Oropharyngeal candidiasis (OPC) is the most common opportunistic fungal infection among patients infected with HIV. Although mucosal infections are seldom lethal, they significantly affect morbidity and cost of treatment. It is known that low CD4 counts and high viral load correlate with OPC. However, this correlation is not absolute and it is recognized that multiple factors will trigger Candida to change from a commensal to pathogenic organism. Careful coordination of different signaling pathways in response to changes in host environmental conditions allows Candida to rapidly invade a variety of mammalian tissues. The proteins that modulate the cross talk between these signaling pathways are poorly described and will be important in coordinating invasion. The 14-3-3 family of proteins is highly conserved in all eukaryotic species. These proteins regulate diverse signaling pathways involved in cell survival, the cell cycle and differentiation, and affect their functions via interactions with phosphorylated serines/ threonines. In C. albicans, the 14-3-3 protein (Bmh1p) is required for vegetative growth, optimal filamentation, chlamydospore formation, and response to rapamycin. We have constructed nine isogenic strains by site directed and random mutagenesis of the BMH1 gene. These strains have allowed us to segregate the multiple functions of Bmh1p including its role in growth from its role in filamentation. Preliminary results demonstrate that a subset of our mutant strains interact significantly different than wild type with cultured macrophages and in the murine disseminated candidiasis model. Thus, a subset of the bmh1 mutant strains will be used to determine: events that are altered during interactions with macrophages and an oral epithelium model; how these interactions may affect the host response; and which of these events influence pathogenesis in the intact animal. We will use a transcriptional approach to identify the cellular processes in C. albicans that are differentially regulated in the bmhl mutants. This will enable us to identify C. albicans cellular functions that respond to the host and dissect which of these are important for pathogenesis. Genes encoding specific regulators of these cellular processes will be knocked out of C.albicans. These strains will then be incubated with host cells to confirm the importance of these pathways. These studies will allow us to begin to identify cellular processes in Candida that are required for its survival within and invasion of different host environments. Inhibitors of mammalian pathways that are regulated by 14-3-3 proteins are now being tested as potential anti-cancer drugs. Consequently, identification of inhibitors of the processes identified in this proposal may prove to be a line of antifungal drug discovery. ? ? ?
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