2.1. Microorganism and culturing conditions

P. lutzii, Pb01, (ATCC MYA-826) was used in the experiments conducted in this study. The cells were cultured in Fava Netto's medium [0.3% (w/v) proteose peptone, 1% (w/v) peptone, 0.5% (w/v) meat extract, 0.5% (w/v) yeast extract, 4% (w/v) glucose, and 0.5% (w/v) NaCl, pH 7.2] at 36 C and 22°C, for yeast cells and the mycelia phase, respectively. Yeast cell viability and growth were evaluated in triplicate every 6 hours. A trypan blue dye exclusion test was performed to ensure over 95% cell viability during the incubation process.

2.2. Preparation of extracellular protein extracts

The yeast and mycelia extracellular proteins were prepared by inoculating 50 µg/mL of wet weight cells in Fava Netto's liquid medium, and the cells were maintained while shaking (200 rpm) for 24 hours at 36°C and 22°C, respectively. After incubation, the cells were removed by centrifugation at 10,000 g for 30 min at 4°C. The culture supernatant was sequentially filtered through 0.45 µm-pore and 0.22 µm-pore membrane filters. Culture filtrates were concentrated and subsequently washed three times with ultrapure water via ultracentrifugation through a 10-kDa molecular weight cut off in ultracel regenerated membrane (Amicon Ultra centrifugal filter, Millipore, Bedford, MA, USA). The protein concentrations were determined via Bradford assay [28]. For proteomic analysis, three biological replicates of protein samples obtained from three different experiments were performed for yeast cells and mycelia. The three yeast and mycelia cell-free supernatant samples were assessed for the presence of Paracoccidioides DNA via PCR, which if positive indicated fungal cellular lyses in the samples as described below.

2.3. The Polymerase Chain Reaction (PCR) analysis

The genomic DNA isolated from Paracoccidiodes mycelia and yeast cells was obtained according to standard protocol [29]. The PCR reactions were performed with cell-free supernatant (2 µL) and genomic DNA samples as follow: 40 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 1 min. A 1622-bp PCR product was generated using sense S2 (5′- ATGGGTCTCAAGGGAATTC-3′) and antisense At2 (5′-CATCCCCTACTTCATTC-3′) oligonucleotides for the gene encoding formamidase (GenBank accession number AY163575). The PCR amplicons were detected via 1% (w/v) agarose gel electrophoresis with ethidium bromide staining. PCR sensitivity was assessed using Paracoccidioides Pb01 genomic DNA (at five dilutions) as a template (50 ng to 1 pg).

2.4. Two-dimensional gel electrophoresis (2-DE)

For the first dimension, each sample containing 500 µg of total protein was treated with a 2D-Clean-up Kit (GE Healthcare, Uppsala, Sweden) according to the manufacturer's instructions. The amount of protein (500 µg) was diluted to 200 µL of the final sample volume and corresponded to 30 g of wet weight cells (Section 2.2). The proteins samples were diluted in 250 μL of rehydration solution containing 7 M urea, 2 M thiourea, 2% (w/v) 3-[(3-Cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS), 0.002% (w/v) dithiothreitol (DTT), 0.5% (v/v) ampholyte 3–11 and trace amounts of bromophenol blue [30]. These samples were loaded onto a 13 cm Immobiline™ DryStrip gel with a pH linear range of 3–11 in a Multiphor-II Electrophoresis System (GE Healthcare, Uppsala, Sweden). The samples were separated according to their isoelectric points at 20°C with a current of 50 µA/strip. The following program was applied: 30 V for 14 hours; 500 V for 500 Vh (step); 1 kV for 800 Vh (gradient); 8 kV for 11.3 kVh (gradient) and 8 kV for 2.9 kVh (step). After isoelectric focusing, the strips were equilibrated twice for 40 min in equilibration buffer [50 mM Tris-HCl pH 8.8, 6 M urea, 30% (v/v) glycerol, 2% (w/v) SDS and 0.002% (w/v) bromophenol blue] containing 18 mM DTT and 135 mM iodoacetamide [31]. The second dimension (SDS-PAGE) was performed with 12% polyacrylamide gels using a vertical system (GE Healthcare) and standard Tris/glycine/SDS buffer for one hour at 150 V and 250 V until the end of the run at 12°C. The gels were stained with Coomassie brilliant blue (PlusOne Coomassie Tablets PhastGel Blue R-350, GE Healthcare) according to the manufacturer's instructions.

2.5. Image analysis

The 2-D gel images were obtained using an Image Scanner III (GE Healthcare). 2D gel spot detection, matching and intensity calculations were performed with Image Master 2D Platinum v7.0 (GE Healthcare). Three independent samples were prepared for each fungal phase to ensure reproducibility. The final set contained six gel images. Software matching between the images was performed, and the matching process was thoroughly assessed via visual inspection. The volume percentage of the spots was used for statistical calculations and the determination of overexpressed proteins.

2.6. 2D-gel statistical analysis

To compare the differences in protein expression between Paracoccidioides mycelia and yeast cells the ANOVA test was applied considering statistically significant p-value ≤0.05. To compare the proteins with multiple isoforms, the sum of the percentage of the volumes (in relation to the total proteins) of each isoform was first obtained in triplicate. Next, the sum of the percentage of volumes for the proteins was used for statistical analysis, which was performed to determine the significant differences in expression profiles between Paracoccidioides mycelia and yeast proteins. All statistical calculations were performed using the software STATISTICA version 7.0. (Statsoft Inc., 2005). The spectrometry analyses were performed on spots displaying significant alterations (≥ fold change of 2.5) between yeast and mycelia samples and spots displaying similar expression levels (common spots).

2.7. Mass spectrometry analysis

Spots of interest were manually excised and digested as previously described [32], [33]. Briefly, the gel pieces were resuspended in 100 μL acetonitrile (ACN) and dried in a speed vacuum. The gel pieces were then reduced with 10 mM DTT and alkylated with 55 mM iodoacetamide. The supernatant was then removed, and the gels were washed with 100 μL ammonium bicarbonate by vortexing for 10 min. The supernatant was removed, and the gel pieces were dehydrated in 100 μL of a solution containing 25 mM ammonium bicarbonate/50% (v/v) ACN, vortexed for 5 min, and centrifuged. This step was then repeated once. Next, the gel pieces were dried in a speed vacuum and 12.5 ng/mL trypsin (sequencing grade modified trypsin, Promega, Madison, WI, USA) solution was added followed by a rehydration step performed on ice at 4°C for 10 min. The supernatant was removed, 25 μL of 25 mM ammonium bicarbonate was added and the supernatant was then incubated at 37°C for 16 hours. Following digestion, the supernatant was placed in a clean tube. Next, 50 μL 50% (v/v) ACN and 5% (v/v) trifluoroacetic acid (TFA) were then added to the gel pieces. The samples were vortexed for 30 min, sonicated for 5 min, and the solution was then combined with the aqueous extraction above. The samples were dried in a speed vacuum, the peptides were solubilized in 10 µL ultrapure water, and the samples were subsequently purified in ZipTip® Pipette Tips (ZipTips® C18 Pipette Tips, Millipore, Bedford, MA, USA). Two microliters of each peptide sample were deposited onto a matrix-assisted laser desorption ionization quadrupole time-of-flight mass spectrometry (MALDI-Q-TOF MS) target plate and dried at room temperature. Next, the peptide mixture was covered with 2 μL of matrix solution (10 mg/ml α-cyano-4-hydroxyciannamic acid matrix in 50% (v/v) ACN and 5% (v/v) trifluoroacetic acid). The mass spectra were recorded in the positive reflectron mode on a MALDI-Q-TOF mass spectrometer (SYNAPT, Waters Corporation, Manchester, UK).

The search against the NCBI non-redundant database using the MS/MS data was performed using Mascot software v. 2.4 (http://www.matrixscience.com) (Matrix Science, Boston, USA). The Mascot MS/MS ion search parameters were as follows: tryptic peptides with one missed cleavage allowed; fungi taxonomic restrictions; fixed modifications: carbamidomethylation of Cys residues; variable modifications: oxidation of methionine; and an MS/MS tolerance of 0.6 Da. The identified proteins were described in functional categories according to the MIPS Functional Catalogue Database (http://fsd.riceblast.snu.ac.kr).

For the identification of the MS spectra using the NCBI database, we included the analysis of post-translational modifications (PTMs) for multiple identified proteins/isoforms. We included variable modifications in the search as follows: the acetylation of lysine and the phosphorylation of serine/tyrosine/tryptophan. All proteins/isoforms that presented matches with predicted modified peptides were selected for manual spectral analysis.

2.8. In silico analyses

A number of programs available online were used for the characterization of the identified extracellular proteins. The identified proteins were analyzed using SignalP 3.0 software (http://www.cbs.dtu.dk/services/SignalP/) for the prediction of signal peptides. For cases when the signal sequence was detected, the protein was considered to be secreted via a classical pathway. For the prediction of secreted proteins by non-classical pathways, the software SecretomeP 2.0 (http://www.cbs.dtu.dk/services/SecretomeP/) was employed. Using the prediction methods, a score ranging between 0 and 1 was assigned to each protein in which a score equal or higher than 0.5 was considered indicative of secretion.

The Fungal Secretome Database (FSD) (http://fsd.riceblast.snu.ac.kr) was used to analyze our results. The FSD provides a summary of putative secretory proteins by in silico analysis based on prediction programs for 158 fungal/oomycete genomes, including Paracoccidioides Pb01 [34]. Each protein sequence identified in this study was also analyzed for adhesin function using the Faapred web server (http://bioinfo.icgeb.res.in/faap/query.html) and for GPI anchor properties using big-PI Fungal Predictor software (http://mendel.imp.univie.ac.at/gpi/fungi/gpi_fungi.htm). The Faapred web server allows for the prediction of adhesins in the fungal proteomes using an accurate method with scores higher than or equal to 0.8 [35]. The big-PI Fungal Predictor is a sensitive prediction tool used for the recognition of C-terminal motifs capable of GPI lipid anchoring in fungal sequences [36].

The ORF sequences of the identified proteins of Paracoccidioides Pb01 yeast cells were submitted for Blast analysis using the NCBI website (http://www.ncbi.nlm.nih.gov/) against orthologues previously reported for Paracoccidioides Pb18 [27] as well the secretomes of the pathogenic fungi Histoplasma capsulatum [10], [18], Cryptococcus neoformans [19] and Aspergillus fumigatus [37].

2.9. Enzymatic activity assay

To determine the activity of formamidase (FMD), ammonia formation was assessed as previously described [23]. Protein extracts were obtained as described in Section 2.2. Next, 1 μg of total protein extract was added to 200 μL of 100 mM formamide substrate solution in 100 mM phosphate buffer containing 10 mM EDTA, pH 7.4. The samples were incubated at 37°C for 30 min. Next, 400 μL phenol-nitroprusside and 400 μL alkaline hypochlorite were added to the samples. The samples were then incubated for 6 min at 50°C, and the absorbance was measured at 625 nm. The amount of ammonia released for each sample was determined using a standard curve. One unit (U) of FMD activity was defined as the amount of enzyme required to hydrolyze 1 μmol formamide per mg total protein per minute (µmol/mg/min). The levels of superoxide dismutase (SOD) and glutathione S-transferase (GST) activity were measured using commercially available kits (Superoxide dismutase Assay Kit and Glutathione-S-Transferase (GST) Assay Kit, Sigma-Aldrich, Co., St. Louis, MO, respectively). The SOD Assay Kit includes Dojindo's highly water-soluble tetrazolium salt (WST-1), which produces a water-soluble formazan (WST-1 formazan) dye upon reduction with a superoxide anion. The reaction product (WST-1 formazan) absorbs at 440 nm, and it is proportional to the amount of superoxide anion. The levels of SOD activity, which were defined as inhibition rate %, were quantified by measuring the decrease in the color development at 440 nm. The GST Assay Kit employs 1-Chloro-2,4-dinitrobenzene (CDNB) to produce GS-DNB by conjugation of the thiol group of glutathione (GSH). The reaction product (GS-DNB) absorbs at 340 nm, and the rate of increase in the absorption is directly proportional to the GST activity of the sample. The GST-specific activity is defined as µmol of GS-DNB per mg of total protein per minute (µmol/mg/min). The enzymatic activity results represent the mean of three independent determinations, and statistical comparisons were performed using the Student's t test. The samples with p-values ≤0.05 were considered statistically significant.

2.10. Brefeldin A treatments and Paracoccidioides internalization by macrophages

Brefeldin A (BFA, Sigma), was used to evaluate the role of conventional protein secretion on Paracoccidioides internalization by macrophages. Stock solutions (1 mg/mL) of Brefeldin A (BFA) were prepared in methanol and stored at −20°C until use. The yeast cells were cultivated in Fava Netto's liquid medium in either the absence or presence of BFA (6 μg/mL) for 6, 12 and 24 hours. The cell-free supernatant samples were obtained as described above, reduced to an equal final volume (1 mL), and processed for one-dimensional electrophoresis (SDS-PAGE). Next, 30 μL of each cell-free supernatant sample was loaded onto a 12% SDS PAGE gel, and the proteins were separated via electrophoresis. The gels were run at 150 V for approximately 2 hours and visualized using Coomassie brilliant blue staining.

Bone marrow-derived macrophages (BMM) were obtained as previously described from 6 to 8-week-old BALB/c male mice [38]. The macrophages were generated by extracting bone marrow cells from the femurs of BALB/c male mice, and the cell cultures were incubated at 37°C with 5% CO₂ in RPMI medium (RPMI 1640, Vitrocell, Brazil) supplemented with 10% (v/v) fetal bovine serum and GM-CSF (Recombinant Murine Granulocyte Macrophage Colony Stimulating Factor, PeproTech-Brasil FUNPEC, Brazil) at 10 ng/mL for 8–10 days, which prompts differentiated macrophages to adhere to the plastic-bottom plates.

To determine the number of adhered/internalized fungi cells by BMM, the differentiated macrophages were quantified and plated at 5×10⁵ cells per well on glass coverslips in 6-well culture plates and infected with Paracoccidioides yeast cells at a 1:5 ratio macrophage: yeast. The cells were co-cultivated for 12 hours at 37°C in 5% CO₂ to allow for fungi adhesion and/or internalization. The supernatants were then aspirated, the monolayer was gently washed twice with PBS 1X to remove any non-adhered/internalized yeast cells, and the samples were processed for microscopy. The glass coverslips were fixed with methanol and stained with Giemsa (Sigma). A total of 200 macrophages were quantified to determine the average number of adhered/internalized fungal cells [39].

The number of viable fungi after co-cultivation with macrophages was determined by quantifying the number of colony forming units (CFUs). The cells were rinsed twice with PBS 1X to remove any non-internalized yeast cells. The wells were washed with distilled water to promote macrophages lysis, and the suspensions were collected in individual tubes. Fifty microliters of cell homogenate was plated in BHI medium supplemented with 5% (v/v) fetal bovine serum and incubated at 37°C in 5% CO₂ for 15 days_. The number of CFUs was expressed as the mean value ± the standard deviation. A portion of this macrophage homogenate was processed for immunoblot analysis. The experiments were performed in triplicate, and the statistical analyses were performed using the Student's t test.

BFA treatment was performed to evaluate the role of extracellular proteins in the adhesion/internalization of Paracoccidioides by macrophages. Paracoccidioides yeast cells were cultivated in Fava Netto's liquid medium in either the absence (control) or presence of BFA (6 μg/mL) for 24 hours prior to the infection of macrophages. The effect of Brefeldin on macrophages was evaluated by adding BFA posteriorly to the co-culture of macrophages with fungal cells (not in the preliminary culture of Paracoccidioides cells) as an additional control (data not show). To test the biological activity of the secreted proteins released by Paracoccidioides yeast cells, we evaluated whether the addition of concentrated culture supernatant influenced the adhesion/internalization of Paracoccidioides by macrophages. We added 30 µL of extracellular protein extract of Paracoccidioides yeast cells (2 µg/µL) to the macrophage and fungal cell co-culture. The number of adhered/internalized yeast cells by BMM and the survival rate (viable fungi after co-cultivation) were determined. The results were compared with the control (absence of extracellular proteins and BFA). All animal work was conducted in accordance with the international rules for animal experimentation. The animal protocol was approved by the Universidade Federal de Goiás ethical committee of animal treatment (Number: 192/2011).

2.11. Sample preparation and nanoUPLC-MS^E acquisition

In vitro cultured macrophages were infected with Paracoccidioides Pb01 yeast cells, and co-cultivated for 12 hours at 37°C in 5% CO_2. The infected and non-infected macrophages (BMM) were incubated in distilled water to promote macrophages lysis and centrifuged at 10,000× g for 5 min to separate the macrophage cytosol from the pellet containing host nuclei, cytoskeleton, and Paracoccidioides yeast cells. The macrophage lysate supernatant was sequentially filtered through a 0.22 µm-pore-size membrane filter, washed three times with ultrapure water by ultracentrifugation using a 10-kDa molecular weight cut off in ultracel regenerated membrane (Amicon Ultra centrifugal filter, Millipore, Bedford, MA, USA), and concentrated to a final volume of 1 mL. The protein concentrations were determined via Bradford assay [28].

To analyze the expression profile of the Paracoccidioides secreted proteins with in vitro cultured macrophages, the samples (obtained as described above) were analyzed using nanoscale liquid chromatography coupled with tandem mass spectrometry. Sample aliquots (100 µg) were prepared for nanoLC-MS/MS as previously described [40]. The digested peptides were further separated via nanoUPLC-MS^E and analyzed using a nanoACQUITY^TM system (Waters Corporation, Manchester, UK). The MS data obtained via UPLC-MS were processed and examined using the ProteinLynx Global Server (PLGS) version 2.4 (Waters Corporation, Manchester, UK). For protein identification and quantification level analysis, the observed intensity measurements were normalized with the identified peptides of the digested internal standard.

2.12. Immunoblot analysis of proteins secreted by Paracoccidioides in macrophages

For immunoblot analysis, the same samples used for nanoUPLC mass spectrometry analysis were probed using triosephosphate isomerase and enolase antibodies [22], [41]. Thirty micrograms of protein sample was loaded onto a 12% SDS-PAGE gel and separated by electrophoresis. The gels were run at 150 V for approximately 2 hours. The proteins were transferred from the gels to nitrocellulose membranes at 30 V for 16 h in a buffer containing 25 mM Tris-HCl (pH 8.8), 190 mM glycine and 20% (v/v) methanol. The gels were stained with Ponceau red to verify complete protein transfer. Next, each membrane was incubated in blocking buffer (1X PBS, 1.4 mM KH2PO4, 8 mM Na2HPO4, 140 mM NaCl, 2.7 mM KCl (pH 7.3), 5% (w/v) nonfat dried milk, and 0.1% (v/v) Tween 20) for 2 h. The membranes were washed with buffer (1X PBS and 0.1% (v/v) Tween 20) and incubated with primary antibodies for 2 h at room temperature. The primary antibodies were used at a dilution of 1 antibody/40000 buffer (v/v). The primary polyclonal antibody employed was either anti-enolase [22] or anti-triosephosphate isomerase [41]. The membranes were then washed in blocking buffer three ×15 min. The membranes were incubated with the appropriate conjugated secondary antibody, either a horseradish-peroxidase conjugated anti-rabbit or anti-mouse IgG, at a 1/5000 (v/v) ratio, and the blots were developed using an enhanced chemioluminescence detection system (ECL, GE Healthcare).

3.1. Validation of the extracellular protein extraction method

Viability analysis was performed with the yeast cells. Using trypan blue staining, we observed yeast cell viability over 95% during a 24-h cultivation in liquid medium (Figure 1A). To standardize sample acquiring, PCR analysis was performed to assess for the Paracoccidioides formamidase gene. The sensitivity of the PCR assay was assessed using FMD specific primers with a serial dilution of the genomic DNA sample (ranging from 50 ng to 1 pg). The smallest quantity of DNA that was amplified by PCR was 5 pg (Figure 1C, lane 4). This result implies that technique is able to amplify the FMD gene with only 5 pg DNA present in the cell-free supernatant. As shown in Figure 1D, the FMD gene was not detected in the yeast and mycelia cell-free supernatants. These data both contribute to the hypothesis that the cell lyses failed to influence the extracellular protein profiles of the extracts obtained for proteomic analysis and validate the extraction method of extracellular proteins expressed by Paracoccidioides. Additionally, none of the secreted had motifs for GPI-anchor (data not shown), as evidenced by in silico, suggesting that the presence of the proteins in the secretome was not due to the accidental release from the fungal surface.

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Figure 1. Validation of the extracellular protein extraction method.

A- The viability of Paracoccidioides yeast cells incubated in Fava Netto's liquid medium (dark gray square) and the incubation of yeast cells in Fava Netto's liquid medium containing 6 µg/mL Brefeldin A (light gray square). Viability was assessed using trypan blue staining. The error bars represent the standard deviation of three biological replicates. B- The growth of Paracoccidioides yeast cells in liquid medium in either the absence (dark line) or presence of 6 µg/mL Brefeldin A (light gray line). Culture growth was evaluated by quantifying the number of yeast cells per mL. The error bars represent the standard deviation of three biological replicates. C- PCR sensitivity for the formamidase gene was assessed using Paracoccidiodes Pb01 genomic DNA (at five dilutions) as a template (50 ng to 1 pg). Lanes: 1 −50 ng; 2 −5 ng; 3 −50 pg; 4 −5 pg; 5 −1 pg; 6 - negative control (without genomic DNA). The formamidase PCR amplicons were assessed via 1% agarose gel electrophoresis and stained with ethidium bromide. D- The yeast and mycelia cell-free supernatant samples (2 µL) were assessed for the presence of Paracoccidiodes DNA via PCR using oligonucleotides specific for the formamidase gene.

https://doi.org/10.1371/journal.pone.0052470.g001

3.2. Analysis of secreted proteins in Paracoccidioides Pb01 yeast cells and mycelia

In this study, we applied a proteomic strategy to identify proteins constitutively secreted by Paracoccidioides Pb01 and analyzed the proteins preferentially secreted by yeast cells and mycelia. Figure 2 depicts the representative two-dimensional gel of both phases performed in biological triplicates. Proteins were distributed in a molecular mass range from 14.10 to 105.25 kDa and an experimental pI value ranging from 3.76 to 10.18 was observed. The figure displays the presence of constitutive and differentially expressed proteins in both secretomes.

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Figure 2. Proteins detected in the secretome of yeast cells and mycelia of Paracoccidiodes via 2D-gel analysis.

Protein profile generated after the separation of the secreted fraction of proteins by yeast cells (A) and mycelia (B) using 2D-eletrophoresis (first dimension: IEF pH range 3 –11 non-linear, second dimension: 12% (w/v) SDS-PAGE) and visualized using Coomassie brilliant blue staining. The 2-D gel images of three biological replications of each phase were compared to identify the differential expression levels of proteins using Image master 2D Platinum software. The protein spots that were identified via MS/MS are numbered and listed in Table S1. The pH gradient is shown above the gel, and the molecular mass protein standards (kDa) are indicated to the left of the gels.

https://doi.org/10.1371/journal.pone.0052470.g002

Figure 3 depicts the graphic summation of the proteomic analysis, which revealed an average of 356 spots for the mycelia and 388 spots for the yeast secretome, which were matched and assessed for statistical significance using an ANOVA test to compare the differences in protein expression between the two fungal phases. After performing the statistical analysis, it was determined that 123 and 146 spots (i.e., proteins) were differentially secreted by mycelia and yeast cells, respectively (Figure 3A).

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Figure 3. Graphic summation of proteomics analysis.

A- The comparative analysis using Image master 2D Platinum software displays the analyzed spots and the preferentially expressed spots in mycelia and yeast secretomes, while the mass spectrometry analyses displays the identified differentially expressed spots. B- The Venn diagram shows the number of identified proteins via MS/MS. The preferentially secreted extracellular proteins include those with statistically significant alteration in the mycelia and yeast secretome, while the constitutive proteins refer to those with similar expression patterns.

https://doi.org/10.1371/journal.pone.0052470.g003

Coomassie G-250 Blue Stained maps were used for MS identification of the individual protein spots. A total of 160 proteins/spots were identified unambiguously (displayed in Table S1), which displays all proteins and isoforms identified in the secretome analysis. The identified proteins/isoforms were analyzed using several prediction programs. While not all secreted proteins/isoforms have canonical signal peptides, 20 of the 160 (12.5%) contained a potential N-terminal secretion signal (Mean S score ≥0.5, Table S1). The proteins/isoforms that presented the signal peptide consensus included enzymes such as glycosyl hydrolase, beta glucosidase, enoyl-CoA hydratase, aminopeptidase and disulfide isomerase. Using the Secretome P algorithm, 84 of 160 (52.5%) proteins/isoforms were predicted to be secreted molecules, including aconitase, enolase, fructose-biphosphate aldolase, 2-methylcitrate synthase, glyceraldehyde-3-phosphate dehydrogenase, dipeptidyl-peptidase, thioredoxin-like proteins and peroxisomal catalase.

Regarding the proteins identified in the secretomes, fungal adhesin prediction analysis using the Faapred web server revealed that approximately 21% of the Paracoccidioides secretome (33 proteins/isoforms) was predicted to be adhesin-like proteins, such as enolase, glyceraldehyde-3-phosphate dehydrogenase, and triosephosphate isomerase (Table S1). All identified proteins/isoforms were grouped into Enzyme Classification (EC) according to guidelines of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB) as listed in Table S1. More than half of the Paracoccidioides Pb01 secretome comprises enzyme-like proteins (64%), and the most prevalent enzyme classes found were oxidoreductases (17%), transferases (17%), and hydrolases (14%) as depicted in Figure S1.

3.3. Constitutive proteins in the secretome of Paracoccidioides Pb01

In this report, we described the proteins constitutively present in the secretome of Paracoccidioides Pb01 mycelia and yeast cells. For this analysis, the isoforms of each protein were combined to perform the statistical analysis. Twenty eight proteins were constitutive to mycelia and yeast secretomes (Figure 3B). Most of these proteins were grouped into the functional category of metabolism, energy and protein fate. The enzymes in the energy category, including enolase, fructose-bifophosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase and phosphogycerate kinase, were abundant in both secretomes as depicted in Table 1.

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Table 1. Constitutive proteins secreted by Paracoccidioides Pb01 yeast and mycelia.

https://doi.org/10.1371/journal.pone.0052470.t001

3.4. Extracellular proteins upregulated in Paracoccidioides Pb01 mycelia

A comparative analysis was performed between the extracellular protein profiles of mycelia and yeast cells. Based on protein categorization, 30 proteins were preferentially secreted by mycelia compared with yeast cells (Figure 3B and Table 2). The proteins identified are implicated in a variety of biological processes, such as cell rescue, cell defense, cell virulence, metabolism, energy, cell cycle, DNA processing, protein fate and protein synthesis (Table 2). Proteins that play a role in cell rescue, defense and virulence include the heat shock protein SSC1, disulfide isomerase, glutathione reductase, peroxisomal catalase, thioredoxin reductase and the TCTP family of proteins. Enzymes involved in the metabolism of amino acids were abundant in the mycelia secretome. Some of the proteins expressed in the mycelia secretome were significantly upregulated compared with the yeast cells, including the Cofilin/tropomyosin-type actin-binding family of proteins (fold change of 1911.74), peroxisomal catalase (fold change of 61.29), glutamate carboxypeptidase (fold change of 51.03), aconitase (fold change of 56.98) and heat shock protein SSC1 (fold change of 35.47) (Table 2). Interestingly, several proteins were detected exclusively in the mycelia secretome, including detoxification proteins such as thioredoxin reductase and disulfide-isomerase tigA, proteins that play a role in the metabolism of amino acids, and proteins involved in energy production. The proteins expressed exclusively in the secretome of mycelia (compared with the yeast phase) are summarized in Table S2.

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Table 2. Preferentially secreted proteins by Paracoccidioides Pb01 mycelia.

https://doi.org/10.1371/journal.pone.0052470.t002

3.5. Extracellular proteins upregulated in Paracoccidioides Pb01 yeast cells

When comparing the secretome of mycelia and yeast cells, 24 proteins were identified that are preferentially secreted by yeast cells into the extracellular environment as depicted in Table 3 and Figure 3B. The differentially expressed proteins displayed fold changes ranging from 2.72 to 70672.37 when compared with mycelia. Most of these proteins play a role in cell rescue, cell defense, cell virulence, cell cycle, DNA processing, and energy production (Table 3). The proteins that play a role in cell rescue, defense and virulence, including heat shock protein 60 (fold change of 6.20), Hsp90 binding co-chaperone (fold change of 20.97), disulfide isomerase Pdi1 (fold change of 5.76), superoxide dismutase (fold change of 2.72), thioredoxin-like protein (fold change of 8.40) and hsp70-like protein (fold change of 7.91), were more abundant in the yeast secretome. The 2-methylcitrate synthase protein was significantly expressed in the yeast phase secretome with a fold change of 70672. Interestingly, several proteins were detected exclusively in the yeast phase, including glutathione S-transferase, energy production-associated enzymes such as pyruvate kinase and phosphoglycerate mutase, proteins that play a role in cell cycle and DNA processing, and proteins involved in cellular transport such as the vesicular-fusion protein, SEC17. The proteins expressed exclusively in the yeast secretome (compared with the mycelia) are summarized in Table S2.

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Table 3. Preferentially secreted proteins by Paracoccidioides Pb01 yeast cells.

https://doi.org/10.1371/journal.pone.0052470.t003

3.6. Enzymatic activity correlates with proteomic data

To validate the significance of the proteomic results, enzymatic assays were performed with extracellular extracts for formamidase (FMD), superoxide dismutase (SOD) and glutathione S-transferase (GST). The differences observed with the 2D-gel analysis correlate with the enzymatic assays (Figure 4). The protein levels and enzymatic activity of FMD was increased in the mycelia secretome compared with the yeast phase as depicted in Table 2 and Figure 4, panel A. SOD and GST presented higher levels of activity in the yeast parasitic phase (Table 3 and Figure 4, panels B and C, respectively). For all the analyzed enzymes, the activity levels correlated with the proteomics data (Tables 2 and 3).

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Figure 4. Enzymatic activity analysis validates the secretome data for Paracoccidioides mycelia and yeast cells.

Activity assay results of (A) formamidase (FMD), (B) superoxide dismutase (SOD) and (C) glutathione S-transferase (GST) assessed for mycelia and yeast protein extracts. FMD activity was assessed by measuring the levels of ammonia released using a standard curve. The SOD and GST Assay Kit were used to determine SOD and GST enzymatic activity, respectively. The student's t test was used for statistical comparisons, and the observed differences were statistically significants (p≤0.05). The erros bars represent the standard deviation of three biological replicates.

https://doi.org/10.1371/journal.pone.0052470.g004

3.7. Protein isoforms and post-translational modifications

The 160 proteins/isoforms identified in the secretome of Paracoccidioides Pb01 are encoded by 86 different genes. Protein isoforms were identified for 37 of these gene products as depicted in Table S1. This observation suggests that many secreted proteins undergo post-translational modification. Table S3 presents the predicted post-translational modifications for the proteins identified in the mycelia and yeast secretomes. Hsp-70 like protein, heat shock SSC1, and 2-methylcitrate synthase represent the molecules displaying the highest number of identified isoforms (seven, eight and nine, respectively). The presence of the same protein displaying differential relative migration with 2D analysis may be associated with post-translational modification (PTM). To predict the putative PTMs, which explain the high isoform frequency, the most common S-T phosphorylation and lysine acetylation was included as a variable modification in the MASCOT search. The predicted PTMs were confirmed via manual spectral analysis as previously described [33]. In spite of the limitations associated with the MASCOT search for the PTM study, this strategy has been used previously to perform such analysis in organisms [42]. We observed an increase in peptide matches and sequence coverage when using those search criteria. Table S3 presents the predicted post-translational modifications for proteins in the mycelia and yeast secretomes. A total of 116 isoforms were analyzed using such criteria, and peptides displaying phosphorylation, acetylation or both PTMs were detected for 44 isoforms (Table S3). Serine/threonine phosphorylation was observed for 31 isoforms, while acetylation was observed for 30 isoforms (Table S4). The spectral analysis of isoform 46 of the heat shock protein, SSC1, revealed twelve phosphorylation and six acetylation sites. Many isoforms identified in the secretome displayed similar molecular weights and different pIs shifts. In contrast, a series of isoforms differed in both molecular weight and pI. This observation may be due to other PTMs, such as glycosylation, which were not analyzed because of the high complexity spectrum of the structures that glycan branches assume [43]. Further studies using analytical tools more appropriate for PTM analysis are required to confirm these putative findings.

3.8. Conventional and Non-conventional protein secretion by Paracoccidiodes

The in silico analysis indicated that 65% of the Paracoccidioides secretome included proteins/isoforms with known classical or non-classical secretion signals and that are actively exported to the extracellular space. Using the SignalP program, we found that 12.5% of the extracellular proteins/isoforms displayed putative signal peptide sequences (Table S1). In contrast, 52.5% of the identified extracellular proteins/isoforms were predicted, using the SecretomeP program, to be secreted by mycelia and yeast via a non-classical mechanism. Proteins/isoforms that were not predicted to be secreted by yeast and mycelia represented 56 of the 160 identified spots (35%) (Table S1). These data are in agreement with those reported by the Fungal Secretome Database (FSD), which describes that 58% of the 9,136 proteins expressed by the Paracoccidioides Pb01 genome are predicted to be Class NS (non-classically secreted), while 14% display a classical secretion signal (Class SP) and 28% of the proteins have not been classified (Figure S2).

3.9. Blocking canonical protein secretion in Paracoccidioides Pb01 yeast cells influences fungal adhesion/internalization by macrophages

Experiments were performed to investigate the effect of protein secretion on the adhesion/internalization of Paracoccidioides yeast cells by macrophages. As demonstrated in Figure 1A, yeast cell viability was over 90% during 24-hour incubation in the presence of the protein secretion inhibitor, Brefeldin A (BFA). Additionally, the growth rate of yeast cells in the presence of BFA was not altered during the incubation as depicted in Figure 1B. The yeast cell protein profile upon incubation with BFA was analyzed via SDS-PAGE using equal volumes of each extract (30 µL). As depicted in Figure 5A, BFA reduced the levels of proteins secreted by yeast cells starting from 12 hours with a stronger effect after 24 hours of incubation. Therefore, treatment with BFA at 24 hours was used for the adhesion/internalization experiments involving Paracoccidioides Pb01 and macrophages.

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Figure 5. Blocking the conventional protein secretion pathway leads to a decrease in Paracoccidioides yeast cell phagocytosis.

A- The protein profile of the cell-free supernatant samples reveals the effect of blocking the protein secretion pathway on yeast cells. Paracoccidioides yeast cells were cultivated in Fava Netto's liquid medium in either the absence (control) (lanes 1, 3 and 5) or presence of Brefeldin A (BFA) at 6 μg/mL (lanes 2, 4 and 6) for 6, 12 and 24 hours, respectively. The cell-free supernatant samples were prepared (as described in the Materials and Methods section), reduced to equal final volumes (1 mL), and processed for one-dimensional electrophoresis (SDS-PAGE). Thirty microliters of each sample was separated via SDS-PAGE and visualized using Coomassie brilliant blue staining. The numbers on the left side correspond to the molecular mass standard. B- The average number of internalized/adhered Paracoccidioides cells by macrophages was determined. Macrophages were infected with Paracoccidioides yeast cells, which were pre-cultivated previously without BFA (control), in the presence of BFA or the presence of concentrated culture supernatant containing extracellular proteins (EP). The adhered/internalized cells were analyzed as described in the materials and methods section. C- The number of viable yeast cells after phagocytosis by macrophages was evaluated by counting the number of colony forming units (CFUs). The results are representative of triplicate biological samples. Statistical significance (* p≤0.05) was determined by comparing the results with the control group.

https://doi.org/10.1371/journal.pone.0052470.g005

The number of internalized/adhered Paracoccidioides cells by macrophages was significantly decreased after yeast treatment with BFA for 24 hours compared with the control as shown in Figure 5, panel B. Additionally, the yeast cell treatment with BFA resulted in a decreased recovery of viable yeast cells in macrophages, (Figure 5, panel C). This finding reflects the consequence of the decrease in the number of internalized/adhered Paracoccidioides cells by macrophages with the addition of BFA, thereby suggesting that secretion inhibition decreases the association of macrophages with yeast cells. In contrast, the addition of concentrated culture supernatant containing Paracoccidioides extracellular proteins resulted in an increase in the number of internalized/adhered fungal cells by macrophages, as well as in increase in the recovery of viable yeast cells into macrophages (Figure 5, panels B and C, respectively).

The Brefeldin's effect on macrophages, which was evaluated by adding BFA to the co-cultivation, failed to cause a significant difference in the average number of internalization/adherence yeast cells by macrophages (data not shown). Figure S3 depicts microscopy analysis of Paracoccidioides adhesion and internalization by macrophages, which was used to determine the average number of internalized/adhered fungi cells in the control (panels A and B), in the presence of BFA (panels C and D) and in the presence of concentrated culture supernatant (panels E and F). The BFA treatment reduced the number of yeast cells adhered/internalized by macrophages, while the addition of extracellular proteins increased the rate of phagocytosis.

3.10. Paracoccidioides Pb01 yeast cell secreted proteins identified in macrophages

A nanoLC-MS/MS-based proteomics approach was employed to identify Paracoccidioides Pb01 yeast cell secreted proteins in in vitro cultured macrophages. A total of 18 Paracoccidioides proteins were identified in the cytoplasm of infected macrophages (Table 4), including enolase, heat shock proteins, translation elongation factors, DNA damage checkpoint proteins, and peptidyl-propyl cis-trans isomerase D. No Paracoccidioides proteins were identified in the negative control.

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Table 4. Paracoccidiodes, Pb01 yeast cells secreted proteins in macrophages by nanoLC-MS/MS.

https://doi.org/10.1371/journal.pone.0052470.t004

3.11. Immunoblot analysis of macrophage lysates

We aimed to determine whether Paracoccidioides expresses constituents of the extracellular proteome during infection in cultured macrophages by examining the secretion of candidate proteins via immunoblot analysis. The immunoblot analysis of macrophage lysates revealed that Paracoccidioides yeast cells secrete enolase (PbEno) and triosephosphate isomerase (PbTpi) in infected macrophages (Figure Suplementary 4, line 2, panels A and B, respectively). The pre-incubation of Paracoccidioides yeast cells with Brefeldin A, a protein secretion inhibitor, displayed a lack of PbEno and PbTpi expression in macrophages (Figure Suplementary 4, line 1, panels A and B, respectively).This finding is most likely due to the reduced number of internalized/adhered Paracoccidioides cells by macrophages during BFA treatment as showed in Figure S3. The negative control represents the non-infected macrophage lysate which indicated that those antibodies did not target mouse proteins (Figure Suplementary 4, line 3).

3.12. Comparative analysis between Paracoccidioides Pb01 extracellular proteins with orthologue proteins found in pathogenic fungi secretomes

We compared the ORFs sequences of the extracellular proteins identified in Paracoccidioides Pb01 yeast cells with the orthologues previously reported in the secretome of Paracoccidioides Pb18 [27], H. capsulatum [10], [18], C. neoformans [19] and A. fumigatus [37] as demonstrated in Table S5 and Figure S5. P. brasiliensis (Pb18) and P. lutzii (Pb01) displayed specific variations in the yeast phase secretome. A total of 63 proteins/isoforms detected in the secretome of yeast cells in Pb01 (57%) have also been described for the secretome of Pb18. Among the common proteins, several enzymes were identified including those involved in the metabolism of carbohydrates and lipids and heat shock proteins. Additionally, 47 proteins/isoforms, from a total of 110, were not common to Pb01 and Pb18, thereby suggesting differences in gene expression/secretion between the two species of Paracocidioides. Proteins exclusively identified in the secretome of Pb01 yeast cells (compared with Pb18) included glutathione reductase and glutathione S transferase. Eighty eight identified extracellular proteins in Paracoccidioides Pb01 (80%) had orthologues that have been previously described in fungal secretomes (Table S5 and Figure S5), while 22 extracellular proteins were exclusively found in the Pb01 yeast cells. We found that 59% of the extracellular proteins identified in Pb01 (65 proteins/isoforms) had orthologues in H. capsulatum (Suplementary Figure 5). Among these proteins, 45 proteins/isoforms were also found in Pb18 (Table S5). Concerning to C. neoformans and A. fumigatus the small number of proteins described in the secretomes may account to the reduced number in orthologues to Pb01.