Drug-induced regulation of the MDR1 promoter in Candida albicans

Abstract

Resistance of Candida albicans to azole antifungal drugs is mediated by two types of efflux pumps, encoded by the MDR1 gene and the CDR gene family. MDR1 mRNA levels in a susceptible clinical isolate are induced by benomyl (BEN) but not by other drugs previously shown to induce MDR1. To monitor MDR1 expression under several conditions, the MDR1 promoter was fused to the Renilla reniformis luciferase reporter gene (RLUC). The promoter was monitored for its responses to four oxidizing agents, five toxic hydrophobic compounds, and an alkylating agent, all shown to induce major facilitator pumps in other organisms. Deletion constructs of the MDR1 promoter were used to analyze the basal transcription of the promoter and its responses to the toxic compound BEN and the oxidizing agent tert-butyl hydrogen peroxide (T-BHP). The cis-acting elements in the MDR1 promoter responsible for induction by BEN were localized between -399 and -299 upstream of the start codon. The cis-acting elements responsible for MDR1 induction by T-BHP were localized between -601 and -500 upstream of the start codon. The T-BHP induction region contains a sequence that resembles the YAP1-responsive element (YRE) in Saccharomyces cerevisiae. This Candida YRE was placed upstream of a noninducible promoter in the luciferase construct, resulting in an inducible promoter. Inversion or mutation of the 7-bp YRE eliminated induction. Many of the drugs used in this analysis induce the MDR1 promoter at concentrations that inhibit cell growth. These analyses define cis-acting elements responsible for drug induction of the MDR1 promoter.

FIG. 1.

Site of integration for MDR1 promoter fusion. The MDR1 promoter (PRO; black box) fused to the RLUC reporter gene (diagonal hatched box) was flanked by the backbone from plasmid pCRW3 (thin lines). pCRW3 also contains a functional ADE2 gene (shaded boxes). The plasmid containing the promoter was cut at the HindIII site for integration into the ADE2 genomic locus (white boxes) containing a deletion of part of the coding region (vertical stripes) that makes the parental strain CAI8 Ade⁻. The correct single integration is shown, and the linear order of the genes is shown (the figure is not drawn to scale).

FIG. 2.

Northern blot assay of MDR1 mRNAs from strains 1 and 17 in the absence and presence of drugs. Cultures of strains 1 and 17 were grown to an OD₆₀₀ of 1.0 and then treated with BEN (B; 75 μg/ml), MTX (M; 100 μg/ml preincubated for 10 min with 200 μg/ml sulfanilamide), NQ (N; 10 μg/ml), OP (20 μg/ml), or no drug for 1 h. Total RNA was prepared, electrophoresed, blotted, and hybridized with probes for MDR1 and ACT1 as a control. The MTX lane for isolate 17 was overloaded in this experiment.

FIG. 3.

(A) Relative intensities of MDR1 promoter in the presence and absence of BEN. Relative luciferase activities in the presence (black boxes) and absence (white boxes) of BEN are shown on the y axis. Specific activities for all constructs were made relative to the specific activity of construct 100 in the absence of the drug so that activities could be easily compared. Constructs are listed across the x axis, including the 2692 constructs from both isolates 1 and 17. (B) Relative (n-fold) induction of the MDR1 promoter in the presence of drug compared to the absence of drug. n-fold induction (grey squares) is the ratio of the relative intensities from panel A (with drug/without drug). Constructs are indicated above the panel, the same as in panel A.

FIG. 4.

(A) Relative intensities of MDR1 promoter in the presence and absence of T-BHP. Relative luciferase activities in the presence (black boxes) and absence (white boxes) of T-BHP are shown on the y axis. Specific activities for all constructs were made relative to the specific activity of construct 100 in the absence of the drug so that activities could be easily compared. Constructs are listed across the x axis, including the 2692 constructs from both isolates 1 and 17. (B) Relative (n-fold) induction of the MDR1 promoter in the presence of drug compared to the absence of drug. n-fold induction (grey squares) is the ratio of the relative intensities from panel A (with drug/without drug). Constructs are indicated above the panel, the same as panel A.

FIG. 5.

Drug induction of full-length MDR1 promoter. The full-length (1,498-bp) MDR1 promoter was assayed in the presence and absence of drugs. Cells were grown to an OD of 1.0 before drug addition (conditions similar to Fig. 2). The x axis represents the time after drug addition. The y axis represents the n-fold induction in the presence of drug, calculated as the specific activity of luciferase from cells in the presence of drug divided by the specific activity of luciferase from cells in the absence of drug. In all panels except the last, solid symbols represent cultures grown in different drug concentrations and open circles represent a time course analysis of cultures grown in a single drug concentration. In the last panel, shading of symbols indicates different drugs and symbol shapes indicate different concentrations. The 1-h time points for concentrations of DA, MMS, T-BHP, MTX, and OP are offset on the graph for clarity. The graphs are all drawn to the same scale. The MMS value at 24 h (1,603-fold induction) does not fit on that scale. Drug concentrations are as follows (time course analysis concentrations are in bold): BEN, 25 μ g/ml (square) and 75 μg/ml (circle); DA, 1 mM (triangle), 3 mM (square), and 9 mM (circle); DEM, 12 mM (square) and 24 mM (circle); FLC, 1 μg/ml (gray square), 10 μg/ml (gray circle), and 100 μg/ml (gray diamond); H₂O₂, 0.044 mM (inverted triangle), 0.44 mM (triangle), and 4.4 mM (square); MMS, 12 mM (square) and 24 mM (circle); MTX, 1 mM (black square), 10 mM (black circle), and 20 mM (black diamond) after preincubation with 200 μg/ml sulfanilamide; NQ, 1 μg/ml (square), 10 μg/ml (circle), and 20 μg/ml (diamond); OP, 0.2 μg/ml (open square), 2 μg/ml (open circle), and 20 μg/ml (open diamond); T-BHP, 0.05 mM (triangle), 0.5 mM (square), and 5 mM (circle).

FIG. 6.

(A) Relative intensities of YRE promoter constructs in the presence and absence of T-BHP. Relative intensities in the presence (solid bars) and absence (open bars) of T-BHP are shown on the y axis. Specific activities for all constructs were made relative to the specific activity of YRE-F in the absence of the drug so that activities could be easily compared. Constructs are listed across the x axis. (B) Relative (n-fold) induction of the MDR1 promoter in the presence of the drug compared to the absence of the drug. n-fold induction is the ratio of the relative intensities from panel A (with drug/without drug). Constructs are indicated above the panel, the same as panel A.