Exploring Potential Virulence Regulators in Paracoccidioides

Aug 22, 2014 - Instituto de Ciência e Tecnologia, Universidade Federal de São Paulo, Campus São José dos Campos, Rua Talim, 330, São José dos Campos ...
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Exploring potential virulence regulators in Paracoccidioides brasiliensis isolates of varying virulence through quantitative proteomics Daniele Gonçalves Castilho, Alison Felipe Alencar Chaves, Patricia Xander, André Zelanis, Eduardo S. Kitano, Solange M.T. Serrano, Alexandre K. Tashima, and Wagner Luiz Batista J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/pr5002274 • Publication Date (Web): 22 Aug 2014 Downloaded from http://pubs.acs.org on August 26, 2014

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Exploring potential virulence regulators in Paracoccidioides brasiliensis isolates of varying virulence through quantitative proteomics

Daniele G. Castilho1; Alison F. A. Chaves1; Patricia Xander2; André Zelanis3; Eduardo S. Kitano4; Solange M. T. Serrano4; Alexandre K. Tashima5 *; Wagner L. Batista1,2 *

1

Departamento de Microbiologia, Imunologia e Parasitologia, Escola Paulista de

Medicina - Universidade Federal de São Paulo, SP, Brazil. 2

Departamento de Ciências Biológicas - Universidade Federal de São Paulo/Campus

Diadema, SP, Brazil. 3

Instituto de Ciência e Tecnologia, Universidade Federal de São Paulo/Campus São

José dos Campos. SP, Brazil. 4

Laboratório Especial de Toxinologia Aplicada – CeTICS, Instituto Butantan, São

Paulo, SP, Brazil. 5

Departamento de Bioquímica, Escola Paulista de Medicina, Universidade Federal de

São Paulo, SP, Brazil.

Corresponding Author: * Wagner L. Batista, phone: +55-11-3319-3594, fax: +55-11-3319-3300, e-mail: [email protected]. Alexandre K. Tashima, phone: +55-11-5576-4848, fax +55-113319-3300, e-mail: [email protected]

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ABSTRACT Few virulence factors have been identified for Paracoccidioides brasiliensis, the agent of paracoccidioidomycosis. In this study, we quantitatively evaluated the protein composition of P. brasiliensis in the yeast phase using minimal and rich media to obtain a better understanding of its virulence and to gain new insights into pathogen adaptation strategies. This analysis was performed on two isolates of the Pb18 strain showing distinct infection profiles in B10.A mice. Using liquid chromatography/tandem mass spectrometry (LC-MS/MS) analysis, we identified and quantified 316 proteins in minimal medium, 29 of which were overexpressed in virulent Pb18. In rich medium, 29 out of 295 proteins were overexpressed in the virulent fungus. Three proteins were found up-regulated in both media, suggesting potential roles of these proteins in virulence regulation in P. brasiliensis. Moreover, genes up-regulated in virulent Pb18 showed increase in its expression after the recovery of virulence of attenuated Pb18. Proteins up-regulated in both isolates were grouped according to their functional categories. Virulent Pb18 undergoes metabolic reorganization and increased expression of proteins involved in fermentative respiration. This approach allowed us to identify potential virulence regulators and provided a foundation for achieving a molecular understanding of how Paracoccidioides modulates the host-pathogen interaction to its advantage.

Keywords: Paracoccidioides brasiliensis; pathogenic fungus; proteomic analysis; virulence regulators.

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INTRODUCTION The dimorphic fungal pathogens Paracoccidioides brasiliensis and P. lutzii cause an important systemic mycosis, paracoccidioidomycosis (PCM), which is prevalent in Latin America. The thermo-dimorphism of P. brasiliensis reflects both a change in morphology and a switch from a saprophytic to a parasitic lifestyle. In the environment, Paracoccidioides exist as mycelia (at temperatures below 26°C). Inhalation of mycelial-produced conidia into the lungs exposes the conidia to mammalian body temperatures, which triggers their differentiation into pathogenic yeast cells.1 The sudden environmental change associated with infection requires the capacity to rapidly adjust to survive and invade the host. This phase transition (from mycelium to yeast) is an important event in the biology of the fungus and results in the expression of virulence factors required for the establishment of infection in others fungi.2 This process involves a series of transcriptional changes, especially in genes related to virulence and pathogenicity,3 such as those involved in the synthesis and remodeling of the cell wall, protein metabolism and oxidative/thermal stress.3-9 The majority of fungal pathogens that cause systemic diseases are acquired via the respiratory tract through the inhalation of spores or conidia.10 P. brasiliensis uses a sequence of different mechanisms to establish a successful infection, from the first contact with host cells through the later stages of the disease. The major protective host immune response against P. brasiliensis is mediated by cells, as demonstrated by granuloma formation.11 The yeast pathogenic form of P. brasiliensis is a facultative intracellular pathogen that is able to survive and replicate within the phagosomes of unactivated murine and human macrophages.12 Paracoccidioides combat the antimicrobial effects of phagocyte-produced reactive oxygen species (ROS)8 and most likely establish a replication-permissive niche inside the phagocyte; however, the mechanistic details of how Paracoccidioides establish a successful infection and thrive within host phagocytes are still unclear.

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Few virulence factors have been identified in the Paracoccidioides genus thus far. Such factors are difficult to identify because Paracoccidioides have been refractory to classical genetic analysis. Despite the difficulty of genetically manipulating this fungus, Torres et al.13 were able to use antisense-RNA technology and Agrobacterium tumefaciens-mediated transformation to deplete the gene encoding gp43 (PbGP43), the major P. brasiliensis antigen. These researchers showed a decrease in pathogenicity of the gp43-depleted fungus in animal models, suggesting that gp43 plays an important role in Paracoccidioides virulence. Another known virulence factor is PbCdc42, an important molecule for the control of yeast cell growth. Down-regulation of PbCDC42 promotes more organized and controlled cellular growth, which markedly decreases P. brasiliensis virulence.14 In addition, in other fungi, α-1,3-glucan, the main component of the yeast cell wall, is closely related to virulence. The ability of this molecule to block the host’s pathogen recognition mechanism promotes fungal escape from the host’s defenses, which contributes to fungal pathogenesis, as reported for Histoplasma capsulatum15 and Aspergillus fumigatus.16 Differences in the virulence of P. brasiliensis have also been associated with the level of α-1,3-glucan in the fungal cell wall (reviewed by San-Blas and Niño-Vega17). Pep04, a peptide identified using a phage-display technique, is a biomarker of virulence in P. brasiliensis. This peptide was identified in variants of the same isolate with different degrees of virulence (virulent Pb18 and attenuated Pb18).18 In addition, Pep04 was observed to block the establishment of fungal infection in mice, and preincubation of the virulent Pb18 isolate with this peptide resulted in significant inhibition of lung infection. In the case of P. brasiliensis, maintenance in culture for long periods leads to attenuation of virulence,19,20 which in turn can be restored after repeated passages of the fungus in animals.21,22 Therefore, single isolates with different degrees of virulence may serve as useful models for identifying potential virulence regulators.

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In this study, we performed a quantitative proteomic analysis of P. brasiliensis in the yeast phase (pathogenic phase) using a Pb18 strain with distinct infection profiles in B10.A mice (virulent Pb18 and attenuated Pb18). This experimental model allowed the exclusion of genomic variations observed in other strains of P. brasiliensis that are commonly used in studies of virulence factors. Thus, comparing Pb18 proteomes of virulent and attenuated Pb18 strains during growth in rich or minimal media allowed for the identification of possible virulence regulators and provided a foundation for understanding the host-pathogen interaction and the establishment of P. brasiliensis infection.

MATERIALS AND METHODS Fungal Strain and Growth Conditions We used P. brasiliensis Pb18 isolates (with different degrees of virulence) in our experiments. Yeast cells were cultured and maintained in solid medium at 37°C using rich medium (RM; modified YPD, modYPD – 0.5% yeast extract, 1% casein peptone, and 0.5% glucose, pH 6.5). After 3 passages on solid medium, the virulent Pb18 (vPb18) isolate was used to infect mice (B10.A) and then re-isolated. The attenuated isolate (aPb18) was maintained in culture for at least 3 years. Yeast cells were cultured in liquid medium at 37ºC with constant stirring in minimal medium (MM; Ham’s F12 medium (Invitrogen, Carlsbad, CA, USA) not supplemented) or RM for 4 to 7 days for subsequent experiments. Ham’s F12 medium is a chemically defined medium containing mainly pure inorganic compounds and simple organic compounds, amino acids and vitamins and has been used in studies of nutrient utilization, stress adaptation and pathogenicity in P. brasiliensis.23-25

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Survival Curve B10.A male mice were used to assess the mortality associated with vPb18 and aPb18 isolates. All procedures and experiments were performed in accordance with the regulations of the Institutional Ethics Committee for Animal Experimentation of the Universidade Federal de São Paulo, Brazil. The animals were handled according to the US National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals (http://oacu.od.nih.gov/ARAC/index.htm). Briefly, the fungal viability of four-day cultures (generally over 98%) was verified via trypan blue exclusion in a Neubauer chamber. Three groups of six mice each (6 to 8 weeks of age) were separated. All groups were infected intra-tracheally (i.t.) with 106 viable yeast cells/animal. The mortality of the mice was recorded for 250 days after infection.26

Growth Assay of P. brasiliensis Growth curves were produced by evaluating fungal counts during days of growth using trypan blue dye. P. brasiliensis yeast cells (vPb18 and a Pb18 isolates) were cultivated in MM or RM for 5 days at 37°C in biological duplicates for each 5

medium. Yeast cells (1×10 – viability > 90%) were seeded in a 12-well culture plate with 2 mL of mYPD or MM and maintained at 37°C with constant stirring for 48 h. After 48 h, the number of viable cells was determined using a Neubauer chamber every day for 11 days. The viable-cell counts were performed in triplicate. Growth curves and statistical analyses were performed using GraphPad Prism software (San Diego, CA, USA).

Protein Extraction

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Proteins were extracted from P. brasiliensis (vPb18 and aPb18 isolates) according the protocol of Villén et al.27 with some modifications. Yeast grown in MM or RM for 5 days at 37°C were collected via centrifugation at 2,000 x g for 15 min at 4°C and were then washed 3 times with cold PBS, distributed into 2 mL tubes and subjected to centrifugation at 10,000 x g for 5 min at 4°C. The supernatant was subsequently removed, and cytoplasmic proteins were prepared by homogenizing the yeast cells with glass beads (425-600 µm - Sigma, St. Louis, MO, USA) in 700 µL of cold lysis buffer (50 mM Tris-HCl [pH 7.5], 2 mM EDTA, 2 mM dithiothreitol [DTT], 50 mM KCl, 0.2% Triton X-100, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride [PMSF], 10 mg/ml aprotinin and 10 mg/mL leupeptin). Then, the yeast cells were disrupted mechanically using a Mini BeadBeater (Biospec Products, Bartlesville, OK, USA) and were centrifuged at 1,000 x g for 3 min at 4°C to separate the glass beads from the lysate. The supernatant was collected and centrifuged at 15,000 x g for 10 min at 4°C. The protein concentration in the supernatant was determined using the Bradford protein assay (Bio-Rad Laboratories, Hercules, CA, USA) according to the manufacturer’s protocol. The samples were aliquoted and stored at -80°C.

Protein Reduction, Alkylation and Enzymatic Digestion Proteins were processed according to the protocol developed by Kleifeld et al.,28 with some modifications. Briefly, 200 µg of protein from each sample (vPb18 or aPb18 cultivated in MM or RM) was denatured with 4.0 M GuHCl (guanidine hydrochloride). After the reduction of disulfide bonds with 5 mM DTT for 1 h at 65°C and alkylation of cysteine with 15 mM iodoacetamide (IAA) for 1 h at room temperature in the dark, the proteins were precipitated with 8 volumes of cold acetone and dried in a SpeedVac (Thermo Scientific, Bremen, GA, USA). The proteins were subsequently dissolved in 5 µL of 100 mM NaOH and digested with 2 µg of sequencing-grade

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modified trypsin (Promega, Madison, WI, USA) in 195 µL of 50 mM HEPES buffer (pH 7.5) overnight at 37°C.

Dimethyl Labeling Tryptic peptides were differentially labeled via stable-isotope dimethyl labeling as previously described.28 Briefly, tryptic peptides (pH 6-7) from each sample (vPb18 and aPb18 cultivated in MM or RM) were incubated overnight at 37°C with 1 M NaBD3CN (to a final concentration of 20 mM) and labeled with 2 M 12CH2O (light formaldehyde) for the aPb18 samples or 13CD2O (heavy formaldehyde) for the vPb18 samples to a final concentration of 40 mM light/heavy formaldehyde, resulting in mass differences of +30.04 and +36.08 Da for the light and heavy formaldehyde, respectively, for each completely labeled N-terminal or Lys site. We repeated the labeling with light or heavy formaldehyde (to strengthen the labeling) via incubation for an additional 2 h at 37°C with 1 M NaBD3CN (to a final concentration of 10 mM) and labeling with 2 M 12CH2O or 13CD2O at a final concentration of 20 mM. The reaction was terminated by adding 1 M Tris (pH 6.8; to a final concentration of 200 mM) to each sample and incubating for 2 h at 37°C. Then, the samples that were cultivated in the same medium and labeled with the light and heavy isotopes were combined (1:1) and cleaned using StageTips (C18-SCX-C18) according to the protocol described by Rappsilber et al.29 After desalting, the samples were finally dried in a SpeedVac and redissolved in 0.1% formic acid prior to nano-liquid chromatography/tandem mass spectrometry (LC-MS/MS) analysis.

Mass Spectrometric Analysis

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All of the samples were analyzed on an LTQ-Orbitrap Velos (Thermo Fisher Scientific, Bremen, GA, USA) connected to an Easy-nLCII (Thermo Fisher Scientific, Bremen, GA, USA). All columns were packed in-house. The trap column (50 mm × 100 µm I.D.) was packed with Jupiter C18 resin (10 µm, Phenomenex Inc., Torrance, CA, USA), and the analytical column (100 mm × 75 µm I.D.) was packed with ACQUA C18 resin (5 µm, Phenomenex Inc., Torrance, CA, USA). The sample was delivered to the trap column at 2 µL/min in 100% solvent A (0.1% formic acid [Sigma, St. Louis, MO, USA]). Solvent B consisted of 0.1% formic acid in acetonitrile. Gradient elution was performed as follows: 7-45% solvent B over 160 min; 45-85% solvent B over 15 min; 85% solvent B for 3 min; followed by a return to the initial condition (7% solvent B over 13 min), at a flow rate of 200 nL/min. The source was operated in positive-ionization mode, with the voltage and temperature adjusted to 1.8 kV and 200°C, respectively. The mass spectrometer was programmed in data-dependent acquisition mode, with a scanning mass of 350-1,600 Th (with a target value of 106 ions) using the LTQ-Orbitrap analyzer with a resolution of 60,000 (in m/z 400), followed by collision-induced dissociation (CID using the ion-trap analyzer) of the 10 most intense ions, with a dynamic exclusion time of 90 sec. The window for the isolation of precursor ions was set to 2 Da, and the minimum count of ions to trigger events (MS2) was 10,000. Each biological replicate was analyzed in technical triplicates, totalizing 12 LC-MS/MS runs.

Data Analysis Two independently produced biological replicates for each medium (MM or RM) were used to prepare the virulent and attenuated samples for proteomic analysis, and each sample was analyzed in technical triplicate via LC-MS/MS. The raw MS data were processed in PEAKS Studio 7 (Bioinformatics Solutions Inc., Waterloo, Canada) against the Paracoccidioides brasiliensis strain Pb18 database (8,741 entries,

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Paracoccidioides brasiliensis Sequencing Project, Broad Institute of Harvard and MIT, http://www.broadinstitute.org). Searches were performed with a precursor mass tolerance of 15 ppm, a fragment mass tolerances of 0.5 Da, carbamidomethylation of Cys as a fixed modification, and oxidation of Met, acetylation of protein N-termini, the deamidation of Asn/Gln and light- or heavy-dimethyl labeling of N-termini and Lys (+30 Da and +36 Da, respectively) as variable modifications and within a false discovery rate (FDR) of 1%. Two missed cleavages were allowed for trypsin, and a maximum of four variable PTMs was allowed per peptide. Relative quantification results were evaluated using PEAKS Q. The quantified proteins were accepted if they contained both light and heavy formaldehyde labeling and if it was present in at least two out of three technical replicates for both biological duplicates. Protein ratios were calculated as vPb18/aPb18 intensities. To assess the accuracy and reliability of the quantification results, the averages of the two quantified ratios (R1 and R2) for the same protein obtained in the two biological replicates were compared according to the method described in Sui et al.30 If the ratios were very different (i.e., |log2(R1/R2)| » 0), the quantification of that particular protein was not repeatable; therefore, it was excluded from the dataset as an outlier. Outliers were excluded by applying Grubb’s test (http://graphpad.com/quickcalcs/Grubbs1.cfm) with a significance level of 0.01 to the set of log2(R1/R2) values.30 Ratios ≤ 0.5 and ≥ 2 were considered to represent differentially expressed proteins. Data are available via ProteomeXchange Consortium with identifier PXD000804.

Paracoccidioides RNA Isolation and Analysis Paracoccidioides yeast cells grown to exponential phase in MM or RM were collected via centrifugation (2,000 x g), washed 4 times and resuspended in TRIzol (Invitrogen, Carlsbad, CA, USA). RNA was released from yeast cells mechanically by

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beating with 0.5-mm-diameter glass beads for 10 min in the presence of TRIzol reagent (Invitrogen, Carlsbad, CA, USA). RNA was purified through extraction with CHCl3, followed by alcohol precipitation of the aqueous phase. Reverse transcription was performed using RevertAid Premium Reverse Transcriptase (Thermo Scientific, Bremen, GA, USA), 5 µg of total RNA and 22-mer oligo-dT primers. Quantitative PCR assays were assembled using a SYBR-Green-based PCR master mix (Applied Biosystems) with diluted reverse-transcribed templates (1:200 final) and a 0.5 µM concentration of each gene-specific primer (Table S1). Amplification was performed in an ABI Prism 7500 Sequence Detection System (Applied Biosystems Inc., Carlsbad, CA, USA) using the following conditions: 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min. The dissociation curve was determined with an additional cycle of 95°C (15 sec), 60°C (20 sec) and 95°C (15 sec). DNA contamination was evaluated via PCR amplification of the PbGP43 gene (Accession No. U26160). Negative controls did not contain DNA or RNA. The relative expression ratio (experimental/control) was determined based on the 2-∆∆Ct method31 after normalization to the level of the α-tubulin (α-TUB) and aldehyde dehydrogenase (ALDH) transcript.

RESULTS Virulence of P. brasiliensis Isolates To identify possible virulence regulators in P. brasiliensis, we initially obtained two isolates: virulent Pb18 that had been reisolated from susceptible B10.A mice and attenuated Pb18 maintained via continuous in vitro subcultivation. The virulence of the P. brasiliensis isolates was compared following intra-tracheal-administered infection. After 250 days, virulent Pb18 had killed all infected animals, while attenuated Pb18 had killed only one of six infected mice. All animals in the PBS group (control) survived

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(Figure 1A). These results confirmed that the two Pb18 isolates exhibit distinct virulence profiles in an animal model.

Growth Assay of P. brasiliensis Isolates To assess the differences between growth stages of the P. brasiliensis isolates, different media (MM or RM) were used, and the growth profiles of vPb18 and aPb18 were evaluated. Significant differences were detected between vPb18 and aPb18 yeast cells during culture in MM and RM. MM yielded better growth of vPb18, while RM yielded better growth of aPb18. In MM, the vPb18 isolate showed a progressive increase in the number of viable cells in log phase, achieving a steady state at 6 days. The cells remained in stationary phase until day 9, ending in a decline or death phase with a sharp decrease in the number of cells (Figure 1B, left panel). However, aPb18 exhibited slow growth compared to vPb18, decreasing drastically after 8 days (Figure 1B, left panel). On RM, aPb18 grew faster than vPb18 (Figure 1B, right panel). These results indicated that vPb18 is better adapted to a nutrient-poor environment, while aPb18 prefers a rich environment.

Protein Quantification in P. brasiliensis Isolates For the quantitative proteomic analysis of aPb18 (maintained for long periods in culture) and vPb18 (freshly isolated from animals), both isolates were cultivated in MM or RM at 37°C for 4 to 7 days. Then, proteins were extracted, denatured, subjected to tryptic digestion and labeled with light or heavy dimethyl labels. The samples (vPb18 and aPb18) were subsequently pooled and analyzed using a nanoscale LC coupled to a high-resolution hybrid mass spectrometer (LTQ-Orbitrap Velos). Two independent

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biological replicates for each medium were produced to prepare the virulent and attenuated samples for proteomic analysis. After data analysis, a total of 6,601 peptides corresponding to 1,773 proteins were identified in the proteomes of both isolates grown in MM, and 28.8% (512) of the identified proteins were quantified with 1% FDR. Only proteins that were quantified in both biological replicates were considered in this study. Under these criteria, 316 proteins were identified and quantified in MM (Table S2 and S3). From RM, 5,319 peptides corresponding to 1,414 proteins were identified in the proteomes of both isolates, and 30.9% (437) of the identified proteins were quantified (1% FDR). Proteins quantified in both biological replicates were selected. The same approach was also used to identify and quantify 295 proteins in RM (Table S4 and S5). Based on the criteria to validate protein identifications and quantifications, exclusive proteins from vPb18 in MM and RM were not found, because the proteins were never exclusive in both biological replicates for each medium. The accuracy and reliability of quantification in MM and RM were plotted in a heat map (Figure 2) and represented according to Pearson’s correlation (Figure S1). The heat map represents proteins that were up-regulated in vPb18 and aPb18 in MM and RM, based on the criteria described in Material and Methods. The relative protein abundances for each biological replicate showed high similarity in both conditions (Figure 2). A scatterplot was generated using the data derived from logarithmized values (base 2) for the ratios (heavy dimethylation/light dimethylation) for each protein measured in the two independent biological replicates, which were plotted against each other (Figure S1). Pearson’s correlation (R) was sufficiently high in both conditions (R = 0.92 for MM an R = 0.93 for RM), indicating high similarity between the biological replicates.

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Following the statistical analyses, we compared the levels of the proteins that were differentially expressed in vPb18 and aPb18 isolates in MM and RM (ratios ≥ 2 or ratios ≤ 0.5). In MM, this approach resulted in the identification of 29 proteins that were up-regulated in vPb18 and 46 proteins that were up-regulated in aPb18, while 241 proteins showed no differences in expression based on the established criteria (Figure 3A). In RM, we observed 29 proteins that were up-regulated in vPb18 and 42 proteins that were up-regulated in aPb18, while 224 proteins showed no differences in expression (Figure 3A). Three proteins were found up-regulated in vPb18 isolate in both media, namely alcohol dehydrogenase (PADG_04701), vacuolar ATP synthase catalytic subunit A (PADG_05789) and mitochondrial peroxiredoxin Prx1 (PADG_03095). The proteins that were up-regulated in vPb18 (ratios ≥ 2) and aPb18 (ratios ≤ 0.5) cultivated in MM or RM were classified into functional categories using the FunCat2 tool32 (http://pedant.gsf.de/pedant3htmlview/pedant3view?Method=analysis&Db=p3_p28733 _Par_brasi_Pb18; Figure 3B). Following this classification, most proteins up-regulated in vPb18 were related to metabolism (48.27% in MM and 24.13% in RM). Enzymes involved in alcoholic fermentation (e.g., alcohol dehydrogenase) were overexpressed in vPb18 in both growth conditions. These data suggest that fermentative respiration has an important role in the virulence of the fungus. Other proteins that were up-regulated in vPb18 were involved in protein fate (13.79% in MM and 17.24% in RM), protein synthesis (13,79% in RM), and defense and virulence (13.79% in RM). A small fraction of the proteins that were up-regulated in vPb18 belong to categories such as the cell cycle and DNA processing (3.44% in MM and RM), cell fate (3.44% in MM), energy (6.89% in MM and 3.44% in RM) and protein synthesis (6.89% in MM and 3.44% in RM; Figure 3B). The proteins that were up-regulated in vPb18 cultivated in MM and RM and their functional categories are summarized in Tables 1 and 2, respectively.

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The most up-regulated proteins in the attenuated isolate were related to metabolism (51.11% in MM and 48.64% in RM), especially carbohydrate metabolism. Many of these proteins are key components of glycolysis and the Krebs cycle, such as enolase (PADG_04059), acetyl-coenzyme A synthetase (PADG_01677) and triosephosphate isomerase (PADG_06906). In addition, proteins involved in protein synthesis (15.5% in MM and 8.1% in RM), protein fate (8.8% in MM and 14.81% in RM), and transport compounds (8.8% in MM) were identified. A minority of the proteins that were up-regulated in aPb18 and belonged to functional categories were related to processes such as energy (2.22% in MM and 5.4% in RM), cellular communication and signal transduction mechanisms (2.22% in MM), biogenesis of cellular components (2.22% in MM) and cell cycle and DNA processing (2.7% in RM; Figure 3B, Tables S6 and S7). Finally, functional analysis revealed significant differences in metabolism (both quantitatively and qualitatively) between the vPb18 isolate and its attenuated counterpart, and the metabolic profiles of the isolates (vPb18 or aPb18) were similar in both growth conditions. It is important to note that many proteins that were upregulated in isolate vPb18 cultivated in MM and/or RM are virulence regulators in other pathogenic fungi, such as carbonic anhydrase (PADG_00315), formamidase (PADG_06490), vacuolar protease A (PADG_00634) and disulfide-isomerase tigA (PADG_0562). Alcohol dehydrogenase (PADG_04701), mitochondrial peroxiredoxin Prx1 (PADG_03095) and protein vacuolar ATP synthase catalytic subunit A (PADG_05789) were up-regulated in vPb18 grown in both conditions (MM or RM). These data suggest that these identified proteins could also be potential virulence regulators in P. brasiliensis (Table 3).

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Validation of Proteomic Data Via qRT-PCR We examined the expression profiles of the protein-coding genes of P. brasiliensis yeast from the vPb18 isolate compared to the aPb18 isolate. We selected ten protein-coding genes that were up-regulated in vPb18 or aPb18 cultivated in MM or RM and performed quantitative reverse-transcription PCR (qRT-PCR) analyses of these genes. The levels of individual transcripts were normalized to the expression of the α-TUB and ALDH genes, which shows equivalent expression in all conditions (data not shown). The specificity for the virulence isolate was determined via comparison of the relative level of expression in vPb18 versus aPb18 for each selected protein-coding gene. The alcohol dehydrogenase (ADH) gene showed more than 49-fold enrichment in vPb18 compared to aPb18 in both media (Figure 4). Other transcripts that were significantly up-regulated in vPb18 grown in RM included those of the vacuolar protease A (PbSAP, 3.2-fold), disulfide-isomerase (TIGA, 3.2-fold) and CAP20 (7.4fold) genes. In MM, transcription of both the carbonic anhydrase (CA) and formamidase (FMD) genes were substantially higher (approximately 60-fold) in vPb18 cells than in aPb18. In contrast, decreased levels of the protein-coding transcripts for fumarate reductase (FDR, 2.9-fold in RM and 4.5-fold in MM) and HSP (2-fold) were correlated with decreased expression of these proteins in vPb18 (Figure 4A). These data confirm those obtained by quantitative proteomic analysis. To determine whether such genes are important during animal infection, we evaluated their expression before and after two passages of aPb18 in mice. The ADH gene showed more than 9-fold increase in expression after the recovery of virulence of aPb18. Other genes as PbSAP (2-fold), PRX (3.5-fold), FMD (2.5-fold) and CA (1.8fold) also showed a significant increase in its expression (Figure 4B). These results demonstrate that the expression of these genes can be modulated by the host environment and therefore, could play an important role as virulence regulators.

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DISCUSSION P. brasiliensis virulence represents a relatively unexplored field of inquiry, and few virulence factors have been characterized. Thus, to obtain a better understanding of the key elements involved in the virulence of this fungus, it is necessary to generate gene-deletion strains. Gene-deletion strains can be developed using different strategies, including RNA interference and mutagenesis. Although antisense-RNA techniques have been developed for P. brasiliensis, they have not been widely used, hindering the identification of virulence factors in this fungus. In this study, the global proteomes of a pair of P. brasiliensis isolates (vPb18 and aPb18) were analyzed to identify potential virulence regulators. vPb18 showed a strong capacity to promote infection in the host and host death, while the attenuated form (aPb18) showed lower lethality. By using isolates of the same P. brasiliensis strain (Pb18) with different degrees of virulence, genomic differences that are commonly observed in different isolates of P. brasiliensis were excluded; thus, the protein expression changes observed in the vPb18 strain compared to its attenuated counterpart are likely related to virulence. The genes of the proteins that we identified as virulence regulators may be good candidates for gene-deletion studies in this fungus. Kioshima et al.18 demonstrated that the vPb18 isolate is not only more pathogenic than its aPb18 counterpart but also shows significant morphological differences that might be associated with survival in the host. Our experiments revealed differences in the growth profiles of these isolates cultivated in MM and RM (Figure 1B). The growth profiles may be associated with adaptation of these isolates in vivo or in vitro. We believe that the faster growth of aPb18 in RM is directly related to the better adaptation of this fungus to the culture medium, explaining its increased activity in glycolysis and the Krebs cycle. Nevertheless, the rapid growth of the vPb18

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isolate in MM suggests that this isolate is more adapted to adverse or limited conditions. Moreover, we detected in our studies an accumulation of alcohol dehydrogenase in vPb18, key protein in fermentative metabolism, suggesting a connection between metabolic flexibility and virulence in mice. Although vPb18 and aPb18 were derived from the same strain, we have demonstrated significant dynamic differences in their proteomes. Some of the proteins that are up-regulated in vPb18, such as carbonic anhydrase (PADG_00315), vacuolar protease A (PADG_05704) and formamidase (PADG_06490), have been implicated in pathogenesis in other organisms (Table 3). In P. brasiliensis, an expressed sequence tag (EST) approach revealed increased levels of carbonic-anhydrase mRNA in yeastphase cells recovered from infected mice.7 Carbonic anhydrase activity is required for fatty acid biosynthesis in C. neoformans.33 Transcriptional profiling of C. neoformans suggests that fatty acid metabolism may be important for the virulence of this pathogen in mammals.34 Carbonic anhydrase also seems to play a role in epithelial invasion in C. albicans.35 The protein vacuolar protease A is analogous to aspartyl proteinase (Sap) in C. albicans. The secretion of aspartyl proteinases is associated with virulence in C. albicans.36-39 C. albicans produces Saps to aid in invasion, adhesion and tissue destruction. In our study, vacuolar protease A (PbSap) was up-regulated only in RM, most likely because of the absence of protein in the MM composition. Sap1 expression in C. albicans varies significantly; the use of different experimental setups has a significant impact on the dependence on protease activity. SAP1-3 play essential roles in the growth of C. albicans in media containing protein.37,40-42 Formamidase has been detected in the extracellular vesicles secreted by H. capsulatum,43 and EST analysis showed that this enzyme was highly expressed in both P. brasiliensis yeast cells44 and yeast cells recovered from the livers of infected mice.45

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Borges et al.46 characterized formamidase from P. brasiliensis and demonstrated that formamidase reacts with immune sera from patients with PCM, indicating that this molecule is potentially associated with the fungus–host interaction. Proteins that were up-regulated in vPb18 in both media (MM and RM) in this study, such as alcohol dehydrogenase (PADG_04701), mitochondrial peroxiredoxin Prx1 (PADG_03095) and vacuolar ATP synthase catalytic subunit A (PADG_05789), are potential contributors to virulence in P. brasiliensis. The capacity of pathogenic fungi to cause disease is related to their capacity to survive in the host. The phagosome interior is an environment of extreme nutritional deficiency and oxidative stress that requires intracellular pathogens to have adaptive mechanisms to survive.47 The attributes that allow the pathogens to colonize, infect, and cause disease in a wide range of hosts are related to metabolic adaptability and flexibility. Microorganisms utilize fermentation as a potential metabolic mechanism for dealing with hostile host conditions.48 Felipe et al.4 observed that P. brasiliensis yeast cells showed increased expression of genes encoding alcohol dehydrogenase (Adh1) and pyruvate dehydrogenase, suggesting an increase in anaerobic metabolism compared to cells of the mycelium. In C. albicans, Adh1 participates in fluconazole resistance through a mechanism that may involve efflux pumps,49 and Adh1 is up-regulated in fluconazoleresistant C. albicans.50 Guo et al.49 demonstrated a close relationship between ADH1 expression and drug resistance in C. albicans. In P. brasiliensis, Adh1 is considered a putative immunogenic protein.51 Proteins associated with the detoxification of ROS are strongly associated with virulence factors.52,53 Felipe et al.4 described genes that could potentially be involved in the response of a fungus to oxidative stress, such as peroxiredoxins, enzymatic antioxidants that promote the reduction of hydrogen peroxide, peroxynitrite, and other hydroperoxides to water and alcohol.54,55 In S. cerevisiae, Prxp1 protects cells against heat shock and endogenously and exogenously produced peroxides. Prxp1 mutants

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are sensitive to oxidizing conditions.56 An orthologous mitochondrial peroxiredoxin (Prx1; PADG_03095) is localized to the cell surface of C. albicans in the pathogenic hyphal phase57 and to the cell wall of P. brasiliensis yeasts (Longo et al., unpublished data). Antioxidative systems are induced when P. brasiliensis yeast cells are exposed to human plasma58 or when they infect macrophages.59 The pathogen P. brasiliensis is exposed to ROS / reactive nitrogen species (RNS) during infection;60 thus, the stress response may be a major virulence factor.61

CONCLUSIONS In this study, we used quantitative proteomics to identify proteins exhibiting dynamic variations in the P. brasiliensis proteome and suggest that they are potential virulence regulators. A pair of P. brasiliensis isolates (vPb18 and aPb18) showing different degrees of virulence were used to identify possible virulence regulators, such as carbonic anhydrase, vacuolar protease A, formamidase, alcohol dehydrogenase, mitochondrial peroxiredoxin Prx1 and vacuolar ATP synthase catalytic subunit A. Some of the identified proteins were related to beta-oxidation and fermentation. This study is the first to use mass spectrometry to identify potential virulence factors using isolates from a single fungal strain (pathogenic phase) that exhibit different degrees of virulence. Further analysis of the candidate virulence proteins identified in this study will lead to a better understanding of P. brasiliensis pathogenesis and the development of novel antifungal agents.

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ASSOCIATED CONTENT Supporting Information Supplementary figure and tables contains the complete set of results. This material is available free of charge via the Internet at http://pubs.acs.org. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository 62 with the dataset identifier PXD000804.

Figure 1. P. brasiliensis maintained in culture or freshly isolated from animals displayed clear differences in virulence and growth curves. Figure 2. Representative heat map showing the reproducibility of the biological duplicates for both conditions (MM and RM). Figure 3. Proteomic identification of the major cytoplasmic constituents of P. brasiliensis yeast from the vPb18 isolate compared to the aPb18 isolate in MM and RM. Figure 4. Relative expression of protein-encoding genes of vPb18 or aPb18 in Paracoccidioides brasiliensis yeast cells. Table 1. Proteins identified and quantified from P. brasiliensis up-regulated in virulent Pb18 isolate cultivated in MM and their predicted biological functional. Table 2. Proteins identified and quantified from P. brasiliensis up-regulated in virulent Pb18 isolate cultivated in RM and their predicted biological functional. Table 3. Proteins up-regulated in virulent Pb18 cultivated in MM and RM related to fungal virulence regulators in others pathogenic microorganism.

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Figure S1. Reproducibility of protein quantification between the biological replicates in MM and RM. Table S1. Primers used in qRT-PCR. Table S2. All proteins identified and quantified in virulent and attenuated isolates cultivated in MM. Table S3. Peptides from LC-MS/MS runs from all replicates of virulent and attenuated isolates cultivated in MM. Table S4. All proteins identified and quantified in virulent and attenuated isolates cultivated in RM. Table S5. Peptides from LC-MS/MS runs from all replicates of virulent and attenuated isolates cultivated in RM. Table S6. Proteins identified and quantified from P. brasiliensis up-regulated in attenuated Pb18 isolate cultivated in MM and their predicted biological functional. Table S7. Proteins identified and quantified from P. brasiliensis up-regulated in attenuated Pb18 isolate cultivated in RM and their predicted biological functional.

ACKNOWLEDGMENTS We are thankful to Geisa Ferreira Fernandes Sovegni and Rosana Puccia’s group for their generous and competent technical assistance and to Rosana Puccia for helpful discussions. The authors acknowledge financial support from CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico/Brazil) Proc. 471015/2011-3, FAPESP (Fundação de Amparo a Pesquisa do Estado de São Paulo/Brazil) Proc. 2011/14392-2 to WLB and Proc. 2012/19321-9 to AKT, and CAPES (Coordenação

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Aperfeiçoamento de Pessoal de Nível Superior). The authors thank the PRIDE Team for the support.

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FIGURE LEGENDS Figure 1. P. brasiliensis maintained in culture or freshly isolated from animals displayed clear differences in virulence and growth curves. A) Survival curves for B10.A mice injected intra-tracheally with 1x106 viable yeast from the different isolates of P. brasiliensis (aPb18 - maintained only in culture medium, or vPb18 - freshly isolated from animals) or with PBS as a control for 250 days (n = 6 animals for each group, p < 0.001 by the log-rank test for significance). Low lethality was observed among animals in the group injected with the aPb18 isolate. B) Cultures of 1x106 viable yeast from the different isolates of P. brasiliensis (vPb18 or aPb18) were grown for 12 days in MM (A) or RM (B), and viable cell counts using trypan blue were performed at specific points during cultivation. All of the data shown in this figure were analyzed using Student’s t-test. Error bars correspond to the standard deviations of measurements performed in triplicate, and asterisks indicate statistically significant differences in expression (p < 0.05).

Figure 2. Representative heat map showing the reproducibility of the biological duplicates for both conditions (MM and RM). The heat map was generated using the data derived from logarithmized values (base 2) for the ratios (heavy dimethylation/light dimethylation) for proteins up-regulated in vPb18 and aPb18 in MM or RM, based on the criteria described in Materials and Methods. Up-regulated proteins in vPb18 are shown in red to yellow, and up-regulated proteins in aPb18 are shown in green to blue.

Figure 3. Proteomic identification of the major cytoplasmic constituents of P. brasiliensis yeast from the vPb18 isolate compared to the aPb18 isolate in MM and RM. A) Venn diagram comparing the number of proteins that were differentially expressed in the virulent and attenuated isolates in MM and RM. In total, 242 (in MM)

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and 225 (in RM) proteins showed no differences in expression based on the established criteria. B) Proteins identified as up-regulated in the virulent and attenuated isolates in MM and RM, according to functional categories. The functional categories of both isolates are listed according to FunCat2 classification (http://pedant.gsf.de/pedant3htmlview/pedant3view?Method=analysis&Db=p3_p28733 _Par_brasi_Pb18).

Figure 4. Relative expression of protein-encoding genes of vPb18 or aPb18 in Paracoccidioides brasiliensis yeast cells. Quantitative RT-PCR of RNA isolated from vPb18 and aPb18 grown in MM (A, right panel) or RM (A, left panel). B) Data represents changes in transcription between the aPb18 before and after two passages through animals. The aPb18 expression was used as a calibrator. Gene-specific primers were used for each reaction, and the change in transcriptional levels was calculated via the ∆∆Ct method, with two housekeepers (α-TUB and ALDH). Fold change values (2∆∆CT) were logarithmized to the base 2 (log2). Data are presented as fold change in gene expression levels in the sample of interest normalized to the housekeepers and relative to the aPb18 sample. All of the data shown in this figure were analyzed using Student’s t-test. Error bars correspond to the standard deviation of measurements performed in triplicate, and asterisks indicate statistically significant differences in expression (p < 0.05).

Figure S1. Reproducibility of protein quantification between the biological replicates in MM and RM. Logarithmized values (base 2) for the H/L ratios (heavy dimethylation/light dimethylation) found for each protein measured in the two independent biological replicates were plotted against each other in both conditions. Correlation between the measurements was determined by Pearson correlation coefficient.

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Table 1. Proteins identified and quantified from P. brasiliensis up-regulated in virulent Pb18 isolate cultivated in MM and their predicted biological functional. Average for quantification of duplicates³

Standard deviation

log2(R1/R2)

2.661 2.347 2.641 3.504 15.108 2.229 2.111

0.068 0.944 0.643 0.043 1.729 0.464 0.549

0.0519 -0.8435 0.5016 -0.0253 0.2340 -0.4282 0.5368

2.548

0.793

0.6458

10.690

2.217

0.4262

6.274

1.590

0.5227

3.248 3.885 3.364 2.085

0.713 0.389 0.803 0.475

0.4519 0.2046 -0.4919 -0.4687

2.278 5.457

0.718 2.557

-0.6536 0.9935

2.066

0.277

0.2742

2.009

0.489

0.5015

CELL CYCLE AND DNA PROCESSING DNA synthesis and replication PADG_05798 conserved hypothetical protein (135 aa)

2.919

0.751

-0.5306

CELL FATE Cell growth / morphogenesis osmotic and salt stress response cell wall PADG_07756 F-actin-capping protein subunit beta (267 aa)

6.376

0.186

-0.0596

Functional category¹

Acession number/Protein description²

METABOLISM C-compound and carbohydrate metabolism PADG_00714 pyruvate decarboxylase (575 aa) PADG_01209 enoyl-CoA hydratase (294 aa) PADG_02271 alcohol dehydrogenase I (328 aa) PADG_04939 3-ketoacid-coenzyme A transferase subunit B (517 aa) PADG_04701 alcohol dehydrogenase (391 aa) PADG_04718 2-methylcitrate dehydratase (547 aa) PADG_07213 pyruvate dehydrogenase protein X component (488 aa) Metabolism of urea PADG_00734 acetyl-/propionyl-coenzyme A carboxylase alpha chain (1245 aa) Nitrogen, sulfur and selenium metabolism PADG_06490 formamidase (414 aa) Lipid, fatty acid and isoprenoid metabolism PADG_00315 carbonic anhydrase (186 aa) Amino acid metabolism PADG_00888 argininosuccinate synthase (414 aa) PADG_02913 adenylyl-sulfate kinase (215 aa) PADG_05277 serine hydroxymethyltransferase (536 aa) PADG_06805 acyl-CoA dehydrogenase (440 aa) PROTEIN FATE Protein folding and stabilization PADG_07599 peptidylprolyl isomerase B (208 aa) PADG_05094 T-complex protein 1 subunit zeta (541 aa) Protein modification PADG_07422 subtilase-type proteinase psp3 (496 aa) Protein targeting, sorting and translocation PADG_05789 vacuolar ATP synthase catalytic subunit A (1519 aa)

PROTEIN SYNTHESIS Ribosome biogenesis PADG_07685 40S ribosomal protein S13-1 (152 aa) Translation PADG_00692 elongation factor 1-alpha (461 aa)

2.591

1.611

1.3611

2.224

0.665

0.6195

ENERGY Electron transport and membrane-associated energy conservation PADG_02561 ATPase alpha subunit (557 aa) PADG_08367 cytochrome c oxidase polypeptide V (207 aa)

2.231 2.007

0.314 0.049

0.2879 0.0494

CELL RESCUE, DEFENSE AND VIRULENCE Stress response PADG_03095 mitochondrial peroxiredoxin PRX1 (223 aa)

5.057

0.854

0.3462

1.978 0.336 0.336 0.317

1.5016 0.1525 0.1525 0.2273

UNCHARACTERIZED PROTEIN PADG_01867 conserved hypothetical protein (325 aa) 2.926 PADG_02338 conserved hypothetical protein (174 aa) 4.501 PADG_02764 conserved hypothetical protein (214 aa) 4.501 PADG_00366 NAD dependent epimerase/dehydratase (255 aa) 2.847 ¹ Functional category by FunCat2 (http://pedant.gsf.de/pedant3htmlview/pedant3view?Method=analysis&Db=p3_p28733_Par_brasi_Pb18). ² Acession number of matched protein from Paracoccidioides brasiliensis (Pb18) database (http://www.broadinstitute.org/annotation/genome/paracoccidioides_brasiliensis/MultiHome.html). ³ Average expression ratios between vPb18 and aPb18.

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Table 2. Proteins identified and quantified from P. brasiliensis up-regulated in virulent Pb18 isolate cultivated in RM and their predicted biological functional. Average for quantification of duplicates³

Standard deviation

log2(R1/R2)

2.347 3.943

0.065 0.393

-0.0566 0.2036

4.634

1.307

0.5833

4.172

0.517

-0.2534

2.477

0.046

0.0379

2.625

0.820

-0.6479

2.311

0.366

-0.3244

2.004 2.221

0.013 0.420

-0.0128 0.3882

4.790 2.098

0.882 0.616

0.3781 0.6075

2.208

0.561

0.5246

2.368

0.246

0.2127

60S ribosomal protein L3 (393 aa) 40S ribosomal protein S2 (261 aa) 60S ribosomal protein L43 (93 aa)

2.074 2.143 2.761

0.221 0.194 0.596

0.2179 0.1850 0.4434

elongation factor 3 (1060 aa)

5.073

2.378

-0.9941

3.420

0.197

0.1178

CELL RESCUE, DEFENSE AND VIRULENCE Stress response PADG_00765 heat shock protein HSP98 (772 aa) PADG_02761 heat shock protein SSB1 (616 aa) PADG_03095 mitochondrial peroxiredoxin PRX1 (223 aa) Virulence, disease factors PADG_00999 pathogenesis associated protein Cap20 (189 aa)

2.516 2.916 2.577

0.169 0.883 1.194

0.1368 0.6277 0.9814

6.468

0.757

0.2394

TRANSPORTED COMPOUNDS Cation transport (H+, Na+, K+, Ca2+ , NH4+, etc.) PADG_05789 vacuolar ATP synthase catalytic subunit A (1519 aa) Protein transport PADG_04241 coatomer subunit alpha (1209 aa)

4.919

1.014

0.4234

4.193

0.699

-0.3417

2.979

1.179

0.8295

2.883

0.612

0.4362

Functional category¹

Acession number/Protein description²

METABOLISM C-compound and carbohydrate metabolism PADG_08119 fumarate hydratase (364 aa) PADG_04701 alcohol dehydrogenase (391 aa) Biosynthesis of tryptophan PADG_01653 ribose-phosphate pyrophosphokinase (321 aa) Assimilation of ammonia, metabolism of the glutamate group PADG_02728 sulfite oxidase (422 aa) Nucleotide/nucleoside/nucleobase metabolism PADG_04869 hypothetical protein (138 aa) Biosynthesis of vitamins, cofactors, and prosthetic groups PADG_08457 biotin synthase (385 aa) Degradation of arginine PADG_08465 fumarylacetoacetase (429 aa) PROTEIN FATE Protein folding and stabilization PADG_05628 disulfide-isomerase tigA (374 aa) PADG_00207 conserved hypothetical protein (446 aa) Protein processing (proteolytic) PADG_00634 vacuolar protease A (401 aa) PADG_00051 26S protease regulatory subunit 8 (440 aa) Protein modification PADG_06155 palmitoyl-protein thioesterase (337 aa) CELL CYCLE AND DNA PROCESSING Mitotic cell cycle and cell cycle control PADG_04016 eukaryotic translation initiation factor 3 (984 aa) PROTEIN SYNTHESIS Ribosome biogenesis PADG_07173 PADG_08602 PADG_01026 Translation PADG_07229

ENERGY Electron transport and membrane-associated energy conservation PADG_06196 NADPH dehydrogenase (376 aa)

CELLULAR COMMUNICATIONS/SIGNAL TRANSDUCTION MECHANISM MAPKKK cascade PADG_04559 progesterone binding protein (123 aa) Cellular signalling PADG_03219 myosin regulatory light chain cdc4 (140 aa)

UNCHARACTERIZED PROTEIN PADG_02092 conserved hypothetical protein (485 aa) 3.350 0.101 -0.0617 PADG_08467 conserved hypothetical protein (259 aa) 5.259 0.295 -0.1146 PADG_03544 ser/Thr protein phosphatase family protein (755 aa) 2.313 0.582 0.5191 ¹ Functional category by FunCat2 http://pedant.gsf.de/pedant3htmlview/pedant3view?Method=analysis&Db=p3_p28733_Par_brasi_Pb18 ² Acession number of matched protein from Paracoccidioides brasiliensis (Pb18) database (http://www.broadinstitute.org/annotation/genome/paracoccidioides_brasiliensis/MultiHome.html). ³ Average expression ratios between vPb18 and aPb18.

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Table 3. Proteins up-regulated in virulent Pb18 cultivated in MM and RM related to fungal virulence regulators in others pathogenic microorganism.

Acession number¹

Possible virulence factor

Average for quantification in MM and/or RM

MM/RM²

Described role in others pathogenic fungi

Reference

4.8

-/+

Required for C. albicans virulence.

38

PADG_00634

vacuolar protease A (PbSap)

PADG_06490

formamidase

10.7/1.7

+/+

Play a central role in the pathogenesis of human pathogens

63

PADG_05789

vacuolar ATP synthase catalytic subunit A

2.01/4.92

+/+

Assist in pathogenesis of Aspergillus niger.

64

PADG_00315

carbonic anhydrase

6.3

+/-

Essential for the pathogenesis of C. albicans in skin infection.

35

PADG_03095

mitochondrial peroxiredoxin Prx1

5.06/2.6

+/+

Protects cells against heat shock and peroxides in S. cerevisiae and C. albicans.

56

PADG_00999

pathogenesis associated protein Cap20

6.5

-/+

Required for Colletotrichum gloeosporioides virulence.

65

PADG_04701

alcohol dehydrogenase (Adh1)

15.11/3.94

+/+

Adh1p participates in fluconazole resistance in C. albicans.

49

PADG_05628

disulfide-isomerase tigA (pdiA)

2.05

-/+

Assist in pathogenesis of Aspergillus niger.

66

PADG_06196

NADPH dehydrogenase

3.42

-/+

Assist in pathogenesis de M. tuberculosis.

67

¹ Acession number of matched protein from Paracoccidioides brasiliensis (Pb18) database (http://www.broadinstitute.org/annotation/genome/paracoccidioides_brasiliensis/MultiHome.html). ² Presence (+) or absence (-) of the protein up-regulated in vPb18 cultivated in MM and RM

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Figure 1 1129x873mm (82 x 82 DPI)

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Figure 2 748x1317mm (82 x 82 DPI)

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Figure 3 1156x1072mm (79 x 79 DPI)

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Figure 4 966x930mm (86 x 86 DPI)

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TOC 514x288mm (300 x 300 DPI)

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