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

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.

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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01655-18 ◽

2018 ◽

Vol 62 (12) ◽

Cited By ~ 1

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.

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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

10.1101/2021.05.07.443069 ◽

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.

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

Eukaryotic Cell ◽

10.1128/ec.05100-11 ◽

2011 ◽

Vol 10 (8) ◽

pp. 1110-1121 ◽

Cited By ~ 18

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

PLoS Genetics ◽

10.1371/journal.pgen.1009582 ◽

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.

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

10.1101/2021.06.23.449691 ◽

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.

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Induction of Candida albicans Drug Resistance Genes by Hybrid Zinc Cluster Transcription Factors

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.04448-14 ◽

2014 ◽

Vol 59 (1) ◽

pp. 558-569 ◽

Cited By ~ 11

Author(s):

Sabrina Schneider ◽

Joachim Morschhäuser

Keyword(s):

Candida Albicans ◽

Transcription Factors ◽

Dna Binding ◽

Target Genes ◽

Efflux Pumps ◽

Pathogenic Yeast ◽

Fluconazole Resistance ◽

Dna Binding Domains ◽

Zinc Cluster ◽

Binding Domains

ABSTRACTThe pathogenic yeastCandida albicanscan develop resistance to azole antifungal drugs by overexpressingERG11, which encodes the drug target, or the multidrug efflux pumpsMDR1andCDR1/CDR2. The constitutive upregulation of these genes is usually caused by gain-of-function mutations in the zinc cluster transcription factors Upc2, Mrr1, and Tac1, respectively. These transcription factors are also required for the induction of their target genes in drug-susceptible strains in the presence of specific stimuli. By swapping the DNA-binding domains of Mrr1, Tac1, and Upc2 we investigated if the hybrid transcription factors could activate their new target genes in response to the same signals. When Tac1 was targeted to theMDR1andERG11promoters, the expression of these genes became inducible by fluphenazine. Similarly,MDR1andCDR2were strongly upregulated by fluconazole when Upc2 was fused to the DNA-binding domains of Mrr1 and Tac1, respectively. In contrast, Mrr1 was unable to promote gene expression in response to benomyl when it was targeted to theCDR2andERG11promoters instead of theMDR1promoter. These results suggest that Tac1 and Upc2 themselves are activated by the inducers fluphenazine and fluconazole, respectively, whereas benomyl does not activate Mrr1 itself but a coregulatory factor that is present at the promoters of Mrr1 target genes. Strains in which the expression levels of Mrr1 and Tac1 target genes were controlled by Upc2 exhibited increased fluconazole resistance, demonstrating that the ability to efficiently upregulate the expression of efflux pumps in the presence of the drug results in enhanced intrinsic fluconazole resistance.

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Relative Contribution of the ABC Transporters Cdr1, Pdh1, and Snq2 to Azole Resistance inCandida glabrata

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01070-18 ◽

2018 ◽

Vol 62 (10) ◽

Cited By ~ 10

Author(s):

Sarah G. Whaley ◽

Qing Zhang ◽

Kelly E. Caudle ◽

P. David Rogers

Keyword(s):

Multidrug Resistance ◽

Minor Role ◽

Azole Resistance ◽

Content Type ◽

Azole Antifungals ◽

Zinc Cluster ◽

Relative Contribution ◽

Genes Encoding ◽

High Level ◽

A Minor

ABSTRACTThe utility of the azole antifungals for the treatment of invasive candidiasis is severely hampered by azole resistance inCandida glabrata. This resistance is mediated almost exclusively by activating mutations in the zinc cluster transcription factor Pdr1, which controls the genes encoding the multidrug resistance transporters Cdr1, Pdh1, and Snq2. However, the specific relative contributions of these transporters to resistance are not known. To address this question, theSAT1flipper method was used to deleteCDR1,PDH1, andSNQ2in a strain ofC. glabrataengineered to carry a clinically relevant activating mutation inPDR1. Susceptibility testing was performed according to the CLSI guidelines, with minor modifications, and confirmed with Etest strips. Of the single-transporter-deletion strains, only theCDR1deletion resulted in a decreased azole MIC. The deletion ofPDH1in combination withCDR1resulted in a moderate decrease in MIC compared to that observed with the deletion ofCDR1alone.SNQ2deletion only decreased the MIC in the triple-deletion strain in the absence of bothCDR1andPDH1. The deletion of all three transporters in combination decreased the MIC to the level observed in thePDR1deletion strains for some, but not all, azoles tested, which indicates that additional Pdr1 targets likely play a minor role in this process. These results indicate that while Cdr1 is the most important Pdr1-mediated multidrug resistance transporter for azole resistance in this clinical isolate, all three of these transporters contribute to its high-level resistance to the azole antifungals.

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

mBio ◽

10.1128/mbio.00934-19 ◽

2019 ◽

Vol 10 (3) ◽

Cited By ~ 7

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.

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Mutations in TAC1B: a Novel Genetic Determinant of Clinical Fluconazole Resistance in Candida auris

mBio ◽

10.1128/mbio.00365-20 ◽

2020 ◽

Vol 11 (3) ◽

Cited By ~ 9

Author(s):

Jeffrey M. Rybak ◽

José F. Muñoz ◽

Katherine S. Barker ◽

Josie E. Parker ◽

Brooke D. Esquivel ◽

...

Keyword(s):

Transcription Factor ◽

Clinical Isolate ◽

Multidrug Resistant ◽

Clinical Isolates ◽

Genetic Determinant ◽

Candida Auris ◽

Fluconazole Resistance ◽

Content Type ◽

Fluconazole Resistant

ABSTRACT Candida auris has emerged as a multidrug-resistant pathogen of great clinical concern. Approximately 90% of clinical C. auris isolates are resistant to fluconazole, the most commonly prescribed antifungal agent, and yet it remains unknown what mechanisms underpin this fluconazole resistance. To identify novel mechanisms contributing to fluconazole resistance in C. auris, fluconazole-susceptible C. auris clinical isolate AR0387 was passaged in media supplemented with fluconazole to generate derivative strains which had acquired increased fluconazole resistance in vitro. Comparative analyses of comprehensive sterol profiles, [3H]fluconazole uptake, sequencing of C. auris genes homologous to genes known to contribute to fluconazole resistance in other species of Candida, and relative expression levels of C. auris ERG11, CDR1, and MDR1 were performed. All fluconazole-evolved derivative strains were found to have acquired mutations in the zinc-cluster transcription factor-encoding gene TAC1B and to show a corresponding increase in CDR1 expression relative to the parental clinical isolate, AR0387. Mutations in TAC1B were also identified in a set of 304 globally distributed C. auris clinical isolates representing each of the four major clades. Introduction of the most common mutation found among fluconazole-resistant clinical isolates of C. auris into fluconazole-susceptible isolate AR0387 was confirmed to increase fluconazole resistance by 8-fold, and the correction of the same mutation in a fluconazole-resistant isolate, AR0390, decreased fluconazole MIC by 16-fold. Taken together, these data demonstrate that C. auris can rapidly acquire resistance to fluconazole in vitro and that mutations in TAC1B significantly contribute to clinical fluconazole resistance. IMPORTANCE Candida auris is an emerging multidrug-resistant pathogen of global concern, known to be responsible for outbreaks on six continents and to be commonly resistant to antifungals. While the vast majority of clinical C. auris isolates are highly resistant to fluconazole, an essential part of the available antifungal arsenal, very little is known about the mechanisms contributing to resistance. In this work, we show that mutations in the transcription factor TAC1B significantly contribute to clinical fluconazole resistance. These studies demonstrated that mutations in TAC1B can arise rapidly in vitro upon exposure to fluconazole and that a multitude of resistance-associated TAC1B mutations are present among the majority of fluconazole-resistant C. auris isolates from a global collection and appear specific to a subset of lineages or clades. Thus, identification of this novel genetic determinant of resistance significantly adds to the understanding of clinical antifungal resistance in C. auris.

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Candida albicans Swi/Snf and Mediator Complexes Differentially Regulate Mrr1-Induced MDR1 Expression and Fluconazole Resistance

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01344-17 ◽

2017 ◽

Vol 61 (11) ◽

Cited By ~ 11

Author(s):

Zhongle Liu ◽

Lawrence C. Myers

Keyword(s):

Transcription Factor ◽

Candida Albicans ◽

Transcriptional Activation ◽

Fungal Pathogens ◽

Efflux Pump ◽

Open Chromatin ◽

Drug Efflux ◽

Fluconazole Resistance ◽

Content Type ◽

Zinc Cluster

ABSTRACT Long-term azole treatment of patients with chronic Candida albicans infections can lead to drug resistance. Gain-of-function (GOF) mutations in the transcription factor Mrr1 and the consequent transcriptional activation of MDR1, a drug efflux coding gene, is a common pathway by which this human fungal pathogen acquires fluconazole resistance. This work elucidates the previously unknown downstream transcription mechanisms utilized by hyperactive Mrr1. We identified the Swi/Snf chromatin remodeling complex as a key coactivator for Mrr1, which is required to maintain basal and induced open chromatin, and Mrr1 occupancy, at the MDR1 promoter. Deletion of snf2, the catalytic subunit of Swi/Snf, largely abrogates the increases in MDR1 expression and fluconazole MIC observed in MRR1 GOF mutant strains. Mediator positively and negatively regulates key Mrr1 target promoters. Deletion of the Mediator tail module med3 subunit reduces, but does not eliminate, the increased MDR1 expression and fluconazole MIC conferred by MRR1 GOF mutations. Eliminating the kinase activity of the Mediator Ssn3 subunit suppresses the decreased MDR1 expression and fluconazole MIC of the snf2 null mutation in MRR1 GOF strains. Ssn3 deletion also suppresses MDR1 promoter histone displacement defects in snf2 null mutants. The combination of this work with studies on other hyperactive zinc cluster transcription factors that confer azole resistance in fungal pathogens reveals a complex picture where the induction of drug efflux pump expression requires the coordination of multiple coactivators. The observed variations in transcription factor and target promoter dependence of this process may make the search for azole sensitivity-restoring small molecules more complicated.

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