Dataset Information

Transcriptional regulation of MDR1, encoding a drug efflux determinant, in fluconazole-resistant Candida albicans strains through an Mcm1p binding site.

ABSTRACT: Constitutive, high-level transcription of the gene encoding the drug efflux facilitator Mdr1p is commonly observed in laboratory and clinical strains of Candida albicans that are resistant to the antifungal drug fluconazole (FLC). In five independently isolated FLC(R) laboratory strains, introduction of a wild-type MDR1 promoter fragment fused to the yeast enhanced green fluorescent protein (yEGFP) reporter gene resulted in high-level expression of GFP, demonstrating that overexpression of MDR1 is dependent on a trans-acting factor. This study identified a 35-bp MDR1 promoter element, termed the MDRE, that mediates high-level MDR1 transcription. When inserted into a heterologous promoter, the MDRE was sufficient to mediate high-level expression of the yEGFP reporter gene specifically in MDR1 trans-activation strains. The MDRE promoted transcription in an orientation-independent and dosage-dependent manner. Deletion of the MDRE in the full-length promoter did not abolish MDR1 trans-activation, indicating that elements upstream of the MDRE also contribute to transcription of MDR1 in these overexpression strains. Analysis of the MDRE sequence indicated that it contains an Mcm1p binding site very similar in organization to the site seen upstream of the Saccharomyces cerevisiae MFA1 and STE2 genes. Electrophoretic mobility shift analysis demonstrated that both wild-type, FLC-sensitive and MDR1 trans-activated, FLC-resistant strains contain a factor that binds the MDRE. Depletion of Mcm1p, by use of a strain in which MCM1 expression is under the control of a regulated promoter (44), resulted in a loss of MDRE binding activity. Thus, the general transcription factor Mcm1p participates in the regulation of MDR1 expression.

SUBMITTER: Riggle PJ

PROVIDER: S-EPMC1694824 | biostudies-literature | 2006 Dec

REPOSITORIES: biostudies-literature

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Transcriptional regulation of MDR1, encoding a drug efflux determinant, in fluconazole-resistant Candida albicans strains through an Mcm1p binding site.

Riggle Perry J PJ Kumamoto Carol A CA

Eukaryotic cell 20061013 12

Constitutive, high-level transcription of the gene encoding the drug efflux facilitator Mdr1p is commonly observed in laboratory and clinical strains of Candida albicans that are resistant to the antifungal drug fluconazole (FLC). In five independently isolated FLC(R) laboratory strains, introduction of a wild-type MDR1 promoter fragment fused to the yeast enhanced green fluorescent protein (yEGFP) reporter gene resulted in high-level expression of GFP, demonstrating that overexpression of MDR1 ...[more]

PMID: 17041190

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Project description:Among the several mechanisms of multidrug resistance (MDR), overexpression of drug efflux pumps CaCdr1p and CaMdr1p belonging to ATP binding cassette (ABC) and major facilitator superfamily (MFS) respectively remain the predominant mechanisms of candidal infections. Therefore inhibiting or modulating the function of these transporters continues to draw attention as effective strategy to combat MDR. We have previously reported the antifungal potential of Geraniol (Ger), a natural monoterpenoid from Palmarosa oil, against Candida albicans. Herein, we explored the fungicidal nature of Ger. The Rhodamine 6G (R6G) and Nile red accumulation confirms the specific effect on CaCdr1p. Mechanistic insights with Candida cells overexpressing CaCdr1p and CaMdr1p revealed that Ger specifically modulates CaCdr1p activity. Kinetic studies further unraveled the competitive inhibition of Ger for R6G efflux as evident from increased apparent Km without affecting Vmax value. The effect of Ger on CaCdr1p was substantiated by molecular docking analyses, which depicted in-silico binding affinity of Ger with CaCdr1p and explored that Ger binds to the active site of CaCdr1p with higher binding energy. Although RT-PCR and western blot revealed no change in expressions of CDR1 and CaCdr1p, confocal microscopy images however depicted CaCdr1p mislocalization in presence of Ger. Interestingly, Ger was synergistic (FICI<0.5) with fluconazole (FLC) which is a well known antifungal drug. Furthermore, Ger sensitizes the FLC sensitive and resistant clinical matched pair of isolates Gu4/Gu5 and led to abrogated R6G efflux and depleted ergosterol. Furthermore, Rhodamine B labeling demonstrates altered mitochondrial potential with Ger which suggest possible linkage of dysfunctional mitochondria with CaCdr1p activity. We also estimated phenotypic virulence marker extracellular phospholipase activity which was considerably diminished along with inhibited cell adherence and biofilm biomass. Lastly, antifungal efficacy of Ger was demonstrated by enhanced survival of Caenorhabditis elegans model and negligible hemolytic activity (20%). Together, modulation of efflux pump activity by Ger and FLC synergism represent a promising approach for combinatorial treatment of candidiasis.

Project description: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 upregulated with MDR1 in drug-resistant, clinical C. albicans isolates. Inactivation of MRR1 in two such drug-resistant isolates abolished both MDR1 expression and multidrug resistance. Sequence analysis of the MRR1 alleles of two matched drug-sensitive and drug-resistant C. albicans isolate pairs showed that the resistant isolates had become homozygous for MRR1 alleles that contained single nucleotide substitutions, resulting in a P683S exchange in one isolate and a G997V substitution in the other isolate. Introduction of these mutated alleles into a drug-susceptible C. albicans strain resulted in constitutive MDR1 overexpression and multidrug resistance. By comparing the transcriptional profiles of drug-resistant C. albicans isolates and mrr1Delta mutants derived from them and of C. albicans strains carrying wild-type and mutated MRR1 alleles, we defined the target genes that are controlled by Mrr1p. Many of the Mrr1p target genes encode oxidoreductases, whose upregulation in fluconazole-resistant isolates may help to prevent cell damage resulting from the generation of toxic molecules in the presence of fluconazole and thereby contribute to drug resistance. The identification of MRR1 as the central regulator of the MDR1 efflux pump and the elucidation of the mutations that have occurred in fluconazole-resistant, clinical C. albicans isolates and result in constitutive activity of this trancription factor provide detailed insights into the molecular basis of multidrug resistance in this important human fungal pathogen.

Dataset Information

Transcriptional regulation of MDR1, encoding a drug efflux determinant, in fluconazole-resistant Candida albicans strains through an Mcm1p binding site.

Publications

Transcriptional regulation of MDR1, encoding a drug efflux determinant, in fluconazole-resistant Candida albicans strains through an Mcm1p binding site.

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