1887

Abstract

We previously identified Irs4p as an epidermal growth factor substrate 15 homology (EH) domain-containing protein that is reactive with antibodies in the sera of patients with candidiasis and contributes to cell wall integrity, hyphal formation and virulence. In this study, we use a yeast two-hybrid method and co-immunoprecipitation to show that Irs4p physically interacts with the phosphatase Inp51p. Disruption of the Inp51p Asn-Pro-Phe (NPF) motif eliminates the interaction, suggesting that this motif is targeted by Irs4p. Both and null mutants exhibit significantly increased levels of phosphatidylinositol-4,5-bisphosphate [PI(4,5)P] without changes in levels of other phosphoinositides. Like the mutant, the mutant demonstrates increased susceptibility to cell wall-active agents, impaired hyphal formation and abnormal chitin distribution along hyphal walls during growth within solid agar. Moreover, the and mutants overactivate the cell wall integrity pathway as measured by Mkc1p phosphorylation. As anticipated, mortality due to disseminated candidiasis is significantly attenuated among mice infected with the mutant, and tissue burdens and inflammation within the kidneys are reduced. Hyphal formation and chitin distribution are also impaired, consistent with observations of embedded growth . All phenotypes exhibited by the and mutants are rescued by complementation with the respective genes. In conclusion, our findings suggest that Irs4p binds and activates Inp51p to negatively regulate PI(4,5)P levels and the cell integrity pathway, and that PI(4,5)P homeostasis is important for coordinating cell wall integrity, hyphal growth and virulence under conditions of cell wall stress.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.2008/018002-0
2008-11-01
2019-10-23
Loading full text...

Full text loading...

/deliver/fulltext/micro/154/11/3296.html?itemId=/content/journal/micro/10.1099/mic.0.2008/018002-0&mimeType=html&fmt=ahah

References

  1. Badrane, H., Cheng, S., Nguyen, M. H., Jia, H. Y., Zhang, Z., Weisner, N. & Clancy, C. J. ( 2005; ). Candida albicans IRS4 contributes to hyphal formation and virulence after the initial stages of disseminated candidiasis. Microbiology 151, 2923–2931.[CrossRef]
    [Google Scholar]
  2. Barelle, C. J., Priest, C. L., MacCallum, D. M., Gow, N. A. R., Odds, F. C. & Brown, A. J. P. ( 2006; ). Niche-specific regulation of central metabolic pathways in a fungal pathogen. Cell Microbiol 8, 961–971.[CrossRef]
    [Google Scholar]
  3. Bickle, M., Delley, P. A., Schmidt, A. & Hall, M. N. ( 1998; ). Cell wall integrity modulates RHO1 activity via the exchange factor ROM2. EMBO J 17, 2235–2245.[CrossRef]
    [Google Scholar]
  4. Bonangelino, C. J., Nau, J. J., Duex, J. E., Brinkman, M., Wurmser, A. E., Gary, J. D., Emr, S. D. & Weisman, L. S. ( 2002; ). Osmotic stress-induced increase of phosphatidylinositol 3,5-bisphosphate requires Vac14p, an activator of the lipid kinase Fab1p. J Cell Biol 156, 1015–1028.[CrossRef]
    [Google Scholar]
  5. Böttcher, C., Wicky, S., Schwarz, H. & Singer-Kruger, B. ( 2006; ). Sjl2p is specifically involved in early steps of endocytosis intimately linked to actin dynamics via the Ark1p/Prk1p kinases. FEBS Lett 580, 633–641.[CrossRef]
    [Google Scholar]
  6. Brand, A., Shanks, S., Duncan, V. M. S., Yang, M., Mackenzie, K. & Gow, N. A. R. ( 2007; ). Hyphal orientation of Candida albicans is regulated by a calcium-dependent mechanism. Curr Biol 17, 347–352.
    [Google Scholar]
  7. Calderone, R. A. & Fonzi, W. A. ( 2001; ). Virulence factors of Candida albicans. Trends Microbiol 9, 327–335.[CrossRef]
    [Google Scholar]
  8. Cheng, S., Clancy, C. J., Zhang, Z., Hao, B., Wang, W., Iczkowski, K. A., Pfaller, M. A. & Nguyen, M. H. ( 2007; ). Uncoupling of oxidative phosphorylation enables Candida albicans to resist killing by phagocytes and persist in tissue. Cell Microbiol 9, 492–501.[CrossRef]
    [Google Scholar]
  9. Churukian, C. J. & Schenk, E. A. ( 1977; ). Rapid Grocott's methenamine-silver nitrate method for fungi and Pneumocystis carinii. Am J Clin Pathol 68, 427–428.
    [Google Scholar]
  10. Churukian, C. J., Schenk, E. A. & Clark, G. ( 1986; ). Dilute ammoniacal silver as a substitute for methenamine silver to demonstrate Pneumocystis carinii and fungi. Lab Med 17, 87–90.
    [Google Scholar]
  11. Confalonieri, S. & Di Fiore, P. P. ( 2002; ). The Eps15 homology (EH) domain. FEBS Lett 513, 24–29.[CrossRef]
    [Google Scholar]
  12. Daum, G., Lees, N. D., Bard, M. & Dickson, R. ( 1998; ). Biochemistry, cell biology and molecular biology of lipids of Saccharomyces cerevisiae. Yeast 14, 1471–1510.[CrossRef]
    [Google Scholar]
  13. de Beer, T., Carter, R. E., Lobel-Rice, K. E., Sorkin, A. & Overduin, M. ( 1998; ). Structure and Asn-Pro-Phe binding pocket of the Eps15 homology domain. Science 281, 1357–1360.[CrossRef]
    [Google Scholar]
  14. Delley, P. A. & Hall, M. N. ( 1999; ). Cell wall stress depolarizes cell growth via hyperactivation of RHO1. J Cell Biol 147, 163–174.[CrossRef]
    [Google Scholar]
  15. Dove, S. K., Cooke, F. T., Douglas, M. R., Sayers, L. G., Parker, P. J. & Michell, R. H. ( 1997; ). Osmotic stress activates phosphatidylinositol-3,5-bisphosphate synthesis. Nature 390, 187–192.[CrossRef]
    [Google Scholar]
  16. Ernst, J. F. ( 2000; ). Transcription factors in Candida albicans – environmental control of morphogenesis. Microbiology 146, 1763–1774.
    [Google Scholar]
  17. Fonzi, W. A. & Irwin, M. Y. ( 1993; ). Isogenic strain construction and gene mapping in Candida albicans. Genetics 134, 717–728.
    [Google Scholar]
  18. Fradin, C., Kretschmar, M., Nichterlein, T., Gaillardin, C., d'Enfert, C. & Hube, B. ( 2003; ). Stage-specific gene expression of Candida albicans in human blood. Mol Microbiol 47, 1523–1543.[CrossRef]
    [Google Scholar]
  19. 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. coli pyrF mutations. Mol Gen Genet 198, 179–182.[CrossRef]
    [Google Scholar]
  20. Hausauer, D. L., Gerami-Nejad, M., Kistler-Anderson, C. & Gale, C. A. ( 2005; ). Hyphal guidance and invasive growth in Candida albicans require the Ras-like GTPase Rsr1p and its GTPase-activating protein Bud2p. Eukaryot Cell 4, 1273–1286.[CrossRef]
    [Google Scholar]
  21. 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]
  22. Kumamoto, C. A. ( 2005; ). A contact-activated kinase signals Candida albicans invasive growth and biofilm development. Proc Natl Acad Sci U S A 102, 5576–5581.[CrossRef]
    [Google Scholar]
  23. Kumamoto, C. A. & Vinces, M. D. ( 2005; ). Contributions of hyphae and hypha-co-regulated genes to Candida albicans virulence. Cell Microbiol 7, 1546–1554.[CrossRef]
    [Google Scholar]
  24. Kuranda, K., Leberre, V., Sokol, S., Palamarczyk, G. & Francois, J. ( 2006; ). Investigating the caffeine effects in the yeast Saccharomyces cerevisiae brings new insights into the connection between TOR, PKC and Ras/cAMP signalling pathways. Mol Microbiol 61, 1147–1166.[CrossRef]
    [Google Scholar]
  25. Lemmon, M. A. ( 2003; ). Phosphoinositide recognition domains. Traffic 4, 201–213.[CrossRef]
    [Google Scholar]
  26. Li, C. R., Wang, Y. M., De Zheng, X., Liang, H. Y., Tang, J. C. W. & Wang, Y. ( 2005; ). The formin family protein CaBni1p has a role in cell polarity control during both yeast and hyphal growth in Candida albicans. J Cell Sci 118, 2637–2648.[CrossRef]
    [Google Scholar]
  27. Lorenz, M. C., Bender, J. A. & Fink, G. R. ( 2004; ). Transcriptional response of Candida albicans upon internalization by macrophages. Eukaryot Cell 3, 1076–1087.[CrossRef]
    [Google Scholar]
  28. Mahan, M. J., Heithoff, D. M., Sinsheimer, R. L. & Low, D. A. ( 2000; ). Assessment of bacterial pathogenesis by analysis of gene expression in the host. Annu Rev Genet 34, 139–164.[CrossRef]
    [Google Scholar]
  29. Martin, T. F. ( 1998; ). Phosphoinositide lipids as signaling molecules: common themes for signal transduction, cytoskeletal regulation, and membrane trafficking. Annu Rev Cell Dev Biol 14, 231–264.[CrossRef]
    [Google Scholar]
  30. Martin, S. W. & Konopka, J. B. ( 2004; ). Lipid raft polarization contributes to hyphal growth in Candida albicans. Eukaryot Cell 3, 675–684.[CrossRef]
    [Google Scholar]
  31. Morales-Johansson, H., Jenoe, P., Cooke, F. T. & Hall, M. N. ( 2004; ). Negative regulation of phosphatidylinositol 4,5-bisphosphate levels by the INP51-associated proteins TAX4 and IRS4. J Biol Chem 279, 39604–39610.[CrossRef]
    [Google Scholar]
  32. Navarro-Garcia, F., Alonso-Monge, R., Rico, H., Pla, J., Sentandreu, R. & Nombela, C. ( 1998; ). A role for the MAP kinase gene MKC1 in cell wall construction and morphological transitions in Candida albicans. Microbiology 144, 411–424.[CrossRef]
    [Google Scholar]
  33. Nguyen, M. H., Cheng, S. & Clancy, C. J. ( 2004; ). Assessment of Candida albicans genes expressed during infections as a tool to understand pathogenesis. Med Mycol 42, 293–304.[CrossRef]
    [Google Scholar]
  34. Oberholzer, U., Nantel, A., Berman, J. & Whiteway, M. ( 2006; ). Transcript profiles of Candida albicans cortical actin patch mutants reflect their cellular defects: contribution of the Hog1p and Mkc1p signaling pathways. Eukaryot Cell 5, 1252–1265.[CrossRef]
    [Google Scholar]
  35. Ozaki, K., Tanaka, K., Imamura, H., Hihara, T., Kameyama, T., Nonaka, H., Hirano, H., Matsuura, Y. & Takai, Y. ( 1996; ). Rom1p and Rom2p are GDP/GTP exchange proteins (GEPs) for the Rho1p small GTP binding protein in Saccharomyces cerevisiae. EMBO J 15, 2196–2207.
    [Google Scholar]
  36. Park, J. I., Collinson, E. J., Grant, C. M. & Dawes, I. W. ( 2005; ). Rom2p, the Rho1 GTP/GDP exchange factor of Saccharomyces cerevisiae, can mediate stress responses via the Ras-cAMP pathway. J Biol Chem 280, 2529–2535.[CrossRef]
    [Google Scholar]
  37. Reuss, O., Vik, A., Kolter, R. & Morschhauser, J. ( 2004; ). The SAT1 flipper, an optimized tool for gene disruption in Candida albicans. Gene 341, 119–127.[CrossRef]
    [Google Scholar]
  38. Rubin-Bejerano, I., Fraser, I., Grisafi, P. & Fink, G. R. ( 2003; ). Phagocytosis by neutrophils induces an amino acid deprivation response in Saccharomyces cerevisiae and Candida albicans. Proc Natl Acad Sci U S A 100, 11007–11012.[CrossRef]
    [Google Scholar]
  39. Salcini, A. E., Confalonieri, S., Doria, M., Santolini, E., Tassi, E., Minenkova, O., Cesareni, G., Pelicci, P. G. & di Fiore, P. P. ( 1997; ). Binding specificity and in vivo targets of the EH domain, a novel protein–protein interaction module. Genes Dev 11, 2239–2249.[CrossRef]
    [Google Scholar]
  40. Santolini, E., Salcini, A. E., Kay, B. K., Yamabhai, M. & di Fiore, P. P. ( 1999; ). The EH network. Exp Cell Res 253, 186–209.[CrossRef]
    [Google Scholar]
  41. Singer-Kruger, B., Nemoto, Y., Daniell, L., Ferro-Novick, S. & De Camilli, P. ( 1998; ). Synaptojanin family members are implicated in endocytic membrane traffic in yeast. J Cell Sci 111, 3347–3356.
    [Google Scholar]
  42. Sprong, H., van der Sluijs, P. & van Meer, G. ( 2001; ). How proteins move lipids and lipids move proteins. Nat Rev Mol Cell Biol 2, 504–513.[CrossRef]
    [Google Scholar]
  43. Staib, P., Kretschmar, M., Nichterlein, T., Kohler, G. & Morschhauser, J. ( 2000; ). Expression of virulence genes in Candida albicans. Adv Exp Med Biol 485, 167–176.
    [Google Scholar]
  44. Stefan, C. J., Audhya, A. & Emr, S. D. ( 2002; ). The yeast synaptojanin-like proteins control the cellular distribution of phosphatidylinositol (4,5)-bisphosphate. Mol Biol Cell 13, 542–557.[CrossRef]
    [Google Scholar]
  45. Stefan, C. J., Padilla, S. M., Audhya, A. & Emr, S. D. ( 2005; ). The phosphoinositide phosphatase Sjl2 is recruited to cortical actin patches in the control of vesicle formation and fission during endocytosis. Mol Cell Biol 25, 2910–2923.[CrossRef]
    [Google Scholar]
  46. Stolz, L. E., Huynh, C. V., Thorner, J. & York, J. D. ( 1998a; ). Identification and characterization of an essential family of inositol polyphosphate 5-phosphatases (INP51, INP52 and INP53 gene products) in the yeast Saccharomyces cerevisiae. Genetics 148, 1715–1729.
    [Google Scholar]
  47. Stolz, L. E., Kuo, W. J., Longchamps, J., Sekhon, M. K. & York, J. D. ( 1998b; ). INP51, a yeast inositol polyphosphate 5-phosphatase required for phosphatidylinositol 4,5-bisphosphate homeostasis and whose absence confers a cold-resistant phenotype. J Biol Chem 273, 11852–11861.[CrossRef]
    [Google Scholar]
  48. Tang, H. Y., Xu, J. & Cai, M. ( 2000; ). Pan1p, End3p, and S1a1p, three yeast proteins required for normal cortical actin cytoskeleton organization, associate with each other and play essential roles in cell wall morphogenesis. Mol Cell Biol 20, 12–25.[CrossRef]
    [Google Scholar]
  49. Verna, J., Lodder, A., Lee, K., Vagts, A. & Ballester, R. ( 1997; ). A family of genes required for maintenance of cell wall integrity and for the stress response in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A 94, 13804–13809.[CrossRef]
    [Google Scholar]
  50. Walther, A. & Wendland, J. ( 2004; ). Polarized hyphal growth in Candida albicans requires the Wiskott-Aldrich syndrome protein homolog Wal1p. Eukaryot Cell 3, 471–482.[CrossRef]
    [Google Scholar]
  51. Zhao, R., Lockhart, S. R., Daniels, K. & Soll, D. R. ( 2002; ). Roles of TUP1 in switching, phase maintenance, and phase-specific gene expression in Candida albicans. Eukaryot Cell 1, 353–365.[CrossRef]
    [Google Scholar]
  52. Zheng, X. D., Wang, Y. M. & Wang, Y. ( 2003; ). CaSPA2 is important for polarity establishment and maintenance in Candida albicans. Mol Microbiol 49, 1391–1405.[CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.2008/018002-0
Loading
/content/journal/micro/10.1099/mic.0.2008/018002-0
Loading

Data & Media loading...

Supplements

[PDF file](29 KB)

PDF

[PDF file](676 KB)

PDF
This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error