Activation of the Multiple Drug Resistance Gene MDR1 in Fluconazole-Resistant, Clinical Candida albicans Strains Is Caused by Mutations in a trans -Regulatory Factor

Abstract

Resistance of Candida albicans against the widely used antifungal agent fluconazole is often due to active drug efflux from the cells. In many fluconazole-resistant C. albicans isolates the reduced intracellular drug accumulation correlates with constitutive strong expression of the MDR1 gene, encoding a membrane transport protein of the major facilitator superfamily that is not detectably expressed in vitro in fluconazole-susceptible isolates. To elucidate the molecular changes responsible for MDR1 activation, two pairs of matched fluconazole-susceptible and resistant isolates in which drug resistance coincided with stable MDR1 activation were analyzed. Sequence analysis of the MDR1 regulatory region did not reveal any promoter mutations in the resistant isolates that might account for the altered expression of the gene. To test for a possible involvement of trans-regulatory factors, a GFP reporter gene was placed under the control of the MDR1 promoter from the fluconazole-susceptible C. albicans strain CAI4, which does not express the MDR1 gene in vitro. This MDR1P-GFP fusion was integrated into the genome of the clinical C. albicans isolates with the help of the dominant selection marker MPA R developed for the transformation of C. albicans wild-type strains. Integration was targeted to an ectopic locus such that no recombination between the heterologous and resident MDR1 promoters occurred. The transformants of the two resistant isolates exhibited a fluorescent phenotype, whereas transformants of the corresponding susceptible isolates did not express the GFP gene. These results demonstrate that the MDR1 promoter was activated by a trans-regulatory factor that was mutated in fluconazoleresistant isolates, resulting in deregulated, constitutive MDR1 expression.Candida albicans is an important opportunistic fungal pathogen of humans and is the major cause of oropharyngeal candidiasis (OPC) in patients with AIDS (21). The azole antifungal agent fluconazole is a widely used compound to treat OPC. In recent years, however, the incidence of treatment failures has been rising. Especially in patients with AIDS who have recurrent OPC and who are receiving prolonged fluconazole therapy, treatment failures are due to the emergence of fluconazole-resistant strains (10, 22). Resistant C. albicans isolates frequently exhibit reduced intracellular drug accumulation that correlates with enhanced expression of certain multiple drug resistance genes, the ATP-binding cassette (ABC) transporters CDR1 and CDR2, and the major facilitator MDR1 (8,14,24,25,29). Fluconazole resistance is usually a stable phenotype that is maintained in the absence of selection pressure by the drug. This implies that genetic alterations have occurred in the resistant isolates that result in a constitutive overexpression of the drug efflux pumps. The MDR1 gene is not detectably expressed in vitro in fluconazole-susceptible C. albicans isolates but is strongly activated in many strains after the development of fluconazole resistance. The molecular chang...

Transcriptional Regulation of MDR1 , Encoding a Drug Efflux Determinant, in Fluconazole-Resistant Candida albicans Strains through an Mcm1p Binding Site

“…Wirsching et al demonstrated that in two independent FLC R clinical strains of C. albicans, MDR1 overexpression was due to mutations in an unidentified trans-regulatory factor(s) (54). We have demonstrated that in five independently isolated FLC R laboratory strains, MDR1 overexpression was also achieved through trans-activation of MDR1, suggesting that this may be the most common mechanism for MDR1 overexpression.…”

“…In some FLC R strains, high-level expression of MDR1 is due to the effects of an undefined trans-acting factor(s) (54). Recent work by Hiller et al indicates that three independent regions of the MDR1 promoter are capable of contributing to MDR1 expression in a FLC R strain overexpressing MDR1 and that the portion of the MDR1 promoter they term region 2 (Ϫ588 to Ϫ500) mediates benomyl induction of MDR1 transcription in the FLC-sensitive (FLC S ) laboratory strain CAI4 (29).…”

Population genomics of drug resistance in Candida albicans

“…In our experimental populations, CAP1 was not significantly altered in expression and no mutations were detected in the DNA sequence (data not shown). In both yeasts, the interactions in the transcriptional networks regulating multidrug transporters have yet to be experimentally dissected (27,29). In addition to transcriptional coregulation, adaptive mutations could have indirect pleiotropic effects (30) on the expression of suites of genes through interactions among various gene products (8), signaling activity across different pathways (31), or changes in cell physiology (17).…”

Identification of promoter elements responsible for the regulation of MDR1 from Candida albicans, a major facilitator transporter involved in azole resistance

“…However, a dramatic transient upregulation of MDR1 can be observed in azole-susceptible strains after treatment with toxic hydrophobic agents (benomyl, methotrexate, 4-NQO, o-phenanthroline), oxidizing agents [diamide, H 2 O 2 , tert-butyl hydrogen peroxide (T-BHP) and diethylmaleate] and the alkylating agent methyl methanesulfonate (Gupta et al, 1998;Harry et al, 2005). Clinical isolates with increased MDR1 expression have not undergone amplification of the MDR1 gene (Wirsching et al, 2000a). Sequence analysis of MDR1 promoters from clinical isolates compared to wild-type promoters did not reveal cis-acting mutations (Harry et al, 2005;Wirsching et al, 2000a).…”