Proteins and Peptidases from Conidia and Mycelia of Scedosporium apiospermum Strain HLPB

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Mycopathologia (2009) 167:25–30 DOI 10.1007/s11046-008-9147-7

Proteins and Peptidases from Conidia and Mycelia of Scedosporium apiospermum Strain HLPB Martha Machado Pereira Æ Bianca Alcaˆntara Silva Æ Marcia Ribeiro Pinto Æ Eliana Barreto-Bergter Æ Andre´ Luis Souza dos Santos

Received: 30 April 2008 / Accepted: 15 July 2008 / Published online: 23 August 2008 Ó Springer Science+Business Media B.V. 2008

Abstract The conidia–mycelia transformation is an essential step during the life cycle of the fungal human pathogens of the Pseudallescheria boydii complex. In the present study, we have analyzed the protein and peptidase profiles in two distinct morphological stages, conidia and mycelia, of Scedosporium apiospermum sensu stricto. Proteins synthesized by the mycelia, migrating at the ranges of 62–48 and 22–18 kDa, were not detected from the conidial extract. Conidia produced a single cellular peptidase of 28 kDa able to digest copolymerized albumin, while mycelia yielded 6 distinct peptidases ranging from 90 to 28 kDa. All proteolytic enzymes were active at acidic pH and fully inhibited by 1,10-phenanthroline, characterizing these activities as metallo-type peptidases. Quantitative peptidase assay, using soluble albumin, showed a high

Martha Machado Pereira and Bianca Alcaˆntara Silva contributed equally to this work. M. M. Pereira  B. A. Silva  E. Barreto-Bergter  A. L. S. Santos (&) Departamento de Microbiologia Geral, Instituto de Microbiologia Prof. Paulo de Go´es (IMPPG), Centro de Cieˆncias da Sau´de (CCS), Bloco I, Universidade Federal do Rio de Janeiro (UFRJ), Ilha do Funda˜o, Avenida Carlos Chagas Filho, 373, Rio de Janeiro 21941-902, Brazil e-mail: [email protected] M. R. Pinto Departamento de Microbiologia, Instituto de Cieˆncias Biolo´gicas, Universidade de Sa˜o Paulo, Sa˜o Paulo, Brazil

metallopeptidase production in mycelial cells in comparison with conidia. The regulated expression of proteins and peptidases in different morphological stages of S. apiospermum represents a potential target for isolation of stage-specific markers for biochemical and immunological analysis. Keywords Scedosporium apiospermum  Conidia  Mycelia  Proteins  Peptidases

Introduction Fungi of the Pseudallescheria boydii complex are ubiquitous filamentous fungi commonly found in soil, sewage, and polluted waters. In the last decades, they have emerged as important human pathogens, particularly in immunocompromised patients. In nature, these fungi grow as saprobes, presenting a filamentous form and reproducing asexually by conidium production and sexually by cleistothecium formation [1]. The conidium is critical in the life cycle of many fungi, because it is the primary means for dispersion and serves as a ‘safe house’ for the fungal genome in adverse environmental conditions [2]. P. boydii conidia serve at first for the dispersion, while ascomata and ascospores are normally much more resistant to adverse environmental influences [1]. Several factors are known to influence the fungal cell morphogenesis. Investigation in this area is important because it can lead to the isolation of specific cell

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types for biochemical and immunological analysis [2]. In this context, our laboratory characterized glucosylceramides localized on the cell surface of P. boydii with a potential role in fungal differentiation [3]. Germination of conidia is an early and crucial step in the invasive pathogenic lifestyle of numerous fungi. In P. boydii, this common concept is still poorly understood, since it is an opportunist microorganism, which infects vertebrates as a result of an accident. Recently, we studied the interaction between P. boydii and HEp2 cells and showed that conidia of P. boydii attached to, and were ingested by, HEp2 cells in a time-dependent process. After 2 h of interaction, the conidia produced a germ-tube like projection, which was able to penetrate the epithelial cell membrane [4], suggesting a possible participation of hydrolytic enzymes (such as phospholipases and peptidases) in this process. Proteolytic enzymes have often been suggested to be involved in fungal morphogenesis and virulence of several human pathogens including Candida albicans [5], Aspergillus fumigatus [6], Paraccocidioides brasiliensis [7], Sporothrix schenckii [8], and Fonsecaea pedrosoi [9, 10]. Peptidase production is considered to enhance the organism’s ability to colonize and penetrate host tissues, and to evade the host immune system by degrading a number of proteins that are important in host defense [5, 11, 12]. In this context, P. boydii was able to release two distinct metallo-type peptidases (28 and 35 kDa) into the extracellular environment, which hydrolyzes several proteinaceous compounds, including human serum proteins, sialylated polypeptides and extracellular matrix components [13, 14]. The present work aims to identify the protein and proteolytic activity profiles synthesized by two distinct morphological stages (conidia and mycelia) of S. apiospermum strain HLPB.

Methods Chemicals Proteolytic inhibitors (phenylmethylsulphonyl fluoride [PMSF], 1,10-phenanthroline, trans-epoxysucciny-Lleucylamido-(4-guanidino) butane [E-64] and pepstatin A), dithiothreitol (DTT) and bovine serum albumin (BSA) were purchased from Sigma Chemical Co. (St

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Louis, MO, USA). Glucose, bacteriological peptone, yeast extract and bacteriological agar were obtained from Oxoid Ltd (Hampshire, England). Reagents used in electrophoresis were obtained from BioRad (Hercules, CA, USA). All other reagents were purchased from Merck (Darmstadt, Germany). Microorganism and Growth Conditions Scedosporium apiospermum strain HLPB was kindly supplied by Dr. Bodo Wanke (Hospital Evandro Chagas, Instituto Oswaldo Cruz, Rio de Janeiro, Brazil) and was recently identified by means of molecular approaches by Dr. Kathrin Tintelnot (Robert Koch-Institut, Berlin, Germany). The sequencing of the ITS regions revealed that this strain belongs to clade 4 (S. apiospermum sensu stricto) according to the taxonomy proposed by Gilgado et al. [15]. Conidia were grown on Petri plates containing modified Sabouraud-dextrose agar (2% glucose, 1% peptone, 0.5% yeast extract and 2% agar) at 25°C. After 7 days in culture, conidia were harvested and washed with sterile 10 mM phosphatebuffered saline (PBS, pH 7.2). The cells were separated by gauze filtration, then collected by centrifugation and washed twice in PBS. Cell growth was estimated by counting the conidia in a Neubauer chamber. In order to obtain mycelial forms, cells grown on Sabouraud-dextrose solid slants were inoculated into Erlenmeyer flasks (500 ml) containing this liquid culture medium (200 ml) and incubated for 7 days at 25°C with constant shaking (200 rpm) [16]. Fungal Extracts Conidia (5 9 108 cells) and mycelia (5 g) were resuspended in 1 ml PBS supplemented with 0.1% Triton X-100. An equivalent volume of glass beads (0.3 mm in diameter) was then added to the suspensions, and cells were broken in a cell homogenizer (Braun Biotech International) by alternating 2-min shaking periods and 2 min cooling intervals (total of 20 cycles). Cell disruption was assessed in a light microscope (Zeiss, Germany). After removal of the glass beads and centrifugation, the protein concentration in the supernatants (whole fungal extracts) [17] was determined using BSA as the standard [18].

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Quantitative Peptidase Assay

protein) and, following electrophoresis at 120 V at 4°C for 90 min, they were incubated in 10 volumes of 1% Triton X-100 for 1 h at room temperature under constant agitation, to remove the SDS. The gels were then incubated for 40 h at 37°C in either 10 mM sodium citrate (pH 2.5), 50 mM sodium phosphate (pH 5.5), 10 mM sodium phosphate (pH 7.0) or 20 mM glycine–NaOH (pH 10.0), in the absence or in the presence of proteolytic enzyme inhibitors (10 mM PMSF, 10 mM 1,10-phenanthroline, 10 lM pepstatin A or 10 lM E-64). The gels were stained overnight with 0.2% Coomassie brilliant blue R-250 in methanol–acetic acid–water (50:10:40) and destained in methanol–acetic acid–water (5:10:85), to intensify the digestion halos [17]. Molecular masses of the peptidases were calculated by the comparison of the mobility of GIBCO BRL SDSPAGE standards (Grand Island, NY, USA). The gels were photographed and digitally processed.

Proteolytic activity was measured spectrophotometrically using BSA as substrate, according to the method described by Buroker-Kilgore and Wang [19]. Initially, fungal extracts were incubated for 30 min at 37°C in the presence or in the absence of 10 mM 1,10-phenanthroline. For assaying the remaining proteolytic activity, BSA (0.5 mg/ml), 8 mM DTT, 50 mM sodium phosphate (pH 5.5) and 20 ll of the fungal extracts were added to a microcentrifuge tube (350 ll) and incubated for 2 h at 37°C. After this incubation, three aliquots (100 ll each) of each reaction mixture were transferred to wells of a microtiter plate containing 50 ll of water and 100 ll of a Coomassie solution (0.025% Coomassie brilliant blue G-250, 11.75% ethanol, and 21.25% phosphoric acid). After 10 min to allow color development, the plate was read at 595 nm on a Thermomax Molecular Device microplate reader. One unit of proteolytic activity was defined as the amount of enzyme that caused a 0.001 unit increase in the absorbance, under standard assay conditions. The proteolytic activity is expressed as arbitrary unit (AU) 9 lg of protein [13]. Protein Profiles Fungal extracts (equivalent to 20 lg of protein) were treated with an equal volume of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) sample buffer (125 mM Tris, pH 6.8, 4% SDS, 20% glycerol and 0.002% bromophenol blue) supplemented with 10% b-mercaptoethanol, followed by heating at 100°C for 5 min. Proteins were analyzed on 10% SDS-PAGE by the method described by Laemmli [20]. Electrophoresis was carried out at 100 V for 90 min at 4°C, and the gels were silver stained. Prior to electrophoresis, Gibco BRL (Grand Island, NY, USA) molecular mass standards were boiled in SDS-PAGE sample buffer and then applied in the same gel. Substrate-SDS-PAGE Proteolytic enzymes were assayed and characterized by electrophoresis on 10% SDS-PAGE copolymerized with 0.1% BSA [21]. The gels were loaded with 20 ll of fungal extracts (equivalent to 20 lg of

Statistical Analysis All the experiments were performed in triplicate and repeated at least three times. Representative images of these experiments are shown. The data were analyzed statistically using Student’s t-test. P values of 0.05 or less were considered statistically significant.

Results and Discussion The development of the conidia germination is as diverse and varied as the number of fungal species that produce conidia [2]. Adaptations to changing environments might occur very rapidly by the upregulation and down-regulation of distinct genes [12]. Recent findings suggest that fungi sense local environmental changes and rapidly respond to these changes by modifying their transcriptional and translational profiles, which helps them to adapt to the host environment and to actively change the local microenvironment. Consequently, numerous metabolic activities, including respiration, RNA and protein synthesis as well as differences in the composition of the cell wall can be detected during the differentiation process [2]. In this context, the conidia–mycelia transformation is an essential step during the life cycle of S. apiospermum, which led us to analyze the protein profile in these two distinct

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morphological stages (Fig. 1A). The separation of soluble whole proteins by SDS-PAGE revealed a noticeable difference in the conidial and mycelial forms (Fig. 1B). Proteinaceous components synthesized by the mycelia, migrating at the ranges of 62– 48 and 22–18 kDa, were not detected in the conidial extracts (Fig. 1B, lanes a, b). These results suggest an intense protein turnover during the in vitro conidia into mycelia transformation in S. apiospermum. Since peptidases are key enzymes implicated in the microbial metabolism, we investigated the expression of cell-associated proteolytic enzymes in S. apiospermum (Fig. 2A). Under our experimental conditions, conidia produced a single peptidase of 28kDa able to digest copolymerized BSA (Fig. 2A, lane a). On the other hand, mycelia yielded at least 6 distinct proteolytic bands with apparent molecular masses ranging from 90 to 28 kDa (Fig. 2A, lane b). The production of a 28 kDa peptidase was the major activity observed in the cellular extract of mycelial form (Fig. 2A, lane b). Peptidases catalyze the hydrolysis of peptide bonds in proteins, but can differ markedly in specificity and mechanism of catalysis [22]. All the proteolytic enzymes evidenced herein were active at pH 5.5; however, in extreme acidic conditions (pH 2.5) as well as in neutral-alkaline values of pH (7.0 and 10.0), no proteolysis was observed (data not shown). Additionally, these peptidases were completely inhibited by

10 mM 1,10-phenanthroline, suggesting that these proteolytic enzymes are metallopeptidases or metal dependent peptidases (Fig. 2B). Conversely, two other metallo-type peptidase inhibitors (EDTA and EGTA) as well as E-64 (a cysteine peptidase inhibitor), pepstatin A (an aspartyl peptidase inhibitor) and PMSF (a serine peptidase inhibitor) did not alter the enzymatic activities (data not shown). The major 28 kDa peptidase was early characterized by our group as an acidic extracellular metallopeptidase depending on divalent cations, such as Zn2+, Co2+, and Mg2+, for its full hydrolytic activity. In addition, this metallo-type peptidase was able to cleave relevant host proteins such as human albumin, immunoglobulin G, hemoglobin, mucin, laminin and fibronectin [13]. As previously suggested, the 28-kDa metallopeptidase could help the fungus to obtain amino acids for its growth, to escape from the immunological host response and to invade host tissues after degrading extracellular matrix components [13]. As not all proteolytic enzymes detected as bands in substrate-SDS-PAGE can be refolded into their active forms, we also measured the proteolytic activity in the whole extracts from conidia and mycelium of S. apiospermum by a quantitative method using BSA as substrate (Table 1). By means of this protocol, we showed a high peptidase production in mycelial cells in comparison with conidia. Moreover, the metallopeptidase inhibitor 1,10-phenanthroline at 10 mM

B A 83 62 48 33 25

a

b 17

a Fig. 1 Morphological stages observed in S. apiospermum strain HLPB: conidia (a) and mycelia (b). (A) Optical microscopy. (B) Protein electrophoretic profiles after silver

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staining. The numbers on the left indicate apparent molecular mass of the protein markers expressed in kilodaltons

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A

B

90

70 45

20

a

b

b

a

Fig. 2 Peptidases observed in whole extracts from conidia (a) and mycelia (b) of S. apiospermum strain HLPB. The proteolytic activities were evidenced after BSA-SDS-PAGE, in which the gel strips were incubated at 37°C, for 40 h, in 50 mM sodium phosphate buffer, pH 5.5, supplemented with 2 mM DTT, in the absence (A) or in the presence of 10 mM 1,10-phenanthroline (B). The numbers on the left indicate apparent molecular mass of the protein markers expressed in kilodaltons Table 1 Proteolytic activities (AU 9 lg protein) detected in two distinct morphological stages of S. apiospermum strain HLPB Stages

1,10-phenanthroline (10 mM) Without

With

Conidia

1636 ± 33*

102 ± 12

Mycelia

2592 ± 78*

136 ± 25

*The difference between proteolytic activities of conidial and mycelial extracts was statistically significant (P \ 0.05, Student’s t-test)

restrained the proteolytic activities in both fungal extracts by approximately 95%, suggesting that metallopeptidases were the most prominent class of peptidases produced by these two distinct morphological forms (Table 1). The number of identified metallopeptidases has increased in the last decades and there are promising industrial and medical applications of metallo-type peptidases based on the diverse biological functions of this superfamily of proteolytic enzymes [22, 23]. Metallopeptidases are involved in processes as diverse as embryonic development and bone formation, production of tetanus and botulism toxins, reproduction, arthritis, and cancer development [23, 24]. In this sense, this class of peptidases

emerges as promising targets for the development of antifungal chemotherapy [11, 12, 22] and metalbased drugs, including 1,10-phenanthroline and its metal-phenanthroline complexes, represent a novel group of antifungal compounds with potential applications either alone or in combination with conventional antifungals [25]. The P. boydii complex comprises important human pathogens, and a crucial feature of these microorganisms is their versatility; this goes beyond the fact that they are able to survive at and infect several anatomically distinct sites, each with its own specific set of environmental pressures [26, 27]. It is also evident that the dynamics of the interaction between the fungus and the cells of the immune system may significantly affect fungal survival and virulence at different body sites. Therefore, defining the extent to which morphogenesis impacts on pathogenesis calls for further studies aimed at discovering new virulence factors that is an attractive direction for future study, including the isolation of stage-specific markers with potential application in biochemical and immunological analysis. Acknowledgments This study was supported by grants from Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq), Financiadora de Estudos e Projetos (FINEP), Fundac¸a˜o de Amparo a` Pesquisa do Estado do Rio de Janeiro (FAPERJ), Fundac¸a˜o Universita´ria Jose´ Bonifa´cio (FUJB), Programa de Apoio a Nu´cleos de Exceleˆncia (PRONEX) and Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP, grant 05/ 02776-0). Marcia Ribeiro Pinto is supported by a FAPESP fellowship (grant 05/56161-6).

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