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

2017 ◽  
Vol 61 (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.

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.

scholarly journals SAGA/ADA Complex Subunit Ada2 Is Required for Cap1- but Not Mrr1-Mediated Upregulation of the Candida albicans Multidrug Efflux PumpMDR1

2014 ◽  
Vol 58 (9) ◽  
pp. 5102-5110 ◽  
Author(s):  
Bernardo Ramírez-Zavala ◽  
Selene Mogavero ◽  
Eva Schöller ◽  
Christoph Sasse ◽  
P. David Rogers ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Candida Albicans ◽  
Efflux Pump ◽  
Wild Type ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Gain Of Function ◽  
Content Type ◽  
Zinc Cluster ◽  
Fluconazole Resistant

ABSTRACTOverexpression of the multidrug efflux pumpMDR1is one mechanism by which the pathogenic yeastCandida albicansdevelops resistance to the antifungal drug fluconazole. The constitutive upregulation ofMDR1in fluconazole-resistant, clinicalC. albicansisolates is caused by gain-of-function mutations in the zinc cluster transcription factor Mrr1. It has been suggested that Mrr1 activatesMDR1transcription by recruiting Ada2, a subunit of the SAGA/ADA coactivator complex. However,MDR1expression is also regulated by the bZIP transcription factor Cap1, which mediates the oxidative stress response inC. albicans. Here, we show that a hyperactive Mrr1 containing a gain-of-function mutation promotesMDR1overexpression independently of Ada2. In contrast, a C-terminally truncated, hyperactive Cap1 causedMDR1overexpression in a wild-type strain but only weakly in mutants lackingADA2. In the presence of benomyl or H2O2, compounds that induceMDR1expression in an Mrr1- and Cap1-dependent fashion,MDR1was upregulated with the same efficiency in wild-type andada2Δ cells. These results indicate that Cap1, but not Mrr1, recruits Ada2 to theMDR1promoter to induce the expression of this multidrug efflux pump and that Ada2 is not required forMDR1overexpression in fluconazole-resistantC. albicansstrains containing gain-of-function mutations in Mrr1.

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 Factors Supporting Cysteine Tolerance and Sulfite Production in Candida albicans

2013 ◽  
Vol 12 (4) ◽  
pp. 604-613 ◽  
Author(s):  
Florian Hennicke ◽  
Maria Grumbt ◽  
Ulrich Lermann ◽  
Nico Ueberschaar ◽  
Katja Palige ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Candida Albicans ◽  
Efflux Pump ◽  
Phenotypic Analysis ◽  
Pathogenic Yeast ◽  
Content Type ◽  
Zinc Cluster ◽  
Enhanced Sensitivity ◽  
Hypha Formation

ABSTRACTThe amino acid cysteine has long been known to be toxic at elevated levels for bacteria, fungi, and humans. However, mechanisms of cysteine tolerance in microbes remain largely obscure. Here we show that the human pathogenic yeastCandida albicansexcretes sulfite when confronted with increasing cysteine concentrations. Mutant construction and phenotypic analysis revealed that sulfite formation from cysteine inC. albicansrelies on cysteine dioxygenase Cdg1, an enzyme with similar functions in humans. Environmental cysteine induced not only the expression of theCDG1gene inC. albicans, but also the expression ofSSU1, encoding a putative sulfite efflux pump. Accordingly, the deletion ofSSU1resulted in enhanced sensitivity of the fungal cells to both cysteine and sulfite. To study the regulation of sulfite/cysteine tolerance in more detail, we screened aC. albicanslibrary of transcription factor mutants in the presence of sulfite. This approach and subsequent independent mutant analysis identified the zinc cluster transcription factor Zcf2 to govern sulfite/cysteine tolerance, as well as cysteine-inducibleSSU1andCDG1gene expression.cdg1Δ andssu1Δ mutants displayed reduced hypha formation in the presence of cysteine, indicating a possible role of the newly proposed mechanisms of cysteine tolerance and sulfite secretion in the pathogenicity ofC. albicans. Moreover,cdg1Δ mutants induced delayed mortality in a mouse model of disseminated infection. Since sulfite is toxic and a potent reducing agent, its production byC. albicanssuggests diverse roles during host adaptation and pathogenicity.

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 Differential Requirement of the Transcription Factor Mcm1 for Activation of the Candida albicans Multidrug Efflux PumpMDR1by Its Regulators Mrr1 and Cap1

2011 ◽  
Vol 55 (5) ◽  
pp. 2061-2066 ◽  
Author(s):  
Selene Mogavero ◽  
Arianna Tavanti ◽  
Sonia Senesi ◽  
P. David Rogers ◽  
Joachim Morschhäuser
Keyword(s):  
Transcription Factor ◽  
Candida Albicans ◽  
Transcription Factors ◽  
Molecular Mechanisms ◽  
Efflux Pump ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Gain Of Function ◽  
Pathogenic Yeast ◽  
Content Type

ABSTRACTOverexpression of the multidrug efflux pump Mdr1 causes increased fluconazole resistance in the pathogenic yeastCandida albicans. The transcription factors Mrr1 and Cap1 mediateMDR1upregulation in response to inducing stimuli, and gain-of-function mutations in Mrr1 or Cap1, which render the transcription factors hyperactive, result in constitutiveMDR1overexpression. The essential MADS box transcription factor Mcm1 also binds to theMDR1promoter, but its role in inducible or constitutiveMDR1upregulation is unknown. Using a conditional mutant in which Mcm1 can be depleted from the cells, we investigated the importance of Mcm1 forMDR1expression. We found that Mcm1 was dispensable forMDR1upregulation by H2O2but was required for fullMDR1induction by benomyl. A C-terminally truncated, hyperactive Cap1 could upregulateMDR1expression both in the presence and in the absence of Mcm1. In contrast, a hyperactive Mrr1 containing a gain-of-function mutation depended on Mcm1 to causeMDR1overexpression. These results demonstrate a differential requirement for the coregulator Mcm1 for Cap1- and Mrr1-mediatedMDR1upregulation. When activated by oxidative stress or a gain-of-function mutation, Cap1 can induceMDR1expression independently of Mcm1, whereas Mrr1 requires either Mcm1 or an active Cap1 to cause overexpression of theMDR1efflux pump. Our findings provide more detailed insight into the molecular mechanisms of drug resistance in this important human fungal pathogen.

scholarly journals The C2H2 Transcription Factor SltA Contributes to Azole Resistance by Coregulating the Expression of the Drug Target Erg11A and the Drug Efflux Pump Mdr1 in Aspergillus fumigatus

2021 ◽  
Author(s):  
Wenlong Du ◽  
Pengfei Zhai ◽  
Tingli Wang ◽  
Michael J Bromley ◽  
Yuanwei Zhang ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Aspergillus Fumigatus ◽  
Fungal Pathogens ◽  
Efflux Pump ◽  
Antifungal Drugs ◽  
Ergosterol Biosynthesis ◽  
Azole Resistance ◽  
Drug Efflux ◽  
Mobility Shift ◽  
Drug Efflux Pump

The emergence of azole-resistant fungal pathogens has posed a great threat to public health worldwide. Although the molecular mechanism of azole resistance has been extensively investigated, the potential regulators of azole resistance remain largely unexplored. Here we identified a new function of the fungal specific C2H2 zinc finger transcription factor SltA (involved in salt-tolerance pathway) in the regulation of azole resistance of the human fungal pathogen Aspergillus fumigatus. Lack of SltA results in an itraconazole hypersusceptibility phenotype. Transcriptional profiling combined with LacZ reporter analysis and electrophoretic mobility shift assays (EMSA) demonstrate that SltA is involved in its own transcriptional regulation and also regulates the expression of genes related to ergosterol biosynthesis (erg11A, erg13A and erg24A) and drug efflux pumps (mdr1, mfsC and abcE) by directly binding to the conserved 5’-AGGCA-3’ motif in their promoter regions, and this binding is dependent on the conserved cysteine and histidine within the C2H2 DNA binding domain of SltA. Moreover, overexpression of erg11A or mdr1 rescues sltA deletion defects under itraconazole conditions, suggesting that erg11A and mdr1 are related to sltA-mediated itraconazole resistance. Most importantly, deletion of SltA in laboratory-derived and clinical azole-resistant isolates significantly attenuates drug resistance. Collectively, we have identified a new function of the transcription factor SltA in regulating azole resistance by coordinately mediating the key azole target Erg11A and the drug efflux pump Mdr1, and targeting SltA may provide a potential strategy for intervention of clinical azole-resistant isolates to improve the efficiency of currently approved antifungal drugs.

scholarly journals Inducible and Constitutive Activation of Two Polymorphic Promoter Alleles of the Candida albicans Multidrug Efflux PumpMDR1

2012 ◽  
Vol 56 (8) ◽  
pp. 4490-4494 ◽  
Author(s):  
Christoph Sasse ◽  
Rebecca Schillig ◽  
Alexandra Reimund ◽  
Julia Merk ◽  
Joachim Morschhäuser
Keyword(s):  
Candida Albicans ◽  
Transcription Factors ◽  
Loss Of Heterozygosity ◽  
Efflux Pump ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Fluconazole Resistance ◽  
Content Type ◽  
Constitutive Activation ◽  
Strain Sc5314

ABSTRACTOverexpression of the multidrug efflux pumpMDR1confers resistance to the antifungal drug fluconazole onCandida albicans. It has been reported that two types ofMDR1promoters exist inC. albicansand that homozygosity for the allele with higher activity may promote fluconazole resistance. We found that the twoMDR1promoter alleles in strain SC5314 were equally well activated by inducing chemicals or hyperactive forms of the transcription factors Mrr1 and Cap1, which controlMDR1expression. In addition, no loss of heterozygosity at theMDR1locus was observed inMDR1-overexpressing clinicalC. albicansstrains that developed fluconazole resistance during therapy.

scholarly journals Contribution of Clinically Derived Mutations in the Gene Encoding the Zinc Cluster Transcription Factor Mrr2 to Fluconazole Antifungal Resistance andCDR1Expression inCandida albicans

2019 ◽  
Vol 63 (5) ◽  
Author(s):  
Andrew T. Nishimoto ◽  
Qing Zhang ◽  
Brandon Hazlett ◽  
Joachim Morschhäuser ◽  
P. David Rogers
Keyword(s):  
Amino Acid ◽  
Efflux Pump ◽  
Antifungal Resistance ◽  
Clinical Isolates ◽  
Amino Acid Substitutions ◽  
Gene Encoding ◽  
Fluconazole Resistance ◽  
Content Type ◽  
Zinc Cluster ◽  
Artificial Activation

ABSTRACTMutations in genes encoding zinc cluster transcription factors (ZCFs) such asTAC1,MRR1, andUPC2play a key role inCandida albicansazole antifungal resistance. Artificial activation of the ZCF Mrr2 has shown increased expression of the gene encoding the Cdr1 efflux pump and resistance to fluconazole. Amino acid substitutions in Mrr2 have recently been reported to contribute to fluconazole resistance in clinical isolates. In the present study, 57 C. albicansclinical isolates with elevated fluconazole MICs were examined for mutations inMRR2and expression ofCDR1. Mutations inMRR2resulting in 15 amino acid substitutions were uniquely identified among resistant isolates, including 4 substitutions (S466L, A468G, S469T, T470N) previously reported to reduce fluconazole susceptibility. Three additional, novel amino acid substitutions (R45Q, A459T, V486M) were also discovered in fluconazole-resistant isolates. When introduced into a fluconazole-susceptible background, no change in fluconazole MIC orCDR1expression was observed for any of the mutations found in this collection. However, introduction of an allele leading to artificial activation of Mrr2 increased resistance to fluconazole as well asCDR1expression. Moreover, Mrr2 amino acid changes reported previously to have the strongest effect on fluconazole susceptibility andCDR1expression also exhibited no differences in fluconazole susceptibility orCDR1expression relative to the parent strain. While all known fluconazole resistance mechanisms are represented within this collection of clinical isolates and contribute to fluconazole resistance to different extents, mutations inMRR2do not appear to alterCDR1expression or contribute to resistance in any of these isolates.

scholarly journals An A643T Mutation in the Transcription Factor Upc2p Causes Constitutive ERG11 Upregulation and Increased Fluconazole Resistance in Candida albicans

2009 ◽  
Vol 54 (1) ◽  
pp. 353-359 ◽  
Author(s):  
Clemens J. Heilmann ◽  
Sabrina Schneider ◽  
Katherine S. Barker ◽  
P. David Rogers ◽  
Joachim Morschhäuser
Keyword(s):  
Transcription Factor ◽  
Candida Albicans ◽  
Acquired Resistance ◽  
Amino Acid Position ◽  
Resistant Isolate ◽  
Ergosterol Biosynthesis ◽  
Common Mechanism ◽  
Gain Of Function ◽  
Fluconazole Resistance ◽  
Zinc Cluster

ABSTRACT The zinc cluster transcription factor Upc2p mediates upregulation of ergosterol biosynthesis genes in response to ergosterol depletion in the fungal pathogen Candida albicans. One mechanism of acquired resistance to the antifungal drug fluconazole, which inhibits ergosterol biosynthesis, is constitutively increased expression of the ERG11 gene encoding the drug target enzyme. A G648D mutation in Upc2p has recently been shown to cause hyperactivity of the transcription factor, resulting in overexpression of ergosterol biosynthesis genes and increased fluconazole resistance. In order to investigate if gain-of-function mutations in Upc2p are a common mechanism of ERG11 upregulation and fluconazole resistance, we sequenced the UPC2 alleles of four ERG11-overexpressing, fluconazole-resistant C. albicans isolates and matched susceptible isolates from the same patients. In three of the isolate pairs, no differences in the UPC2 alleles were found, suggesting that mechanisms other than Upc2p mutations can cause ERG11 overexpression. One resistant isolate had become homozygous for a UPC2 allele containing a G1927A substitution that caused an alanine-to-threonine exchange at amino acid position 643 of Upc2p. Replacement of one of the endogenous UPC2 alleles in a fluconazole-susceptible strain by the UPC2 A643T allele resulted in ERG11 overexpression and increased fluconazole resistance, which was further elevated when the A643T mutation was also introduced into the second UPC2 allele. These results further establish gain-of-function mutations in UPC2, which can be followed by loss of heterozygosity for the mutated allele, as a mechanism of ERG11 overexpression and increased fluconazole resistance in C. albicans, but other mechanisms of ERG11 upregulation also exist.

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