Biology subjects

Moye-Rowley, W. S.

Publications and source records attributed to Moye-Rowley, W. S..

5 recordsLinked to original sources

bioRxiv2022

Complex and critical roles for the AtrR transcription factor in control of cyp51A expression in Aspergillus fumigatus

Aspergillus fumigatus is the major filamentous fungal pathogen in humans. The gold standard treatment of A. fumigatus is based on azole drug use but the appearance of azole-resistant isolates is increasing at an alarming rate. The cyp51A gene encodes the enzymatic target of azole drugs and azole-resistant alleles of cyp51A often have an unusual genetic structure containing a duplication of a 34 or 46 bp region in the promoter causing enhanced gene transcription. These tandem repeats are called TR34/TR46 and produce duplicated binding sites for the SrbA and AtrR transcription factors. Using site-directed mutagenesis, we demonstrate that both the SrbA (sterol response element: SRE) and AtrR binding sites (AtrR response element: ATRE) are required for normal cyp51A gene expression. Loss of either the SRE or ATRE from the distal 34 bp repeat of the TR34 promoter (further 5 from the transcription start site) caused loss of expression of cyp51A and decreased voriconazole resistance. Surprisingly, loss of these same binding sites from the proximal 34 or 46 bp repeat led to increased cyp51A expression and voriconazole resistance. These data indicate that these duplicated regions in the cyp51A promoter function differently. Our findings suggest that the proximal 34 or 46 bp repeat in cyp51A recruits a corepressor that requires multiple factors to act while the distal repeat is free of this repression and provides the elevated cyp51A expression caused by these promoter duplications. ImportanceAspergillus fumigatus is the most common human filamentous fungal pathogen. Azole drugs are the current therapy of choice for A. fumigatus but the prevalence of azole resistance is increasing. The main genetic alteration causing azole resistance is a change in the cyp51A gene that encodes the target of these drugs. Azole-resistant cyp51A alleles routinely contain duplications in their promoter regions that cause increased gene transcription. Here, we demonstrate that clinical isolates containing a 34 or 46 bp duplication in the cyp51A promoter required the presence of the transcription factor-encoding atrR gene to exhibit elevated azole resistance. Elimination of transcription factor binding sites in the cyp51A gene have differential actions on expression of the resulting mutant allele. These data dissect the molecular inputs to cyp51A transcription and reveal a complicated function of the promoter of this gene that is critical in azole resistance.

microbiology↗

bioRxiv2021

Aspergillus fumigatus ffmA encodes a C2H2-containing transcriptional regulator that modulates azole resistance and is required for normal growth

The production of a collection of disruption mutant strains corresponding to a large number of transcription factors from the filamentous fungal pathogen Aspergillus fumigatus has permitted rapid identification of transcriptional regulators involved in a range of different processes. Here we characterize a gene designated ffmA (favors fermentative metabolism) as an C2H2-containing transcription factor that is required for azole drug resistance and normal growth. Loss of ffmA caused cells to exhibit significant defects in growth, either under untreated or azole-challenged conditions. Loss of FfmA caused a reduction in expression of the AbcG1 ATP-binding cassette transporter, previousy shown to contribute to azole resistance. Strikingly, overproduction of the AtrR transcription factor gene restored a wild-type growth phenotype to a ffmA{Delta} strain. Overexpression of AtrR also suppressed the defect in AbcG1 expression caused by loss of FfmA. Replacement of the ffmA promoter with a doxycycline-repressible promoter restored near normal growth in the absence of doxycycline. Finally, chromatin immunoprecipitation experiments indicated that FfmA bound to its own promoter as well as to the abcG1 promoter. These data imply that FfmA and AtrR interact both with respect to abcG1 expression and also more broadly to regulate hyphal growth. ImportanceInfections associated with azole-resistant forms of the primary human pathogen, Aspergillus fumigatus, are associated with poor outcomes in patient populations. This makes analysis of the mechanisms underlying azole resistance of A. fumigatus a high priority. In this work, we describe characterization of a gene designated ffmA that encodes a sequence-specific transcriptional regulator. We identified ffmA in a screen of a collection of gene disruption mutant strains made in A. fumigatus. Loss of ffmA caused sensitivity to azole drugs and also a large reduction in normal growth. We found that overproduction of the AtrR transcription factor was able to restore growth to ffmA null cells. We provide evidence that FfmA can recognize promoters of genes involved in azole resistance as well as the ffmA promoter itself. Our data indicate that FfmA and AtrR interact to support azole resistance and normal growth.

microbiology↗

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↗

bioRxiv2021

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

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.

genetics↗