Isolates from the Paracoccidioides complex, Pb01 (P. lutzii), Pb02 (P. americana), Pb18 (P. brasiliensis), and PbEpm83 (P. restrepiensis), were used in this study. The P. venezuelensis species was not used during the study because it belongs to a monophyletic population restricted to Venezuela and cases of the disease caused by this species in other regions of Latin America have not been described (Theodoro et al., 2012; Turissini et al., 2017). The yeast phase was maintained in vitro at 36°C in a Fava Netto’s semisolid medium for 72 h (Fava-Netto, 1955). The components of the Fava Netto’s culture medium were 0.5% (w/v) yeast extract, 1% (w/v) peptone, 0.5% (w/v) meat extract, 0.3% (w/v) proteose-peptone, 0.5% (w/v) NaCl, 4% (w/v) glucose, 1% (w/v) agar, pH 7.2.

Yeasts of each Paracoccidioides spp. were transferred to the Fava Netto’s liquid medium and maintained at 36°C under agitation at 120 rpm for 96 h. After this period, cells were washed in phosphate buffered saline (PBS) and counted in Neubauer’s chamber, and the viability was measured using Trypan blue dye. For inoculum, 5 × 10⁶ viable cells/ml were used in liquid medium McVeigh-Morton (MMcM) (Restrepo and Jimenez, 1980) and incubated at 120 rpm for 24 h at 36°C. After incubation, the cells were centrifuged at 2,000 × g for 20 min. The supernatant obtained after centrifugation was subjected to vacuum filtration on 0.22-μm membranes (Millipore, United States). The filtered samples were concentrated using a 10-kDa ultrafiltration system (Amicon^®, Millipore, United States) and then washed three times with PBS (Weber et al., 2012). The quantification of extracellular extracts was determined by the Bradford method (Bradford, 1976).

The genomic DNA of the isolates was obtained according to the protocol described by Sambrook and Russell (2001). For the PCR reactions, the supernatant of sample secretomes (2 μl) and the genomic DNA obtained were used. The reactions were performed in 40 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 2 min. The 681 base pair PCR products were generated using oligonucleotides (sense 5′-GACATGCGTGATATCGACTTG-3′ and antisense 5′-GTGCGCCATGCCATTCT-3′) for the formamidase coding gene (GenBank accession number AY163575). PCR amplicons were detected by 1% (w/v) agarose gel electrophoresis using the GelRed intercalating agent (Biotium^TM, United States). To verify the sensitivity of PCR, a genomic DNA curve containing five dilutions (50 ng, 5 ng, 50 pg, 5 pg, and 1 pg) was constructed for each Paracoccidioides species, as described by Weber et al. (2012).

The integrity of the extracellular extracts was verified by 1-D SDS-PAGE (Laemmli, 1970). Thirty micrograms of extracellular extracts from each isolate were prepared with sample buffer [0.2 M Tris–HCl, pH 6.8, 40% (w/v) SDS, 2% (w/v) β-mercaptoethanol, and bromophenol blue traces] and heated in the thermoblock at 100°C for 10 min. Subsequently, the samples were submitted to 12% 1-D SDS-PAGE. As reference, the low-molecular-weight marker (GE Healthcare, United Kingdom) was used. Soon after this step, the gels were stained by Coomassie Blue. ImageQuant 300 (GE Healthcare, United Kingdom) was used to obtain the images.

Balb/c mice from 8 to 12 weeks old were provided by bioterium of the Instituto de Patologia Tropical e Saúde Pública, da Universidade Federal de Goiás (IPTSP-UFG). The animals were handled according to Conselho Nacional de Controle de Experimentação Animal (CONCEA-MCTIC) and submitted to the Ethics Committee of Comissão de Ética no Uso de Animais (CEUA-UFG) under the registry number 030/2016. Four groups containing five animals each were immunized with extracellular extract samples to induce the production of polyclonal antibodies. Separately, each group was immunized using proteins secreted by P. lutzii, P. americana, P. brasiliensis, and P. restrepiensis. For the immunizations, 50 μg of extracellular extracts were used in three doses with intervals of 15 days using the complete Freund’s adjuvant (Sigma-Aldrich, United States) for the first dose and the incomplete Freund’s adjuvant (Sigma-Aldrich, United States) for the second and third immunizations. The negative control was obtained from immunized mice only with Freund’s adjuvant as previously described. After the immunizations, the animals were euthanized, and the whole blood of the animals was collected. Whole blood was incubated at 37°C for 10 min, 4°C for 10 min, and centrifuged at 500 × g for 10 min. After centrifugation, the immunized sera containing the polyclonal antibodies were collected.

Immunoblotting was performed as described by de Oliveira et al. (2018) with some modifications. The extracellular extract samples (30 μg) were submitted to 1-D SDS-PAGE at 12% with subsequent transfer to nitrocellulose membranes. The primary antibodies (1:500) were incubated for 2 h, and the secondary antibody (1:20,000) (alkaline phosphatase-labeled mouse anti-IgG; Sigma-Aldrich, United Kingdom) was incubated for 2 h. The membranes were then washed twice with PBS and once with alkaline phosphatase buffer for 15 min. The chromogenic substrate solution for alkaline phosphatase containing BCIP (5-bromo-4-chloro-3-indolyl phosphate) and NBT (tetrazolium-nitroblue chloride) (Sigma-Aldrich, United Kingdom) was used.

Protein G-Sepharose^® 4B resin (Invitrogen, United States) was used for affinity chromatography. The resin was washed three times with PBS and 1.5 ml of immunized or control serum at a concentration of 1.5 μg/μl. The resin was subsequently added to the affinity columns, incubated at 4°C for 30 min to promote the binding of the antibodies to protein G, which is covalently linked to Sepharose beads. After ligation, the columns were washed three times with PBS. Then, 1.5 mg extracellular extracts from each Paracoccidioides species were added to the columns, maintained for 16 h at 4°C for the antigen-antibody binding. Afterward, the columns were washed three times using PBS, then the elution solution buffer [glycine 0.1 M, 0.02% sodium azide (w/v), pH 2.6] was added and incubated for 10 min. Subsequently, the supernatants containing antigens were obtained. The experimental procedure of immunoprecipitation is summarized in Supplementary Figure S1.

Antigens of the Paracoccidioides species obtained by immunoprecipitation were subjected to tryptic digestion. Briefly, 10 μl of 50 mM ammonium bicarbonate pH 8.5 were added to 50 μg of the protein extracts and subsequently subjected to tryptic digestion as previously described (Murad and Rech, 2012; Bailao et al., 2014; de Curcio et al., 2017; de Oliveira et al., 2018). Then, 25 μl of 0.2% (w/v) RapiGest SF^TM surfactant (Waters Corporation, United Kingdom) was added to the protein extracts and vortexed with subsequent incubation at 80°C for 15 min. After this incubation, 2.5 μl of 100 mM dithiothreitol (GE Healthcare, United States) was added and incubated at 60°C for 30 min. Then, 2.5 μl of 300 mM iodoacetamide (GE Healthcare, United States) was added and maintained at room temperature for 30 min. Subsequently, 10 μl of 50 mM trypsin (Promega, United States) solution was added and incubated for 16 h at 37°C. To precipitate the RapiGest SF^TM, 10 μl of 5% trifluoroacetic acid (Sigma-Aldrich, United Kingdom) was added and incubated for 90 min at 37°C. The samples were centrifuged at 20,000 × g for 30 min at 6°C, and the supernatants were collected. The supernatants were dried by using speed vacuum (Eppendorf, GER). All peptides obtained were resuspended in 80 μl of a solution containing 20 mM ammonium formate and Fosforilase MassPREP^TM Digestion Standard (100 fmol/μl for PbEpm83, 400 fmol/μl for Pb01, 100 fmol/μl for Pb02, and 100 fmol/μl for Pb18) as the endogenous standard.

After tryptic digestion of the purified exoantigens, the samples were submitted to nanoUPLC-MS^E. For the chromatographic analyses of peptides, the nanoACQUITY^TM M-Class system (Waters Corporation, United Kingdom) was used. For the first dimension, the fragmentation of peptides was performed through a 5-μm UPLC M-Class Peptide BEH C18, 130 Å (300 μm × 50 mm; Waters Corporation, Milford, MA, United States). The fragmented peptides were then submitted to five different acetonitrile/0.1% (v/v) formic acid concentrations (F1, 11.4%; F2, 14.7%; F3, 17.4%; F4, 20.7%; and F5, 50%). Each fraction eluted was trapped in a 5-μm Acquity UPLC M-Class Symmetry C18 Trap Column, 100 Å (180 μm × 20 mm; Waters Corporation, Milford, MA, United States). For the second dimension, for the separation of peptides, an Acquity UPLC M-Class HSS T3 1.8 μm (75 μm × 150 mm) Analytical Column was used. For mass calibration, a solution of 200 fmol/μl of the precursor ion [Glu1]-Fibronopeptide B human (m/z 785.8426) (GFP) (Sigma-Aldrich, St. Louis, MO, United States) was used with a constant flow of 0.5 μl/min, and it was measured every 30 s. The eluted peptides were analyzed by Synapt G1 HDMS^TM (Waters Corporation, United Kingdom) mass spectrometer. This device is equipped with nano-electrospray ion source and two mass analyzers, one quadrupole and one time-of-flight (Q-TOF) called nanoESI-Q-TOF (Waters Corporation, United Kingdom). This equipment operates in MS^E mode, alternating at 6 V (low power) and 40 V (high power) in each acquisition mode (0.4 s). Three biological replicates were performed for P. lutzii (Pb01), P. americana (Pb02), P. brasiliensis (Pb18), and P. restrepiensis (PbEmp83) samples.

After nanoUPLC-MS^E acquisition, the data processing was performed by using Protein Lynx Global Server version 3.0.2 software (Waters Corporation, United Kingdom). The obtained spectra were compared with sequences deposited in the database of Pb01 (P. lutzii), Pb03 (P. americana), Pb18 (P. brasiliensis), and PbCnh (P. restrepiensis)¹ to identify the peptides.

After data processing and protein identification, the identified proteins were subjected to in silico analyses. The identified proteins were functionally categorized based on UniProt² database. To predict the subcellular localization of the secreted antigens, WoLF PSORT software³ was employed. Expasy software⁴ was used for isoelectric point prediction. For the prediction of proteins secreted by classical and non-classical pathways, SignalP 4.1⁵ and SecretomeP 2.0⁶ were, respectively, used. Regarding SignalP and SecretomeP software, values greater than or equal to 0.5 were considered indicative of secretion. For prediction of linear B-cell epitopes, we used the software BCPREDS⁷ and ABCpred⁸.

To analyze and generate the Venn diagram, we used OrthoVenn software⁹ and Draw Venn Diagram¹⁰. BLASTp (Basic Local Alignment Search Tool) software¹¹ and ClustalX 2.0 and R software¹² were used for the homology analysis of the identified exoantigens and heat map generation, respectively. ProtScale software¹³ was used for prediction of hydrophilic peptides. The three-dimensional (3-D) modeling of the peptides was performed using I-Tasser¹⁴ and the figures were visualized by using PyMol version 2.3 software¹⁵. To verify energy minimization and total structural refinement of 3-D models, the GalaxyWEB¹⁶ and ModRefiner¹⁷ servers were used.

To verify if cell lysis would influence the profile of proteins secreted by the P. lutzii (Pb01), P. americana (Pb02), P. brasiliensis (Pb18), and P. restrepiensis (PbEpm83) species, the PCR technique was used, as described by Weber et al. (2012), with some modifications.

The formamidase gene of Paracoccidioides species was analyzed. The sensitivity of the technique was attested to by constructing a standard curve using genomic DNA (samples from 50-ng to 1-pg dilutions) and primers to formamidase gene, generating a 681 base pair amplicon, which was evaluated by agarose gel electrophoresis.

These results demonstrate that this assay was able to amplify extremely low amounts of genomic DNA (1 pg); nevertheless, no amplifications were detected in the supernatant of secretome samples of the isolates under study (Supplementary Figure S2). It was possible to observe that the extracellular extracts did not present PCR-detectable contamination and, accordingly, there are no detectable contaminants from the intracellular compartment since the gene that encodes the formamidase was not amplified in the samples analyzed during the PCR tests (Supplementary Figure S2). This demonstrates that even in the case of any cell lysis, it was undetectable by PCR and thus cannot influence the profile of samples during the proteomics analyses.

Extracellular extracts of P. lutzii, P. americana, P. brasiliensis, and P. restrepiensis were analyzed by 12% 1-D SDS-PAGE. After separation, the proteins of isolates depicted a molecular mass distribution between 103 and 16 kDa (Supplementary Figure S3).

High abundance of extracellular protein species by P. lutzii, P. Americana, and P. restrepiensis species was found in the 45-kDa range, demonstrating that the profile of the secreted proteins between the analyzed species presented some similarities (Supplementary Figure S3). Among these, the one with the highest abundance of proteins in the 45-kDa range was the P. americana species. The P. brasiliensis species did not present high expression of these proteins in the 45-kDa range (Supplementary Figure S3).

Analyses were performed with the objective of obtaining the profile of exoantigens of Paracoccidioides species. For this, the extracellular extracts of the P. lutzii, P. americana, P. brasiliensis, and P. restrepiensis species were used. Initially, extracellular extracts were subjected to 1-D SDS-PAGE with subsequent transfer to nitrocellulose membranes. Subsequently, immunoblotting assays were performed using antibodies obtained from control and immunized mice (Figure 1).

These results demonstrated that immunoblottings probed with antibodies obtained from immunized mice only with Freund’s adjuvant (controls) did not show any reactivity with the proteins of Paracoccidioides species. On the other hand, the serum obtained from mice immunized with secreted protein extracts from P. lutzii, P. americana, P. brasiliensis, and P. restrepiensis depicts reactivity against Paracoccidioides exoproteomes (Figure 1). In addition, by means of these data, it was possible to observe differences in antigenic profiles among the species representative of the Paracoccidioides complex. The antigenic profile of P. lutzii showed a wide range of antigenic proteins between 105 and 19 kDa. For the P. americana species, a range of antigens between 99 and 14 kDa were presented. Antigens in the range from 99 to 37 kDa were observed in samples from P. brasiliensis species. Regarding P. restrepiensis, it is possible to observe that the profile of the antigenic proteins varies between 105 and 21 kDa (Figure 1). As a result, it was possible to verify that the secreted protein extracts from Paracoccidioides species present antigenic molecules that induce the production of different antibodies.

After analyzing the differences in the antigenic profiles between Paracoccidioides species, we used immunoprecipitation approach to purify the antibody-reactive exoantigens (Supplementary Figure S1) by using the secretome samples from P. lutzii, P. americana, P. brasiliensis, and P. restrepiensis species. The immunoprecipitated samples were separated by 12% 1-D SDS-PAGE and demonstrate the presence of immunoreactive proteins, which proves the antigen-antibody binding (Figure 2). These profile differences are observed according to the molecular weight of the bands of proteins that are visualized in the electrophoresis (Figure 2). However, unlike what was seen regarding the immunized serum, the control sample showed no differences in protein profiles. The absence of immunoreactive proteins in the control sample demonstrates that there was no anti-secretome antibody production (Figure 2).

To identify the immunoprecipitated exoantigens, proteomic strategies were used. Initially, the samples obtained from the Paracoccidioides species were digested with trypsin and submitted to nanoUPLC-MS^E, resulting in the identification of 15 exoantigens for P. lutzii (Supplementary Table S1), 14 for P. americana (Supplementary Table S2), 17 for P. brasiliensis (Supplementary Table S3), and 33 for isolate P. restrepiensis (Supplementary Table S4). The immunoprecipitated exoantigens identified in both the immunized and the control serum were removed from the analysis.

The exoantigens identified in Paracoccidioides species were functionally classified in biological processes based on UniProt database. In P. lutzii, the most abundant functional class was metabolism (27%), followed by protein fate (20%), energy (6%), transcription (7%), protein synthesis (7%), cell cycle and DNA processing (6%), and hypothetical proteins (27%) (Figure 3A). The most abundant functional classes of P. americana after the functional classification were protein fate (43%), metabolism (22%), energy (14%), protein with binding function or cofactor requirement (7%), and hypothetical proteins (14%) (Figure 3B). For P. brasiliensis, metabolism (59%) was the most represented functional class, followed by protein fate (17%), energy (12%), and protein synthesis (12%) (Figure 3C). The functional classes of P. restrepiensis exoantigens were represented by metabolism (37%), protein fate (18%), protein synthesis (18%), cellular transport (3%), cell cycle and DNA processing (6%), cell rescue, defense, and virulence (3%), energy (6%), and hypothetical proteins (9%) (Figure 3D).

To identify signal peptides related to secretion pathways to the extracellular environment by alternative or classic routes, we used SecretomeP and SignalP software, respectively (Supplementary Tables S1–S4). The data obtained by the SecretomeP software revealed that 53.33% (P. lutzii), 28.57% (P. americana), 23.52% (P. brasiliensis), and 39.39% (P. restrepiensis) of identified exoantigens were predicted for secretion by non-classics pathways (Figures 4A–D). Using the SignalP software, it was revealed that 6.66, 14.20, 11.76, and 03.03% exoantigens of P. lutzii, P. americana, P. brasiliensis, and P. restrepiensis, respectively, showed signal peptides for secretion by the classical route (Figures 4A–D).

When comparing the exoantigens identified in Paracoccidioides species by software OrthoVenn, seven proteins from P. lutzii were exclusives in comparison with other fungus species described in our study. Four, seven, and 17 exoantigens of P. americana, P. brasiliensis, and P. restrepiensis were exclusive to these isolates, respectively (Figure 5 and Supplementary Table S5). The proteins 2-methylcitrate synthase mitochondrial, malate dehydrogenase NAD-dependent, and HSP 70-like protein were identified in all species during the immunoproteomics analyses (Supplementary Table S6). Other proteins, such as endo-1,3(4)-beta-glucanase, dihydrolipoyl dehydrogenase, and glutamate carboxypeptidase, were identified in common between P. americana, P. brasiliensis, and P. restrepiensis species (Supplementary Table S6).

A comparative analysis was carried out to verify which molecules were identified as exclusive and conserved among the studied Paracoccidioides species. BLASTp-NCBI algorithm was used, and the genome of P. venezuelensis was inserted to analyze its exoantigens homology within the Paracoccidioides complex. The exoantigens hypothetical protein (PAAG_05807) and glutamate-1-semialdehyde 2,1-aminomutase (PAAG_06925) were exclusive to P. lutzii when their homology was compared to the other species of Paracoccidioides (Table 1). In addition, the exoantigen hypothetical protein (PAAG_12630) of P. lutzii showed low homology to P. brasiliensis (72%) and P. restrepiensis (76%), but the molecule was not homologous to P. americana and P. venezuelensis. All other exoantigens analyzed have strong homology between the species of Paracoccidioides, demonstrating the presence of these conserved proteins among Paracoccidioides species (Supplementary Table S7). In addition, the homology of the Paracoccidioides spp. exoantigens was analyzed against proteins of other species and pathogens annotated in the NCBI genome, such as Homo sapiens (taxid:9606), Cryptococcus neoformans (taxid:5207), Histoplasma capsulatum (taxid:5037), S. schenckii (taxid:29908), Coccidioides immitis (taxid:5501), C. albicans (taxid:5476), A. fumigatus (taxid:746128), Mycobacterium tuberculosis (taxid:1773), Escherichia coli (taxid:562), Leishmania braziliensis (taxid:5660), and Streptococcus pneumoniae (taxid:1313). During the analyses of P. lutzii exoantigens, the hypothetical protein (PAAG_12701) and hypothetical protein (PAAG_05807) were not homologous to any of the analyzed species, making these molecules excellent biomarkers to be used for the epidemiological monitoring and diagnosis of PCM caused by P. lutzii. The exoantigens hypothetical protein (PAAG_12630) presented homology of 92% to H. capsulatum, and glutamate-1-semialdehyde 2,1-aminomutase (PAAG_06925) presented low homology to H. capsulatum (62%), C. immitis (39%), and C. neoformans (39%) (Figure 6A). In P. americana, the exoantigen serine protease (PABG_00534) showed homology to H. capsulatum (74%) and C. immitis (51%). In addition, endo-1,3(4)-beta-glucanase (PABG_12341), although homologous to H. capsulatum (66%), C. immitis (64%), A. fumigatus (54%), S. schenckii (42%), and C. albicans (41%), presented low homology to the antigens of these species (Figure 6B). In P. brasiliensis, the exoantigen ribosome biogenesis protein BMS1 (PADG_00459) was homologous to H. capsulatum (66%), C. immitis (63%), and A. fumigatus (65%). In addition, PTH1 family peptidyl-tRNA hydrolase (PADG_05841) was present in several other pathogens, but exhibited low homology (Figure 6C). For the P. restrepiensis species, the exoantigen single-strand binding protein family (A0A1E2Y9T2) presented low homology to H. capsulatum (73%), C. immitis (33%), A. fumigatus (45%), S. schenckii (60%), and E. coli (39%). Another exoantigen in P. restrepiensis that showed low homology was oxidoreductase (A0A1D2JMM5). This molecule was homologous to H. capsulatum (61%), C. immitis (46%), A. fumigatus (45%), S. schenckii (28%), C. neoformans (35%), and C. albicans (24%) (Figure 6D). All comparisons of homology between Paracoccidioides spp. and other species of pathogens analyzed during the study are listed in Supplementary Table S8.

Epitopes are specific regions of an antigen to which an antibody binds. These peptide regions are presented by MHC molecules to T lymphocytes (Abbas et al., 2008). Bioinformatics tools were used during analyses to predict and characterize which B-cell epitopes are present in the exoantigens identified by our immunoproteomics analysis. BCPREDs and ABCpred software were used together to verify if the predicted epitopes would be identified by both tools, leading to confirmation and strengthening of the experimentally obtained data. Initially, the proteins were submitted to BCPREDS software, where the epitopes with the highest score were selected (Supplementary Table S9). Predicted epitopes were also counted for each identified exoantigen, totaling 93 epitopes present in 15 proteins of P. lutzii and 158 epitopes present in 14 proteins of P. americana. For P. brasiliensis, 143 epitopes were predicted in 17 proteins, and there were 232 epitopes in 33 proteins of P. restrepiensis. All of these predicted epitopes are listed in Supplementary Table S9.

Subsequently, the epitopes were analyzed using ABCpred software, where the sequences predicted by this tool were selected and compared to the sequences predicted by BCPREDS software. This analysis was performed to verify if these two software tools share the same results, strengthening our experimental data (Supplementary Table S9). Thus, all epitopes predicted by both tools were verified and showed high score values, except for the exoantigens PAAG_12701 of P. lutzii, which did not show epitope prediction in any software.

In addition, the amount of predicted epitopes for each identified exoantigen was also evaluated. Interestingly, one of the exoantigens that had the largest number of predicted epitopes was endo-1,3 (4) beta-glucanase with 23 epitopes. This antigen was identified in P. americana, P. brasiliensis, and P. restrepiensis species. Next, aminopeptidase 2 presented 18 epitopes and was identified in P. americana and P. brasiliensis species. Heat shock proteins such as HSP70 and HSP60 presented 17 and 10 predicted epitopes, respectively, and were identified in all species in this study. The glutamate carboxypeptidase was another exoantigen that showed large numbers of predicted epitopes. A total of 11 epitopes were predicted in this molecule and identified in P. brasiliensis and P. restrepiensis species. Also, in P. brasiliensis and P. restrepiensis, the exoantigen dihydrolipoyl dehydrogenase was analyzed and presented nine predicted epitopes (Supplementary Table S9).

After verifying which exoantigens were homologous to antigenic molecules in other organisms, the homology of the B lymphocyte epitopes predicted by BCPREDS and ABCpred software was verified to identify which antigenic sequences were conserved or not in other organisms. For this, all sequences of the epitopes listed in Supplementary Table S9 were analyzed against homologous epitopes of other important fungi and pathogenic bacteria. Proteins with homology to human molecules were also analyzed to identify and avoid the process of cross-reactivity during future tests. Initially, species such as C. neoformans, H. capsulatum, S. schenckii, C. immitis, C. albicans, A. fumigatus, M. tuberculosis, E. coli, S. pneumoniae, L. braziliensis, and H. sapiens were selected. All sequences of proteins homologous to the exoantigens identified during the immunoproteomic were obtained from the UniProt database. ClustalX software was used to perform the alignment and all of the sequences were manually checked. After analysis of all identified epitopes, we selected only those that did not present homology with other analyzed organisms. In addition, the epitope topology in the protein structure is important. To check which epitopes were hydrophilic and can be present in the protein surface, we used ProtScale software, as performed by Ejazi et al. (2018). Thus, we selected only the hydrophilic, non-homologous to other related organisms and exclusive epitopes of the Paracoccidioides complex (Table 2). Therefore, five, 11, 10, and 18 epitopes of P. lutzii, P. americana, P. brasiliensis, and P. restrepiensis, respectively, were obtained (Table 2). In addition, the homology of all epitopes among Paracoccidioides species was evaluated to confirm its presence in all species of this fungus.

All exoantigens that showed exclusive epitopes common to all Paracoccidioides species (Table 2) had their 3-D structure built by using I-TASSER software and were refined by ModRefiner and GalaxyWEB algorithms. The position of each identified epitope was evaluated in exoantigen models and whether they were present in the surface of the model or not, which possibly facilitates antigen-antibody binding. The epitopes used for the analysis are displayed in Table 2, totaling two, four, three, and five exoantigens for P. lutzii, P. americana, P. brasiliensis, and P. restrepiensis, respectively. For P. lutzii, the exoantigen nucleic acid-binding protein (PAAG_04814 – 3 epitopes) was evaluated. During the analysis of the 3-D model of this exoantigen, it was possible to verify that one epitope was on the surface (Figure 7A). In P. americana, molecular modeling of four exoantigens was made: serine protease (PABG_00534, one epitope), aminopeptidase 2 (PABG_00710, one epitope), endonuclease G, mitochondrial (PABG_04696, two epitopes), and endo-1,3(4)-beta-glucanase (PABG_12341, seven epitopes). After analysis, it was verified that all epitopes analyzed in the exoantigens were present externally to the analyzed models (Figure 7B). Regarding P. brasiliensis predicted models, the exoantigens aminopeptidase 2 (PADG_03149, one epitope), carbonic anhydrase (PADG_07674, two epitopes), and endo-1,3(4)-beta-glucanase (PADG_12370, seven epitopes) had their epitopes analyzed in the 3-D models obtained, and it was also possible to verify the presence of the antigens on the surface (Figure 7C). In P. restrepiensis, five exoantigens had their epitopes analyzed in generated 3-D models. Thus, the exoantigens oxidoreductase (A0A1D2JMM5, 3 epitopes), endonuclease G mitochondrial (A0A1E2XUM2, one epitope), endo-1,3(4)-beta-glucanase (A0A1E2Y5K2, seven epitopes), ATP synthase subunit D (A0A1E2YCS7, five epitopes), and carbonic anhydrase (A0A1E2YFV5, two epitopes) were analyzed, evidencing that all predicted epitopes were present in the exposed regions in the 3-D models (Figure 7D).

After identification of the exclusive epitopes of Paracoccidioides, the level of homology of these epitopes was investigated among species. Thus, the FASTA sequences were obtained of proteins of the Paracoccidioides species that were homologous to exoantigens identified during the immunoproteomics assays. For this analysis, homologous molecules of the species P. lutzii, P. americana, P. brasiliensis, P. restrepiensis, and P. venezuelensis were selected. ClustalX software was used to perform the alignment and the homology of these epitopes in the Paracoccidioides complex was manually analyzed. Consequently, non-homologous epitopes to other pathogens were identified that were exclusive to each species of Paracoccidioides (Table 3). In P. lutzii, seven epitopes from two proteins, hypothetical protein (PAAG_05807) and hypothetical protein (PAAG_12630), were identified (Table 3). In P. brasiliensis, three predicted epitopes were exclusive. Two epitopes were present in malate dehydrogenase NAD-dependent (PADG_08054) and one in endo-1,3(4)-beta-glucanase (PADG_12370) (Table 3). For P. restrepiensis, two epitopes were identified and belong to ER lumen protein retaining receptor 2 (A0A1E2Y490) (Table 3). Identification of these epitopes needs to be tested to be used during epidemiological monitoring of the disease, supporting the recognition of which species of Paracoccidioides is causing PCM.