1887

Abstract

Many small G proteins require post-translational modification to allow functional association to the cell membrane. This process often involves the enzymic addition of hydrophobic prenyl groups to a conserved cysteine residue near the C-terminus of the protein. The enzymes that catalyse these reactions include protein farnesyltransferase and protein geranylgeranyltransferases. The human fungal pathogen requires functional Ras and Rho proteins in order to undergo normal growth and differentiation. Since farnesylation and geranylgeranylation are likely required for the proper function of these small G proteins, we hypothesized that inhibition of these prenylation events would alter the growth and cellular morphogenesis of this fungus. We cloned the gene encoding the single protein-farnesyltransferase -chain homologue in . Using a gene-disruption strategy in a diploid strain, we demonstrated that this gene encodes an essential function, in contrast to the case in , in which the homologous gene is not essential for growth. Pharmacological inhibition of farnesyltransferase activity resulted in dose-dependent cytostasis of , as well as prevention of hyphal differentiation. Simultaneous inhibition of farnesylation and calcineurin signalling results in a synthetic effect on growth. Protein farnesylation is required for the growth and cellular differentiation of and may provide novel targets for antifungal therapy.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.27030-0
2004-06-01
2019-09-15
Loading full text...

Full text loading...

/deliver/fulltext/micro/150/6/mic1501925.html?itemId=/content/journal/micro/10.1099/mic.0.27030-0&mimeType=html&fmt=ahah

References

  1. Alspaugh, J. A., Perfect, J. R. & Heitman, J. ( 1997; ). Cryptococcus neoformans mating and virulence are regulated by the G-protein gamma subunit GPA1 and cAMP. Genes Dev 11, 3206–3217.[CrossRef]
    [Google Scholar]
  2. Alspaugh, J. A., Cavallo, L. M., Perfect, J. R. & Heitman, J. ( 2000; ). RAS1 regulates filamentation, mating and growth at high temperature of Cryptococcus neoformans. Mol Microbiol 36, 352–365.[CrossRef]
    [Google Scholar]
  3. Casey, P. J. & Seabra, M. C. ( 1996; ). Protein prenyltransferases. J Biol Chem 271, 5289–5292.[CrossRef]
    [Google Scholar]
  4. Casey, P. J., Solski, P. A., Der, C. J. & Buss, J. E. ( 1989; ). p21ras is modified by a farnesyl isoprenoid. Proc Natl Acad Sci U S A 86, 8323–8327.[CrossRef]
    [Google Scholar]
  5. Chang, Y. C. & Penoyer, L. A. ( 2000; ). Properties of various Rho1 mutant alleles of Cryptococcus neoformans. J Bacteriol 182, 4987–4991.[CrossRef]
    [Google Scholar]
  6. Cox, G. M., Toffaletti, D. L. & Perfect, J. R. ( 1996; ). Dominant selection system for use in Cryptococcus neoformans. J Med Vet Mycol 34, 385–391.[CrossRef]
    [Google Scholar]
  7. Davidson, R. C., Cruz, M. C., Sia, R. A., Allen, B., Alspaugh, J. A. & Heitman, J. ( 2000a; ). Gene disruption by biolistic transformation in serotype D strains of Cryptocococus neoformans. Fungal Genet Biol 29, 38–48.[CrossRef]
    [Google Scholar]
  8. Davidson, R. C., Moore, T. D., Odom, A. R. & Heitman, J. ( 2000b; ). Characterization of the MFalpha pheromone of the human fungal pathogen Cryptocococus neoformans. Mol Microbiol 38, 1017–1026.
    [Google Scholar]
  9. Davidson, R. C., Blankenship, J. R., Kraus, P. R., de Jesus, B. M., Hull, C. M., D'Souza, C., Wang, P. &, Heitman. J. ( 2002; ). A PCR-based strategy to generate integrative targeting alleles with large regions of homology. Microbiology 148, 2607–2615.
    [Google Scholar]
  10. Davidson, R. C., Nichols, C. B., Cox, G. M., Perfect, J. R. & Heitman, J. ( 2003; ). A MAP kinase cascade composed of cell type specific and non-specific elements controls mating and differentiation of the fungal pathogen Cryptococcus neoformans. Mol Microbiol 49, 469–485.[CrossRef]
    [Google Scholar]
  11. 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]
  12. Galgiani, J. N., Bartlett, M. S., Ghannoum, M. A., Espinel-Ingroff, A., Lancaster, M. V., Odds, F. C., Pfaller, M. A., Rinaldi, M. G. & Walsh, T. J. ( 1997; ). Reference method for broth dilution antifungal susceptibility testing of yeasts; approved standard. National Committee for Clinical Laboratory Standards 17 No. 9, 1–29.
  13. Hancock, J. F., Magee, A. I., Childs, J. E. & Marshall, C. J. ( 1989; ). All ras proteins are polyisoprenylated but only some are palmitoylated. Cell 57, 1167–1177.[CrossRef]
    [Google Scholar]
  14. He, B., Chen, P., Chen, S. Y., Vancura, K. L., Michaelis, S. & Powers, S. ( 1991; ). RAM2, an essential gene of yeast, and RAM1 encode the two polypeptide components of the farnesyltransferase that prenylates a-factor and Ras proteins. Proc Natl Acad Sci U S A 88, 11373–11377.[CrossRef]
    [Google Scholar]
  15. Hill, B. T., Perrin, D. & Kruczynski, A. ( 2000; ). Inhibition of RAS-targeted prenylation: protein farnesyl transferase inhibitors revisited. Crit Rev Oncol/Hematol 33, 7–23.[CrossRef]
    [Google Scholar]
  16. Jiang, Y., Rossi, G. & Ferro-Novick, S. ( 1993; ). Bet2p and Mad2p are components of a prenyltransferase that adds geranylgeranyl onto Ypt1p and Sec4p. Nature 366, 84–86.[CrossRef]
    [Google Scholar]
  17. Kelly, R., Card, D., Register, E. & 8 other authors ( 2000; ). Geranylgeranyltransferase I of Candida albicans: null mutants or enzyme inhibitors produce unexpected phenotypes. J Bacteriol 182, 704–713.[CrossRef]
    [Google Scholar]
  18. Kinsella, B. T., Erdman, R. A. & Maltese, W. A. ( 1991; ). Posttranslational modification of Ha-ras p21 by farnesyl versus geranylgeranyl isoprenoids is determined by the COOH-terminal amino acid. Proc Natl Acad Sci U S A 88, 8934–8938.[CrossRef]
    [Google Scholar]
  19. Kohl, N. E., Omer, C. A., Conner, M. W. & 7 other authors ( 1995; ). Inhibition of farnesyltransferase induces regression of mammary and salivary carcinomas in ras transgenic mice. Nat Med 1, 792–797.[CrossRef]
    [Google Scholar]
  20. 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]
  21. Kwon-Chung, K. J. ( 1975; ). Filobasidiella the perfect state of Cryptococcus neoformans. Mycologia 67, 1197–1200.[CrossRef]
    [Google Scholar]
  22. Kwon-Chung, K. J., Edman, J. C. & Wickes, B. L. ( 1992; ). Genetic association of mating types and virulence in Cryptococcus neoformans. Infect Immun 60, 602–605.
    [Google Scholar]
  23. Lerner, E. C., Qian, Y., Hamilton, A. D. & Sebti, S. M. ( 1995; ). Disruption of oncogenic K-Ras4B processing and signaling by a potent geranylgeranyltransferase I inhibitor. J Biol Chem 270, 26770–26773.[CrossRef]
    [Google Scholar]
  24. Mayer, M. L., Caplin, B. E. & Marshall, M. S. ( 1992; ). CDC43 and RAM2 encode the polypeptide subunits of a yeast type I protein geranylgeranyltransferase. J Biol Chem 267, 20589–20593.
    [Google Scholar]
  25. McGeady, P., Logan, D. A. & Wansley, D. L. ( 2002; ). A protein-farnesyl transferase inhibitor interferes with the serum-induced conversion of Candida albicans from a cellular yeast form to a filamentous form. FEMS Microbiol Lett 213, 41–44.[CrossRef]
    [Google Scholar]
  26. Moore, T. D. & Edman, J. C. ( 1993; ). The alpha-mating type locus of Cryptococcus neoformans contains a peptide pheromone gene. Mol Cell Biol 13, 1962–1970.
    [Google Scholar]
  27. Odom, A., Muir, S., Lim, E., Toffaletti, D. L., Perfect, J. & Heitman, J. ( 1997; ). Calcineurin is required for virulence of Cryptococcus neoformans. EMBO J 16, 2576–2589.[CrossRef]
    [Google Scholar]
  28. Ohya, Y., Qadota, H., Anraku, Y., Pringle, J. R. & Botstein, D. ( 1993; ). Suppression of yeast geranylgeranyl transferase I defect by alternative prenylation of two target GTPases, Rho1p and Cdc42p. Mol Biol Cell 4, 1017–1025.[CrossRef]
    [Google Scholar]
  29. Patel, D. V., Schmidt, R. J., Biller, S. A., Gordon, E. M., Robinson, S. S. & Manne, V. ( 1995; ). Farnesyl diphosphate-based inhibitors of Ras farnesyl protein transferase. J Med Chem 38, 2906–2921.[CrossRef]
    [Google Scholar]
  30. Perfect, J. R., Lang, S. D. R. & Durack, D. T. ( 1980; ). Chronic cryptococcal meningitis: a new experimental model in rabbits. Am J Pathol 101, 177–194.
    [Google Scholar]
  31. Pitkin, J. W., Panaccione, D. G. & Walton, J. D. ( 1996; ). A putative cyclic peptide efflux pump encoded by the TOXA gene of the plant-pathogenic fungus Cochliobolus carbonum. Microbiology 142, 1557–1565.[CrossRef]
    [Google Scholar]
  32. Prendergast, G. C. ( 2000; ). Farnesyltransferase inhibitors: antineoplastic mechanism and clinical prospects. Curr Opin Cell Biol 12, 166–173.[CrossRef]
    [Google Scholar]
  33. Reiss, Y., Goldstein, J. L., Seabra, M. C., Casey, P. J. & Brown, M. S. ( 1990; ). Inhibition of purified p21ras farnesyl:protein transferase by Cys-AAX tetrapeptides. Cell 62, 81–88.[CrossRef]
    [Google Scholar]
  34. Rocha, C. R., Schroppel, K., Harcus, D., Marcil, A., Dignard, D., Taylor, B. N., Thomas, D. Y., Whiteway, M. & Leberer, E. ( 2001; ). Signaling through adenylyl cyclase is essential for hyphal growth and virulence in the pathogenic fungus Candida albicans. Mol Biol Cell 12, 3631–3643.[CrossRef]
    [Google Scholar]
  35. Sambrook, J. & Russel, D. W. ( 2001; ). Molecular Cloning: a Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  36. Schafer, W. R., Kim, R., Sterne, R., Thorner, J., Kim, S. H. & Rine, J. ( 1989; ). Genetic and pharmacological suppression of oncogenic mutations in ras genes of yeast and humans. Science 245, 379–385.[CrossRef]
    [Google Scholar]
  37. Seabra, M. C., Reiss, Y., Casey, P. J., Brown, M. S. & Goldstein, J. L. ( 1991; ). Protein farnesyltransferase and geranylgeranyltransferase share a common alpha subunit. Cell 65, 429–434.[CrossRef]
    [Google Scholar]
  38. Sherman, F. ( 1991; ). Getting started with yeast. Methods Enzymol 194, 3–21.
    [Google Scholar]
  39. Sia, R. A., Lengeler, K. B. & Heitman, J. ( 2000; ). Diploid strains of the pathogenic basidiomycete Cryptococcus neoformans are thermally dimorphic. Fungal Genet Biol 29, 153–163.[CrossRef]
    [Google Scholar]
  40. Song, J. L. & White, T. C. ( 2003; ). RAM2: an essential gene in the prenylation pathway of Candida albicans. Microbiology 149, 249–259.[CrossRef]
    [Google Scholar]
  41. Wang, P., Perfect, J. R. & Heitman, J. ( 2000; ). The G-protein beta subunit GPB1 is required for mating and haploid fruiting in Cryptococcus neoformans. Mol Cell Biol 20, 352–362.[CrossRef]
    [Google Scholar]
  42. Waugh, M. S., Vallim, M. A., Heitman, J. & Alspaugh, J. A. ( 2003; ). Ras1 controls pheromone expression and response during mating in Cryptococcus neoformans. Fungal Genet Biol 38, 110–121.[CrossRef]
    [Google Scholar]
  43. Wickes, B. L., Mayorga, M. E., Edman, U. & Edman, J. C. ( 1996; ). Dimorphism and haploid fruiting in Cryptococcus neoformans: association with the alpha-mating type. Proc Natl Acad Sci U S A 93, 7327–7331.[CrossRef]
    [Google Scholar]
  44. Wickes, B. L., Edman, U. & Edman, J. C. ( 1997; ). The Cryptococcus neoformans STE12 α gene: a putative Saccharomyces cerevisiae STE12 homologue that is mating type specific. Mol Microbiol 26, 951–960.[CrossRef]
    [Google Scholar]
  45. Zhang, F. L. & Casey, P. J. ( 1996; ). Protein prenylation: molecular mechanisms and functional consequences. Annu Rev Biochem 65, 241–269.[CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.27030-0
Loading
/content/journal/micro/10.1099/mic.0.27030-0
Loading

Data & Media loading...

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