Cryptococcus neoformans produces a life-threatening meningoencephalitis in both immunosuppressed and normal hosts. Despite treatments with amphotericin B and triazoles, the management of this infection remains suboptimal because of the lack of fungicidal regimens and development of drug resistance. The foundation for this proposal, Genetic controls for host temperature growth of Cryptococcus neoformans, is based on the hypothesis that C. neoformans adapt to 37 degrees Celsius in the host by expressing or repressing certain genes which are essential for its survival and growth under this environmental stress. This concept of studying environmental stresses such as temperature regulating virulence potential and the microorganism has been very effectively used in pathogenic bacteria to elegantly elucidate molecular mechanisms of disease. Recently, we have proven that C. neoformans bacteria to elegantly elucidate molecular mechanisms of disease. Recently, we have proven that C. neoformans can similarly act as a model system for this strategy. For instance, we discovered that C. neoformans uses the calcineurin A (CNAl) gene in the signaling pathway for 37 degrees Celsius, pH, pCO2 growth and virulence. CNAl is the first gene identified with elevated temperature growth in C. neoformans and its importance could not have been predicted by classical model fungi such as S. cerevisiae. Along with previous genetic data and animal studies it is clear that the temperature growth phenotype is essential to this pathogen and concepts from these studies can probably by extrapolated to other fungal pathogens. In this proposal, we will use the following strategies: 1) insertional mutagenesis, 2) regulated promoter trap, 3) cDNA library subtraction, and 4) differential display RT-PCR to capture a series of elevated-temperature regulated genes and/or essential genes for growth at 37 degrees Celsius. Our focus will use these strategies for identification but then we will isolate, sequence, and validate the importance of these genes by knockout mutants in animal models. The direct phenotype study allows us to focus on fungicidal targets (i.e. inability to survive at 37 degrees Celsius) Our strategies of combing the C. neoformans genome for elevated temperature growth genes can represent the potential for validation of antifungal targets for new drugs.
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