Loss-of-Function Mutations Suppress the Fluconazole Susceptibility of Δ Mutation in Candida glabrata, Implicating Additional Positive Regulators of Ergosterol Biosynthesis.

Two of the major classes of antifungal drugs in clinical use target ergosterol biosynthesis. Despite its importance, our understanding of the transcriptional regulation of ergosterol biosynthesis genes in pathogenic fungi is essentially limited to the role of hypoxia and sterol-stress-induced transcription factors such as Upc2 and Upc2A as well as homologs of sterol response element binding (SREB) factors. To identify additional regulators of ergosterol biosynthesis in Candida glabrata, an important human fungal pathogen with reduced susceptibility to ergosterol biosynthesis inhibitors relative to other spp., we used a serial passaging strategy to isolate suppressors of the fluconazole hypersusceptibility of a Δ deletion mutant. This led to the identification of loss-of-function mutations in two genes: , the homolog of a hypoxia gene transcriptional suppressor in Saccharomyces cerevisiae, and , a transcription factor that is involved in the regulation of carbon dioxide response in C. glabrata. Here, we describe a detailed analysis of the genetic interaction of and . In the presence of fluconazole, loss of Rox1 function restores expression to the Δ mutant and inhibits the expression of and , leading to increased levels of ergosterol and decreased levels of the toxic sterol 14α methyl-ergosta-8,24(28)-dien-3β, 6α-diol, relative to the Δ mutant. Our observations establish that Rox1 is a negative regulator of gene biosynthesis and indicate that a least one additional positive transcriptional regulator of gene biosynthesis must be present in C. glabrata. Candida glabrata is one of the most important human fungal pathogens and has reduced susceptibility to azole-class inhibitors of ergosterol biosynthesis. Although ergosterol is the target of two of the three classes of antifungal drugs, relatively little is known about the regulation of this critical cellular pathway. Sterols are both essential components of the eukaryotic plasma membrane and potential toxins; therefore, sterol homeostasis is critical for cell function. Here, we identified two new negative regulators in C. glabrata of ergosterol () biosynthesis gene expression. Our results also indicate that in addition to Upc2A, the only known activator of genes, additional positive regulators of this pathway must exist.