Vu, B. | Search bioRxiv

bioRxiv2021

Loss-of-function ROX1 mutations suppress the fluconazole susceptibility of upc2AΔ 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 Candida spp., we used a serial passaging strategy to isolate suppressors of the fluconazole hypersusceptibility of a upc2A{Delta} 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 upc2A{Delta} mutant and inhibits the expression of ERG3 and ERG6, leading to increased levels or ergosterol and decreased levels of the toxic sterol, 14 methyl-ergosta-8,24(28)-dien-3{beta}, 6-diol, relative to upc2A{Delta}. 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. ImportanceCandida 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 of C. glabrata of ergosterol (ERG) biosynthesis gene expression. Our results also indicate that in addition to Upc2A, the only known activator of ERG genes, additional positive regulators of this pathway must exist.s

microbiology↗

bioRxiv2021

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

Azoles, the most commonly used antifungal drugs, specifically inhibit the fungal lanosterol -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. ImportanceAzole 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.

genetics↗