Biology subjects

Moye-Rowley, W. S.

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

16 recordsLinked to original sources

bioRxiv2026

Candida glabrata YPK2 is a multidrug susceptibility locus

The biological conservation between fungi and mammals due to a common ancestor has made development of selective antifungal drugs a difficult challenge. Further complicating this situation is the selection of antifungal drug-resistant organisms during drug treatment. The pathogenic yeast Nakaseomyces glabratus (called here Candida glabrata) presents an especially challenging organism due to its tendency to frequently lose susceptibility to the major antifungal drug class the azoles. Additionally, C. glabrata develops resistance to echinocandin drugs, a second, more recently described antifungal agent at 10 times the rate of other organisms. Previous work has established that the sterol responsive transcriptional regulator Upc2A is a key determinant of azole susceptibility in C. glabrata and plays a role in echinocandin resistance. We used a biochemical approach to identify proteins that co-purified with Upc2A and identified the Ypk2 AGC kinase as an interacting protein. Strains lacking YPK2 exhibited increased susceptibility to fluconazole and the echinocandin caspofungin. A ypk2{Delta} strain failed to normally induce transcription of several ERG genes but exhibited normal induction of the CDR1 ATP-binding cassette transporter gene. Isogenic ypk2{Delta} strains were also highly susceptible to the three major classes of antifungal drugs, indicating that this kinase behaves as a multidrug susceptibility factor. RNA-seq analyses indicated that the transcriptional response to exposure is different for each drug and each response is differentially altered upon loss of Ypk2. Our data indicate that Ypk2 plays an important role in coordinating gene expression that impacts susceptibility to all major antifungal drug classes.

molecular biology↗

bioRxiv2025

The HosA histone deacetylase regulates stress resistance, host cell interactions, and virulence in Aspergillus fumigatus

The capacity of Aspergillus fumigatus to cause invasive pulmonary aspergillosis depends on its ability to adapt to dynamic and stressful microenvironments within the host. Epigenetic regulation, including histone deacetylation, plays a critical role in fungal adaptation to stress. Here, we investigated the role of the class I histone deacetylase (HDAC) HosA in A. fumigatus stress resistance, host cell interactions, and virulence. A {Delta}hosA mutant had increased susceptibility to intracellular oxidant stress induced by menadione. It also had impaired capacity to invade and damage two pulmonary epithelial cell lines in vitro. In a corticosteroid-immunosuppressed mouse model of invasive aspergillosis, mice infected with the {Delta}hosA mutant survived significantly longer than those infected with the wild-type strain, despite having similar pulmonary fungal burden. The {Delta}hosA mutant also induced a weaker inflammatory response than the wild-type strain. Transcriptomic analysis revealed that HosA regulates genes involved in secondary metabolite biosynthesis and energy metabolism, functioning as both an activator and repressor of distinct gene sets. Collectively, these results indicate that HosA is a key epigenetic regulator that governs A. fumigatus interactions with host cells and virulence during invasive pulmonary aspergillosis. ImportanceEpigenetic modifications in A. fumigatus can be induced by environmental changes and stresses such as those induced by interaction with host cells. HosA, a class I histone deacetylase, has been shown to play a key role in regulating secondary metabolism in several Aspergillus species, but its function in A. fumigatus was previously unknown. We found that deletion of hosA increased susceptibility to intracellular, but not extracellular, oxidative stress. The {Delta}hosA mutant also exhibited significantly reduced pulmonary epithelial cell invasion and host cell damage, as well as attenuated virulence in immunosuppressed mice. Together, these findings indicate that HosA functions as a key epigenetic regulator that governs stress resistance, secondary metabolism, and fungal-host interactions. Defining the functions of HosA could provide critical insight into the epigenetic mechanisms that control fungal pathogenicity and may reveal a potential therapeutic target for invasive aspergillosis.

microbiology↗

bioRxiv2025

Regulated proteolysis of green fluorescent protein fusion proteins in Aspergillus fumigatus

Aspergillosis caused by Aspergillus fumigatus is a clinical issue of such severity that the World Health Organization has designated this organism as 1 of the 4 most critical fungi to study. Progress in A. fumigatus has been limited by the availability of genetic tools with which to study this filamentous fungus. Currently available means of altering the dosage of genes and gene products include construction of disruption mutants as well as regulated promoters. These are powerful techniques but somewhat limited for the analysis of essential genes. Here we describe a new method that permits regulated proteolysis of any A. fumigatus protein that can be made as a fusion protein to the well-described Green Fluorescent Protein (GFP) of Aequorea victoria. A GFP fusion protein of interest can be targeted for degradation using a single-chain antibody called a nanobody that recognizes GFP (GFPNb). This GFPNb is in turn fused to an E3 ligase protein called Rnf4 from rat that efficiently ubiquitinates target proteins. A fusion gene was constructed under control of a doxycycline-inducible promoter that produced a GFPNb-Rnf4 fusion protein in A. fumigatus. Here we show that production of this GFPNb-Rnf4 protein led to the rapid proteolysis of a variety of GFP fusion proteins. Additionally, we found that some GFP fusion proteins triggered a corresponding genomic response when their degradation was induced while others were simply degraded. These studies provide a new means to directly regulate protein levels in A. fumigatus and generate new alleles of genes exposing the underlying regulatory circuitry. ImportanceAspergillus fumigatus is the major filamentous fungal pathogen of such importance in infectious disease that it has been designated as one of the 4 most critical fungal organisms to study. One of the limiting capabilities in the analysis of A. fumigatus is the limiting genetic toolbox that can be employed in this fungus. Here we describe the first system that allows the regulated degradation of a protein of interest using a recombinant single protein chain antibody (nanobody) directed against Green Fluorescent Protein (GFP). GFP is routinely used to construct fusion proteins that in turn allow localization of the fluorescent protein and immunological detection. Here we use this nanobody to deliver a ubiquitin ligase that in turn causes rapid depletion of GFP fusion proteins. This allows direct perturbation of protein levels in A. fumigatus, a feature not previously accessible to experimentation.

molecular biology↗

bioRxiv2024

Similarities and distinctions in the activation of the Candida glabrata Pdr1 regulatory pathway by azole and non-azole drugs

Incidences of fluconazole (FLC) resistance among Candida glabrata clinical isolates is a growing issue in clinics. The pleiotropic drug response (PDR) network in C. glabrata confers azole resistance and is defined primarily by the Zn2Cys6 zinc cluster-containing transcription factor Pdr1 and target genes such as CDR1, that encodes an ATP-binding cassette transporter protein thought to act as a FLC efflux pump. Mutations in the PDR1 gene that render the transcription factor hyperactive are the most common cause of fluconazole resistance among clinical isolates. The phenothiazine class drug fluphenazine and a molecular derivative, CWHM-974, which both exhibit antifungal properties, have been shown to induce the expression of Cdr1 in Candida spp. We have used a firefly luciferase reporter gene driven by the CDR1 promoter to demonstrate two distinct patterns of CDR1 promoter activation kinetics: gradual promoter activation kinetics that occur in response to ergosterol limitations imposed by exposure to azole and polyene class antifungals and a robust and rapid CDR1 induction occurring in response to the stress imposed by fluphenazines. We can attribute these different patterns of CDR1 induction as proceeding through the promoter region of this gene since this is the only segment of the gene included in the luciferase reporter construct. Genetic analysis indicates that the signaling pathways responsible for phenothiazine and azole induction of CDR1 overlap but are not identical. The short time course of phenothiazine induction suggests that these compounds may act more directly on the Pdr1 protein to stimulate its activity. ImportanceCandida glabrata has emerged as the second-leading cause of candidiasis due in part to its ability to acquire high level resistance to azole drugs, a major class of antifungal, that acts to block the biosynthesis of the fungal sterol ergosterol. The presence of azole drugs causes the induction of a variety of genes involved in controlling susceptibility to this drug class including drug transporters and ergosterol biosynthetic genes such as ERG11. We found that the presence of azole drugs leads to an induction of genes encoding drug transporters and ERG11, while exposure of C. glabrata cells to antifungals of the phenothiazine class of drugs caused a much faster and larger induction of drug transporters but not ERG11. Coupled with further genetic analyses of the effects of azole and phenothiazine drugs, our data indicate that these compounds are sensed and responded to differentially in the yeast cell.

molecular biology↗

bioRxiv2024

Overlapping coactivator function is required for transcriptional activation by the Candida glabrata Pdr1 transcription factor

Azole resistance in the pathogenic yeast Candida glabrata is a serious clinical complication and increasing in frequency. The majority of resistant organisms have been found to contain a substitution mutation in the Zn2Cys6 zinc cluster-containing transcription factor Pdr1. These mutations typically lead to this factor driving high, constitutive expression of target genes like the ATP-binding cassette transporter-encoding gene CDR1. Overexpression of Cdr1 is required for the observed elevated fluconazole resistance exhibited by strains containing one of these hyperactive PDR1 alleles. While the identity of hyperactive PDR1 alleles has been extensively documented, the mechanisms underlying how these gain-of-function (GOF) forms of Pdr1 lead to elevated target gene transcription are not well understood. We have used a tandem affinity purification (TAP)-tagged form of Pdr1 to identify coactivator proteins that biochemically purify with the wild-type and two different GOF forms of Pdr1. Three coactivator proteins were found to associate with Pdr1: the SWI/SNF complex Snf2 chromatin remodeling protein and two different components of the SAGA complex, Spt7 and Ngg1. We found that deletion mutants lacking either SNF2 or SPT7 exhibited growth defects, even in the absence of fluconazole challenge. To overcome these issues, we employed a conditional degradation system to acutely deplete these coactivators and determined that loss of either coactivator complex, SWI/SNF or SAGA, caused defects in Pdr1-dependent transcription. A double degron strain that could be depleted for both SWI/SNF and SAGA exhibited a profound defect in PDR1 autoregulation, revealing that these complexes work together to ensure high level Pdr1-dependent gene transcription.

genetics↗

bioRxiv2024

Loss of a conserved C-terminal region of the Aspergillus fumigatus AtrR transcriptional regulator leads to a gene-specific defect in target gene expression

Treatment of fungal infections associated with the filamentous fungus Aspergillus fumigatus is becoming more problematic as this organism is developing resistance to the main chemotherapeutic drug at an increasing rate. Azole drugs represent the current standard-of-care in treatment of aspergillosis with this drug class acting by inhibiting a key step in biosynthesis of the fungal sterol ergosterol. Azole compounds block the activity of the lanosterol -14 demethylase, encoded by the cyp51A gene. A common route of azole resistance involves an increase in transcription of cyp51A. This transcriptional increase requires the function of a Zn2Cys6 DNA-binding domain-containing transcription activator protein called AtrR. AtrR was identified through its action as a positive regulator of expression of an ATP-binding cassette transporter (abcC/cdr1B here called abcG1). Using both deletion and alanine scanning mutagenesis, we demonstrate that a conserved C-terminal domain in A. fumigatus is required for expression of abcG1 but dispensable for cyp51A transcription. This domain is also found in several other fungal pathogen AtrR homologues consistent with a conserved gene-selective function of this protein segment being conserved. Using RNA-seq, we find that this gene-specific transcriptional defect extends to several other membrane transporter-encoding genes including a second ABC transporter locus. Our data reveal that AtrR uses at least two distinct mechanisms to induce gene expression and that normal susceptibility to azole drugs cannot be provided by maintenance of wild-type expression of the ergosterol biosynthetic pathway when ABC transporter expression is reduced. ImportanceAspergillus fumigatus is the primary human filamentous fungal pathogen. The principal chemotherapeutic drug used to control infections associated with A. fumigatus are the azole compounds. These drugs are well-tolerated and effective but resistance is emerging at an alarming rate. Most resistance is associated with mutations that lead to overexpression of the azole target enzyme, lanosterol -14 demethylase, encoded by the cyp51A gene. A key regulator of cyp51A gene expression is the transcription factor AtrR. Very little is known of the molecular mechanisms underlying the effect of AtrR on gene expression. Here we use deletion and clustered amino acid substitution mutagenesis to map a region of AtrR that confers gene-specific activation on target genes of this transcription factor. This region is highly conserved across AtrR homologues from other pathogenic species arguing that its importance in transcriptional regulation is maintained across evolution.

genetics↗

bioRxiv2023

Calcineurin is required for Candida glabrata Pdr1 transcriptional activation

Fluconazole is the most commonly used antifungal today. A result of this has been the inevitable selection of fluconazole resistant organisms. This is an especially acute problem in the pathogenic yeast Candida glabrata. Elevated minimal inhibitory concentrations (MICs) for fluconazole in C. glabrata are frequently associated with substitution mutations within the Zn2Cys6 zinc cluster-containing transcription factor-encoding gene PDR1. These mutant Pdr1 regulators drive constitutively high expression of target genes like CDR1 that encodes an ATP-binding cassette transporter thought to act as a drug efflux pump. Exposure of C. glabrata to fluconazole induced expression of both Pdr1 and CDR1, although little is known of the molecular basis underlying the upstream signals that trigger Pdr1 activation. Here, we show that the protein phosphatase calcineurin is required for fluconazole-dependent induction of Pdr1 transcriptional regulation. Calcineurin catalytic activity is required for normal Pdr1 regulation and a hyperactive form of this phosphatase can increase resistance to the echinocandin caspofungin but does not show a similar elevation for fluconazole resistance. Loss of calcineurin from strains expressing two different gain-of-function forms of Pdr1 also caused a decrease in CDR1 expression and fluconazole resistance, demonstrating that even these hyperactive Pdr1 regulatory mutants cannot bypass the requirement for calcineurin. Our data implicate calcineurin activity as a link tying azole and echinocandin resistance together via the control of transcription factor activity. ImportanceWhile drug resistant microorganisms are a problem in treatment of all infectious disease, this is an especially acute problem with fungi due to the existence of only 3 classes of antifungal drugs, including the azole drug fluconazole. In the pathogenic yeast Candida glabrata, mutant forms of a transcription factor called Pdr1 are commonly associated with fluconazole resistance and poor clinical outcomes. Here we identify a protein phosphatase called calcineurin that is required for fluconazole-dependent induction of Pdr1 transcriptional activation and associated drug resistance. Gain-of-function mutant forms of Pdr1 still required the presence of calcineurin to confer normally elevated fluconazole resistance. Previous studies showed that calcineurin is required for resistance to the echinocandin class of antifungal drugs and our data demonstrate this protein phosphatase is also required for azole drug resistance. Calcineurin plays a central role in resistance to two of the three major classes of antifungal drugs in C. glabrata.

microbiology↗

bioRxiv2023

Transcription factor FfmA interacts both physically and genetically with AtrR to properly regulate gene expression in the fungus Aspergillus fumigatus

Transcriptional regulation of azole resistance in the filamentous fungus Aspergillus fumigatus is a key step in development of this problematic clinical phenotype. We and others have previously described a C2H2-containing transcription factor called FfmA that is required for normal levels of voriconazole susceptibility and expression of an ATP-binding cassette transporter gene called abcG1. Null alleles of ffmA exhibit a strongly compromised growth rate even in the absence of any external stress. Here we employ an acutely repressible doxycycline-off form of ffmA to rapidly deplete FfmA protein from the cell. Using this approach, we carried out RNA-seq analyses to probe the transcriptome of A. fumigatus cells that have been deprived of normal FfmA levels. We found that 2000 genes were differentially expressed upon depletion of FfmA, consistent with the wide-ranging effect of this factor on gene regulation. Chromatin immunoprecipitation coupled with high throughput DNA sequencing analysis (ChIP-seq) identified 530 genes that were bound by FfmA using two different antibodies for immunoprecipitation. More than 300 of these genes were also bound by AtrR demonstrating the striking regulatory overlap with FfmA. However, while AtrR is clearly an upstream activation protein with clear sequence specificity, our data suggest that FfmA is a chromatin-associated factor that may bind to DNA in a manner dependent on other factors. We provide evidence that AtrR and FfmA interact in the cell and can influence one anothers expression. This interaction of AtrR and FfmA is required for normal azole resistance in A. fumigatus.

genetics↗

bioRxiv2023

Rapid, efficient auxin-inducible protein degradation in Candida pathogens

A variety of inducible protein degradation (IPD) systems have been developed as powerful tools for protein functional characterization. IPD systems provide a convenient mechanism for rapid inactivation of almost any target protein of interest. Auxin-inducible degradation (AID) is one of the most common IPD systems and has been established in diverse eukaryotic research model organisms. Thus far, IPD tools have not been developed for use in pathogenic fungal species. Here, we demonstrate that the original AID and the second generation AID2 systems work efficiently and rapidly in the human pathogenic yeasts Candida albicans and Candida glabrata. We developed a collection of plasmids that support AID system use in laboratory strains of these pathogens. These systems can induce >95% degradation of target proteins within minutes. In the case of AID2, maximal degradation was achieved at low nanomolar concentrations of the synthetic auxin analog 5-adamantyl-indole-3-acetic acid (5-Ad-IAA). Auxin-induced target degradation successfully phenocopied gene deletions in both species. The system should be readily adaptable to other fungal species and to clinical pathogen strains. Our results define the AID system as a powerful and convenient functional genomics tool for protein characterization in fungal pathogens.

molecular biology↗

bioRxiv2023

CWHM-974 is a fluphenazine derivative with improved antifungal activity against Candida albicans due to reduced susceptibility to multidrug transporter-mediated resistance mechanisms

Multidrug resistance (MDR) transporters such as ATP Binding Cassette (ABC) and Major Facilitator Superfamily (MFS) proteins are important mediators of antifungal drug resistance, particularly with respect to azole class drugs. Consequently, identifying molecules that are not susceptible to this mechanism of resistance is an important goal for new antifungal drug discovery. As part of a project to optimize the antifungal activity of clinically used phenothiazines, we synthesized a fluphenazine derivative (CWHM-974) with 8-fold higher activity against Candida spp. compared to the fluphenazine and with activity against Candida spp. with reduced fluconazole susceptibility due to increased multidrug resistance transporters. Here, we show that the improved C. albicans activity is because fluphenazine induces its own resistance by triggering expression of CDR transporters while CWHM-974 induces expression but does not appear to be a substrate for the transporters or is insensitive to their effects through other mechanisms. We also found that fluphenazine and CWHM-974 are antagonistic with fluconazole in C. albicans but not in C. glabrata, despite inducing CDR1 expression to high levels. Overall, CWHM-974 represents a unique example of a medicinal chemistry-based conversion of chemical scaffold from MDR-sensitive to MDR-resistant and, hence, active against fungi that have developed resistance to clinically used antifungals such as the azoles.

microbiology↗

bioRxiv2022

Biochemical identification of a nuclear coactivator protein required for AtrR-dependent gene regulation in Aspergillus fumigatus

Azole drugs represent the primary means of treating infections associated with the filamentous fungal pathogen Aspergillus fumigatus. A central player in azole resistance is the Zn2Cys6 zinc cluster-containing transcription factor AtrR. This factor stimulates expression of the both the cyp51A gene encoding the azole drug target enzyme as well as an ATP-binding cassette transporter-encoding gene called abcG1 (aka cdr1B). We have used a fusion protein between AtrR and the tandem affinity purification (TAP) moiety to purify proteins that associate with AtrR from A. fumigatus. Protein fractions associated with AtrR-TAP were subjected to MudPIT mass spectrometry and one of the proteins identified was encoded by the AFUA_6g08010 gene. We have designated this protein NcaA (Nuclear Co-Activator of AtrR). Loss of ncaA caused a reduction in voriconazole resistance and drug-induced abcG1 expression, although did not impact induction of cyp51A transcription. We confirmed association of AtrR and NcaA by co-immunoprecipitation from otherwise wild-type cells. Expression of fusion proteins between AtrR and NcaA with green fluorescent protein allowed determination that these two proteins were localized in the A. fumigatus nucleus. Together, these data support the view that NcaA is required for nuclear gene transcription controlled by AtrR. ImportanceAspergillus fumigatus is the major filamentous fungal pathogen in humans and is susceptible to the azole antifungal class of drugs. However, loss of azole susceptibility has been detected with increasing frequency in the clinic and infections associated with these azole resistant isolates linked to treatment failure and worse outcomes. Many of these azole resistant mutant strains contain mutant alleles of the cyp51A gene encoding the azole drug target. A transcription factor essential for cyp51A gene transcription has been identified and designated AtrR. AtrR is required for azole inducible cyp51A transcription but we know little of the regulation of this transcription factor. Using a biochemical approach, we identify a new protein called NcaA that is involved in regulation of AtrR at certain target gene promoters. Understanding the mechanisms controlling AtrR function is an important goal in preventing or reversing azole resistance in this pathogen.

microbiology↗

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↗