DNA Repair Genes and Acquired Drug Resistance in Candida

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DESCRIPTION (provided by applicant): Candida albicans is the most common fungal pathogen of humans. Normally a commensal, candidiasis may result when the host becomes debilitated or immunosuppressed. In hematogenously disseminated infections, mortality can reach 50%, even with treatment using various antifungal drugs. Acquired antifungal drug resistance is becoming a serious clinical problem, but the mechanisms by which resistance develops are unknown. The genetic changes leading to drug resistance may involve mutation, homozygosis, chromosome translocations, and alterations of repeat sequences. We propose to examine the roles of C. albicans homologs of genes known to be involved in a number of DNA repair pathways, to identify those genes that are required for the genomic changes resulting in antifungal drug resistance in Candida. We will construct strains with null mutations in both copies of genes involved in each of the primary DNA repair processes - base excision repair, nucleotide excision repair, mismatch repair and double-strand break repair. We will use a series of assays to characterize the DNA repair activities of each of the deletion mutants. We will also determine the rate at which the various mutant strains acquire resistance to commonly used antifungal agents, and the type of lesions found in the resistant strains, to identify those DNA repair processes having direct roles in drug resistance development in Candida. The results of these experiments will allow us to determine the ways in which antifungal resistance phenotypes arise and suggest targets for combined therapy that would greatly decrease the problem of acquired resistance. Our data may also address the mechanisms underlying the striking genomic instability of C. albicans observed in vivo. Karyotypic rearrangements arise relatively rarely in the laboratory but are very common in clinical isolates. To date, a rigorous analysis of the mechanisms underlying these observations has not been attempted in Candida. Since we expect that we will find altered karyotypes among the drug-resistant mutants we isolate, our proposed experiments will point to some of these mechanisms, providing a solid framework for future work on drug resistance, Candida genome stability, and associated processes.