Cloning of Candida albicans genes conferring resistance to azole antifungal agents: characterization of CDR2, a new multidrug ABC transporter gene

Abstract

Resistance to azole antifungal agents in Candida albicans can be mediated by multidrug efflux transporters. In a previous study, w e identified a t least two such transporters, C d r l p and Benp, which belong to the class of ATP-binding -cassette (ABC) transporters and of major facilitators, respectively. To isolate additional factors potentially responsible for resistance to azole antifungal agents in C. albicans, the hypersusceptibility of a Saccharomyces cerevisiae multidrug transporter mutant, Apdr5, to these agents was complemented with a C. albicans genomic library. Several new genes were isolated, one of which was a new ABC transporter gene called CDR2 (Candida drug Eesistance). The protein Cdr2p encoded b y this gene exhibited 84% identity with C d r l p and could confer resistance to azole antif ungal agents, to other antifungals (terbinafine, amorolfine) and to a variety of metabolic inhibitors. The disruption of CDR2 in the C. albicans strain CAM-2 did not render cells more susceptible to these substances. When the disruption of CDR2 was performed in the background of a mutant in which CDR7 was deleted, the resulting double Acdrl Acdr2 mutant was more susceptible to these agents than the single Acdrl mutant. The absence of hypersusceptibility of the single Acdr2 mutant could be explained by the absence of CDR2 mRNA in azole-susceptible C. albicans strains. CDR2 was overexpressed, however, in clinical C. albicans isolates resistant to azole antifungal agents as described previously for CDRI, but to levels exceeding or equal to those reached b y CDRl. Interestingly, CDR2 expression was restored in Acdr7 mutants reverting spontaneously to wildtype levels of susceptibility t o azole antifungal agents. These data demonstrate that CDR2 plays an important role in mediating the resistance of C. albicans to azole antifungal agents.

Fcr1p Inhibits Development of Fluconazole Resistance in Candida albicans by Abolishing CDR1 Induction

“…Two major mechanisms of azole resistance have been identified: One common resistance mechanism is the reduced intracellular accumulation of drugs, which is correlated with the increased expression of the CDR genes, members of the ABC efflux pump family, and/or of the MDR1 gene, a member of the MFS efflux pump family. 13,31,32) Other common mechanisms are increased expression of the ERG11 gene that encodes the target enzyme of azole drugs, sterol 14a-demethylase, and alteration of the enzyme due to point mutations in the gene. [32][33][34][35][36][37][38] C. albicans FCR1 gene has been reported characterization by encoding a putative 517-amino-acid protein with an N-terminal Zn 2 C 6 -type zinc finger motif homologous to that found in fungal zinc cluster regulatory factors including S. cerevisiae Pdr1p and Pdr3p.…”

“…Cdr1p could mediate azole resistance by causing active energy-dependent extrusion of the drug out of cells. 31,39) In the present study, we used a fluorescent dye, rhoamine 6 G (R6G), which is a substrate of CDR1, to identify CDR activity because a previous study showed that the efflux of rhoamine 6 G is positively correlated with the expression of CDR1. 40) Our results for rhodamine 6 G active efflux also showed that with the susceptibilities of the strains to FLC decreased, and the extrusion of rhodamine 6 G by C. albicans increased in the presence of glucose providing energy for rhodamine 6 efflux (Fig.…”

A novel polyamide SL-A92 as a potential fungal resistance blocker: synthesis and bioactivities in Candida albicans

“…Four kinds of CDR genes in C albicans have been cloned [13,[40][41][42] , but only over-expression of CDR1 and CDR2 resulted in azole resistance [43] . Tac1p is the major transcription factor needed for the regulation of CDR1 and CDR2, and is characterized by a highly conserved Zn(II) 2 Cys 6 zinc finger motif formed by six cysteines that coordinate two zinc atoms within the DNAbinding domain [11,44,45] .…”

“…These transcription factors, as well as the transcription processes, represent potential targets against MDR. Among the MDR genes regulated by zinc cluster transcription factors, the expression of CDR1 and CDR2 has received considerable attention, because their encoding ABC transporter proteins, Cdr1p and Cdr2p, could pump azoles out of cells to reduce azole accumulation as a self-defense mechanism [12][13][14][15] . Our previous studies have also demonstrated that elevated CDR1 and CDR2 levels are associated with the progression of MDR in antifungal treatment [16,17] .…”

The SCR1 gene from Schwanniomyces occidentalis encodes a highly hydrophobic polypeptide, which confers ribosomal resistance to cycloheximide

“…The genome sequence from S. cerevisiae has revealed the presence of as many as 28 ORFs homologous to either the ABC or the MFS family (Goffeau et al, 1997). Yeast multidrug transporters include the ABCs protein CDR1-3 from Candida albicans (Sanglard et al, 1997;Balan et al, 1997) and the MSF BEN r (CaMDR1) that renders cells resistant to cyh, benomyl, methotrexate, 4-nitro-quinoline-N-oxide, or terbinafine (Ben-Yaacov et al, 1994;Goldway et al, 1995;Sanglard et al, 1997;Gupta et al, 1998). The C. maltosa CYR R and the C. dubliniensis CdMDR1, homologous to CaMDR1, confer resistance to cyh (Sasnauskas et al, 1992) or to fluconazole, cyh, benomyl or sulphometuron methyl, respectively (Moran et al, 1998).…”