Similarities and distinctions in the activation of the Candida glabrata Pdr1 regulatory pathway by azole and non-azole drugs.

Abstract

Incidences of fluconazole (FLC) resistance among Candida glabrata clinical isolates are a growing issue in clinics. The pleiotropic drug response network in C. glabrata confers azole resistance and is defined primarily by the Zn₂Cys₆ zinc cluster-containing transcription factor Pdr1 and target genes such as CDR1, which encodes an ATP-binding cassette transporter protein thought to act as an FLC efflux pump. Mutations in the PDR1 gene that render the transcription factor hyperactive are the most common cause of fluconazole resistance among clinical isolates. The phenothiazine class drug fluphenazine and a molecular derivative, CWHM-974, which both exhibit antifungal properties, have been shown to induce the expression of Cdr1 in Candida spp. We have used a firefly luciferase reporter gene driven by the CDR1 promoter to demonstrate two distinct patterns of CDR1 promoter activation kinetics: gradual promoter activation kinetics that occur in response to ergosterol limitations imposed by exposure to azole and polyene class antifungals and a robust and rapid CDR1 induction occurring in response to the stress imposed by fluphenazines. We can attribute these different patterns of CDR1 induction as proceeding through the promoter region of this gene since this is the only segment of the gene included in the luciferase reporter construct. Genetic analysis indicates that the signaling pathways responsible for phenothiazine and azole induction of CDR1 overlap but are not identical. The short time course of phenothiazine induction suggests that these compounds may act more directly on the Pdr1 protein to stimulate its activity.

Importance: Candida glabrata has emerged as the second-leading cause of candidiasis due, in part, to its ability to acquire high-level resistance to azole drugs, a major class of antifungal that acts to block the biosynthesis of the fungal sterol ergosterol. The presence of azole drugs causes the induction of a variety of genes involved in controlling susceptibility to this drug class, including drug transporters and ergosterol biosynthetic genes such as ERG11. We found that the presence of azole drugs leads to an induction of genes encoding drug transporters and ERG11, while exposure of C. glabrata cells to antifungals of the phenothiazine class of drugs caused a much faster and larger induction of drug transporters but not ERG11. Coupled with further genetic analyses of the effects of azole and phenothiazine drugs, our data indicate that these compounds are sensed and responded to differentially in the yeast cell.