Institut de Microbiologie, Centre Hospitalier Universitaire Vaudois (CHUV), Rue de Bugnon, CH-1011 Lausanne, Switzerland1
Author for correspondence: Dominique Sanglard. Tel: +41 21 314 40 83. Fax: +41 21 314 40 60. e-mail: Dominique.Sanglard{at}chuv.hospvd.ch
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ABSTRACT |
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Keywords: multidrug efflux transporters, azole antifungal agents, Candida albicans
Abbreviations: MF, major facilitator; MFS, major facilitator superfamily; OPC, oropharyngeal candidiasis
The GenBank accession number for the sequence reported in this paper is AF188621.
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INTRODUCTION |
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The repeated use of fluconazole to treat OPC episodes has led to the appearance of clinical resistance which had been often correlated with in vitro resistance (Odds et al., 1996 ; Rex et al., 1995
; Troillet et al., 1993
). It was demonstrated in a recent study investigating the mechanisms of resistance to fluconazole in C. albicans isolates that the majority of resistant C. albicans isolates failed to accumulate the levels of fluconazole measured in susceptible isolates. This phenomenon was due to an enhanced efflux of fluconazole. Multidrug efflux transporters of two different classes were up-regulated in C. albicans azole-resistant isolates, including ABC transporters and major facilitators (MFs), and were identified as possible mediators for this effect (Sanglard et al., 1995
). Each of the genes for these two transporters classes, CDR1 (Prasad et al., 1995
) and CaMDR1 (Fling et al., 1991
), were shown to be up-regulated in individual C. albicans isolates resistant to azole antifungal agents.
Additional mechanisms have been demonstrated to participate in azole resistance in C. albicans clinical isolates. These separate mechanisms involve alterations in the cellular target of azole antifungals (Sanglard et al., 1998 ) or alterations in the ergosterol biosynthetic pathway (Marichal & Vanden Bossche, 1995
). However, other mechanisms of azole resistance may be still found in clinical isolates. We therefore attempted the cloning of azole resistance genes putatively involved in the resistance of clinical isolates by a functional complementation strategy. The screening of a C. albicans genome library in a Saccharomyces cerevisiae mutant lacking the ABC transporter gene PDR5 allowed the cloning of several azole resistance genes. Previously isolated genes (CDR1 and CaMDR1) (Fling et al., 1991
; Prasad et al., 1995
) were found in this screening. A new ABC transporter gene, CDR2, was also isolated and was shown to be co-ordinately up-regulated with CDR1 in several C. albicans azole-resistant clinical isolates (Sanglard et al., 1997
). Three additional genes were also cloned, among them ERG11 (encoding the cytochrome P450 lanosterol 14
-demethylase), FLU1 and FLU2, the latter now referred to as CAP1 and encoding a transcription factor (Alarco et al., 1997
; Sanglard et al., 1997
). Here we report the characterization of FLU1 and show that it encodes a new multidrug efflux transporter with similarity to the MF transporters.
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METHODS |
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Accumulation of [3H]fluconazole in C. albicans isolates.
Fluconazole accumulation experiments in C. albicans were performed with 3H-labelled fluconazole (Amersham) as described previously (Sanglard et al., 1995 ), except that a single 20 min incubation was used. Each [3H]fluconazole accumulation experiment was repeated twice.
Construction of plasmids.
For the disruption of FLU1 in C. albicans, a 2·7 kb SphIBglII fragment from pDS255 (Sanglard et al., 1997 ) was subcloned into pMTL21 to yield pDIS1. A blunt-ended 3·7 kb SalIBglII fragment from pMB-7 (Fonzi & Irwin, 1993
) containing the hisG-URA3-hisG disruption cassette was inserted in the single SnaBI site of pDIS1 to create pDIS2. The linear 6·4 kb SphIBglII fragment from pDIS2 was used for FLU1 disruption experiments.
Transformation of C. albicans and S. cerevisiae.
C. albicans CAF4-2 and other related strains were transformed by a LiAc procedure developed by Sanglard et al. (1996 ). After transformation with linear DNA fragments the cells were plated in YNB selective medium and incubated for 23 d at 30 °C. S. cerevisiae YKKB-13 was also transformed with plasmids by the same method, except that incubation time on selective medium was 34 d.
Northern blotting and signal quantification.
Total RNA from yeasts was extracted and electrophoresed following the method described by Sanglard et al. (1995 ). Transfer of RNA was performed by capillary action on GeneScreen Plus membranes (NEN). Membranes were pre-hybridized at 42 °C, with a buffer consisting of 50% formamide, 1% SDS, 4x SSC (1x SSC is 0·15 M sodium chloride, 0·015 M sodium citrate), 10% dextran sulfate and 100 µg salmon sperm DNA ml-1. DNA probes were labelled with [
-32P]dATP by random priming (Feinberg & Vogelstein, 1984
) and added to the hybridization solution overnight at 42 °C. Washing steps were at high stringency, identical to those recommended by the supplier (NEN). The TEF3 mRNAs were analysed using a 0·7 kb EcoRIPstI fragment from pDC1 as described by Hube et al. (1994
). Probes were stripped off in sequential hybridizations by boiling membranes for 10 min in TE buffer with 0·1% SDS.
Southern blotting.
Genomic DNA from C. albicans strains was isolated from 5 ml cultures grown overnight in YEPD medium. Cells were collected by centrifugation and washed twice in TE. Pellets were resuspended in 5 ml PRO-Buffer (1 M sorbitol, 25 mM EDTA, 20 mM Tris/HCl, pH 7·5) and 50 µg 100T Zymolyase ml-1 (Seikagaku) and 0·1% ß-mercaptoethanol (Sigma) were added. The mixture was incubated at 37 °C until complete cell wall digestion occurred (up to 30 min). After centrifugation the cell pellets were slowly resuspended in 2 ml lysing solution (0·1 M EDTA, 0·8% SDS, 50 µg ml-1 proteinase K, 0·1 M Tris/HCl, pH 7·5) and were incubated on ice for 10 min. After centrifugation (10 min at 5500 r.p.m.), supernatants were transferred into new tubes and DNA was precipitated with 5 ml ethanol. DNA pellets were gently resuspended in 0·5 ml TE containing 100 µg RNase A ml-1 (Roche) and incubated at 37 °C for 15 min. DNA from each culture was then precipitated with 2-propanol, transferred into Eppendorf tubes, washed with 70% ethanol and finally resuspended in TE. DNA was digested by restriction enzymes and size-fractionated by 1 % agarose gel electrophoresis. The digested DNA was vacuum-blotted on Gene Screen Plus membranes. Prehybridization and hybridization of the membrane with labelled DNA probe were performed at 42 °C in a solution containing 50% formamide, 5x SSC, 1% SDS and 100 µg denatured salmon sperm DNA ml-1. The probe was prepared by random priming as described above. The membrane was washed as recommended by the manufacturer and exposed at -70 °C on a Fuji X-ray film with an intensifying screen.
PCR amplifications.
PCR buffers and Taq polymerase were from Boehringer Mannheim. Briefly, PCR was carried out in a Thermal Cycler 480 (Perkin Elmer) with a first cycle of denaturation for 4 min at 94 °C followed by 30 cycles of annealing at 54 °C for 2 min, elongation at 72 °C for 2 min and denaturation at 94 °C for 30 s. PCR was completed by a final elongation step at 72 °C for 10 min. Primers for PCR are described in the legend to Fig. 4. Yeast DNA templates for PCR were prepared from overnight cultures by mechanical breakage with glass beads as described previously (Sanglard et al., 1996
).
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RESULTS |
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The C. albicans DNA insert of pDS255 was subcloned into different fragments. As summarized in Fig. 1, a minimal fragment of 4200 bp in pDS255-7 was still able to confer resistance to fluconazole and cycloheximide, but not to other azole derivatives such as itraconazole and ketoconazole. The nucleotide sequence of this fragment was determined and an uninterrupted ORF of 1833 bp, starting from the most upstream ATG codon, was detected. The gene encoding this ORF was named FLU1 (fluconazole resistance). The 5' flanking region starting from this ATG codon displayed the typical structural organization of yeast promoters: an A at position -3 and a TATA box consisting of two overlapping TATA consensus sequences (TATATA and TATAAA) at position -127 (Chen & Struhl, 1988
). A+T-rich regions (80% between -236 and -1, and 95% in a 28 bp tract between -109 and -82) were also observed and could serve as upstream promoter elements for the constitutive expression of FLU1, as suggested by Struhl et al. (1985
). Chromosomal mapping of FLU1 revealed its presence on chromosome 7 of the C. albicans genome. FLU1 is situated on the published physical map in a location hybridizing to fosmid 11H9 (Chibana et al., 1998
).
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Expression of FLU1 in C. albicans clinical isolates
Several matched pairs of C. albicans clinical isolates were selected to address the possible expression of FLU1 in C. albicans. Six isolates were taken from a previously published study (Sanglard et al., 1995 ) and six additional isolates were from separate patients. Each pair contained an azole-susceptible and an azole-resistant isolate. The susceptibility to azole derivatives of these 12 isolates is given in Table 2
. These clinical strains were sequential isolates from a group of HIV-positive patients and were characterized by different genotyping methods (Boerlin et al., 1996
). Each of these isolates was chosen on the basis of their already known azole resistance mechanisms. Total RNA from these isolates was extracted and subjected to Northern blotting analysis with labelled probes corresponding to the multidrug efflux transporters FLU1, CaMDR1, CDR1 and CDR2. The expression of these multidrug efflux transporter genes is presented in Fig. 2
. The azole-resistant isolates from three patients (II, III and V) showed up-regulation of CDR1, correlating well with a decrease in azole susceptibility. The expression of CDR2 mirrored the increased expression of CDR1 in agreement with previously published results (Sanglard et al., 1997
). The azole-resistant isolates from patients I, IV and VI showed up-regulation of CaMDR1 (Sanglard et al., 1995
). In contrast, FLU1 expression did not parallel azole resistance as illustrated in Fig. 2(b)
. FLU1 expression was higher in the azole-resistant isolate of one pair (isolate 742); however, FLU1 expression decreased in the azole-resistant isolate of a separate pair (isolate 91). In other pairs of isolates, no variation of FLU1 expression could be measured, although the basal level of expression differed between each pair. Taken together, these results suggested that FLU1, in contrast to CDR1, CDR2 and CaMDR1, was not up-regulated in azole-resistant strains, at least in the isolates investigated here.
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Accumulation of [3H]fluconazole in C. albicans
Since the expression of FLU1 in S. cerevisiae mediates resistance to fluconazole, therefore assigning fluconazole as a substrate for Flu1p, we addressed the accumulation of this azole in C. albicans flu1/
flu1 mutants. A similar experiment with the mutant DSY448 revealed a decrease in fluconazole accumulation compared to the wild-type (Sanglard et al., 1996
). However, a decrease in fluconazole accumulation could not be measured between
flu1/
flu1 mutants and the wild-type, even in the genetic background of multidrug transporter mutants (data not shown). This experiment suggests either a low efflux capacity of Flu1p for fluconazole or low expression levels of this transporter in C. albicans, thus making the measurement of accumulation differences between mutants and wild-type cells difficult.
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DISCUSSION |
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When expressed in S. cerevisiae, FLU1 mediated specific resistance to fluconazole among the different azole antifungal agents tested. The absence of cross-resistance to all three azole derivatives in S. cerevisiae already suggested that the protein encoded by FLU1 could be more related to the MF CaMdr1p than to the ABC transporters Cdr1p and Cdr2p, since the expression of CaMDR1 resulted in specific resistance to fluconazole (Sanglard et al., 1995 , 1997
). In fact, the sequencing of FLU1 revealed that this gene encoded a putative MF transporter. The structural organization of Flu1p was characteristic of MF efflux transporters. MFs have been grouped into three clusters (I, II and III) based on hydropathy analyses. As deduced from this analysis FLU1 belongs to cluster I, since this transporter has 12 putative transmembrane domains (Goffeau et al., 1997
). Further studies should confirm the localization of this protein to the cytoplasmic membrane. Flu1p has the highest similarity with YLL028wp from S. cerevisiae. This latter transporter might be therefore functional in the efflux of fluconazole or cycloheximide. We attempted to demonstrate this function for YLL028wp; however, although the gene encoding the protein could be expressed under the control of the ADHI promoter, no drug resistance phenotype was obtained (data not shown).
In our study, when FLU1 was up-regulated in the S. cerevisiae pdr5 mutant, it increased the resistance of this yeast only to fluconazole and cycloheximide (see Fig. 1a
). On the other hand, the disruption of FLU1 in C. albicans did not lead to an increased susceptibility to cycloheximide, but to a small increase in susceptibility to fluconazole. Disruption of FLU1 did not affect drug susceptibility to a great extent, except in the case of mycophenolic acid. A surprising effect resulting from the disruption of FLU1 was that a slight increase in susceptibility to all azoles was observed compared to the wild-type, whereas expression of FLU1 in S. cerevisiae could only confer resistance to fluconazole. The most likely explanation of these results could be that the absence of Flu1p in C. albicans affects either the expression or the activity of other transporters accepting other azoles (itraconazole or ketoconazole) as substrates. Taken together, the results of drug susceptibility assays reveal differences in two distinct approaches used to assign substrates to specific multidrug transporters. While the activity of transporters in C. albicans may be masked by the presence of other transporters responsible for intrinsic resistance of this yeast to various drugs and metabolic inhibitors, these activities may be better demonstrated using S. cerevisiae in which the PDR5 gene was deleted. Alternatively, the differences observed with the two experimental systems may be due to differences in subcellular localization of Flu1p between both yeast species or differences in the expression of FLU1 in both yeast species. We observed that in C. albicans, FLU1 mRNA signals were comparatively very low as opposed to CDR1 or TEF3. In S. cerevisiae, FLU1 was expressed on the support of a YEp24-derived multicopy plasmid. Therefore, expression levels of FLU1 may have been higher in S. cerevisiae and thus could influence the results of the susceptibility assays.
Some ORFs of cluster I MF transporters from different yeast species have known functions as multidrug resistance pumps. Car1p of Schizosaccharomyces pombe transports cycloheximide and amiloride (Jia et al., 1993 ), Cyhrp from Candida maltosa is specific for cycloheximide and hydrophobic drugs (Ben-Yaacov et al., 1994
), and CaMDR1 expression renders S. cerevisiae resistant to methotrexate, benomyl, cycloheximide, 4-nitroquinoline-N-oxide, sulfomethuron methyl, terbinafine, amorolfine, fluphenazine, 1,10-phenantroline, cerulenin and benzotriazoles (Ben-Yaacov et al., 1994
; Fling et al., 1991
; Sanglard et al., 1997
). In S. cerevisiae, several MF transporters have been characterized, being functional as multidrug resistant pumps: Atr1p transports aminotriazoles and 4-nitroquinoline-N-oxide (Gompel-Klein & Brendel, 1990
; Kanazawa et al., 1988
), Sge1p is specific for crystal violet and ethidium bromide (Amakasu et al., 1993
; Ehrenhofer-Murray et al., 1994
) and Flr1p, which is the functional homologue of CaMdr1p in S. cerevisiae, can take at least fluconazole and cycloheximide (Alarco et al., 1997
) or cerulenin (Oskouian & Saba, 1999
) as substrate. The expression of the S. cerevisiae MF gene YOR273c conferred resistance to the antimalarial drug quinidine (Delling et al., 1998
).
Even though FLU1 encodes a transporter for fluconazole, the expression of this gene in C. albicans clinical isolates could not be related to their degree of azole resistance. We observed some variations in the expression of FLU1 among the clinical isolates investigated in this study, the basis of which remains unknown. This is in contrast to CaMDR1, which is up-regulated from almost non-detectable levels to high levels in some azole-resistant isolates. Some indirect evidence could establish a link between the expression of CaMDR1 and CAP1, a recently cloned transcription factor with a basic leucine zipper motif similar to the S. cerevisiae YAP1 gene. CAP1 was shown to activate the transcription of the MF transporter FLR1 (Alarco et al., 1997 ), which is functionally similar to CaMDR1 in C. albicans. Interestingly, the CaMDR1 promoter contains YAP1-like DNA recognition elements: a sequence, 5'-TGACTCA-3', at position -737 to -731 upstream of the ATG codon, which is the optimal YAP1 binding site (Ellenberger et al., 1992
; Kim & Struhl, 1995
; Oliphant et al., 1989
) and three other sequences that differ at position ±2 from TGACTCA. Multidrug resistance in C. albicans resulting from up-regulation of CaMDR1 could therefore also be regulated by CAP1. FLU1 does not contain any conventional YAP1-like recognition element in its available promoter sequence and therefore its expression is not likely to be dependent on CAP1. These observations are in agreement with the differences in expression between CaMDR1 and FLU1 in clinical isolates.
In conclusion, we have shown here that a gene cloned in S. cerevisiae by functional complementation for a drug resistance phenotype was not coupled to azole resistance in C. albicans. Development of azole resistance can be explained in most azole-resistant strains by known alterations. However, among the growing number of characterized azole-resistant isolates, some of them still acquire resistance by unknown mechanisms. By using a prospective functional screening of C. albicans drug resistance genes in suitable yeast genetic backgrounds, these additional remaining azole resistance mechanisms may be revealed in the future.
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ACKNOWLEDGEMENTS |
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Received 31 March 2000;
revised 30 June 2000;
accepted 20 July 2000.