The zinc cluster transcription factor Tac1p regulates
            PDR16
            expression in
            Candida albicans

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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.03065-14 ◽

2014 ◽

Vol 58 (9) ◽

pp. 5102-5110 ◽

Cited By ~ 16

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.

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A Gain-of-Function Mutation in the Transcription Factor Upc2p Causes Upregulation of Ergosterol Biosynthesis Genes and Increased Fluconazole Resistance in a Clinical Candida albicans Isolate

Eukaryotic Cell ◽

10.1128/ec.00103-08 ◽

2008 ◽

Vol 7 (7) ◽

pp. 1180-1190 ◽

Cited By ~ 152

Author(s):

Nico Dunkel ◽

Teresa T. Liu ◽

Katherine S. Barker ◽

Ramin Homayouni ◽

Joachim Morschhäuser ◽

...

Keyword(s):

Gene Expression ◽

Transcription Factor ◽

Candida Albicans ◽

Clinical Isolates ◽

Resistant Isolate ◽

Ergosterol Biosynthesis ◽

Gain Of Function ◽

Zinc Cluster ◽

Fluconazole Resistant ◽

Lanosterol Demethylase

ABSTRACT In the pathogenic yeast Candida albicans, the zinc cluster transcription factor Upc2p has been shown to regulate the expression of ERG11 and other genes involved in ergosterol biosynthesis upon exposure to azole antifungals. ERG11 encodes lanosterol demethylase, the target enzyme of this antifungal class. Overexpression of UPC2 reduces azole susceptibility, whereas its disruption results in hypersusceptibility to azoles and reduced accumulation of exogenous sterols. Overexpression of ERG11 leads to the increased production of lanosterol demethylase, which contributes to azole resistance in clinical isolates of C. albicans, but the mechanism for this has yet to be determined. Using genome-wide gene expression profiling, we found UPC2 and other genes involved in ergosterol biosynthesis to be coordinately upregulated with ERG11 in a fluconazole-resistant clinical isolate compared with a matched susceptible isolate from the same patient. Sequence analysis of the UPC2 alleles of these isolates revealed that the resistant isolate contained a single-nucleotide substitution in one UPC2 allele that resulted in a G648D exchange in the encoded protein. Introduction of the mutated allele into a drug-susceptible strain resulted in constitutive upregulation of ERG11 and increased resistance to fluconazole. By comparing the gene expression profiles of the fluconazole-resistant isolate and of strains carrying wild-type and mutated UPC2 alleles, we identified target genes that are controlled by Upc2p. Here we show for the first time that a gain-of-function mutation in UPC2 leads to the increased expression of ERG11 and imparts resistance to fluconazole in clinical isolates of C. albicans.

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The zinc cluster transcription factor Rha1 is a positive filamentation regulator in Candida albicans

Genetics ◽

10.1093/genetics/iyab155 ◽

2021 ◽

Author(s):

Raha Parvizi Omran ◽

Bernardo Ramírez-Zavala ◽

Walters Aji Tebung ◽

Shuangyan Yao ◽

Jinrong Feng ◽

...

Keyword(s):

Transcription Factor ◽

Candida Albicans ◽

Transcription Factors ◽

Systemic Infection ◽

Hyphal Growth ◽

Growth Conditions ◽

Hyphal Development ◽

Zinc Cluster ◽

Function Blocks ◽

External Signals

Abstract Zinc cluster transcription factors are essential fungal regulators of gene expression. In the pathogen Candida albicans, the gene orf19.1604 encodes a zinc cluster transcription factor regulating filament development. Hyperactivation of orf19.1604, which we have named RHA1 for Regulator of Hyphal Activity, generates wrinkled colony morphology under non-hyphal growth conditions, triggers filament formation, invasiveness, and enhanced biofilm formation and causes reduced virulence in the mouse model of systemic infection. The strain expressing activated Rha1 shows up-regulation of genes required for filamentation and cell-wall-adhesion-related proteins. Increased expression is also seen for the hyphal-inducing transcription factors Brg1 and Ume6, while the hyphal repressor Nrg1 is downregulated. Inactivation of RHA1 reduces filamentation under a variety of filament-inducing conditions. In contrast to the partial effect of either single mutant, the double rha1 ume6 mutant strain is highly defective in both serum- and Spider-medium-stimulated hyphal development. While the loss of Brg1 function blocks serum-stimulated hyphal development, this block can be significantly bypassed by Rha1 hyperactivity, and the combination of Rha1 hyperactivity and serum addition can generate significant polarization even in brg1 ume6 double mutants. Thus, in response to external signals, Rha1 functions with other morphogenesis regulators including Brg1 and Ume6, to mediate filamentation.

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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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Factors Supporting Cysteine Tolerance and Sulfite Production in Candida albicans

Eukaryotic Cell ◽

10.1128/ec.00336-12 ◽

2013 ◽

Vol 12 (4) ◽

pp. 604-613 ◽

Cited By ~ 25

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.

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The zinc cluster transcription factor Ahr1p directs Mcm1p regulation of Candida albicans adhesion

Molecular Microbiology ◽

10.1111/j.1365-2958.2010.07504.x ◽

2010 ◽

Vol 79 (4) ◽

pp. 940-953 ◽

Cited By ~ 31

Author(s):

Christopher Askew ◽

Adnane Sellam ◽

Elias Epp ◽

Jaideep Mallick ◽

Hervé Hogues ◽

...

Keyword(s):

Transcription Factor ◽

Candida Albicans ◽

Zinc Cluster

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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01102-09 ◽

2009 ◽

Vol 54 (1) ◽

pp. 353-359 ◽

Cited By ~ 80

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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The zinc cluster transcription factor Czf1 regulates cell wall architecture and integrity in Candida albicans

Molecular Microbiology ◽

10.1111/mmi.14727 ◽

2021 ◽

Author(s):

Austin Mottola ◽

Bernardo Ramírez‐Zavala ◽

Kerstin Hünniger ◽

Oliver Kurzai ◽

Joachim Morschhäuser

Keyword(s):

Cell Wall ◽

Transcription Factor ◽

Candida Albicans ◽

Zinc Cluster ◽

Cell Wall Architecture

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The zinc cluster transcription factor Rha1 is a positive filamentation regulator in Candida albicans

10.1101/2020.01.21.901744 ◽

2020 ◽

Cited By ~ 1

Author(s):

Raha Parvizi Omran ◽

Chris Law ◽

Vanessa Dumeaux ◽

Joachim Morschhäuser ◽

Malcolm Whiteway

Keyword(s):

Cell Wall ◽

Transcription Factor ◽

Candida Albicans ◽

Transcription Factors ◽

Transcriptional Profiling ◽

Hyphal Growth ◽

Growth Conditions ◽

Hyphal Development ◽

Zinc Cluster ◽

Genes Encoding

AbstractZinc cluster transcription factors are essential fungal specific regulators of gene expression. In the dimorphic pathogen Candida albicans, they control processes ranging from metabolism and stress adaptation to mating, virulence, and antifungal resistance. Here, we have identified the gene CaORF19.1604 as encoding a zinc cluster transcription factor that acts as a regulator of filament development. Hyperactivation of CaORF19.1604, which we have named RHA1 for Regulator of Hyphal Activity, leads to a wrinkled colony morphology under non-hyphal growth conditions, to pseudohyphal growth and filament formation, to invasiveness and enhanced biofilm formation.  Cells with activated Rha1 are sensitive to cell wall modifying agents such as Congo red and the echinocandin drug caspofungin but show normal sensitivity to fluconazole. RNA-sequencing-based transcriptional profiling of the activated Rha1 strain reveals the up-regulation of genes for core filamentation and cell-wall-adhesion-related proteins such as Als1, Als3, Ece1, and Hwp1. Upregulation is also seen for the genes for the hyphal-inducing transcription factors Brg1 and Ume6 and genes encoding several enzymes involved in arginine metabolism, while downregulation is seen for the hyphal repressor Nrg1. The deletion of BRG1 blocks the filamentation caused by activated Rha1, while null mutants of UME6 result in a partial block. Deletion of RHA1 can partially reduce healthy hyphal development triggered by environmental conditions such as Spider medium or serum at 37°C.In contrast to the limited effect of either single mutant, the double rha1 ume6 deletion strain is totally defective in both serum and Spider medium stimulated hyphal development. While the loss of Brg1 function blocks serum-stimulated hyphal development, this block can be significantly bypassed by Rha1 hyperactivity, and the combination of Rha1 hyperactivity and serum addition can generate significant polarization in even brg1 ume6 double mutants. Our results thus suggest that in response to external signals, Rha1 functions to facilitate the switch from an Nrg1 controlled yeast state to a Brg1/Ume6 regulated hyphal state.Author SummaryCandida albicans is the predominant human fungal pathogen, generating a mortality rate of 40% in systemically infected patients. The ability of Candida albicans to change its morphology is a determinant of its tissue penetration and invasion in response to variant host-related stimuli. The regulatory mechanism for filamentation includes a complex network of transcription factors that play roles in regulating hyphae associated genes. We identify here a new regulator of filamentation from the zinc cluster transcription factor family. We present evidence suggesting that this transcription factor assists the Nrg1/Brg1 switch regulating hyphal development.

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