Cryptococcal infections are an increasing source of mortality and morbidity in immunocompromised hosts, particularly those with AIDS. It is the most common invasive fungal infection in AIDS patients, who do not respond optimally to conventional therapy. Our strategy is to study and understand the yeast virulence factors which favor invasion of normal and compromised hosts. The basis for this proposal is to study C. neoformans virulence factors through molecular manipulations. Our 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. This concept has been used extensively in studies of plant pathogens, and recently in pathogenic bacteria, to elucidate molecular mechanisms of virulence. By identifying and characterizing specific virulence genes, development of strategies to interrupt these genes, their proteins or protein receptors can be performed. Our plan is to apply molecular techniques to isolate genes of C. neoformans which are important to the establishment of infection. Specifically, the investigation involves the following strategies: (1) Identification of potentially important genes by their differential expression under exposure to certain host factors at the site of infection in both animals and humans; (2) Screening of these unique, cloned genes for time of expression at the site of infection; (3) Site-directed mutagenesis of in vivo expressed genes to produce mutants of a virulent C neoformans strain, and determination of the mutants virulence in vivo. This project will primarily focus on gene(s) required to maintain C. neoformans infection in the subarachnoid space. This site remains the primary concern of medical therapeutics. Virulence genes identified by the above protocol will be sequenced and compared to other known genes to determine possible function of encoded proteins or receptors. Although the molecular domestication of C. neoformans is in its early stages, our preliminary studies have been successful and more advanced techniques such as subtractive cDNA libraries and an efficient transformation system will be refined. The potential benefits of this proposal are: (1) to establish basic molecular knowledge about the pathogenic fungi and their responses to mammalian host factors; (2) predict mechanisms which can interrupt invasion of the host by these pathogens and thus devise new strategies for treatment.
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