MDR1 -Mediated Drug Resistance in Candida dubliniensis

Abstract

Candida dubliniensis is a recently described opportunistic fungal pathogen that is closely related to Candida albicans. Candida dubliniensis readily develops resistance to the azole antifungal agent fluconazole, both in vitro and in infected patients, and this resistance is usually associated with upregulation of the CdMDR1 gene, encoding a multidrug efflux pump of the major facilitator superfamily. To determine the role of CdMDR1 in drug resistance in C. dubliniensis, we constructed an mdr1 null mutant from the fluconazole-resistant clinical isolate CM2, which overexpressed the CdMDR1 gene. Sequential deletion of both CdMDR1 alleles was performed by the MPA R -flipping method, which is based on the repeated use of a dominant mycophenolic acid resistance marker for selection of integrative transformants and its subsequent deletion from the genome by FLP-mediated, site-specific recombination. In comparison with its parental strain, the mdr1 mutant showed decreased resistance to fluconazole but not to the related drug ketoconazole. In addition, we found that CdMDR1 confers resistance to the structurally unrelated drugs 4-nitroquinoline-N-oxide, cerulenin, and brefeldin A, since the enhanced resistance to these compounds of the parent strain CM2 compared with the matched susceptible isolate CM1 was abolished in the mdr1 mutant. In contrast, CdMDR1 inactivation did not cause increased susceptibility to amorolfine, terbinafine, fluphenazine, and benomyl, although overexpression of CdMDR1 in a hypersusceptible Saccharomyces cerevisiae strain had previously been shown to confer resistance to these compounds. The effect of CdMDR1 inactivation was identical to that seen in two similarly constructed C. albicans mdr1 mutants. Therefore, despite species-specific differences in the amino acid sequences of the Mdr1 proteins, overexpression of CaMDR1 and CdMDR1 in clinical C. albicans and C. dubliniensis strains seems to confer the same drug resistance profile in both species.

Evolutionary Dynamics of Candida albicans during In Vitro Evolution

“…The mechanisms of resistance to azole drugs have been studied extensively (18,19,21,27,28,32,36,37,40,42,43). However, a large percentage (50%) of drug-resistant clinical isolates from a reported collection have unknown mechanisms of resistance (40).…”

TheCandida dubliniensis CdCDR1Gene Is Not Essential for Fluconazole Resistance

“…Molecular analysis of fluconazoleresistant C. dubliniensis isolates and in vitro-generated fluconazole-resistant derivatives has shown that in each case, the fluconazole resistance phenotype is associated with increased expression of the CdMDR1 gene encoding the multidrug transporter CdMdr1p (13). Wirsching et al demonstrated that targeted deletion of both copies of CdMDR1 in a C. dubliniensis clinical isolate with reduced susceptibility to fluconazole was sufficient to render the null mutant susceptible to fluconazole (35).…”

“…This allowed amplification of the 5Ј and 3Ј ends of the CdCDR1 ORF and the entire plasmid vector (21). These primers contained SacII and XhoI restriction endonuclease recognition sequences that enabled the insertion of the SacII/XhoI fragment of pSFI1 (containing the site-specific recombinase FLP and the MPA resistance gene) (34,35), thereby replacing the central region of the CdCDR1 ORF to create plasmid pCDR⌬1. To create the second construct, the internal AccI/ BamHI fragment of CdCDR1 was cloned into pUC19 and subjected to inverse PCR with the primer pair 6F-6R (Table 2; Fig.…”

“…Targeted disruption of the CdCDR1 gene in C. dubliniensis CD57R was carried out by using the MPA R -flipper technique (34,35). Two different deletion constructs were made to inactivate both alleles of the CdCDR1 gene.…”

Azole susceptibility and resistance in Candida dubliniensis

“…Overexpression of CdMDR1 has been shown to be involved in mediating a reduced accumulation of drug in fluconazole-resistant clinical isolates and in-vitro-generated derivatives [38]. Furthermore, the role of CdMDR1 in fluconazole resistance has been confirmed by the disruption of both alleles of the gene in a fluconazoleresistant clinical isolate overexpressing CdMDR1 [39].…”