C. neoformans causes life threatening human mycoses in severely immunocompromised hosts such as cancer and AIDS patients. Despite amphotericin B and newer triazoles for treatment, the management of this infection remains problematic. The investigators believe that understanding the basic molecular mechanisms for C. neoformans survival in the host will aid in the development of new drugs. The basis for this renewal remains the identification and characterization of C. neoformans virulence factors through genetic manipulations. The investigator's primary hypothesis is that under certain environmental stresses, C. neoformans will adapt to these new conditions by expressing genes which are essential to its survival and growth. Although a more general approach than directed studies on certain phenotypic markers of virulence such as phenoloxidase production or capsule synthesis, it is the investigator's opinion that the yeast can reveal unique molecular mechanisms during infection which cannot be predicted from phenotype analysis. This concept of environmental stresses regulating virulence genes is not new and in fact, has been used very effectively in plant pathogens and pathogenic bacteria to elegantly elucidate molecular mechanisms of virulence. However, it has not been well-developed for pathogenic fungi. It is the investigator's approach is this proposal to fully test this hypothesis and through it identify and characterize specific genes and their regulators associated with virulence. Over the last two years the molecular foundation of C. neoformans has significantly expanded. The investigator has developed an effective transformation system for C. neoformans which will be a technological cornerstone for this proposal, and recently he has produced gene knockouts. His initial studies in an animal model of cryptococcal meningitis support the hypothesis that differential gene expression occurs at the site of infection and uniquely expressed genes have been isolated. The first specific aim of the investigation focuses on isolating and identifying these up-regulated and down-regulated genes during a CNS infection, b differential gene hybridizations and cDNA subtractive library techniques. These genes in rabbits will be screened for infection site specificity and human CNS specificity. Selected genes will be identified by sequence analysis and through site- directed mutagenesis, mutants will have their virulence determined. For the second specific aim these genes will also allow the investigators to test a second hypothesis that these genes are under a regulatory gene(s). There is significant precedent for a wide domain regulatory arrangement in pathogenic bacteria and nonpathogenic fungi, but it has not been proven in human fungal pathogens. Manipulation of these regulatory gene(s) could have major consequences on the pathobiology of the fungus, and could reveal a major target(s) for antifungals, complementing the present pharmaceutical approach with natural products.
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