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Antimicrobial Agents and Chemotherapy, July 2005, p. 2785-2792, Vol. 49, No. 7
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.7.2785-2792.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Drug-Induced Regulation of the MDR1 Promoter in Candida albicans

Jo Beth Harry,2,{dagger} Brian G. Oliver,1,2 Jia L. Song,1,2,{ddagger} Peter M. Silver,1,2 John T. Little,2,§ Jake Choiniere,2 and Theodore C. White1,2*

Department of Pathobiology, School of Public Health and Community Medicine, University of Washington,1 Seattle Biomedical Research Institute, Seattle, Washington2

Received 31 December 2004/ Returned for modification 10 March 2005/ Accepted 8 April 2005


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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.


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INTRODUCTION
 
The pathogenic yeast Candida albicans causes oral, systemic, or vaginal infections in immunocompromised patients or immunocompetent women (reviewed in reference 27). Oral candidiasis is one of the first and one of the most frequent opportunistic infections in immunocompromised, human immunodeficiency virus-seropositive (HIV+) individuals (12). Resistance to treatment with the azole antifungal drug fluconazole (FLC) occurs in these HIV+ patient populations and in bone marrow transplant populations (19, 23). The molecular mechanisms that result in azole resistance include overexpression of (i) the major facilitator efflux pump gene MDR1, (ii) the ABC transporter efflux pump CDR gene family, and (iii) the azole target enzyme gene ERG11 (reviewed in reference 37). Point mutations in ERG11 are also important for resistance. These molecular mechanisms of resistance can occur simultaneously or independently of each other and exist in different combinations.

In Candida, the MDR1 gene is a multidrug resistance gene that confers resistance to benomyl (BEN), benztriazoles, cycloheximide (CHX), methotrexate (MTX), 4-nitroquinoline-N-oxide (NQ), and sulfometuron methyl (2, 8). Disruption of the MDR1 gene led to increased susceptibility to MTX, NQ, and CHX (11, 29). In clinical isolates from HIV+ patients, there is a correlation between azole drug resistance and overexpression of the MDR1 gene (10, 20, 24, 30, 35, 36).

There is little information concerning the transcriptional regulation of MDR1. In C. albicans, MDR1 was transcriptionally activated by treatment with MTX, NQ, BEN, o-phenanthroline (OP), hydrogen peroxide (H2O2), and sulfometuron methyl but not by CHX, chloramphenicol, pH, temperature, or FLC (6). In one study, an azole-sensitive isolate expressed MDR1 mRNA only early in exponential growth while the matched azole-resistant isolate expressed MDR1 mRNA in logarithmically growing cells and at low levels during diauxic shift (21). Nuclear run-on analyses with these isolates demonstrated that increased mRNAs for MDR1 are the result of an increased transcription rate due to increased mRNA synthesis rather than the lack of mRNA degradation (21). This specifically implicates the MDR1 promoter as the cause of increased mRNA levels.

In Saccharomyces cerevisiae, an MDR1 analogue, FLR1, was shown to be induced by the oxidizing agents H2O2, diamide (DA), diethylmaleate (DEM), and T-BHP and the alkylating agent methyl methanesulfonate (MMS) (3, 15, 26). These FLR1 inductions were mediated by the transcription factor Yap1p (26, 33) that is involved in the fungal oxidative stress response (4, 17, 18). Genes that respond to oxidative stress through Yap1p have a YRE (Yap1p response element) in their promoters. In Saccharomyces, the consensus YRE is TTAC/GTAA, a palindromic sequence with two identical TTA half sites (7). The sequence has been shown to act in only one orientation, suggesting that it is not an enhancer element (5, 34). A YAP1-related gene, CAP1, that mediates the oxidative stress response in C. albicans has been identified (1, 40). Alarco and Raymond found that disruption of CAP1 led to an increase in MDR1 expression, while DeMicheli et al. did not observe this increase (1, 6). Thus, CAP1 may be a negative transcriptional regulator of MDR1 in Candida.

In this report, MDR1 expression was monitored using both Northern blot analysis and the luciferase reporter gene. The induction of the MDR1 promoter was monitored at various stages of cell growth and after exposure to 10 drugs at various concentrations. MDR1 promoter deletions were used to analyze basal transcription, as well as to determine which regions of the promoter are important for induction by BEN and by T-BHP. Induction by T-BHP was shown to be directly the result of a YRE that is both necessary and sufficient for induction by T-BHP.


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MATERIALS AND METHODS
 
Strain maintenance and manipulation and chemicals. Strain CAI8 (ade2::hisG/ade2::hisG {Delta}ura3::imm434/{Delta}ura3::imm434), kindly provided by William A. Fonzi (9), was used in all transformation experiments. C. albicans isolates 1 (2-76; azole sensitive) and 17 (12-99; azole resistant) from a series of 17 oral isolates from a single HIV+ patient (38) were also studied. Strains were maintained on YEPD (10 g of yeast extract, 20 g of peptone, 20 g of dextrose, with or without 15 g of Bacto Agar per liter) or on CSM-ade medium (0.75 g CSM-ADE [Bio 101, Inc., Vista, CA], 1.7 g yeast nitrogen base without amino acids or ammonium sulfate, 5 g ammonium sulfate, 20 g dextrose, and 50 mg uridine per liter). All C. albicans isolates were stored at –80°C in CSM-ade or YEPD containing 10% glycerol. Medium components were obtained from Fisher Scientific (Pittsburgh, PA) or Bio 101, Inc. Chemicals were obtained from Fisher, Sigma (St. Louis, MO), or Aldrich (Milwaukee, WI).

Construction of the MDR1 promoter deletions in Renilla luciferase reporter plasmid pCRW3. Renilla reniformis luciferase (RLUC) reporter plasmid pCRW3 was generously provided by David Soll (32). Reporter plasmids based on pCRW3 were constructed as previously described (31), inserting MDR1 promoter fragments between the ClaI and XmaI restriction sites upstream of the RLUC reporter gene. Oligonucleotides (Gibco-BRL, Rockville, MD; Table 1) were designed to generate nested fragments of the MDR1 promoter (MDR1-1R at the ATG start codon of MDR1 and an upstream oligonucleotide). These nested promoter fragments consisted of 2,692 bp, 1,997 bp, 1,498 bp, 999 bp, 799 bp, 601 bp, 500 bp, 399 bp, 299 bp, 200 bp, and 100 bp upstream of the ATG start codon of MDR1. The fragments were PCR amplified from genomic DNAs of isolates 1 and 17 using Pfu DNA polymerase (Stratagene, La Jolla, CA) (38). Each of the pCRW3 constructs containing an MDR1 promoter fragment was sequenced using an ABI automated DNA sequencer with Taq dye primer and dye terminator chemistries (Applied Biosystems, Foster City, CA).


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  		TABLE 1. Oligonucleotides used in the analyses in this studya

The constructs were linearized within the ADE2 gene at the HindIII restriction site and electroporated into C. albicans CAI8 cells (31). Transformants were analyzed by PCR and Southern blotting as previously described (31) to confirm that a single copy of the plasmid was integrated at the ADE locus (diagrammed in Fig. 1). CAI8 transformants with a single integration of the upstream fragments from the MDR1 promoter region fused to the RLUC reporter were designated constructs 2692, 1997, 1498, 999, 799, 601, 500, 399, 299, 200, and 100. Construct 1997 behaved like constructs 2692 and 1498 and will not be discussed further. Construct 799 behaved like constructs 601 and 999 and will not be discussed further.



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  		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).

Additional constructs were created that encode either the YRE (TTAGTAA) within a 49-bp fragment or a mutated fragment in which the YRE was mutated (YME = TAGATCT, substituting a BglII site). These fragments were inserted upstream of construct 500 described above. The fragment was prepared by annealing two oligonucleotide pairs (YRE+-YRE– and YME+-YME–). The resulting fragment had a 5' CG overhang and was cloned at the ClaI site upstream of the construct 500 promoter. The 49-bp fragments were inserted at the ClaI site in either direction, creating YRE-F, YRE-R, YME-F, and YME-R, where the fragment insertion designated F (forward) matched the sequence of the coding strand and R matched the sequence of the noncoding strand of the promoter.

RNA extraction and Northern blot analysis. For Northern blot analysis, cells were grown to an optical density at 600 nm (OD600) of 1.0 and RNA was isolated. Total RNA preparation, gel electrophoresis, Northern blotting, oligonucleotide labeling with polynucleotide kinase, and random priming for radioactive probe preparation were performed according to standard published methods (22, 28). DNA probes for the Northern blot assay consisted of actin oligonucleotide ACT50 (Table 1) and a plasmid with the MDR1 coding region. mRNA signals were quantified using the Storm Phosphorimager and ImageQuant software (Molecular Dynamics, Sunnyvale, CA). MDR1 mRNA signals were normalized to ACT1 mRNA signals.

In vitro assay of RLUC activity. Luciferase assays were performed as previously described (31). Specific activity was defined as the relative luminescence per 10 s per µg of protein extract. Protein concentrations were determined by the Bradford assay (Bio-Rad Laboratories, Hercules, CA).

Drug induction. For MDR1 promoter induction by BEN, CAI8 transformants were grown in YEPD to an OD600 of 1.0 and then 25 to 75 µg/ml of BEN was added and the culture incubated at 30°C for the appropriate time. For all other MDR1 induction, CAI8 transformants were grown in CSM-ade to an OD600 of 1.0 and a range of concentrations was tested for a specified amount of time. The ranges of concentrations tested for the potential MDR1 inducers were as follows: DA, 1 mM to 9 mM; DEM, 12 mM to 24 mM; FLC, 1 µg/ml to 100 µg/ml; H2O2, 0.04 mM to 4.4 mM; MTX, 1 mM to 20 mM in the presence of 200 µg/ml of sulfanilamide (8); MMS, 12 mM to 24 mM; NQ, 1 mg/ml to 20 mg/ml; OP, 0.2 µg/ml to 20 µg/ml; T-BHP, 0.05 mM to 5 mM.

MIC analysis. Drug susceptibility was determined using the standard NCCLS broth microdilution protocol. The range of drug concentrations tested differed for each drug, based on preliminary tests. The MIC was defined as the concentration of a drug needed to inhibit 80% of cell growth, consistent with standard protocols (25). RPMI 1640 medium buffered with morpholinepropanesulfonic acid (MOPS) to pH 7 and supplemented to 2% glucose was used for MIC testing.


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RESULTS
 
Northern blot analysis of isolates 1 and 17. In order to understand the transcription profile of the MDR1 promoter, Northern blot analysis was performed on total RNA from azole-susceptible isolate 1 and azole-resistant isolate 17 in the presence and absence of selected compounds that have been shown to affect MDR1 expression in other strains. The drugs used included BEN, MTX, NQ, and OP, all of which had previously been shown to stimulate MDR1 promoter activity in C. albicans strain ATCC 10261 (13). The MDR1 mRNA from azole-resistant isolate 17 was highly expressed under all conditions (Fig. 2). The MDR1 mRNA from azole-susceptible isolate 1 was overexpressed only when induced by BEN and was undetectable in the uninduced state or when treated with MTX, NQ, or OP at concentrations previously used by others (Fig. 2). The lack of MDR1 mRNA from azole-susceptible isolate 1 in the absence of drug and the high-level expression of MDR1 mRNA in azole-resistant isolate 17 in the absence of drug was consistent with previously published reports from our laboratory (14, 21). In Fig. 2, the four drugs did not induce expression of MDR1 in isolate 17 above levels that were already expressed in the absence of drug, suggesting that MDR1 expression was maximal in this resistant isolate.



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  		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.

Comparison of MDR1 promoter sequences. To compare the promoter sequences from the susceptible and resistant isolates, full-length promoter fragments (2,692 bp; reference 8) were amplified from isolates 1 and 17 and cloned into plasmid pCRW3, which contains the R. reniformis luciferase gene (RLUC) as a reporter (31, 32). This plasmid contains RLUC and the C. albicans selectable marker ADE2. Constructs were integrated as a single copy at the ade2 locus of strain CAI8 (ura3/ura3 ade2/ade2; reference 9) (Fig. 1). Detailed descriptions of the promoter constructs and transformations are given in Materials and Methods and a previous report (31).

When the promoters from each clone were sequenced, 13 MDR1 sequence differences were identified between the clones produced from isolates 1 and 17 (A306G, G341C, T343A, A448T, T565G, T655C, A682G, G724C, A739G, C755T, AAACAC811-816CC, A832C, and G890A, where the number refers to a position upstream of the ATG start codon based on the genome sequence [16], the base[s] before the number is the sequence from isolate 1, and the base[s] after the number is from isolate 17). Additional clones were sequenced with similar results. When these two constructs (2692 [isolate 1] and 2692 [isolate 17]) were analyzed for basal transcription or for induction with BEN or T-BHP, no significant differences in promoter activity were observed between the two MDR1 promoter constructs in a CAI8 background (Fig. 3 and 4). A small difference was seen for basal transcription of the two clones in the BEN experiments. However, this difference was not observed for basal transcription of the two clones in the T-BHP experiments.



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  		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.



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  		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.

Constructs for the identification of cis-acting elements within the MDR1 promoter. Since the promoters behaved similarly in a common strain background, it is likely that azole-resistant isolates upregulate MDR1 using trans-acting factors. Therefore, it was important to identify regions of the promoter (cis-acting elements) that are important for that regulation. To identify these cis-acting elements, deletions of the isolate 1 MDR1 promoter (2692, 1498, 999, 601, 500, 399, 299, 200, and 100) were constructed and luciferase activity was monitored. Each designation refers to the distance in base pairs from the ATG start codon of the MDR1 coding region in the genome sequence.

The MDR1 construct originally sequenced by Fling et al. included a 2,697-bp region upstream of the ATG codon (8). Promoter fragments of 2,692 bp (16) which correspond to the 2,697-bp region for the other strain were used in comparing promoters from isolates 1 and 17. However, detailed analysis of the contig containing MDR1 revealed another upstream open reading frame that included the first 1,490 bp at the 5' end of the MDR1 promoter, leaving a 1,207-bp MDR1 promoter (16). When analyzing the MDR1 promoter deletions, the 1,498-bp MDR1-RLUC construct (1498) was used throughout. There was no significant difference in promoter activity between 2692 and 1498 as determined by luciferase assays (Fig. 3 and Fig. 4).

Basal transcription of the MDR1 promoter. Basal transcription of the MDR1 promoter (in the absence of drug) was studied in logarithmic-phase growing cells (open squares, Fig. 3 and Fig. 4). All constructs showed increased basal promoter activity compared to pCRW3, including construct 100 (19- to 33-fold). The specific activities of all constructs were compared to the specific activity of construct 100, since absolute values differed in repeated luciferase experiments and since the pCRW3 value was consistently background (Fig. 3A and 4A, open squares). This defines a very minimal promoter consistent with previous work that defined the transcription initiation site (65 bp upstream of the ATG start codon), a consensus TATA sequence (135 bp upstream of the MDR1 transcription initiation site), and a nonconsensus TATA sequence (96 bp upstream from the MDR1 start site) (14). Basal transcription increased 22-fold as the size of the promoter increased to 1,498 bp. However, no one region of the promoter from bp 100 to 1,498 showed a large increase in basal transcription.

The MDR1 promoter is induced by several hydrophobic or toxic compounds. To better analyze MDR1 promoter induction in the presence of various drugs, the full-length (1,498-bp) MDR1 promoter in the SC5314 (CAI8) background was examined using RLUC as a reporter. n-fold induction, which is the specific activity of the drug-induced promoter relative to the specific activity of the uninduced promoter, is reported for 10 drugs in Fig. 5. For each drug, several concentrations were tested at one time point (usually 1 h). Subsequently, a time course analysis (0.5 to 24 h) was performed at one concentration for drugs that induced the promoter.



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  		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).

The drugs selected for this analysis include those shown to have an effect on C. albicans MDR1 mRNA levels (8, 13), including BEN, MTX, OP, NQ, and FLC, as well as drugs shown to have an effect on the MDR1 homolog in S. cerevisiae, FLR1 (26), including DA, DEM, H2O2, MMS, and T-BHP.

Three drugs (FLC, MTX, and OP) did not induce the MDR1 promoter. MTX and OP were tested at 1 h under the same conditions as the other drugs. FLC was tested at 5, 24, and 48 h as previous analysis for the effect of FLC on cells had shown that the effect of FLC was maximal at 24 to 48 h (31).

Six drugs (DEM, DA, MMS, NQ, H2O2, and T-BHP) showed greater than 10-fold induction (10- to 421-fold) by 0.5 to 1 h, and that induction remained high for 5 to 24 h. MMS and NQ inductions increased at 24 h compared to 1 h, while induction by the other drugs was reduced but remained high. One drug, BEN, showed 347-fold induction at 0.5 h and was reduced 46-fold by 5 h. The compounds that induced MDR1 promoter activity include hydrophobic, toxic compounds (MTX, NQ, OP, and BEN), oxidizing agents (DEM, DA, T-BHP, and H2O2), and the alkylating agent MMS. The regions of the MDR1 promoter that respond to the hydrophobic compound BEN and the oxidizing agent T-BHP were identified and characterized (see below). These two drugs were selected based on the level of induction and the drugs' effects in Candida and in other systems. The regions of the promoter that respond to the alkylating agent MMS were not identified or further characterized.

MDR1 promoter induction as monitored by RLUC activity occurred quite rapidly, within 30 min. For example, BEN induction was induced 347-fold by 0.5 h and was subsequently reduced 46-fold by 5 h. This reduction in luciferase activity over 5 h (Fig. 5) allows an estimation of luciferase half-life at less than 1.8 h.

BEN induction of the MDR1 promoter. The MDR1 promoter deletion constructs were used to define the regions of the MDR1 promoter that are important for induction by BEN (Fig. 3). A 15-fold increase in induction by BEN was observed in construct 399 compared to 299. An adjacent region of the promoter (bp 200 to 299) may also have contributed to BEN induction of the MDR1 promoter (3.5-fold).

T-BHP induction of the MDR1 promoter. T-BHP was used to characterize the region of the promoter that responds to oxidizing agents, using the MDR1 promoter deletion constructs (Fig. 4). The region between bp 500 and 601 was responsible for a 23-fold induction in promoter activity in response to T-BHP. No other regions of the promoter altered promoter activity in response to T-BHP (all less than threefold). The bp 500 to 601 region was also important for induction by the other drugs, DA, DEM, and H2O2 (data not shown).

YRE in Candida. In S. cerevisiae, the YRE is the sequence TTAC/GTAA (7). That sequence is present as TTAGTAA at positions –525 to –532 within the bp 500 to 601 region of the MDR1 promoter shown to be important for the T-BHP response. The sequence is not found at any other location in the MDR1 promoter.

A 49-bp fragment, encoding positions –550 to –508 flanked by ClaI sites, was inserted in both directions at the ClaI site upstream of construct 500. The 6-bp ClaI site replaces positions –501 to –507 of the promoter. When the 49-bp fragment containing the intact YRE is inserted at the ClaI site, the construct (YRE-F) is induced by T-BHP 104-fold, levels equivalent to construct 1498 (Fig. 6). When the same fragment is inserted in the opposite direction (YRE-R), which changes the sequence but not the spacing to construct 500, there is no induction. Equally important, the YRE sequence TTACTAA was changed to AGATCTA (a BglII site) and inserted upstream of construct 500 in both directions (YME-F and YME-R; M = mutant), while the rest of the fragment sequence was unchanged. Neither construct was induced by T-BHP (Fig. 6).



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  		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.

Effect of MDR1 induction on cell growth and FLC susceptibility. While these drugs are known to induce MDR1 promoter activity (Fig. 5), the susceptibility of Candida to these drugs has not been reported. MICs for drugs that induce MDR1 expression were determined for strains SC5314 (parent of CAI8), CAI8, and 1 (Table 2). The MIC of BEN was determined in YEPD, as it is not soluble in standard medium (RPMI 1640). The MICs of four drugs (DEM, H2O2, MMS, and T-BHP) were within 2 dilutions (fourfold) of the inducing concentration; essentially, the inducing concentration was equivalent to the MIC. The MIC was considerably lower than the inducing concentration for DA (5.4-fold) and NQ (16-fold). BEN was the only drug whose MIC was significantly higher than the concentration that induced MDR1 promoter activity.


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  		TABLE 2. MICs and effective concentrations of MDR1 inducers

Concentrations of these drugs two- to eightfold below the MICs of the drugs were tested for their effect on FLC susceptibility in strain SC5314. Most of the drugs had no effect (data not shown). However, in the presence of DEM at 0.25 times the MIC, the FLC MIC was increased 8-fold at 24 h (0.21 to 1.7 µg/ml) and 16-fold at 48 h (0.42 to 6.7 µg/ml). However, when this lower concentration of DEM was used in luciferase assays, it failed to induce the MDR1 promoter at either 1 or 48 h (data not shown).


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DISCUSSION
 
MDR1 promoter activity has been analyzed in the presence of 10 drugs known to have an effect on related pumps, including oxidizing agents (DA, DEM, H2O2, and T-BHP), toxic hydrophobic compounds (BEN, NQ, OP, MTX, and FLC), and an alkylating agent (MMS) (Fig. 5). Deletion analysis of the promoter identified a region important for induction by BEN between positions –299 and –399 (Fig. 3) and a region important for induction by T-BHP between positions –500 and –601 (Fig. 4) that includes a YRE. When this YRE sequence was added to a noninducible promoter, it caused induction of the promoter (Fig. 6). Many of the drug concentrations that induce MDR1 were found to correspond to the MIC of the drug for the strain (Table 2). Finally, at least one MDR1 pump inducer, DEM, can alter the FLC susceptibility of a strain at concentrations below the MIC of DEM.

Northern blot analysis (Fig. 2) was used to test four drugs (BEN, MTX, NQ, and OP) for induction of the MDR1 promoter in susceptible isolate 1 and matching resistant isolate 17 under conditions that were used by others to analyze susceptible strain ATCC 10261 (13). For each of the four drugs, the Northern blot analyses (Fig. 2) were consistent with the luciferase assays (Fig. 5). However, three of the drugs (MTX, OP, and NQ) that induced MDR1 expression in strain ATCC 10261 did not induce MDR1 expression in strain 1 at the same drug concentration. (NQ did induce MDR1 expression at a lower concentration [1 µg/ml] in luciferase assays [Fig. 5].) BEN is the one drug that induces MDR1 expression in both isolate 1 and strain ATCC 10261. Thus, there are differences in responsiveness between strain ATCC 10261 and isolate 1 that may be the result of strain, laboratory, or technical differences.

Sequence analysis of allelic PCR clones from isolates 1 and 17 revealed 13 differences within the 1,498-bp region. These 13 differences most likely represent allelic variation, since these constructs were PCR amplified from the diploid C. albicans genome. Additional clones from the two isolates had similar allelic differences (data not shown). Since the MDR1 promoters were amplified by PCR, it is also possible that the base pair differences are the result of PCR error although Pfu polymerase was used. Regardless of the origin, there are no functional differences between the two cloned sequences (Fig. 3 and 4). This suggests that a mutation or alteration in a trans-acting regulatory factor is likely responsible for the increased MDR1 promoter activity in azole-resistant isolate 17. These results are consistent with the previous analysis of an unrelated resistant isolate (39).

Ten different drugs known to affect MDR1-like pumps in other systems were tested against the MDR1 promoter-luciferase construct at different concentrations and times of induction (Fig. 5). The use of luciferase as a reporter makes such analyses much simpler than Northern blot analysis. The rapid induction by BEN (within 30 min) is consistent with previous reports (13). In addition, the 46-fold reduction in induction by BEN within 5 h suggests that the cells adapt to the drug or the drug is degraded. This reduction in induction by BEN is important, as C. albicans was found to be resistant to the transcription inhibitor actinomycin D and to the translation inhibitors verrucarin, CHX, and trichodermin-trichothecin (data not shown). Because C. albicans is resistant to these translation and transcription inhibitors, the half-life of luciferase could not be determined by standard methods. The reduction in induction by BEN allows an estimation of the luciferase half-life at less than 1.8 h. Unlike the short-term induction seen with BEN, the induction by oxidizing agents DA, DEM, H2O2, and T-BHP persists for up to 24 h after rapid induction in less than 30 min.

FLC does not induce the MDR1 promoter (Fig. 5), consistent with previous studies with C. albicans (13) and S. cerevisiae (26). FLC was tested at several concentrations and time points, as maximal FLC induction of the ERG11 promoter only occurs after 24 to 48 h (31).

Deletion fragments of the MDR1 promoter were used to identify the region of the promoter responsible for induction by BEN, –399 to –299. An analysis of this region did not identify any repetitive or palindromic sequence that might be involved in induction by BEN (data not shown). BEN induction mediated by the –399 to –299 region, separate from the YRE at –532 to –525, contradicts previous work with S. cerevisiae showing that BEN activates the FLR1 promoter through a YRE (26). BEN does not show induction correlated with the YRE at –532 to –525, and a YRE sequence is not present in the BEN inducing region (–399 to –299). The trans-acting factor that might be important for induction of the MDR1 promoter by BEN has not been identified, and the region has not been defined further.

Deletion fragments were also used to identify the region responsible for T-BHP induction, –601 to –500, containing the YRE (–532 to –525). The YRE inserted upstream of the noninducible 500 construct (YRE-F) causes induction, while the mutant (YME-F) and inverted orientation of the YRE (YRE-R) did not, consistent with the orientation-specific function of YRE in S. cerevisiae (5, 34). This lack of activity of YRE-R is not due to phasing of the DNA (orientation of the DNA on the helix), as YRE-F and YRE-R have the same position relative to the reporter gene. The YRE constructs exhibit a similar but reduced effect with other oxidizers, including DA and DEM, although the induction with H2O2 was not observed (data not shown), consistent with previous work with S. cerevisiae (40). MMS induction is also regulated by the YRE (data not shown), consistent with results from S. cerevisiae (26). Additional elements important for full induction with DA, DEM, H2O2, MMS, and NQ were identified between –600 and –1000 but have not been characterized further (data not shown).

To determine the effect of MDR1 inducers on the growth of the cells, MICs were determined (Table 2). The MICs were consistently equal to or lower than the concentration that induced the MDR1 drug pump, except for BEN. These results are consistent with the induction of MDR1 as a specific stress response of the cells. The response is specific, as other drugs, such as FLC, did not induce MDR1 expression at levels up to 100-fold above the MIC. Induction at or above the MICs of the drugs in Table 2 raises significant questions about the clinical significance of oxidative stress induction of MDR1, since the inducing concentrations would also result in cell death or stasis.

One drug, DEM, had an effect on FLC susceptibility. DEM increased resistance to FLC 8- to 16-fold at DEM concentrations 0.25 times the MIC, where cell growth is not inhibited. DEM might induce expression of MDR1, resulting in increased efflux of FLC and increased resistance. However, this concentration of DEM did not alter promoter activity as monitored by RLUC. The differences between DEM concentrations that induce the MDR1 promoter and concentrations that alter FLC susceptibility may have several explanations. First, it is possible that DEM is altering resistance by a mechanism unrelated to MDR1 expression. Second, these differences may be due to technical issues such as timing, inoculum sizes, medium differences, or the half-life of DEM in culture. The half-life of the Mdr1p protein in the plasma membrane may be longer than the half-life of the MDR1 mRNA, which may explain why the FLC MICs are increased at concentrations that have a limited effect on the promoter.

This work details the response of the MDR1 promoter to many drugs. It separates induction of the promoter by BEN from its induction by oxidizing agents. It also suggests that these observed induction effects might have a (subtle) clinical consequence for FLC susceptibility in vivo. Future work will be directed to a better understanding of the trans-acting transcription factors that regulate these cis-acting elements in the MDR1 promoter.


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ACKNOWLEDGMENTS
 
We thank William Fonzi (Georgetown University, Washington, D.C.) for providing the CAI8 strain and David Soll (University of Iowa, Iowa City) for the Renilla luciferase reporter pCRW3 plasmid. We thank members of the White laboratory for valuable comments on and support in preparing the manuscript.

This research was funded by NIH NIDR grants R01 DE-11367 and R01 DE14161. J.B.H. was supported by NIAID NRSA grant 1F32 AI10497-01. J.L.S. and B.G.O. were supported by NIH Pathobiology Training Grant T32 AI 07509. T.C.W. is the recipient of a New Investigator Award in Molecular Pathogenic Mycology from the Burroughs Wellcome Fund.


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FOOTNOTES
 
* Corresponding author. Mailing address: Seattle Biomedical Research Institute, 307 Westlake Ave., N., Suite 500, Seattle, WA 98109-5219. Phone: (206) 256-7344. Fax: (206) 256-7229. E-mail: ted.white{at}sbri.org. Back

{dagger} Present address: National Institute on Aging, NIH, Baltimore, Md. Back

{ddagger} Present address: Brown University, Providence, R.I. Back

§ Present address: University of California at Santa Clara, Santa Clara, Calif. Back


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Antimicrobial Agents and Chemotherapy, July 2005, p. 2785-2792, Vol. 49, No. 7
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.7.2785-2792.2005
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