Novartis Research Institute, Brunner Strasse 59, A-1235 Vienna, Austria1
Author for correspondence: Neil S. Ryder. Tel: +43 1 86 634 324. Fax: +43 1 86 634 354. e-mail: neil.ryder{at}pharma.novartis.com
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ABSTRACT |
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Keywords: azoles, fluconazole resistance, Candida albicans, lanosterol 14-demethylase
Abbreviations: 14DM, lanosterol 14-demethylase
a Present address: Department of Dermatology, Hôpital Cantonal Universitaire de Genève, 24 Rue Micheli-du-Crest, CH-1211 Geneva 14, Switzerland
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INTRODUCTION |
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The prolonged and repeated periods of treatment needed by AIDS patients have led to the increasing occurrence of candidiasis resistant to fluconazole therapy. Drug susceptibility testing of C. albicans isolated from these patients revealed in many cases that the organism had acquired resistance to fluconazole (Denning et al., 1997 ; Law et al., 1994
; Rex et al., 1995
; Tumbarello et al., 1996
; White et al., 1998
). Several mechanisms, independent of 14DM, have been reported which could contribute at least partially to fluconazole resistance in Candida species, including increased ergosterol biosynthesis (Vanden Bossche et al., 1992
), a lesion in
5,6 sterol desaturase (Kelly et al., 1997
), and lower intracellular accumulation of the drug (Marichal et al., 1995
; Parkinson et al., 1995
; Sanglard et al., 1995
; Vanden Bossche et al., 1992
; Venkateswarlu et al., 1996
, 1997
). This last mechanism seems to be the most common and is associated with the overexpression of two types of multidrug transporters, the ATP-binding cassette (ABC) transporters (Albertson et al., 1996
; Miyazaki et al., 1998
; Moran et al., 1998
; Prasad et al., 1995
; Sanglard et al., 1995
, 1996
, 1997
; van Veen & Konings, 1998
; White, 1997a
) and the major facilitators (Albertson et al., 1996
; Goldway et al., 1995
; Moran et al., 1998
; Sanglard et al., 1995
, 1996
; White, 1997a
). Recently, additional C. albicans genes able to directly or indirectly confer resistance to fluconazole when expressed in Saccharomyces cerevisiae have been identified (Alarco et al., 1997
; Sanglard et al., 1997
).
An obvious mechanism for resistance to azoles would be alteration of their primary target, 14DM. Based on indirect biochemical evidence, both overexpression and mutation of 14DM have been suggested to explain acquired resistance to azoles in several fungi (Ishida et al., 1988 ; Joseph-Horne et al., 1995
; Lamb et al., 1995
; Orozco et al., 1998
; Vanden Bossche et al., 1990
, 1992
, 1994
; Venkateswarlu et al., 1997
; White, 1997a
, b
). In S. cerevisiae, overexpression of 14DM encoded by ERG11 leads to resistance to azoles, but the amplitude of resistance is rather weak, not exceeding fivefold (Doignon et al., 1993
; Kalb et al., 1986
; Kelly et al., 1993
). Several studies have supported the concept that azole resistance can be due to mutation(s) in 14DM (Lamb et al., 1997
; Loeffler et al., 1997
; White, 1997b
). Substitution of the highly conserved residue T315A in the C. albicans 14DM gene (CaERG11, also called ERG16 and CYP51A1) conferred a fivefold resistance to azoles when this gene was expressed in S. cerevisiae (Lamb et al., 1997
). Sequencing of CaERG11 from a number of clinical isolates revealed the existence of numerous amino acid substitutions in the gene amplified from resistant isolates, suggesting a possible link with the resistant phenotype (Loeffler et al., 1997
). Lower sensitivity of sterol biosynthesis to fluconazole in cell-free extracts from a fluconazole-resistant C. albicans isolate correlates with the amino acid substitution R467L in CaERG11 at both loci in the diploid genome resulting from a loss of allelic variation (White, 1997b
). Very recently, Sanglard et al. (1998)
have formally demonstrated that several mutations in CaERG11 can confer azole resistance upon S. cerevisiae. Interestingly, a single point mutation does not seem to drastically affect azole sensitivity of CaERG11 but combinations of point mutations, principally G129A with G464S, Y132H with S405F, or Y132H with G484S, have cooperative effects (Sanglard et al., 1998
).
The present study was performed as part of an ongoing project to develop novel approaches to overcoming fungal drug resistance. Using a collection of fluconazole-resistant clinical isolates, we first identified potentially altered 14DM by measuring sensitivity to azole of sterol synthesis in cell-free extracts. Cloning and sequencing of CaERG11 from the four isolates from which cell-free sterol synthesis was insensitive to fluconazole revealed the presence of several amino acid substitutions in each of them. Five of the 12 identified amino acid substitutions have not been previously reported and all their combinations are novel. Finally, we show that expression of altered CaERG11 in S. cerevisiae resulted in increased resistance to fluconazole and, to a variable extent, also to ketoconazole.
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METHODS |
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MIC determinations.
Fluconazole, itraconazole and ketoconazole were prepared by Novartis Pharmaceuticals. Drug susceptibility testing of Candida species was performed in a broth macrodilution assay slightly modified from the NCCLS system (Galgiani et al., 1995 ), as previously described (Ryder et al., 1998
). Drugs were prepared according to the recommendations of the NCCLS method. MIC was defined as the lowest drug concentration causing 80% inhibition of fungal growth. The assay was validated with the standard drugs fluconazole, amphotericin B (Bristol-Myers Squibb) and 5-fluorocytosine (Sigma) in three reference strains of C. albicans: ATCC 90028, ATCC 90029 and ATCC 24433 (Galgiani et al., 1995
). The reference strains of C. krusei (ATCC 6258) and C. parapsilosis (ATCC 22019) were used to validate the assay with itraconazole and ketoconazole (Rex et al., 1996
). To ensure reproducibility, strain ATCC 24433 was run in parallel with each set of assays; MICs with this strain never varied by more than one dilution.
A microdilution assay using a 96-well plate with a flat bottom and low-evaporation lid (Costar) was developed to test drug susceptibility of S. cerevisiae. Under the same conditions, the micro assay gave similar results to those initially obtained with the macro assay. Briefly, to 200 µl per well of a cell suspension at 2·5x103 c.f.u. ml-1, prepared in the same medium as for the macro assay but supplemented with 1·8% glucose (final concn 2%) and 0·2 mg adenine ml-1 (as well as 0·2 mg uracil ml-1 for untransformed cells), 2 µl of a serial twofold dilution of fluconazole or ketoconazole in DMSO was added. The plate was shaken for 10 min at 900 r.p.m. and incubated for 48 h at 32 °C in maximum relative humidity. Growth was monitored spectrophotometrically at 620 nm with a Digiscan microtitre plate reader (Asys Hitech) after having shaken the plates for 30 min at 900 r.p.m. MICs were defined as the minimal drug concentration reducing growth by 80%, in comparison with solvent controls.
Sterol biosynthesis assay in whole cells or cell-free extracts.
Whole-cell ergosterol biosynthesis was measured by incorporation of [1,2-14C]acetate into nonsaponifiable lipids as described previously (Ryder, 1985 ; Ryder et al., 1984
). Sterol synthesis in cell-free extracts, prepared as in Favre & Ryder (1997b
) and Ryder (1987)
, was assayed in a final volume of 500 µl, consisting of 50 mM sodium/potassium phosphate pH 7·4, 0·5 mM DTT, 1 mM NAD, 1 mM NADP, 0·1 mM FAD, 3 mM glucose 6-phosphate, 5 mM ATP, 3 mM glutathione, 2·7 mM MnCl2, 4 mM MgCl2 and 0·5 µCi (1·85x104 Bq) (RS)-[2-14C]mevalonate, supplemented with 1·25 mg protein from cell-free extracts and the appropriate concentration of antimycotic, added from a 50-fold concentrated stock prepared in DMSO. After 2 h incubation at 30 °C in a shaking water bath, reactions were terminated by the addition of 1 ml 15% KOH in 90% ethanol. Nonsaponifiable lipids were extracted and analysed by TLC (Ryder, 1985
; Ryder et al., 1984
); the 14C-radioactivity present in each fraction was measured with an Instant Imager (Packard). Reproducibility of extraction was regularly checked by adding 15000 d.p.m. of [1
,2
(n)-3H]cholesterol, as internal standard, cutting the region corresponding to 4-desmethyl sterols and measuring the 3H-radioactivity by scintillation counting (Favre & Ryder, 1996
). IC50 values were calculated from a sterol biosynthesis inhibition curve determined with at least four different concentrations of the antimycotics.
Cloning and sequencing of CaERG11.
Genomic DNA from C. albicans was isolated according to Philippsen et al. (1991) . PCR amplification of CaERG11 from genomic DNA was performed with the forward primer 5'-CTCGGATCCTATATATATAAGTTTCTTTTCAAGAAGATCATAACTCAAT-3' and the reverse primer 5'-CCGAATTCATCAGAACACTGAATCGAAAGAAAGTTGCCGTTTTA-3', including a BamHI and an EcoRI site, respectively. PCR was conducted using the polymerases mixture Expand High Fidelity (Boehringer Mannheim) and an annealing temperature of 55 °C under standard conditions. For direct sequencing, the PCR amplicons were purified with the PCR product purification kit from Qiagen. For cloning, PCR products were digested with BamHI and EcoRI and ligated into the vector pBS+ (Stratagene). Sequencing on both strands of two independent clones or PCR amplicons was performed with a Li-Cor automatic sequencer, model 4000, using primers labelled with the infrared dye IRD40 (MWG Biotech) and Thermosequenase (Amersham).
Expression of c-myc-tagged CaERG11 in S. cerevisiae.
An NcoI site containing the translational initation codon was introduced at the 5' end, whereas a c-myc tag sequence and an XbaI site were inserted at the 3' end of CaERG11 ORF by two PCR amplifications from the cloned genes in pBS+, using the forward primer 5'-AACGTGCCATGGCTATTGTTGAAACTGTCATTG-3' and sequentially the following reverse primers: (1) 5'-CTTCAGAAATAAGTTTTTGTTCAAACATACAAGTTTCTCTTTTTTCC-3' and (2) 5'-GGTCTAGATTATTAAAGATCTTCTTCAGAAATAAGTTTTTGTTC-3'. A similar strategy was applied to introduce the same c-myc sequence at the 5' end of CaERG11 ORF, between the translational initiation codon ATG and the triplet encoding the second amino acid. The promoter of S. cerevisiae ERG11 was PCR-amplified from genomic DNA isolated from the strain HRI with the following forward and reverse primers, which contain an XbaI and an NcoI site, respectively: 5'-CCTCTAGAGACAATACAACCAACCTTCCTCC-3' and 5'-CTTGGTAGCAGCCATGGTTGTATT ACTCGTTTG-3'. The identity of the amplified fragment of approximately 1 kbp including the ERG11 promoter (accession no. U10555) was confirmed by restriction analysis and partial sequencing. The terminator of S. cerevisiae CYC1 was amplified by PCR from the plasmid pYES2 (Invitrogen) with the forward and reverse primers, which contain an XbaI and a XhoI site, respectively: 5'-ATGCATCTAGAGGGCCGCATC-3' and 5 '-CACCTGCTCGAGGGCCGCAAATTAAAGCCTT CGA-3'. Amplified CaERG11 genes, digested with NcoI and XbaI, and the CYC1 terminator, digested with XbaI and XhoI, were subcloned together into the plasmid Litmus 28 (BioLabs). Finally, the ERG11 promoter, digested with XbaI and NcoI, and the previous insert in Litmus 28, isolated by digesting the plasmid with NcoI and XhoI, were cloned into the vector pRS416 (Stratagene). For each final construct of c-myc-tagged CaERG11 in pRS416, the ORF was completely sequenced to make sure that no mutation had been introduced by the PCR amplifications and the various cloning steps.
The triplet 787CTG in CaERG11 ORF (encoding Ser in C. albicans: Santos & Tuite, 1995 ) was replaced by the triplet TCT with the QuickChange site-directed mutagenesis kit (Stratagene) using the primer 5'-CTTATATGAAAGAAATTAAATCTAGAAGAGAACTGTGTG-3' and its reversed, complementary primer, which contain an XbaI site. To preserve the amino acid substitution E266D in CaERG11 from the strain NFI 2007, the same primers as above, except for the substitution A32
C, were used, whereas the standard primers were applied to suppress the alteration. After site-directed mutagenesis, CaERG11 ORFs were again completely sequenced to confirm the replacement of 787CTG by TCT (identified by restriction analysis with XbaI) and to ensure that no other mutations had been introduced.
S. cerevisiae INVSC2 was transformed with pRS416 containing or not containing CaERG11 using the lithium acetate method (Becker & Guarente, 1991 ). Two colonies from each transformation were isolated and independently analysed.
Antibodies and Western blotting.
Whole-cell extracts were prepared according to Favre & Ryder (1997a ). Cell lysates from S. cerevisiae transformants were prepared from cultures grown in conditions as similar as possible to those for MIC determinations (medium, temperature and cell density) in 250 ml Erlenmeyer flasks containing 100 ml medium for 24 h without agitation, harvested by centrifugation, washed with phosphate-buffered saline and either immediately processed or frozen at -70 °C. Protein concentration was determined with the Bradford reagent and detergent-compatible (DC) protein assay (Bio-Rad), for samples without and with detergents, respectively. Western blot analysis with the monoclonal anti-c-myc antibodies (clone 9E10, Boehringer Mannheim), 10 µg ml-1, was performed as previously described (Favre & Ryder, 1997a
). Membranes were exposed to Hyperfilm ECL (Amersham).
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RESULTS |
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Biological and biochemical characterization of four selected azole-resistant isolates
Susceptibility of the four selected fluconazole-resistant strains to several azoles was measured and compared with that of a set of four control strains, known to be susceptible to these drugs (NFI 0124, ATCC 90028, ATCC 90029 and ATCC 24433). Most of the fluconazole-resistant strains showed some degree of cross-resistance to ketoconazole (especially, as expected, the Darlington strain) and itraconazole, although to various extents (Table 1).
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S. cerevisiae INVSC2 was transformed with the vector containing CaERG11, or with vector alone, and then tested for susceptibility to fluconazole and ketoconazole (this strain was primarily resistant to itraconazole) (Table 5). Expression of CaERG11 from the Darlington strain conferred an increased resistance to fluconazole in comparison with that from NFI 0124. However, the position of the c-myc tag influenced the susceptibility of the transformants to both fluconazole and ketoconazole whatever the source of CaERG11 (Table 5
). Western blotting with anti-c-myc tag antibody revealed that this variation in susceptibility correlated with the apparent level of expression of 14DM. Proteins tagged at the carboxyl (C) terminus were more abundant than those tagged at the amino (N) terminus (Fig. 1
; compare lanes 27 with lanes 8 and 9). The c-myc tag at the N-terminus might interfere with the targeting of 14DM to the membrane of the endoplasmic reticulum, which depends on a transmembrane domain and also probably on clusters of positively charged residues, both situated toward the N-terminus of P450 proteins (Boscott & Grant, 1994
; Chen et al., 1988
; Sanglard et al., 1993
). CaERG11 from NFI 2007 and NFI 2021 were therefore exclusively tagged at the C-terminus. A cryptic alteration of NFI 2013 CaERG11 introduced an XbaI site at position 409 of the ORF (data not shown), precluding its cloning into the expression vector, which was based on a strategy involving two digestions with XbaI. Since codon CUG is decoded as Ser in C. albicans instead of Leu (Santos & Tuite, 1995
), expression of CaERG11 in S. cerevisiae introduces the substitution S263L. To ensure that this would not influence the sensitivity of 14DM to inhibitors, the triplet 787CTG was systematically replaced by TCT (a Ser codon) in CaERG11 tagged at the 3' end. Results obtained with these last plasmids are presented in Table 5
. The substitution S263L in CaERG11 gene from the sensitive strain NFI 0124 resulted in a slight decrease (one step dilution) in fluconazole and ketoconazole resistance. Transformants expressing the CaERG11 genes from fluconazole-resistant strains were 32-fold more resistant to fluconazole than transformants expressing CaERG11 from the sensitive strain (Table 5
). The identity of residue 263, Ser or Leu, did not affect MICs to fluconazole of transformants expressing the CaERG11 genes from resistant strains (data not shown). The amino acid substitutions present in the Darlington CaERG11 gene simultaneously conferred a potent cross-resistance to ketoconazole (16-fold more than the control), in the other cases cross-resistance to ketoconazole was weak (4-fold) (Table 5
). Transformants expressing NFI 2007 CaERG11 without the amino acid substitution E266D were indistinguishable from those expressing CaERG11 containing all four amino acid substitutions identified in this gene (not shown).
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DISCUSSION |
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Sixteen different mutations leading to amino acid substitutions have previously been identified in CaERG11 from fluconazole-resistant isolates (Loeffler et al., 1997 ; Sanglard et al., 1998
; White, 1997b
). We report here five new, previously undescribed substitutions, to give a total of 21 which are now known. This multiplicity of mutations in CaERG11 potentially leading to fluconazole resistance, and their widespread distribution along the primary structure, might be surprising at first glance. However, a molecular model for C. albicans 14DM has been derived from the crystallographic structure of bacterial (Pseudomonas putida) soluble P450 camphor hydroxylase (P450cam) (Boscott & Grant, 1994
; Poulos et al., 1987
) (see also Loeffler et al., 1997
; Sanglard et al., 1998
). The active site of P450 proteins is buried in the core of the protein and is constituted by a complex three-dimensional arrangement of various secondary structures involving several parts of the linear protein sequence. It is thus not surprising that several amino acid substitutions along the primary structure of 14DM can affect the affinity of azoles, which act as competitive inhibitors by binding to the active site.
Fourteen of these 21 known amino acid substitutions involve residues which are conserved in all the yeast 14DM genes which have been sequenced so far. Six of these are located in a segment spanning residues 126147, three are clustered at positions 448450, and two are in the proximity of the thiolate (5th) ligand of the haem (Cys470) (Fig. 2). Amino acid substitutions from position 126 to 147 are close to or part of the entrance port for the substrate leading to the active site. For instance, Lys143, substituted by Glu in CaERG11 from isolate NFI 2021, is thought to be important for membrane binding and the correct entrance of the substrate (Boscott & Grant, 1994
; Loeffler et al., 1997
). Amino acid substitutions between residues 405 to 450 are located in the helix-poor domain connecting helices K to L, which are involved in the substrate-binding site in P450cam (Boscott & Grant, 1994
; Poulos et al., 1987
). Surprisingly, despite the selectivity of fluconazole for fungal versus mammalian 14DM, 10 out of the 14 residues substituted in CaERG11 from azole-resistant isolates are conserved in both fungal and mammalian 14DMs (Fig. 2
).
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Mutations in CaERG11 from strains Darlington, NFI 2007 and NFI 2013 negatively affected not only the affinity for azoles but also the enzymic activity (Table 2), as expected from the competitive inhibition of 14DM by azoles. Therefore, the apparent lack of effect on 14DM activity of the new amino acid substitutions identified in CaERG11 from NFI 2021, K143E and T229A is intriguing, although a similar phenomenon has been suggested to occur in azole-resistant Ustilago maydis strains (Joseph-Horne et al., 1995
). It is conceivable that these mutations somehow affect the binding of fluconazole without influencing enzyme activity, since there was no indication of any abnormal accumulation of ergosterol pathway intermediates in NFI 2021 (Table 3
). An alternative explanation is that reduced substrate binding of 14DM is compensated by an up-regulation of 14DM and possibly other enzymes in the pathway. In support of the latter explanation, evidence has been found for overexpression of both 14DM and squalene epoxidase in this strain (authors unpublished data), which would be compatible with the normal profile seen in Table 3
. The lack of marked cross-resistance to itraconazole and ketoconazole in NFI 2021, together with the similar extent of reduction in sensitivity to azoles in the cell-free and whole-cell ergosterol biosynthesis assays (Table 2
), suggests that resistance in this strain may be accounted for solely by these changes in the ergosterol pathway.
Amino acid substitutions in CaERG11 probably do not constitute the sole mechanism of resistance to antimycotics present in any of the four strains examined. In contrast to NFI 2021 discussed above, strains Darlington, NFI 2007 and NFI 2013 show cross-resistance to other azoles and a more marked reduction of sensitivity of ergosterol biosynthesis in whole cells than in cell-free assays (Table 2). For example, NFI 2013 is strongly resistant to itraconazole but its sterol synthesis in cell-free extracts is very sensitive to the same azole. Therefore, these three strains probably also express one or more transporters which reduce the intracellular concentration of the antimycotics. In support of this, reduced cellular accumulation of fluconazole was observed in strains Darlington, NFI 2007 and NFI 2013, but not in NFI 2021 (authors unpublished data). A similar diversity of resistance mechanisms expressed in a single isolate has previously been observed (Lopez-Ribot et al., 1998
; Sanglard et al., 1998
; White, 1997a
).
In conclusion, azole resistance in C. albicans is a complex phenomenon which can involve several different mechanisms. In addition to overexpression of the target enzyme or drug transporters, which have been widely reported, mutations in CaERG11 can significantly contribute to a clinically occurring resistant phenotype induced by long-term treatment of patients with azoles. Our data suggest that amino acid substitutions in 14DM may contribute to resistance in about 20% of fluconazole-resistant C. albicans strains. The diversity of the mutations able to influence the affinity of 14DM for azoles precludes any simple analysis to detect them. Moreover, selection of more than one resistance mechanism by prolonged treatment with azoles also seems to be very common (Lopez-Ribot et al., 1998 ; Sanglard et al., 1998
; White, 1997a
), excluding a simple therapeutic approach to combat azole resistance. Combination of antimycotics with different modes of action, for example azole and allylamine (Barchiesi et al., 1998
; Ryder & Favre, 1997
), is one potential approach to this problem.
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ACKNOWLEDGEMENTS |
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Received 8 February 1999;
revised 21 May 1999;
accepted 28 May 1999.