The immunosuppressants cyclosporin A (CsA), FK506, and rapamycin block signaling events required for T-cell activation. CsA and FK506 prevent responses to antigen; rapamycin inhibits responses to IL-2. These three natural products also have antifungal activities, and previous studies reveal a remarkable conservation of action from microorganisms to vertebrates in which the drugs diffuse into the cell, associate with an immunophilin protein, cyclophilin A for CsA and FKBP12 for FK506/rapamycin, and are toxic to other proteins. A conserved calcium-regulated phosphatase, calcineurin, is the target of the cyclophilin A - CsA and FKBP12-FK506 complexes. The targets of rapamycin, the TOR proteins, are conserved proteins likely to transduce growth-promoting signals. Although touted as potential antifungal agents, little is known about the actions or targets of these agents in pathogenic organisms. The investigator proposes to study the antifungal properties of immunosuppressants in the pathogenic basidiomycete Cryptococcus neoformans, whose incidence has risen, especially in HIV infection, to become the leading cause of fungal meningitis. The investigator discovered that C. neoformans is resistant to CsA or FK506 at 22C but sensitive to both drugs at 37C. Genetic studies suggest immunophilin-drug complexes inhibit calcineurin to prevent growth at 37C. Because growth at 37C is required for infection, the Investigator s hypothesis is that calcineurin is required for pathogenicity. The C. neoformans calcineurin A gene was cloned, sequenced, and disrupted by homologous recombination. As predicted, calcineurin mutants are viable at 24C but not at 37C. Two calcineurin mutants are nonpathogenic in an animal model. Reintroduction of the calcineurin A gene restored growth at 37C, and the Investigator will test whether virulence is also restored. A nonimmunosuppressive antifungal FK506 analog was identified. The Investigator will analyze drug-resistant C. neoformans mutants and clone C. neoformans FKBP12, cyclophilin, and TOR genes. The Investigator s goals are to identify immunophilins, calcineurin, and their functions in C. neoformans to delineate a signal transduction pathway regulating pathogenesis and to define novel antifungal drug targets.
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