Fungal infections are increasing as a result of AIDS, transplantation, and high dose chemotherapy. Yet the armamentarium of antifungal agents is scare--amphotericin B, azoles, flucytosine--drug resistant isolates are emerging, and a molecular understanding of fungal virulence is limited. Dr. Heitman and his group propose to elucidate signal transduction cascades regulating virulence of Cryptococcus neoformans, the leading cause of fungal meningitis and a common opportunistic infection in AIDS patients. Several features make C. neoformans ideal for studies of fungal pathogenesis. The organism exists as a stable haploid with a defined sexual life cycle. Gene disruption by homologous recombination is now possible in this organism, and animal models have been established to test both mutant strains and candidate drugs in a viurlence setting. These advances provide tools to establish the molecular basis of traits associated with virulence: including growth at elevated temperature, capsule, melalin, and urease production, and mating type. This group recently identified a molecular factor regulating C. neoformans virulence as calcineurin, a conserved Ca++-calmodulin activated protein phosphatase. They cloned the C. neoformans gene encoding the enzymatic domain (A) of calcineurin, disrupted the gene, and found that calcineurin mutant strains are viable but sensitive to conditions in the infected host: elevated temperature, alkaline pH and 5% CO2. As a consequence, strains lacking calcineurin are nonpathogenic in animals. Calcineurin is the target of the immunosuppressants cyclosporin A (CsA) and FK506, which inhibit |T cells and revolutionalized therapy of transplant patients. Much is known on calcineurin biochemistry and structure, including the crystal structures of calcineurin alone and bound to FK506,a multitude of CsA and FK506 analogs have been developed, and simple in vitro assays have become accessible techniques. Based on known signalling roles in other organisms, the investigator hypothesizes that calcineurin regulates C. neoformans virulence by dephosphorylating proteins required for temperature-, pH- and CO2- resistant growth. The group proposes to delineate this signalling cascade to understand the regulation of pathogenesisis and to provide targets for therapeutic intervention. One of several strong features of this submission is the solid track record of this Howard Hughes Assistant Investigator (Dr. Heitman) in uncovering calcineurin-dependent phenotypic changes in S. cerevisiae in response to immunosuppressants, and exposing the catalytic subunit of this Ca++ and calmodulin dependent protein phosphatase in C. neoformans as a key player in temperature, pH and CO2 resistant growth and a virulence determinant. The focus of this proposal rivets on calcineurin as the investigator seeks first to identify and further characterize the molecular components of calcineurin by cloning the regulatory (B) component of the calcineurin heterodimer, and then shifts to identify the substrates for calcineurin phosphatase by a combined approaches using affinity chromatography coupled with the two hybrid system to identify proteins that bind calcineurin. These efforts will be supported by classical genetic suppressor analysis to identify multicopy suppressors of calcineurin mutants and thus identify the genes that function in signal transduction downstream of the Ca++/CaM protein phosphatase. Earlier events prior to calcineurin activation will also be examined in this proposal, as the C neoformans calmodulin gene will be cloned, mutated in several domains (including the Ca++ binding domains, and the calcineurin interaction site) to generate clones with altered interaction or activation capabilities specifically with calcineurin and potentially with other Ca++/CaM dependent proteins involved in growth phenotype and virulence. In addition, the group will examine the role of extracellular Ca++ on calcineurin dependent growth, and Dr. Heitman plans to establish aquarian as a reporter protein for cellular conditions in which intracellular Ca++ levels fluctuate in C. neoformans. The soundness of the molecular biology of this proposal, the pivotal position of Dr. Heitman as an investigator into the biology of calcineurin, the strong line-up of co-investigators (Dr. Maria Cardenas; biochemical and protein purification support and Dr. John Perfect; transformation of C. neoformans and virulence analysis) combine for a highly enthusiastic rating of this application.
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