Multiple cis-acting sequences mediate upregulation of the MDR1 efflux pump in a fluconazole-resistant clinical Candida albicans isolate

Abstract

Overexpression of the MDR1 gene, which encodes a multidrug efflux pump of the major facilitator superfamily, is a frequent cause of resistance to the antimycotic agent fluconazole and other metabolic inhibitors in clinical Candida albicans strains. Constitutive MDR1 overexpression in such strains is caused by mutations in as yet unknown trans-regulatory factors. In order to identify the cis-acting sequences in the MDR1 regulatory region that mediate constitutive MDR1 upregulation, we performed a promoter deletion analysis in the genetic background of an MDR1-overexpressing clinical C. albicans isolate. We found that several different regions in the MDR1 promoter can mediate MDR1 overexpression in this isolate. In contrast, deletion of one of these regions abolished benomyl-induced MDR1 expression in a C. albicans laboratory strain. These results suggest that multiple transcription factors control expression of the MDR1 efflux pump in C. albicans and that the mutation(s) that causes constitutive MDR1 overexpression and drug resistance in clinical C. albicans isolates affects the activities of several of these transcription factors.

FIG. 1.

MDR1 promoter analysis. The structure of the insert of plasmid pMPG2 containing the P_MDR1-GFP reporter fusion is shown on top. The MDR1 promoter is symbolized by the bent arrow; the GFP gene, which is fused to the transcription termination sequence of the ACT1 gene (T_ACT1; filled circle), is symbolized by the gray arrow; and the URA3 selection marker is symbolized by the hatched arrow. The ACT1 flanking sequences (position numbers are with respect to the position of the ACT1 start codon) used for ectopic integration of the P_MDR1-GFP fusion into the ACT1 locus are represented by the white arrows, which also indicate the orientation of the ACT1 gene at the integration site. The relevant restriction sites used for the plasmid constructions are shown. Enlarged representations of the MDR1 regulatory region and deletion derivatives are shown below, and the names of the corresponding plasmids are indicated to the left. The extent of the MDR1 promoter sequences contained in the various plasmids is given. The transcriptional start site mapped at position −65 by Harry et al. (12) is indicated by the small bent arrow, and activating regions 1 to 3 identified in the present study are indicated by the black bars in pMPG2. The YRE is indicated by the small gray box. The fluorescence micrographs to the right of each construct show cells of strain F5U4 carrying the corresponding reporter fusion for illustration, and the mean fluorescence of the cells measured by flow cytometry is given. The inducibilities of the various MDR1 promoter derivatives by benomyl were also determined by measuring the fluorescence of transformants of strain CAI4 carrying the same reporter fusions in the presence of benomyl. All values are the means of results obtained for two independently constructed reporter strains.

FIG. 2.

Structures of MDR1 promoter derivatives carrying internal deletions (indicated by the dashed lines). The extent of the MDR1 promoter sequences in each derivative is indicated, and the names of the plasmids containing the corresponding P_MDR1-GFP reporter fusions are given to the left. The constitutive and benomyl-inducible activities of the MDR1 promoter derivatives in strains F5U4 and CAI4, respectively, were determined as explained in the legend to Fig. 1. The results obtained with strains carrying the P_MDR1-GFP fusion contained in pMPG2 are taken from Fig. 1 and are included for comparison.

FIG. 3.

Structures and activities of truncated MDR1 promoter derivatives with internal deletions. For explanations, see the legend to Fig. 1. The results obtained with strains carrying the P_MDR1-GFP fusions contained in pMPG2 and pMPG5 are taken from Fig. 2 and are included for comparison.

FIG. 4.

Structures and activities of truncated MDR1 promoter derivatives with internal deletions in the region between positions −313 and −239. For explanations, see the legend to Fig. 1. The results obtained with strains carrying the P_MDR1-GFP fusions contained in pMPG2 and pMPG13 are taken from Fig. 1 and are included for comparison.