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

Abstract

Candida albicans frequently develops resistance to treatment with azole drugs due to the acquisition of gain-of-function mutations in the transcription factor Tac1p. Tac1p hyperactivation in azole-resistant isolates results in the constitutive overexpression of several genes, including CDR1 and CDR2, which encode two homologous transporters of the ATP-binding cassette family. Functional studies of Cdr1p and Cdr2p have been carried out so far by heterologous expression in the budding yeast Saccharomyces cerevisiae and by gene deletion or overexpression in azole-sensitive C. albicans strains in which CDR1 expression is low and CDR2 expression is undetectable. Thus, the direct demonstration that CDR1 and CDR2 overexpression causes azole resistance in clinical strains is still lacking, as is our knowledge of the relative contribution of each transporter to clinical azole resistance. In the present study, we used the SAT1 flipper system to delete the CDR1 and CDR2 genes from clinical isolate 5674. This strain is resistant to several azole derivatives due to a strong hyperactive mutation in Tac1p and expresses high levels of Cdr1p and Cdr2p. We found that deleting CDR1 had a major effect, reducing resistance to fluconazole (FLC), ketoconazole (KTC), and itraconazole (ITC) by 6-, 4-, and 8-fold, respectively. Deleting CDR2 had a much weaker effect, reducing FLC or KTC resistance by 1.5-fold, and had no effect on ITC resistance. These results demonstrate that Cdr1p is a major determinant of azole resistance in strain 5674 and potentially in other clinical strains overexpressing Cdr1p and Cdr2p, while Cdr2p plays a more minor role.Candida albicans is one of the leading causes of fungal infections affecting immunocompromised individuals. Candida infections range from chronic superficial infections of the skin and mucosal surfaces to invasive, life-threatening systemic infections (21, 38). Many antifungal drugs used to treat Candida infections target the biosynthesis of ergosterol, the major sterol in the fungal cell membranes (24). Polyenes, such as amphotericin B (AMB), directly bind to ergosterol and form pores in the cell membrane, resulting in low selectivity and high toxicity (24). Azoles, a class of well-tolerated antifungal drugs that includes fluconazole (FLC), ketoconazole (KTC), itraconazole (ITC), and new-generation derivatives such as voriconazole and posaconazole, target the enzyme lanosterol 14␣-demethylase (Erg11p), which is involved in ergosterol biosynthesis, blocking the production of ergosterol and causing the accumulation of toxic intermediate sterol species (24). As a consequence, the fluidity and permeability of the fungal cell membrane are changed and the activity of membrane-bound proteins, such as enzymes involved in cell wall synthesis, is altered (24).However, the fungistatic rather than fungicidal action of azole drugs leads to the frequent emergence of azole-resistant (A r ) C. albicans strains (1, 44). One mechanism of azole resistance consists of increased levels of ERG11 RNA, resulting in i...

The Monoamine Oxidase A Inhibitor Clorgyline Is a Broad-Spectrum Inhibitor of Fungal ABC and MFS Transporter Efflux Pump Activities Which Reverses the Azole Resistance of Candida albicans and Candida glabrata Clinical Isolates

“…Given that there are very few other antifungal drug classes (5), there is a need to develop drugs that counter such resistance and extend the utility of azoles such as FLC. The flow cytometry-based screen for FLC-enhancing agents using S. cerevisiae strains expressing either CaCdr1p or CaCdr2p was developed because these ABC transporters are major contributors to the clinical resistance of C. albicans to azoles (13,47). The PCL was selected as the compound library because it is compact (1,200 compounds) and also because published data which would aid interpretation of the results are available on each compound.…”

“…There are two classes of efflux pumps: ATP-binding cassette (ABC) transporters that use the hydrolysis of ATP as their energy source, and major facilitator superfamily (MFS) pumps that utilize membrane potential to drive efflux. The C. albicans ABC transporter Cdr1p (CaCdr1p) is reported to be a major contributor to azole resistance in clinical isolates of fluconazole (FLC [Diflucan])-resistant strains of the opportunistic pathogen C. albicans (13,47). ABC efflux pumps are often responsible for azole resistance in other pathogenic fungi, such as CneMdr1p in Cryptococcus neoformans (23).…”

Fluconazole Transport into Candida albicans Secretory Vesicles by the Membrane Proteins Cdr1p, Cdr2p, and Mdr1p

“…For example, it has been shown that intracellular [ 3 H]fluconazole levels decreased when CDR1 (20,33) and MDR1 (37) were overexpressed in S. cerevisiae and also that fluconazole MICs increased when CDR1, CDR2 (36,53), and MDR1 (31) were overexpressed in C. albicans. The levels to which CDR1, CDR2, and MDR1 were expressed in C. albicans in the present study did not approach the expression levels that others achieved in S. cerevisiae or C. albicans; this may have been due to our use of episomal rather than integrating vectors and our use of the ACT1 and GAL1 promoters instead of fluconazole-inducible promoters.…”

The ABCs of Candida albicans Multidrug Transporter Cdr1

“…The inability to accumulate detrimental concentrations of antifungal agents is attributed mainly to the ATP binding cassette (ABC) and major facilitator superfamily (MFS) classes of efflux proteins, which are overexpressed in resistant fungal isolates (3). Among the ABC transporters, Cdr1p and Cdr2p are the ones with the utmost clinical implications (4) and of the two, Cdr1p is the major determinant of azole resistance (5).…”