Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Genome-wide expression and location analyses of the Candida albicans Tac1p regulon

ABSTRACT: A major mechanism of azole resistance in Candida albicans is the over-expression of the genes encoding the ABC transporters Cdr1p and Cdr2p. Constitutive over-expression of these efflux pumps is due to mutations in the gene encoding Tac1p, resulting in hyperactivity of this zinc cluster transcription factor. In order to identify the transcriptional targets of Tac1p, we examined four matched sets of clinical isolates representing the development of CDR1- and CDR2-mediated azole resistance, using gene expression profiling analysis. We identified 31 genes that were consistently up-regulated with CDR1 and CDR2, including TAC1 itself, as well as 12 consistently down-regulated genes. When a resistant strain deleted for TAC1 was similarly examined, the expression of almost all of these genes was returned to levels similar to those in the matched azole-susceptible isolate. Keywords: gene expression profiling Four clinical match isolates (azole susceptible and resistant) were grown as three independent replicates for each set to mid-log phase. A TAC1 knockout derived from resistant isolate 5674 was also grown as three independent replicates. Each resistant isolate was compared to its susceptible parent, and the TAC1 knockout was compared to the susceptible parent (5457) of the resistant isolate, 5674.

ORGANISM(S): Candida albicans

SUBMITTER: Sadri Znaidi

PROVIDER: E-GEOD-8727 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

The transcription factor Mrr1p controls expression of the MDR1 efflux pump and mediates multidrug resistance in Candida albicans.

Morschhäuser Joachim J Barker Katherine S KS Liu Teresa T TT BlaB-Warmuth Julia J Homayouni Ramin R Rogers P David PD

PLoS pathogens 20071101 11

Constitutive overexpression of the MDR1 (multidrug resistance) gene, which encodes a multidrug efflux pump of the major facilitator superfamily, is a frequent cause of resistance to fluconazole and other toxic compounds in clinical Candida albicans strains, but the mechanism of MDR1 upregulation has not been resolved. By genome-wide gene expression analysis we have identified a zinc cluster transcription factor, designated as MRR1 (multidrug resistance regulator), that was coordinately upregulat ...[more]

PMID: 17983269

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Project description:In Candida albicans, the transcription factor Upc2 is central to the regulation of ergosterol biosynthesis. UPC2-activating mutations contribute to azole resistance, whereas disruption increases azole susceptibility. In the present study, we investigated the relationship of UPC2 to fluconazole susceptibility, particularly in azole-resistant strains. In addition to the reduced fluconazole MIC previously observed with UPC2 disruption, we observed a lower minimum fungicidal concentration (MFC) for a upc2M-NM-^T/M-NM-^T mutant than for its azole-susceptible parent, SC5314. Moreover, the upc2M-NM-^T/M-NM-^T mutant was unable to grow on a solid medium containing 10 M-BM-5g/ml fluconazole and exhibited increased susceptibility and a clear zone of inhibition by Etest. Time-kill analysis showed higher fungistatic activity against the upc2M-NM-^T/M-NM-^T mutant than against SC5314. UPC2 disruption in strains carrying specific resistance mutations also resulted in reduced MICs and MFCs. UPC2 disruption in a highly azole resistant clinical isolate containing multiple resistance mechanisms likewise resulted in a reduced MIC and MFC. This mutant was unable to grow on a solid medium containing 10 M-BM-5g/ml fluconazole and exhibited increased susceptibility and a clear zone of inhibition by Etest. Time-kill analysis showed increased fungistatic activity against the upc2M-NM-^T/M-NM-^T mutant in the resistant background. Microarray analysis showed attenuated induction by fluconazole of genes involved in sterol biosynthesis, iron transport, or iron homeostasis in the absence of UPC2. Taken together, these data demonstrate that the UPC2 transcriptional network is universally essential for azole resistance in C. albicans and represents an attractive target for enhancing azole antifungal activity. We examined the genome-wide gene expression profiles of the wild-type parent strain SC5314 and its upc2M-NM-^T/M-NM-^T derivative in response to fluconazole in order to identify genes whose expression in response to fluconazole is influenced by Upc2.

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.

Dataset Information

Genome-wide expression and location analyses of the Candida albicans Tac1p regulon

Publications

The transcription factor Mrr1p controls expression of the MDR1 efflux pump and mediates multidrug resistance in Candida albicans.

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