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. Author manuscript; available in PMC: 2014 Apr 1.
Published in final edited form as: FEMS Microbiol Lett. 2013 Apr;341(2):87–95. doi: 10.1111/1574-6968.12097

Identification of human plasma proteins associated to the cell wall of the pathogenic fungus Paracoccidioides brasiliensis

LVG Longo a, ES Nakayasu b,1, AL Matsuo a, R Peres da Silva a, TJP Sobreira c, MC Vallejo a,2, L Ganiko b, IC Almeida b,#, R Puccia a,*
PMCID: PMC3619194  NIHMSID: NIHMS444251  PMID: 23398536

Abstract

Paracoccidioides brasiliensis and P. 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.

Keywords: Paracoccidioides brasiliensis, cell wall, human plasma proteins

Introduction

Paracoccidioides brasiliensis and P. lutzii are thermodimorphic species responsible for paracoccidioidomycosis (PCM), a prevalent systemic granulomatous mycosis in Latin America. The active disease occurs in 1 to 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 non-covalently 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 1,175 proteins have been described in human plasma (reviewed in (Anderson, et al., 2004)), 95% of protein abundance is represented by only ten (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 microbes (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, since 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 (unpublished data).

1. Materials and methods

1.1. P. brasiliensis isolate and growth conditions

P. 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 (6,000xg, 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.

1.2. 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 saline buffer (PBS) and mechanically disrupted with glass beads (425-600 µm, Sigma Aldrich) in B. Braun (6 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 (8,000×g for 45 min at 25°C) in 85% sucrose (Kanetsuna, et al., 1969). Non-specifically 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.

1.3. 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-micron 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.

1.4. 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 ten 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 3,500 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/dimorph_collab.1/MultiHome) sequences, in both correct and reverse orientations, using TurboSequest (Bioworks 3.3.1, Thermo Fisher Scientific). The database search parameters included: i) trypsin cleavage in both peptide termini with one missed cleavage site allowed; ii) carbamidomethylation of cysteine residues as a fixed modification; iii) oxidation of methionine residues as a variable modification; and iv) 2.0 Da 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.

1.5. In vitro phagocytosis assay

Phagocytosis assays were carried out with macrophage cell lineage J774.16 cultured in DMEM/10% inactivated FBS. 2x105 cells were activated with 50 U/ml 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 non-internalized yeasts bound via mannose receptor. Cells were fixed with methanol, 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 done by the Student t-test.

2. Results and discussion

In order to identify human plasma proteins that interact with P. brasiliensis yeast surface, carefully isolated cell wall preparations were exhaustedly washed with salt to remove non-specifically bound proteins. Non-covalently interacting plasma proteins were extracted with hot SDS, and tryptic peptides were analyzed by LC-MS/MS (for raw data, see Supplemental Files). 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 since 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 P. 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 Code Cellular Process emPAI mass%
cell wall		 Plasma mass%
(as in Pieper, et al., 2003)
Complement activation / regulation 38.6
IPI00783987 Complement C3 10
IPI00887739 Similar to complement C3 7.4
IPI00739237 Complement C3 9.3
IPI00478003 Alpha-2-macroglobulin 6.9 3.6
IPI00887154 Complement component 4B 1.4
IPI00291262 Clusterin 1.3
IPI00921523 Complement factor B 1.1
IPI00021727 C4b-binding protein alpha chain 0.8
IPI00029739 Complement factor H 0.4
Transport 19.3
IPI00384697 Serum albumin 7.0 54
IPI00022434 Serum albumin 6.0
IPI00022488 Hemopexin 2.6 1.1
IPI00878282 Serum albumin 1
IPI00940791 Transthyretin 0.7 0.3
IPI00017601 Ceruloplasmin 2.1
Coagulation pathway 14.7
IPI00790784 Alpha-1-antitrypsin 2.3 3.8
IPI00032179 Antithrombin-III 1.4 0.3
IPI00298971 Vitronectin 1.4
IPI00877703 Fibrinogen gama chain 1.1
IPI00298497 Fibrinogen beta chain 1.1
IPI00019568 Prothrombin 1.1
IPI00022418 Fibronectin splice variant E 1
IPI00339226 Fibronectin 3.4
IPI00022371 Histidine-rich glycoprotein 0.7
IPI00029717 Fibrinogen alpha chain 1
IPI00019580 Plasminogen 0.4
Immunoglobulins (immune response) 9.7 22.5
IPI00852577 Ig lambda-1 chain C regions 0.7
IPI00154742 Ig lambda-2 chain C regions 0.6
IPI00386879 Immunoglobulin heavy constant alpha 1 2.3
IPI00827560 HRV Fab N28-VL 0.5
IPI00896380 Ig mu chain C region 1.8
IPI00739205 Ig heavy chain V-I region HG3 0.5
IPI00384407 Myosin-reactive Ig heavy chain variable region 0.4
IPI00384409 Myosin-reactive Ig heavy chain variable region 0.4
IPI00784950 Immunoglobulin heavy constant alpha 2 1.1
IPI00785067 Immunoglobulin heavy constant alpha 2 1.1
IPI00470652 Single-chain Fv 0.5
Lipid metabolism 9.3
IPI00021841 Apolipoprotein A-I 1.4 3
IPI00847635 Alpha-1-antichymotrypsin 0.6 0.6
IPI00022229 Apolipoprotein B-100 6.8
IPI00218732 Serum paraoxonase/arylesterase 1 0.5
Others/Unknown 4.6
IPI00796830 UNKNOWN 0.6
IPI00646384 UNKNOWN 0.5
IPI00940494 Uncharacterized protein 0.5
IPI00022895 Alpha-1B-glycoprotein 1.3
IPI00879931 Serpin peptidase inhibitor 0.9
IPI00292530 Inter-alpha-trypsin inhibitor heavy chain H1 0.7
IPI00935352 Uncharacterized protein 0.2
Acute-phase response 2
IPI00218192 Inter-alpha-trypsin inhibitor heavy chain H4 1.5
IPI00022431 Alpha-2-HS-glycoprotein 0.5 0.8
Homeostasis 1.7
IPI00032220 Angiotensinogen 1.3
IPI00477597 Haptoglobin-related protein 0.4 3
Protein Code Cellular Process emPAI Mr (Da) emPAI*Mr emPAI mass% emPAI mol% Plasma mass% (as in [21])
Immunoglobulins (immune response) 9.72 25.48 22.50
IPI00852577 Ig lambda-1 chain C regions P0CG04 0.70125428 11.35 7.96 0.67 3.92
IPI00154742 Ig lambda-2 chain C regions P0CG05 0.649648074 11.29 7.34 0.62 3.63
IPI00386879 immunoglobulin heavy constant alpha 1 Q96K68 0.505836354 53.09 26.85 2.27 2.83
IPI00827560 HRV Fab N28-VL A2IPI3 0.467799268 12.28 5.75 0.49 2.61
IPI00896380 Ig mu chain C region P01871 0.435035831 49.31 21.45 1.81 2.43
IPI00739205 Ig heavy chain V-I region HG3 P01743 0.42510267 12.95 5.50 0.47 2.37
IPI00384407 Myosin-reactive immunoglobulin heavy chain variable region Q9UL92 0.333521432 13.58 4.53 0.38 1.86
IPI00384409 Myosin-reactive immunoglobulin heavy chain variable region Q9UL94
Q9UL92 		0.333521432 13.21 4.40 0.37 1.86
IPI00784950 immunoglobulin heavy constant alpha 2 Q6MZV6
Q9UL92 		0.251875026 51.64 13.01 1.10 1.41
IPI00785067 immunoglobulin heavy constant alpha 2 Q6P089 0.245197085 52.00 12.75 1.08 1.37
IPI00470652 Single-chain Fv Q65ZC8 0.211527659 26.13 5.53 0.47 1.18
Transport Q9UL92 19.34 22.02
IPI00384697 Serum albumin P02768 1.187761624 69.37 82.39 6.96 6.64 54
IPI00022434 Serum albumin Q56G89 1.020949938 69.08 70.53 5.96 5.70
IPI00022488 Hemopexin P02790 0.59985872 51.68 31.00 2.62 3.35 1.1
IPI00878282 Serum albumin Q9UL92 0.519911083 22.86 11.88 1.00 2.90
IPI00940791 Transthyretin E7EW61 0.412537545 20.29 8.37 0.71 2.30 0.3
IPI00017601 Ceruloplasmin P00450 0.201284899 122.21 24.60 2.08 1.12
Complement activation / regulation Q9UL92 38.63 18.42
IPI00783987 Complement C3 P01024 0.632172129 187.15 118.31 10.00 3.53
IPI00887739 Similar to complement C3 Q9UL92 0.605016318 144.81 87.61 7.40 3.38
IPI00739237 Complement C3 Q9UL92 0.584893192 187.15 109.46 9.25 3.27
IPI00478003 Alpha-2-macroglobulin P01023 0.502551161 163.29 82.06 6.94 2.81 3.6
IPI00887154 Complement component 4B Q6U2L1 0.342078063 47.45 16.23 1.37 1.91
IPI00291262 Clusterin P10909 0.291549665 52.50 15.30 1.29 1.63
IPI00921523 Complement factor B P00751
Q9UL92 		0.154781985 85.53 13.24 1.12 0.86
IPI00021727 C4b-binding protein alpha chain P04003
Q9UL92 		0.149756995 67.03 10.04 0.85 0.84
IPI00029739 Complement factor H P08603
Q9UL92 		0.034700871 139.10 4.83 0.41 0.19
Coagulation pathway Q9UL92 14.74 13.87
IPI00790784 Alpha-1-antitrypsin P01009
Q9UL92 		0.584893192 46.74 27.34 2.31 3.27 3.8
IPI00032179 Antithrombin-III P01008 0.304321387 52.60 16.01 1.35 1.70 0.3
IPI00298971 Vitronectin P04004 0.299081397 54.31 16.24 1.37 1.67
IPI00877703 Fibrinogen gama chain C9JC84 0.24782547 52.34 12.97 1.10 1.38
IPI00298497 Fibrinogen beta chain P02675 0.232846739 55.93 13.02 1.10 1.30
IPI00019568 Prothrombin P00734 0.184484581 70.04 12.92 1.09 1.03
IPI00022418 Fibronectin splice variant E A6YID6 0.163561851 70.22 11.48 0.97 0.91
IPI00339226 Fibronectin P02751 0.151650644 262.63 39.83 3.37 0.85
IPI00022371 Histidine-rich glycoprotein P04196 0.142068906 59.58 8.46 0.72 0.79
IPI00029717 Fibrinogen alpha chain P02671 0.119902793 94.97 11.39 0.96 0.67
IPI00019580 Plasminogen P00747 0.051908639 90.57 4.70 0.40 0.29
Lipid metabolism 9.28 5.49
IPI00021841 Apolipoprotein A-I P02647 0.528306733 30.78 16.26 1.37 2.95 3
IPI00847635 Alpha-1-antichymotrypsin P01011 0.158323286 47.65 7.54 0.64 0.88 0.6
IPI00022229 Apolipoprotein B-100 P04114 0.156051281 515.61 80.46 6.80 0.87
IPI00218732 Serum paraoxonase/arylesterase 1 P27169 0.140624924 39.73 5.59 0.47 0.79
Acute-phase response 1.97 1.62
IPI00218192 Inter-alpha-trypsin inhibitor heavy chain H4 Q14624 0.171190257 103.36 17.69 1.50 0.96
IPI00022431 Alpha-2-HS-glycoprotein F5H0Q5 0.118872212 46.60 5.54 0.47 0.66 0.8
Homeostasis 1.71 2.31
IPI00032220 Angiotensinogen P01019 0.291549665 53.15 15.50 1.31 1.63
IPI00477597 Haptoglobin-related protein P00739 0.122018454 39.03 4.76 0.40 0.68 3
Others/Unknown 4.61 10.81
IPI00796830 UNKNOWN 0.519911083 12.993 6.76 0.57 2.90
IPI00646384 UNKNOWN 0.42510267 13.16 5.59 0.47 2.37
IPI00940494 Uncharacterized protein F5GXM8 0.389495494 14.08 5.48 0.46 2.18
IPI00022895 Alpha-1B-glycoprotein P04217 0.291549665 54.25 15.82 1.34 1.63
IPI00879931 Serpin peptidase inhibitor E9PGN7 0.182298865 59.49 10.85 0.92 1.02
IPI00292530 Inter-alpha-trypsin inhibitor heavy chain H1 P19827 0.079775162 101.39 8.09 0.68 0.45
IPI00935352 Uncharacterized protein F8W967 0.047615753 41.90 2.00 0.17 0.27
Not identified 1183.21
transferrin 3.3
alpha-1-acid glycoprotein 1.3
Protein Code Cellular Process emPAI mass% Plasma mass% (as in [21]) cw/pl pl/cw
Immunoglobulins (immune response) 9.72 22.5 0.432 2.314815
IPI00852577 Ig lambda-1 chain C regions 0.672563076
IPI00154742 Ig lambda-2 chain C regions 0.620103392
IPI00386879 immunoglobulin heavy constant alpha 1 2.269575171
IPI00827560 HRV Fab N28-VL 0.485626254
IPI00896380 Ig mu chain C region 1.81289135
IPI00739205 Ig heavy chain V-I region HG3 0.465122774
IPI00384407 Myosin-reactive immunoglobulin heavy chain variable region 0.382790971
IPI00384409 Myosin-reactive immunoglobulin heavy chain variable region 0.372220528
IPI00784950 immunoglobulin heavy constant alpha 2 1.099261708
IPI00785067 immunoglobulin heavy constant alpha 2 1.077556648
IPI00470652 Single-chain Fv 0.467083877
Transport 19.34
IPI00384697 Serum albumin 6.963384401
IPI00022434 Serum albumin 5.961013305
IPI00878282 Serum albumin 1.004441092
3 serum albumin 13.9288388 54 0.257941 3.876849
IPI00022488 Hemopexin 2.619847634 1.1 2.38168 0.419872
IPI00940791 Transthyretin 0.707569825 0.3 2.358566 0.423986
IPI00017601 Ceruloplasmin 2.078922683
Complement activation / regulation 38.63
IPI00783987 Complement C3 9.999049156
IPI00887739 Similar to complement C3 7.404535366
IPI00739237 Complement C3 9.251239525
IPI00478003 Alpha-2-macroglobulin 6.935546654 3.6 1.926541 0.519065
IPI00887154 Complement component 4B 1.371943478
IPI00291262 Clusterin 1.293506619
IPI00921523 Complement factor B 1.118902604
IPI00021727 C4b-binding protein alpha chain 0.848425949
IPI00029739 Complement factor H 0.407937086
Coagulation pathway 14.74
IPI00790784 Alpha-1-antitrypsin 2.310338244 3.8 0.607984 1.644781
IPI00032179 Antithrombin-III 1.352922438 0.3 4.509741 0.221742
IPI00298971 Vitronectin 1.372699212
IPI00877703 Fibrinogen gama chain 1.096228858
IPI00298497 Fibrinogen beta chain 1.100620553
Fibrinogen alpha chain 0.962426618
3.159276029
IPI00019568 Prothrombin 1.092007895
IPI00022418 Fibronectin splice variant E 0.97063572
IPI00339226 Fibronectin 3.366033953
IPI00022371 Histidine-rich glycoprotein 0.715357485
IPI00029717 Fibrinogen alpha chain 0.962426618
IPI00019580 Plasminogen 0.397335516
Lipid metabolism 9.28
IPI00021841 Apolipoprotein A-I 1.37424672 3 0.458082 2.183014
IPI00847635 Alpha-1-antichymotrypsin 0.6376098 0.6 1.062683 0.941014
IPI00022229 Apolipoprotein B-100 6.800214728
IPI00218732 Serum paraoxonase/arylesterase 1 0.47220433
Acute-phase response 1.97
IPI00218192 Inter-alpha-trypsin inhibitor heavy chain H4 1.495399077
IPI00022431 Alpha-2-HS-glycoprotein 0.46818096 0.8 0.585226 1.708741
Homeostasis 1.71
IPI00032220 Angiotensinogen 1.309744753
IPI00477597 Haptoglobin-related protein 0.402496621 3 0.134166 7.453479
Others/Unknown 4.61
IPI00796830 UNKNOWN 0.570921874
IPI00646384 UNKNOWN 0.472631708
IPI00940494 Uncharacterized protein 0.46342725
IPI00022895 Alpha-1B-glycoprotein 1.336849378
IPI00879931 Serpin peptidase inhibitor 0.916601794
IPI00292530 Inter-alpha-trypsin inhibitor heavy chain H1 0.68359158
IPI00935352 Uncharacterized protein 0.168609529
Not identified
transferrin 3.3 0 3.3
alpha-1-acid glycoprotein 1.3 0 1.3
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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) versus 6.9% of cell wall-bound proteins (Fig. 1A).

Fig. 1.

Fig. 1

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Relative abundance (relative emPAI mass%) of plasma proteins presently identified in P. 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).

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(s) 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, C3g, 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 [32]. 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.

Fig. 2.

Fig. 2

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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 (P < 0.05) comparing with F12 control.

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). P. 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 and coworkers (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). P. brasiliensis extracellular thiol-dependent subtilysin-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, since it was linked to capsule production and inhibition of phagocytosis (Chun & Madhani, 2010).

In conclusion, by 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 non-covalently 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 will be available online upon the acceptance of the present manuscript

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Acknowledgments

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.

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