Loss-of-function ROX1 mutations suppress the fluconazole susceptibility of upc2AΔ mutation in Candida glabrata, implicating additional positive regulators of ergosterol biosynthesis

Abstract

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 Candida spp., we used a serial passaging strategy to isolate suppressors of the fluconazole hypersusceptibility of a upc2AΔ deletion mutant. This led to the identification of loss of function mutants in two genes: ROX1, the homolog of a hypoxia gene transcriptional suppressor in Saccharomyces cerevisiae, and CST6, 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 ROX1 and UPC2A. In the presence of fluconazole, loss of Rox1 function restores ERG11 expression to the upc2AΔ mutant and inhibits the expression of ERG3 and ERG6, leading to increased levels or ergosterol and decreased levels of the toxic sterol, 14α methyl-ergosta-8,24(28)-dien-3β, 6α-diol, relative to upc2AΔ. Our observations establish that Rox1 is a negative regulator of ERG gene biosynthesis and indicate that a least one additional positive transcriptional regulator of ERG gene biosynthesis must be present in C. glabrata.

Modulation of yeast Erg1 expression and terbinafine susceptibility by iron bioavailability

“…Here, we show that ROX1 deletion leads to an increase in ERG1 transcript and protein levels under both iron‐sufficient and iron‐deficiency conditions (Figure 4 ), strongly suggesting that Rox1 also limits ERG1 gene expression when iron bioavailability decreases. The regulation of ERG genes by Rox1 has just been reported to be conserved in the pathogen Candida glabrata (Ollinger et al, 2021 ). On the other hand, the mRNA‐binding proteins Cth1 and Cth2 post‐transcriptionally inhibit the expression of multiple ARE‐containing mRNAs during adaptation to iron deficiency (Puig et al, 2005 ; Puig et al, 2008 ; Ramos‐Alonso et al, 2018 ).…”