yeasts K. marxiannus, C. parapsilosis, S. cerevisiae, P. membranifaciens and C. tropicalis. The IC50 values are shown in Table 4. We also tested the inhibitory effect of the peptides CaTI and CaNap from the RP3 and RP4 peaks, respectively, at a concentration of 16 mg mL1, against the yeasts S. cerevisiae (Fig. 3A) and C. albicans (Fig. 3B). It is possible to observe a significant effect of CaTI, especially against S. cerevisiae yeast. This diversity found in the growth of these different yeast species may be influenced mainly by the differentiated development of its cells. Recent reports have related that trypsin inhibitor and 2S albumins inhibit the growth of several fungi (Guidici et al., 2000; Agizzio et al., 2003, 2006; Pelegrini et al., 2006). For example, an antifungal protein designated as Psc-AFP, with an Table 4 Antifungal activity of the F3 fraction Yeasts IC50 values (mg mL1)a Saccharomyces cerevisiae (CE1038) C. parapsilosis (CE0002) C. tropicalis (CE0017) Pichia membranifaciens (CE0015) Kluyveromyces marxiannus (CE0025) Candida albicans (CE0022) Candida guilliermondii (CE0013) o32 o64 o64 o16 16 o16 o16 a Protein concentrations required for 50% growth inhibition after 42 h at 301. apparent molecular mass of 18 kDa, was isolated from a traditional Chinese herb, Malaytea scurfpea. Automated Edman degradation determined that the partial N-terminal sequence of Psc-AFP displayed homology with plant trypsin inhibitors. Psc-AFP at 10 mM inhibited the mycelial growth of Alternaria brassicae, Aspergillus niger, Fusarium oxysporum and Rhizoctonia cerealis, suggesting that Psc-AFP also has a role in defense against pathogens (Yang et al., 2006). We further analyzed, through optical and scanning electron microscopy, possible alterations in yeast morphology caused by the F3 fraction. Initial photomicrographs of S. cerevisiae were taken after 42 h of yeast growth in the presence of the F3 fraction. Normal growth development was observed for control cells (Fig. 4A and C). S. cerevisiae cells treated with the F3 fraction (16 mg mL1) showed a notable inhibition of growth, and several morphological alterations such as cellular agglomeration, cytoplasmatic deformation and reduction of the cell number were observed in these cells (Fig. 4B and D). ARTICLE IN PRESS 607 S.F.F. Ribeiro et al. / Toxicon 50 (2007) 600–611 A 900 800 Absorbance, 600 nm 700 600 500 400 300 200 100 0 0 10 20 30 40 30 40 Time, h B 700 Absorbance, 600 nm 600 500 400 300 200 100 0 0 10 20 Time, h Fig. 3. Inhibition growth assay of the yeast S. cerevisiae (A) and C. albicans (B) in the presence of RP3 and RP4 fractions, obtained after chromatography in HPLC in an RP C2/C18 column. Growth was observed until 42 h. (-K-) Control; (-m-) RP3, 16 mg mL1; (-’-) RP4, 16 mg mL1. Experiments were performed in triplicate and the standard errors (coefficients of variation were less than 20%) were omitted for clarity. Further tests to evaluate the inhibition of the growth of different yeast cells were analyzed through scanning electron microscopy to verify possible alterations in yeast morphology. Photomicrographs of the yeast cells were taken after 18 h of growth in the presence of the F3 fraction. Normal growth development was observed for all control cells (Fig. 5A, C, E, G, I and K). Cultures (C. guilliermondii, S. cerevisiae and K. marxiannus) treated with the F3 fraction exhibited notable alterations in bud formation, as well as difficulty in liberating buds (Figs. 5B, L and H). In addition, dimorphic transitions with the development of pseudohyphae were observed during the growth of the cultured C. tropicalis (Fig. 5F) in the presence of the F3 fraction. Interestingly, this cellular dimorphism may occur as a result of alterations in the pH of the medium in which cells are grown and can be ARTICLE IN PRESS 608 S.F.F. Ribeiro et al. / Toxicon 50 (2007) 600–611 confirmed by the fact that the F3 fraction probably acts on the plasma membrane of these yeasts. Osborn et al. (1995) related that plant defensins isolated from different seeds cause morphological alterations that are often very distinct in some, but not all, tested fungi. Diz et al. (2006) also showed that peptides present in the F1 fraction of chilli pepper seeds promote a yeast-pseudohyphae transition state in the C. albicans yeast following the permeabilization of the plasma membrane that is promoted by F1 fraction peptides. 3.4. Effect on the glucose-stimulated acidification of the medium of yeast cells Fig. 4. Optical microscopy of S. cerevisiae cells in the presence of the F3 fraction. (A, C) Control (absence of the F3 fraction); (B, D) presence of the F3 fraction (16 mg mL1). Arrows shows cellular agglomeration and cytoplasmatic deformation. Bars: (A–D), 7.5 mm. The plasma membrane H+-ATPase plays an essential role in fungal cell physiology. Interference in the function of H+-ATPase in fungi by antagonists commonly leads to cell death. In this study, we investigated whether the F3 fraction could interfere with the yeast H+-ATPase. For this, we monitored the glucose-stimulated acidification of the incubation medium by S. cerevisiae cells in the presence of various concentrations of the F3 fraction; this Fig. 5. Scanning electron microscopy of yeast cells in the presence of Capsicum annuum seed antimicrobial peptides. (A, C, E, G, I, K) Control, absence of the F3 fraction; (B, D, F, H, J, L) presence of the F3 fraction (16 mg mL1). (A, B) C. guilliermondii; (C, D) C. parapsilosis; (E, F) C. tropicalis; (G, H) K. marxiannus; (I, J) P. membranifaciens; (K, L) S. cerevisiae. Arrows show difficulty in liberating buds. Bars: (A–D), 1 mm; (E, F), 3 mm; (G–L), 1 mm. ARTICLE IN PRESS 609 S.F.F. Ribeiro et al. / Toxicon 50 (2007) 600–611 A 100 90 80 % Acidification 70 60 50 40 30 20 10 0 B 100 90 80 % Acidification 70 60 50 40 30 20 10 0 C 100 90 % Acidification 80 70 60 50 40 30 20 10 0 Control 2µg/mL-1 4µg/mL-1 8µg/mL-1 16µg/mL-1 Fig. 6. Effect of the F3 fraction from Capsicum annuum seeds on the glucose-dependent acidification of the medium by S. cerevisiae cells. Glucose was added after 10, 30 and 60 min of cell incubation with the F3 fraction, to a final concentration of 0.15 M. (A) 60 min; (B) 30 min; (C) 10 min. ARTICLE IN PRESS 610 S.F.F. Ribeiro et al. / Toxicon 50 (2007) 600–611 phenomenon is dependent on the activity of the H+-ATPase. The F3 fraction inhibited the glucosestimulated acidification of the medium by S. cerevisiae by 100% when using concentrations that varied from 2 to 16 mg mL1 for the three different times of incubation utilized (Fig. 6). Different peptides have been studied and analyses have been performed to test the ability that some of these peptides have to act on the plasma membrane. Thevissen et al. (1999) demonstrated, for example, that when the fungi Neurospora crassa and Fusarium culmorum were tested with the plant defensins RsAFP2 and DmAMP1, an ion flux across the fungal plasma membrane was also observed. The antifungal activity of osmotin has also been suspected to involve specific target components of the fungal plasma membrane. Yun et al. (1997) showed that osmotin induces spore lysis, inhibits spore germination or reduces germtube viability in several fungal species that exhibit some degree of sensitivity in hyphal growth inhibition tests. The species-specific growth inhibition was correlated with the ability of osmotin to dissipate the fungal membrane pH gradient. In addition, 2S albumins present in passion fruit seeds may present the ability to inhibit glucose-dependent acidification by yeast cells of S. cerevisiae and also by phytopathogenic fungi (Agizzio et al., 2003, 2006). Diz et al. (2006) demonstrated that the peptides present in chilli pepper seeds inhibited glucose-dependent acidification of yeast cells of S. cerevisiae by 100%, utilizing a 160 mg mL1 concentration of the F1 fraction that, when in contact with the yeast cells, was able to promote a permeabilization across the plasma membrane. Interestingly, dimorphic transition has been found to be controlled by pH in C. albicans. Mycelium growth, for example, is favored by incubation at neutral pH; the fungus grows in a yeast-like manner under acidic conditions, but only in a glucose-containing medium (Soll, 1985). Thus, the yeast-to-mycelium transition observed in a yeast culture grown in the presence of F3 fraction could be due to the inhibition of medium acidification promoted by F3 peptides. Acknowledgments This study forms part of the M.Sc. degree thesis of SFFR, carried out at the Universidade Estadual do Norte Fluminense. We acknowledge the financial support of the Brazilian agencies CNPq, FAPERJ and TECNORTE/FENORTE. We are grateful to G.A. Moraes for the preparation of samples for microscopy analysis. References Abad, L.R., D’Urso, M.P., Liu, D., Narasimhan, M.L., Reuveni, M., Zhu, J.K., Niu, X., Singh, N.K., Hasegawa, P.M., Bressan, P.A., 1996. Antifungal activity of tobacco osmotin has specificity and involves plasma membrane permeabilization. Plant Sci. 118, 11–23. 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