Azole resistance is mediated by integration of sterol gene regulation and membrane transporter production by the zinc cluster-containing transcription factor Upc2A in Candida glabrata

Abstract

The most commonly used antifungal drugs are the azole compounds that interfere with biosynthesis of the fungal-specific sterol: ergosterol. The pathogenic yeast Candida glabrata commonly acquires resistance to azole drugs like fluconazole via mutations in a gene encoding a transcription factor called PDR1. These PDR1 mutations lead to overproduction of drug transporter proteins like the ATP-binding cassette transporter Cdr1. In other Candida species, mutant forms of a transcription factor called Upc2 are associated with azole resistance, owing to the important role of this protein in control of expression of genes encoding enzymes involved in the ergosterol biosynthetic pathway. Recently, the C. glabrata Upc2A factor was demonstrated to be required for normal azole resistance, even in the presence of a hyperactive mutant form of PDR1. Using genome-scale approaches, we define the network of genes bound and regulated by Upc2A. By analogy to a previously described hyperactive UPC2 mutation found in Saccharomyces cerevisiae, we generated a similar form of Upc2A in C. glabrata called G898D Upc2A. Chromatin immunoprecipitation coupled with Next Generation Sequencing (ChIP-seq) demonstrated that wild-type Upc2A binding to target genes was strongly induced by fluconazole while G898D Upc2A bound similarly, irrespective of drug treatment. We also carried out RNA-seq analysis to determine the genes that were direct or indirect targets of Upc2A transcriptional control. In addition to the well-described ERG genes as Upc2A transcriptional targets, we found a large group of genes encoding components of the translational apparatus along with membrane proteins. These Upc2A-regulated membrane protein-encoding genes are often targets of the Pdr1 transcription factor, demonstrating the high degree of overlap between these two regulatory networks. Finally, we provide evidence that Upc2A impacts the Pdr1-Cdr1 system during the anaerobic response and also modulates resistance to caspofungin. These studies provide a new perspective of Upc2A as a master regulator of lipid and membrane protein biosynthesis. Author summaryIn the pathogenic yeast Candida glabrata, expression of the genes encoding enzymes in the ergosterol biosynthetic pathway is controlled by the transcription factor Upc2A. C. glabrata has a low intrinsic susceptibility to azole therapy and acquires fluconazole resistance at high frequency. These azole resistant mutants typically contain substitution mutations in a gene encoding the transcription factor Pdr1. Pdr1 does not appear to regulate ergosterol genes and instead induces expression of genes encoding drug transport proteins like CDR1. Here we establish that extensive overlap exists between the regulatory networks defined by Upc2A and Pdr1. Genomic approaches are used to describe the hundreds of genes regulated by Upc2A that far exceed the well-described impact of this factor on genes involved in ergosterol biosynthesis. The overlap between Upc2A and Pdr1 is primarily described by co-regulation of genes encoding membrane transporters like CDR1. We provide evidence that Upc2A impacts the transcriptional control of the FKS1 gene, producing a target of a second major class of antifungal drugs, the echinocandins. Our data are consistent with Upc2A playing a role as a master regulator coordinating the synthesis of membrane structural components, both at the level of lipids and proteins, to produce properly functional biological membranes.