scholarly journals 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

2021 ◽  
Author(s):  
Bao Gia Vu ◽  
Mark A. Stamnes ◽  
Yu Li ◽  
P. David Rogers ◽  
W. Scott Moye-Rowley
Keyword(s):  
Transcription Factor ◽  
Membrane Protein ◽  
Candida Glabrata ◽  
Target Genes ◽  
Antifungal Drugs ◽  
Azole Resistance ◽  
Mutant Form ◽  
Pathogenic Yeast ◽  
Zinc Cluster ◽  
Genes Encoding

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.

scholarly journals The Candida glabrata Upc2A transcription factor is a global regulator of antifungal drug resistance pathways

PLoS Genetics ◽  
2021 ◽  
Vol 17 (9) ◽  
pp. e1009582
Author(s):  
Bao Gia Vu ◽  
Mark A. Stamnes ◽  
Yu Li ◽  
P. David Rogers ◽  
W. Scott Moye-Rowley
Keyword(s):  
Transcription Factor ◽  
Membrane Protein ◽  
Candida Glabrata ◽  
Target Genes ◽  
Protein Biosynthesis ◽  
Antifungal Drugs ◽  
Azole Resistance ◽  
Mutant Form ◽  
Pathogenic Yeast ◽  
Genes Encoding

The most commonly used antifungal drugs are the azole compounds, which 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. Analysis of Upc2A genomic binding sites demonstrated that wild-type Upc2A binding to target genes was strongly induced by fluconazole while G898D Upc2A bound similarly, irrespective of drug treatment. Transcriptomic analyses revealed that, in addition to the well-described ERG genes, a large group of genes encoding components of the translational apparatus along with membrane proteins were responsive to Upc2A. 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 and also modulates resistance to caspofungin. These studies provide a new perspective of Upc2A as a master regulator of lipid and membrane protein biosynthesis.

scholarly journals A Zinc Cluster Transcription Factor Contributes to the Intrinsic Fluconazole Resistance of Candida auris

mSphere ◽  
2020 ◽  
Vol 5 (2) ◽  
Author(s):  
Eva-Maria Mayr ◽  
Bernardo Ramírez-Zavala ◽  
Ines Krüger ◽  
Joachim Morschhäuser
Keyword(s):  
Transcription Factor ◽  
Efflux Pumps ◽  
Azole Resistance ◽  
Drug Resistant ◽  
Candida Auris ◽  
Pathogenic Yeast ◽  
Fluconazole Resistance ◽  
Content Type ◽  
Zinc Cluster ◽  
Genes Encoding

ABSTRACT The recently emerged pathogenic yeast Candida auris is a major concern for human health, because it is easily transmissible, difficult to eradicate from hospitals, and highly drug resistant. Most C. auris isolates are resistant to the widely used antifungal drug fluconazole due to mutations in the target enzyme Erg11 and high activity of efflux pumps, such as Cdr1. In the well-studied, distantly related yeast Candida albicans, overexpression of drug efflux pumps also is a major mechanism of acquired fluconazole resistance and caused by gain-of-function mutations in the zinc cluster transcription factors Mrr1 and Tac1. In this study, we investigated a possible involvement of related transcription factors in efflux pump expression and fluconazole resistance of C. auris. The C. auris genome contains three genes encoding Mrr1 homologs and two genes encoding Tac1 homologs, and we generated deletion mutants lacking these genes in two fluconazole-resistant strains from clade III and clade IV. Deletion of TAC1b decreased the resistance to fluconazole and voriconazole in both strain backgrounds, demonstrating that the encoded transcription factor contributes to azole resistance in C. auris strains from different clades. CDR1 expression was not or only minimally affected in the mutants, indicating that Tac1b can confer increased azole resistance by a CDR1-independent mechanism. IMPORTANCE Candida auris is a recently emerged pathogenic yeast that within a few years after its initial description has spread all over the globe. C. auris is a major concern for human health, because it can cause life-threatening systemic infections, is easily transmissible, and is difficult to eradicate from hospital environments. Furthermore, C. auris is highly drug resistant, especially against the widely used antifungal drug fluconazole. Mutations in the drug target and high activity of efflux pumps are associated with azole resistance, but it is not known how drug resistance genes are regulated in C. auris. We have investigated the potential role of several candidate transcriptional regulators in the intrinsic fluconazole resistance of C. auris and identified a transcription factor that contributes to the high resistance to fluconazole and voriconazole of two C. auris strains from different genetic clades, thereby providing insight into the molecular basis of drug resistance of this medically important yeast.

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

2021 ◽  
Author(s):  
Bao Vu ◽  
W. Scott Moye-Rowley
Keyword(s):  
Transcription Factor ◽  
Candida Glabrata ◽  
Double Mutant ◽  
Antifungal Drugs ◽  
Cross Resistance ◽  
Ergosterol Biosynthesis ◽  
Azole Resistance ◽  
Pathogenic Yeast ◽  
Fluconazole Resistance ◽  
Growth Defects

Azoles remain the most common used antifungal drugs for invasive candidiasis worldwide. They specifically inhibit the fungal lanosterol a-14 demethylase enzyme, which is commonly referred to as Erg11 in fungi. 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 azole resistance. Candida glabrata is also a pathogenic yeast that has a low intrinsic susceptibility to azole drugs and easily develops elevated resistance. These azole resistant mutations are almost exclusively found to 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 associated mutations in 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. The azole resistance caused by the DM allele of ERG11 imposed a fitness cost that was not observed with hyperactive PDR1 alleles. These data support the view that C. glabrata does not typically acquire ERG11 mutations owing to growth defects associated with these lesions while hyperactive PDR1 alleles have no obvious growth issues. 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.

Azole-resistant alleles of ERG11 in Candida glabrata trigger activation of the Pdr1 and Upc2A transcription factors.

2022 ◽  
Author(s):  
Bao Gia Vu ◽  
W. Scott Moye-Rowley
Keyword(s):  
Transcription Factor ◽  
Candida Albicans ◽  
Candida Glabrata ◽  
Double Mutant ◽  
Antifungal Drugs ◽  
Cross Resistance ◽  
Ergosterol Biosynthesis ◽  
Azole Resistance ◽  
Pathogenic Yeast ◽  
Cellular Effects

Azoles, the most commonly used antifungal drugs, specifically inhibit the fungal lanosterol α-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. In C. glabrata , 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 .

scholarly journals A Hyperactive Form of the Zinc Cluster Transcription Factor Stb5 CausesYOR1Overexpression and Beauvericin Resistance inCandida albicans

2018 ◽  
Vol 62 (12) ◽  
Author(s):  
Bernardo Ramírez-Zavala ◽  
Hannah Manz ◽  
Frank Englert ◽  
P. David Rogers ◽  
Joachim Morschhäuser
Keyword(s):  
Transcription Factor ◽  
Target Genes ◽  
Efflux Pump ◽  
Hyphal Growth ◽  
Pathogenic Yeast ◽  
Fluconazole Resistance ◽  
Content Type ◽  
Activating Mutations ◽  
Zinc Cluster ◽  
Constitutive Overexpression

ABSTRACTGain-of-function mutations in the zinc cluster transcription factors Mrr1, Tac1, and Upc2, which result in constitutive overexpression of their target genes, are a frequent cause of fluconazole resistance in the pathogenic yeastCandida albicans. In this study, we show that an activated form of another zinc cluster transcription factor, Stb5, confers resistance to the natural compound beauvericin via the overexpression ofYOR1, encoding an efflux pump of the ATP-binding cassette transporter superfamily. Beauvericin was recently shown to potentiate the activity of azole drugs againstC. albicans. Although Yor1 did not contribute to fluconazole resistance whenC. albicanscells were treated with the drug alone, Stb5-mediatedYOR1overexpression diminished the synergistic effect of the fluconazole-beauvericin combination, thereby enhancing fluconazole resistance in beauvericin-treatedC. albicanscells. Stb5-mediatedYOR1overexpression also suppressed the inhibition of hyphal growth, an important virulence trait ofC. albicans, by beauvericin. Therefore, activating mutations in Stb5, which result in constitutiveYOR1overexpression, may enableC. albicansto acquire resistance to beauvericin and thereby overcome both the sensitization to azole drugs and the inhibition of morphogenesis caused by this compound.

scholarly journals Candida glabrata Drug:H+Antiporter CgQdr2 Confers Imidazole Drug Resistance, Being Activated by Transcription Factor CgPdr1

2013 ◽  
Vol 57 (7) ◽  
pp. 3159-3167 ◽  
Author(s):  
Catarina Costa ◽  
Carla Pires ◽  
Tânia R. Cabrito ◽  
Adeline Renaudin ◽  
Michiyo Ohno ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Transcription Factor ◽  
Multidrug Resistance ◽  
Candida Glabrata ◽  
Antifungal Drugs ◽  
Antifungal Drug ◽  
Major Facilitator Superfamily ◽  
Pathogenic Yeast ◽  
Content Type ◽  
Antifungal Drug Resistance

ABSTRACTThe widespread emergence of antifungal drug resistance poses a severe clinical problem. Though predicted to play a role in this phenomenon, the drug:H+antiporters (DHA) of the major facilitator superfamily have largely escaped characterization in pathogenic yeasts. This work describes the first DHA from the pathogenic yeastCandida glabratareported to be involved in antifungal drug resistance, theC. glabrata QDR2(CgQDR2) gene (ORFCAGL0G08624g). The expression ofCgQDR2inC. glabratawas found to confer resistance to the antifungal drugs miconazole, tioconazole, clotrimazole, and ketoconazole. By use of a green fluorescent protein (GFP) fusion, the CgQdr2 protein was found to be targeted to the plasma membrane inC. glabrata. In agreement with these observations,CgQDR2expression was found to decrease the intracellular accumulation of radiolabeled clotrimazole inC. glabrataand to play a role in the extrusion of this antifungal from preloaded cells. Interestingly, the functional heterologous expression ofCgQDR2in the model yeastSaccharomyces cerevisiaefurther confirmed the role of this gene as a multidrug resistance determinant: its expression was able to complement the susceptibility phenotype exhibited by itsS. cerevisiaehomologue,QDR2, in the presence of imidazoles and of the antimalarial and antiarrhythmic drug quinidine. In contrast to the findings reported for Qdr2, CgQdr2 expression does not contribute to the ability of yeast to grow under K+-limiting conditions. Interestingly,CgQDR2transcript levels were seen to be upregulated inC. glabratacells challenged with clotrimazole or quinidine. This upregulation was found to depend directly on the transcription factor CgPdr1, the major regulator of multidrug resistance in this pathogenic yeast, which has also been found to be a determinant of quinidine and clotrimazole resistance inC. glabrata.

scholarly journals Evidence that Ergosterol Biosynthesis Modulates Activity of the Pdr1 Transcription Factor inCandida glabrata

mBio ◽  
2019 ◽  
Vol 10 (3) ◽  
Author(s):  
Bao Gia Vu ◽  
Grace Heredge Thomas ◽  
W. Scott Moye-Rowley
Keyword(s):  
Transcription Factor ◽  
Candida Glabrata ◽  
Ergosterol Biosynthesis ◽  
Azole Resistance ◽  
Atp Binding ◽  
Pathogenic Yeast ◽  
Content Type ◽  
Atp Binding Cassette Transporter ◽  
Encoding Gene ◽  
Atp Binding Cassette

ABSTRACTA crucial limitation in antifungal chemotherapy is the limited number of antifungal drugs currently available. Azole drugs represent the most commonly used chemotherapeutic, and loss of efficacy of these drugs is a major risk factor in successful treatment of a variety of fungal diseases.Candida glabratais a pathogenic yeast that is increasingly found associated with bloodstream infections, a finding likely contributed to by its proclivity to develop azole drug resistance.C. glabrataoften acquires azole resistance via gain-of-function (GOF) mutations in the transcription factor Pdr1. These GOF forms of Pdr1 drive elevated expression of target genes, including the ATP-binding cassette transporter-encodingCDR1locus. GOF alleles ofPDR1have been extensively studied, but little is known of how Pdr1 is normally regulated. Here we test the idea that reduction of ergosterol biosynthesis (as occurs in the presence of azole drugs) might trigger activation of Pdr1 function. Using two different means of genetically inhibiting ergosterol biosynthesis, we demonstrated that Pdr1 activity and target gene expression are elevated in the absence of azole drug. Blocks at different points in the ergosterol pathway lead to Pdr1 activation as well as to induction of other genes in this pathway. Delivery of the signal from the ergosterol pathway to Pdr1 involves the transcription factor Upc2A, anERGgene regulator. We show that Upc2A binds directly to thePDR1andCDR1promoters. Our studies argue for a physiological link between ergosterol biosynthesis and Pdr1-dependent gene regulation that is not restricted to efflux of azole drugs.IMPORTANCEA likely contributor to the increased incidence of non-albicanscandidemias involvingCandida glabratais the ease with which this yeast acquires azole resistance, in large part due to induction of the ATP-binding cassette transporter-encoding geneCDR1. Azole drugs lead to induction of Pdr1 transactivation, with a central model being that this factor binds these drugs directly. Here we provide evidence that Pdr1 is activated without azole drugs by the use of genetic means to inhibit expression of azole drug target-encoding geneERG11. These acute reductions in Erg11 levels lead to elevated Pdr1 activity even though no drug is present. A key transcriptional regulator of theERGpathway, Upc2A, is shown to directly bind to thePDR1andCDR1promoters. We interpret these data as support for the view that Pdr1 function is responsive to ergosterol biosynthesis and suggest that this connection reveals the normal physiological circuitry in which Pdr1 participates.

scholarly journals Transcription Factor ADS-4 Regulates Adaptive Responses and Resistance to Antifungal Azole Stress

2015 ◽  
Vol 59 (9) ◽  
pp. 5396-5404 ◽  
Author(s):  
Kangji Wang ◽  
Zhenying Zhang ◽  
Xi Chen ◽  
Xianyun Sun ◽  
Cheng Jin ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Efflux Pump ◽  
Fungal Infections ◽  
Antifungal Drugs ◽  
Bzip Transcription Factor ◽  
Azole Resistance ◽  
Homologous Gene ◽  
Adaptive Responses ◽  
Transcriptional Responses ◽  
Content Type

ABSTRACTAzoles are commonly used as antifungal drugs or pesticides to control fungal infections in medicine and agriculture. Fungi adapt to azole stress by rapidly activating the transcription of a number of genes, and transcriptional increases in some azole-responsive genes can elevate azole resistance. The regulatory mechanisms that control transcriptional responses to azole stress in filamentous fungi are not well understood. This study identified a bZIP transcription factor, ADS-4 (antifungaldrugsensitive-4), as a new regulator of adaptive responses and resistance to antifungal azoles. Transcription ofads-4inNeurospora crassacells increased when they were subjected to ketoconazole treatment, whereas the deletion ofads-4resulted in hypersensitivity to ketoconazole and fluconazole. In contrast, the overexpression ofads-4increased resistance to fluconazole and ketoconazole inN. crassa. Transcriptome sequencing (RNA-seq) analysis, followed by quantitative reverse transcription (qRT)-PCR confirmation, showed that ADS-4 positively regulated the transcriptional responses of at least six genes to ketoconazole stress inN. crassa. The gene products of four ADS-4-regulated genes are known contributors to azole resistance, including the major efflux pump CDR4 (Pdr5p ortholog), an ABC multidrug transporter (NcAbcB), sterol C-22 desaturase (ERG5), and a lipid transporter (NcRTA2) that is involved in calcineurin-mediated azole resistance. Deletion of theads-4-homologous gene Afads-4inAspergillus fumigatuscaused hypersensitivity to itraconazole and ketoconazole, which suggested that ADS-4 is a functionally conserved regulator of adaptive responses to azoles. This study provides important information on a new azole resistance factor that could be targeted by a new range of antifungal pesticides and drugs.

scholarly journals Functional Dissection of a Candida albicans Zinc Cluster Transcription Factor, the Multidrug Resistance Regulator Mrr1

2011 ◽  
Vol 10 (8) ◽  
pp. 1110-1121 ◽  
Author(s):  
Sabrina Schubert ◽  
Christina Popp ◽  
P. David Rogers ◽  
Joachim Morschhäuser
Keyword(s):  
Transcription Factor ◽  
Candida Albicans ◽  
Transcriptional Activation ◽  
Efflux Pump ◽  
Major Facilitator Superfamily ◽  
Constitutive Activity ◽  
Activation Domain ◽  
Pathogenic Yeast ◽  
Content Type ◽  
Zinc Cluster

ABSTRACTThe overexpression of theMDR1gene, which encodes a multidrug efflux pump of the major facilitator superfamily, is a frequent cause of resistance to the widely used antimycotic agent fluconazole and other toxic compounds in the pathogenic yeastCandida albicans. The zinc cluster transcription factor Mrr1 controlsMDR1expression in response to inducing chemicals, and gain-of-function mutations inMRR1are responsible for the constitutiveMDR1upregulation in fluconazole-resistantC. albicansstrains. To understand how Mrr1 activity is regulated, we identified functional domains of this transcription factor. A hybrid protein consisting of the N-terminal 106 amino acids of Mrr1 and the transcriptional activation domain of Gal4 fromSaccharomyces cerevisiaeconstitutively inducedMDR1expression, demonstrating that the DNA binding domain is sufficient to target Mrr1 to theMDR1promoter. Using a series of C-terminal truncations and systematic internal deletions, we could show that Mrr1 contains multiple activation and inhibitory domains. One activation domain (AD1) is located in the C terminus of Mrr1. When fused to the tetracycline repressor TetR, this distal activation domain induced gene expression from a TetR-dependent promoter. The deletion of an inhibitory region (ID1) located near the distal activation domain resulted in constitutive activity of Mrr1. The additional removal of AD1 abolished the constitutive activity, but the truncated Mrr1 still could activate theMDR1promoter in response to the inducer benomyl. These results demonstrate that the activity of Mrr1 is regulated in multiple ways and provide insights into the function of an important mediator of drug resistance inC. albicans.

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