Multiple mechanisms impact fluconazole resistance of mutant Erg11 proteins in Candida glabrata

Abstract

Azoles, the most commonly used antifungal drugs, specifically inhibit the fungal lanosterol α-14 demethylase enzyme, which is referred to as Erg11. Inhibition of Erg11 ultimately leads to a reduction in ergosterol production, an essential fungal membrane sterol. Many Candida species, such as Candida albicans, develop mutations in this enzyme which reduces the azole binding affinity and results in increased resistance. Candida glabrata is also a pathogenic yeast that has low intrinsic susceptibility to azole drugs and easily develops elevated resistance. These azole resistant mutations typically cause hyperactivity of the Pdr1 transcription factor and rarely lie within the ERG11 gene. Here, we generated C. glabrata ERG11 mutations that were analogous to azole resistance alleles from C. albicans ERG11. Three different Erg11 forms (Y141H, S410F, and the corresponding double mutant (DM)) conferred azole resistance in C. glabrata with the DM Erg11 form causing the strongest phenotype. The DM Erg11 also induced cross-resistance to amphotericin B and caspofungin. Resistance caused by the DM allele of ERG11 imposed a fitness cost that was not observed with hyperactive PDR1 alleles. Crucially, the presence of the DM ERG11 allele was sufficient to activate the Pdr1 transcription factor in the absence of azole drugs. Our data indicate that azole resistance linked to changes in ERG11 activity can involve cellular effects beyond an alteration in this key azole target enzyme. Understanding the physiology linking ergosterol biosynthesis with Pdr1-mediated regulation of azole resistance is crucial for ensuring the continued efficacy of azole drugs against C. glabrata. Importance Azole drugs target the Erg11 enzyme and lead to a reduction in fungal ergosterol, a vital sterol in yeast. Mutations in Erg11 are common among azole resistant Candida albicans clinical isolates, but not in C. glabrata, a major human pathogen. In this study, we showed that ERG11 mutations were tolerated in C. glabrata, and these mutations could confer azole resistance. We found that the strongest azole-resistant allele of ERG11 led to induction of the Pdr1 transcription factor and Cdr1 ATP-binding cassette transporter protein in the absence of drug. ERG11 mutations can cause azole resistance via altered enzymatic properties but also by triggering induction of other resistance systems owing to impacts on ergosterol biosynthesis. These data illustrate the deep connections between ergosterol biosynthesis and regulation of membrane transporter proteins via Pdr1 and the ergosterol-responsive transcription factor Upc2A.