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Volume 150, Issue 10

Systematic identification of covalently bound cell wall proteins and analysis of protein–polysaccharide linkages of the human pathogen Free

Abstract

is an important cause of systemic candidiasis in humans. This paper reports a systematic analysis of the putative glycosylphosphatidylinositol-modified (GPI) proteins of , a large part of which are covalently bound to the cell wall glucan network and the remainder of which are retained in the plasma membrane, and of cell wall proteins (CWPs) which are covalently bound in a mild-alkali-sensitive manner. genomic analysis revealed 106 putative GPI proteins. Fifty-one of these GPI proteins could be categorized as adhesive proteins, potentially implicated in fungus–host interactions or biofilm formation during the development of fungal infections. Eleven proteins belonged to well-known GPI protein families of glycoside hydrolases, probably involved in cell wall expansion and remodelling during growth. Other identified GPI proteins included phospholipases, aspartic proteases, homologues of Ecm33p and Kre1p, and structural CWPs. Interestingly, the GPI algorithm predicted three orthologues of an abundant CWP in , Cwp1p, which is absent in . To evaluate the predictions, isolated cell walls were extracted using HF-pyridine, which specifically cleaves phosphodiester bonds, to release GPI-CWPs. Immunological analysis of the extract using one-dimensional SDS-PAGE and anti-Cwp1p antiserum indicated the presence of a Cwp1p homologue in cell walls. Further analysis by two-dimensional gel electrophoresis and electrospray ionization tandem mass spectrometry (ESI-MS/MS) confirmed the presence of two of the predicted Cwp1p proteins, Cwp1.1p and Cwp1.2p. Crh1p, a putative 1,3--glucan remodelling enzyme, was also identified. genomic analysis further revealed five putative Pir proteins (Pir1–5p) and five members of the Bgl2 glycoside hydrolase family 17, belonging to a class of putative CWPs that can be extracted with NaOH. Immunological analysis of mild-alkali-extracted CWPs showed the presence of a Pir2p homologue. Together, these experimental data and predictions represent the first systematic analysis of the cell wall proteome.

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Keyword(s): 1D, one-dimensional , 2D, two-dimensional , CAI, codon adaptation index , ConA, concanavalin A , CWP, cell wall protein , ER, endoplasmic reticulum , ESI-MS/MS, electrospray ionization tandem mass spectrometry and GPI, glycosylphosphatidylinositol (-modified)
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2004-10-01
2024-04-20
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References

  1. Bailey, D. A., Feldmann, P. J., Bovey, M., Gow, N. A. & Brown, A. J.(1996). The Candida albicansHYR1 gene, which is activated in response to hyphal development, belongs to a gene family encoding yeast cell wall proteins. J Bacteriol 178, 5353–5360. [Google Scholar]
  2. Bom, I. J., Dielbandhoesing, S. K., Harvey, K. N., Oomes, S. J., Klis, F. M. & Brul, S.(1998). A new tool for studying the molecular architecture of the fungal cell wall: one-step purification of recombinant trichoderma beta-(1-6)-glucanase expressed in Pichia pastoris. Biochim Biophys Acta 1425, 419–424.[CrossRef] [Google Scholar]
  3. Boone, C., Sommer, S. S., Hensel, A. & Bussey, H.(1990). Yeast KRE genes provide evidence for a pathway of cell wall beta-glucan assembly. J Cell Biol 110, 1833–1843.[CrossRef] [Google Scholar]
  4. Breinig, F., Tipper, D. J. & Schmitt, M. J.(2002). Kre1p, the plasma membrane receptor for the yeast K1 viral toxin. Cell 108, 395–405.[CrossRef] [Google Scholar]
  5. Calcagno, A. M., Bignell, E., Warn, P., Jones, M. D., Denning, D. W., Muhlschlegel, F. A., Rogers, T. R. & Haynes, K.(2003).Candida glabrataSTE12 is required for wild-type levels of virulence and nitrogen starvation induced filamentation. Mol Microbiol 50, 1309–1318.[CrossRef] [Google Scholar]
  6. Cappellaro, C., Mrsa, V. & Tanner, W.(1998). New potential cell wall glucanases of Saccharomyces cerevisiae and their involvement in mating. J Bacteriol 180, 5030–5037. [Google Scholar]
  7. Castillo, L., Martínez, A. I., Garcera, A., Elorza, M. V., Valentin, E. & Sentandreu, R.(2003). Functional analysis of the cysteine residues and the repetitive sequence of Saccharomyces cerevisiae Pir4/Cis3: the repetitive sequence is needed for binding to the cell wall beta-1,3-glucan. Yeast 20, 973–983.[CrossRef] [Google Scholar]
  8. Chaffin, W. L., Lopez-Ribot, J. L., Casanova, M., Gozalbo, D. & Martinez, J. P.(1998). Cell wall and secreted proteins of Candida albicans: identification, function, and expression. Microbiol Mol Biol Rev 62, 130–180. [Google Scholar]
  9. Cheng, S., Clancy, C. J., Checkley, M. A., Handfield, M., Hillman, J. D., Progulske-Fox, A., Lewin, A. S., Fidel, P. L. & Nguyen, M. H.(2003). Identification of Candida albicans genes induced during thrush offers insight into pathogenesis. Mol Microbiol 48, 1275–1288.[CrossRef] [Google Scholar]
  10. Cormack, B. P., Ghori, N. & Falkow, S.(1999). An adhesin of the yeast pathogen Candida glabrata mediating adherence to human epithelial cells. Science 285, 578–582.[CrossRef] [Google Scholar]
  11. Coutinho, P. M. & Henrissat, B.(1999). Carbohydrate-active enzymes: an integrated database approach. In Recent Advances in Carbohydrate Bioengineering, pp. 3–12. Edited by H. J. Gilbert, G. Davies, B. Henrissat & B. Svensson. Cambridge: Royal Society of Chemistry.
  12. Csank, C. & Haynes, K.(2000).Candida glabrata displays pseudohyphal growth. FEMS Microbiol Lett 189, 115–120.[CrossRef] [Google Scholar]
  13. De Groot, P. W., Ruiz, C., Vázquez de Aldana, C. R. & 14 other authors(2001). A genomic approach for the identification and classification of genes involved in cell wall formation and its regulation in Saccharomyces cerevisiae. Comp Funct Genomics 2, 124–142.[CrossRef] [Google Scholar]
  14. De Groot, P. W., Hellingwerf, K. J. & Klis, F. M.(2003). Genome-wide identification of fungal GPI proteins. Yeast 20, 781–796.[CrossRef] [Google Scholar]
  15. De Groot, P. W., De Boer, A. D., Cunningham, J., Dekker, H. L., De Jong, L., Hellingwerf, K. J., De Koster, C. & Klis, F. M.(2004). Proteomic analysis of Candida albicans cell walls reveals covalently bound carbohydrate-active enzymes and adhesins. Eukaryot Cell 3, 955–965.[CrossRef] [Google Scholar]
  16. De Las Penas, A., Pan, S. J., Castano, I., Alder, J., Cregg, R. & Cormack, B. P.(2003). Virulence-related surface glycoproteins in the yeast pathogen Candida glabrata are encoded in subtelomeric clusters and subject to RAP1- and SIR-dependent transcriptional silencing Genes Dev 17, 2245–2258.[CrossRef] [Google Scholar]
  17. Eisenhaber, B., Schneider, G., Wildpaner, M. & Eisenhaber, F.(2004). A sensitive predictor for potential GPI lipid modification sites in fungal protein sequences and its application to genome-wide studies for Aspergillus nidulans, Candida albicans, Neurospora crassa, Saccharomyces cerevisiae and Schizosaccharomyces pombe. J Mol Biol 337, 243–253.[CrossRef] [Google Scholar]
  18. Fidel, P. L., Jr, Vazquez, J. A. & Sobel, J. D.(1999).Candida glabrata: review of epidemiology, pathogenesis, and clinical disease with comparison to C. albicans. Clin Microbiol Rev 12, 80–96. [Google Scholar]
  19. Frieman, M. B. & Cormack, B. P.(2003). The omega-site sequence of glycosylphosphatidylinositol-anchored proteins in Saccharomyces cerevisiae can determine distribution between the membrane and the cell wall. Mol Microbiol 50, 883–896.[CrossRef] [Google Scholar]
  20. Frieman, M. B., McCaffery, J. M. & Cormack, B. P.(2002). Modular domain structure in the Candida glabrata adhesin Epa1p, a beta1,6 glucan-cross-linked cell wall protein. Mol Microbiol 46, 479–492.[CrossRef] [Google Scholar]
  21. Garcia, R., Bermejo, C., Grau, C., Perez, R., Rodriguez-Pena, J. M., Francois, J., Nombela, C. & Arroyo, J.(2004). The global transcriptional response to transient cell wall damage in Saccharomyces cerevisiae and its regulation by the cell integrity signaling pathway. J Biol Chem 279, 15183–15195.[CrossRef] [Google Scholar]
  22. Garcia-Sanchez, S., Aubert, S., Iraqui, I., Janbon, G., Ghigo, J. M. & D'Enfert, C.(2004). Biofilms of Candida albicans: a developmental state associated with specific and stable gene expression patterns. Eukaryot Cell 3, 536–545.[CrossRef] [Google Scholar]
  23. Gomez, M. J., Torosantucci, A., Arancia, S., Maras, B., Parisi, L. & Cassone, A.(1996). Purification and biochemical characterization of a 65-kilodalton mannoprotein (MP65), a main target of anti-Candida cell-mediated immune responses in humans. Infect Immun 64, 2577–2584. [Google Scholar]
  24. Gozalbo, D., Gil-Navarro, I., Azorin, I., Renau-Piqueras, J., Martinez, J. P. & Gil, M. L.(1998). The cell wall-associated glyceraldehyde-3-phosphate dehydrogenase of Candida albicans is also a fibronectin and laminin binding protein. Infect Immun 66, 2052–2059. [Google Scholar]
  25. Halme, A., Bumgarner, S., Styles, C. & Fink, G. R.(2004). Genetic and epigenetic regulation of the FLO gene family generates cell-surface variation in yeast. Cell 116, 405–415.[CrossRef] [Google Scholar]
  26. Hamada, K., Terashima, H., Arisawa, M. & Kitada, K.(1998). Amino acid sequence requirement for efficient incorporation of glycosylphosphatidylinositol-associated proteins into the cell wall of Saccharomyces cerevisiae. J Biol Chem 273, 26946–26953.[CrossRef] [Google Scholar]
  27. Hamada, K., Terashima, H., Arisawa, M., Yabuki, N. & Kitada, K.(1999). Amino acid residues in the omega-minus region participate in cellular localization of yeast glycosylphosphatidylinositol-attached proteins. J Bacteriol 181, 3886–3889. [Google Scholar]
  28. Hoj, P. B., Condron, R., Traeger, J. C., McAuliffe, J. C. & Stone, B. A.(1992). Identification of glutamic acid 105 at the active site of Bacillus amyloliquefaciens 1,3-1,4-beta-d-glucan 4-glucanohydrolase using epoxide-based inhibitors. J Biol Chem 267, 25059–25066. [Google Scholar]
  29. Hoyer, L. L.(2001). The ALS gene family of Candida albicans. Trends Microbiol 9, 176–180.[CrossRef] [Google Scholar]
  30. Hoyer, L. L., Payne, T. L., Bell, M., Myers, A. M. & Scherer, S.(1998).Candida albicansALS3 and insights into the nature of the ALS gene family. Curr Genet 33, 451–459.[CrossRef] [Google Scholar]
  31. Hoyer, L. L., Clevenger, J., Hecht, J. E., Ehrhart, E. J. & Poulet, F. M.(1999). Detection of Als proteins on the cell wall of Candida albicans in murine tissues. Infect Immun 67, 4251–4255. [Google Scholar]
  32. Hube, B. & Naglik, J.(2001).Candida albicans proteinases: resolving the mystery of a gene family. Microbiology 147, 1997–2005. [Google Scholar]
  33. Jiang, B., Sheraton, J., Ram, A. F., Dijkgraaf, G. J., Klis, F. M. & Bussey, H.(1996).CWH41 encodes a novel endoplasmic reticulum membrane N-glycoprotein involved in beta 1,6-glucan assembly. J Bacteriol 178, 1162–1171. [Google Scholar]
  34. Kapteyn, J. C., Montijn, R. C., Dijkgraaf, G. J., Van den, E. H. & Klis, F. M.(1995). Covalent association of beta-1,3-glucan with beta-1,6-glucosylated mannoproteins in cell walls of Candida albicans. J Bacteriol 177, 3788–3792. [Google Scholar]
  35. Kapteyn, J. C., Montijn, R. C., Vink, E., de la, Cruz. J., Llobell, A., Douwes, J. E., Shimoi, H., Lipke, P. N. & Klis, F. M.(1996). Retention of Saccharomyces cerevisiae cell wall proteins through a phosphodiester-linked beta-1,3-/beta-1,6-glucan heteropolymer. Glycobiology 6, 337–345.[CrossRef] [Google Scholar]
  36. Kapteyn, J. C., Van Egmond, P., Sievi, E., Van den Ende, H., Makarow, M. & Klis, F. M.(1999). The contribution of the O-glycosylated protein Pir2p/Hsp150 to the construction of the yeast cell wall in wild-type cells and beta 1,6-glucan-deficient mutants. Mol Microbiol 31, 1835–1844.[CrossRef] [Google Scholar]
  37. Kapteyn, J. C., ter Riet, B., Vink, E., Blad, S., De Nobel, H., Van den Ende, H. & Klis, F. M.(2001). Low external pH induces HOG1-dependent changes in the organization of the Saccharomyces cerevisiae cell wall. Mol Microbiol 39, 469–479.[CrossRef] [Google Scholar]
  38. Klis, F. M., Ram, A. F. J., Montijn, R. C., Kapteyn, J. C., Caro, L. H. & Vossen, J. H.(1998). Posttranslational modifications of secretory proteins. Methods Microbiol 26, 223–238. [Google Scholar]
  39. Klis, F. M., de Groot, P. & Hellingwerf, K.(2001). Molecular organization of the cell wall of Candida albicans. Med Mycol 39 Suppl 1, 1–8. [Google Scholar]
  40. Klis, F. M., Mol, P., Hellingwerf, K. & Brul, S.(2002). Dynamics of cell wall structure in Saccharomyces cerevisiae. FEMS Microbiol Rev 26, 239–256.[CrossRef] [Google Scholar]
  41. Lachke, S. A., Joly, S., Daniels, K. & Soll, D. R.(2002). Phenotypic switching and filamentation in Candida glabrata. Microbiology 148, 2661–2674. [Google Scholar]
  42. Li, F. & Palecek, S. P.(2003).EAP1, a Candida albicans gene involved in binding human epithelial cells. Eukaryot Cell 2, 1266–1273.[CrossRef] [Google Scholar]
  43. Lo, H. J., Kohler, J. R., DiDomenico, B., Loebenberg, D., Cacciapuoti, A. & Fink, G. R.(1997). Nonfilamentous Candida albicans mutants are avirulent. Cell 90, 939–949.[CrossRef] [Google Scholar]
  44. Martínez, A. I., Castillo, L., Garcerá, A., Elorza, M. V., Valentín, E. & Sentandreu, R.(2004). Role of Pir1 in the construction of the Candida albicans cell wall. Microbiology 150, 3151–3161.[CrossRef] [Google Scholar]
  45. Mouyna, I., Monod, M., Fontaine, T., Henrissat, B. & Lechenne, B., Latgé, J. P.(2000). Identification of the catalytic residues of the first family of beta(1-3)glucanosyltransferases identified in fungi. Biochem J 347, 741–747.[CrossRef] [Google Scholar]
  46. Mrsa, V. & Tanner, W.(1999). Role of NaOH-extractable cell wall proteins Ccw5p, Ccw6p, Ccw7p and Ccw8p (members of the Pir protein family) in stability of the Saccharomyces cerevisiae cell wall. Yeast 15, 813–820.[CrossRef] [Google Scholar]
  47. Mrsa, V., Seidl, T., Gentzsch, M. & Tanner, W.(1997). Specific labelling of cell wall proteins by biotinylation. Identification of four covalently linked O-mannosylated proteins of Saccharomyces cerevisiae. Yeast 13, 1145–1154.[CrossRef] [Google Scholar]
  48. Nakayama, K., Feng, Y., Tanaka, A. & Jigami, Y.(1998). The involvement of mnn4 and mnn6 mutations in mannosylphosphorylation of O-linked oligosaccharide in yeast Saccharomyces cerevisiae. Biochim Biophys Acta 1425, 255–262.[CrossRef] [Google Scholar]
  49. Neuhoff, V., Arold, N., Taube, D. & Ehrhardt, W.(1988). Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie Brilliant Blue G-250 and R-250. Electrophoresis 9, 255–262.[CrossRef] [Google Scholar]
  50. Nisini, R., Romagnoli, G., Gomez, M. J., La Valle, R., Torosantucci, A., Mariotti, S., Teloni, R. & Cassone, A.(2001). Antigenic properties and processing requirements of 65-kilodalton mannoprotein, a major antigen target of anti-Candida human T-cell response, as disclosed by specific human T-cell clones. Infect Immun 69, 3728–3736.[CrossRef] [Google Scholar]
  51. Pfaller, M. A., Messer, S. A., Hollis, R. J., Jones, R. N. & Diekema, D. J.(2002). In vitro activities of ravuconazole and voriconazole compared with those of four approved systemic antifungal agents against 6,970 clinical isolates of Candida spp. Antimicrob Agents Chemother 46, 1723–1727.[CrossRef] [Google Scholar]
  52. Pfaller, M. A., Diekema, D. J., Messer, S. A., Boyken, L. & Hollis, R. J.(2003a). Activities of fluconazole and voriconazole against 1,586 recent clinical isolates of Candida species determined by Broth microdilution, disk diffusion, and Etest methods: report from the ARTEMIS Global Antifungal Susceptibility Program, 2001. J Clin Microbiol 41, 1440–1446.[CrossRef] [Google Scholar]
  53. Pfaller, M. A., Messer, S. A., Boyken, L., Tendolkar, S., Hollis, R. J. & Diekema, D. J.(2003b). Variation in susceptibility of bloodstream isolates of Candida glabrata to fluconazole according to patient age and geographic location. J Clin Microbiol 41, 2176–2179.[CrossRef] [Google Scholar]
  54. Pitarch, A., Sanchez, M., Nombela, C. & Gil, C.(2002). Sequential fractionation and two-dimensional gel analysis unravels the complexity of the dimorphic fungus Candida albicans cell wall proteome. Mol Cell Proteomics 12, 967–982. [Google Scholar]
  55. Rex, J. H., Walsh, T. J., Sobel, J. D., Filler, S. G., Pappas, P. G., Dismukes, W. E. & Edwards, J. E.(2000). Practice guidelines for the treatment of candidiasis. Infectious Diseases Society of America. Clin Infect Dis 30, 662–678.[CrossRef] [Google Scholar]
  56. Rodriguez-Pena, J. M., Cid, V. J., Arroyo, J. & Nombela, C.(2000). A novel family of cell wall-related proteins regulated differently during the yeast life cycle. Mol Cell Biol 20, 3245–3255.[CrossRef] [Google Scholar]
  57. Roemer, T. & Bussey, H.(1995). Yeast Kre1p is a cell surface O-glycoprotein. Mol Gen Genet 249, 209–216.[CrossRef] [Google Scholar]
  58. Russo, P., Kalkkinen, N., Sareneva, H., Paakkola, J. & Makarow, M.(1992). A heat shock gene from Saccharomyces cerevisiae encoding a secretory glycoprotein. Proc Natl Acad Sci U S A 89, 3671–3675.[CrossRef] [Google Scholar]
  59. Scherrer, R., Louden, L. & Gerhardt, P.(1974). Porosity of the yeast cell wall and membrane. J Bacteriol 118, 534–540. [Google Scholar]
  60. Sharp, P. M. & Li, W. H.(1987). The Codon Adaptation Index – a measure of directional synonymous codon usage bias, and its potential applications. Nucleic Acids Res 15, 1281–1295.[CrossRef] [Google Scholar]
  61. Shimoi, H., Iimura, Y. & Obata, T.(1995). Molecular cloning of CWP1: a gene encoding a Saccharomyces cerevisiae cell wall protein solubilized with Rarobacter faecitabidus protease I. J Biochem 118, 302–311. [Google Scholar]
  62. Shimoi, H., Sakamoto, K., Okuda, M., Atthi, R., Iwashita, K. & Ito, K.(2002). The Awa1 gene is required for the foam-forming phenotype and cell surface hydrophobicity of sake yeast. Appl Environ Microbiol 68, 2018–2025.[CrossRef] [Google Scholar]
  63. Staab, J. F., Bradway, S. D., Fidel, P. L. & Sundstrom, P.(1999). Adhesive and mammalian transglutaminase substrate properties of Candida albicans Hwp1. Science 283, 1535–1538.[CrossRef] [Google Scholar]
  64. Sundstrom, P.(2002). Adhesion in Candida spp. Cell Microbiol 4, 461–469.[CrossRef] [Google Scholar]
  65. Tabernero, C., Coll, P. M., Fernandez-Abalos, J. M., Perez, P. & Santamaria, R. I.(1994). Cloning and DNA sequencing of bgaA, a gene encoding an endo-beta-1,3-1,4-glucanase, from an alkalophilic Bacillus strain (N137). Appl Environ Microbiol 60, 1213–1220. [Google Scholar]
  66. Terashima, H., Hamada, K. & Kitada, K.(2003). The localization change of Ybr078w/Ecm33, a yeast GPI-associated protein, from the plasma membrane to the cell wall, affecting the cellular function. FEMS Microbiol Lett 218, 175–180.[CrossRef] [Google Scholar]
  67. Van der Vaart, J. M., Caro, L. H., Chapman, J. W., Klis, F. M. & Verrips, C. T.(1995). Identification of three mannoproteins in the cell wall of Saccharomyces cerevisiae. J Bacteriol 177, 3104–3110. [Google Scholar]
  68. Viladot, J. L., de Ramon, E., Durany, O. & Planas, A.(1998). Probing the mechanism of Bacillus 1,3-1,4-beta-d-glucan 4-glucanohydrolases by chemical rescue of inactive mutants at catalytically essential residues. Biochemistry 37, 11332–11342.[CrossRef] [Google Scholar]
  69. Vossen, J. H., Muller, W. H., Lipke, P. N. & Klis, F. M.(1997). Restrictive glycosylphosphatidylinositol anchor synthesis in cwh6/gpi3 yeast cells causes aberrant biogenesis of cell wall proteins. J Bacteriol 179, 2202–2209. [Google Scholar]
  70. Weig, M., Haynes, K., Rogers, T. R., Kurzai, O., Frosch, M. & Muhlschlegel, F. A.(2001). A GAS-like gene family in the pathogenic fungus Candida glabrata. Microbiology 147, 2007–2019. [Google Scholar]
  71. Zhao, X., Pujol, C., Soll, D. R. & Hoyer, L. L.(2003). Allelic variation in the contiguous loci encoding Candida albicansALS5, ALS1 and ALS9. Microbiology 149, 2947–2960.[CrossRef] [Google Scholar]
  72. Zlotnik, H., Fernandez, M. P., Bowers, B. & Cabib, E.(1984).Saccharomyces cerevisiae mannoproteins form an external cell wall layer that determines wall porosity. J Bacteriol 159, 1018–1026. [Google Scholar]
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Systematic identification in silico of covalently bound cell wall proteins and analysis of protein–polysaccharide linkages of the human pathogen Candida glabrata
Microbiology 150, 3129 (2004); https://doi.org/10.1099/mic.0.27256-0
/content/journal/micro/10.1099/mic.0.27256-0
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