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Keywords:

  • Paracoccidioides brasiliensis ;
  • cell wall;
  • human plasma proteins

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Paracoccidioides brasiliensis and Paracoccidioides lutzii are thermodimorphic species that cause paracoccidioidomycosis. The cell wall is the outermost fungal organelle to form an interface with the host. A number of host effector compounds, including immunologically active molecules, circulate in the plasma. In the present work, we extracted cell-wall-associated proteins from the yeast pathogenic phase of P. brasiliensis, isolate Pb3, grown in the presence of human plasma and analyzed bound plasma proteins by liquid chromatography–tandem mass spectrometry. Transport, complement activation/regulation, and coagulation pathway were the most abundant functional groups identified. Proteins related to iron/copper acquisition, immunoglobulins, and protease inhibitors were also detected. Several human plasma proteins described here have not been previously reported as interacting with fungal components, specifically, clusterin, hemopexin, transthyretin, ceruloplasmin, alpha-1-antitrypsin, apolipoprotein A-I, and apolipoprotein B-100. Additionally, we observed increased phagocytosis by J774.16 macrophages of Pb3 grown in plasma, suggesting that plasma proteins interacting with P. brasiliensis cell wall might be interfering in the fungal relationship with the host.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Paracoccidioides brasiliensis and Paracoccidioides lutzii are thermodimorphic species responsible for paracoccidioidomycosis (PCM), a prevalent systemic granulomatous mycosis in Latin America. The active disease occurs in 1–2% of infected individuals, whose number is estimated in 10 million throughout endemic areas (San-Blas et al., 2002). Once in the pulmonary alveolar epithelium, inhaled infectious particles can establish infection as long as they transform into the pathogenic yeast form.

The cell wall is the outermost fungal structure in contact with the host, and its dynamic structure can rapidly change to adapt to the environment (Kapteyn et al., 2000). The yeast phase of Paracoccidioides cell wall is composed mainly of α-1,3-glucan and chitin, with a small proportion of β-1,3-glucan and galactomannan (Kanetsuna et al., 1972). Typical covalently linked structural proteins have not yet been described in Paracoccidioides; however, numerous noncovalently linked proteins have been shown in this compartment (Puccia et al., 2011).

Human plasma is composed by a large number of proteins, including both typical plasma proteins, such as albumin and lipoproteins, and tissue molecules that can be used in diagnosis and therapeutic monitoring (Anderson & Anderson, 2002). Although 1175 proteins have been described in human plasma (reviewed in Anderson et al., 2004), 95% of protein abundance is represented by only 10 (Putnam, 1984; Pieper et al., 2003): albumin (54%), immunoglobulin G (17%), alpha-1-antitrypsin (3.8%), alpha-2-macroglobulin (3.6%), immunoglobulin A (3.5%), transferrin (3.3%), haptoglobin (3%), apolipoprotein A-1 (3%), immunoglobulin M (2%), and alpha-1-acid-glycoprotein (1.3%).

Many plasma compounds, such as complement components and immunoglobulins, are immunologically active molecules and compose major defense lines of the host against invading microorganisms (Zipfel et al., 2007). Therefore, a better knowledge of the interactions between fungal cell wall and host plasma proteins may help us to understand infection development and host defense (Cottier & Pavelka, 2012).

The aim of the present work was to identify human plasma proteins that interact with P. brasiliensis yeast cell wall, because they might interfere in the host–pathogen relationship. For this, we employed liquid chromatography–tandem mass spectrometry (LC-MS/MS)-based proteomic analysis to identify proteins extracted with hot sodium dodecyl sulfate (SDS) from Pb3 cell wall, carefully isolated from yeasts cultivated in plasma-containing defined medium. We chose Pb3 as model because it represents P. brasiliensis cryptic species PS2, whose members are less virulent in B10. A mice (Carvalho et al., 2005). In this model, Pb3 evokes a predominant Th1-type protective immune response enriched in IgG2a, IgG2b, and IgG3 and high amounts of INF-γ (Carvalho K. & Puccia, unpublished data).

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Paracoccidioides brasiliensis isolate and growth conditions

Paracoccidioides brasiliensis isolate Pb3 was maintained in the yeast phase at 36 °C in solid modified YPD medium (0.5% yeast extract, 0.5% casein peptone, 1.5% glucose, pH 6.5). For cell wall isolation, yeast cells were cultivated in defined Ham's F12 medium (Invitrogen) added of 1.5% glucose (F12/Glc) and supplemented or not with 2% heat-inactivated (56 °C, 30 min) human plasma, obtained from healthy donors of Hospital São Paulo (UNIFESP Ethics Committee, approval protocol number 0366/07). Although we started with 2% plasma, we observed protein precipitation, which was discarded by centrifugation (6000 g, 30 min, 4 °C). Cells were transferred from 7-day-old slants into F12/Glc (200 mL) and cultivated at 36 °C for 4 days (pre-inoculum). Yeast cells from four pre-inoculums were transferred to 500 mL of fresh medium and cultivated for 2 days for cell wall purification. Yeast cells were analyzed for viability (> 95%) with trypan blue.

Cell wall purification

Yeast cells cultivated in the presence (Pb3pl) or absence (Pb3) of heat-inactivated human plasma were harvested by centrifugation, washed three times with phosphate-buffered saline (PBS), and mechanically disrupted with glass beads (425–600 μm; Sigma Aldrich) in B. Braun (six times for 10 min, alternating with 10 min in ice) in the presence of PBS with protease inhibitors [100 mM ethylenediamine tetraacetic acid, EDTA, 10 mM 1,10-phenanthroline, 1 mM phenylmethylsulfonyl fluoride, PMSF, 1 μM pepstatin A, and 15 μM trans-epoxysuccinyl-l-leucylamido(4-guanidino)butane, E-64]. Cell wall was isolated from cytoplasmic contents and membranous structures by three sequential centrifugations (8000 g for 45 min at 25 °C) in 85% sucrose (Kanetsuna et al., 1969). Nonspecifically bound components were eliminated by five sequential washes with each of the ice-cold solutions: deionized water, 5% NaCl, 2% NaCl, 1% NaCl, and 1 mM PMSF (Pitarch et al., 2002); final cell wall preparation was lyophilized.

SDS extraction of cell surface-associated proteins

Isolated cell wall (100 mg) was extracted twice by boiling with SDS for 5 min in extraction buffer (100 mM EDTA, 50 mM Tris–HCl pH 7.8, 2% SDS). The SDS extracts were centrifuged, filtrated through a sterile 0.22-μm filter, and precipitated in ice-cold acetone (1 h at −20 °C). After a 30-min centrifugation (16 000 g at 4 °C), the protein pellet was removed, washed in acetone, and dried at room temperature.

Proteomic analysis

Protein digestion was carried out using the ammonium bicarbonate/methanol method (Russell et al., 2001). Tryptic peptides were desalted in POROS R2 microcolumns (Jurado et al., 2007) and dried in an Eppendorf vacuum centrifuge concentrator. Peptides were then dissolved in 0.1% formic acid (FA), loaded onto a reversed-phase trap column (1 cm × 75 μm, Luna C18, 5 μm; Phenomenex), and separated in a capillary column (20 cm × 75 μm, Luna C18, 5 μm; Phenomenex) coupled to a nanoHPLC (1D Plus; Eksigent). Peptides were eluted in a linear gradient from 8.75% to 35% acetonitrile in 0.1% FA over 200 min and directly analyzed in an electrospray linear ion trap mass spectrometer (LTQ XL/ETD; Thermo Fisher Scientific) equipped with a TriVersa NanoMate nanospray source (Advion). The nanospray was set at 1.45 kV and 0.25 psi N2 pressure using a chip A (Advion). MS spectra were collected in centroid mode at the 400–1700 m/z range, and the 10 most intense ions were subjected twice to collision-induced dissociation with 35% normalized collision energy, before being dynamically excluded for 60s.

MS/MS spectra from peptides with 800 to 3500 Da, more than 10 counts, and at least 15 fragments were converted into DTA files using bioworks v.3.3.1 (Thermo Fisher) and searched against human (IPI v), porcine trypsin (GenBank), and Paracoccidioides (http://www.broadinstitute.org/annotation/genome/paracoccidioides_brasiliensis/MultiHome.html) sequences, in both correct and reverse orientations, using TurboSequest (Bioworks 3.3.1; Thermo Fisher Scientific). The database search parameters included: (1) trypsin cleavage in both peptide termini with one missed cleavage site allowed; (2) carbamidomethylation of cysteine residues as a fixed modification; (3) oxidation of methionine residues as a variable modification; and (4) 2.0 and 1.0 Da for peptide and fragment mass tolerance, respectively. TurboSequest outputs were filtered with DCn ≥ 0.05, peptide probability ≤ 0.05, and Xcorr ≥ 1.5, 2.0, and 2.5 for singly-, doubly-, and triply charged peptides, respectively. After filtering, the files were exported into XML formats, and the peptide sequences were assembled into proteins using an in-house written script (Nakayasu et al., 2012). The protein hits were refiltered with the sum of peptide Xcorr ≥ 3.5. The false discovery rate (FDR) was estimated as described previously (Rodrigues et al., 2008). Only proteins detected by at least two peptides exclusively in the Pb3pl cell wall were considered.

Functions and processes in which identified proteins are involved were analyzed using the Database for Annotation, Visualization and Integrated Discovery (DAVID) v6.7 (http://david.abcc.ncifcrf.gov; Dennis et al., 2003), and Blast2GO (http://www.blast2go.org/; Conesa et al., 2005) for Gene Ontology (GO). The exponentially modified protein abundance index (emPAI; Ishihama et al., 2005) was used for protein abundance comparison considering the protein molecular masses.

In vitro phagocytosis assay

Phagocytosis assays were carried out with macrophage cell lineage J774.16 cultured in DMEM/10% inactivated FBS. 2 × 105 cells were activated with 50 U mL−1 IFN-γ (PeproTech, Rock Hill, NJ) at 37 °C overnight and incubated with P. brasiliensis yeasts at a ratio of 5 : 1 macrophages/fungi for 6 h at 37 °C. Yeasts were cultivated in plasma-containing F12 medium. When grown in F12 alone, they were incubated with plasma (37 °C, 1 h) before the assay. Fresh and heat-inactivated plasma (56 °C, 1 h) were used. Three washes with 0.15 M α-methyl-mannopyranoside were performed to remove noninternalized yeasts bound via mannose receptor. Cells were fixed with methanol and stained with Giemsa (1 : 2 for 30 min), and phagocytosed yeasts were counted under light microscopy. Phagocytic index (PI) was defined as infected macrophages/counted macrophages, and pairwise comparison between groups was carried out by the Student's t-test.

Results and discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

To identify human plasma proteins that interact with P. brasiliensis yeast surface, carefully isolated cell wall preparations were exhaustedly washed with salt to remove nonspecifically bound proteins. Noncovalently interacting plasma proteins were extracted with hot SDS, and tryptic peptides were analyzed by LC-MS/MS (for raw data, see Supporting Information, Tables S1–S3). We identified 52 plasma proteins with two or more peptides present only in Pb3pl cell wall, annotated them into functional categories, and quantified them by relative emPAI (mass%; Table 1). We chose the emPAI method for protein quantification because it provides an absolute abundance value that enabled us to compare our data with the literature. Proteins categorized as transport, complement activation/regulation, and coagulation pathways were the most abundant. Proteins related to lipid metabolism, immune response, acute-phase response, and homeostasis were identified at lower relative amounts.

Table 1. Plasma proteins detected by LC-MS/MS in Paracoccidioides brasiliensis (Pb3)-derived cell wall. Distribution into functional groups was performed according to Gene Ontology classification. Protein relative abundance in the sample (relative emPAI mass%) and mass percentage in plasma (Pieper et al., 2003) are shown
Protein codeCellular processemPAI mass% cell wallPlasma mass%(as in Pieper et al., 2003)
Complement activation/regulation38.6 
IPI00783987Complement C310 
IPI00887739Similar to complement C37.4 
IPI00739237Complement C39.3 
IPI00478003Alpha-2-macroglobulin6.93.6
IPI00887154Complement component 4B1.4 
IPI00291262Clusterin1.3 
IPI00921523Complement factor B1.1 
IPI00021727C4b-binding protein alpha chain0.8 
IPI00029739Complement factor H0.4 
Transport19.3 
IPI00384697Serum albumin7.054
IPI00022434Serum albumin6.0 
IPI00022488Hemopexin2.61.1
IPI00878282Serum albumin1 
IPI00940791Transthyretin0.70.3
IPI00017601Ceruloplasmin2.1 
Coagulation pathway14.7 
IPI00790784Alpha-1-antitrypsin2.33.8
IPI00032179Antithrombin-III1.40.3
IPI00298971Vitronectin1.4 
IPI00877703Fibrinogen gama chain1.1 
IPI00298497Fibrinogen beta chain1.1 
IPI00019568Prothrombin1.1 
IPI00022418Fibronectin splice variant E1 
IPI00339226Fibronectin3.4 
IPI00022371Histidine-rich glycoprotein0.7 
IPI00029717Fibrinogen alpha chain1 
IPI00019580Plasminogen0.4 
Immunoglobulins (immune response)9.722.5
IPI00852577Ig lambda-1 chain C regions0.7 
IPI00154742Ig lambda-2 chain C regions0.6 
IPI00386879Immunoglobulin heavy constant alpha 12.3 
IPI00827560HRV Fab N28-VL0.5 
IPI00896380Ig mu chain C region1.8 
IPI00739205Ig heavy chain V-I region HG30.5 
IPI00384407Myosin-reactive Ig heavy chain variable region0.4 
IPI00384409Myosin-reactive Ig heavy chain variable region0.4 
IPI00784950Immunoglobulin heavy constant alpha 21.1 
IPI00785067Immunoglobulin heavy constant alpha 21.1 
IPI00470652Single-chain Fv0.5 
Lipid metabolism9.3 
IPI00021841Apolipoprotein A-I1.43
IPI00847635Alpha-1-antichymotrypsin0.60.6
IPI00022229Apolipoprotein B-1006.8 
IPI00218732Serum paraoxonase/arylesterase 10.5 
Others/Unknown4.6 
IPI00796830Unknown0.6 
IPI00646384Unknown0.5 
IPI00940494Uncharacterized protein0.5 
IPI00022895Alpha-1B-glycoprotein1.3 
IPI00879931Serpin peptidase inhibitor0.9 
IPI00292530Inter-alpha-trypsin inhibitor heavy chain H10.7 
IPI00935352Uncharacterized protein0.2 
Acute-phase response2 
IPI00218192Inter-alpha-trypsin inhibitor heavy chain H41.5 
IPI00022431Alpha-2-HS-glycoprotein0.50.8
Homeostasis1.7 
IPI00032220Angiotensinogen1.3 
IPI00477597Haptoglobin-related protein0.43

We also correlated the relative emPAI of cell-wall-associated plasma proteins with their relative mass percentages in plasma (Pieper et al., 2003), as shown in Table 1 and Fig. 1. Note that proteins of the coagulation pathway (antithrombin-III), transport (hemopexin and transthyretin), and complement activation/regulation (alpha-2-macroglobulin) were abundantly enriched in the fungal cell wall. Of them, only the latter is among the most abundant in plasma, representing 3.6% of total plasma proteins mass (Pieper et al., 2003) vs. 6.9% of cell-wall-bound proteins (Fig. 1a).

image

Figure 1. Relative abundance (relative emPAI mass%) of plasma proteins presently identified in Paracoccidioides brasiliensis (Pb3) isolated cell wall. Their percentage relative to total plasma proteins (Pieper et al., 2003) is shown in parallel. The figures show proteins relatively more abundant in the cell wall (a) or in plasma (b).

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Albumin, which is the most abundant plasma protein (54%), was responsible for only 13.9% of cell-wall-associated protein mass (Table 1; Fig. 1b). Alpha-1-acid and alpha-2-HS glycoproteins, haptoglobin, transferrin, apolipoprotein A-1, alpha-1-antitrypsin, and immunoglobulins were also relatively more abundant in plasma (Fig. 1b). Together, these observations suggest that plasma proteins have not randomly bound to the cell wall and that our analysis generally identified specifically bound proteins.

The presence of albumin interacting with cell wall components is speculative, and unspecific binding cannot be disregarded in this particular case. However, it has already been shown that Candida albicans Ala1/Ala5 adhesin is able to bind to BSA-coated beads, probably because of free threonine, serine, or alanine patches (Gaur et al., 2002). Although an Ala1/Ala5 adhesin ortholog has not been found in Paracoccidioides genome, there could be other albumin-binding protein(s) not yet described. In Paracoccidioides, many proteins colocalize to the surface and bind to extracellular matrix-associated proteins (reviewed in Puccia et al., 2011), but none has apparently been tested to bind to BSA.

Many immunoglobulin chains were found on the cell wall; however, they were twice more abundant in plasma than among cell-wall-associated plasma proteins (Fig. 1b). That is not surprising, considering that only a small amount of the total immunoglobulin repertoire would be able to recognize fungal surface antigens, leading to opsonization and activation of both the classical complement pathway and phagocytosis (Ehrnthaller et al., 2011).

Complement activation/regulation components, such as C3-, C4b-binding protein alpha-chain (C4BP), factors B and H were responsible for 38.6% of the cell-wall-bound plasma protein mass. That corroborates with previously reported immunofluorescence data showing that C3, C3a, C3d, C3 g, C4, C5b-9, and factors H and B are present on the P. brasiliensis yeast cell surface (Munk & Da Silva, 1992). The results in Fig. 2 showed that Pb3 cultivated in plasma-containing medium was 31% more internalized by J774.16 macrophages than Pb3 grown in the absence of plasma, while incubation in pure plasma caused a 78% increase in phagocytosis, corroborating previous data about the effect of serum in phagocytosis of a distinct isolate (Gonzaies et al., 2012). The effect was probably related to complement binding, considering that controls with inactivated plasma (both to grow and to assay the yeasts) were similar to a negative control with medium alone.

image

Figure 2. Phagocytic index for Pb3 yeast cells after 6 h of incubation with J774.16 macrophages. The assay was carried out with yeasts grown in F12 (control), F12-containing either inactivated (F12pl 56 °C) or fresh human plasma (F12pl) and also with yeasts grown in F12, but previously incubated for 1 h at 37 °C in heat-inactivated (pl 56 °C) or fresh (pl) human plasma. Values are averages of three measurements with standard deviations. *Significant differences (< 0.05) comparing with F12 control.

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In C. albicans, C3b binds directly to the yeast surface or via mannan-specific antibodies (Zhang & Kozel, 1998), opsonizing and mediating recognition by host immune effector cells for phagocytosis (van Lookeren Campagne et al., 2007). To avoid an excessive response and subsequent self-damage to host tissues, the complement system is tightly regulated by soluble and membrane-bound proteins, such as factor-I, factor-H, C4BP, vitronectin, and clusterin (Carroll, 2004), presently identified. Complement regulators would help the pathogen to evade the immune system by down-regulating complement activation. C4BP is a major plasma inhibitor of the classical and mannose-binding lectin-mediated complement pathways, and its alpha-chain is responsible for binding to C. albicans cell wall (Meri et al., 2004). Some microorganism surface ligands of complement factors have already been elucidated, such as Pra1 and Gpm1 in C. albicans (Zipfel et al., 2007). In this fungus, interaction with vitronectin increased binding to and phagocytosis by macrophages (Limper & Standing, 1994).

The complement cascade is intimately connected to the blood coagulation system, and their activation occurs simultaneously (Markiewski et al., 2007), thus explaining why we identified members of the coagulation cascade on P. brasiliensis cell wall preparations. In C. albicans, plasminogen bound to surface CaGpm1p was accessible for activation and was converted to active plasmin, which is a key enzyme of intravascular fibrinolysis and acts in the degradation of the host extracellular matrix (Poltermann et al., 2007). Paracoccidioides brasiliensis Pb3 has two CaGpm1p orthologs: fructose-2,6-biphosphatase (PABG_05093) and conserved hypothetical protein PABG_05096, whose localization and affinity for plasminogen remain unknown. Fibrinogen chains were detected in high abundance (3.1% emPAI mass%) among cell-wall-associated plasma proteins. Als3p adhesin in C. albicans binds to fibrinogen (Nobbs et al., 2010), and although an ortholog in P. brasiliensis has not been found, other protein(s) might have similar functions.

Transport proteins such as hemopexin (discussed below) and transthyretin were more represented in the cell wall than in plasma (Table 1 and Fig. 1a). Transthyretin, involved in thyroxine and retinol transport, had altered expression in plasma during experimental invasive pulmonary aspergillosis (Gonzales et al., 2010). It presents adhesive properties and binds to many compounds including plant flavonoids (Green et al., 2005). Possibly, transthyretin may bind to P. brasiliensis cell wall components via disulfide bridges (Ruiz-Herrera et al., 2006), considering it can form disulfide bonds with a thiol-Sepharose 4B column (Fex et al., 1977).

Extracellular proteases can play important roles in pathogenic fungal nutrition, tissue invasion, and host immune system evasion (Naglik et al., 2003). Recently, Maza et al. (2012) showed that P. brasiliensis extracellular proteases degrade proinflammatory cytokines. Therefore, host protease inhibitors would be an obvious defense mechanism by neutralizing fungal proteases involved in infection. On the cell wall of P. brasiliensis grown in plasma-containing medium, we identified plasma proteins with serine protease inhibitor activity, such as alpha-1-antitrypsin, inter-alpha-trypsin inhibitor, alpha-2-macroglobulin, and angiotensinogen (Table 1). Paracoccidioides brasiliensis extracellular thiol-dependent subtilisin-like protease (Carmona et al., 1995) and a secreted 66-kDa serine protease (Parente et al., 2010) could possibly be neutralized by the human plasma protease inhibitors during infection. These fungal serine protease activities cleave extracellular matrix-associated proteins in vitro and could play a role in tissue damage and dissemination.

Both iron and copper are key regulators of host–pathogen interactions (Doherty, 2007; Kim et al., 2008). We presently identified hemopexin and ceruloplasmin bound to P. brasiliensis cell wall. Hemopexin tightly binds to heme groups and scavenges the free heme in order to protect the body from oxidative damage. Ceruloplasmin is responsible for carrying about 70% of the total copper in human plasma and exhibits a copper-dependent oxidase activity, which possibly oxidizes Fe2+ into Fe3+, thus participating in iron transport. Microorganism receptors for host Fe-binding proteins and ligands have been described (Nevitt, 2011). The presence of plasma iron and copper carriers in P. brasiliensis cell wall may be due to an attempt to accumulate these nutrients during growth. Iron availability is important for fungal growth (Arango & Restrepo, 1988), and the presence of siderophores has been demonstrated (Castaneda et al., 1988). In silico analysis showed that P. brasiliensis also has a high-affinity copper transport protein (Ctr3p) ortholog (Silva et al., 2011). The importance of copper homeostasis in Cryptococcus neoformans virulence was demonstrated, because it was linked to capsule production and inhibition of phagocytosis (Chun & Madhani, 2010).

In conclusion, using a careful protocol employing sucrose centrifugation and successive washes with different NaCl concentrations, we isolated cell wall from Pb3 yeasts cultivated in the presence of human plasma. The noncovalently associated plasma proteins were extracted with boiling SDS, and a proteomic analysis by LC/MS-MS was applied. Complement pathway components were identified, and their role in the phagocytosis was suggested. Several human plasma proteins described here have not been previously reported as interacting with fungal components, specifically clusterin, hemopexin, transthyretin, ceruloplasmin, alpha-1-antitrypsin, apolipoprotein A-I, and apolipoprotein B-100. This report represents an initial step to understanding the P. brasiliensis cell wall interaction with host components and the possible role of plasma proteins in the host–parasite relationship and infection, especially in a low-virulence isolate.

Data availability

Proteomic data is available online (Supporting Information, Data S1 and Data S2).

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

This work has been funded by FAPESP, CNPq, and NIH (grants # 5G12RR008124-16A1, 5G12RR008124-16A1S1, and 8G12MD007592). We thank the Biomolecule Analysis Core Facility at UTEP, supported by the Research Centers in Minority Institutions (RCMI) program, grant # 8G12MD007592, to the Border Biomedical Research Center (BBRC), from the National Institutes on Minority Health and Health Disparities (NIMHD), a component of the NIH, for the access to the LC-MS instrument.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References
  8. Supporting Information
FilenameFormatSizeDescription
fml12097-sup-0001-DataS1.RAWplain text document156421KData S1. LC-MS/MS run of proteins extracted from cell walls of P. brasiliensis grown in the absence of human plasma.
fml12097-sup-0002-DataS2.RAWplain text document153692KData S2. LC-MS/MS run of proteins extracted from cell walls of P. brasiliensis grown in the presence of human plasma.
fml12097-sup-0003-TableS1-S3.xlsapplication/msexcel2914K

Table S1. Abundance estimation of identified proteins by emPAI.

Table S2. Protein and peptide identification of cell wall preparation from Pb3 isolate.

Table S3. Protein and peptide identification of cell wall preparation from Pb3 isolate grown in the presence of human plasma.

fml12097-sup-0004-SuppLegend.docWord document23K 

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