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Regulation of efflux pump expression and drug resistance by the transcription factors Mrr1, Upc2, and Cap1 in Candida albicans

ABSTRACT: Constitutive overexpression of the Mdr1 efflux pump is an important mechanism of acquired drug resistance in the yeast Candida albicans. The zinc cluster transcription factor Mrr1 is a central regulator of MDR1 expression, but other transcription factors have also been implicated in MDR1 regulation. To better understand how MDR1-mediated drug resistance is achieved in this important fungal pathogen, we studied the interdependence of Mrr1 and two other MDR1 regulators, Upc2 and Cap1, in the control of MDR1 expression. A mutated, constitutively active Mrr1 could upregulate MDR1 and confer drug resistance in the absence of Upc2 or Cap1. On the other hand, Upc2 containing a gain-of-function mutation only slightly activated the MDR1 promoter, and this activation depended on the presence of a functional MRR1 gene. In contrast, a C-terminally truncated, activated form of Cap1 could upregulate MDR1 in a partially Mrr1-independent fashion. The induction of MDR1 expression by toxic chemicals occurred independently of Upc2, but required the presence of Mrr1 and also partially depended on Cap1. Transcriptional profiling and in vivo DNA 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 of MDR1 expression in wild-type C. albicans, but gain-of-function mutations in either of the two transcription factors can independently mediate efflux pump overexpression and drug resistance.

ORGANISM(S): Candida albicans

PROVIDER: GSE23532 | GEO | 2011/07/01

SECONDARY ACCESSION(S): PRJNA131763

REPOSITORIES: GEO

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Project description:Cap1p, a transcription factor of the basic region-leucine zipper family, controls the oxidative stress response in Candida albicans. It was shown that alteration of the C-terminal cysteine-rich domain (CRD) of Cap1p results in nuclear retention and constitutive transcriptional activation. To further characterize the function of Cap1p in C. albicans, we used genome-wide location profiling (ChIP-on-chip), allowing the identification of Cap1p-transcriptional targets in vivo. Location profiling using a tiled-oligonucleotide DNA microarray identified 89 targets that were bound by Cap1p-HA3 or Cap1p-CSE-HA3 (binding ratio â¥ 2-fold, P â¤ 0.01). Strikingly, Cap1p binding was not only detected at the promoter region of its target genes but also at their 3'-end and within their open-reading frame. Overrepresented functional groups of Cap1p targets (P â¤ 0.02) included notably 11 genes involved in response to oxidative stress (CAP1, GLR1, TRX1, others), 13 genes involved in response to drug (PDR16, MDR1, FLU1, others) and 3 genes involved in regulation of nitrogen utilization (orf19.2693, orf19.3121 and GST3). Bioinformatic analyses suggested that Cap1p binds to the DNA motif 5'- MTKASTMA. Transcriptome analyses showed that increased expression of most of Cap1p targets accompanies Cap1p binding at these targets, indicating that Cap1p is a transcriptional activator. We conclude that, in addition to protecting the cell against oxidative stress, Cap1p appears to have other functions including drug resistance and the regulation of nitrogen utilization. The atypical binding pattern of Cap1p suggests that this transcription factor may associate with the transcriptional or the chromatin remodeling machinery to exert its activity. We performed three gene expression profile comparisons: (1) benomyl-induced gene expression in a strain containing wildtype CAP1 (CJD21/PMK-CAP1 +/- benomyl), (2) benomyl-induced gene expression in a strain where CAP1 is disrupted (CJD21/PMK +/- benomyl), and (3) gene expression in a strain containing a hyperactive allele of CAP1 compared to a strain containing wildtype CAP1 (CJD21/PMK-CAP1-CSE vs. CJD21/PMK-CAP1). There were three biological replicates (each drug-treated and untreated strain was grown/treated in three independent experiments). Since our Affymetrix platform requires each sample is hybridized on a separate chip, there is no dye-swapping. For each benomyl-treatment experiment (comparisons 1 and 2 above), the untreated (diluent-treated) samples are the reference samples. For comparison 3, the CJD21-PMK-CAP1 sample is the reference sample.

Project description:In the pathogenic yeast Candida albicans, the zinc cluster transcription factor Upc2p has been shown to regulate 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. Over-expression of UPC2 reduces azole susceptibility, whereas its disruption results in hypersusceptibility to azoles and reduced accumulation of exogenous sterols. Constitutive up-regulation of ERG11 is a major cause of resistance to fluconazole in clinical isolates of C. albicans, yet 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 up-regulated with ERG11 in a fluconazole resistant clinical isolate as 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 up-regulation 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 increased expression of ERG11 and imparts resistance to fluconazole in clinical isolates of C. albicans. Keywords: genome-wide expression profiling Clinical isolates S1 (susceptible) and S2 (resistant), genome strain SC5314, UPC2 disruption strains (UPC2M4A and UPC2M4B), UPC2 re-integrant strains of non-mutated UPC2 allele (UPC2M2K21A and UPC2M2K21B), and UPC2 re-integrant strains of mutated UPC2 allele (UPC2M2K31A and UPC2M2K31B) were grown for 3 hours until log phase. RNA was extracted for each isolate/strain. Experiments were performed in duplicate.

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Regulation of efflux pump expression and drug resistance by the transcription factors Mrr1, Upc2, and Cap1 in Candida albicans

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