1887

Abstract

Immunoscreening of a cDNA library with a polyclonal germ-tube-specific antibody (pAb anti-gt) resulted in the isolation of a gene encoding a lysine/glutamic-acid-rich protein, which was consequently designated . The nucleotide and deduced amino acid sequences of this gene displayed no significant homology with any other known sequence. encodes a 134 kDa lysine (14·5 %)/glutamic acid (16·7 %) protein (Ker1p) that contains two potential transmembrane segments. was expressed in a pH-conditional manner, with maximal expression at alkaline pH and lower expression at pH 4·0, and was regulated by . A Δ null mutant grew normally but was hyperflocculant under germ-tube-inducing conditions, yet this behaviour was also observed in stationary-phase cells grown under other incubation conditions. Western blotting analysis of different subcellular fractions, using as a probe a monospecific polyclonal antibody raised against a highly antigenic domain of Ker1p (pAb anti-Ker1p), revealed the presence of a 134 kDa band in the purified plasma-membrane fraction from the wild-type strain that was absent in the homologous preparation from Δ mutant. The pattern of cell-wall protein and mannoprotein species released by digestion with -glucanases, reactive towards pAbs anti-gt and anti-Ker1p, as well as against concanavalin A, was also different in the Δ mutant. Mutant strains also displayed an increased cell-surface hydrophobicity and sensitivity to Congo red and Calcofluor white. Overall, these findings indicate that the mutant strain was affected in cell-wall composition and/or structure. The fact that the mutant had attenuated virulence in systemic mouse infections suggests that this surface protein is also important in host–fungus interactions.

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2004-08-01
2019-10-14
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References

  1. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A. & Struhl, K. ( 1992; ). Current Protocols in Molecular Biology. New York: Greene Publishing Associates and Wiley-Interscience.
  2. Braun, R. B. & Johnson, A. D. ( 2000; ). TUP1, CPH1 and EFG1 make independent contributions to filamentation in Candida albicans. Genetics 155, 57–67.
    [Google Scholar]
  3. Brown, A. J. P. & Gow, N. A. R. ( 1999; ). Regulatory networks controlling Candida albicans morphogenesis. Trends Microbiol 7, 333–338.[CrossRef]
    [Google Scholar]
  4. Buffo, J., Herman, M. A. & Soll, D. R. ( 1984; ). A characterization of pH-regulated dimorphism in Candida albicans. Mycopathology 85, 21–30.[CrossRef]
    [Google Scholar]
  5. Buurman, E. T., Westwater, C., Hube, B., Brown, A. J., Odds, F. C. & Gow, N. A. R. ( 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]
  6. Calera, J. A. & Calderone, R. A. ( 1999; ). Flocculation of hyphae is associated with a deletion in the putative CaHK1 two-component histidine kinase gene from Candida albicans. Microbiology 145, 1431–1442.[CrossRef]
    [Google Scholar]
  7. Casanova, M., Gil, M. L., Cardeñoso, L., Martínez, J. P. & Sentandreu, R. ( 1989; ). Identification of wall-specific antigens synthesized during germ tube formation by Candida albicans. Infect Immun 57, 262–271.
    [Google Scholar]
  8. Chaffin, W. L., López-Ribot, J. L., Casanova, M., Gozalbo, D. & Martínez, 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. Davis, D., Wilson, R. B. & Mitchell, A. P. ( 2000; ). RIM101-dependent and -independent pathways govern pH responses in Candida albicans. Mol Cell Biol 20, 971–978.[CrossRef]
    [Google Scholar]
  10. De Bernardis, F., Mühlschlegel, 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]
  11. De Groot, P. W. J., 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 Genom 2, 124–142.[CrossRef]
    [Google Scholar]
  12. Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A. & Smith, F. ( 1956; ). Colorimetric method for determination of sugars and related substances. Anal Chem 28, 350–356.[CrossRef]
    [Google Scholar]
  13. Eroles, P., Sentandreu, M., Elorza, M. V. & Sentandreu, R. ( 1997; ). The highly immunogenic enolase and Hsp70 are adventitious Candida albicans cell wall proteins. Microbiology 143, 313–320.[CrossRef]
    [Google Scholar]
  14. Espeso, E. A., Tilburn, J., Sánchez-Pulido, L., Brown, C. V., Valencia, A., Arst, H. N., Jr & Peñalva, M. A. ( 1997; ). Specific DNA recognition by the Aspergillus nidulans three zinc finger transcription factor PacC. J Mol Biol 274, 466–480.[CrossRef]
    [Google Scholar]
  15. Fonzi, W. A. ( 1999; ). PHR1 and PHR2 of Candida albicans encode putative glycosidases required for proper cross-linking of β-1,3- and β-1,6-glucans. J Bacteriol 181, 7070–7079.
    [Google Scholar]
  16. Fonzi, W. A. & Irwin, M. Y. ( 1993; ). Isogenic strain construction and gene mapping in Candida albicans. Genetics 134, 717–728.
    [Google Scholar]
  17. Geourjeon, C. & Deleage, G. ( 1995; ). SOPMA: significant improvements in protein secondary structure prediction by consensus prediction from multiple alignments. Comput Appl Biosci 11, 681–684.
    [Google Scholar]
  18. Gietz, R. D., Schiestl, R. H., Willems, A. R. & Woods, R. A. ( 1995; ). Studies on the transformation of intact yeast cells by the LiAc/SS-DNA/PEG procedure. Yeast 11, 355–360.[CrossRef]
    [Google Scholar]
  19. Gillum, A. M., Tsay, E. Y. & Kirsch, D. R. ( 1984; ). Isolation of Candida albicans gene for orotidine-5′-phosphate decarboxylase by complementation of S. cerevisiae ura3 and E. coli pyrF mutations. Mol Gen Genet 198, 179–182.[CrossRef]
    [Google Scholar]
  20. Gow, N. A. R. & Gooday, G. W. ( 1982; ). Vacuolation, branch production and linear growth of germ tubes of Candida albicans. J Gen Microbiol 128, 2195–2198.
    [Google Scholar]
  21. Hazen, K. C. ( 1990; ). Cell surface hydrophobicity of medically important fungi, specially Candida species. In Microbial Cell Surface Hydrophobicity, pp. 249–295. Edited by R. J. Doyle & M. Rosenberg. Washington, DC: American Society for Microbiology.
  22. Hazen, K. C. & Hazen, B. W. ( 1987; ). A polystyrene microsphere assay for detecting surface hidrophobicity variations within Candida albicans populations. J Microbiol Methods 6, 289–299.[CrossRef]
    [Google Scholar]
  23. Hewitt, C. J. & Nebe-Von-Caron, G. ( 2001; ). An industrial application of multi-parameter flow cytometry: assessment of cell physiological state and its application to the study of microbial fermentations. Cytometry 44, 179–187.[CrossRef]
    [Google Scholar]
  24. Hofmann, K. & Stoffel, W. ( 1992; ). profilegraph: an interactive graphical tool for protein sequence analysis. Comput Appl Biosci 8, 331–337.
    [Google Scholar]
  25. Hoyer, L. L., Scherer, S., Shatzman, A. R. & Livi, G. P. ( 1995; ). Candida albicans ALS1: domains related to a Saccharomyces cerevisiae sexual agglutinin separated by a repeating motif. Mol Microbiol 15, 39–54.[CrossRef]
    [Google Scholar]
  26. Hube, B., Monod, M., Schofield, D. A., Brown, A. J. P. & Gow, N. A. R. ( 1994; ). Expression of seven members of the gene family encoding secretory aspartyl proteinases in Candida albicans. Mol Microbiol 14, 87–99.[CrossRef]
    [Google Scholar]
  27. Jigami, Y. & Odani, T. ( 1999; ). Mannosylphosphate transfer to yeast mannan. Biochim Biophys Acta 1426, 335–345.[CrossRef]
    [Google Scholar]
  28. Kjer-Nielsen, L., Teasdale, R. D., Van Vliet, C. & Gleeson, P. A. ( 1999; ). A novel Golgi-localisation domain shared by a class of coiled-coil peripheral membrane proteins. Curr Biol 9, 385–388.[CrossRef]
    [Google Scholar]
  29. Kun, J. F. K., Waller, K. L. & Coppel, R. L. ( 1999; ). Plasmodium falciparum: structural and functional domains of the mature-parasite-infected erythrocyte surface antigen. Exp Parasitol 91, 258–267.[CrossRef]
    [Google Scholar]
  30. Laemmli, U. K. ( 1970; ). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.[CrossRef]
    [Google Scholar]
  31. Lee, K. L., Buckley, M. R. & Campbel, C. C. ( 1975; ). An amino acid liquid synthetic medium for development of mycelial and yeast forms of Candida albicans. Sabouraudia 13, 148–153.[CrossRef]
    [Google Scholar]
  32. López-Ribot, J. L., Casanova, M., Martínez, J. P. & Sentandreu, R. ( 1991; ). Characterization of cell wall proteins of yeast and hydrophobic mycelial cells of Candida albicans. Infect Immun 62, 742–746.
    [Google Scholar]
  33. Lupas, A., Van Dyke, M. & Stock, J. ( 1991; ). Predicting coiled coils from protein sequences. Science 252, 1162–1164.[CrossRef]
    [Google Scholar]
  34. Lussier, M., Sdicu, A. M., Shahinian, S. & Bussey, H. ( 1998; ). The Candida albicans KRE9 gene is required for cell wall β-1,6-glucan synthesis and is essential for growth on glucose. Proc Natl Acad Sci U S A 95, 9825–9830.[CrossRef]
    [Google Scholar]
  35. Maneu, V. E., Cervera, A. M., Martínez, J. P. & Gozalbo, D. ( 1996; ). Molecular cloning and characterization of a Candida albicans gene (EFB1) coding for the elongation factor EF-1β. FEMS Microbiol Lett 145, 157–162.
    [Google Scholar]
  36. Martínez, J. P., Gil, M. L., López-Ribot, J. L. & Chaffin, W. L. ( 1998; ). Serologic response to cell wall mannoproteins and proteins of Candida albicans. Clin Microbiol Rev 11, 121–141.
    [Google Scholar]
  37. Masuoka, J. & Hazen, K. C. ( 1997; ). Cell wall protein mannosylation determines Candida albicans cell surface hidrophobicity. Microbiology 143, 3015–3021.[CrossRef]
    [Google Scholar]
  38. Masuoka, J. & Hazen, K. C. ( 1999; ). Differences in the acid-labile component of Candida albicans mannan from hydrophobic and hydrophilic yeast cells. Glycobiology 9, 1281–1286.[CrossRef]
    [Google Scholar]
  39. Mekalanos, J. J. ( 1992; ). Environmental signals controlling expresión of virulence determinants in bacteria. J Bacteriol 174, 1–7.
    [Google Scholar]
  40. Monteoliva, L., Lopez-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]
  41. Mühlschlegel, F. & Fonzi, W. A. ( 1997; ). PHR2 of Candida albicans encodes a functional homolog of the pH-regulated gene PHR1 with an inverted pattern of pH-dependent expression. Mol Cell Biol 17, 5960–5967.
    [Google Scholar]
  42. Munro, C. A., Winter, K., Buchan, A., Henry, K., Becker, J. N., Brown, A. J., Bulawa, C. E. & Gow, N. A. ( 2001; ). Chs1 of Candida albicans is an essential chitin synthase required for synthesis of the septum and for cell integrity. Mol Microbiol 39, 1414–1426.
    [Google Scholar]
  43. Murad, A. M. A., Lee, P. R., Broadbent, I. D., Barelle, C. J. & Brown, A. J. P. ( 2000; ). CIp10, an efficient and convenient integrating vector for Candida albicans. Yeast 16, 325–327.[CrossRef]
    [Google Scholar]
  44. Nakajima, H., Hirata, A., Ogawa, Y., Yonehara, T., Yoda, K. & Yamasaki, M. ( 1991; ). A cytoskeleton-related gene, uso1, is required for intracellular protein transport in Saccharomyces cerevisiae. J Cell Biol 113, 245–260.[CrossRef]
    [Google Scholar]
  45. Odds, F. C. ( 1988; ). Candida and Candidosis. A Review and Bibliography, 2nd edn. London: Baillière Tindall.
  46. Peñalva, M. A. & Arst, H. N., Jr ( 2002; ). Regulation of gene exprssion by ambient pH in filamentous fungi and yeasts. Microbiol Mol Biol Rev 66, 426–446.[CrossRef]
    [Google Scholar]
  47. Porta, A., Ramón, A. M. & Fonzi, W. A. ( 1999; ). PRR1, a homolog of Aspergillus nidulans palF, controls pH-dependent gene expression and filamentation in Candida albicans. J Bacteriol 181, 7516–7523.
    [Google Scholar]
  48. Ramón, A. M., Porta, A. & Fonzi, W. A. ( 1999; ). Effect of environmental pH on morphological development of C. albicans is mediated via the PacC-related transcription factor encoded by PRR2. J Bacteriol 181, 7524–7530.
    [Google Scholar]
  49. Rose, M. D., Winston, F. & Hieter, D. ( 1990; ). Methods in Yeast Genetics: a Laboratory Course Manual. Plainview, New York: Cold Spring Laboratory Press.
  50. Saporito-Irwin, S. M., Birse, C. E., Sypherd, P. S. & Fonzi, W. A. ( 1995; ). PHR1, a pH-regulated gene of Candida albicans, is required for morphogenesis. Mol Cell Biol 15, 601–613.
    [Google Scholar]
  51. Sentandreu, M., Elorza, M. V., Sentandreu, R. & Fonzi, W. A. ( 1998; ). Cloning and characterization of PRA1, a gene encoding a novel pH-regulated antigen of Candida albicans. J Bacteriol 180, 282–289.
    [Google Scholar]
  52. Serrano, R. ( 1988; ). H+-ATPase from plasma membranes of Saccharomyces cerevisiae and Avena sativa roots: purification and reconstitution. Methods Enzymol 157, 533–544.
    [Google Scholar]
  53. Southard, S. B., Spetch, C. A., Mishra, C., Chen-Weiner, J. & Robbins, P. W. ( 1999; ). Molecular analysis of the Candida albicans homolog of Saccharomyces cerevisiae MNN9, required for glycosylation of cell wall mannoproteins. J Bacteriol 181, 7439–7448.
    [Google Scholar]
  54. Staab, J. F., Ferrer, C. A. & Sundstrom, P. ( 1996; ). Developmental expression of a tandemly repeated, proline- and glutamine-rich amino acid motif on hyphal surfaces of Candida albicans. J Biol Chem 271, 6298–6305.[CrossRef]
    [Google Scholar]
  55. Strambio-de-Castillia, C., Blobel, G. & Rout, M. P. ( 1999a; ). Proteins connecting the nuclear pore complex with the nuclear interior. J Cell Biol 144, 839–855.[CrossRef]
    [Google Scholar]
  56. Strambio-de-Castillia, C., Blobel, G. & Rout, M. P. ( 1999b; ). Slk19p is a centromere protein that functions to stabilize mitotic spindles. J Cell Biol 146, 415–425.[CrossRef]
    [Google Scholar]
  57. Tebele, N., Skilton, R. A., Katende, J., Wells, C. W., Nene, V., McElwain, T., Morzaria, S. P. & Mosoke, A. J. ( 2000; ). Cloning, characterization and expression of a 200-kilodalton diagnostic antigen of Babesia bigemina. J Clin Microbiol 38, 2240–2247.
    [Google Scholar]
  58. Tilburn, J., Sarkar, S., Widdick, D. A., Espeso, E. A., Orejas, M., Mungroof, J. M., Peñalva, A. & Arst, H. N., Jr ( 1995; ). The Aspergillus PacC zinc finger transcription factor mediates regulation of both acid and alkaline expressed genes by ambient pH. EMBO J 14, 779–790.
    [Google Scholar]
  59. Timpel, C., Strahl-Bolsinger, S., Ziegelbauer, K. & Ernst, J. F. ( 1998; ). Multiple functions of Pmt1p-mediated protein O-mannosylation in the fungal pathogen Candida albicans. J Biol Chem 273, 20837–20846.[CrossRef]
    [Google Scholar]
  60. Timpel, C., Zink, S., Strahl-Bolsinger, S., Schroppel, K. & Ernst, J. F. ( 2000; ). Morphogenesis, adhesive properties, and antifungal resistance depend on the Pmt6 protein mannosyltransferase in the fungal pathogen Candida albicans. J Bacteriol 182, 3063–3071.[CrossRef]
    [Google Scholar]
  61. Warit, S., Zhang, N., Short, A., Walmsley, R. M., Oliver, S. G. & Stateva, L. I. ( 2000; ). Glycosylation deficiency phenotypes resulting from depletion of GDP-mannose pyrophosphorylase in two yeast species. Mol Microbiol 36, 1156–1166.[CrossRef]
    [Google Scholar]
  62. Yanisch-Perron, C., Vieira, J. & Messing, J. ( 1985; ). Improved M13 phage cloning vectors and host strains: nucleotide references of the M13 mp18 and pUC19 vectors. Gene 33, 103–119.[CrossRef]
    [Google Scholar]
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