| Candida glabrata emerged in the last decade as a common cause of mucosal and invasive fungal infection. This is in large part due to its intrinsically low susceptibility or acquired resistance to azole antifungals such as fluconazole. In C. glabrata clinical isolates and laboratory-derived mutants, the predominant mechanism behind azole resistance is upregulated expression of multidrug transporter genes CDR1 and PDH1. One of these laboratory-derived mutants, F15, contains a putative gain-of-function mutation (P927L) that was identified in the single homolog of Saccharomyces cerevisiae transcription factors Pdr1--Pdr3. Disruption of C. glabrata PDR1 using a novel technique (PRODIGE) conferred equivalent fluconazole hypersensitivity to both F15 and 66032 and eliminated both constitutive and fluconazole-induced CDR1-PDH1 expression.; Reintroduction of wild-type or F15 PDR1 fully reversed these effects; together these results demonstrate a role for this gene in both acquired and intrinsic azole resistance. CDR1 disruption had a partial effect, reducing fluconazole trailing in both strains while restoring wild-type susceptibility to F15. In an azole-resistant clinical isolate, PDR1 disruption reduced azole MICs 8 to 64-fold with no effect on sensitivity to other antifungals. To extend this analysis, C. glabrata microarrays were generated and used to analyze genome-wide expression in F15 relative to its parent. Homologs of 10 S. cerevisiae genes previously shown to be Pdr1--Pdr3 targets were upregulated (YOR1, RTA1, RSB1, RPN4, YLR346c, and YMR102c along with CDR1, PDH1, and PDR1 itself) or downregulated (PDR12); roles for these genes include small molecule transport and transcriptional regulation. Additional genes involved in transport (e.g., QDR2, YBT1), lipid metabolism ( ATF2, ARE1), cell stress (HSP12, CTA1), DNA repair (YIM1, MEC3), and cell wall function (MKC7, MNT3 ) showed altered expression. These azole resistance-associated changes could affect C. glabrata tissue-specific virulence; in support of this, we detected differences in F15 oxidant, alcohol, and weak acid sensitivities. Finally, we describe a candidate C. glabrata virulence factor involving catalase-meditated hydrogen peroxide resistance. This organism provides a promising model for studying the genetic basis of multidrug resistance and its impact on virulence. |
