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Eukaryotic Cell, December 2006, p. 1957-1968, Vol. 5, No. 12
1535-9778/06/$08.00+0     doi:10.1128/EC.00243-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Transcriptional Regulation of MDR1, Encoding a Drug Efflux Determinant, in Fluconazole-Resistant Candida albicans Strains through an Mcm1p Binding Site{triangledown}

Perry J. Riggle and Carol A. Kumamoto*

Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts 02111

Received 30 July 2006/ Accepted 29 September 2006


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ABSTRACT
 
Constitutive, high-level transcription of the gene encoding the drug efflux facilitator Mdr1p is commonly observed in laboratory and clinical strains of Candida albicans that are resistant to the antifungal drug fluconazole (FLC). In five independently isolated FLCR laboratory strains, introduction of a wild-type MDR1 promoter fragment fused to the yeast enhanced green fluorescent protein (yEGFP) reporter gene resulted in high-level expression of GFP, demonstrating that overexpression of MDR1 is dependent on a trans-acting factor. This study identified a 35-bp MDR1 promoter element, termed the MDRE, that mediates high-level MDR1 transcription. When inserted into a heterologous promoter, the MDRE was sufficient to mediate high-level expression of the yEGFP reporter gene specifically in MDR1 trans-activation strains. The MDRE promoted transcription in an orientation-independent and dosage-dependent manner. Deletion of the MDRE in the full-length promoter did not abolish MDR1 trans-activation, indicating that elements upstream of the MDRE also contribute to transcription of MDR1 in these overexpression strains. Analysis of the MDRE sequence indicated that it contains an Mcm1p binding site very similar in organization to the site seen upstream of the Saccharomyces cerevisiae MFA1 and STE2 genes. Electrophoretic mobility shift analysis demonstrated that both wild-type, FLC-sensitive and MDR1 trans-activated, FLC-resistant strains contain a factor that binds the MDRE. Depletion of Mcm1p, by use of a strain in which MCM1 expression is under the control of a regulated promoter (44), resulted in a loss of MDRE binding activity. Thus, the general transcription factor Mcm1p participates in the regulation of MDR1 expression.


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INTRODUCTION
 
Candida albicans is a commensal organism of the human gastrointestinal tract and genitourinary tract but can become an important opportunistic pathogen, especially in immunocompromised individuals (32). Depending on the underlying immune defect, C. albicans causes a variety of disease states, including superficial mucosal infections and more serious disseminated infections that exhibit approximately 40% attributable mortality (24). C. albicans has been associated with the AIDS epidemic from the outset (21). Oropharyngeal candidiasis, or thrush, in seemingly healthy individuals is often the first indication of human immunodeficiency virus (HIV) infection, and most AIDS patients have one or more episodes of Candida infection (22, 52). Fluconazole is the drug of choice for treating candidal infection in AIDS patients due to its low toxicity and oral bioavailability (7, 18, 53). However, the widespread use of fluconazole to treat candidal infections has led to the emergence of resistant Candida strains, and resistance is now a clinical problem (22, 51, 52). In one study, approximately one-third of late-stage AIDS patients carried drug-resistant C. albicans in their oral cavities (35). Fluconazole-resistant (FLCR) C. albicans is now seen in both HIV and non-HIV patients who have never been exposed to fluconazole (38).

Commonly seen molecular mechanisms for fluconazole resistance in C. albicans include increased efflux of fluconazole and mutation of ERG11, the gene encoding the target of fluconazole, lanosterol demethylase. Mutations within the ERG11 structural gene reduce fluconazole binding (41, 53). The most common mechanism, occurring in approximately 85% of clinical isolates, is increased efflux mediated by overexpression of the genes encoding the multidrug resistance efflux proteins Mdr1p, Cdr1p, and Cdr2p (41).

Mdr1p is classified in the major facilitator superfamily of efflux transporters and exports a variety of structurally unrelated compounds, including fluconazole, benomyl, methotrexate, cycloheximide, 4-nitroquinoline-N-oxide, and sulfometuron methyl (6). C. albicans cells lacking the MDR1 gene are hypersensitive to most of the drugs listed above (20). In two FLCR clinical isolates, deletion of MDR1 abolished the drug resistance phenotype of these strains, demonstrating that in these strains, fluconazole resistance was dependent on the overexpression of MDR1 (55). Moreover, engineered overexpression of MDR1 confers increased resistance to some compounds (28).

Despite the frequency with which MDR1-overexpressing, FLCR strains are isolated, the molecular mechanisms leading to high-level expression of MDR1 are not well understood (37). In some FLCR strains, high-level expression of MDR1 is due to the effects of an undefined trans-acting factor(s) (54). Recent work by Hiller et al. indicates that three independent regions of the MDR1 promoter are capable of contributing to MDR1 expression in a FLCR strain overexpressing MDR1 and that the portion of the MDR1 promoter they term region 2 (–588 to –500) mediates benomyl induction of MDR1 transcription in the FLC-sensitive (FLCS) laboratory strain CAI4 (29). In another study, analyzing the FLCS C. albicans laboratory strain CAI8, Harry et al. identified two or more cis-acting promoter regions that contribute to MDR1 expression (25). Under the conditions used by Harry et al., the more-proximal cis-acting element (–399 to –299) was responsible for benomyl-induced transcription of MDR1 while the more-distal cis-acting element (–601 to –500) was implicated in MDR1 induction by oxidizing agents. The most-distal region identified by both groups (–601/–588 to –500) contains a sequence that resembles and functions like the YAP1-responsive element of Saccharomyces cerevisiae (25). Yap1p is a member of the bZIP family of transcription factors and regulates the S. cerevisiae FLR1 gene, encoding a multidrug efflux protein that is similar to C. albicans Mdr1p (1). The homologous C. albicans gene is termed CAP1 (for C. albicans AP-1) (2). Surprisingly, deletion of CAP1 in C. albicans did not lead to FLC hypersensitivity (2). When the CAP1 gene was disrupted in a laboratory-derived FLCR strain that overexpresses MDR1, increased levels of MDR1 mRNA were observed, indicating that Cap1p acts as a negative regulator of MDR1 in this strain and is not responsible for the preexisting MDR1 overexpression. Therefore, other uncharacterized protein factors probably bind to elements within the MDR1 promoter and trans-activate the MDR1 gene in FLCR strains.

Expression of the other drug efflux transporters, Cdr1p and Cdr2p, is also regulated through a trans-activating factor that binds a specific sequence within their promoters (10, 11). Cdr1p and Cdr2p are ATP binding cassette (ABC) transporters that are highly similar to each other in sequence (46). Increased expression of these genes has been observed in FLCR clinical strains, and deletion of the CDR1 gene in C. albicans results in hypersensitivity to the azole drugs fluconazole, ketoconazole, and itraconazole and to other drugs such as terbinafine and amorolfine (47). The CDR2 gene also confers resistance to the azoles, terbinafine, and amorolfine (46). Sanglard and coworkers identified a transcription factor, Tac1p, that binds an element within the CDR1/CDR2 promoter, the drug resistance element (DRE), and demonstrated its involvement in the high-level constitutive expression of these genes seen in some FLCR strains (10). Mutations within the TAC1 open reading frame (ORF) that lead to hyperactive trans-activation of CDR1 and CDR2 have been identified in FLCR strains (9). Therefore, mutations that result in increased activity of trans-activators occur commonly in FLCR strains.

In this study, we demonstrate that a promoter element termed the MDRE (for "MDR1 drug resistance element") mediates MDR1 trans-activation in five independent FLCR, MDR1-overexpressing strains, indicating a common mechanism for trans-activation. The MDRE acts in a dosage-dependent and orientation-independent manner to increase transcription of MDR1 in drug-resistant, trans-activation strains. The protein Mcm1p binds the MDRE, and mutations within the MDRE that abolish Mcm1p binding abolish MDR1 trans-activation. Extracts from MDR1 trans-activation strains contain higher levels of MDRE binding activity. The sequence of MCM1 is wild type in these FLCR strains, indicating that the activity rather than the structure of Mcm1p is altered in the trans-activating mutants. Therefore, these results demonstrate that Mcm1p participates in the regulation of expression from the MDR1 promoter at the MDRE.


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MATERIALS AND METHODS
 
Strains, plasmids, and media. C. albicans strains are listed in Table 1. Parent strains were CAI4 ({Delta}ura3/{Delta}ura3) (17) and CAPR306 ({Delta}ura3/{Delta}ura3 {Delta}crd1/{Delta}crd1) (42). The crd1 deletion strain CAPR306 was used as a parent in these studies because the crd1 mutation provides a convenient genetic marker for experiments such as protoplast fusion. To select FLCR strains, independent pools of the parental strains were exposed to increasing levels of fluconazole during liquid growth in batch culture in Sabouraud's dextrose broth (Difco). Briefly, the two drug-sensitive parental strains were grown in six replicate populations each in 2 µg/ml fluconazole. Upon reaching saturation, the cultures were diluted 1:1,000 in fresh medium again containing 2 µg/ml fluconazole. Upon reaching saturation, these populations were diluted into fresh medium containing 4 µg/ml fluconazole. This selection was repeated in 4 µg/ml fluconazole, then 8 µg/ml fluconazole (twice), then 16 µg/ml fluconazole (twice), then 32 µg/ml fluconazole (twice), and finally 64 µg/ml fluconazole (once). A representative isolate from each final population was chosen for detailed analysis. Antifungal susceptibility testing was performed by the standard CLSI (formerly NCCLS) microdilution method (5, 15). Strains were routinely grown in yeast extract-peptone-dextrose, CM-uridine, and synthetic dextrose (SD) (43).


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  		TABLE 1. Candida albicans strains used in this study

DNA manipulations and analysis. Plasmid isolation, PCR, restriction digestion, cloning, gel electrophoresis, and Southern hybridization analysis were performed by standard methods (45). C. albicans genomic DNA was isolated by a glass bead disruption procedure (43). Automated DNA sequencing was performed by Michael Berne and coworkers at the Tufts University Core Facility.

RNA analysis. Cells for RNA isolation were grown in SD liquid medium at 30°C to an optical density at 600 nm (OD600) of 1.0. Uridine was added as needed at 100 µg/ml. For RNA extraction, cells were lysed with glass beads and phenol (4). Twenty micrograms of total RNA per sample was separated on a formaldehyde agarose gel, transferred to a Nytran-plus membrane, and probed by standard Northern hybridization methods (45). The MDR1 probe was generated with a PCR product that corresponded to the MDR1 ORF. The CDR1 and CDR2 probes were generated by PCR and spanned the nucleotide regions –109 to +280 and –112 to +274, respectively, with regard to their ORFs (46). Probes were labeled with [{alpha}-32P]dATP by the random priming method using the Stratagene Prime-IT kit.

Construction of MDR1 and CDR2 promoter transcriptional fusions to the yEGFP reporter gene. The plasmids constructed for this study are described in Table 2. The integrating plasmid pLIB1 (T. Volkert and C. A. Kumamoto, unpublished data) encodes the yeast enhanced green fluorescent protein gene (yEGFP) flanked by 10 bp of the ACT1 5' untranslated region (UTR) and 389 bp of the ACT1 3' UTR. SalI and EcoRV sites upstream of the ACT1 5' UTR sequence were used for cloning (8). pLIB1 also carries a fragment of the CaADE2 gene to provide homology for integration and the CaURA3 gene as a selectable marker.


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  		TABLE 2. yEGFP reporter plasmids used in this study

Template DNA for PCR amplification of MDR1 or CDR2 promoter fragments was from strain CAI4, a naïve strain that had never been exposed to fluconazole (17). Various MDR1 promoter fragments were PCR amplified with the primers listed in Table 3, digested with the restriction enzyme SphI, and ligated into EcoRV-digested pLIB1. CDR2 promoter fusions were generated similarly, except that the restriction enzyme EcoRV was used to digest PCR-amplified CDR2 fragments prior to ligation into the pLIB1 EcoRV site. All MDR1 and CDR2 promoter fusion constructs were sequenced. For the heterologous expression plasmids, the primers MDRSF and MDRSR were annealed, phosphorylated, and ligated into the SalI site of pLIB1. The plasmid pPRC2-4mut contains the region –230 to –7 of the CDR2 promoter but contains a point mutation (underlined) within the DRE (5'-TGGAAATCGG-3') which renders the promoter completely defective in the ability to trans-activate CDR2 expression in a CDR1/CDR2 trans-activation strain. The plasmid pPRC2-5 contains the region of the CDR2 promoter from –187 to –7; the DRE is absent, thereby rendering the plasmid completely defective for CDR2 trans-activation.


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  		TABLE 3. Oligonucleotides used in this study

Transformation of C. albicans. Candida cells were transformed by the lithium acetate method of Gietz et al. and selected for uridine prototrophy on CM-uridine (19). yEGFP reporter plasmids were digested with the restriction enzyme BamHI to direct their integration into the ADE2 gene. Southern hybridization analysis was used to confirm single integration into the ADE2 locus.

Microscopy. Cells were visualized by using an Olympus BX60 microscope with a 1.4NA 100x objective lens. Images were collected with a Hamamatsu (Bridgewater, NJ) model C4742-95 cooled charge-coupled-device camera and analyzed with OpenLab software. Relative fluorescence of the MDR1 trans-activation strains containing a single integrated copy of pPRM1 was set at 100% (++++). Other fluorescence values are indicated as follows: greater than 125%, +++++; between 70% and 40%, +++; between 40% and 20%, ++; below 20% but above background autofluorescence, +; equal to background autofluorescence, –.

EMSAs. Yeast cells were grown to an OD600 of 1.0 and disrupted by the glass bead method in the following lysis buffer: 10 mM MgCl2, 10% glycerol, 20 mM Tris-Cl (pH 7.9), 1 mM dithiothreitol, 1 mM EDTA, 0.3 M (NH4)2SO4, 1 mM phenylmethylsulfonyl fluoride, and the Roche protease inhibitor set at manufacturer-suggested concentrations (4). Mcm1p-depleted extracts were generated by exposing strain MRcan42 to doxycycline at 20 µg/ml for 0, 2, 4, and 6 h, as described previously (44). Whole-cell extracts were frozen in aliquots in liquid nitrogen and stored at –80°C. Protein concentrations were determined with the Pierce Micro BCA kit and the manufacturer's protocol. Probes for electrophoretic mobility shift assays (EMSAs) were generated by annealing oligonucleotides MDRSF and MDRSR (Table 3) and 5'-end labeling the DNA with T4 polynucleotide kinase and [{gamma}-32P]ATP (45). EMSAs were performed in a 20-µl reaction mixture that contained the following components: 12% glycerol, 12 mM HEPES-NaOH (pH 7.9), 4 mM Tris-HCl (pH 7.9), 1 mM EDTA, 1 mM dithiothreitol, 2 ng labeled probe, 2,000 ng poly(dI-dC/dI-dC), 200 ng annealed ACT1-5'F/ACT1-5'R oligonucleotides (Table 3), 15 µg bovine serum albumin, and 1 to 5 µg protein from whole-cell extracts (4). The ACT1-5'F/ACT1-5'R double-stranded DNA (dsDNA) fragment was routinely added to all reactions to compete nonspecific binding to the labeled probe. Reactions were incubated for 20 min prior to loading on a low-ionic-strength gel. For reactions containing unlabeled competitor DNA, a 100-fold molar excess of competitor DNA was incubated for 10 min with protein extract prior to the addition of labeled probe. Competitor oligonucleotides shown in Fig. 6B were generated by annealing the following oligonucleotides (Table 3): GSF and GSR ({Delta}0), D1GSF and D1GSR ({Delta}1), D2GSF and D2GSR ({Delta}2), D3GSF and D3GSR ({Delta}3), D4GSF and D4GSR ({Delta}4), D5GSF and D5GSR ({Delta}5), D6GSF and D6GSR ({Delta}6), D7GSF and D7GSR ({Delta}7), D8GSF and D8GSR ({Delta}8), and D9GSF and D9GSR ({Delta}9). Low-ionic-strength electrophoresis buffer contained 6.75 mM Tris-HCl (pH 7.9), 3.3 mM sodium acetate (pH 7.9), and 1 mM EDTA. Low-ionic-strength gel mix contained 6.75 mM Tris-HCl (ph7.9), 1 mM EDTA (pH 7.9), 3.3 mM sodium acetate (pH 7.9), 4% acrylamide, 0.05% bisacrylamide, and 2.5% glycerol (4). The gel was prerun at 100 V for 90 min at 4°C with buffer recirculation. After sample loading, electrophoresis was continued at 30 to 35 mA for approximately 2 h until the bromphenol blue marker dye exited the gel. After electrophoresis, gels were transferred to Whatman paper, dried, and exposed for autoradiography.


Figure 6
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  		FIG. 6. MDRE binding activity detected in cell extracts. (A) EMSA with whole-cell protein extracts from a FLCS parental strain and a representative MDR1 trans-activation strain, CAPR514. The probe is a 5' end-labeled dsDNA fragment corresponding to bp –299 to –254 with respect to the MDR1 initiation codon and was generated by annealing oligonucleotides MDRSF and MDRSR (Table 3). Lanes 5 and 9 also contain a 100-fold excess of unlabeled probe DNA. The radiolabeled probe was incubated with protein extracts and analyzed on a polyacrylamide gel, and the resulting autoradiogram or phosphorimager scan is shown. Samples were free probe (lane 1); CAPR306 (wild-type [WT]) extract with 1 µg (lane 2), 2 µg (lane 3), 3 µg (lane 4), and 3 µg (lane 5) total protein; and CAPR314 (MDR1 OE) extract with 1 µg (lane 6), 2 µg (lane 7), 3 µg (lane 8), and 3 µg (lane 9) of total protein. (B) EMSA with competitor fragments that have deletions throughout the MDRE region. Reactions were carried out as described for panel A. Full-length unlabeled competitor ({Delta}0) is a 76-bp DNA fragment that contains the 35-bp MDRE plus 21 bp upstream and 20 bp downstream and was generated by annealing oligonucleotides GSF and GSR (Table 3). Competitor fragments ({Delta}2 to {Delta}9) have 7-bp deletions of the numbered regions corresponding to those shown in Fig. 3. Competitor fragments were added at a 100-fold molar excess.


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RESULTS
 
Phenotypic characterization of FLCR strains overexpressing drug efflux transporters. In order to define the molecular events that lead to trans-activation of the drug efflux transporter genes in FLCR strains, C. albicans strains highly resistant to fluconazole (MIC50 > 64 µg/ml) were generated by exposure of strains CAI4 (ura3/ura3) and CAPR306 (ura3/ura3 crd1/crd1) to increasing concentrations of fluconazole during liquid growth, as described in Materials and Methods. The two drug-sensitive parental strains were grown in six replicate populations each and were exposed to stepwise increases of fluconazole. After drug selection, all 12 independent populations exhibited a fluconazole MIC50 of >64 µg/ml, as determined by the CLSI microdilution assay (15). Parental populations cultured in the same manner except without drug exhibited a fluconazole MIC50 of approximately 0.25 µg/ml, identical to that of the original parental isolates. A representative isolate from each of the independent populations was chosen for further analysis.

To determine whether the drug-resistant isolates carried mutations in trans-acting factors that increased expression of MDR1 or CDR2, the promoters of these two genes were PCR amplified from the chromosome of the drug-sensitive strain CAI4 and cloned upstream of the promoterless yEGFP gene in the vector pLIB1 (Fig. 1A). These promoter fusions did not exhibit detectable GFP expression when introduced into the parental drug-sensitive strains (Fig. 1B and Table 4). When the MDR1 fusion plasmid pPRM1 was introduced into the drug-resistant isolates, 5 of the 12 independent isolates exhibited high levels of GFP expression in both the presence and absence of drug (Table 4), indicating that these strains contained trans-acting mutations that increased MDR1 transcription (e.g., strain CAPR514, an MDR1 overexpressor [OE], shown in Fig. 1B). Two of 12 independent drug-resistant isolates exhibited increased levels of PCDR2(841-847)-GFP expression, indicating that these strains contained trans-acting mutations that increased CDR2 expression (Table 4). FLCR and yEGFP expression were stably maintained in the absence of fluconazole selection for 60 generations (Table 4, column 4). To confirm that the strains with increased PMDR1-GFP and PCDR2-GFP expression truly overexpressed MDR1 or CDR2, respectively, the expression of these genes in the FLCR strains was analyzed by Northern blotting. As shown in Fig. 2 and Table 4, each strain exhibiting trans-activation of MDR1 or CDR2 had increased levels of MDR1 mRNA or CDR2 mRNA, respectively. Strains exhibiting CDR2 trans-activation also coordinately overexpressed CDR1, as has been previously described for strains that contain hyperactive TAC1 alleles (9). This report focuses on the mechanism(s) whereby MDR1 is trans-activated in FLCR strains; mutations leading to trans-activation of CDR2 will be described in detail elsewhere.


Figure 1
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  		FIG. 1. Expression of the GFP reporter gene in an MDR1 trans-activation strain. (A) Diagram of the yEGFP reporter plasmid pPRM1 integrated at the ADE2 genomic locus (not drawn to scale). The MDR1 promoter (hatched box) is transcriptionally fused to the yEGFP ORF (white box) plus 389 bp of the ACT1 3' UTR (black box). The URA3 gene (gray box) provides a selectable marker for integration into CAI4 derivatives. The plasmid backbone is denoted as a thin line. The stippled boxes indicate the incomplete fragments of the ADE2 gene generated by the integration with BamHI-digested pPRM1. (B) Cells of strains containing the chromosomally integrated PMDR1(1108)-GFP fusion plasmid pPRM1 were grown on agar medium without fluconazole and resuspended in phosphate-buffered saline. Nomarski (left) and corresponding fluorescence (right) micrographs of cells are shown. Exposure for the fluorescence micrographs was 800 ms.


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  		TABLE 4. Phenotypes of MDR1 and CDR2 trans-activation strainsa


Figure 2
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  		FIG. 2. Northern hybridization analysis of MDR1, CDR2, and CDR1 mRNA in FLCR trans-activation strains. Total RNA was extracted from the strains, and mRNA samples were separated on a 1.2% agarose gel, transferred to a nylon membrane, and hybridized with MDR1-, CDR2-, and CDR1-specific probes. Mobilities of the MDR1, CDR2 and CDR1 transcripts are indicated at right. Ethidium bromide-stained rRNA is shown as a loading control. Samples were as follows: lane 1, CAPR507 (FLCR); lane 2, CAPR510 (FLCR); lane 3, CAPR513 (FLCR); lane 4, CAPR514 (FLCR); lane 5, CAPR515 (FLCR); lane 6, CAPR517 (FLCR); lane 7, CAPR518 (FLCR); lane 8, CAI4 (FLCS); lane 9, CAPR306 (FLCS).

Identification of an MDRE within the MDR1 promoter. To delimit the site(s) within the MDR1 promoter that is important for its expression in the MDR1 trans-activation strains, additional promoter fusions were constructed, as shown in Fig. 3. yEGFP was transcriptionally fused to the following fragments from the MDR1 promoter: –852 to –7, –622 to –7, –423 to –7, and –226 to –7. All of the MDR1 promoter fusion constructs, except for the smallest fusion (–226 to –7), exhibited strong fluorescence when introduced into the five MDR1 trans-activation strains (Fig. 3A). This result indicates that an MDRE(s) lies within 423 bp upstream of the initiation codon. Additionally, the five independently isolated MDR1 overexpression mutants delimited the same region of the MDR1 promoter as important for high-level trans-activation, suggesting that the same mechanism for trans-activation was utilized in all of these strains.


Figure 3
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  		FIG. 3. Deletion of portions of the MDR1 promoter results in defective expression of the GFP reporter. (A) The hatched box labeled MDR1 promoter represents the full-length MDR1 promoter encompassing bp –1108 to –7 relative to the start of the ORF (not drawn to scale). The MDRE (see the text) is shown as a black box. Numbers above and below the hatched box indicate the end points of smaller fusions extending from the numbered line to residue –7. The thick black arrow denotes the transcription initiation site at –65, and the asterisk indicates the putative consensus TATA element at –200 (26). Fusions indicated above the hatched box were fully active in MDR1 trans-activation. Fusions indicated below the box were completely inactive. (B) Internal 7-bp deletions ({Delta}1 to {Delta}9, indicated by white boxes) were constructed in fusions extending from –335, –309, or –295 to –7. Internal deletions correspond to the following regions of the MDR1 promoter: {Delta}1 (–309 to –303), {Delta}2 (–302 to –296), {Delta}3 (–295 to –289), {Delta}4 (–288 to –282), {Delta}5 (–281 to –275), {Delta}6 (–274 to 268), {Delta}7 (–267 to –261), {Delta}8 (–260 to –254), and {Delta}9 (–253 to –246). GFP expression from these constructs is indicated at right. ++++, full activity; +++, 40 to 70% of full activity; +, 5 to 20% of full activity; –, no activity. (C) Internal deletion of region 5 (–281 to –275) in the full-length 1,108-bp MDR1 promoter fragment. ++++ indicates full activity.

To determine the 5' end of the putative MDRE, further promoter fusions were generated that had 5' ends between 429 and 226 bp upstream of the MDR1 initiation codon. Constructs containing promoter fragments with 5' ends at –407, –385, –356, –335, and –309 all exhibited strong fluorescence, similar to the –1108 fusion, when integrated into MDR1 trans-activation strains; however, MDR1 trans-activation strains containing fusions with a 5' end lying at –282 or –254 exhibited a complete loss of fluorescence (Fig. 3A). Harry et al. determined that the MDR1 transcriptional start point was at –65 and identified a putative TATA binding site at –200, based on sequence analysis (26); therefore, the region mediating trans-activation is well upstream of these core promoter elements.

To define the 5' and 3' boundaries of the MDRE, 7-bp deletions were constructed that spanned the region –309 to –245, as shown in Fig. 3B. MDR1 promoter fusions with deletions of –309 to –303 (i.e., {Delta}1) and –302 to –296 ({Delta}2) exhibited expression in MDR1 trans-activation strains similar to the full-length promoter fusion seen in pPRM1. However, deletions in the region from –295 to –261 ({Delta}3, {Delta}4, {Delta}5, {Delta}6, and {Delta}7) led to a drastic drop in the ability of the promoter fragments to mediate reporter gene expression. The promoter construct with a deletion from –260 to –254 ({Delta}8) exhibited a moderate defect in the ability to be trans-activated, while a promoter construct deleted for bp –253 to –247 ({Delta}9) was similar in GFP expression to the full-length promoter construct pPRM1. The ability of the {Delta}9 promoter construct to express GFP as well as the full-length promoter construct pPRM1 defined the 3' end of the MDRE and indicated that these deletions were upstream of the basal transcriptional sites, such as the TATA factor binding site required for promoter function. As shown in Fig. 3C, deletion of a portion of the MDRE in the context of the full-length (–1108) MDR1 promoter did not abolish trans-activation in MDR1 OE strains, indicating that upstream elements were capable of mediating MDR1 trans-activation in the absence of the functional MDRE. These upstream elements are unrelated to the MDRE and may correspond to regions identified by Harry et al. and Hiller et al. (25, 29). The sequence conferring high-level MDR1 trans-activation, termed the MDRE, encompassed bp –295 to –261 relative to the MDR1 initiation codon (Fig. 4). The MDRE is relatively large, between 23 and 35 bp in length, suggesting that it may define more than one protein binding site (i.e., a regulatory protein and its coregulator). Inspection of the sequence indicated an inverted repeat (11 of 12 bp) that matches the Saccharomyces cerevisiae Mcm1p binding site (59).


Figure 4
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  		FIG. 4. The MDRE contains an Mcm1p binding motif. The sequence of the region conferring a strong MDR1 trans-activation phenotype, termed the MDRE, is shown. Regions indicated by brackets and labeled {Delta}3 to {Delta}7 correspond to the deletions shown in Fig. 3 and described in Materials and Methods. The lower sequence represents the {alpha}2p, Mcm1p, and Ste12p binding sites found in the S. cerevisiae STE2 promoter. Sequences in bold correspond to the Mcm1p binding site, 5'-CCYWWNNNGG-3'. Boxed regions correspond to the TGTA sequence of the {alpha}2p binding site. Underlined sequence corresponds to the Ste12p (Cph1p) binding site, 5'-ATGAAACA-3'. Vertical lines between the two sequences indicate identity; dashes indicate matches to the {alpha}2-Mcm1 consensus binding site as described by Zhong and Vershon (5'-TGTANW3/4CCN6GGW4/3NTACA-3'); X indicates deviation from the S. cerevisiae consensus for these binding sites (57).

The MDRE mediates high-level trans-activation when cloned into a heterologous promoter. To determine whether the MDRE can function as an independent element outside of the context of the MDR1 promoter, the MDRE was cloned into a nonfunctional promoter. Derivatives of the CDR2 promoter containing a DRE point mutation or a deletion of the DRE were used because, when fused to yEGFP, these constructs did not express GFP in MDR1 trans-activation strains. The –299 to –255 region of the MDR1 promoter was cloned into the SalI site of pPRC2-4mut (mutation in DRE) and pPRC2-5 (deletion of DRE). The plasmids pPRhetFW and pPR2xhetFW contained one or two copies, respectively, of the MDRE cloned adjacent to the nonfunctional CDR2 promoter of pPRC2-5 (Fig. 5). The plasmid pPR2xhetRV contained two copies of the MDRE cloned adjacent to the nonfunctional CDR2 promoter of pPRC2-4mut, but the MDREs are in the orientation opposite that seen in the MDR1 promoter (Fig. 5).


Figure 5
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  		FIG. 5. The MDRE mediates trans-activation of a heterologous reporter. The hatched rectangle represents the MDR1 promoter. The MDRE region (–299 to –254 with respect to the MDR1 initiation codon) is depicted as a black arrow. White rectangles represent various regions of the CDR2 promoter. The asterisk in line F denotes a DRE mutation (TGGAAATCGG). The depicted promoter fragments were fused to the yEGFP reporter (not drawn to scale). Strains used for assessment of promoter activity are as follows: CAPR306 (FLCS), CAPR518 (CDR1/CDR2 OE), and CAPR514 (MDR1 OE). Integrated reporter fusion plasmids are as follows: A, pPRM1; B, pPRC2-4; C, pPRC2-5; D, pPRhetFW; E, pPR2xhetFW; F, pPR2xhetRV. Plus and minus symbols indicate relative GFP expression in the various strain backgrounds.

All of these constructs failed to express GFP in a FLCS strain background, as expected (Fig. 5). When the previously inactive CDR2 promoter fusion plasmids contained one or more copies of the MDRE (i.e., pPRhetFW, pPR2xhetFW, or pPR2xhetRV), these plasmids specifically mediated trans-activation in the MDR1 trans-activation strain (CAPR514) but not in the FLCS (CAPR306) or CDR1/CDR2 trans-activation strain (CAPR518) (Fig. 5). The MDRE acted in a dosage-dependent manner, as plasmids pPR2xhetFW and pPR2xhetRV (two copies of the MDRE) mediated higher levels of trans-activation in strain CAPR514 (MDR1 OE) than did pPRhetFW or pPRM1, which contained a single copy of the MDRE (Fig. 5). In addition to the dosage-dependent effect, the MDRE also exhibited an orientation-independent effect in that the promoter fusion construct pPR2xhetRV mediated trans-activation to the same high level as did pPR2xhetFW (Fig. 5). This ability of the MDRE to mediate transcriptional enhancement in an orientation-independent manner is similar to that of an upstream activation sequence (23).

MDRE binding activity detected by EMSA. To identify factors that mediate MDR1 trans-activation through binding of the MDRE, EMSA was undertaken with protein extracts from FLCS and MDR1 trans-activation strains. As can be seen in Fig. 6A, lanes 2 to 4 and 6 to 8, whole-cell protein extracts from both CAPR306 (FLCS) and CAPR514 (MDR1 OE) contained a factor(s) that bound to and retarded the mobility of the MDRE probe. This binding was specifically competed with a 100-fold excess of unlabeled MDRE fragment (Fig. 6A, lanes 5 and 9), while a 100-fold excess of an oligonucleotide fragment derived from an irrelevant promoter did not compete (see Materials and Methods). Using equal amounts of protein extract, extracts from strain CAPR514 (MDR1 OE) exhibited a more than fivefold increase in binding activity compared to extracts from strain CAPR306 (FLCS).

The region –288 to –275 of the MDRE is required for factor binding. DNA fragments containing the 7-bp deletions that genetically define the MDRE (Fig. 3B) were tested for the ability to compete the binding of the labeled MDRE probe. The MDRE probe was specifically shifted when no competitor was present (Fig. 6B). However, when a 100-fold excess of a 76-bp ({Delta}0) fragment that spans the MDRE was added to the binding reaction, binding was effectively competed and no shift of the MDRE probe was seen (Fig. 6B). As shown in Fig. 6B, regions 2, 3, 6, 7, 8, and 9 were not required for binding, as 69-bp fragments that were missing these regions still effectively competed with the labeled MDRE probe for factor binding. In contrast, regions 4 and 5 (–288 to –275) were necessary for specific competition with the labeled MDRE probe (Fig. 6B). Therefore, regions 4 and 5 of the MDRE, which are necessary for MDR1 trans-activation, are also necessary for factor binding. The sequence from regions 4 and 5 matches the S. cerevisiae consensus binding site for Mcm1p (Fig. 4) (59).

Whole-cell extracts from a strain depleted for Mcm1p failed to shift the MDRE. The MCM1 gene is an essential gene in C. albicans; however, its function can be studied by placing MCM1 under the control of a regulated promoter (44). In the strain MRcan42, expression of the MCM1 gene is under the control of the doxycycline-repressible promoter PTR. Whole-cell extracts were made from the strain MRcan42 with cells exposed to repressive conditions for 0, 2, 4, or 6 h. Substantial reduction of Mcm1p occurred by 2 h, with Mcm1p no longer detectable by 4 h (44). Whole-cell extracts from cells of strain CAPR514 (MDR1 OE) exposed to doxycycline retained the ability to shift the MDRE probe after 6 h of doxycycline treatment (Fig. 7, lanes 4, 5, and 6), indicating that the presence of doxycycline did not inhibit interaction between the MDRE and the factor(s) that leads to gel mobility shifts. Whole-cell extracts of cells from strain MRcan42 specifically shifted the MDRE under nonrepressive conditions (Fig. 7, lane 7). However, exposure of MRcan42 cells to doxycycline quickly led to the loss of the factor that shifted the MDRE probe. By 2 h of doxycycline exposure, extracts of the MRcan42 cells exhibited a dramatic reduction in the ability to shift the MDRE probe (Fig. 7, lane 8), and by 4 h of doxycycline exposure, extracts from the MRcan42 cells no longer shifted the MDRE probe (Fig. 7, lane 9). These results strongly suggest that Mcm1p is the factor that binds the MDRE.


Figure 7
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  		FIG. 7. MDRE binding activity is reduced in extracts of a strain depleted for Mcm1p. Cells of strain MRcan42, in which expression of MCM1 is under the control of the doxycycline-repressible promoter PTR, or cells of strain CAPR314 (MDR1 OE) were exposed to doxycycline for between 0 and 6 h. Extracts of these cells were incubated with radiolabeled probe and analyzed as described for Fig. 6. Lane 3 contains a 100-fold molar excess of cold competitor fragment (+cc). Lane 1, free MDRE probe; lanes 2 to 6, 3 µg of protein extract from strain CAPR514 (MDR1 OE) exposed to doxycycline for 0, 0, 2, 4, and 6 h, respectively; lanes 7 to 10, 3 µg of protein extract from MRcan42 (PTR-MCM1) cells that had been exposed to doxycycline for 0, 2, 4, and 6 h, respectively.

The MDRE is similar in sequence to regulatory regions in the S. cerevisiae STE2 and MFA1 promoters. Mcm1p is a combinatorial transcription factor which generally binds near and derives regulatory specificity from other DNA binding and/or accessory elements (56). In S. cerevisiae, a-specific promoter elements exhibit a high degree of sequence similarity throughout the extended 16-bp palindrome that comprises the Mcm1p binding site (31, 39). There are also symmetrically placed binding sites for the homeobox protein {alpha}2, which acts in concert with Mcm1p to repress the a genes (33). Ste12p is required for full activation of these genes in a cells exposed to mating pheromone. As shown in Fig. 4, the MDRE is highly similar in sequence to regulatory sequences in the 5' UTRs of the a genes STE2 and MFA1 (59). In Fig. 4, the core TGTA {alpha}2p binding sites that symmetrically flank the Mcm1p binding site are boxed in the S. cerevisiae STE2 promoter sequence (48, 58). The symmetrical boxed regions of the MDRE sequence (GGTA/TACC) are similar to the {alpha}2 binding site and maintain the spacing of 3 or 4 bp that is important for repression in S. cerevisiae (48). However, the 5' T residue that is thought to be critical for {alpha}2p binding is replaced by a G residue (49, 58). Activation of a genes such as STE2 and MFA1 in haploid a cells of S. cerevisiae requires Ste12p, which binds to the consensus sequence ATGAAACA (59), underlined in Fig. 4 (12, 14). A similar sequence, 5'-ATGACACA-3', with one mismatch from the consensus, is shown underlined in the MDRE. The location of this putative Ste12p binding site adjacent to the Mcm1p binding site of the MDRE raises the possibility that the C. albicans homolog of Ste12p, Cph1p, may play a role in MDR1 trans-activation.


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DISCUSSION
 
Wirsching et al. demonstrated that in two independent FLCR clinical strains of C. albicans, MDR1 overexpression was due to mutations in an unidentified trans-regulatory factor(s) (54). We have demonstrated that in five independently isolated FLCR laboratory strains, MDR1 overexpression was also achieved through trans-activation of MDR1, suggesting that this may be the most common mechanism for MDR1 overexpression.

Recent work from the Morschhäuser laboratory has implicated three regions of the MDR1 promoter as important for trans-activation in the MDR1-overexpressing strain F5U4, a Ura– strain derived from a FLCR clinical isolate: region 1 (–397 to –300), region 2 (–588 to –500), and region 3 (–287 to 209) (29). We demonstrated that in our five independently isolated MDR1 trans-activating laboratory strains, a small region of the MDR1 promoter, the MDRE, was sufficient to mediate high-level trans-activation of MDR1. The MDRE (–295 to –261) shares a 26-bp overlap with the 78-bp region 3 (–287 to –209) described by Hiller et al. and may identify the same cis-acting element. These results raise the possibility that the MDRE contributes to MDR1 expression in both laboratory-isolated and clinically isolated FLCR strains.

Although our reporter gene methodologies were quite similar, some differences were seen between the results described in this report and the publication by Hiller et al. Using the MDR1 trans-activation strain F5U4, Hiller and coworkers constructed 5' MDR1 promoter deletions to –495 without any effect on promoter activity while deletion to –397 resulted in an approximately two-thirds reduction of promoter activity but maintained constitutive overexpression. Further 5' deletion to –300 abolished all promoter activity (29). In contrast, we observed that constructs beginning at –295 exhibited full promoter activity in the MDR1 trans-activation strains CAPR510, CAPR513, CAPR514, CAPR515, and CAPR517 (Fig. 3). Hiller et al. observed that small internal deletions in the region from –355 to –209 had little or no effect on promoter activity when sequences upstream of –495 were present (29). Similarly, we found that deletion of the MDRE in the context of the full-length (–1108) promoter did not affect MDR1 expression, indicating that there was a region(s) upstream of the MDRE that was functionally redundant with the MDRE and could contribute to MDR1 trans-activation. If the promoter fusion constructs of Hiller and coworkers contained any two of their three regions, high-level reporter gene expression was detected in MDR1 OE strains (29). In contrast, in the MDR1 trans-activation strains described in this study, promoter fusions containing only the MDRE (–299 to –254), which may correspond to region 3 of Hiller et al., were capable of mediating expression equal to that of full-length (–1108) promoter constructs.

One possible interpretation of these differing results is that multiple mechanisms control the expression of MDR1 and that the importance of the various cis-acting regions for MDR1 overexpression differs depending on the strain. Anderson et al. showed that, in S. cerevisiae, different fluconazole selection regimens led to the selection of different types of resistant mutants (3). Therefore, MDR1 trans-activating strains selected under different conditions may differ in the relative contributions of various transcription factors to the expression of MDR1. In addition, the constructs used by Hiller et al. were integrated at the ACT1 locus while our constructs were integrated at the ADE2 locus. The local context of the integration site may affect expression from the promoter; therefore, the differences in integration sites may contribute to the differences in our results.

The MDRE is distinct from sites identified thus far as important for benomyl-induced expression of MDR1 in the congenic FLCS strains CAI4 and CAI8. The findings of Hiller et al. indicated that region 2 (–588 to –500) of the MDR1 promoter mediates benomyl induction of MDR1 in strain CAI4 (29). In contrast, work by Harry et al., using the related strain CAI8, identified another region of the MDR1 promoter (–399 to –299) as being responsible for benomyl-induced expression (25). It is unclear why these two research groups identified different regions of the MDR1 promoter as important for benomyl induction in these related laboratory strains, but Hiller et al. suggest that this may be due to the differences in benomyl exposure used by the two groups (29). These two regions identified thus far as mediating benomyl induction of MDR1 are clearly distinct from the MDRE identified in this study with FLCR strains; therefore, these findings underscore the conclusion that the MDR1 promoter is complex and subject to regulation through multiple pathways.

Several lines of evidence indicate that the MDRE is bound by the general transcription factor Mcm1p and that this binding is important for MDR1 trans-activation in FLCR strains. The most obvious feature of the MDRE is the exact match of part of the MDRE to the Mcm1p consensus binding site, 5'-CCYWWNNNGG-3' (59). Internal deletions that removed all or portions of the Mcm1p binding site from GFP fusion constructs abolished trans-activation of these constructs. Protein binding detected by EMSA correlated well with the biological activity of yEGFP fusion constructs, and deletions within the MDRE region that disrupted the predicted Mcm1p binding site abolished binding of the factor. When Mcm1p was depleted by growing a strain carrying a tetracycline-repressible MCM1 under repressing conditions, protein binding to the MDRE was lost (44). These results demonstrate that trans-activation is associated with Mcm1p binding.

In S. cerevisiae, Mcm1p is a component of several different protein complexes and, depending on the context, may be involved in activation or repression of gene expression (13, 34, 36, 40). Mcm1p is a general transcription factor that regulates numerous classes of genes and is regulated by internal or external signals or cell cycle events. However, Mcm1p has not been implicated in regulation of S. cerevisiae drug efflux determinants such as Flr1p or the ABC-type transporter Pdr5p. Therefore, the mechanism of fluconazole resistance described in this communication which is mediated through Mcm1p-dependent overexpression of an efflux determinant may not be shared with S. cerevisiae.

In addition to containing an Mcm1p binding site, the MDRE has sequence similarity to regulatory regions upstream of the S. cerevisiae STE2 and MFA1 genes (59). STE2 is one of the most highly characterized S. cerevisiae a genes. Repression of a-specific genes in {alpha} haploid cells and a/{alpha} diploid cells requires cooperative binding of {alpha}2p and Mcm1p to adjacent binding sites and subsequent recruitment of the Ssn6-Tup1 repression complex (27). In contrast, no {alpha}2p is present in a cells and therefore no Ssn6-Tup1 complex is recruited; in this situation, Mcm1p cooperates with the transcription factor Ste12p to activate transcription (30). In S. cerevisiae, the only binding sites for coregulators that flank the Mcm1p binding site and are in opposite orientation are {alpha}2 boxes with the core sequence 5'-TGTA-3'. The 5' T residue is thought to be important for binding, although one of the {alpha}2 boxes of S. cerevisiae ASG7 contains a 5' C residue rather than a T residue (57). However, in the MDRE, these boxes contain the sequence 5'-GGTA-3'. Therefore, these boxes may not represent binding sites for C. albicans MTL{alpha}2p. The Johnson laboratory has suggested that the ability of {alpha}2p to interact with Mcm1p to repress a-specific genes in S. cerevisiae arose after an ancestral split with C. albicans, consistent with the idea that the symmetrical sites flanking the Mcm1p binding site are not {alpha}2p binding sites (50).

A sequence similar to a Ste12p binding site partially overlaps the boundary of the MDRE (59). In some strains of C. albicans, e.g., clinical strain B792, this sequence is identical to the consensus Ste12p binding site (16). These observations suggest that C. albicans Cph1p, the Ste12p homolog, participates in regulating the expression of MDR1. Preliminary analyses indicate that the CPH1 ORFs from the MDR1 trans-activation strains are wild type in sequence (Riggle and Kumamoto, unpublished). Thus, the role, if any, of Cph1p in MDR1 trans-activation requires further study.

The data presented in this paper suggest the working model presented in Fig. 8. Mcm1p is capable of binding to the MDRE of the MDR1 promoter in both wild-type and MDR1 trans-activation strains. In trans-activation strains, the MCM1 ORF is wild type in sequence and is transcriptionally expressed at wild-type levels, as determined by Northern hybridization analysis (Riggle and Kumamoto, unpublished). However, Mcm1p activity is modified in these strains so that Mcm1p binding is increased. This increased binding and/or a unique interaction with a coregulator leads to increased transcription of the MDR1 gene. Future studies will focus on determining the mechanism of enhanced MDR1 trans-activation in FLCR strains.


Figure 8
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  		FIG. 8. Model of MDR1 transcription mediated by the MDRE. In MDR1 trans-activation strains, a trans-acting factor modifies Mcm1p, illustrated by the flag (or its partner, illustrated by the asterisk), such that Mcm1p binds the MDRE with greater affinity and acts as a positive regulator and/or fails to interact with a negative regulator.


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ADDENDUM IN PROOF
 
A similar cis-acting region has recently been described (B. Rognon, Z. Kozovska, A. T. Coste, G. Pardini, and D. Sanglard, Microbiology, in press).


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ACKNOWLEDGMENTS
 
We thank Stephen Rupp for sharing strain MRcan42, Susan Hadley for helpful discussions and the gift of fluconazole, and members of the lab for helpful discussions.

This research was supported by grant AI 052805 (to C.A.K.) from the National Institutes of Health.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Molecular Biology and Microbiology, Tufts University School of Medicine, 136 Harrison Ave., Boston, MA 02111. Phone: (617) 636-0404. Fax: (617) 636-0337. E-mail: carol.kumamoto{at}tufts.edu. Back

{triangledown} Published ahead of print on 13 October 2006. Back


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Eukaryotic Cell, December 2006, p. 1957-1968, Vol. 5, No. 12
1535-9778/06/$08.00+0     doi:10.1128/EC.00243-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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