Glucan Binding to Azole Drugs: A Novel Resistance Mechanism in Candida albicans

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Project Summary The pathogenic yeast Candida albicans (Ca) is a frequent cause of oral and systemic fungal infections in immune-compromised people and of vaginal infections in women. Candida infections are usually treated with azole drugs, and azole resistance arises frequently in these patient populations. The major mechanisms of azole resistance include increased expression of efflux pumps and alterations in enzymes in ergosterol biosynthesis. However, these mechanisms of resistance have not been identified in many resistant clinical isolates. Recently, a third mechanism of azole resistance has been found in Candida biofilms - matrices of cells and extracellular material that forms on mucosal surfaces and implanted medical devices. The high levels of azole resistance in these biofilms is due to increased production of ??1,3 glucan in the cell wall that binds to azole drugs. Our Hypothesis is that ??1,3 glucan binding of azoles is a component of resistance not only in biofilms, but in planktonic clinically resistant isolates. Glucan binding of azoles to the cell wall would prevent the drug from reaching the cytoplasm, increasing resistance. The Overall Goal is to evaluate the contribution of azole binding to ??1,3 glucan as a component of drug resistance in Candida. Specifically, this proposal investigates how ??1,3 glucan binding affects drug accumulation in fungal cells, and it evaluates ??1,3 glucan binding in a collection of clinical isolates, including isolates with no known resistance mechanisms. The Specific Aims of this proposal are: 1. To determine the effect of ??1,3 glucan binding on fluconazole accumulation in Candida albicans. Azole accumulation by fungal cells is likely to be the result of several competing processes, including import and efflux (internal accumulation), and possibly ??1,3 glucan binding (external accumulation). Radiolabeled FLC accumulation in cells will be evaluated under conditions that alter the glucan content of the cells. 2. To assess ??1,3 glucan binding in clinical isolates with altered drug susceptibilities. Azole binding to ??1,3 glucans, and ??1,3 glucan levels in the cells will be assessed in resistant clinical isolates in which no known mechanism of resistance has been identified. The interactions between azoles and fungal cells will continue to be clinically significant issues for the foreseeable future. ??1,3 glucan binding of azole drugs is a new and important aspect of these interactions. A full characterization of this process, together with our understanding of the other facets of drug/cell interactions, has the potential to contribute to improvements in diagnosis, treatment and prevention of fungal infections and of resistance. Narrative The pathogenic yeast Candida albicans causes significant human disease in the mouth, vagina and blood stream. This proposal will analyze the interaction between the antifungal drug fluconazole and C. albicans. In particular, it will test the hypothesis that the fungal cell wall acts as a sponge, soaking up the drug, thus allowing the cells to persist in the presence of drug.