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• W1975086865 abstract "The human pathogenic yeast Candida albicans utilizes host complement regulators for immune evasion. Here we identify the first fungal protein that binds Factor H and FHL-1. By screening a protein array of 4088 proteins of Saccharomyces cerevisiae, phosphoglycerate mutase (ScGpm1p) was identified as a Factor H- and FHL-1-binding protein. The homologous C. albicans Gpm1p (CaGpm1p) was cloned and recombinantly expressed as a 36-kDa His-tagged protein. Purified CaGpm1p binds the host complement regulators Factor H and FHL-1, but not C4BP. The CaGpm1p binding regions in the host proteins were localized; FHL-1 binds via short consensus repeats (SCRs) 6 and 7, and Factor H utilizes two contact regions that are located in SCRs 6 and 7 and in SCRs 19 and 20. In addition, recombinant CaGpm1p binds plasminogen via lysine residues. CaGpm1p is a surface protein as demonstrated by immunostaining and flow cytometry. A C. albicans gpm1-/- mutant strain was generated that did not grow on glucose-supplemented but on ethanol- and glycerol-supplemented medium. Reduced binding of Factor H and plasminogen to the null mutant strain is in agreement with the presence of additional binding proteins. Attached to CaGpm1p, each of the three host plasma proteins is functionally active. Factor H and FHL-1 show cofactor activity for cleavage of C3b, and bound plasminogen is converted by urokinase-type plasminogen activator to proteolytically active plasmin. Thus, the surface-expressed CaGpm1p is a virulence factor that utilizes the host Factor H, FHL-1, and plasminogen for immune evasion and degradation of extracellular matrices. The human pathogenic yeast Candida albicans utilizes host complement regulators for immune evasion. Here we identify the first fungal protein that binds Factor H and FHL-1. By screening a protein array of 4088 proteins of Saccharomyces cerevisiae, phosphoglycerate mutase (ScGpm1p) was identified as a Factor H- and FHL-1-binding protein. The homologous C. albicans Gpm1p (CaGpm1p) was cloned and recombinantly expressed as a 36-kDa His-tagged protein. Purified CaGpm1p binds the host complement regulators Factor H and FHL-1, but not C4BP. The CaGpm1p binding regions in the host proteins were localized; FHL-1 binds via short consensus repeats (SCRs) 6 and 7, and Factor H utilizes two contact regions that are located in SCRs 6 and 7 and in SCRs 19 and 20. In addition, recombinant CaGpm1p binds plasminogen via lysine residues. CaGpm1p is a surface protein as demonstrated by immunostaining and flow cytometry. A C. albicans gpm1-/- mutant strain was generated that did not grow on glucose-supplemented but on ethanol- and glycerol-supplemented medium. Reduced binding of Factor H and plasminogen to the null mutant strain is in agreement with the presence of additional binding proteins. Attached to CaGpm1p, each of the three host plasma proteins is functionally active. Factor H and FHL-1 show cofactor activity for cleavage of C3b, and bound plasminogen is converted by urokinase-type plasminogen activator to proteolytically active plasmin. Thus, the surface-expressed CaGpm1p is a virulence factor that utilizes the host Factor H, FHL-1, and plasminogen for immune evasion and degradation of extracellular matrices. Candida albicans is the most important human pathogenic yeast and causes disseminated infections (1Beck-Sague C. Banerjee S. Jarvis W.R. Am. J. Public Health. 1993; 83: 1739-1742Crossref PubMed Scopus (76) Google Scholar, 2Pfaller M. Wenzel R. Eur. J. Clin. Microbiol. Infect. Dis. 1992; 11: 287-291Crossref PubMed Scopus (225) Google Scholar). The yeast form represents a common saprophyte in healthy individuals, which resides mainly on the skin, oral cavity, urogenital, and gastrointestinal tracts. In addition, C. albicans can cause systemic infections predominantly in immunocompromised or granulocytopenic patients (3Fisher-Hoch S.P. Hutwagner L. Clin. Infect. Dis. 1995; 21: 897-904Crossref PubMed Scopus (142) Google Scholar). C. albicans systemic infections, which are difficult to diagnose and treat, can be lethal (4Edwards Jr., J.E. N. Engl. J. Med. 1991; 324: 1060-1062Crossref PubMed Scopus (212) Google Scholar). Virulence of C. albicans is based on its ability to bind to host cells (5Calderone R.A. Fonzi W.A. Trends Microbiol. 2001; 9: 327-335Abstract Full Text Full Text PDF PubMed Scopus (1005) Google Scholar). Several secretory proteolytic enzymes are involved in tissue invasion of C. albicans (6Schaller M. Borelli C. Korting H.C. Hube B. Mycoses. 2005; 48: 365-377Crossref PubMed Scopus (353) Google Scholar). In addition, morphogenetic transition from yeast to hyphal forms correlates with the infection process and causes adherence to host cells and tissue penetration (7Rooney P.J. Klein B.S. Cell Microbiol. 2002; 4: 127-137Crossref PubMed Scopus (89) Google Scholar). As a commensal, C. albicans has evolved highly effective strategies of immune evasion to survive in the host (8Romani L. Nat. Rev. Immunol. 2004; 4: 1-23Crossref PubMed Scopus (666) Google Scholar). The complement system represents an important part of host innate immunity. Four activation pathways have been described. The alternative pathway, which is initiated spontaneously, and by default, the lectin and classical pathway, which are triggered by antibodies or carbohydrates, are relevant for the recognition and elimination of microbes (9Walport M.J. N. Engl. J. Med. 2001; 344: 1058-1066Crossref PubMed Scopus (2405) Google Scholar). The role of the recently described thrombin pathway (10Huber-Lang M. Sarma J.V. Zetoune F.S. Rittirsch D. Neff T.A. McGuire S.R. Lambris J.D. Warner R.L. Flierl M.A. Hoesel L.M. Gebhard F. Younger J.G. Drouin S.M. Wetsel R.A. Ward P.A. Nat. Med. 2006; 12: 682-687Crossref PubMed Scopus (769) Google Scholar) for immune defense needs to be worked out. C. albicans activates both the alternative and classical pathways of complement (11Kozel T.R. Weinhold L.C. Lupan D.M. Infect. Immun. 1996; 64: 3360-3368Crossref PubMed Google Scholar). C3b molecules bind directly to the C. albicans surface or via mannan-specific IgG antibodies, which occur naturally in human serum (12Zhang M.X. Kozel T.R. Infect. Immun. 1998; 66: 4845-4850Crossref PubMed Google Scholar). Upon entry into a human host, any microbe is attacked by the host complement system. However, pathogenic microbes control complement activation at their surface. This type of complement escape is mediated either by the acquisition of host regulators to the surface of the pathogen or by expression of endogenous complement regulators (13Zipfel P.F. Skerka C. Hellwage J. Jokiranta S.T. Meri S. Brade V. Kraiczy P. Noris M. Remuzzi G. Biochem. Soc. Trans. 2002; 30: 971-978Crossref PubMed Scopus (212) Google Scholar). An increasing number of pathogenic microbes utilize host complement regulators for immune evasion and for down-regulation of complement activation. For the yeast C. albicans, acquisition of the central fluid phase alternative pathway regulators Factor H and FHL-1 and the classical pathway regulator C4BP have been demonstrated (14Meri T. Blom A.M. Hartmann A. Lenk D. Meri S. Zipfel P.F. Infect. Immun. 2004; 72: 6633-6641Crossref PubMed Scopus (95) Google Scholar, 15Meri T. Hartmann A. Lenk D. Eck R. Wurzner R. Hellwage J. Meri S. Zipfel P.F. Infect. Immun. 2002; 70: 5185-5192Crossref PubMed Scopus (116) Google Scholar). Binding of host complement regulators such as Factor H, FHL-1, and C4BP was also shown for Gram-negative bacteria, such as Borrelia burgdorferi (16Alitalo A. Meri T. Ramo L. Jokiranta T.S. Heikkila T. Seppala I.J. Oksi J. Viljanen M. Meri S. Infect. Immun. 2001; 69: 3685-3691Crossref PubMed Scopus (140) Google Scholar, 17Kraiczy P. Skerka C. Kirschfink M. Brade V. Zipfel P.F. Eur. J. Immunol. 2001; 31: 1674-1684Crossref PubMed Scopus (215) Google Scholar), Neisseria gonorrhoeae (18Ram S. McQuillen D.P. Gulati S. Elkins C. Pangburn M.K. Rice P.A. J. Exp. Med. 1998; 188: 671-680Crossref PubMed Scopus (226) Google Scholar), Neisseria meningitidis (19Ram S. Mackinnon F.G. Gulati S. McQuillen D.P. Vogel U. Frosch M. Elkins C. Guttormsen H.K. Wetzler L.M. Oppermann M. Pangburn M.K. Rice P.A. Mol. Immunol. 1999; 36: 915-928Crossref PubMed Scopus (130) Google Scholar), Gram-positive bacteria, like Streptococcus pyogenes (20Johnsson E. Berggard K. Kotarsky H. Hellwage J. Zipfel P.F. Sjobring U. Lindahl G. J. Immunol. 1998; 161: 4894-4901PubMed Google Scholar, 21Kotarsky H. Hellwage J. Johnsson E. Skerka C. Svensson H.G. Lindahl G. Sjobring U. Zipfel P.F. J. Immunol. 1998; 160: 3349-3354PubMed Google Scholar), Streptococcus pneumoniae (22Neeleman C. Geelen S.P. Aerts P.C. Daha M.R. Mollnes T.E. Roord J.J. Posthuma G. van Dijk H. Fleer A. Infect. Immun. 1999; 67: 4517-4524Crossref PubMed Google Scholar), and parasites including Onchocerca volvulus, Echinococcus granulosus (23Diaz A. Ferreira A. Sim R.B. J. Immunol. 1997; 158: 3779-3786PubMed Google Scholar), and the human immunodeficiency virus (24Stoiber H. Clivio A. Dierich M.P. Annu. Rev. Immunol. 1997; 15: 649-674Crossref PubMed Scopus (88) Google Scholar). In their bound configuration these host proteins maintain their regulatory activities and protect the microbes against complement-mediated phagocytosis and lysis. For several pathogens the ligands for the host regulators have been identified and include classical virulence factors like the M protein of S. pyogenes. Factor H- and FHL1-binding proteins of C. albicans have been proposed (13Zipfel P.F. Skerka C. Hellwage J. Jokiranta S.T. Meri S. Brade V. Kraiczy P. Noris M. Remuzzi G. Biochem. Soc. Trans. 2002; 30: 971-978Crossref PubMed Scopus (212) Google Scholar), but so far these proteins have not been isolated. Here we identify the phosphoglycerate mutase Gpm1p of C. albicans (CaGpm1p) as the first fungal Factor H- and FHL-1-binding protein of yeast. Strains and Growth Conditions of C. albicans—The C. albicans strains used in this study are listed in Table 1. Strains were grown in YPD medium (2% (w/v) glucose, 2% (w/v) peptone, 1% (w/v) yeast extract) or YPGE medium (3% (w/v) glycerol, 2% ethanol (w/v), 2% (w/v) peptone, 1% (w/v) yeast extract) at 30 °C. Cells were counted with a hemocytometer (Fein-Optik, Bad Blankenburg, Germany). Hyphal growth was induced in liquid culture by a temperature shift from 30 to 37 °C.TABLE 1C. albicans strains and plasmidsStrain or plasmidGenotype or descriptionRef. or sourceSC5314Wild-type28Fonzi W.A. Irwin M.Y. Genetics. 1993; 134: 717-728Crossref PubMed Google ScholarCAI-4Δura3::imm434/Δura3::imm434, isogenic to SC531428Fonzi W.A. Irwin M.Y. Genetics. 1993; 134: 717-728Crossref PubMed Google ScholarCAP1Δgpm1::hisG-URA3-hisG/GPM1, Δura3::imm434/Δura3::imm434This workCAP2Δgpm1::hisG/GPM1, Δura3::imm434/Δura3::imm434This workCAP3Δgpm1::hisG/Δgpm1::hisG-URA3-hisG, Δura3::imm434/Δura3::imm434This workCAP4Δgpm1::hisG/Δgpm1::hisG, Δura3::imm434/Δura3::imm434This workCAP5Δgpm1::hisG/GPM1::hisG-URA3-hisG, Δura3::imm434/Δura3::imm434This workpMB7C. albicans gene disruption vector28Fonzi W.A. Irwin M.Y. Genetics. 1993; 134: 717-728Crossref PubMed Google Scholar Open table in a new tab Antibodies and Proteins—For specific detection of Factor H and FHL-1 in the protein array, monoclonal antibodies B22 and L20 (25Oppermann M. Manuelian T. Jozsi M. Brandt E. Jokiranta T.S. Heinen S. Meri S. Skerka C. Gotze O. Zipfel P.F. Clin. Exp. Immunol. 2006; 144: 342-352Crossref PubMed Scopus (132) Google Scholar) (directed against SCR 5 and SCR 19 of Factor H) were labeled with Alexa Fluor® 647 (Molecular Probes, Eugene, OR) according to the manufacturer's instructions. Horseradish peroxidase-conjugated rabbit anti-goat antisera and horseradish peroxidase-conjugated rabbit anti-mouse antisera as well as horseradish peroxidase-conjugated swine anti-rabbit antisera were obtained from Dako (Glostrup, Denmark). Polyclonal goat anti-Factor H antiserum (Calbiochem) and a polyclonal goat anti-plasminogen antiserum (Acris, Hiddenhausen, Germany) was used for ELISA. 3The abbreviations used are: ELISAenzyme-linked immunosorbent assayPBSphosphate-buffered salineRTroom temperatureBSAbovine serum albuminSCRsshort consensus repeatsuPAurokinase-type plasminogen activatorϵACAϵ-aminocaproic acid Polyclonal antibodies against CaGpm1p were raised by immunizing rabbits with purified recombinant CaGpm1p. Alexa Fluor® 488-labeled goat anti-rabbit antisera (Molecular Probes), used for fluorescence microscopy, and fluorescein isothiocyanate-labeled swine anti-rabbit antisera (Dako, Glostrup, Denmark), used for flow cytometry, were obtained from Molecular Probes. A monoclonal mouse antibody directed against human γ tubulin (kindly provided by Peter Hemmerich, Jena, Germany) was used for detection of γ tubulin. For detection of C3b degradation products, a polyclonal goat anti-C3 antiserum (Calbiochem) was used. Recombinant CaGpm1p was detected with PentaHis antiserum (Qiagen, Hilden, Germany). Factor H, Factor I, and C3b were obtained from Calbiochem, uPA was from Chemicon (Hofheim, Germany), and plasminogen was from Chromogenix (Milano, Italy). enzyme-linked immunosorbent assay phosphate-buffered saline room temperature bovine serum albumin short consensus repeats urokinase-type plasminogen activator ϵ-aminocaproic acid Expression of Recombinant Proteins—The C. albicans phosphoglycerate mutase gene CaGPM1 was amplified by PCR using genomic DNA from strain SC5314 and primers S1 (5′-GAATTCGTATGCCAAAGTTAGTTTTAGT-3′) and S2 (5′-TCTAGATATTTCTTTTGACCTTGAGCAG-3′); EcoRI and XbaI restriction sites are underlined. The resulting 760-bp DNA fragment contained the complete CaGPM1 coding region flanking an additional EcoRI and a XbaI restriction site. The DNA fragment was subcloned into Escherichia coli cloning vector pCR4Blunt-TOPO (Invitrogen) and subsequently cloned into the EcoRI and XbaI sites of the Pichia pastoris vector pPICZαB (Invitrogen). CaGpm1p was recombinantly expressed as a His-tagged protein in P. pastoris strain X33. Protein expression was induced with 1% methanol. After 3 days of expression the culture supernatant was harvested, and recombinant CaGpm1p was precipitated with 80% ammonium sulfate. FHL-1 (SCR 1-7) and recombinant deletion constructs of Factor H (SCRs 1-5, SCRs 1-6, SCRs 8-11, SCRs 11-15, SCRs 15-18, and SCRs 19 and 20) were expressed in the baculovirus system as described (26Kuhn S. Zipfel P.F. Gene (Amst.). 1995; 162: 225-229Crossref PubMed Scopus (103) Google Scholar). All recombinant proteins were purified by nickel affinity chromatography using HisTrap columns in anÁkta fast protein liquid chromatography system (GE Healthcare) and concentrated in PBS using Vivaspin 15R concentrators with a cut off of 10 kDa (Vivascience, Hannover, Germany). Protein Binding Assays—The ProtoArray Yeast Proteome PPI kit (Invitrogen), representing 4088 purified recombinant yeast proteins of the yeast Saccharomyces cerevisiae, spotted onto a glass slide, was used to identify Factor H- and FHL-1-binding proteins. Arrays were probed either with Factor H or FHL-1 (6 μg each), and bound proteins were detected with fluorescence-labeled antibodies. The arrays were scanned using a fluorescent microarray scanner (Gene Pix 4000B, Molecular Devices). Significant interactions were identified by analyzing the acquired data with the ProtoArray Prospector software (Invitrogen). Additional arrays were probed exclusively with the antibodies to identify background reactivity. For ELISA CaGpm1p (0.25 μg in carbonate-bicarbonate buffer, Sigma) was immobilized onto a microtiter plate (half-area plate, Corning) at RT overnight. Nonspecific binding sites were blocked with PBS containing 2% BSA for 2 h at RT. After incubation with the ligand protein (0.75 μg) for 2 h at RT, wells were washed 3 times with PBS-T buffer (PBS containing 0.05% Tween 20) and incubated with primary antiserum (1:1000 dilution in blocking solution) for 1 h at RT. After washing with PBS-T, horseradish peroxidase-conjugated antisera (1:2000 dilution in blocking solution) were added and incubated for 1 h at RT. After washing, substrate o-phenylenediamine dihydrochloride (Sigma) was added. The reaction was stopped with 2 m H2SO4, and the optical density was measured at 490 nm in an ELISA plate reader (SpektraMax 190, Molecular Devices). Immunofluorescence Assays and Flow Cytometry—C. albicans yeast or hyphal cells (108) were incubated at RT for 30 min in PBS supplemented with 2% BSA. After blocking of nonspecific binding sites, the cells were incubated at 4 °C overnight with rabbit anti-CaGpm1p antiserum or with preimmune serum (1:50 dilution). After incubation, cells were washed 3 times with blocking buffer. A goat anti-rabbit antiserum labeled with Alexa 488 was added at a dilution of 1:200 in blocking buffer at RT. Cells were again washed three times and examined with a laser scanning microscope (LSM 510 META, Zeiss). C. albicans yeast cells were analyzed by flow cytometry. After incubation at 4 °C for 30 min with CaGpm1p antiserum (1:100 dilution in 1% BSA-PBS), C. albicans yeast cells were washed with PBS supplemented with 1% BSA (1% BSA-PBS). Alexa Flu- or ® 488-labeled goat anti-rabbit serum was used as secondary antibody (1:200 dilution in 1% BSA-PBS). After incubation at 4 °C for 30 min, the cells were washed with 1% BSA-PBS and examined by flow cytometry (LSR II, BD Biosciences). Forward and sideward scatters were used for the identification of the cells, and fluorescent events of 10,000 cells were counted. Preparation of C. albicans Cytoplasmic and Cell Wall Fraction—For cell wall fraction preparation C. albicans was grown in a YPD preculture at 30 °C overnight. Afterward, C. al-bicans cells were cultivated in Soll's medium (27Swoboda R.K. Bertram G. Delbruck S. Ernst J.F. Gow N.A. Gooday G.W. Brown A.J. Mol. Microbiol. 1994; 13: 663-672Crossref PubMed Scopus (48) Google Scholar), pH 4.5, at 30 °C overnight followed by growth in Soll's medium, pH 6.5, at 37 °C for 45 min. The cells were harvested by centrifugation and stored at -20 °C. After resuspending the pellet in PBS supplemented with 25 mm DTT and 12.5 mm phenylmethylsulfonyl fluoride (final concentrations), the cells were lysed by glass beads in a Mini-BeadBeater (Biospec Products) followed by three freeze and thaw cycles. For the isolation of cytoplasmic proteins, cell lysate of the C. albicans null mutant and the wild type strain was centrifuged, and the supernatant was collected. Cell debris was resuspended in PBS and again centrifuged. This step was repeated 10 times. Proteins derived from cell wall pellet were extracted by reducing loading buffer Roti-Load1 (Roth, Karlsruhe, Germany) incubated at 99 °C for 5 min and then separated by SDS-PAGE. Cofactor Assay—Cofactor activity of CaGpm1p-bound complement regulators Factor H and FHL-1 was performed as described (15Meri T. Hartmann A. Lenk D. Eck R. Wurzner R. Hellwage J. Meri S. Zipfel P.F. Infect. Immun. 2002; 70: 5185-5192Crossref PubMed Scopus (116) Google Scholar). CaGpm1p was coated to the surface of a microtiter plate (half-area plate, Corning), and Factor H (0.2 μg/well) or FHL-1 (0.2 μg/well) was bound to this matrix. After extensive washing with PBS, C3b (0.4 μg/well) and Factor I (0.8 μg/well) were added. After the incubation at 37 °C the supernatant was separated by SDS-PAGE, and C3 degradation products were analyzed by Western blot using an anti-C3 antiserum and a horseradish peroxidase-conjugated anti-mouse serum as secondary antibody. Disruption and Reintegration of CaGPM1 Gene—The CaGPM1 gene was disrupted using the hisG-URA3-hisG cassette (28Fonzi W.A. Irwin M.Y. Genetics. 1993; 134: 717-728Crossref PubMed Google Scholar). The 5′ region of CaGPM1 was amplified by PCR using chromosomal DNA of C. albicans SC5314 as a template and primers SP1-SacI (5′-ATATATCGAGCTCGTTAGATCACCTTTTACTCTCAAG-3′, position -354 to -330) and SP2-KpnI (5′-ATATATGCGGTACCATCAAAGGATAATTGGAGCAAGG-3′, position -29 to -25) (underlined sequences are complementary to the genomic sequence of the CaGPM1 gene). The resulting 325-bp PCR product was digested with SacI/KpnI and cloned into the disruption vector pMB7, yielding plasmid pG0 (28Fonzi W.A. Irwin M.Y. Genetics. 1993; 134: 717-728Crossref PubMed Google Scholar). Primer SP3 SalI (5′-ATATATGCGTCGACCTTGGCCCGAACTGAAGAATTCA-3′, position +748 to +771) and primer SP4 PstI (5′-ATAAATGACTGCAGGATATAAGTGTCGATGATTCTAG-3′, position +837 to +814) were used to amplify the 3′ region. The 334-bp product was digested with SalI and PstI and cloned into plasmid pG0, resulting in pG1. For gene disruption, plasmid pG1 was treated with SacI and PstI. The 3.3-kilobase SacI/PstI fragment of plasmid pG1 containing the URA-blaster flanked by short sequences from the 5′ and 3′ ends of CaGPM1 and portions of the promotor and terminator, respectively, was used to transform C. albicans Ura- strain CAI-4. Transformation was performed in the presence of lithium acetate. After selection on Sabouraud (1% (w/v) peptone (casein), 2% (w/v) glucose) medium, the resulting Ura+ transformants were examined for gene replacement by PCR and Southern analysis. In the first step one allele of CaGPM1 was replaced by the hisG-URA3-hisG cassette (CAP1). Strain CAP1 was plated on 5-fluoroorotic acid-containing medium for isolation of Ura- segregants (CAP2). A second transformation with the same disruption construct led to the isolation of a Cagpm1 null mutant (CAP3) on Sabouraud-GE (1% (w/v) peptone (casein), 3% (w/v) glycerol, 2% (w/v) ethanol) plates. Again, Ura- segregants were selected (CAP4). For homologous reintegration of the CaGPM1 gene, CaGPM1 was amplified by PCR with primer SP1-SacI (5′-ATATATCGAGCTCGTTAGATCACCTTTTACTCTCAAG-3′, position -354 to -330) and SP5-KpnI (5′-ATATATGCGGTACCCTTAATTTCTGAGTCTAACAGAAC-3′, position +866 to +842). The resulting 1220-bp PCR product was SacI/KpnI-digested and cloned into the disruption vector pMB7, yielding plasmid pG2. Primer SP6 SalI (5′-ATATATGCGTCGACTGTAATGTGGATGTTAGTTTGCT-3′, position +868 to +891) and primer SP7 PstI (5′-ATAAATGACTGCAGCAATTGGTAACTCAATATATACTC-3′, position +1200 to +1176) were used to amplify the downstream region. This resulting 332-bp product was digested with SalI and PstI and cloned into plasmid pG2, yielding pG3. CaGPM1 was reintroduced into the null mutant strain CAP4 by transformation with the 5.6-kilobase SacI/PstI insert of pG3 that harbors the CaGPM1 gene, upstream as well as downstream regions for homologous recombination, and the URA3 gene as a selectable marker, and yielded CAP5. Candida ELISA—For Candida ELISAs, C. albicans yeast cells from an overnight culture were washed with PBS, diluted into carbonate-bicarbonate buffer (Sigma) to 1 × 107 cells/ml and immobilized onto a microtiter plate (MaxiSorb, Nunc) at 4 °C overnight. After washing with PBS-T buffer once, nonspecific binding sites were blocked with PBS containing 1× Roti-Block (Roth) for 2 h at RT. After incubation with 20% normal human serum/EDTA (normal human serum/diluted in blocking solution supplemented with 0.05% Tween 20 (Block-T) and 10 mm EDTA) for 1 h at 37 °C, wells were washed 3 times with PBS-T and incubated with primary antiserum (1:1000 dilution in Block-T) for 1 h at RT. After washing with PBS-T buffer, horseradish peroxidase-conjugated antisera (1:1000 dilution in Block-T) were added and incubated for 1 h at RT. After washing, substrate o-phenylenediamine dihydrochloride (Sigma) was added. The reaction was stopped with 2 m H2SO4, and the optical density was measured at 490 nm in an ELISA plate reader (SpektraMax 190, Molecular Devices). Activation of Plasminogen and Assaying Plasmin Activity—Plasmin activity was measured as described (29Bergmann S. Rohde M. Preissner K.T. Hammerschmidt S. Thromb. Haemostasis. 2005; 94: 304-311PubMed Google Scholar). Briefly, recombinant CaGpm1p (0.25-2 μg) was immobilized onto the surface of a microtiter plate (half-area plate, Corning). After blocking with PBS containing 2% BSA, plasminogen (0.6 μg/well) was added for 2 h at RT. Unbound plasminogen was removed by washing with PBS/substrate buffer. Plasminogen activator uPA (4 ng/well) and chromogenic substrate S-2251 (d-valyl-leucyl-lysine-p-nitroanilide dihydrochloride, Sigma; 150 μg/well) in 0.32 m Tris-HCl, 1.77 m NaCl, pH 7.5, were added. Plasmin activity was measured at 37 °C in intervals of 30 min by recording the absorbance at 405 nm (SpektraMax 190, Molecular Devices). Identification of ScGpm1p as a Factor H- and FHL-1-binding Protein—We have previously shown that host complement regulators Factor H and FHL-1 bind to the pathogenic yeast C. albicans (15Meri T. Hartmann A. Lenk D. Eck R. Wurzner R. Hellwage J. Meri S. Zipfel P.F. Infect. Immun. 2002; 70: 5185-5192Crossref PubMed Scopus (116) Google Scholar). To identify yeast proteins that bind Factor H and FHL-1, a protein microarray representing 4088 recombinant proteins of the related yeast S. cerevisiae was used, and four Factor H-binding (YDR047W, YKL152C, YGR191W, YBL024W) and three FHL-1-binding (YDR047W, YKL152C, YBL024W) proteins were identified. All three FHL-1-binding proteins do also bind Factor H, and YGR191W binds specifically Factor H but not FHL-1. Spot YKL152C, which bound both Factor H and FHL-1, represents the phosphoglycerate mutase ScGpm1p (Fig. 1A). The C. albicans homologue CaGpm1p showed the highest homology to the S. cerevisiae protein (78%). Because this protein was recently identified as a plasminogen-binding protein (30Crowe J.D. Sievwright I.K. Auld G.C. Moore N.R. Gow N.A. Booth N.A. Mol. Microbiol. 2003; 47: 1637-1651Crossref PubMed Scopus (198) Google Scholar), it was selected for further analyses. To confirm this interaction, the Gpm1p of C. albicans was cloned and recombinantly expressed in the yeast P. pastoris. The culture supernatant and various fractions obtained by nickel chelate chromatography were separated by SDS-PAGE and analyzed by silver staining (Fig. 1B). Two prominent bands of 36 and 31 kDa were detected in the supernatant and the elute fraction. Western blotting with α-His antiserum identified the 36-kDa band as the recombinant CaGpm1p protein (Fig. 1B, lane 5), suggesting that the second 31-kDa band represents a degradation product that lacks the His tag. CaGpm1p Binds Human Plasma Proteins—Binding of Factor H and FHL-1 to recombinant CaGpm1p was assayed by ELISA. CaGpm1p was immobilized to the microtiter plate, and purified Factor H and FHL-1 were added. Both immune regulators bound to CaGpm1p. CaGpm1p was recently identified as a plasminogen-binding protein (30Crowe J.D. Sievwright I.K. Auld G.C. Moore N.R. Gow N.A. Booth N.A. Mol. Microbiol. 2003; 47: 1637-1651Crossref PubMed Scopus (198) Google Scholar), and this interaction was confirmed for the recombinant yeast protein. The classical pathway regulator C4BP did not bind to immobilized CaGpm1p (Fig. 2). Factor H and FHL-1 as well as plasminogen bound to recombinant immobilized CaGpm1p. Mapping of the Binding Regions in Factor H and FHL-1—To localize binding sites for CaGpm1p within the two host regulators, recombinant deletion constructs of FHL-1 and of Factor H representing SCRs 1-5, SCRs 1-6, SCRs 1-7/FHL-1, SCRs 8-11, SCRs 11-15, SCRs 15-18, and SCRs 19 and 20 (26Kuhn S. Zipfel P.F. Gene (Amst.). 1995; 162: 225-229Crossref PubMed Scopus (103) Google Scholar) were tested for CaGpm1p binding. FHL-1 (SCRs 1-7) and SCRs 1-6 did bind to immobilized CaGpm1p, but SCRs 1-5 showed rather weak binding. In addition, recombinant deletion constructs of Factor H SCRs 19 and 20 did clearly bind, whereas SCRs 15-18 bound to a smaller extent. Constructs SCRs 8-11 and SCRs 11-15 did not bind (Fig. 3A). Based on these results it is concluded that FHL-1 has one interacting region with CaGpm1p, located in SCRs 6 and 7 (region I). Factor H has two binding regions, one shared with FHL-1 and a second region (region II) located in the C terminus, i.e. SCRs 19 and 20 (Fig. 3B). In the ELISA assays region I binds with higher intensity than region II. Lysine-dependent Binding of Plasminogen to CaGpm1p—To characterize the CaGpm1p plasminogen interaction in more detail we analyzed the role of the lysine analogue ϵ-aminocaproic acid (ϵACA). ϵACA inhibited plasminogen binding to CaGpm1p in a dose-dependent manner. Inhibition of about 20% was observed with 0.1 mm ϵACA, and maximal inhibition (>90%) was observed with 10 mm of the inhibitor (Fig. 4). Thus, binding of plasminogen to CaGpm1p is mediated by lysine residues. CaGpm1p Is a Surface Protein of C. albicans—To confirm CaGpm1p expression on the surface of intact C. albicans, an antiserum was raised against the recombinant yeast protein. Confocal laser scanning microscopy showed expression of CaGpm1p on the surface of intact C. albicans yeast cells (Fig. 5A, upper, left panel). Staining was particularly prominent on the tip of the hyphae (Fig. 5A, lower, left panel). In addition, surface localization of CaGpm1p is demonstrated by flow cytometry (Fig. 5B). Furthermore, the native 27.5-kDa CaGpm1p protein was identified in the cytoplasmic fraction and also in a cell wall extract by Western blotting using the specific antiserum (Fig. 5C, lanes 1 and 2). Preimmune serum showed no reactivity (data not shown). To exclude contamination of the cell wall fraction with intracellular proteins, the presence of the intracellular protein γ tubulin was assayed. Staining for the 53-kDa γ tubulin is detected in the cytoplasmic but not in the cell wall fraction (Fig. 5C, bottom panel), thus, demonstrating that the cell wall extract is free of major cytoplasmic contaminants. In summary, three independent methods show expression of CaGpm1p at the surface of C. albicans. Generation of a C. albicans gpm1 Null Mutant Strain—To verify the role of CaGpm1p in the binding of Factor H, FHL-1, and plasminogen to C. albicans, a Cagpm1 knock-out mutant was generated using the Ur" @default.
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• W1975086865 title "Gpm1p Is a Factor H-, FHL-1-, and Plasminogen-binding Surface Protein of Candida albicans" @default.
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