MDR1-mediated drug resistance in Candida dubliniensis

Candida dubliniensis is a recently described opportunistic fungal pathogen that is closely related to Candida albicans. Candida dubliniensis readily d evelops resistance to the azole antifungal agent fluconazole, both in vitro and in infected patients, and this resistance is usually associated with u pregulation of the CdMDR1 gene, encoding a multidrug efflux pump of the maj or facilitator superfamily. To determine the role of CdMDR1 in drug resista nce in C. dubliniensis, we constructed an mdr1 null mutant from the flucona zole-resistant clinical isolate CM2, which overexpressed the CdMDR1 gene. S equential deletion of both CdMDR1 alleles was performed by the MPA(R)-flipp ing method, which is based on the repeated use of a dominant mycophenolic a cid resistance marker for selection of integrative transformants and its su bsequent deletion from the genome by FLP-mediated, site-specific recombinat ion. In comparison with its parental strain, the mdr1 mutant showed decreas ed resistance to fluconazole but not to the related drug ketoconazole. In a ddition, we found that CdMDR1 confers resistance to the structurally unrela ted drugs 4-nitroquinoline-N-oxide, cerulenin, and brefeldin A, since the e nhanced resistance to these compounds of the parent strain CM2 compared wit h the matched susceptible isolate CM1 was abolished in the mdr1 mutant. In contrast, CdMDR1 inactivation did not cause increased susceptibility to amo rolfine, terbinafine, fluphenazine, and benomyl, although overexpression of CdMDR1 in a hypersusceptible Saccharomyces cerevisiae strain had previousl y been shown to confer resistance to these compounds. The effect of CdMDR1 inactivation was identical to that seen in two similarly constructed C. alb icans mdr1 mutants. Therefore, despite species-specific differences in the amino acid sequences of the Mdr1 proteins, overexpression of CaMDR1 and CdM DR1 in clinical C. albicans and C. dubliniensis strains seems to confer the same drug resistance profile in both species.