1887

Abstract

Glycosylphosphatidyl inositol (GPI)-anchored proteins in are responsible for a vast range of functions, and deletions in certain GPI-anchored proteins severely reduce adhesion and virulence of this organism. In addition, completely modified GPIs are necessary for virulence. GPI anchor biosynthesis is essential for viability and starts with the transfer of -acetylglucosamine to phosphatidylinositol. This step is catalysed by a multi-subunit complex, GPI–-acetylglucosaminyltransferase (GPI–GnT). In this, the first report to our knowledge on a subunit of the GPI–GnT complex, we show that Gpi19p is the functional equivalent of the Gpi19p. An N-terminal truncation mutant of Gpi19p functionally complements a conditionally lethal mutant. Further, we constructed a conditional null mutant of by disrupting one allele and placing the remaining copy under the control of the MET3 promoter. Repression leads to growth defects, cell wall biogenesis aberrations, azole sensitivity and hyperfilamention. In addition, there is a noticeable gene dosage effect, with the heterozygote also displaying intermediate degrees of most phenotypes. The mutants also displayed a reduced susceptibility to the antifungal agent amphotericin B. Collectively, the results suggest that is required for normal morphology and cell wall architecture.

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2010-10-01
2019-12-13
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References

  1. Arthington-Skaggs, B. A., Jradi, H., Desai, T. & Morrison, C. J. ( 1999; ). Quantitation of ergosterol content: novel method for determination of fluconazole susceptibility of Candida albicans. J Clin Microbiol 37, 3332–3337.
    [Google Scholar]
  2. Bahmed, K., Bonaly, R., Wathier, M., Pucci, B. & Coulon, J. ( 2002; ). Change of cell wall chitin content in amphotericin B resistant Kluyveromyces strains. FEMS Microbiol Lett 216, 99–103.[CrossRef]
    [Google Scholar]
  3. Bahmed, K., Bonaly, R. & Coulon, J. ( 2003; ). Relation between cell wall chitin content and susceptibility to amphotericin B in Kluyveromyces, Candida and Schizosaccharomyces species. Res Microbiol 154, 215–222.[CrossRef]
    [Google Scholar]
  4. Brodsky, R. A., Mukhina, G. L., Li, S., Nelson, K. L., Chiurazzi, P. L., Buckley, J. T. & Borowitz, M. J. ( 2000; ). Improved detection and characterization of paroxysmal nocturnal hemoglobinuria using fluorescent aerolysin. Am J Clin Pathol 114, 459–466.
    [Google Scholar]
  5. Care, R. S., Trevethick, J., Binley, K. M. & Sudbery, P. E. ( 1999; ). The MET3 promoter: a new tool for Candida albicans molecular genetics. Mol Microbiol 34, 792–798.[CrossRef]
    [Google Scholar]
  6. Choi, D. K., Suzuki, Y., Yoshimura, S., Togashi, T., Hida, M., Taylor, T. D., Wang, Y., Sugano, S., Hattori, M. & Sakaki, Y. ( 2001; ). Molecular cloning and characterization of a gene expressed in mouse developing tongue, mDSCR5 gene, a homolog of human DSCR5 (Downs Syndrome critical region gene 5). Mamm Genome 12, 347–351.[CrossRef]
    [Google Scholar]
  7. Davierwala, A. P., Haynes, J., Li, Z., Brost, R. L., Robinson, M. D., Yu, L., Mnaimneh, S., Ding, H., Zhu, H. & other authors ( 2005; ). The synthetic genetic interaction spectrum of essential genes. Nat Genet 37, 1147–1152.[CrossRef]
    [Google Scholar]
  8. Deutschbauer, A. M., Jaramillo, D. F., Proctor, M., Kumm, J., Hillenmeyer, M. E., Davis, R. W., Nislow, C. & Giaever, G. ( 2005; ). Mechanisms of Haploinsufficiency revealed by genome-wide profiling in yeast. Genetics 169, 1915–1925.[CrossRef]
    [Google Scholar]
  9. Ernst, J. F. ( 2000; ). Transcription factors in Candida albicans – environmental control of morphogenesis. Microbiology 146, 1763–1774.
    [Google Scholar]
  10. Feng, Q., Summers, E., Guo, B. & Fink, G. ( 1999; ). Ras signaling is required for serum-induced hyphal differentiation in Candida albicans. J Bacteriol 181, 6339–6346.
    [Google Scholar]
  11. Ferguson, M. A. J. ( 2000; ). Glycosylphosphatidylinositol biosynthesis validated as a drug target for African sleeping sickness. Proc Natl Acad Sci U S A 97, 10673–10675.[CrossRef]
    [Google Scholar]
  12. Ferrando-Miguel, R., Cheon, M. S. & Lubec, G. ( 2004; ). Protein levels of genes encoded on chromosome 21 in fetal Down Syndrome brain (Part V): overexpression of phosphatidyl-inositol-glycan class P protein (DSCR5). Amino Acids 26, 255–261.
    [Google Scholar]
  13. Figler, R. A., Omote, H., Nakamoto, R. K. & Al-Shawi, M. K. ( 2000; ). Use of chemical chaperones in the yeast Saccharomyces cerevisiae to enhance heterologous membrane protein expression: high-yield expression and purification of human P-glycoprotein. Arch Biochem Biophys 376, 34–46.[CrossRef]
    [Google Scholar]
  14. Gerami-Nejad, M., Hausauer, D., McClellan, M., Berman, J. & Gale, C. ( 2004; ). Cassettes for the PCR-mediated construction of regulatable alleles in Candida albicans. Yeast 21, 429–436.[CrossRef]
    [Google Scholar]
  15. Grimme, S. J., Colussi, P. A., Taron, C. H. & Orlean, P. ( 2004; ). Deficiencies in the essential Smp3 mannosyl transferase block glycosylphoshatidylinositol assembly and lead to defects in growth and cell wall biogenesis in Candida albicans. Microbiology 150, 3115–3128.[CrossRef]
    [Google Scholar]
  16. Kinoshita, T., Ohishi, K. & Takeda, J. ( 1997; ). GPI anchor synthesis in mammalian cells: genes, their products and a deficiency. J Biochem 122, 251–257.[CrossRef]
    [Google Scholar]
  17. Köhler, 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]
  18. Leidich, S. D. & Orlean, P. ( 1996; ). Gpi1, a Saccharomyces cerevisiae protein that participates in the first step in glycosylphosphatidylinositol anchor synthesis. J Biol Chem 271, 27829–27837.[CrossRef]
    [Google Scholar]
  19. Leidich, S. D., Kostova, Z., Latek, R. R., Costello, L. C., Drapp, D. A., Gray, W., Fassler, J. S. & Orlean, P. ( 1995; ). Temperature-sensitive GPI anchoring mutants gpi2 and gpi3 are defective in the synthesis of N-acetylglucosaminyl phosphatidylinositol. J Biol Chem 270, 13029–13035.[CrossRef]
    [Google Scholar]
  20. Martinez-Lopez, R., Monteoliva, L., Diez-Orejas, R., Nombela, C. & Gil, C. ( 2004; ). The GPI-anchored protein, CaEcm3p, is required for cell wall integrity, morphogenesis and virulence in Candida albicans. Microbiology 150, 3341–3354.[CrossRef]
    [Google Scholar]
  21. Martinez-Lopez, R., Park, H., Myers, C. L., Gil, C. & Filler, S. G. ( 2006; ). Candida albicans Ecm33p is important for normal cell wall architecture and interactions with host cells. Eukaryot Cell 5, 140–147.[CrossRef]
    [Google Scholar]
  22. Miller, J. P., Lo, R. S., Ben-Hur, A., Desmarais, C., Stagljar, I., Noble, W. S. & Fields, S. ( 2005; ). Large scale identification of yeast integral membrane protein interactions. Proc Natl Acad Sci U S A 102, 12123–12128.[CrossRef]
    [Google Scholar]
  23. Murakami, Y., Siripanyaphinyo, U., Hong, Y., Tashima, Y., Maeda, Y. & Kinoshita, T. ( 2005; ). The initial enzyme for glycosylphosphatidylinositol biosynthesis requires PIG-Y, a seventh component. Mol Biol Cell 16, 5236–5246.[CrossRef]
    [Google Scholar]
  24. Nagamune, K., Nozaki, T., Maeda, Y., Ohishi, K., Fukuma, T., Hara, T., Schwarz, R. T., Sutterlin, C., Brun, R. & other authors ( 2000; ). Critical roles of glycosylphosphatidylinositol for Trypanosoma brucei. Proc Natl Acad Sci U S A 97, 10336–10341.[CrossRef]
    [Google Scholar]
  25. Newman, H. A., Romeo, M. J., Lewis, S. E., Yan, B. C., Orlean, P. & Levin, D. E. ( 2005; ). Gpi19, the Saccharomyces cerevisiae homolog of mammalian PIG-P, is a subunit of the initial enzyme for glycosylphosphatidylinositol anchor biosynthesis. Eukaryot Cell 4, 1801–1807.[CrossRef]
    [Google Scholar]
  26. Oswal, N., Sahni, N. S., Bhattacharya, A., Komath, S. S. & Muthuswami, R. ( 2008; ). Unique motifs identify PIG-A proteins from glycosyltransferases of the GT4 family. BMC Evol Biol 8, 168.[CrossRef]
    [Google Scholar]
  27. Pasrija, R., Krishnamurthy, S., Prasad, T., Ernst, J. F. & Prasad, R. ( 2005; ). Squalene epoxidase encoded by ERG1 affects morphogenesis and drug susceptibilities of Candida albicans. J Antimicrob Chemother 55, 905–913.[CrossRef]
    [Google Scholar]
  28. Piłsyk, S. & Paszewski, A. ( 2009; ). The Aspergillus nidulans pigP gene encodes a subunit of GPI–N-acetylglucosaminyltransferase which influences filamentation and protein secretion. Curr Genet 55, 301–309.[CrossRef]
    [Google Scholar]
  29. Plaine, A., Walker, L., Da Costa, G., Mora-Montes, H. M., McKinnon, A., Gow, N. A. R., Gaillardin, C., Munro, C. & Richard, M. L. ( 2008; ). Functional analysis of Candida albicans GPI-anchored proteins: roles in cell wall integrity and caspofungin sensitivity. Fungal Genet Biol 45, 1404–1414.[CrossRef]
    [Google Scholar]
  30. Ram, A. F., Wolters, A., Ten Hoopen, R. & Klis, F. M. ( 1994; ). A new approach for isolating cell wall mutants in Saccharomyces cerevisiae by screening for hypersensitivity to calcofluor white. Yeast 10, 1019–1030.[CrossRef]
    [Google Scholar]
  31. Richard, M. L. & Plaine, A. ( 2007; ). A comprehensive analysis of GPI-anchored proteins in Candida albicans. Eukaryot Cell 6, 119–133.[CrossRef]
    [Google Scholar]
  32. Richard, M., Ibata-Ombetta, S., Dromer, F., Bordon-Pallier, F., Jouault, T. & Gaillardin, C. ( 2002; ). Complete glycosylphosphatidylinositol anchors are required in Candida albicans for full morphogenesis, virulence and resistance to macrophages. Mol Microbiol 44, 841–853.[CrossRef]
    [Google Scholar]
  33. Sanglard, D., Ischer, F., Parkinson, T., Falconer, D. & Bille, J. ( 2003; ). Candida albicans mutations in the ergosterol biosynthetic pathway and resistance to several antifungal agents. Antimicrob Agents Chemother 47, 2404–2412.[CrossRef]
    [Google Scholar]
  34. Seo, K., Akiyoshi, H. & Ohnishi, Y. ( 1999; ). Alteration of cell wall composition leads to amphotericin B resistance in Aspergillus flavus. Microbiol Immunol 43, 1017–1025.[CrossRef]
    [Google Scholar]
  35. Shao, M., Liu, Z., Wang, C., Li, H., Carron, C., Zhang, H. & Shi, D. ( 2009; ). Down syndrome critical region protein 5 regulates membrane localization of Wnt receptors, dishevelled stability and convergent extension in vertebrate embryos. Development 136, 2121–2131.[CrossRef]
    [Google Scholar]
  36. Sobering, A. K., Romeo, M. J., Vay, H. A. & Levin, D. E. ( 2003; ). A novel Ras inhibitor, Eri1, engages yeast Ras at the endoplasmic reticulum. Mol Cell Biol 23, 4983–4990.[CrossRef]
    [Google Scholar]
  37. Sobering, A. K., Watanabe, R., Romeo, M. J., Yan, B. C., Specht, C. A., Orlean, P., Riezman, H. & Levin, D. E. ( 2004; ). Yeast Ras regulates the complex that catalyzes the first step in GPI-anchor biosynthesis at the ER. Cell 117, 637–648.[CrossRef]
    [Google Scholar]
  38. Vats, D., Vishwakarma, R. A., Bhattacharya, S. & Bhattacharya, A. ( 2005; ). Reduction of cell surface glycosylphosphatidylinositol conjugates in Entamoeba histolytica by antisense blocking of E. histolytica GlcNAc phosphatidylinositol deacetylase expression: effect on cell proliferation, endocytosis and adhesion to cell targets. Infect Immun 73, 8381–8392.[CrossRef]
    [Google Scholar]
  39. Vossen, J. H., Ram, A. F. & Klis, F. M. ( 1995; ). Identification of SPT14/CHW6 as the yeast homologue of hPigA, a gene involved in the biosynthesis of GPI anchors. Biochim Biophys Acta 1243, 549–551.[CrossRef]
    [Google Scholar]
  40. Watanabe, R., Kinoshita, T., Masaki, R., Yamamoto, A., Takeda, J. & Inoue, N. ( 1996; ). PIG-A and PIG-H which participate in glycosylphosphatidylinositol anchor biosynthesis form a complex in the endoplasmic reticulum. J Biol Chem 271, 26868–26875.[CrossRef]
    [Google Scholar]
  41. Watanabe, R., Inoue, N., Westfall, B., Taron, C. H., Orlean, P., Takeda, J. & Kinoshita, T. ( 1998; ). The first step of glycosylphosphatidylinositol biosynthesis is mediated by a complex of PIG-A, PIG-H, PIG-C and GPI1. EMBO J 17, 877–885.[CrossRef]
    [Google Scholar]
  42. Watanabe, R., Murakami, Y., Marmor, M. D., Inoue, N., Maeda, Y., Hino, J., Kangawa, K., Julius, M. & Kinoshita, T. ( 2000; ). Initial enzyme for glycosylphosphatidylinositol biosynthesis requires GPI19 and is regulated by DPM2. EMBO J 19, 4402–4411.[CrossRef]
    [Google Scholar]
  43. Wilson, R. B., Davis, D. & Mitchell, A. P. ( 1999; ). Rapid hypothesis testing with Candida albicans through gene disruption with short homology regions. J Bacteriol 181, 1868–1874.
    [Google Scholar]
  44. Yan, B. C., Westfall, B. A. & Orlean, P. ( 2001; ). Ynl038wp (Gpi15) is the Saccharomyces cerevisiae homologue of human pigHp and participates in the first step in glycosylphosphatidylinositol assembly. Yeast 18, 1383–1389.[CrossRef]
    [Google Scholar]
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