Aplicant's Abstract) Current treatments for HIV and AIDS-associated infections, including oral candidiasis, are becoming less effective in part because these pathogens are developing resistance to the currently used drugs. In recurrent oral candidiasis, the use of azole antifungals, including fluconazole, for treatment and/or prophylaxis contributes to the recent, dramatic increase in Candida strains that are resistant to antifungal drugs. In the past several years, the basic molecular mechanisms of azole resistance have been identified including the overexpression of the target enzyme and two types of efflux pumps, and mutations in the target enzyme and other enzymes in the ergosterol biosynthetic pathway. Overexpressed pumps include the CDR pumps which are members of the ATP Binding Cassette (ABC) Transporter family and MDR1, which is a member of the Major Facilitator class of pumps. In surveys of small numbers of clinical isolates, overexpression of CDR pumps, especially CDR1, appears to be the most common mechanism of azole drug resistance. The goals of this proposal are (1) to characterize the mechanisms by which the CDR pumps are overexpressed, focusing on the CDR1 promoter and transcription factors that interact with the promoter and (2) to use molecular techniques to monitor the acquisition of resistance in sensitive isolates and to monitor the maintanance or loss of resistance in resistant isolates in the absence of drug selection. Together, these findings should help in the development of strategies to minimize and treat the emergence of antifungal resistance. The expression of the CDR1 promoter will be monitored under a variety of growth conditions, fungal cell types and known substrates for the ABC transporters. Molecular analyses will demonstrate that increased mRNA levels are the result of overexpression and will identify the CDR1 start of transcription. The CDR1 promoter will be dissected to identify regions of the promoter that are important for transcriptional activity. An inducible promoter linked to CDR1 will be used to prove that overexpression is sufficient to cause resistance. Several genetic screens in Saccharomyces and Candida will identify and characterize transcription factors that interact with the CDR1. Recently developed assays from this laboratory will be used to monitor the molecular mechanisms that are involved in the in vitro development of resistance in sensitive isolates and the loss of resistance in the absence of drug.
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