This proposal has two goals: contribute to our understanding of the biology of Tor signaling in the fungal opportunistic pathogen C. albicans, and identify potential drug targets for synergistic antifungal activity with rapamycin analogs. The Tor pathway promotes growth and proliferation in eukaryotes in response to availability of nutrients and absence of noxious stressors. The centrality of this pathway to many essential cellular processes has emerged from work in model yeasts and in metazoans. In C. albicans pathogenesis, Tor signaling is predicted to play a crucial role for three reasons. Firstly, the Tor pathway co-regulates morphogenesis in C. albicans. Secondly, the fungus encounters starvation conditions in host phagocytes;and thirdly, noxious stresses are imposed by the host immune system. The Tor pathway is also important because it is specifically inhibited by the prokaryotic product rapamycin, opening the door to development of fungal-specific rapamycin analogs for treatment of Candida infections. Partial divergence between the Tor pathway of C. albicans and that of model yeasts has already become apparent. For this reason, an unbiased forward genetic approach is proposed to discover novel components of C. albicans Tor signaling not known from model organisms, and to identify new targets for inhibition by small molecules that can potentiate the cidal activity of rapamycin analogs. We propose a screen of our heterozygous transposon mutant collection because with this reagent, essential genes involved in Tor signaling can be identified. Another advantage of this collection is that haploinsufficiency phenotypes, displayed by heterozygotes, are more likely to be severe in the corresponding homozygotes, biasing the screen against mutations with mild effects only indirectly linked to Tor. A pilot screen using this mutant collection yielded representatives of each of our two goals: a homolog of a kinase regulator, which signals in development of the metazoan immune system, was isolated because it is required for rapamycin sensitivity. Secondly, a mutant in a putative plasma membrane ion pump was found to be exquisitely rapamycin hypersensitive. This ion pump has no human homolog. Its conserved S. cerevisiae homolog has been shown to be inhibited by two different small molecules, supporting the idea that further inhibitors can be found. Characterizing these genes will contribute to our basic understanding of C. albicans Tor signaling and possibly lay the groundwork for a small molecule screen for synergistic candidacidal drugs.
Candida albicans is the most common cause of invasive fungal infections, which even treated appropriately, carry a high mortality. This work will identify new components of a central signaling pathway required for growth and proliferation of C. albicans, and characterize a potential drug target for synergistic antifungal therapy.
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