1887

Abstract

The Ras1 signal transduction pathway controls the ability of the pathogenic fungus to grow at high temperatures and to mate. A second gene was identified in this organism. is expressed at a very low level compared to , and a mutation caused no alterations in vegetative growth rate, differentiation or virulence factor expression. The mutant strain was equally virulent to the wild-type strain in the murine inhalational model of cryptococcosis. Although a double mutant strain is viable, mutation of both genes results in a decreased growth rate at all temperatures compared to strains with either single mutation. Overexpression of the gene completely suppressed the mutant mating defect and partially suppressed its high temperature growth defect. After prolonged incubation at a restrictive temperature, the mutant demonstrated actin polarity defects that were also partially suppressed by overexpression. These studies indicate that the Ras1 and Ras2 proteins share overlapping functions, but also play distinct signalling roles. Our findings also suggest a mechanism by which Ras1 controls growth of this pathogenic fungus at 37 °C, supporting a conserved role for Ras homologues in microbial cellular differentiation, morphogenesis and virulence.

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2002-01-01
2019-09-21
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References

  1. Adams, A. E., Johnson, D. I., Longnecker, R. M., Sloat, B. F. & Pringle, J. R. ( 1990; ). CDC42 and CDC43, two additional genes involved in budding and the establishment of cell polarity in the yeast Saccharomyces cerevisiae. J Cell Biol 111, 131-142.[CrossRef]
    [Google Scholar]
  2. Alspaugh, J. A., Perfect, J. R. & Heitman, J. ( 1997; ). Cryptococcus neoformans mating and virulence are regulated by the G-protein a subunit GPA1 and cAMP. Genes Dev 11, 3206-3217.[CrossRef]
    [Google Scholar]
  3. 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]
  4. Barbacid, M. ( 1987; ). ras genes. Annu Rev Biochem 56, 779-827.[CrossRef]
    [Google Scholar]
  5. Cox, G. M., Mukherjee, J., Cole, G. T., Casadevall, A. & Perfect, J. R. ( 2000; ). Urease as a virulence factor in experimental Cryptococcosis. Infect Immun 68, 443-448.[CrossRef]
    [Google Scholar]
  6. DeFeo-Jones, D., Tatchell, K., Robinson, L. C., Sigal, I. S., Vass, W. C., Lowy, D. R. & Scolnick, E. M. ( 1985; ). Mammalian and yeast ras gene products: biological function in their heterologous systems. Science 228, 179-184.[CrossRef]
    [Google Scholar]
  7. Edman, J. C. ( 1992; ). Isolation of telomere-like sequences from Cryptococcus neoformans and their use in high-efficiency transformation. Mol Cell Biol 12, 2777-2783.
    [Google Scholar]
  8. Fukui, Y., Kozasa, T., Kaziro, Y., Takeda, T. & Yamamoto, M. ( 1986; ). Role of ras homolog in the life cycle of Schizosaccharomyces pombe. Cell 44, 329-336.[CrossRef]
    [Google Scholar]
  9. 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]
  10. Heitman, J., Casadevall, A., Lodge, J. K. & Perfect, J. R. ( 1999; ). The Cryptococcus neoformans genome sequencing project. Mycopathologia 148, 1-7.[CrossRef]
    [Google Scholar]
  11. Ho, J. & Bretscher, A. ( 2001; ). Ras regulates the polarity of the yeast actin cytoskeleton through the stress response pathway. Mol Biol Cell 12, 1541-1555.[CrossRef]
    [Google Scholar]
  12. Ho, S. N., Hunt, H. D., Horton, R. M., Pullen, J. K. & Pease, L. R. ( 1989; ). Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77, 51-59.[CrossRef]
    [Google Scholar]
  13. Kataoka, T., Powers, S., McGill, C., Fasano, O., Strathern, J., Broach, J. & Wigler, M. ( 1984; ). Genetic analysis of yeast RAS1 and RAS2 genes. Cell 37, 437-445.[CrossRef]
    [Google Scholar]
  14. Kataoka, T., Broek, D. & Wigler, M. ( 1985; ). DNA sequence and characterization of the S. cerevisiae gene encoding adenylate cyclase. Cell 43, 493-505.[CrossRef]
    [Google Scholar]
  15. Kopecká, M., Gabriel, M., Takeo, K., Yamaguchi, M., Svoboda, A., Ohkusu, M., Hata, K. & Yoshida, S. ( 2001; ). Microtubules and actin cytoskeleton in Cryptococcus neoformans compared with ascomycetous budding and fission yeasts. Eur J Cell Biol 80, 303-311.[CrossRef]
    [Google Scholar]
  16. Kwon-Chung, K. J. & Bennett, J. E. (1992). Cryptococcosis. In Medical Mycology, pp. 397–446. Malvern, PA: Lea & Febiger.
  17. Kwon-Chung, K. J., Varma, A., Edman, J. C. & Bennett, J. E. ( 1992a; ). Selection of ura5 and ura3 mutants from the two varieties of Cryptococcus neoformans on 5-fluoroorotic acid medium. J Med Vet Mycol 30, 61-69.[CrossRef]
    [Google Scholar]
  18. Kwon-Chung, K. J., Wickes, B. L., Stockman, L., Roberts, G. D., Ellis, D. & Howard, D. H. ( 1992b; ). Virulence, serotype, and molecular characteristics of environmental strains of Cryptococcus neoformans var. gattii. Infect Immun 60, 1869-1874.
    [Google Scholar]
  19. Milburn, M. V., Tong, L., deVos, A. M., Brunger, A., Yamaizumi, Z., Nishimura, S. & Kim, S. H. ( 1990; ). Molecular switch for signal transduction: structural differences between active and inactive forms of protooncogenic ras proteins. Science 247, 939-945.[CrossRef]
    [Google Scholar]
  20. Mösch, H. U., Roberts, R. L. & Fink, G. R. ( 1996; ). Ras2 signals via the Cdc42/Ste20/mitogen-activated protein kinase module to induce filamentous growth in Saccharomyces cerevisiae. Proc Natl Acad Sci USA 93, 5352-5356.[CrossRef]
    [Google Scholar]
  21. Mösch, H. U., Kubler, E., Krappmann, S., Fink, G. R. & Braus, G. H. ( 1999; ). Crosstalk between the Ras2p-controlled mitogen-activated protein kinase and cAMP pathways during invasive growth of Saccharomyces cerevisiae. Mol Biol Cell 10, 1325-1335.[CrossRef]
    [Google Scholar]
  22. Nielsen, O., Davey, J. & Egel, R. ( 1992; ). The ras1 function of Schizosaccharomyces pombe mediates pheromone-induced transcription. EMBO J 11, 1391-1395.
    [Google Scholar]
  23. 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]
  24. 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]
  25. Pruyne, D. & Bretscher, A. ( 2000; ). Polarization of cell growth in yeast. II. The role of the cortical actin cytoskeleton. J Cell Sci 113, 571-585.
    [Google Scholar]
  26. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  27. Sherman, F. ( 1991; ). Getting started with yeast. In Methods in Enzymology , pp. 3-21. Edited by C. Guthrie & G. R. Fink. San Diego, CA:Academic Press.
  28. 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]
  29. Stanhill, A., Schick, N. & Engelberg, D. ( 1999; ). The yeast ras/cyclic AMP pathway induces invasive growth by suppressing the cellular stress response. Mol Cell Biol 19, 7529-7538.
    [Google Scholar]
  30. Tatchell, K., Chaleff, D., Defeo-Jones, D. & Scolnick, E. ( 1984; ). Requirement of either of a pair of ras-related genes of Saccharomyces cerevisiae for spore viability. Nature 309, 523-527.[CrossRef]
    [Google Scholar]
  31. Toda, T., Uno, I., Ishikawa, T. & 7 other authors ( 1985; ). In yeast, RAS proteins are controlling elements of adenylate cyclase. Cell 40, 27–36.[CrossRef]
    [Google Scholar]
  32. Toffaletti, D. L., Rude, T. H., Johnston, S. A., Durack, D. T. & Perfect, J. R. ( 1993; ). Gene transfer in Cryptococcus neoformans by use of biolistic delivery of DNA. J Bacteriol 175, 1405-1411.
    [Google Scholar]
  33. Varma, A. & Kwon-Chung, K. J. ( 1999; ). Characterization of the glyceraldehyde-3-phosphate dehydrogenase gene. Gene 232, 155-163.[CrossRef]
    [Google Scholar]
  34. Vartivarian, S. E., Anaissie, E. J., Cowart, R. E., Sprigg, H. A., Tingler, M. J. & Jacobson, E. S. ( 1993; ). Regulation of cryptococcal capsular polysaccharide by iron. J Infect Dis 167, 186-190.[CrossRef]
    [Google Scholar]
  35. Wang, P., Cardenas, M. E., Cox, G. M., Perfect, J. & Heitman, J. ( 2001; ). Two cyclophilin A homologs with shared and distinct functions important for growth and virulence of Cryptococcus neoformans. EMBO Rep 21, 1-8.
    [Google Scholar]
  36. Wolfe, K. H. & Shields, D. C. ( 1997; ). Molecular evidence for an ancient duplication of the entire yeast genome. Nature 387, 708-713.[CrossRef]
    [Google Scholar]
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