Overexpression of MDR1, which encodes a membrane transport protein of the major facilitator superfamily, is one mechanism by which the human fungal pathogen Candida albicans can develop increased resistance to the antifungal drug fluconazole and other toxic compounds. In clinical C. albicans isolates, constitutive MDR1 overexpression is accompanied by the upregulation of other genes, but it is not known if these additional alterations are required for Mdr1p function and drug resistance. To investigate whether MDR1 overexpression is sufficient to confer a drug-resistant phenotype in C. albicans, we expressed the MDR1 gene from the strong ADH1 promoter in C. albicans laboratory strains that did not express the endogenous MDR1 gene as well as in a fluconazole-resistant clinical C. albicans isolate in which the endogenous MDR1 alleles had been deleted and in a matched fluconazole-susceptible isolate from the same patient. Forced MDR1 overexpression resulted in increased resistance to the putative Mdr1p substrates cerulenin and brefeldin A, and this resistance did not depend on the additional alterations which occurred during drug resistance development in the clinical isolates. In contrast, artificial expression of the MDR1 gene from the ADH1 promoter did not enhance or only slightly enhanced fluconazole resistance, presumably because Mdr1p expression levels in the transformants were considerably lower than those observed in the fluconazole-resistant clinical isolate. These results demonstrate that MDR1 overexpression in C. albicans is sufficient to confer resistance to some toxic compounds that are substrates of this efflux pump but that the degree of resistance depends on the Mdr1p expression level.Candida albicans is an opportunistic fungal pathogen that can cause superficial mucosal infections as well as life-threatening systemic infections, especially in immunocompromised patients. Infections by C. albicans are frequently treated with the antimycotic agent fluconazole, which inhibits the biosynthesis of ergosterol, the major sterol in the fungal cell membrane. C. albicans can develop resistance to fluconazole by different molecular mechanisms, including alterations in the sterol biosynthetic pathway, overexpression of ERG11, which encodes the target enzyme of fluconazole (sterol 14␣-demethylase, or Erg11p), mutations in ERG11 that result in a reduced affinity of Erg11p for fluconazole, and overexpression of genes encoding membrane transport proteins (CDR1, CDR2, and MDR1) that actively transport fluconazole out of the cell. In clinical C. albicans strains, several of these mechanisms are often combined to result in a stepwise development of fluconazole resistance (for a review, see reference 17).The MDR1 gene encodes an efflux pump of the major facilitator superfamily, whose members use the proton gradient across the cytoplasmic membrane as an energy source for transport (1, 4). While MDR1 is normally expressed only at low levels in standard laboratory media, many fluconazole-resistant clinical C. albicans isol...

Overexpression of the MDR1 gene, which encodes a multidrug efflux pump of the major facilitator superfamily, is a frequent cause of resistance to the antimycotic agent fluconazole and other metabolic inhibitors in clinical Candida albicans strains. Constitutive MDR1 overexpression in such strains is caused by mutations in as yet unknown trans-regulatory factors. In order to identify the cis-acting sequences in the MDR1 regulatory region that mediate constitutive MDR1 upregulation, we performed a promoter deletion analysis in the genetic background of an MDR1-overexpressing clinical C. albicans isolate. We found that several different regions in the MDR1 promoter can mediate MDR1 overexpression in this isolate. In contrast, deletion of one of these regions abolished benomyl-induced MDR1 expression in a C. albicans laboratory strain. These results suggest that multiple transcription factors control expression of the MDR1 efflux pump in C. albicans and that the mutation(s) that causes constitutive MDR1 overexpression and drug resistance in clinical C. albicans isolates affects the activities of several of these transcription factors.The yeast Candida albicans is a member of the microflora on mucosal surfaces of the gastrointestinal and urogenitary tracts in many healthy people, but it can also cause superficial as well as life-threatening systemic infections, especially in immunocompromised patients (21). Oropharyngeal candidiasis, which frequently affects patients infected with the human immunodeficiency virus and AIDS patients, is commonly treated with the antimycotic agent fluconazole, which inhibits the biosynthesis of ergosterol, the major sterol in the fungal cell membrane. C. albicans can develop resistance to fluconazole, especially during long-term treatment of oropharyngeal candidiasis (30). Fluconazole resistance is caused by different molecular mechanisms, including alterations in the sterol biosynthetic pathway; overexpression of the ERG11 gene, which encodes the target enzyme of fluconazole, sterol 14␣-demethylase (Erg11p); mutations in the ERG11 gene that result in a reduced affinity of Erg11p to fluconazole; and overexpression of genes encoding membrane transport proteins which actively transport fluconazole out of the cell. In clinical C. albicans strains, several of these mechanisms are often combined to result in the stepwise development of clinically relevant fluconazole resistance (for a review, see reference 18).Two types of efflux pumps that mediate resistance to fluconazole and structurally unrelated toxic compounds have been identified in C. albicans (6,(23)(24)(25). While CDR1 and CDR2 belong to the ATP-binding cassette (ABC) transporters, MDR1 is a member of the major facilitator superfamily which uses the proton gradient across the cytoplasmic membrane as an energy source for transport. In drug-susceptible C. albicans strains, MDR1 is expressed only at low levels in standard laboratory media, but its expression can be induced by some toxic compounds, like benomyl (10, 13, 27). In contras...

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The burden of fungal infections for humans, animals and plants is widely underestimated and comprises deadly infections as well as great conomic costs. Despite that, antifungal drugs are scarce and emergence of resistance in fungal strains contributes to a high mortality. To overcome this shortage, we propose toxic intermediates and their controlling enzymes in metabolic pathways as a resource for new targets and provide a web-service, FunTox-Networks to explore the landscape of toxic intermediates in the metabolic networks of fungal pathogens. The toxicity of metabolites is predicted by a new random forest regression model and is available for over one hundred fungal species. Further, for major fungal pathogens, metabolic networks from the KEGG database were enriched with data of toxicity and regulatory effort for each enzyme to support identification of targets. We determined several toxic intermediates in fungal-specific pathways like amino acid synthesis, nitrogen and sulfur assimilation, and the glyoxylate bypass. For the latter, we show experimentally that growth of the pathogen Candida albicans is inhibited when the detoxifying enzymes Mls1 and Hbr2 are deleted and toxic glyoxylate accumulates in the cell. Thus, toxic pathway intermediates and their controlling enzymes represent an untapped resource of antifungal targets.