Cryptococcus neoformans is a basidiomycetous fungus which can cause life threatening infections. It can be especially serious in AIDS patients who are at the highest risk for infection and often require life-long antifungal prophylaxis. The MATalpha mating type is one of the four known virulence factors of C. neoformans. In addition to being more virulent. MATalpha cells, unlike MATa cells, can also produce an extensive hyphal phase in the haploid state called monokaryotic fruiting. The main objective of this proposal is to identify the molecular and genetic links between the MATalpha mating type, monokaryotic fruiting, and virulence. This objective requires a basic understanding of mating in C. neoformans and will be accomplished in four specific aims.
The first aim of this study will be to understand the function of STE12a, a MATalpha-specific homolog of the S. cerevisiae STE12 gene. STE12a will be studied as a potential key virulence factor since its role as a transcriptional activator allows it to interact and induce expression of other genes. One gene which STE12a can induce is CNLAC1, a proven virulence factor. The role of STE12a in virulence, as well as possible interactions with genes involved in melanin and capsule production, mating, and monokaryotic fruiting will be investigated.
The second aim of this study will be to isolate the MAT alpha mating type locus. Physical mapping of the locus and complementation of sterile mutants will allow the identification MAT alpha-specific genes. These genes will then be tested for their roles in virulence using the mouse model as well as their roles in mating and monokaryotic fruiting.
The third aim of this study will be to investigate the molecular basis of monokaryotic fruiting. This phenotype provides the mechanism for producing infectious particles in the absence of the opposite mating type and explains, in part, the extreme bias in favor of MATalpha cells in the environment. Factors which induce or repress monokaryotic fruiting will be evaluated. In order to improve the phenotype of hyphae production and make this phenomenon more amenable to genetic analysis, strain improvement will be performed to select for strains with an advanced hyphal phenotype. Genetic markers will be introduced into these strains and hyphal-negative mutants will be created by mutagenesis. These mutants will be complemented with genomic DNA libraries in order to isolate monokaryotic fruiting-related genes which can then be tested for their roles in mating and virulence. Finally, the last aim of this study will be to isolate the MATa locus. in light of the reduced virulence of MATa cells and the MATa-specificity of some of the conserved mating type genes, the information contained in MATa will contrast well with the information in MATalpha and will contribute to the understanding of mating and virulence in C. neoformans.
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