1 Department of Oral Sciences, University of Otago, PO Box 647, Dunedin, New Zealand; 2 Department of Bioactive Molecules, National Institute of Infectious Diseases, Tokyo, Japan
Received 29 June 2004; returned 1 August 2004; revised 8 September 2004; accepted 12 September 2004
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Abstract |
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Methods: The C. albicans CDR1 open reading frame was fused to the C. albicans HEX1 promoter and used to complement a CDR1-null mutant to create strain FL3. The effect of inducing the FL3 HEX1 promoter, by growth on medium containing N-acetylglucosamine (GlcNAc) as the carbon source, on CDR1 expression and drug susceptibility was determined.
Results: C. albicans FL3 cells grown on medium containing GlcNAc overexpressed CDR1 mRNA and a 170 kDa plasma membrane protein that reacted with anti-Cdr1p antibodies. Overexpression of Cdr1p in C. albicans FL3 conferred resistance to structurally unrelated chemicals such as terbinafine, brefeldin A, cerulenin and nigericin as well as to azole antifungal agents, but not resistance to polyene antibiotics. FK506, ascomycin and ciclosporin A chemosensitized FL3 to fluconazole. FL3 cells grown on GlcNAc effluxed 5.3 times as much Cdr1p substrate rhodamine 6G, over a 10 min period, as FL3 cells grown on glucose, and this rhodamine 6G efflux was inhibited by including fluconazole in the assay.
Conclusion: This study provides the first direct demonstration of Cdr1p pump activity in C. albicans.
Keywords: MDR , fluconazole , antifungals , efflux pumps
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Introduction |
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C. albicans possesses membrane transporters with homology to proteins of the ATP-binding cassette (ABC) family, such as Cdr1p and Cdr2p,1214 as well as proteins which have homology to the major facilitator superfamily (MFS) class of drug/proton antiport efflux pumps, for example Mdr1p (also referred to as Benrp) and Flu1p.6,1517 The role of MFS pump Mdr1p in C. albicans fluconazole resistance has been indicated by transcriptional analyses8,11,18 and gene inactivation in laboratory strains19 and clinical isolates.20 The C. albicans CDR1 gene is a homologue of Saccharomyces cerevisiae ABC multidrug efflux pump gene PDR5,21 and expression of CDR1 is often associated with energy-dependent drug efflux in fluconazole-resistant clinical isolates.9,18,2224 The C. albicans CDR1 gene was first cloned by the complementation of an S. cerevisiae pdr5 mutant where expression of Cdr1p conferred resistance to cycloheximide, chloramphenicol and miconazole.25 We have also demonstrated functional expression of Cdr1p in an S. cerevisiae strain deficient in seven major membrane transporters, which conferred resistance to several drugs, and the ability to efflux the fluorescent compound rhodamine 6G (R6G).26 Conversely, a C. albicans strain deleted in CDR1 was hypersusceptible to fluconazole, itraconazole, ketoconazole, terbinafine, amorolfine, cycloheximide, brefeldin A and fluphenazine.27 These studies indirectly indicate a drug pumping function for Cdr1p, but there have been no studies that directly examine its role in C. albicans.
The C. albicans HEX1 gene encodes hexosaminidase, a secreted hydrolytic enzyme that preferentially cleaves chitobiose into two molecules of N-acetylglucosamine (GlcNAc). We have previously demonstrated that the C. albicans HEX1 promoter (HEX1p) is induced by growth on GlcNAc as carbon source, and repressed by growth on glucose.28,29 The C. albicans HEX1p has also been used to control the expression of KRE6 (encoding a glucan synthase) in C. albicans.30 In this study we use HEX1p-controlled overexpression of CDR1 in a C. albicans CDR1-deleted strain to elucidate Cdr1p function.
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Materials and methods |
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The C. albicans strains used in this study are listed in Table 1. C. albicans ATCC 10261, A72, CAI4 and CAI4/CDR1 cells were routinely cultured in YPD medium (1% yeast extract, 2% Bacto peptone, 2% glucose) at 30°C with shaking (150 rpm). C. albicans FL3 and DL1 cells were routinely cultured in YNB (0.67% yeast nitrogen base without amino acids) containing 1% glucose and incubated at 30°C with shaking (150 rpm).
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YNB was obtained from Difco (Detroit, MI, USA), CSM-URA was obtained from Bio 101 (Vista, CA, USA) and GlcNAc was obtained from Sigma (Auckland, New Zealand). Fluconazole was obtained from Pfizer Ltd (Sandwich, Kent, UK). Ketoconazole, itraconazole and miconazole were obtained from Janssen Research Foundation (Beerse, Belgium). Amphotericin B was obtained from E.R. Squibb & Sons (Princeton, NJ, USA) and terbinafine was obtained from Novartis Internatonal AG (Basel, Switzerland). Carbonyl cyanide p-chlorophenylhydrazone (CCCP), nigericin, valinomycin, brefeldin A, cerulenin, nystatin, filipin, 4-nitroquinoline N-oxide, 2,4-dinitrophenol, 1,10-phenanthroline, ascomycin, ciclosporin A, verapamil and R6G were obtained from Sigma. FK506 was obtained from Fujisawa Pharmaceutical Company (Osaka, Japan).
Plasmid construction and yeast transformation
The C. albicans CDR1 open reading frame and transcription termination sequences were PCR amplified from C. albicans ATCC 10261 genomic DNA using the following primers: 5'-AAAAATCGATGTCAGATTCTAAGATG-3' (ClaI site underlined) and 5'-GCGAGGATATCGTTCTTTGACAG-3' (EcoRV site underlined). This DNA fragment was digested with ClaI and EcoRV and ligated into pBluescript SK (Stratagene, La Jolla, CA, USA), which had previously been digested with ClaI and EcoRV, to form plasmid pMN9101. The C. albicans A72 HEX1 promoter was PCR amplified from plasmid pRC170828 using the primers 5'-TTGTATGTAGTCGACAACATGG-3' (SalI site underlined) and 5'-TTTATCTAACACCATCGATTTTC-3' (ClaI site underlined). This DNA fragment was digested with SalI and ClaI and ligated into pMN9101, which had previously been digested with SalI and ClaI, to form pMN9121. The HEX1p/CDR1 construct was removed from pMN9121 with SalI and EcoRV, end-filled and cloned into shuttle plasmid pRC2312,32 which had previously been digested with SmaI, to form plasmid pMN9131. C. albicans CAI4/CDR1 was transformed with either pRC2312 or pMN9131 to uracil prototrophy by the spheroplast method, as described previously.33
Effect of azoles on C. albicans growth and MIC determination
To determine the effects of antifungal agents on the growth of C. albicans, yeast cells grown in YNB containing 0.1 M glucose at 30°C for 16 h were used to inoculate 4 mL of YNB containing either 0.1 M glucose or 0.1 M GlcNAc containing fluconazole (80.1 mg/L) in an L-shaped test tube. The tubes were incubated at 30°C with shaking, and cell growth was monitored each hour by reading the optical density at 660 nm in an automated Advantec TN-1506 Bio-Photorecorder (Advantec Toyo Co., Tokyo, Japan). Cultures grew in the yeast morphology under these conditions. The effects of antifungal agents were also determined by a microtitre plate microdilution assay based on the macrodilution reference method for MIC determination of the NCCLS.34 The medium used for the growth assays consisted of 0.67% YNB containing either 0.1 M glucose or 0.1 M GlcNAc. The YNB medium was supplemented with uridine (25 mg/L) for the growth of C. albicans CAI4 and CAI4/CDR1 strains. The microtitre plate wells contained dilutions of antifungal agents (final concentrations: fluconazole, 640.125 mg/L; itraconazole, 80.016 mg/L and ketoconazole, 20.004 mg/L) and were incubated at 35°C for 48 h. The growth of cells in individual wells (optical density at 590 nm) was measured with a microplate reader (EL340; Bio-Tek, Winooski, VT, USA) to determine the final MIC.34 The MIC80 was the lowest concentration of drug that inhibited growth yield by at least 80% compared with a no-drug control.
Agar diffusion drug susceptibility assays
Drug susceptibility was also measured using agar diffusion assays on CSM-URA plates supplemented with 0.1 M GlcNAc, 0.67% YNB and 1.5% agar. Plates were seeded with yeast cells suspended in top agar (5 mL, 105 cells/mL). One to ten microlitres of drug solution, or solvent control, was spotted onto sterile Whatman paper discs which were then dried and placed on the top agar (solvents used for individual drugs are given in brackets following the drug name). The following amounts (µg) of drugs were applied to individual discs: fluconazole (water), 1.0; ketoconazole [dimethyl sulphoxide (DMSO)], 0.03; itraconazole (DMSO), 0.05; miconazole (DMSO), 0.05; amphotericin B (DMSO), 120; CCCP (DMSO), 100; nigericin (ethanol), 50; valinomycin (ethanol), 0.25; terbinafine (DMSO), 5; brefeldin A (ethanol), 2.5; cerulenin (DMSO), 0.5; nystatin (DMSO), 5; filipin (DMSO), 15; 4-nitroquinoline N-oxide (ethanol), 1; 2,4-dinitrophenol (ethanol), 50; 1,10-phenanthroline (ethanol), 50. Agar plates were incubated at 30°C for 48 h or until clear growth inhibition zones were visible. Several other drugs were also examined by a similar agar diffusion assay method for their chemosensitization of C. albicans FL3 to fluconazole. CSM-URA + GlcNAc plates were seeded with yeast cells suspended in top agar supplemented with or without fluconazole at sub-MIC concentrations (FL3, 2 mg/L; DL1, 0.1 mg/L). To test for a synergic effect of each test drug with fluconazole, 110 µL of test drugs was spotted onto sterile Whatman paper discs, which were then dried and placed on the top agar. The following amounts (µg) of drugs were applied to individual discs: FK506 (DMSO), 1; ascomycin (DMSO), 0.1; ciclosporin A (DMSO), 10; and verapamil (ethanol), 200. Agar plates were incubated at 30°C for 48 h or until clear growth inhibition zones were visible.
Northern analysis of RNA extracted from C. albicans
C. albicans cells were used to inoculate 300 mL of YNB containing 1% glucose and incubated at 30°C for 16 h with shaking. Cells were harvested by centrifugation (3000g, 5 min), washed with sterile distilled water, resuspended in 800 mL of distilled water to an OD540 of 3.0 and starved by incubating at 30°C for 3 h with shaking. Starved cells were then harvested by centrifugation (3000g, 5 min) and resuspended to an OD540 of 2.0 in 400 mL of YNB containing either 25 mM glucose or 25 mM GlcNAc. Appropriate culture volumes were withdrawn at the indicated times and total RNA was extracted from the cells as described previously.6 RNA (20 µg) was electrophoresed in agarose gels, vacuum blotted on to Hybond+ nylon membrane (Amersham Pharmacia Biotech New Zealand, Auckland, New Zealand) and fixed with UV radiation. Membranes were hybridized with [-32P]dCTP-labelled probes under high-stringency conditions as described previously.28 A C. albicans CDR1 probe (ORF nt 1497) and an ACT1 probe were generated by PCR amplification of C. albicans ATCC 10261 genomic DNA.6
Immunodetection of Cdr1p
Crude protein extracts were prepared from C. albicans cells grown to mid-exponential phase on either glucose or GlcNAc as described earlier. Plasma membrane fractions of these cells were obtained by sucrose gradient centrifugation as described by Monk et al.35 Protein samples (40 µg) were separated by electrophoresis in 8% SDSpolyacrylamide gels and either stained with Coomassie Blue or electroblotted (100 V, 1 h, 4°C) onto nitrocellulose membranes (Highbond-C; Amersham). Western blots were incubated with a 1:500 dilution of anti-Cdr1p antibodies (kindly provided by Dr D. Sanglard, University Hospital Lausanne, Switzerland). Immunoreactivity was detected using horseradish peroxidase-labelled swine anti-rabbit IgG antibodies at a 1:1000 dilution.
Rhodamine efflux assay
C. albicans cells were grown in CSM-URA medium (containing 1% glucose) at 30°C for 16 h, subcultured into the same medium at an initial OD540 of 1.0 and incubated at 30°C until the OD540 reached 4.0. Cells were then washed three times in sterile water (3000g, 5 min, 20°C), resuspended in sterile water at an OD540 of 3.0 and starved of glucose by incubation at 30°C for 16 h. The cells were resuspended at an OD540 of 2.0 in CSM-URA medium containing either glucose (25 mM) or GlcNAc (25 mM) and incubated at 27°C for 3 h to either repress or induce CDR1, respectively (a temperature of 27°C was used to ensure that all cells were in the yeast morphology; some cells formed germ tubes in medium containing GlcNAc at 30°C). Cells were washed twice in water, once in 50 mM HEPESNaOH (pH 7.0) and then incubated in HEPES buffer at an OD540 of 1.0 in the presence of 5 mM 2-deoxy-D-glucose (Sigma) at 30°C for 1 h with shaking (200 rpm) to de-energize the cells. R6G was then added to the de-energized cells (to 10 µM) and they were incubated at 30°C for a further 1.5 h. The cells were washed twice in 50 mM HEPES buffer and resuspended in HEPES buffer at an OD540 of 10.0 (2.2 x 108 cells/mL). Aliquots of cells (0.4 mL) were pre-incubated at 30°C for 5 min and then R6G efflux from the cells was initiated by adding glucose (to 10 mM), and incubation at 30°C continued. In some experiments fluconazole (10 or 100 µM) was added to the R6G-loaded cells, before the glucose. At specified time-points after the addition of glucose, cells were removed from duplicate assays by centrifugation, and two 100 µL aliquots of each assay supernatant were transferred to the wells of 96-well black flat-bottom microtitre plates (BMG Labtechnologies GmbH, Offenburg, Germany). The fluorescence of R6G in the samples was measured with a POLARstar OPTIMA plate reader (BMG Labtechnologies) using excitation and emission wavelengths of 485 and 520 nm, respectively.
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Results and discussion |
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In order to investigate the function of Cdr1p in C. albicans, CDR1 was expressed under the control of the HEX1 promoter in CDR1-deleted C. albicans strain CAI4/CDR1 (Table 1). This strain was transformed with either pRC2312 (vector alone, which yielded control transformant DL1) or pMN9131 (vector containing the CDR1 open reading frame fused to the HEX1 promoter, which yielded transformant FL3), with selection for uracil prototrophs. The expression of CDR1 in FL3 was examined by northern analysis, immunodetection of Cdr1p and by measuring the effect of Cdr1p expression on cell growth in the presence of fluconazole. C. albicans FL3 cells were grown in medium containing either GlcNAc or glucose as carbon source, to either induce or repress the HEX1 promoter, respectively, and the expression of CDR1 mRNA over a 3 h period was determined by northern analysis (Figure 1). Expression of CDR1 mRNA was induced rapidly (within 0.5 h) by incubation with GlcNAc. The amount of CDR1 mRNA was reduced at the 3 h time-point, but so was the amount of ACT1 mRNA. A small amount of CDR1 mRNA was detected in cells grown on glucose with only a slight increase in the amounts present over the 3 h time course. There was no expression of CDR1 mRNA in the negative control strain DL1 grown on either carbon source. To confirm that the GlcNAc induction of CDR1 mRNA expression in C. albicans FL3 resulted in Cdr1p expression, SDSPAGE and immunoblot analysis of plasma membrane proteins was undertaken (Figure 2). The expression of a protein with a molecular weight equivalent to that expected for Cdr1p (170 kDa) in plasma membrane samples from C. albicans FL3 cells increased during growth on GlcNAc, and was present at a lower concentration in cells grown on glucose (Figure 2a). The protein that was overexpressed in cells grown on GlcNAc was confirmed to be Cdr1p by immunodetection using anti-C. albicans Cdr1p antibodies (Figure 2b). The amount of Cdr1p in the plasma membrane samples increased over the 3 h time course of GlcNAc induction whereas CDR1 mRNA expression peaked at t=0.5 h (Figure 1). There was a low and constant amount of Cdr1p expression in C. albicans FL3 cells grown on glucose.
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The effect of Cdr1p expression on the growth of FL3 and DL1 cells in medium containing glucose or GlcNAc and various concentrations of fluconazole was monitored hourly during the time course (048 h) of usual MIC determinations (Figure 3). GlcNAc is a well-recognized inducer of hyphal growth at 37°C.36 Therefore, to restrict the effects of GlcNAc to CDR1 expression, these growth experiments were undertaken at 30°C; conditions under which cells maintained a yeast morphology. The growth kinetics were strain and carbon source dependent. There was a longer lag phase for cells grown on GlcNAc (20 h) than for those grown on glucose (
15 h). This probably reflects the need for cells grown on GlcNAc to express genes involved in GlcNAc uptake and utilization.37,38 Apart from the difference in the length of the lag phase, the growth kinetics of DL1 cells, and the growth inhibitory effects of fluconazole on this strain, were similar for both GlcNAc- and glucose-grown cells; growth was inhibited by fluconazole concentrations >0.2 mg/L (Figure 3a and b). In contrast, the initial growth responses of FL3 cells to increasing concentrations of fluconazole were markedly different when either GlcNAc or glucose was provided as the carbon source. Strain FL3 grew normally on GlcNAc in the presence of up to 2.0 mg/L fluconazole (Figure 3d) and only showed a slightly increased lag phase in the presence of 3 or 4 mg/L fluconazole, whereas growth on glucose showed a marked lag in the presence of 28 mg/L fluconazole (Figure 3c). These results are consistent with the rapid induction of Cdr1p in GlcNAc-grown FL3 cells, the low level of Cdr1p expression in glucose-grown FL3 cells and the absence of detectable Cdr1p expression in DL1 cells, confirming a direct role for Cdr1p in mediating C. albicans resistance to fluconazole early during the growth of FL3 cells, but this was not evident at the typical end-point for MIC assays. When growth inhibition by fluconazole (and other azoles) was measured using a typical end-point MIC assay, where growth was measured only after 48 h of incubation in the presence of different antifungal drug concentrations, glucose-grown FL3 cells appeared to be more resistant to fluconazole, and other azoles, than cells grown on GlcNAc (Table 2). The data presented in Figure 3 show that this was due to slow growth in the presence of fluconazole permitted by a small amount of Cdr1p expression in the glucose-grown cells, resulting in sufficient cells after 48 h to indicate resistance. The MIC data confirmed, however, that the transformation of CAI4/
CDR1 with pMN9131 (to form FL3) conferred decreased azole susceptibility compared with the control strain (DL1) transformed with pRC2312, or the untransformed strain CAI4/
CDR1. This CDR1 disruptant, CAI4/
CDR1, was, in turn, more susceptible to azoles than its parental strain (CAI4), as expected,27 and transformation of this strain with pRC2312 (DL1) did not significantly affect the susceptibility of cells to any azole tested.
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Drug susceptibility phenotype of C. albicans FL3 cells overexpressing Cdr1p
The drug susceptibilities of C. albicans FL3 cells overexpressing Cdr1p and its counterpart DL1 containing vector plasmid alone were measured using agar diffusion assays (Figure 4). FL3 cells growing on GlcNAc, and overexpressing Cdr1p, were less susceptible than the control DL1 cells to the following drugs: azole antifungal agents such as fluconazole, ketoconazole, itraconazole and miconazole; terbinafine, a specific inhibitor of squalene epoxidase; brefeldin A, a fungal metabolite which is a macrocyclic lactone exhibiting a wide range of antibiotic activity and an inhibitor of LDH-mediated cholesterol efflux; cerulenin, an antifungal antibiotic that inhibits sterol and fatty acid biosynthesis; and nigericin, a polyether ionophore which disrupts membrane potential. Overexpression of Cdr1p in FL3 cells did not confer resistance to polyene antifungal agents nystatin, amphotericin B and filipin; uncouplers of oxidative phosphorylation in mitochondria, 4-nitroquinoline N-oxide, CCCP and 2,4-dinitrophenol; proteinase inhibitor 1,10-phenanthroline; or potassium ionophore valinomycin.
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Acknowledgements |
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Footnotes |
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* Present address. Department of Bioactive Molecules, Institute of Infectious Diseases, Tokyo, Japan.
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