Targeted gene disruption in Candida albicans wild‐type strains: the role of the MDR1 gene in fluconazole resistance of clinical Candida albicans isolates

Abstract

Resistance of the pathogenic yeast Candida albicans to the antifungal agent fluconazole is often caused by active drug efflux out of the cells. In clinical C. albicans strains, fluconazole resistance frequently correlates with constitutive activation of the MDR1 gene, encoding a membrane transport protein of the major facilitator superfamily that is not expressed detectably in fluconazole‐susceptible isolates. However, the molecular changes causing MDR1 activation have not yet been elucidated, and direct proof for MDR1 expression being the cause of drug resistance in clinical C. albicans strains is lacking as a result of difficulties in the genetic manipulation of C. albicans wild‐type strains. We have developed a new strategy for sequential gene disruption in C. albicans wild‐type strains that is based on the repeated use of a dominant selection marker conferring resistance against mycophenolic acid upon transformants and its subsequent excision from the genome by FLP‐mediated, site‐specific recombination (MPAR‐flipping). This mutagenesis strategy was used to generate homozygous mdr1/mdr1 mutants from two fluconazole‐resistant clinical C. albicans isolates in which drug resistance correlated with stable, constitutive MDR1 activation. In both cases, disruption of the MDR1 gene resulted in enhanced susceptibility of the mutants against fluconazole, providing the first direct genetic proof that MDR1 mediates fluconazole resistance in clinical C. albicans strains. The new gene disruption strategy allows the generation of specific knock‐out mutations in any C. albicans wild‐type strain and therefore opens completely novel approaches for studying this most important human pathogenic fungus at the molecular level.

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

“…Overexpression and gene inactivation studies have shown that MDR1 confers specific resistance to fluconazole, benomyl, cycloheximide, benzotriazole, methotrexate, 4-nitroquinoline-N-oxide (4-NQO) and sulfomethuron methyl (Ben-Yaacov et al, 1994;Fling et al, 1991;Goldway et al, 1995;Wirsching et al, 2000b). Disruption of MDR1 causes an increased susceptibility to methotrexate, cycloheximide and 4-NQO (Goldway et al, 1995;Sanglard et al, 1996).…”

Berberine Inhibits Fluphenazine-Induced Up-Regulation of <i>CDR1</i> in <i>Candida albicans</i>

“…4,5) Several genes have been confirmed to contribute to the development of multidrug resistance including ERG11, CDR1, CDR2, MDR1, RTA2, AOX1 and AOX2. [6][7][8][9][10][11][12] Among them, upregulation of CDR1, a member of the ATP-binding cassette (ABC) transporter super-family in C. albicans, is a common occurrence in clinical C. albicans isolates resistant to antifungal azoles. 13) Cdr1p pumps azoles out of C. albicans cells to reduce intracellular accumulation, which can be indirectly identified by the efflux of rhodamine 6G (R6G), which is a fluorescent substrate transported by Cdr1p.…”

Relative Contributions of the Candida albicans ABC Transporters Cdr1p and Cdr2p to Clinical Azole Resistance

“…The overexpression of transporter genes in A r isolates suggested that a reduced accumulation of azoles in the cell was responsible for the observed azole resistance phenotype (1,44). By using a dominant selectable marker, it was shown that deleting MDR1 from A r clinical isolates overexpressing this gene reduced the resistance of the cells to FLC, providing a direct demonstration that MDR1 is involved in clinical FLC resistance (45). However, the direct contribution of CDR1 and CDR2 to clinical azole resistance remained to be determined.…”