Overexpression of the MDR1 Gene Is Sufficient To Confer Increased Resistance to Toxic Compounds in Candida albicans
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《抗菌试剂及化学方法》
1.Institut für Molekulare Infektionsbiologie, Universit?t Würzburg, R?ntgenring 11, D-97070 Würzburg, Germany,2.Institute of Microbiology, University Hospital Lausanne, Lausanne, Switzerland
ABSTRACT
Overexpression of MDR1, which encodes a membrane transport protein of the major facilitator superfamily, is one mechanism by which the human fungal pathogen Candida albicans can develop increased resistance to the antifungal drug fluconazole and other toxic compounds. In clinical C. albicans isolates, constitutive MDR1 overexpression is accompanied by the upregulation of other genes, but it is not known if these additional alterations are required for Mdr1p function and drug resistance. To investigate whether MDR1 overexpression is sufficient to confer a drug-resistant phenotype in C. albicans, we expressed the MDR1 gene from the strong ADH1 promoter in C. albicans laboratory strains that did not express the endogenous MDR1 gene as well as in a fluconazole-resistant clinical C. albicans isolate in which the endogenous MDR1 alleles had been deleted and in a matched fluconazole-susceptible isolate from the same patient. Forced MDR1 overexpression resulted in increased resistance to the putative Mdr1p substrates cerulenin and brefeldin A, and this resistance did not depend on the additional alterations which occurred during drug resistance development in the clinical isolates. In contrast, artificial expression of the MDR1 gene from the ADH1 promoter did not enhance or only slightly enhanced fluconazole resistance, presumably because Mdr1p expression levels in the transformants were considerably lower than those observed in the fluconazole-resistant clinical isolate. These results demonstrate that MDR1 overexpression in C. albicans is sufficient to confer resistance to some toxic compounds that are substrates of this efflux pump but that the degree of resistance depends on the Mdr1p expression level.
INTRODUCTION
Candida albicans is an opportunistic fungal pathogen that can cause superficial mucosal infections as well as life-threatening systemic infections, especially in immunocompromised patients. Infections by C. albicans are frequently treated with the antimycotic agent fluconazole, which inhibits the biosynthesis of ergosterol, the major sterol in the fungal cell membrane. C. albicans can develop resistance to fluconazole by different molecular mechanisms, including alterations in the sterol biosynthetic pathway, overexpression of ERG11, which encodes the target enzyme of fluconazole (sterol 14-demethylase, or Erg11p), mutations in ERG11 that result in a reduced affinity of Erg11p for fluconazole, and overexpression of genes encoding membrane transport proteins (CDR1, CDR2, and MDR1) that actively transport fluconazole out of the cell. In clinical C. albicans strains, several of these mechanisms are often combined to result in a stepwise development of fluconazole resistance (for a review, see reference 17).
The MDR1 gene encodes an efflux pump of the major facilitator superfamily, whose members use the proton gradient across the cytoplasmic membrane as an energy source for transport (1, 4). While MDR1 is normally expressed only at low levels in standard laboratory media, many fluconazole-resistant clinical C. albicans isolates constitutively overexpress MDR1 (6, 7, 14, 19, 23, 26). Deletion of the MDR1 gene from MDR1-overexpressing C. albicans isolates resulted in decreased fluconazole resistance of the mutants, confirming that MDR1 overexpression contributed to the resistant phenotype of these isolates (28). Expression of MDR1 from a plasmid in the heterologous host Saccharomyces cerevisiae resulted in increased resistance of the transformants to fluconazole and a variety of structurally unrelated toxic compounds, demonstrating that MDR1 encodes a multidrug resistance protein (1, 23). However, clinical C. albicans isolates that overexpress MDR1 exhibit increased resistance to only some of these compounds, e.g., cerulenin, brefeldin A, or 4-nitroquinoline-N-oxide (4-NQO), but not to others (29), indicating that the role of MDR1 in drug resistance may be better studied in C. albicans itself. The constitutive overexpression of MDR1 in clinical C. albicans isolates has been shown to be caused by mutations in as yet unidentified regulatory factors (27). Transcription profiling experiments and proteomic analyses of matched pairs of fluconazole-susceptible and MDR1-overexpressing, fluconazole-resistant isolates have demonstrated that a common set of additional genes is upregulated together with MDR1 in fluconazole-resistant isolates (11, 13, 21). These findings raised the possibility that the functionality of Mdr1p in mediating drug resistance in C. albicans may depend on additional alterations occurring in drug-resistant strains. A straightforward approach to address this question would be to force expression of MDR1 from a strong promoter in a drug-susceptible strain and study the effect of this defined genetic manipulation on the susceptibility of the strain to putative Mdr1p substrates. In the present study, we have taken this approach to find out whether MDR1 overexpression alone is sufficient to confer increased resistance to such compounds or if additional alterations are required for the function of this efflux pump.
MATERIALS AND METHODS
Strains and growth media. The C. albicans strains used in this study are listed in Table 1. All strains were stored as frozen stocks with 15% glycerol at –80°C. Strain CAI4 was propagated on SD agar plates containing 6.7 g of yeast nitrogen base without amino acids (BIO101, Vista, Calif.), 20 g of glucose, 0.77 g of complete supplement medium without uracil (BIO101), 100 μg ml–1 uridine, and 15 g of agar per liter. All other strains were propagated on YPD agar plates (20 g of peptone, 10 g of yeast extract, 20 g of glucose, 15 g of agar per liter). Strains were routinely grown in YPD liquid medium at 30°C. To support growth of the ura3 mutant strain CAI4, 100 μg ml–1 uridine was added to the medium.
Plasmid construction. Plasmid pADH1G2, containing the GFP gene under control of the ADH1 promoter, has been described previously (13) (Fig. 1C). To express MDR1 from the ADH1 promoter, the MDR1 open reading frame (ORF) was amplified by PCR from genomic DNA of strain CAI4 with the primer pair MDR27 (5'-AGAACTCGAGAATGCATTACAGATTTTTGAGAGAT-3') and MDR28 (5'-ATGACGGATCCTAATTAGCATACTTAGATCTTGC-3'). The PCR product was digested at the XhoI and BamHI sites (underlined) introduced in front of the start codon (bold) and behind the stop codon (in bold in the reverse sequence), respectively, cloned into the vector pBluescript, and confirmed by sequencing. The XhoI-BamHI MDR1 fragment from the resulting plasmid, pMDR1E1, was then cloned together with a BamHI-EcoRI fragment from pYPR127E2 (13) containing the ACT1 transcription termination sequence (TACT1) and URA3 sequences into SalI/EcoRI-digested pADH1G2 to generate pMDR1E2 (Fig. 1A). To substitute the caSAT1 (Candida-adapted SAT1) selection marker for the URA3 marker, a fragment from the ADH1 downstream region was first amplified by PCR from CAI4 genomic DNA with the primers ADH8 (5'-GGTGCTGAACCAAACTGCAGTGAAGCTGAC-3') and ADH11 (5'-GAACCTTTGATTTCCGCGGATTTGACAACAGC-3'), digested at the introduced PstI and SacII sites (underlined), and cloned together with an XhoI-PstI fragment containing the caSAT1 marker (20) into the XhoI/SacII-digested vector pBluescript to produce pSAT2. An XhoI-SacI caSAT1-3'ADH1 fragment from pSAT2 was then cloned behind the ACT1 transcription termination sequence in the SalI/SacI-digested plasmid pCBF1M4 (3) to generate pSAT3. Finally, a KpnI-SalI fragment from pMDR1E2 containing the ADH1 promoter and the N-terminal part of MDR1 was cloned together with a SalI-BamHI fragment containing the remainder of MDR1 into KpnI/BglII-digested pSAT3 to produce pMDR1E4 (Fig. 1B). A control construct without MDR1 was generated by cloning a BamHI-SalI GFP-TACT1 fragment from pMEP2G2 (2) together with the XhoI-SacI caSAT1-3'ADH1 fragment from pSAT2 into BamHI/SacI-digested pADH1G2 to produce pADH1G3 (Fig. 1D).
Candida albicans transformation. C. albicans strains were transformed by electroporation (12) with the following gel-purified linear DNA fragments: the XbaI-SacI fragment from pMDR1E2 containing the PADH1-MDR1 fusion and the URA3 selection marker, the XbaI-SacII fragment from pMDR1E4 containing the PADH1-MDR1 fusion and the caSAT1 selection marker, and the XbaI-SacII fragment from the control construct pADH1G3 containing the GFP gene and the caSAT1 selection marker. Uridine-prototrophic transformants were selected on SD agar plates, and nourseothricin-resistant transformants were selected on YPD agar plates containing 200 μg ml–1 nourseothricin (Werner Bioagents, Jena, Germany), as described previously (20). Single-copy integration of all constructs was confirmed by Southern hybridization with probes from the ADH1 upstream and downstream regions.
Isolation of genomic DNA and Southern hybridization. Genomic DNAs from C. albicans strains were isolated as described previously (15). DNA (10 μg) was digested with SpeI, separated in 1% (wt/vol) agarose gels, and, after ethidium bromide staining, transferred by vacuum blotting onto nylon membranes and fixed by UV cross-linking. Southern hybridization with enhanced chemiluminescence (ECL)-labeled probes was performed with an ECL labeling and detection kit from Amersham (Braunschweig, Germany) according to the manufacturer's instructions.
Drug susceptibility tests. Stock solutions of drugs were prepared as follows. Fluconazole (1 mg ml–1) was dissolved in water, and cerulenin (5 mg ml–1), brefeldin A (5 mg ml–1), and 4-NQO (0.2 mg ml–1) were dissolved in dimethyl sulfoxide. In the assays, serial twofold dilutions in assay medium were prepared from the following initial concentrations: fluconazole, 100 μg ml–1; cerulenin, 50 μg ml–1; brefeldin A, 500 μg ml–1; and 4-NQO, 4 μg ml–1. Susceptibility tests were carried out in high-resolution medium (14.67 g HR medium [Oxoid GmbH, Wesel, Germany], 1 g NaHCO3, 0.2 M phosphate buffer, pH 7.2), using a previously described microdilution method (22). Readings were carried out after 24 h. The tests were performed four times independently on different occasions, usually producing identical results. Minor variations of one dilution step were occasionally observed, and the results of representative experiments are shown.
Western blot analysis. Crude protein extracts were prepared from C. albicans cells grown to log phase (optical density at 540 nm = 0.4) in YPD at 30°C. Cells from a 5-ml volume of each culture were pelleted by centrifugation and resuspended in 1 ml of sterile distilled water. The cells were lysed by the addition of 150 μl 1.85 M NaOH-7.5% (vol/vol) ?-mercaptoethanol and then incubated on ice for 10 min. Proteins were precipitated by the addition of 150 μl of ice-cold 50% (vol/vol) trichloroacetic acid and incubation on ice for 10 min; this was followed by centrifugation at 10,000 x g for 5 min at 4°C. Each sample was resuspended in 100 μl of sample buffer (40 mM Tris-HCl, 8 M urea, 5% [wt/vol] sodium dodecyl sulfate, 0.1 mM EDTA, 1% [vol/vol] ?-mercaptoethanol, 0.1 mg of bromophenol blue per ml), incubated for 30 min at 37°C, and then centrifuged at 10,000 x g for 5 min to remove cell debris. Ten microliters of each sample (approximately 20 μg of protein) was loaded into a sodium dodecyl sulfate-10% [wt/vol] polyacrylamide gel and electrophoresed in a Mini-PROTEAN II electrophoresis cell (Bio-Rad). After electrophoresis, proteins were transferred to nitrocellulose membranes by Western blotting using a Bio-Rad Mini Trans-Blot electrophoretic transfer cell according to the manufacturer's instructions. Immunodetection of Mdr1p was performed using a polyclonal rabbit anti-Mdr1p antiserum (16) and horseradish peroxidase-conjugated anti-rabbit antiserum (Jackson Immunoresearch, West Grove, PA) as a secondary antibody. Signals were detected using an ECL kit from Amersham.
RESULTS
Forced overexpression of MDR1 in C. albicans laboratory strains. To investigate the effect of forced MDR1 overexpression in a drug-susceptible C. albicans laboratory strain, we placed the MDR1 ORF under control of the strong ADH1 promoter (Fig. 1A) and integrated the PADH1-MDR1 fusion into the genome of the uridine-auxotrophic strain CAI4, which is widely used for genetic manipulations in C. albicans. Two independent uridine-prototrophic transformants carrying single copies of the PADH1-MDR1 fusion in one of the ADH1 alleles were used for phenotypic analyses. As controls, two previously constructed derivatives of strain CAI4 containing an otherwise identical construct with a GFP reporter gene instead of MDR1 (13) (Fig. 1C) were used. The susceptibility of the strains to various putative Mdr1p substrates was then tested by determining the MICs of these compounds, using a previously published microdilution method (22). Compared with the control strains, the transformants expressing MDR1 from the ADH1 promoter exhibited a fourfold reduced susceptibility to cerulenin and brefeldin A, demonstrating that overexpression of MDR1 alone is sufficient to confer increased resistance to these metabolic inhibitors in C. albicans (Fig. 2A). In contrast, only a twofold reduced susceptibility to fluconazole was observed in the strains expressing MDR1 from the ADH1 promoter, and the susceptibility of the strains to 4-NQO remained unchanged compared with the controls, suggesting that MDR1 overexpression in these strains had no or only a marginal effect on their susceptibilities to these compounds.
To confirm that MDR1 overexpression would produce the same effects in a prototrophic C. albicans wild-type strain, we replaced the URA3 selection marker with the caSAT1 marker, which confers resistance to nourseothricin (20) (Fig. 1B), and integrated the PADH1-MDR1 fusion and a control construct without MDR1 (Fig. 1D) in strain SC5314, the progenitor of strain CAI4. In each case, two independent transformants were kept and used for further analysis. Like the CAI4 derivatives, transformants of strain SC5314 expressing MDR1 from the ADH1 promoter showed a fourfold increased resistance to cerulenin and brefeldin A compared with the parental strain or control transformants (Fig. 2B). Again, no or only a minor effect of MDR1 overexpression on the susceptibility to fluconazole or 4-NQO was observed in this series of strains. Western immunoblotting with an anti-Mdr1p antiserum showed that similar amounts of Mdr1p were produced in all transformants carrying the PADH1-MDR1 fusion (Fig. 2C), whereas Mdr1p was not detected in transformants carrying a control construct. The minor but reproducible effects of MDR1 expression from the ADH1 promoter on resistance to fluconazole (only in CAI4) and 4-NQO (only in SC5314) therefore depend on other differences between these two strains that allow a slight effect on resistance to be detectable or not.
Forced overexpression of the MDR1 gene in clinical C. albicans isolates. Fluconazole-resistant clinical C. albicans isolates overexpressing MDR1 show additional, conserved alterations in their gene expression patterns compared with matched fluconazole-susceptible isolates from the same patients (11, 13, 21). Therefore, we considered the possibility that these additional alterations in MDR1-overexpressing clinical C. albicans isolates may be required for optimal Mdr1p function. To address this question, we introduced the PADH1-MDR1 fusion into a derivative of the drug-resistant clinical isolate G5 from which the endogenous, overexpressed MDR1 alleles had been deleted (strain G5M432) (28). MDR1 expressed from the ADH1 promoter would therefore be the only MDR1 copy in the corresponding transformants, but the additional alterations occurring in the drug-resistant progenitor should be preserved. In the same way, the PADH1-MDR1 fusion was introduced into the matched, drug-susceptible isolate G2, which did not express the endogenous MDR1 alleles at detectable levels and did not exhibit the additional alterations in gene expression. Both parental strains were also transformed with the control construct not containing MDR1, and two independent transformants were kept for phenotypic analysis in each case. As reported previously, the clinical isolate G5 exhibited a strong increase in resistance to fluconazole compared with that of the matched isolate G2 which was caused by multiple resistance mechanisms (6), and this resistance was reduced after deletion of MDR1 in strain G5M432 (28) (Fig. 3, upper left panel). In neither transformants of strain G2 nor transformants of G5M432 did expression of MDR1 from the ADH1 promoter result in a detectable increase of resistance to fluconazole compared with the parental strains or control transformants. In contrast, MDR1 expression from the ADH1 promoter caused a fourfold increased resistance to cerulenin and brefeldin A compared with the strains containing the control construct (Fig. 3, upper right and lower left panels). In addition, strains carrying the PADH1-MDR1 fusion in these backgrounds also showed a fourfold increased resistance to 4-NQO (Fig. 3, lower right panel). No difference in the effect of MDR1 expression from the ADH1 promoter on drug resistance was observed between the two matched isolates, indicating that Mdr1p function did not depend on the additional alterations that had occurred in isolate G5. Note, however, that the strains expressing the PADH1-MDR1 fusion did not reach the same level of resistance to cerulenin, brefeldin A, and 4-NQO as the clinical isolate G5, although it was previously demonstrated that MDR1 overexpression was the sole cause of resistance to these compounds in this strain (29) (also compare strains G2, G5, and the mdr1 mutant G5M432 in the corresponding panels in Fig. 3). To reveal a possible explanation for the differences in drug resistance of the MDR1-overexpressing clinical isolate G5 and the strains expressing MDR1 from the ADH1 promoter, we compared Mdr1p expression levels in the various strains by Western immunoblotting with the anti-Mdr1p antiserum (Fig. 4). As expected from previous work, no Mdr1p was detected in the fluconazole-susceptible clinical isolate G2 (Fig. 4, lane 1), but a specific doublet band reacting with the antiserum was observed after treatment of the cells with benomyl, which is known to induce the MDR1 promoter (lane 2) (10, 25). The MDR1-overexpressing isolate G5 showed a strongly reacting band (Fig. 4, lane 4), which disappeared after deletion of both MDR1 alleles from this strain (strain G5M432; lanes 3 and 9). Mdr1p was constitutively expressed in transformants carrying the PADH1-MDR1 fusion (Fig. 4, lanes 7, 8, 12, and 13), whereas Mdr1p was not detected in strains carrying the control construct (lanes 5, 6, 10, and 11). It was particularly interesting that expression of MDR1 from the ADH1 promoter did not result in the same high Mdr1p expression levels as in the clinical isolate G5, suggesting that the degree of Mdr1p-mediated resistance depends on the level of Mdr1p overexpression.
DISCUSSION
The function of MDR1 as a multidrug resistance gene in C. albicans is well established by several lines of evidence, including the strong correlation between MDR1 overexpression and resistance to different toxic compounds in matched pairs of clinical C. albicans isolates (6, 7, 14, 19, 23, 26), the partial or complete loss of the resistant phenotype after deletion of MDR1 from MDR1-overexpressing C. albicans strains (9, 28, 29), and the ability of the gene to confer drug resistance in the heterologous host S. cerevisiae (1, 23). Nevertheless, the differences in the spectra of drugs to which MDR1 conferred resistance in S. cerevisiae transformants carrying MDR1 on a plasmid and in MDR1-overexpressing clinical C. albicans isolates raised questions about the ability of this efflux pump to confer resistance to specific compounds in C. albicans. In addition, the observation that a conserved group of additional genes was concomitantly upregulated together with MDR1 in drug-resistant clinical C. albicans isolates (11, 13, 21) pointed to the possibility that overexpression of MDR1 alone may not be sufficient to mediate resistance to some drugs and that resistance may depend on these additional alterations. Therefore, we addressed this issue by artificially overexpressing MDR1 from a strong promoter in a drug-susceptible C. albicans laboratory strain that did not express the endogenous MDR1 alleles at detectable levels. A similar approach has recently been taken by other groups to directly study the function of the ABC transporter genes CDR1 and CDR2 in drug resistance in C. albicans (18, 24). Forced MDR1 overexpression from the ADH1 promoter in the C. albicans laboratory strain CAI4 or its prototrophic parental strain, SC5314, was sufficient to confer increased resistance to the putative Mdr1p substrates cerulenin and brefeldin A but had only a minor effect on the susceptibilities of the strains to fluconazole and 4-NQO. The degree of resistance to all drugs, as measured by the MIC, was lower in the laboratory strains expressing MDR1 from the ADH1 promoter than in the clinical isolate G5, which overexpressed the endogenous MDR1 alleles, suggesting that optimal functioning of the Mdr1p efflux pump might indeed depend on other cellular alterations occurring in drug-resistant clinical isolates. However, a direct comparison of the relative increases in resistance after expressing the PADH1-MDR1 fusion in a derivative of such a drug-resistant clinical C. albicans isolate, in which the overexpressed endogenous MDR1 alleles had been deleted, and a matched, drug-sensitive isolate showed no significant differences, demonstrating that the additional alterations that had occurred in the drug-resistant isolate were not required for Mdr1p function. Nevertheless, the degree of Mdr1p-mediated resistance to specific compounds seems to depend on the strain background, since MDR1 overexpression from the ADH1 promoter resulted in a fourfold increased resistance to 4-NQO in the clinical isolates but had no or only a minor effect on 4-NQO resistance in the laboratory strains. In contrast, the relative increases in resistance to cerulenin and brefeldin A were similar in all strains expressing the PADH1-MDR1 fusion.
Western immunoblot analysis showed that the amount of Mdr1p was significantly lower in cells expressing the MDR1 gene from the ADH1 promoter than in the clinical isolate G5, which overexpressed the endogenous MDR1 alleles. Although the ADH1 promoter is a strong promoter, this does not necessarily result in correspondingly high protein levels. For example, we previously observed that MEP2 transcript levels were higher when the gene was expressed from the ADH1 promoter than from its own promoter, but despite this, higher protein levels were observed when MEP2 was expressed from its own promoter (2). Therefore, this result suggested that the degree of Mdr1p-mediated drug resistance depends on the Mdr1p expression level. For apparently good substrates, such as cerulenin and brefeldin A, relatively low Mdr1p expression levels are already sufficient to confer significant resistance, whereas fluconazole seems to be transported less efficiently by Mdr1p, with significant resistance being seen only in strains that strongly overexpress the MDR1 gene, such as many clinical C. albicans isolates.
ACKNOWLEDGMENTS
We thank Bill Fonzi and Fritz Mühlschlegel for the gift of strains SC5314 and CAI4. Sequence data for Candida albicans were obtained from the Stanford Genome Technology Center website at http://www-sequence.stanford.edu/group/candida.
This study was supported by the Deutsche Forschungsgemeinschaft (DFG grants MO846/3 and SFB630) and the European Community (EC project QLK2-CT-2001-02377). Sequencing of Candida albicans was accomplished with the support of the NIDR and the Burroughs Wellcome Fund.
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ABSTRACT
Overexpression of MDR1, which encodes a membrane transport protein of the major facilitator superfamily, is one mechanism by which the human fungal pathogen Candida albicans can develop increased resistance to the antifungal drug fluconazole and other toxic compounds. In clinical C. albicans isolates, constitutive MDR1 overexpression is accompanied by the upregulation of other genes, but it is not known if these additional alterations are required for Mdr1p function and drug resistance. To investigate whether MDR1 overexpression is sufficient to confer a drug-resistant phenotype in C. albicans, we expressed the MDR1 gene from the strong ADH1 promoter in C. albicans laboratory strains that did not express the endogenous MDR1 gene as well as in a fluconazole-resistant clinical C. albicans isolate in which the endogenous MDR1 alleles had been deleted and in a matched fluconazole-susceptible isolate from the same patient. Forced MDR1 overexpression resulted in increased resistance to the putative Mdr1p substrates cerulenin and brefeldin A, and this resistance did not depend on the additional alterations which occurred during drug resistance development in the clinical isolates. In contrast, artificial expression of the MDR1 gene from the ADH1 promoter did not enhance or only slightly enhanced fluconazole resistance, presumably because Mdr1p expression levels in the transformants were considerably lower than those observed in the fluconazole-resistant clinical isolate. These results demonstrate that MDR1 overexpression in C. albicans is sufficient to confer resistance to some toxic compounds that are substrates of this efflux pump but that the degree of resistance depends on the Mdr1p expression level.
INTRODUCTION
Candida albicans is an opportunistic fungal pathogen that can cause superficial mucosal infections as well as life-threatening systemic infections, especially in immunocompromised patients. Infections by C. albicans are frequently treated with the antimycotic agent fluconazole, which inhibits the biosynthesis of ergosterol, the major sterol in the fungal cell membrane. C. albicans can develop resistance to fluconazole by different molecular mechanisms, including alterations in the sterol biosynthetic pathway, overexpression of ERG11, which encodes the target enzyme of fluconazole (sterol 14-demethylase, or Erg11p), mutations in ERG11 that result in a reduced affinity of Erg11p for fluconazole, and overexpression of genes encoding membrane transport proteins (CDR1, CDR2, and MDR1) that actively transport fluconazole out of the cell. In clinical C. albicans strains, several of these mechanisms are often combined to result in a stepwise development of fluconazole resistance (for a review, see reference 17).
The MDR1 gene encodes an efflux pump of the major facilitator superfamily, whose members use the proton gradient across the cytoplasmic membrane as an energy source for transport (1, 4). While MDR1 is normally expressed only at low levels in standard laboratory media, many fluconazole-resistant clinical C. albicans isolates constitutively overexpress MDR1 (6, 7, 14, 19, 23, 26). Deletion of the MDR1 gene from MDR1-overexpressing C. albicans isolates resulted in decreased fluconazole resistance of the mutants, confirming that MDR1 overexpression contributed to the resistant phenotype of these isolates (28). Expression of MDR1 from a plasmid in the heterologous host Saccharomyces cerevisiae resulted in increased resistance of the transformants to fluconazole and a variety of structurally unrelated toxic compounds, demonstrating that MDR1 encodes a multidrug resistance protein (1, 23). However, clinical C. albicans isolates that overexpress MDR1 exhibit increased resistance to only some of these compounds, e.g., cerulenin, brefeldin A, or 4-nitroquinoline-N-oxide (4-NQO), but not to others (29), indicating that the role of MDR1 in drug resistance may be better studied in C. albicans itself. The constitutive overexpression of MDR1 in clinical C. albicans isolates has been shown to be caused by mutations in as yet unidentified regulatory factors (27). Transcription profiling experiments and proteomic analyses of matched pairs of fluconazole-susceptible and MDR1-overexpressing, fluconazole-resistant isolates have demonstrated that a common set of additional genes is upregulated together with MDR1 in fluconazole-resistant isolates (11, 13, 21). These findings raised the possibility that the functionality of Mdr1p in mediating drug resistance in C. albicans may depend on additional alterations occurring in drug-resistant strains. A straightforward approach to address this question would be to force expression of MDR1 from a strong promoter in a drug-susceptible strain and study the effect of this defined genetic manipulation on the susceptibility of the strain to putative Mdr1p substrates. In the present study, we have taken this approach to find out whether MDR1 overexpression alone is sufficient to confer increased resistance to such compounds or if additional alterations are required for the function of this efflux pump.
MATERIALS AND METHODS
Strains and growth media. The C. albicans strains used in this study are listed in Table 1. All strains were stored as frozen stocks with 15% glycerol at –80°C. Strain CAI4 was propagated on SD agar plates containing 6.7 g of yeast nitrogen base without amino acids (BIO101, Vista, Calif.), 20 g of glucose, 0.77 g of complete supplement medium without uracil (BIO101), 100 μg ml–1 uridine, and 15 g of agar per liter. All other strains were propagated on YPD agar plates (20 g of peptone, 10 g of yeast extract, 20 g of glucose, 15 g of agar per liter). Strains were routinely grown in YPD liquid medium at 30°C. To support growth of the ura3 mutant strain CAI4, 100 μg ml–1 uridine was added to the medium.
Plasmid construction. Plasmid pADH1G2, containing the GFP gene under control of the ADH1 promoter, has been described previously (13) (Fig. 1C). To express MDR1 from the ADH1 promoter, the MDR1 open reading frame (ORF) was amplified by PCR from genomic DNA of strain CAI4 with the primer pair MDR27 (5'-AGAACTCGAGAATGCATTACAGATTTTTGAGAGAT-3') and MDR28 (5'-ATGACGGATCCTAATTAGCATACTTAGATCTTGC-3'). The PCR product was digested at the XhoI and BamHI sites (underlined) introduced in front of the start codon (bold) and behind the stop codon (in bold in the reverse sequence), respectively, cloned into the vector pBluescript, and confirmed by sequencing. The XhoI-BamHI MDR1 fragment from the resulting plasmid, pMDR1E1, was then cloned together with a BamHI-EcoRI fragment from pYPR127E2 (13) containing the ACT1 transcription termination sequence (TACT1) and URA3 sequences into SalI/EcoRI-digested pADH1G2 to generate pMDR1E2 (Fig. 1A). To substitute the caSAT1 (Candida-adapted SAT1) selection marker for the URA3 marker, a fragment from the ADH1 downstream region was first amplified by PCR from CAI4 genomic DNA with the primers ADH8 (5'-GGTGCTGAACCAAACTGCAGTGAAGCTGAC-3') and ADH11 (5'-GAACCTTTGATTTCCGCGGATTTGACAACAGC-3'), digested at the introduced PstI and SacII sites (underlined), and cloned together with an XhoI-PstI fragment containing the caSAT1 marker (20) into the XhoI/SacII-digested vector pBluescript to produce pSAT2. An XhoI-SacI caSAT1-3'ADH1 fragment from pSAT2 was then cloned behind the ACT1 transcription termination sequence in the SalI/SacI-digested plasmid pCBF1M4 (3) to generate pSAT3. Finally, a KpnI-SalI fragment from pMDR1E2 containing the ADH1 promoter and the N-terminal part of MDR1 was cloned together with a SalI-BamHI fragment containing the remainder of MDR1 into KpnI/BglII-digested pSAT3 to produce pMDR1E4 (Fig. 1B). A control construct without MDR1 was generated by cloning a BamHI-SalI GFP-TACT1 fragment from pMEP2G2 (2) together with the XhoI-SacI caSAT1-3'ADH1 fragment from pSAT2 into BamHI/SacI-digested pADH1G2 to produce pADH1G3 (Fig. 1D).
Candida albicans transformation. C. albicans strains were transformed by electroporation (12) with the following gel-purified linear DNA fragments: the XbaI-SacI fragment from pMDR1E2 containing the PADH1-MDR1 fusion and the URA3 selection marker, the XbaI-SacII fragment from pMDR1E4 containing the PADH1-MDR1 fusion and the caSAT1 selection marker, and the XbaI-SacII fragment from the control construct pADH1G3 containing the GFP gene and the caSAT1 selection marker. Uridine-prototrophic transformants were selected on SD agar plates, and nourseothricin-resistant transformants were selected on YPD agar plates containing 200 μg ml–1 nourseothricin (Werner Bioagents, Jena, Germany), as described previously (20). Single-copy integration of all constructs was confirmed by Southern hybridization with probes from the ADH1 upstream and downstream regions.
Isolation of genomic DNA and Southern hybridization. Genomic DNAs from C. albicans strains were isolated as described previously (15). DNA (10 μg) was digested with SpeI, separated in 1% (wt/vol) agarose gels, and, after ethidium bromide staining, transferred by vacuum blotting onto nylon membranes and fixed by UV cross-linking. Southern hybridization with enhanced chemiluminescence (ECL)-labeled probes was performed with an ECL labeling and detection kit from Amersham (Braunschweig, Germany) according to the manufacturer's instructions.
Drug susceptibility tests. Stock solutions of drugs were prepared as follows. Fluconazole (1 mg ml–1) was dissolved in water, and cerulenin (5 mg ml–1), brefeldin A (5 mg ml–1), and 4-NQO (0.2 mg ml–1) were dissolved in dimethyl sulfoxide. In the assays, serial twofold dilutions in assay medium were prepared from the following initial concentrations: fluconazole, 100 μg ml–1; cerulenin, 50 μg ml–1; brefeldin A, 500 μg ml–1; and 4-NQO, 4 μg ml–1. Susceptibility tests were carried out in high-resolution medium (14.67 g HR medium [Oxoid GmbH, Wesel, Germany], 1 g NaHCO3, 0.2 M phosphate buffer, pH 7.2), using a previously described microdilution method (22). Readings were carried out after 24 h. The tests were performed four times independently on different occasions, usually producing identical results. Minor variations of one dilution step were occasionally observed, and the results of representative experiments are shown.
Western blot analysis. Crude protein extracts were prepared from C. albicans cells grown to log phase (optical density at 540 nm = 0.4) in YPD at 30°C. Cells from a 5-ml volume of each culture were pelleted by centrifugation and resuspended in 1 ml of sterile distilled water. The cells were lysed by the addition of 150 μl 1.85 M NaOH-7.5% (vol/vol) ?-mercaptoethanol and then incubated on ice for 10 min. Proteins were precipitated by the addition of 150 μl of ice-cold 50% (vol/vol) trichloroacetic acid and incubation on ice for 10 min; this was followed by centrifugation at 10,000 x g for 5 min at 4°C. Each sample was resuspended in 100 μl of sample buffer (40 mM Tris-HCl, 8 M urea, 5% [wt/vol] sodium dodecyl sulfate, 0.1 mM EDTA, 1% [vol/vol] ?-mercaptoethanol, 0.1 mg of bromophenol blue per ml), incubated for 30 min at 37°C, and then centrifuged at 10,000 x g for 5 min to remove cell debris. Ten microliters of each sample (approximately 20 μg of protein) was loaded into a sodium dodecyl sulfate-10% [wt/vol] polyacrylamide gel and electrophoresed in a Mini-PROTEAN II electrophoresis cell (Bio-Rad). After electrophoresis, proteins were transferred to nitrocellulose membranes by Western blotting using a Bio-Rad Mini Trans-Blot electrophoretic transfer cell according to the manufacturer's instructions. Immunodetection of Mdr1p was performed using a polyclonal rabbit anti-Mdr1p antiserum (16) and horseradish peroxidase-conjugated anti-rabbit antiserum (Jackson Immunoresearch, West Grove, PA) as a secondary antibody. Signals were detected using an ECL kit from Amersham.
RESULTS
Forced overexpression of MDR1 in C. albicans laboratory strains. To investigate the effect of forced MDR1 overexpression in a drug-susceptible C. albicans laboratory strain, we placed the MDR1 ORF under control of the strong ADH1 promoter (Fig. 1A) and integrated the PADH1-MDR1 fusion into the genome of the uridine-auxotrophic strain CAI4, which is widely used for genetic manipulations in C. albicans. Two independent uridine-prototrophic transformants carrying single copies of the PADH1-MDR1 fusion in one of the ADH1 alleles were used for phenotypic analyses. As controls, two previously constructed derivatives of strain CAI4 containing an otherwise identical construct with a GFP reporter gene instead of MDR1 (13) (Fig. 1C) were used. The susceptibility of the strains to various putative Mdr1p substrates was then tested by determining the MICs of these compounds, using a previously published microdilution method (22). Compared with the control strains, the transformants expressing MDR1 from the ADH1 promoter exhibited a fourfold reduced susceptibility to cerulenin and brefeldin A, demonstrating that overexpression of MDR1 alone is sufficient to confer increased resistance to these metabolic inhibitors in C. albicans (Fig. 2A). In contrast, only a twofold reduced susceptibility to fluconazole was observed in the strains expressing MDR1 from the ADH1 promoter, and the susceptibility of the strains to 4-NQO remained unchanged compared with the controls, suggesting that MDR1 overexpression in these strains had no or only a marginal effect on their susceptibilities to these compounds.
To confirm that MDR1 overexpression would produce the same effects in a prototrophic C. albicans wild-type strain, we replaced the URA3 selection marker with the caSAT1 marker, which confers resistance to nourseothricin (20) (Fig. 1B), and integrated the PADH1-MDR1 fusion and a control construct without MDR1 (Fig. 1D) in strain SC5314, the progenitor of strain CAI4. In each case, two independent transformants were kept and used for further analysis. Like the CAI4 derivatives, transformants of strain SC5314 expressing MDR1 from the ADH1 promoter showed a fourfold increased resistance to cerulenin and brefeldin A compared with the parental strain or control transformants (Fig. 2B). Again, no or only a minor effect of MDR1 overexpression on the susceptibility to fluconazole or 4-NQO was observed in this series of strains. Western immunoblotting with an anti-Mdr1p antiserum showed that similar amounts of Mdr1p were produced in all transformants carrying the PADH1-MDR1 fusion (Fig. 2C), whereas Mdr1p was not detected in transformants carrying a control construct. The minor but reproducible effects of MDR1 expression from the ADH1 promoter on resistance to fluconazole (only in CAI4) and 4-NQO (only in SC5314) therefore depend on other differences between these two strains that allow a slight effect on resistance to be detectable or not.
Forced overexpression of the MDR1 gene in clinical C. albicans isolates. Fluconazole-resistant clinical C. albicans isolates overexpressing MDR1 show additional, conserved alterations in their gene expression patterns compared with matched fluconazole-susceptible isolates from the same patients (11, 13, 21). Therefore, we considered the possibility that these additional alterations in MDR1-overexpressing clinical C. albicans isolates may be required for optimal Mdr1p function. To address this question, we introduced the PADH1-MDR1 fusion into a derivative of the drug-resistant clinical isolate G5 from which the endogenous, overexpressed MDR1 alleles had been deleted (strain G5M432) (28). MDR1 expressed from the ADH1 promoter would therefore be the only MDR1 copy in the corresponding transformants, but the additional alterations occurring in the drug-resistant progenitor should be preserved. In the same way, the PADH1-MDR1 fusion was introduced into the matched, drug-susceptible isolate G2, which did not express the endogenous MDR1 alleles at detectable levels and did not exhibit the additional alterations in gene expression. Both parental strains were also transformed with the control construct not containing MDR1, and two independent transformants were kept for phenotypic analysis in each case. As reported previously, the clinical isolate G5 exhibited a strong increase in resistance to fluconazole compared with that of the matched isolate G2 which was caused by multiple resistance mechanisms (6), and this resistance was reduced after deletion of MDR1 in strain G5M432 (28) (Fig. 3, upper left panel). In neither transformants of strain G2 nor transformants of G5M432 did expression of MDR1 from the ADH1 promoter result in a detectable increase of resistance to fluconazole compared with the parental strains or control transformants. In contrast, MDR1 expression from the ADH1 promoter caused a fourfold increased resistance to cerulenin and brefeldin A compared with the strains containing the control construct (Fig. 3, upper right and lower left panels). In addition, strains carrying the PADH1-MDR1 fusion in these backgrounds also showed a fourfold increased resistance to 4-NQO (Fig. 3, lower right panel). No difference in the effect of MDR1 expression from the ADH1 promoter on drug resistance was observed between the two matched isolates, indicating that Mdr1p function did not depend on the additional alterations that had occurred in isolate G5. Note, however, that the strains expressing the PADH1-MDR1 fusion did not reach the same level of resistance to cerulenin, brefeldin A, and 4-NQO as the clinical isolate G5, although it was previously demonstrated that MDR1 overexpression was the sole cause of resistance to these compounds in this strain (29) (also compare strains G2, G5, and the mdr1 mutant G5M432 in the corresponding panels in Fig. 3). To reveal a possible explanation for the differences in drug resistance of the MDR1-overexpressing clinical isolate G5 and the strains expressing MDR1 from the ADH1 promoter, we compared Mdr1p expression levels in the various strains by Western immunoblotting with the anti-Mdr1p antiserum (Fig. 4). As expected from previous work, no Mdr1p was detected in the fluconazole-susceptible clinical isolate G2 (Fig. 4, lane 1), but a specific doublet band reacting with the antiserum was observed after treatment of the cells with benomyl, which is known to induce the MDR1 promoter (lane 2) (10, 25). The MDR1-overexpressing isolate G5 showed a strongly reacting band (Fig. 4, lane 4), which disappeared after deletion of both MDR1 alleles from this strain (strain G5M432; lanes 3 and 9). Mdr1p was constitutively expressed in transformants carrying the PADH1-MDR1 fusion (Fig. 4, lanes 7, 8, 12, and 13), whereas Mdr1p was not detected in strains carrying the control construct (lanes 5, 6, 10, and 11). It was particularly interesting that expression of MDR1 from the ADH1 promoter did not result in the same high Mdr1p expression levels as in the clinical isolate G5, suggesting that the degree of Mdr1p-mediated resistance depends on the level of Mdr1p overexpression.
DISCUSSION
The function of MDR1 as a multidrug resistance gene in C. albicans is well established by several lines of evidence, including the strong correlation between MDR1 overexpression and resistance to different toxic compounds in matched pairs of clinical C. albicans isolates (6, 7, 14, 19, 23, 26), the partial or complete loss of the resistant phenotype after deletion of MDR1 from MDR1-overexpressing C. albicans strains (9, 28, 29), and the ability of the gene to confer drug resistance in the heterologous host S. cerevisiae (1, 23). Nevertheless, the differences in the spectra of drugs to which MDR1 conferred resistance in S. cerevisiae transformants carrying MDR1 on a plasmid and in MDR1-overexpressing clinical C. albicans isolates raised questions about the ability of this efflux pump to confer resistance to specific compounds in C. albicans. In addition, the observation that a conserved group of additional genes was concomitantly upregulated together with MDR1 in drug-resistant clinical C. albicans isolates (11, 13, 21) pointed to the possibility that overexpression of MDR1 alone may not be sufficient to mediate resistance to some drugs and that resistance may depend on these additional alterations. Therefore, we addressed this issue by artificially overexpressing MDR1 from a strong promoter in a drug-susceptible C. albicans laboratory strain that did not express the endogenous MDR1 alleles at detectable levels. A similar approach has recently been taken by other groups to directly study the function of the ABC transporter genes CDR1 and CDR2 in drug resistance in C. albicans (18, 24). Forced MDR1 overexpression from the ADH1 promoter in the C. albicans laboratory strain CAI4 or its prototrophic parental strain, SC5314, was sufficient to confer increased resistance to the putative Mdr1p substrates cerulenin and brefeldin A but had only a minor effect on the susceptibilities of the strains to fluconazole and 4-NQO. The degree of resistance to all drugs, as measured by the MIC, was lower in the laboratory strains expressing MDR1 from the ADH1 promoter than in the clinical isolate G5, which overexpressed the endogenous MDR1 alleles, suggesting that optimal functioning of the Mdr1p efflux pump might indeed depend on other cellular alterations occurring in drug-resistant clinical isolates. However, a direct comparison of the relative increases in resistance after expressing the PADH1-MDR1 fusion in a derivative of such a drug-resistant clinical C. albicans isolate, in which the overexpressed endogenous MDR1 alleles had been deleted, and a matched, drug-sensitive isolate showed no significant differences, demonstrating that the additional alterations that had occurred in the drug-resistant isolate were not required for Mdr1p function. Nevertheless, the degree of Mdr1p-mediated resistance to specific compounds seems to depend on the strain background, since MDR1 overexpression from the ADH1 promoter resulted in a fourfold increased resistance to 4-NQO in the clinical isolates but had no or only a minor effect on 4-NQO resistance in the laboratory strains. In contrast, the relative increases in resistance to cerulenin and brefeldin A were similar in all strains expressing the PADH1-MDR1 fusion.
Western immunoblot analysis showed that the amount of Mdr1p was significantly lower in cells expressing the MDR1 gene from the ADH1 promoter than in the clinical isolate G5, which overexpressed the endogenous MDR1 alleles. Although the ADH1 promoter is a strong promoter, this does not necessarily result in correspondingly high protein levels. For example, we previously observed that MEP2 transcript levels were higher when the gene was expressed from the ADH1 promoter than from its own promoter, but despite this, higher protein levels were observed when MEP2 was expressed from its own promoter (2). Therefore, this result suggested that the degree of Mdr1p-mediated drug resistance depends on the Mdr1p expression level. For apparently good substrates, such as cerulenin and brefeldin A, relatively low Mdr1p expression levels are already sufficient to confer significant resistance, whereas fluconazole seems to be transported less efficiently by Mdr1p, with significant resistance being seen only in strains that strongly overexpress the MDR1 gene, such as many clinical C. albicans isolates.
ACKNOWLEDGMENTS
We thank Bill Fonzi and Fritz Mühlschlegel for the gift of strains SC5314 and CAI4. Sequence data for Candida albicans were obtained from the Stanford Genome Technology Center website at http://www-sequence.stanford.edu/group/candida.
This study was supported by the Deutsche Forschungsgemeinschaft (DFG grants MO846/3 and SFB630) and the European Community (EC project QLK2-CT-2001-02377). Sequencing of Candida albicans was accomplished with the support of the NIDR and the Burroughs Wellcome Fund.
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