Our studies define signal transduction cascades regulating physiology and virulence of the pathogen Cryptococcus neoformans. The immunosuppressants cyclosporin A (CsA), FK506, and rapamycin block signaling cascades required for T-cell activation. These compounds also have antifungal activities, and we have identified fungal homologs of the mammalian target proteins calcineurin A, calcineurin B, cyclophilin A, FKBP12, TOR1, and TOR2. Our genetic and biochemical studies demonstrate that CsA and FK506 antifungal affects are mediated by cyclophilin and FKBP12 dependent inhibition of the fungal calcineurin phosphatase homologue. In addition, rapamycin action is mediated by FKBP12 dependent inhibition of the fungal TOR kinase homologs. Our studies demonstrate that in C. neoformans, calcineurin is required for growth at 37 C and other stress conditions and, as a consequence, is required for virulence of both serotype A and D strains in two different animal models. Recently, we discovered that calcineurin is required for mating and haploid fruiting in C. neoformans, which may provide a molecular link between signaling pathways regulating the differentiation and virulence of this pathogen. The lifecycle and sexual cycle of C. neoformans have been defined. Interestingly, the MATalpha mating type is more prevalent in the environment and in patients, and MATalpha strains are more virulent than congenic MATa strains. In addition, in response to nitrogen starvation, strains of the MATalpha mating type filament and sporulate (haploid fruiting). Because cryptococcal infection requires inhalation into the alveoli of the lung, the spores produced by mating or haploid fruiting may represent the infectious particle. We have discovered that, in addition to its role in virulence, calcineurin is also required for mating and haploid fruiting. This discovery may link the known or suspected roles of calcineurin, mating type, and haploid fruiting in virulence. In this proposal, we will first establish which steps in mating and fruiting are regulated by calcineurin. Second, we will identify targets of calcineurin that regulate mating and fruiting. Using the two-hybrid assay, we have identified a conserved calcineurin binding protein and will test a possible role in mating and fruiting. Third, we will analyze the functions of cyclophilin A in C. neoformans and test the hypothesis that cyclophilin A regulates calcineurin. We have discovered diploid strains and are developing these tools to study calcineurin roles in mating and essential genes. Finally, we will characterize two transposable elements discovered during these studies, whose transposition may be regulated by calcineurin and which will be used as insertional mutagens to define other components of the calcineurin pathways.
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