Fungi may respond to the stress of antifungal chemotherapy in specific ways that ultimately reduce the efficacy of the therapy. This """"""""antifungal response"""""""" would be analogous to, but mechanistically distinct from, the heat shock response. The focus of this proposal is one such antifungal response, termed MDR induction, involving the rapid drug-dependent transcriptional induction of genes encoding transporters involved in multidrug resistance (MDR). Specifically, a 15-20 min exposure of Candida albicans, Candida krusei, or Saccharomyces cerevisiae to certain drugs (including the antifungal azoles miconazole and clotrimazole, the benzimidazole albendazole, and sulfadiazine) induced a 3 to > 10-fold increase in mRNAs encoding specific MDR transporters. initial studies indicate that MDR induction is the most likely explanation for the intrinsic resistance of Candida species to benzimidazoles and for the antagonistic activity between compounds that induce MDR (albendazole and sulfadiazine) and compounds that are substrates for the MDR transporters (azoles). Hypothetically, MDR induction could also contribute to phenotypic adaptation to azoles (reflected in the """"""""trailing effect""""""""), to the variable sensitivity of different fungi to different azoles, and to acquired antifungal drug resistance.
The Specific Aims of this proposal are to examine the: (1) Spectrum of MDR induction. New antifungal agents and anti-infective agents commonly used in immunocompromised patients will be tested for C. albicans MDR induction and antagonism of azole activity. MDR induction and antagonism will be compared in azole-sensitive and resistant isolates, and in C. albicans yeast and hyphal forms. MDR gene fragments will be amplified from Candida glabrata, Aspergillus fumigatus, and Cryptococcus neoformans and used as probes for MDR induction studies in those fungi. (2) Transcriptional activators of MDR induction in C. albicans. Initial studies in Saccharomyces cerevisiae have implicated two classes of transcriptional activators in azole-dependent MDR induction, PDR1/PDR3 and YAP1/YAP2. Candida homologues of these factors will be identified by PCR or expression in S. cerevisiae. Their role in MDR induction will be examined by gene disruption.
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