Mechanisms of fluconazole resistance in Candida albicans isolates from Japanese AIDS patients

Kazunori Maebashia,*, Masakazu Niimib, Michinari Kudoha, Frank J. Fischerb, Koichi Makimuraa, Kyoko Niimib, R. Jane Piperb, Katsuhisa Uchidaa, Mikio Arisawac, Richard D. Cannonb and Hideyo Yamaguchia

a Teikyo University Institute of Medical Mycology, 359 Otsuka, Hachioji, Tokyo 192-0395, Japan; b Molecular Microbiology Laboratory, Department of Oral Sciences and Orthodontics, School of Dentistry, University of Otago, PO Box 647, Dunedin, New Zealand; c Department of Mycology, Nippon Roche Research Center, 200 Kajiwara, Kamakura, Kanagawa 247-0063, Japan


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Four Candida albicans isolates, TIMM 3163, TIMM 3164, TIMM 3165 and TIMM 3166, with reduced fluconazole susceptibility were obtained from three AIDS patients in Japan, and the mechanisms of their drug resistance were studied. All isolates showed lower levels of intracellular accumulation of fluconazole than ATCC 10231, a susceptible control strain of C. albicans. Increased amounts of CDR1 and CDR2 mRNA encoding putative ATP binding cassette (ABC) transporters were associated with the azole resistance of all TIMM isolates, apart from TIMM 3164. In addition, increased Cdr1p levels were immunodetected in the cell membrane fractions of all the TIMM strains except for TIMM 3164. Gene amplification was not responsible for CDR1 overexpression and there were no significant differences in the mRNA levels of CDR3 or CDR4 (ABC transporters) in the azole-susceptible and -resistant cells. CaMDR1 (a major facilitator superfamily) gene expression was not observed in any of the resistant isolates or the control strain. These results suggest that energy-dependent drug efflux associated with increased expression of CDR1 and CDR2 is involved in the fluconazole resistance mechanisms in two of the four isolates, TIMM 3165 and TIMM 3166. TIMM 3164 demonstrated energy-dependent drug efflux without overexpression of CDR1-4 or CaMDR1, indicating that some other pump may be operating. Despite showing low levels of drug efflux and overexpression of CDR1 and CDR2, efflux in TIMM 3163 was not energy dependent, suggesting that the expressed Cdr1p non-functional Cdr1p and that other resistance mechanisms may operate in this strain.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Candida albicans is an important opportunistic fungal pathogen of humans and the major cause of oropharyngeal candidosis (OPC) in AIDS patients.1 The triazole antifungal agent fluconazole has been widely used to treat patients with OPC. However, emergence of C. albicans strains resistant to azole antifungal drugs owing to prolonged fluconazole treatment is now a recognized problem in AIDS patients with OPC.1,2

Several possible mechanisms of resistance to azole antifungal agents in C. albicans have been reported.3 Azole drugs inhibit ergosterol biosynthesis in fungal cells by binding to their cytochrome P-450 sterol 14{alpha}-demethylase (Erg11p).4 Three mechanisms of azole resistance have been demonstrated involving fungal ergosterol biosynthetic pathways: (i) a change in Erg11p affinity for azole drugs;5,6 (ii) overexpression of Erg11p;7,8 and (iii) a change in {triangleup}5,6-sterol desaturase.9,10 Recently, point mutations in the ERG11 gene have been identified as a cause of reduced affinity of Erg11p in several azole-resistant isolates.11,12

In a greater number of azole-resistant C. albicans isolates, however, resistance is associated with reduced cellular influx and/or increased efflux of azole drugs, in particular fluconazole.13,14 The CDR1, CDR2 and CaMDR1 genes are proposed to encode cell membrane-associated transporters, which act as possible mediators to enhance fluconazole efflux.7,8,1517 The CDR1 and CDR2 genes have homology to genes of the ATP binding cassette (ABC) family, which contains genes involved in multiple drug resistance in other organisms.18 It has also been demonstrated that ABC-type transporters are responsible for the export of several other azole drugs such as itraconazole and ketoconazole in addition to fluconazole.15,17 The CaMDR1 gene belongs to the major facilitator superfamily (MFS) and confers azole resistance only to fluconazole.15,19 Recent studies of serial C. albicans isolates from AIDS patients with OPC demonstrated that the different mechanisms described above often contribute to the final resistant phenotype.7,8

The majority of previous research has been restricted to an analysis of drug-resistant clinical isolates from the USA or Europe. Recently, fluconazole-resistant C. albicans strains have also been isolated from Japanese AIDS patients with OPC.20 This paper describes biochemical and molecular biological studies that revealed novel mechanisms of fluconazole resistance.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Materials

Fluconazole (Pfizer, NY, USA), itraconazole (Janssen Research Foundation, Beerse, Belgium) and terbinafine (Novartis, Basel, Switzerland) were obtained from commercial sources. Radioisotopes were purchased from Amersham (UK). [3H]Fluconazole (specific radioactivity 740 GBq/mmol) was prepared by unilabelling with tritium at Amersham. Unless specified, other chemicals were purchased from Sigma Chemical Co. (St Louis, MO, USA) or Wako Pure Chemical Industries (Tokyo, Japan).

Strains

Four C. albicans strains, TIMM 3163, TIMM 3164, TIMM 3165 and TIMM 3166, with reduced susceptibility to fluconazole were used in this study. These strains were originally isolated from Japanese AIDS patients with OPC receiving fluconazole therapy.20 TIMM 3163 was obtained from a patient who had recurrent oral candidosis and had received >60 g fluconazole over a 34 month period. TIMM 3164 was isolated from the pharynx of a patient who had received 22 g fluconazole over a 14 month period, and TIMM 3165 was isolated from the blood of the same patient at post-mortem 10 months and 4 g fluconazole later. TIMM 3166 was isolated from a third patient suffering from recurrent OPC. C. albicans ATCC 10231 was used as the susceptible control strain. C. albicans strains were maintained on Sabouraud dextrose agar and subcultured in YPG broth (1% yeast extract, 1% Bacto peptone, 2% glucose) before use. To prepare a cell-free extract for in vitro sterol biosynthesis studies, the glucose concentration of YPG broth was increased to 4%.

Susceptibility testing

The MICs of antifungal agents for C. albicans isolates were determined according to NCCLS guidelines.21 The MICs of Cdrp and/or CaMdr1p pump substrates, except terbinafine, were also determined by the same procedures and defined as the lowest drug concentrations to prevent any discernible growth. The MICs of terbinafine, fluconazole, itraconazole and miconazole were determined as the lowest drug concentrations to show an 80% inhibition of drug-free control growth.

Measurement of sterol 14{alpha}-demethylase activity of growing cells

Cells were grown in YPG broth at 30°C for 16 h on a rotary shaker, harvested by centrifugation at 1500g for 5 min, washed and then suspended in fresh SAAMF medium (Nippon Bio-Supp Center, Tokyo, Japan) at a cell density of 1 x 106 cells/mL. Aliquots (9.89 mL) of the cell suspensions were incubated at 30°C for 6 h, and then 10 µL of sodium [14C]acetate (185 kBq, 2.146 MBq/mmol) and 0.1 mL of a fluconazole solution at various concentrations, or 1% dimethylsulphoxide (DMSO) as a control, were added. After incubation at 30°C for 3 h, cells were saponified in a 90% ethanol solution containing 15% potassium hydroxide and 0.1% pyrogallol at 90°C for 1 h. Non-saponifiable lipids were extracted and separated by thin-layer chromatography (TLC) using pre-coated silica gel plates (Merck, Darmstadt, Germany) and heptane-diisopropylether-acetic acid-ethyl acetate (60:40:4:34.7 vols) as a developing solvent. The radioactivity of the ergosterol fraction was quantified using a BAS2000 bioimage analyser (Fuji Film, Tokyo, Japan). The IC50 of fluconazole for ergosterol biosynthesis was calculated by comparison with the radioactivity of the ergosterol fraction from the drug-free control.

Measurement of sterol 14{alpha}-demethylase activity of cell-free extracts

Cells were grown semi-anaerobically to the logarithmic growth phase at 30°C for 13 h in YPG broth containing 4% glucose. The incorporation of [14C]mevalonate into ergosterol was measured as described previously.22 Cells were harvested, washed and suspended in a minimum volume of ice-cold 0.1 M phosphate buffer (pH 7.4) supplemented with 30 nM nicotinamide, 5 mM MgCl2 and 5 mM reduced glutathione. Cells were disrupted in a cooled Braun cell homogenizer (B. Braun, Melsungen, Germany) with glass beads (diameter 0.45–0.5 mm). All subsequent procedures were conducted at 4°C. Cell debris was removed by centrifugation at 1500g for 5 min, and the supernatant was further centrifuged twice at 8000g for 20 min each time, finally yielding a cell-free extract. Its protein concentration was determined by the Lowry method23 and the extract adjusted to 11 mg protein/mL. It was then mixed with 1/100 volume of a MgCl2–MnCl2 solution (61.0 and 39.6 mg/mL, respectively). To this mixture (0.88 mL), 10 µL of a [14C]mevalonic acid dibenzethylenediamine salt solution (9.25 kBq, 2.109 MBq/mmol) was added alongside 10 µL of a fluconazole solution at various concentrations (or 0.05% DMSO as a control), together with 0.1 mL of 0.1 mM potassium phosphate buffer (pH 7.4) containing 6 mmol of ATP, 3 µmol of glucose-6-phosphate, 1 µmol of NAD and 1 µmol of NADP. The reaction mixture was incubated at 30°C for 3 h, and then the reaction stopped by the addition of 1 mL of 15% KOH in 90% ethanol, and heating at 85°C for 1 h. The radioactivity in the ergosterol fraction was quantified as described in the experiments with growing cells.

Accumulation of [3H]fluconazole by C. albicans isolates

Cells were grown in YPG broth at 37°C for 12 h to the logarithmic growth phase, harvested by centrifugation at 1500g for 5 min, washed with Dulbecco's phosphate-buffered saline (PBS) (Nissui Pharmaceutical Co., Ltd, Tokyo, Japan), and finally suspended in the same buffer to a cell density of 2 x 108 cells/mL. Carbon-starved cells were prepared by incubating YPG-grown log phase cells in 0.67% yeast nitrogen base (Difco Laboratories, Detroit, MI, USA) for an additional 2 h. The cell suspension was incubated at 37°C for 30 min, and after the addition of [3H]fluconazole (100 nM, 14.8 kBq/mL), incubation was continued. At different time intervals, 2 mL aliquots of samples were filtered with Whatman GF/C filters, and washed three times with 4 mL of PBS containing 50 µM unlabelled fluconazole. The radiolabelled cells were suspended in liquid scintillation cocktail and the radioactivity was counted in an Aloka scintillation counter. Where indicated, 0.5 mM sodium azide or 60 mM 2-deoxyglucose as energy inhibitors, or 20 mM glucose as additional energy source, were added, 15 min before the addition of [3H]fluconazole.

Quantification of CDR and CaMDR1 mRNA in C. albicans isolates

Quantification of mRNA was based on an amplification by reverse transcriptase–polymerase chain reaction (RT–PCR). Total cellular RNA was extracted by a glass beads/phenol-chloroform method24 from C. albicans strains grown to logarithmic growth phase in YPG broth at 37°C for 12 h. RNA was then isolated using a Quick Prep Micro mRNA Purification Kit (Pharmacia Biotech, Uppsala, Sweden) according to the manufacturer's instructions, to prevent contamination with genomic DNA. Seventy nanograms of purified mRNA was reverse transcribed using a Ready To Go Kit (Pharmacia Biotech). cDNA was PCR amplified by adding 1 µL of each sample to 99 µL of a solution containing 50 mM KCl, 1.5 mM MgCl2, 10 mM Tris–HCl (pH 7.5), 0.2 mM deoxynucleoside triphosphates and 2.5 U of Taq DNA polymerase (Pharmacia Biotech) and primers. PCR was performed in a Gene Amp PCR System 9600 (Perkin-Elmer, Foster City, CA, USA) with a first cycle of denaturation for 4 min at 94°C, which was followed by 25 or 30 cycles of annealing at 55°C for 1 min, elongation at 72°C for 90 s and denaturation at 94°C for 1 min. PCR was completed by a final elongation step at 72°C for 10 min. After the completion of amplification, PCR products were analysed by 1.2% agarose gel electrophoresis and stained with ethidium bromide. The amount of each PCR product was quantified by using NIH image software (version 1.55, Wayne Rasbard, NIH, MD, USA). The primers for CDR mRNA (5'-GTGCTGGTTGTTCCACATTGT-3' and 5'-AACCCTCTAGTGGCATTATCC-3') were designed based on comparison with the sequences of CDR1 (DDBJ/ EMBL/GenBank accession no. X53823). Primers for CaMDR1 mRNA (5'-CCTAAGATTTTGCACTCGGA-3' and 5'-AGATGTATTGCTATTGGGGC-3') were constructed using the registered sequence of CaMDR1 (DDBJ/EMBL/GenBank accession no. X77589). Primers for ACT1 mRNA were prepared as described by Kan25 as follows: 5'-GCCGGTGACGACGCTCCAAGAGCTG-3' and 5'-CCGTGTTCAATTGGGTATCTCAAGGTC-3'.

Analysis of mRNA in C. albicans strains

C. albicans cells were grown in YPG broth at 30°C with shaking. RNA samples were prepared from exponentially growing cells by the methods described previously.16 CDR1, CaMDR1, ERG11 and ACT1 probes were synthesized by PCR amplification of C. albicans ATCC 10261 genomic DNA.16 CDR2 probe was PCR-amplified from C. albicans ATCC 10261.17 The probes for CDR3 and CDR4 were 2.7 kb EcoRI-digested and 1.2 kb EcoRI-digested fragments of the genes, respectively, cloned from a lambda EMBL4 library of C. albicans ATCC 10261 genomic DNA. 32P-radiolabelled probes were hybridized with dot blots of RNA under high stringency conditions.16 The amounts of RNA on the dot blots were standardized with respect to the amount of ACT1 mRNA.

Preparation of plasma membranes and Western blot analysis

Gradient-purified C. albicans plasma membranes were prepared by a modification of the method described by Monk et al.26 Exponential phase cells grown in 100 mL YPG were harvested by centrifugation (3000g for 5 min at 4°C), washed with homogenizing buffer (50 mM Tris–HCl pH 7.5, 2 mM EDTA and 2% glucose supplemented with 0.1 mL of 100 mM phenylmethylsulphonyl fluoride per 10 mL homogenizing buffer) and resuspended in 2 mL of the same buffer. The suspension was vortex-mixed vigorously with 1.5 g glass beads (0.5 mm diameter) for five 1 min periods, alternating with 1 min periods on ice. The undisrupted cells and cell debris were removed by centrifugation at 7500g for 10 min at 4°C. The supernatant was centrifuged at 30000g for 45 min. The pellet was then resuspended in GTED-20 (10 mM Tris pH 7.0, 0.5 mM EDTA and 20% glycerol) and the centrifugation repeated. The pellet (crude membranes) was resuspended in GTED-20. Purified plasma membranes were recovered at the 54/44% (w/w) sucrose interface of a step gradient containing 2 mM EDTA and 20 mM Tris (pH 7.2) after centrifugation for 3 h at 100000g in an SW41 rotor. The material located at the interface was recovered, diluted to 20 mL with GTED-20 and centrifuged at 150000g for 45 min. Pellets of purified plasma membrane were resuspended in 0.2 mL of GTED-20 and stored at –80°C until use. The protein concentrations of the purified plasma membranes were determined according to the method of Bradford using {gamma}-globulin as a standard.

The plasma membrane fractions (20 µg) were separated on 8% SDS–PAGE gels. One gel was stained with Coommassie Blue R250, and the proteins in the other were transferred on to a nitrocellulose membrane by electroblotting in 25 mM Tris, 192 mM glycine, 20% (v/v) methanol at 100 V for 45 min. For immunodetection, blots were incubated with anti-Cdr1p antibodies (donated by D. Sanglard, Centre Hospitalier Universitaire Vaudois, Switzerland) diluted 1:500, and antibody binding was revealed by using peroxidase-conjugated swine immunoglobulins to rabbit immunoglobulin G (Dako Corp., Carpinteria, CA, USA) at a 1:1000 dilution.


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 Materials and methods
 Results
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 References
 
Susceptibility of C. albicans isolates to antifungal drugs

The MICs of antifungal agents against a fluconazolesusceptible C. albicans strain (ATCC 10231) and four C. albicans isolates (TIMM 3163, TIMM 3164, TIMM 3165 and TIMM 3166) with reduced fluconazole susceptibility were determined according to the NCCLS M27-A guidelines. TIMM 3165 was resistant to fluconazole, with an MIC of 64 mg/L (Table IGo). This strain was cross-resistant to miconazole (if the interpretive breakpoints for itraconazole are extrapolated to miconazole) and susceptible dose-dependent (SDD) to itraconazole according to the interpretive breakpoints proposed by Rex et al.27 TIMM 3163 was resistant to itraconazole and miconazole, and SDD to fluconazole. TIMM 3164 and TIMM 3166 were both SDD to miconazole and, although classified as susceptible to fluconazole, their MICs were eight- and 16-fold higher, respectively, than the fluconazole-susceptible strain ATCC 10231. All four TIMM isolates were susceptible to amphotericin B and 5-fluorocytosine.


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Table I. In vitro activity of antifungal agents for the fluconazole-susceptible control strain (ATCC 10231) and four isolates of C. albicans with reduced susceptibility to azole antifungal agents, as determined by the NCCLS M27-A method
 
Effect of fluconazole on sterol biosynthesis by resistant isolates

The sterol compositions of C. albicans membrane fractions were determined by gas-chromatography mass spectrometry.10 The ergosterol composition of all strains was between 92.1 and 97% of sterols, except for TIMM 3166 where ergosterol accounted for 85.8% of sterols. TIMM 3166 showed a correspondingly higher 4,14{alpha}-dimethyl zymosterol content (10.8%) than the other strains (0–2.2%). There was no significant difference in the proportion of other 14-methyl sterols between the strains. The effect of fluconazole on ergosterol biosynthesis by both growing cells and cell-free extracts was compared between the susceptible control strain and each TIMM isolate. As shown in Table IIGo, the extent of fluconazole inhibition of sterol biosynthesis, as represented by the IC50, in growing cells of each TIMM isolate was lower than that of the susceptible control strain, which correlated with their susceptibility to fluconazole. However, IC50 values obtained with cell-free extracts from the TIMM isolates did not correlate with their fluconazole susceptibility; the IC50s for TIMM 3163, TIMM 3164 and TIMM 3165 were only slightly higher than the value for the susceptible control strain, and the IC50 for cell-free extracts of TIMM 3166 was lower than that for the control strain. It should be noted that differences in IC50 values between strains may not be reflected in different resistance phenotypes as yeast can tolerate moderate changes in sterol composition. When the TIMM strains were exposed to sub-MIC levels of fluconazole there was a decrease in the ergosterol content of membranes and a concomitant increase in amounts of the 14-methyl sterols lanosterol and 24-methylene-dihydrolanosterol.


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Table II. Effect of fluconazole on ergosterol synthesis by growing cells or cell-free extracts of a fluconazolesusceptible control strain (ATCC 10231) and four isolates of C. albicans with reduced susceptibility to azole antifungal agents
 
Fluconazole accumulation in TIMM isolates

To investigate whether a change of permeability and/or intracellular accumulation of fluconazole was responsible for reduced drug susceptibility, the time-courses of [3H]fluconazole accumulation by growing cells were compared for each TIMM isolate and the susceptible control strain. As shown in Figure 1Go, [3H]fluconazole accumulation by the control strain ATCC 10231 proceeded linearly for 10 min, with an initial rate of 41.8 fmol/min/108 cells, and reached 669 fmol/108 cells after 30 min incubation. The amounts of fluconazole retained by the four isolates, TIMM 3163, TIMM 3164, TIMM 3165 and TIMM 3166, during the initial 30 min were lower relative to the control strain (60.9, 51.9, 23.9 and 30.5%, respectively). Fluconazole accumulation by the SDD isolate TIMM 3163 continued for the 60 min of incubation, while that by the other three isolates levelled off after 20 min. The rates of fluconazole accumulation by isolates TIMM 3163, TIMM 3164, TIMM 3165 and TIMM 3166 during the initial 5 min were calculated to be 30.6, 38.2, 13.6 and 18.3 fmol/min/108 cells, respectively, indicating that the initial rates of fluconazole accumulation for the isolates TIMM 3165 and TIMM 3166 were remarkably lower than the values for the control strain and the other two TIMM isolates.



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Figure 1. [3H]Fluconazole accumulation by a fluconazole-susceptible control strain (ATCC 10231) and four isolates of C. albicans with reduced susceptibility to azole antifungals. The value at each time point is the mean of triplicate experiments, which did not vary by more than 15%. Symbols: {square}, ATCC 10231; {diamondsuit}, TIMM 3163; {blacktriangleup}, TIMM 3164; •, TIMM 3165; {blacktriangledown}, TIMM 3166.

 
The reduced intracellular accumulation of fluconazole observed for TIMM 3165 and TIMM 3166 is likely to be caused by an increased energy-dependent drug efflux that is known to be coupled with ATP hydrolysis or a change in membrane potential. To examine this possibility, experiments were conducted to see whether the energy inhibitors sodium azide and 2-deoxyglucose, carbon starvation and glucose as an energy supplier, influenced the accumulation of fluconazole by the control strain and the four isolates. As shown in Figure 2Go, the intracellular level of fluconazole was scarcely affected in the control ATCC 10231 strain in the presence of 0.5 mM of sodium azide, 60 mM of 2-deoxyglucose, 20 mM glucose, or under carbon-starved conditions. In contrast, sodium azide, 2-deoxyglucose and carbon starvation increased fluconazole accumulation in the three isolates TIMM 3164, TIMM 3165 and TIMM 3166. Supplementation of cells with glucose decreased accumulation in these isolates. The fluconazole accumulation in strain TIMM 3163, however, was different from the other TIMM isolates. Sodium azide, 2-deoxyglucose and glucose did not affect the accumulation of fluconazole, and fluconazole accumulation decreased in the carbon-starved TIMM 3163 cells.



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Figure 2. Effect of respiratory inhibitors, starvation and glucose on [3H]fluconazole accumulation by a fluconazole-susceptible control strain (ATCC 10231) and four isolates of C. albicans with reduced susceptibility to azole antifungals. Cells were pre-incubated in the presence of 0.5 mM sodium azide ({blacksquare}), 60 mM 2-deoxyglucose ({blacksquare}) or 20 mM glucose ({blacksquare}) at 37°C for 15 min, and then incubation was continued for 30 min after the addition of [3H]fluconazole ({square} = starvation). The values are the means of triplicate experiments, which did not vary by more than 15%.

 
Expression of CDR and CaMDR1 genes

Cdrp multi-drug efflux transporters and CaMdr1p have been identified as drug resistance mediators in C. albicans. In the present study, we used RT–PCR to semi-quantitatively determine the expression of the CDR family of genes and of CaMDR1. The basis of this assay is that at an optimal number of PCR cycles, the quantity of amplified product of CDR genes or CaMDR1 reflects the expression level of the corresponding gene. We used primers corresponding to the conserved walker ATP binding motifs of CDR1, which should amplify mRNA from all of the CDR family of genes.17,18,28,29 The intensity of signals representing the amplified products of CDR and ACT1 genes was measured densitometrically after PCR amplifications of 25 cycles for ATCC 10231, TIMM 3164, TIMM 3165 and TIMM 3166, and that of 30 cycles for the isolate TIMM 3163. Comparison of the level of CDR product relative to that of ACT1 product for each TIMM isolate with the value for the susceptible strain demonstrated that the extent of CDR gene expression was only 1.3-fold higher in TIMM 3164, and 2.1- to 4.4-fold higher in the other TIMM isolates than the susceptible strain (Table IIIGo). However, under the experimental conditions where genomic DNA from the susceptible strain was amplified by a PCR run of 30 cycles, the presence of CaMDR1 mRNA was not detected in the susceptible strain or any TIMM isolates (data not shown).


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Table III. Comparison of the level of CDR mRNA in four isolates with reduced susceptibility to azole antifungal agents with that in the susceptible control strain (ATCC 10231) of C. albicans
 
Comparison of amounts of mRNA for multi-drug resistance genes in C. albicans isolates

In order to determine which CDR genes were overexpressed in TIMM 3163, TIMM 3165 and TIMM 3166, the amounts of mRNAs for C. albicans genes related to multi-drug resistance (CDR1, CDR2, CDR3, CDR4, CaMDR1 and ERG11) were determined for the clinical isolates and the standard laboratory strain. The amounts of CDR1 mRNA were significantly higher in all TIMM strains except TIMM 3164 than in ATCC 10231 (Figure 3Go). CDR2 mRNA was also expressed at a significant level in those clinical isolates, while the laboratory strain showed no CDR2 mRNA expression. The increase in the amounts of CDR1 and CDR2 mRNA in TIMM 3163, TIMM 3165 and TIMM 3166 correlated well with the MICs of fluconazole. In contrast, the amounts of ERG11 mRNA and CDR4 mRNA did not correlate directly with azole susceptibility for any clinical isolates (fluconazole-susceptible strain ATCC 10261 had a similar amount of CDR4 mRNA to TIMM 3163, data not shown). CaMDR1 and CDR3 mRNAs were not detected in the RNA extracts of any strains tested.



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Figure 3. Amount of CDR1, CDR2, CDR4 and ERG11 mRNA relative to ACT1 mRNA in a fluconazole-susceptible control strain (ATCC 10231, {square}) and four isolates of C. albicans ({blacksquare}, TIMM 3163; {blacksquare}, TIMM 3164; {blacksquare}, TIMM 3165; {blacksquare}, TIMM 3166) with reduced susceptibility to azole antifungals.

 
Immunodetection of Cdr1p proteins in plasma membranes

In order to demonstrate that CDR1 mRNA levels correlated with Cdr1p protein expression, Western blotting analysis was performed using anti-Cdr1p antibodies. As shown in Figure 4Go, CDR1 products were detected as a single band of 170 kDa in the plasma membrane fractions isolated from all four TIMM strains and the susceptible ATCC strain. However, the intensities of Cdr1p bands for all TIMM strains except TIMM 3164 were significantly higher than that for ATCC 10231.



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Figure 4. Plasma membrane proteins from a fluconazole-susceptible control strain (ATCC 10231) and four isolates of C. albicans with reduced susceptibility to antifungal azoles. Plasma membrane fractions were prepared by fractionating with sucrose gradient centrifugation. (a) SDS–PAGE of plasma membrane proteins (25 µg) stained with Coomassie Blue. (b) Immunoblot of gel in (a), incubated with anti-Cdr1p antibodies.

 
Southern blot analysis of CDR1 gene

Southern blot analysis of genomic DNA from Candida strains indicated that gene amplification was not responsible for the reduced susceptibility of TIMM strains. The signal intensities of BamHI fragments that hybridized with the CDR1 probe were similar for all strains (data not shown). A similar result was obtained for XhoI-digested chromosomal DNA, except that there was a restriction fragment polymorphism in TIMM 3163 that gave a band of 3.45 kb that hybridized with the probe in addition to the 6.8 kb band common to all strains. Therefore, other mechanisms such as transcriptional regulation must be responsible for overexpression of CDR1.30

Susceptibility of C. albicans isolates to Cdrp and/or CaMdr1p pump substrates

It has been demonstrated that Cdrp pumps of C. albicans can accept not only fluconazole but also various other unrelated compounds, such as rhodamine 6G, cerulenin, brefeldin A and terbinafine as substrates, and that the CaMdr1p pump of this yeast also acts as an efflux pump for fluconazole, cerulenin, brefeldin A, terbinafine, 4-nitroquinoline-N-oxide and phenanthroline.17 To examine whether the function of either or both of these two efflux pump specificities were enhanced in the TIMM isolates, some structurally unrelated efflux pump substrates were chosen and their growth inhibitory activities against the TIMM isolates and the susceptible control strain were determined. The results are summarized in Table IVGo. MICs of rhodamine 6G, a Cdrp pump substrate, for the four TIMM isolates were two- to four-fold higher than the value for the susceptible strain. The MICs of cerulenin, brefeldin A and terbinafine, which are substrates for both Cdrp and CaMdr1p pumps, were also greater for the TIMM isolates compared with the susceptible strain. In contrast, MICs of two CaMdr1p pump substrates, 4-nitroquinoline-N-oxide and phenanthroline, for the TIMM isolates were almost equal to the corresponding values for the susceptible strain.


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Table IV. Growth-inhibitory activities of several Cdrp and/or CaMdr1p efflux pump substrates against a fluconazole-susceptible control C. albicans strain (ATCC 10231) and four isolates with reduced susceptibility to antifungal azoles
 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The incidence of OPC caused by fluconazole-resistant C. albicans in AIDS patients has created a problem in the management of these patients.1 The emergence of fluconazole-resistant C. albicans has also been observed among Japanese AIDS patients with OPC who have repeatedly received fluconazole.20,31 The mechanisms of fluconazole resistance have been studied extensively in resistant C. albicans strains, which were isolated predominantly from AIDS patients with OPC in the USA or several European countries.7,8,12,1517 However, no information is available on the mechanisms of fluconazole resistance for resistant strains isolated in Japan.

Therefore, we attempted to elucidate the fluconazole-resistance mechanisms using four C. albicans isolates with reduced susceptibility to fluconazole, TIMM 3163, 3164, 3165 and 3166, which were originally isolated by Oka from three Japanese AIDS patients with OPC in 1994.20 Pre-treatment isolates from these patients were not available. While it would have been interesting to know how matched pre-treatment isolates responded to fluconazole, our experience of examining commensal fluconazole-susceptible isolates with low MIC values is that they invariably demonstrate the characteristics shown by C. albicans ATCC 10231. While TIMM 3164 and 3166 were classified as susceptible to fluconazole, they had elevated MICs and it is recognized that a significant proportion (20%) of isolates with these MICs will cause treatment failure, even if fluconazole dosages exceed 100 mg/day. 27 The target of the antifungal fluconazole, and other azole antifungals, is known to be sterol 14{alpha}-demethylase (encoded by ERG11), a key enzyme in the fungal ergosterol synthetic pathway.4 This was confirmed to be the case with not only the susceptible control strain but also all of the four TIMM isolates, because the inhibitory activity of fluconazole against ergosterol synthesis by growing cells of these five strains correlated with that against their growth when assessed by the IC50 value. Compared with the fluconazole-susceptible control strain, all of the TIMM isolates tested showed 4.7- to 61.2-fold greater values of IC50 for ergosterol synthesis by growing cells. However, the extent of the inhibitory activity of fluconazole against ergosterol synthesis by cell-free extracts from the resistant isolates was markedly decreased, and the IC50 value for the TIMM isolates was not significantly different from the value for the susceptible control strain. Our preliminary data suggest, therefore, that Erg11p is not involved in the fluconazole resistance of the TIMM isolates, and that some other biological mechanism or combination of mechanisms is responsible for the fluconazole resistance in all of the four TIMM isolates.

The most common fluconazole resistance mechanism in C. albicans has been reported to be a reduced intracellular accumulation of the drug as a result of reduced influx and/or increased efflux.13,14 Recently, evidence has been presented that efflux of azole drugs from fungal cells is due to energy-dependent pumps.32,33 Several investigators have correlated increased expression of the CDR genes (specifically CDR1 and CDR2), which are members of the ABC superfamily, and the CaMDR1 gene of the MFS superfamily, with the development of fluconazole resistance.7,8,1517 We therefore used a comprehensive range of approaches to investigate the possibility that the fluconazole resistance of the clinical isolates was due to energy-dependent drug efflux. We measured the fluconazole accumulation by strains and determined the effect of energy supply on this accumulation. We investigated expression of the CDR family of drug pumps and of CaMDR1 by RT–PCR, dot blot analysis and immunodetection. We also characterized the nature of the drug resistance by measuring susceptibility to a range of toxic putative pump substrates.

Strains TIMM 3165 and TIMM 3166 demonstrated a typical ABC drug efflux pump phenotype. The rate and extent of fluconazole accumulation by these strains was much lower than that of the susceptible strain. This effect was potentiated by glucose but reversed by metabolic inhibitors and by starvation. The strains were resistant to a range of toxic ABC pump substrates, and overexpressed CDR1 and CDR2 mRNA and Cdr1p. Therefore it is likely that the fluconazole resistance of TIMM 3165 and TIMM 3166 is due to Cdr2p- and/or Cdr1p-mediated drug efflux.

The resistance phenotypes of TIMM 3163 and TIMM 3164 are more intriguing and may be due to novel mechanisms. Strain TIMM 3164 demonstrated energy-dependent drug efflux in a similar fashion to TIMM 3165 and TIMM 3166, but did not overexpress CDR1-4, CaMDR1 or ERG11. Furthermore, RT–PCR using sequences conserved between ABC efflux pumps failed to demonstrate expression of this class of transporter in these strains. Therefore, the fluconazole resistance in TIMM 3164 may be due to a novel energy-dependent efflux pump distantly related to CDR1. Strain TIMM 3163, in contrast, over-expressed CDR1 and CDR2 but did not show an energy-dependent efflux phenotype, and accumulated 66% as much fluconazole as susceptible strain ATCC 10231. This suggests that Cdr1p, although expressed, is not functional as has been suggested for the well-studied resistant C. albicans strain Darlington.16 Unlike Darlington, however, TIMM 3163 was not resistant to the polyene amphotericin B, and cell-free sterol biosynthesis was as susceptible to fluconazole as the control strain. These results indicate that the azole resistance of TIMM 3163 was not due to energy-dependent drug efflux or altered sterol biosynthesis, and so may be due to an entirely new mechanism. Clearly the resistance of this clinical isolate warrants further investigation.


    Acknowledgments
 
We thank Dr Shinichi Oka (International Medical Center of Japan) for supplying fluconazole-resistant C. albicans isolates. We also thank Dr Dominique Sanglard of Centre Hospitalier Universitaire Vaudois (Switzerland) for the kind gift of anti-C. albicans Cdr1p antibodies. M. N. and R. D. C. gratefully acknowledge financial support from the Health Research Council of New Zealand.


    Notes
 
* Tel: +81-45-545-3178; Fax: +81-45-541-2359; E-mail: Kazunori_Maebashi{at}meiji.co.jp Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Rex, J. H., Rinaldi, M. G. & Pfaller, M. A. (1995). Resistance of Candida species to fluconazole. Antimicrobial Agents and Chemotherapy 39, 1–8.[Free Full Text]

2 . Ghannoum, M. A., Rex, J. H. & Galgiani, J. N. (1996). Susceptibility testing of fungi: current status of correlation of in vitro data with clinical outcome. Journal of Clinical Microbiology 34, 489–95.[Abstract]

3 . White, T. C., Marr, K. A. & Bowden, R. A. (1998). Clinical, cellular, and molecular factors that contribute to antifungal drug resistance. Clinical Microbiology Reviews 11, 382–402.[Abstract/Free Full Text]

4 . Vanden Bossche, H., Marichal, P. & Odds, F. C. (1994). Molecular mechanisms of drug resistance in fungi. Trends in Microbiology 2, 393–400.[Medline]

5 . Vanden Bossche, H., Marichal, P., Gorrens, J., Bellens, D., Moereels, H. & Janssen, P. A. (1990). Mutation in cytochrome P-450-dependent 14{alpha}-demethylase results in decreased affinity for azole antifungals. Biochemical Society Transactions 18, 56–9.[ISI][Medline]

6 . Venkateswarlu, K., Denning, D. W., Manning, N. J. & Kelly, S. L. (1996). Comparison of D0870, a new triazole antifungal agent, to fluconazole for inhibition of Candida albicans cytochrome P-450 by using in vitro assays. Antimicrobial Agents and Chemotherapy 40,1382–6.[Abstract]

7 . White, T. C. (1997). Increased mRNA levels of ERG16, CDR, and MDR1 correlate with increases in azole resistance in Candida albicans isolates from a patient infected with human immunodeficiency virus. Antimicrobial Agents and Chemotherapy 41, 1482–7.[Abstract]

8 . Franz, R., Kelly, S. L., Lamb, D. C., Kelly, D. E., Ruhnke, M. & Morschhauser, J. (1998). Multiple molecular mechanisms contribute to a stepwise development of fluconazole resistance in clinical Candida albicans strains. Antimicrobial Agents and Chemotherapy 42, 3065–72.[Abstract/Free Full Text]

9 . Hitchcock, C. A., Barrett-Bee, K. J. & Russell, N. J. (1987). Inhibition of 14{alpha}-sterol demethylase activity in Candida albicans Darlington dose not correlate with resistance to azole. Journal of Medical and Veterinary Mycology 25, 329–33.[ISI][Medline]

10 . Howell, S. A., Mallet, A. I. & Noble, W. C. (1990). A comparison of the sterol content of multiple isolates of the Candida albicans Darlington strain with other clinically azole-sensitive and -resistant strains. Journal of Applied Bacteriology 69, 692–6.[ISI][Medline]

11 . Lamb, D. C., Kelly, D. E., Schunck, W. H., Shyadehi, A. Z., Akhtar, M., Lowe, D. J. et al. (1997). The mutation T315A in Candida albicans sterol 14{alpha}-demethylase causes reduced enzyme activity and fluconazole resistance through reduced affinity. Journal of Biological Chemistry 272, 5682–8.[Abstract/Free Full Text]

12 . Sanglard, D., Ischer, F., Koymans, L. & Bille, J. (1998). Amino acid substitutions in the cytochrome P-450 lanosterol 14{alpha}-demethylase (CYP51A1) from azole-resistant Candida albicans clinical isolates contribute to resistance to azole antifungal agents. Antimicrobial Agents and Chemotherapy 42, 241–53.[Abstract/Free Full Text]

13 . Ryley, J. F., Wilson, R. G. & Barrett-Bee, K. J. (1984). Azole resistance in Candida albicans. Sabouraudia 22, 53–63.[ISI][Medline]

14 . Venkateswarlu, K., Denning, D. W., Manning, N. J. & Kelly, S. L. (1995). Resistance to fluconazole in Candida albicans from AIDS patients correlated with reduced intracellular accumulation of drug. FEMS Microbiology Letters 131, 337–41.[ISI][Medline]

15 . Sanglard, D., Kuchler, K., Ischer, F., Pagani, J. L., Monod, M. & Bille, J. (1995). Mechanisms of resistance to azole antifungal agents in Candida albicans isolates from AIDS patients involve specific multidrug transporters. Antimicrobial Agents and Chemotherapy 39, 2378–86.[Abstract]

16 . Albertson, G. D., Niimi, M., Cannon, R. D. & Jenkinson, H. F. (1996). Multiple efflux mechanisms are involved in Candida albicans fluconazole resistance. Antimicrobial Agents and Chemotherapy 40, 2835–41.[Abstract]

17 . Sanglard, D., Ischer, F., Monod, M. & Bille, J. (1997). Cloning of Candida albicans genes conferring resistance of azole antifungal agents: characterization of CDR2, a new multidrug ABC transporter gene. Microbiology 143, 405–16.[Abstract]

18 . Prasad, R., De Wergifosse, P., Goffeau, A. & Balzi, E. (1995). Molecular cloning and characterization of a novel gene of Candida albicans, CDR1, conferring multiple resistance to drugs and antifungals. Current Genetics 27, 320–9.[ISI][Medline]

19 . Fling, M. E., Kopf, J., Tamarkin, A., Gorman, J. A., Smith, H. A. & Koltin, Y. (1991). Analysis of a Candida albicans gene that encodes a novel mechanism for resistance to benomyl and methotrexate. Molecular and General Genetics 227, 318–29.[Medline]

20 . Oka, S. (1994). Fungal infections in AIDS patients. Japanese Journal of Medical Mycology 35, 241–5.

21 . National Committee for Clinical Laboratory Standards. (1997). Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts: Approved Standard M27-A. NCCLS, Villanova, PA.

22 . Vanden Bossche, H., Marichal, P., Odds, F. C., Le Jeune, L. & Coene, M. C. (1992). Characterization of an azole-resistant Candida glabrata isolate. Antimicrobial Agents and Chemotherapy 36, 2602–10.[Abstract]

23 . Lowry, O. H., Resebrough, N. J., Farr, A. L. & Randall, R. J. (1951). Protein measurement with Folin phenol reagent. Journal of Biological Chemistry 193, 265–75.[Free Full Text]

24 . Brown, A. J. P. (1994). RNA extraction and mRNA quantitation in Candida albicans. In Molecular Biology of Pathogenic Fungi, (Maresca, B. & Kobayashi, G. S., Eds), pp. 127–31. Telos Press, New York.

25 . Kan, V. L. (1993). Polymerase chain reaction for the diagnosis of candidemia. Journal of Infectious Diseases 168, 779–83.[ISI][Medline]

26 . Monk, B. C., Kurtz, M. B., Marrinan, J. A. & Perlin, D. S. (1991). Cloning and characterization of the plasma membrane H+-ATPase from Candida albicans. Journal of Bacteriology 173, 6826–36.[ISI][Medline]

27 . Rex, J. H., Pfaller, M. A., Galgiani, J. N., Bartlett, M. S., EspinelIngroff, A., Ghannoum, M. A. et al. (1997). Development of interpretive breakpoints for antifungal susceptibility testing: conceptual framework and analysis of in vitro–in vivo correlation data for fluconazole, itraconazole, and Candida infections. Subcommittee on Antifungal Susceptibility Testing of the National Committee for Clinical Laboratory Standards. Clinical Infectious Diseases 24, 235–47.[ISI][Medline]

28 . Balan, I., Alarco, A.-M. & Raymond, M. (1997). The Candida albicans CDR3 gene codes for an opaque-phase ABC transporter. Journal of Bacteriology 179, 7210–8.[Abstract]

29 . Franz, R., Michel, S. & Morschhauser, J. (1998). A fourth gene from the Candida albicans CDR family of ABC transporters. Gene 220, 91–8.[ISI][Medline]

30 . Talibi, D. & Raymond, M. (1999). Isolation of a putative Candida albicans transcriptional regulator involved in pleiotropic drug resistance by functional complementation of a pdr1 pdr3 mutation in Saccharomyces cerevisiae. Journal of Bacteriology 181, 231–40.[Abstract/Free Full Text]

31 . Sudo, T., Makimura, K., Kawata, K., Ito, A., Oka, S., Uchida, K. et al. (1997). Evaluation of antifungal susceptibility testing by the broth microdilution method against Candida species: activities of 5 antifungal agents against Candida species isolated from oral candidiasis or other candidal infectious diseases and correlation of vitro data and clinical outcome of fluconazole therapy. Japanese Journal of Chemotherapy 45, 115–22.

32 . Clark, F. S., Parkinson, T., Hitchcock, C. A. & Gow, N. A. (1996). Correlation between rhodamine 123 accumulation and azole sensitivity in Candida species: possible role for drug efflux in drug resistance. Antimicrobial Agents and Chemotherapy 40, 419–25.[Abstract]

33 . Odds, F. C. (1996). Resistance of clinically important yeasts to antifungal agents. International Journal of Antimicrobial Agents 6, 145–7.[ISI]

Received 31 July 2000; returned 4 October 2000; revised 14 November 2000; accepted 2 January 2001