The GPI-anchored protein CaEcm33p is required for cell wall integrity, morphogenesis and virulence in Free

Abstract

Ecm33p is a widely distributed fungal protein with functional relevance, clearly demonstrated by Δ mutant phenotypes, mainly related to the cell wall. Homology searches with genes identified Ecm33p, as well as the two other proteins of its family: Pst1p and the product of . Ecm33p is a 423 aa protein which has the typical features of cell-surface GPI proteins and is able to complement Δ cell wall defects. Heterozygous (RML1) and homozygous (RML2) mutants of were obtained, as well as a single and a double reintegrant (RML3 and RML4, respectively). mutant strains displayed an aberrant morphology, being more rounded and bigger than the wild-type, suggesting morphogenetic defects. They also exhibited cell wall defects, with enhanced sensitivity to different compounds that interfere in polymerization of cell wall components (Calcofluor white, Congo red and hygromycin B) and a marked tendency to flocculate extensively. In addition, CaEcm33p is required for normal yeast-to-hyphae transition . In liquid medium (5 % serum), the transition was delayed in mutants, and after 24 h the culture contained very abnormal large and rounded cells. On solid medium (10 % serum, Spider or SLADH) RML2 failed to produce hyphae and media invasiveness. showed a gene dosage effect, demonstrated by the intermediate phenotype of the heterozygous mutants RML1 and confirmed by Northern blot analysis. Furthermore, CaEcm33p is also involved in virulence. In a murine systemic model of infection, 100 % mouse survival and no kidney or brain colonization were obtained 30 days after infection with 10 cells of any homozygous or heterozygous Δ mutant tested. In contrast, all mice infected with parental or RML4 (two Ca copies reintegrated) strains died in a few days, showing that, in these conditions, two copies were required for virulence.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.27320-0
2004-10-01
2024-03-28
Loading full text...

Full text loading...

/deliver/fulltext/micro/150/10/mic1503341.html?itemId=/content/journal/micro/10.1099/mic.0.27320-0&mimeType=html&fmt=ahah

References

  1. Aramayo, R. & Metzenberg, R. L.(1996). Meiotic transvection in fungi. Cell 86, 103–113.[CrossRef] [Google Scholar]
  2. Ausubel, F. M., Kingston, R. E., Brent, R., Moore, D. D., Seidman, J. G., Smith, J. A. & Struhl, K.(1993).Current Protocols in Molecular Biology. New York: Greene Publishing Associates & Wiley Interscience.
  3. Bidlingmaier, S. & Snyder, M.(2002). Large-scale identification of genes important for apical growth in Saccharomyces cerevisiae by directed allele replacement technology (DART) screening. Funct Integr Genomics 1, 345–356.[CrossRef] [Google Scholar]
  4. Biswas, S., Roy, M. & Datta, A.(2003).N-Acetylglucosamine-inducible CaGAP1 encodes a general amino acid permease which co-ordinates external nitrogen source response and morphogenesis in Candida albicans. Microbiology 149, 2597–2608.[CrossRef] [Google Scholar]
  5. Boeke, J. D., LaCroute, F. & Fink, G. R.(1984). A positive selection for mutants lacking orotidine-5′-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance. Mol Gen Genet 197, 345–346.[CrossRef] [Google Scholar]
  6. Braun, B. R. & Johnson, A. D.(2000).TUP1, CPH1 and EFG1 make independent contributions to filamentation in Candida albicans. Genetics 155, 57–67. [Google Scholar]
  7. Bruneau, J. M., Magnin, T., Tagat, E., Legrand, R., Bernard, M., Diaquin, M., Fudali, C. & Latge, J. P.(2001). Proteome analysis of Aspergillus fumigatus identifies glycosylphosphatidylinositol-anchored proteins associated to the cell wall biosynthesis. Electrophoresis 22, 2812–2823.[CrossRef] [Google Scholar]
  8. Buurman, E. T., Westwater, C., Hube, B., Brown, A. J., Odds, F. C. & Gow, N. A.(1998). Molecular analysis of CaMnt1p, a mannosyl transferase important for adhesion and virulence of Candida albicans. Proc Natl Acad Sci U S A 95, 7670–7675.[CrossRef] [Google Scholar]
  9. Cabib, E. & Bowers, B.(1971). Chitin and yeast budding. Localization of chitin in yeast bud scars. J Biol Chem 246, 152–159. [Google Scholar]
  10. Caro, L. H., Tettelin, H., Vossen, J. H., Ram, A. F., van den, E. H. & Klis, F. M.(1997). In silicio identification of glycosyl-phosphatidylinositol-anchored plasma-membrane and cell wall proteins of Saccharomyces cerevisiae. Yeast 13, 1477–1489.[CrossRef] [Google Scholar]
  11. Carotti, C., Ferrario, L., Roncero, C., Valdivieso, M. H., Duran, A. & Popolo, L.(2002). Maintenance of cell integrity in the gas1 mutant of Saccharomyces cerevisiae requires the Chs3p-targeting and activation pathway and involves an unusual Chs3p localization. Yeast 19, 1113–1124.[CrossRef] [Google Scholar]
  12. Casanova, M., Lopez-Ribot, J. L., Martinez, J. P. & Sentandreu, R.(1992). Characterization of cell wall proteins from yeast and mycelial cells of Candida albicans by labelling with biotin: comparison with other techniques. Infect Immun 60, 4898–4906. [Google Scholar]
  13. 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]
  14. Csank, C., Schroppel, K., Leberer, E., Harcus, D., Mohamed, O., Meloche, S., Thomas, D. Y. & Whiteway, M.(1998). Roles of the Candida albicans mitogen-activated protein kinase homolog, Cek1p, in hyphal development and candidiasis. Infect Immun 66, 2713–2721. [Google Scholar]
  15. De Bernardis, F., Muhlschlegel, F. A., Cassone, A. & Fonzi, W. A.(1998). The pH of the host niche controls gene expression in and virulence of Candida albicans. Infect Immun 66, 3317–3325. [Google Scholar]
  16. 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]
  17. Eisenhaber, B., Bork, P. & Eisenhaber, F.(1998). Sequence properties of GPI-anchored proteins near the omega-site: constraints for the polypeptide binding site of the putative transamidase. Protein Eng 11, 1155–1161.[CrossRef] [Google Scholar]
  18. Elorza, M. V., Murgui, A. & Sentandreu, R.(1985). Dimorphism in Candida albicans: contribution of mannoproteins to the architecture of yeast and mycelial cell walls. J Gen Microbiol 131, 2209–2216. [Google Scholar]
  19. Fonzi, W. A. & Irwin, M. Y.(1993). Isogenic strain construction and gene mapping in Candida albicans. Genetics 134, 717–728. [Google Scholar]
  20. Garcia, R., Bermejo, C., Grau, C., Perez, R., Rodriguez-Peña, 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]
  21. Gillum, A. M., Tsay, E. Y. & Kirsch, D. R.(1984). Isolation of the Candida albicans gene for orotidine-5′-phosphate decarboxylase by complementation of S. cerevisiae ura3 and E. colipyrF mutations. Mol Gen Genet 198, 179–182.[CrossRef] [Google Scholar]
  22. Gimeno, C. J., Ljungdahl, P. O., Styles, C. A. & Fink, G. R.(1992). Unipolar cell divisions in the yeast S. cerevisiae lead to filamentous growth: regulation by starvation and RAS. Cell 68, 1077–1090.[CrossRef] [Google Scholar]
  23. Gonzalez, M. M., Diez-Orejas, R., Molero, G., Alvarez, A. M., Pla, J., Nombela, C. & Sanchez-Perez, M.(1997). Phenotypic characterization of a Candida albicans strain deficient in its major exoglucanase. Microbiology 143, 3023–3032.[CrossRef] [Google Scholar]
  24. Gow, N. A.(1997). Germ tube growth of Candida albicans. Curr Top Med Mycol 8, 43–55. [Google Scholar]
  25. Gow, N. A., Robbins, P. W., Lester, J. W., Brown, A. J., Fonzi, W. A., Chapman, T. & Kinsman, O. S.(1994). A hyphal-specific chitin synthase gene (CHS2) is not essential for growth, dimorphism, or virulence of Candida albicans. Proc Natl Acad Sci U S A 91, 6216–6220.[CrossRef] [Google Scholar]
  26. Groll, A. H., De Lucca, A. J. & Walsh, T. J.(1998). Emerging targets for the development of novel antifungal therapeutics. Trends Microbiol 6, 117–124.[CrossRef] [Google Scholar]
  27. Hanahan, D.(1983). Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166, 557–580.[CrossRef] [Google Scholar]
  28. Hube, B., Monod, M., Schofield, D. A., Brown, A. J. & Gow, N. A.(1994). Expression of seven members of the gene family encoding secretory aspartyl proteinases in Candida albicans. Mol Microbiol 14, 87–99.[CrossRef] [Google Scholar]
  29. Hurtrel, B., Lagrange, P. H. & Michel, J. C.(1980). Systemic candidiasis in mice. I. Correlation between kidney infection and mortality rate. Ann Immunol 131C, 93–104. [Google Scholar]
  30. Jung, U. S. & Levin, D. E.(1999). Genome-wide analysis of gene expression regulated by the yeast cell wall integrity signalling pathway. Mol Microbiol 34, 1049–1057.[CrossRef] [Google Scholar]
  31. Kapteyn, J. C., Montijn, R. C., Dijkgraaf, G. J. & Klis, F. M.(1994). Identification of beta-1,6-glucosylated cell wall proteins in yeast and hyphal forms of Candida albicans. Eur J Cell Biol 65, 402–407. [Google Scholar]
  32. Kapteyn, J. C., Montijn, R. C., Dijkgraaf, G. J., van den, E. H. & Klis, F. M.(1995a). 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]
  33. Kapteyn, J. C., Dijkgraaf, G. J., Montijn, R. C. & Klis, F. M.(1995b). Glucosylation of cell wall proteins in regenerating spheroplasts of Candida albicans. FEMS Microbiol Lett 128, 271–277.[CrossRef] [Google Scholar]
  34. Kapteyn, J. C., Hoyer, L. L., Hecht, J. E., Muller, W. H., Andel, A. Verkleij A. J., Makarow, M., van den, E. H. & Klis, F. M.(2000). The cell wall architecture of Candida albicans wild-type cells and cell wall-defective mutants. Mol Microbiol 35, 601–611. [Google Scholar]
  35. Kinneberg, K. M., Bendel, C. M., Jechorek, R. P., Cebelinski, E. A., Gale, C. A., Berman, J. G., Erlandsen, S. L., Hostetter, M. K. & Wells, C. L.(1999). Effect of INT1 gene on Candida albicans murine intestinal colonization. J Surg Res 87, 245–251.[CrossRef] [Google Scholar]
  36. 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.[CrossRef] [Google Scholar]
  37. 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]
  38. Kohler, J. R. & Fink, G. R.(1996).Candida albicans strains heterozygous and homozygous for mutations in mitogen-activated protein kinase signaling components have defects in hyphal development. Proc Natl Acad Sci U S A 93, 13223–13228.[CrossRef] [Google Scholar]
  39. Leberer, E., Harcus, D., Broadbent, I. D. & 7 other authors(1996). Signal transduction through homologs of the Ste20p and Ste7p protein kinases can trigger hyphal formation in the pathogenic fungus Candida albicans. Proc Natl Acad Sci U S A 93, 13217–13222.[CrossRef] [Google Scholar]
  40. Lipke, P. N. & Ovalle, R.(1998). Cell wall architecture in yeast: new structure and new challenges. J Bacteriol 180, 3735–3740. [Google Scholar]
  41. Liu, H., Kohler, J. & Fink, G. R.(1994). Suppression of hyphal formation in Candida albicans by mutation of a STE12 homolog. Science 266, 1723–1726.[CrossRef] [Google Scholar]
  42. Lopez-Ribot, J. L., Casanova, M., Gil, M. L. & Martinez, J. P.(1996). Common and form-specific cell wall antigens of Candida albicans as released by chemical and enzymatic treatments. Mycopathologia 134, 13–20.[CrossRef] [Google Scholar]
  43. Martin, H., Arroyo, J., Sanchez, M., Molina, M. & Nombela, C.(1993). Activity of the yeast MAP kinase homologue Slt2 is critically required for cell integrity at 37 degrees C. Mol Gen Genet 241, 177–184. [Google Scholar]
  44. Martin, H., Rodriguez-Pachon, J. M., Ruiz, C., Nombela, C. & Molina, M.(2000). Regulatory mechanisms for modulation of signaling through the cell integrity Slt2-mediated pathway in Saccharomyces cerevisiae. J Biol Chem 275, 1511–1519.[CrossRef] [Google Scholar]
  45. Massari, M. E. & Murre, C.(2000). Helix-loop-helix proteins: regulators of transcription in eucaryotic organisms. Mol Cell Biol 20, 429–440.[CrossRef] [Google Scholar]
  46. McCreath, K. J., Specht, C. A. & Robbins, P. W.(1995). Molecular cloning and characterization of chitinase genes from Candida albicans. Proc Natl Acad Sci U S A 92, 2544–2588.[CrossRef] [Google Scholar]
  47. Mitchell, A. P.(1998). Dimorphism and virulence in Candida albicans. Curr Opin Microbiol 1, 687–692.[CrossRef] [Google Scholar]
  48. Monteoliva, L., Sánchez, M., Pla, J., Gil, C. & Nombela, C.(1996). Cloning of Candida albicans SEC14 gene homologue coding for a putative essential function. Yeast 12, 1097–1105.[CrossRef] [Google Scholar]
  49. Monteoliva, L., Matas, M. L., Gil, C., Nombela, C. & Pla, J.(2002). Large-scale identification of putative exported proteins in Candida albicans by genetic selection. Eukaryot Cell 1, 514–525.[CrossRef] [Google Scholar]
  50. Mormeneo, S., Rico, H., Iranzo, M., Aguado, C. & Sentandreu, R.(1996). Study of supramolecular structures released from the cell wall of Candida albicans by ethylenediamine treatment. Arch Microbiol 166, 327–335.[CrossRef] [Google Scholar]
  51. Navarro-Garcia, F., Sanchez, M., Nombela, C. & Pla, J.(2001). Virulence genes in the pathogenic yeast Candida albicans. FEMS Microbiol Rev 25, 245–268.[CrossRef] [Google Scholar]
  52. Nielsen, H., Engelbrecht, J., Brunak, S. & von Heijne, G.(1997). A neural network method for identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Int J Neural Syst 8, 581–599.[CrossRef] [Google Scholar]
  53. Odds, F. C.(1988).Candida and Candidosis. London: Baillière Tindall.
  54. Odds, F. C.(2003). Reflections on the question: what does molecular mycology have to do with the clinician treating the patient? Med Mycol 41, 1–6. [Google Scholar]
  55. Orlean, P.(1997). Biogenesis of yeast cell wall and surface components. In The Molecular and Cellular Biology of the Yeast Saccharomyces. Cell Cycle and Biology, pp. 229–362. Edited by J. R. Pringle, J. R. Broach & E. W. Jones. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  56. Pardo, M., Monteoliva, L., Pla, J., Sanchez, M., Gil, C. & Nombela, C.(1999). Two-dimensional analysis of proteins secreted by Saccharomyces cerevisiae regenerating protoplasts: a novel approach to study the cell wall. Yeast 15, 459–472.[CrossRef] [Google Scholar]
  57. Pardo, M., Ward, M., Bains, S., Molina, M., Blackstock, W., Gil, C. & Nombela, C.(2000). A proteomic approach for the study of Saccharomyces cerevisiae cell wall biogenesis. Electrophoresis 21, 3396–3410.[CrossRef] [Google Scholar]
  58. Pardo, M., Monteoliva, L., Vázquez, P., Martínez, R., Molero, G., Nombela, C. & Gil, C.(2004).PST1 and ECM33 encode two yeast cell surface GPI proteins important for cell wall integrity. Microbiology 150 (in press). [Google Scholar]
  59. 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 1, 967–982.[CrossRef] [Google Scholar]
  60. Pitarch, A., Sanchez, M., Nombela, C. & Gil, C.(2003). Analysis of the Candida albicans proteome. I. Strategies and applications. J Chromatogr B Analyt Technol Biomed Life Sci 787, 101–128.[CrossRef] [Google Scholar]
  61. Robinson, K. A. & Lopes, J. M.(2000).Saccharomyces cerevisiae basic helix-loop-helix proteins regulate diverse biological processes. Nucleic Acids Res 28, 1499–1505.[CrossRef] [Google Scholar]
  62. Roncero, C. & Duran, A.(1985). Effect of Calcofluor white and Congo red on fungal cell wall morphogenesis: in vivo activation of chitin polymerization. J Bacteriol 163, 1180–1185. [Google Scholar]
  63. Ruiz-Herrera, J., Mormeneo, S., Vanaclocha, P., Font-de-Mora, J., Iranzo, M., Puertes, I. & Sentandreu, R.(1994). Structural organization of the components of the cell wall from Candida albicans. Microbiology 140, 1513–1523.[CrossRef] [Google Scholar]
  64. Rymond, B. C. & Rosbash, M.(1985). Cleavage of 5′ splice site and lariat formation are independent of 3′ splice site in yeast mRNA splicing. Nature 317, 735–737.[CrossRef] [Google Scholar]
  65. Rymond, B. C. & Rosbash, M.(1986). Differential nuclease sensitivity identifies tight contacts between yeast pre-mRNA and spliceosomes. EMBO J 20, 3517–3523. [Google Scholar]
  66. Sambrook, J., Fritsch, E. F. & Maniatis, T.(1989).Molecular Cloning: a Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  67. Sanjuan, R., Zueco, J., Stock, R., Font, d. M. & Sentandreu, R.(1995). Identification of glucan-mannoprotein complexes in the cell wall of Candida albicans using a monoclonal antibody that reacts with a (1,6)-beta-glucan epitope. Microbiology 141, 1545–1551.[CrossRef] [Google Scholar]
  68. Sarthy, A. V., McGonigal, T., Coen, M., Frost, D. J., Meulbroek, J. A. & Goldman, R. C.(1997). Phenotype in Candida albicans of a disruption of the BGL2 gene encoding a 1,3-beta-glucosyltransferase. Microbiology 143, 367–376.[CrossRef] [Google Scholar]
  69. Sharkey, L. L., McNemar, M. D., Saporito-Irwin, S. M., Sypherd, P. S. & Fonzi, W. A.(1999).HWP1 functions in the morphological development of Candida albicans downstream of EFG1, TUP1, and RBF1. J Bacteriol 181, 5273–5279. [Google Scholar]
  70. Sohn, K., Urban, C., Brunner, H. & Rupp, S.(2003).EFG1 is a major regulator of cell wall dynamics in Candida albicans as revealed by DNA microarrays. Mol Microbiol 47, 89–102. [Google Scholar]
  71. Souciet, J., Aigle, M., Artiguenave, F. & 21 other authors(2000). Genomic exploration of the hemiascomycetous yeasts: 1. A set of yeast species for molecular evolution studies. FEBS Lett 487, 3–12.[CrossRef] [Google Scholar]
  72. 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]
  73. 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]
  74. Teunissen, A. W. & Steensma, H. Y.(1995a). Review: the dominant flocculation genes of Saccharomyces cerevisiae constitute a new subtelomeric gene family. Yeast 11, 1001–1013.[CrossRef] [Google Scholar]
  75. Teunissen, A. W., van den Berg, J. A. & Steensma, H. Y.(1995b). Localization of the dominant flocculation genes FLO5 and FLO8 of Saccharomyces cerevisiae. Yeast 11, 735–745.[CrossRef] [Google Scholar]
  76. Teunissen, A. W., van den Berg, J. A. & Steensma, H. Y.(1995c). Transcriptional regulation of flocculation genes in Saccharomyces cerevisiae. Yeast 11, 435–446.[CrossRef] [Google Scholar]
  77. Urban, C., Sohn, K., Lottspeich, F., Brunner, H. & Rupp, S.(2003). Identification of cell surface determinants in Candida albicans reveals Tsa1p, a protein differentially localized in the cell. FEBS Lett 544, 228–235.[CrossRef] [Google Scholar]
  78. 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]
  79. Vincent, J. L., Anaissie, E., Bruining, H. & 12 other authors(1998). Epidemiology, diagnosis and treatment of systemic Candida infection in surgical patients under intensive care. Intensive Care Med 24, 206–216.[CrossRef] [Google Scholar]
  80. Walther, A. & Wendland, J.(2003). An improved transformation protocol for the human fungal pathogen Candida albicans. Curr Genet 42, 339–343.[CrossRef] [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.27320-0
Loading
/content/journal/micro/10.1099/mic.0.27320-0
Loading

Data & Media loading...

Most cited Most Cited RSS feed