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 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 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 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 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 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 Gain-of-Function Mutations in the Transcription Factor MRR1 Are Responsible for Overexpression of the MDR1 Efflux Pump in Fluconazole-Resistant Candida dubliniensis Strains

2008 ◽  
Vol 52 (12) ◽  
pp. 4274-4280 ◽  
Author(s):  
Sabrina Schubert ◽  
P. David Rogers ◽  
Joachim Morschhäuser
Keyword(s):  
Transcription Factor ◽  
Efflux Pump ◽  
Candida Dubliniensis ◽  
Major Facilitator Superfamily ◽  
Multidrug Efflux Pump ◽  
Nucleotide Substitutions ◽  
Gain Of Function ◽  
Zinc Cluster ◽  
Fluconazole Resistant

ABSTRACT Candida dubliniensis, a yeast that is closely related to Candida albicans, can rapidly develop resistance to the commonly used antifungal agent fluconazole in vitro and in vivo during antimycotic therapy. Fluconazole resistance in C. dubliniensis is usually caused by constitutive overexpression of the MDR1 gene, which encodes a multidrug efflux pump of the major facilitator superfamily. The zinc cluster transcription factor Mrr1p has recently been shown to control MDR1 expression in C. albicans in response to inducing stimuli, and gain-of-function mutations in the MRR1 gene result in constitutive upregulation of the MDR1 efflux pump. We identified a gene with a high degree of similarity to C. albicans MRR1 (CaMRR1) in the C. dubliniensis genome sequence. When C. dubliniensis MRR1 (CdMRR1) was expressed in C. albicans mrr1Δ mutants, it restored benomyl-inducible MDR1 expression, demonstrating that CdMRR1 is the ortholog of CaMRR1. To investigate whether MDR1 overexpression in C. dubliniensis is caused by mutations in MRR1, we sequenced the MRR1 alleles from a fluconazole-resistant, clinical C. dubliniensis isolate and a matched, fluconazole-susceptible isolate from the same patient as well as those from four in vitro-generated, fluconazole-resistant C. dubliniensis strains derived from two different C. dubliniensis isolates. We found that all five resistant strains contained single nucleotide substitutions or small in-frame deletions that resulted in amino acid changes in Mrr1p. Expression of these mutated alleles in C. albicans resulted in the constitutive activation of the MDR1 promoter and multidrug resistance. Therefore, mutations in MRR1 are the major cause of MDR1 upregulation in both C. albicans and C. dubliniensis, demonstrating that the transcription factor Mrr1p plays a central role in the development of drug resistance in these human fungal pathogens.

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 Regulation of Efflux Pump Expression and Drug Resistance by the Transcription Factors Mrr1, Upc2, and Cap1 in Candida albicans

2011 ◽  
Vol 55 (5) ◽  
pp. 2212-2223 ◽  
Author(s):  
Sabrina Schubert ◽  
Katherine S. Barker ◽  
Sadri Znaidi ◽  
Sabrina Schneider ◽  
Franziska Dierolf ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Candida Albicans ◽  
Transcription Factors ◽  
Target Genes ◽  
Efflux Pump ◽  
Binding Studies ◽  
Gain Of Function ◽  
Content Type ◽  
Zinc Cluster ◽  
Constitutively Active

ABSTRACTConstitutive overexpression of the Mdr1 efflux pump is an important mechanism of acquired drug resistance in the yeastCandida albicans. The zinc cluster transcription factor Mrr1 is a central regulator ofMDR1expression, but other transcription factors have also been implicated inMDR1regulation. To better understand howMDR1-mediated drug resistance is achieved in this fungal pathogen, we studied the interdependence of Mrr1 and two otherMDR1regulators, Upc2 and Cap1, in the control ofMDR1expression. A mutated, constitutively active Mrr1 could upregulateMDR1and confer drug resistance in the absence of Upc2 or Cap1. On the other hand, Upc2 containing a gain-of-function mutation only slightly activated theMDR1promoter, and this activation depended on the presence of a functionalMRR1gene. In contrast, a C-terminally truncated, activated form of Cap1 could upregulateMDR1in a partially Mrr1-independent fashion. The induction ofMDR1expression by toxic chemicals occurred independently of Upc2 but required the presence of Mrr1 and also partially depended on Cap1. Transcriptional profiling andin vivoDNA binding studies showed that a constitutively active Mrr1 binds to and upregulates most of its direct target genes in the presence or absence of Cap1. Therefore, Mrr1 and Cap1 cooperate in the environmental induction ofMDR1expression in wild-typeC. albicans, but gain-of-function mutations in either of the two transcription factors can independently mediate efflux pump overexpression and drug resistance.

scholarly journals The Quorum-Sensing Molecule Farnesol Is a Modulator of Drug Efflux Mediated by ABC Multidrug Transporters and Synergizes with Drugs in Candida albicans

2011 ◽  
Vol 55 (10) ◽  
pp. 4834-4843 ◽  
Author(s):  
Monika Sharma ◽  
Rajendra Prasad
Keyword(s):  
Candida Albicans ◽  
Quorum Sensing ◽  
Efflux Pump ◽  
Major Facilitator Superfamily ◽  
Simultaneous Increase ◽  
Drug Efflux ◽  
Multidrug Efflux Pump ◽  
Content Type ◽  
Multidrug Transporters ◽  
Quorum Sensing Molecule

ABSTRACTOverexpression of theCaCDR1-encoded multidrug efflux pump protein CaCdr1p (Candidadrug resistance protein 1), belonging to the ATP binding cassette (ABC) superfamily of transporters, is one of the most prominent contributors of multidrug resistance (MDR) inCandida albicans. Thus, blocking or modulating the function of the drug efflux pumps represents an attractive approach in combating MDR. In the present study, we provide first evidence that the quorum-sensing molecule farnesol (FAR) is a specific modulator of efflux mediated by ABC multidrug transporters, such as CaCdr1p and CaCdr2p ofC. albicansand ScPdr5p ofSaccharomyces cerevisiae. Interestingly, FAR did not modulate the efflux mediated by the multidrug extrusion pump protein CaMdr1p, belonging to the major facilitator superfamily (MFS). Kinetic data revealed that FAR competitively inhibited rhodamine 6G efflux in CaCdr1p-overexpressing cells, with a simultaneous increase in an apparentKmwithout affecting theVmaxvalues and the ATPase activity. We also observed that when used in combination, FAR at a nontoxic concentration synergized with the drugs at their respective nonlethal concentrations, as was evident from their <0.5 fractional inhibitory concentration index (FICI) values and from the drop of 14- to 64-fold in the MIC80values in the wild-type strain and in azole-resistant clinical isolates ofC. albicans. Our biochemical experiments revealed that the synergistic interaction of FAR with the drugs led to reactive oxygen species accumulation, which triggered early apoptosis, and that both could be partly reversed by the addition of an antioxidant. Collectively, FAR modulates drug extrusion mediated exclusively by ABC proteins and is synergistic to fluconazole (FLC), ketoconazole (KTC), miconazole (MCZ), and amphotericin (AMB).

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