Department of Anti-infectives Research1, Center for Molecular Design2, Department of Biotechnology3 and Department of Functional Genomics4, Janssen Research Foundation, Turnhoutseweg 30, B2340 Beerse, Belgium
Department of Molecular Cell Biology and Genetics, University of Maastricht, The Netherlands5
Author for correspondence: Patrick Marichal. Tel: +32 14 60 31 97. Fax: +32 14 60 54 03. e-mail: pmaricha{at}janbe.jnj.com
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ABSTRACT |
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Keywords: itraconazole, fluconazole, resistance, Erg11p, modelling
The GenBank accession numbers for the sequences reported in this paper are AF153844AF153850.
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INTRODUCTION |
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Over the last 5 years multiple biochemical studies have been published to elucidate the underlying causes of azole resistance in pathogenic fungi. These studies resulted in a series of reviews dedicated to this subject (Bodey, 1997 ; De Muri & Hostetter, 1995
; Denning et al., 1997
; Dupont, 1995
; Frosco & Barrett, 1998
; Hartman & Sanglard, 1997
; Johnson & Warnock, 1995
; Joseph-Horn & Hollomon, 1997
; Marichal & Vanden Bossche, 1995
; Odds, 1998
; Rex et al., 1995
; Sanglard et al., 1998a
; Vanden Bossche, 1997
; Vanden Bossche et al., 1994
, 1998
; White et al., 1998
). Several mechanisms have been identified that contribute to azole resistance. Probably the most common mechanism is to effect a diminution in the concentration of active compound at the target site. In the majority of cases studied recently, this was the result of overexpression of efflux pumps. So far, two types of efflux transporters have been reported in resistant isolates. The ATP-binding-cassette type (ABC transporters; e.g. CDR1 and CDR2) can export a wide variety of azoles and unrelated chemicals (Kolaczkowski & Goffeau, 1997
), and the so-called major facilitators, (e.g. CaMDR1, previously described as BENr) with a much narrower substrate spectrum (Sanglard et al., 1995
).
The second way in which fungi achieve effective resistance to azoles is to circumvent or compensate for the toxic consequences of the changes in sterol composition. Azole-induced growth inhibition results from both the depletion of sterol molecules able to perform some sparking hormonal function (Rodriguez et al., 1985 ), e.g. ergosterol, and coincidental accumulation of membrane-disturbing 14-methylated precursors. As well as ergosterol, sufficient quantities of 14-methylfecosterol can also fulfil this sparking function. (Watson et al., 1989
). Accumulation of 14-methylfecosterol is achieved if cells are deficient in
8,7 isomerase and or
5,6 desaturase. This effect has been described in an azole-resistant C. albicans isolate (Kelly et al., 1997
) as well as in Saccharomyces cerevisiae (Bard et al., 1993
).
A third general type of mechanism for azole resistance involves changes at the level of the antifungal target. The primary target for the azole class of antifungals is the cytochrome-P450-catalysed 14-demethylation of ergosterol precursors. This enzyme is encoded by ERG11 (also described as ERG16, CYP51). Overexpression of this enzyme, induced either by enhanced transcription or by gene or chromosomal amplification, results in a decreased sensitivity for this class of antifungals (Marichal et al., 1997
; Doignon et al., 1993
). Point mutations in Erg11p [suggested by biochemical data (Vanden Bossche et al., 1990
) or by sequence analysis], such as Y132H (tyrosine 132 is replaced by a histidine; Sanglard et al., 1998b
), T315A (threonine 315 replaced by alanine; Lamb et al., 1997
) or R476K (arginine 476 replaced by lysine; White, 1997
), have been shown to decrease the affinity of the target for an azole. Numerous publications have listed other Erg11p mutations, but unfortunately the effect of the mutation on azole sensitivity was not always tested.
In this study, we sequenced the ERG11 gene from a selection of five azole-resistant isolates and two sensitive isolates and evaluated the effects of amino acid substitutions on subcellular sterol biosynthesis and azole sensitivity. Computer modelling and sequence analysis were used to position the mutations found in the 3D model available and to predict their possible importance for resistance.
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METHODS |
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MIC determination.
MICs were determined spectrophotometrically by a broth microdilution method (Odds et al., 1995 ) based on the NCCLS M27A protocol (National Committee for Clinical Laboratory Standards, 1995
). In brief, cells were inoculated in RPMI buffered to pH 7·0 with 1·65 M MOPS. Results were read after 48 h at 37 °C in a microplate reader (model 3550; Bio-Rad). The MIC was the lowest concentration that inhibited growth by more than 50%; this end point showed the best reproducibility and correlation with results from the NCCLS broth macrodilution method. Quality control yeasts Candida krusei ATCC 6258 and Candida parapsilosis ATCC 22019 were tested in parallel and were inhibited at MICs in the correct ranges for the antifungals tested (National Committee for Clinical Laboratory Standards, 1995
).
Sterol synthesis by subcellular fractions.
To prepare the subcellular fractions, the C. albicans isolates were grown for 8 h in static culture and then for 8 h in a reciprocating shaker set at 100 strokes min-1 in 200 ml PYG2 medium (l-1: 10 g polypeptone, 10 g yeast extract, 40 g glucose) in 500 ml Erlenmeyer flasks at 30 °C. This method yields cells in the late exponential phase. Cells were collected by centrifugation at 1500 g, washed and resuspended at a density of 109 cells ml-1 in cold homogenization buffer containing 0·1 M potassium phosphate buffer, 30 nM nicotinamide, 5 mM MgCl2, 5 mM reduced glutathione and 1 mM PMSF. Cells were broken in a 350 ml Bead-Beater receptacle (Biospec Products) filled with 200 g prechilled acid-washed glass beads (diam. 0·400·45 mm) by three cycles of 1 min agitation with intermittent cooling. To prevent heating of the samples, the outer jacket of the Bead-Beater was filled with ice-cold water. The homogenate was differentially centrifuged at 4 °C in a Sorvall SS34 rotor, first for 5 min at 1500 g and then for 20 min at 8000 g. The protein content of the S8000 supernatant fraction was measured according to the Bio-Rad method using bovine serum albumin as a standard (Bradford, 1976 ). To measure sterol biosynthesis, 900 µl of the resultant S8000 fraction was added to 100 µl cofactor buffer to achieve final concentrations of 1 µM NADP, 1 µM NAD, 3 µM glucose 6-phosphate, 5 µM ATP, 2 µM MnCl2, 3 µM MgCl2 and 0·25 µCi [14C]mevalonic acid. One microlitre of inhibitor stock solution and/or DMSO was added to the incubation tubes prior to the addition of the S8000 extract. Tubes were incubated for 2 h in an orbital New Brunswick shaker set at 300 r.p.m. at 30 °C. Reactions were stopped by the addition of 1 ml 15% KOH dissolved in 90% ethanol and were saponified for 1 h at 84 °C. Non-saponifiable lipids were extracted with 3 ml n-heptane. Heptane extracts were then dried with a stream of N2 and sterols were separated by TLC as described previously (Vanden Bossche et al., 1992
). All experiments were repeated at least three times with different cell extracts.
Spectrophotometric analysis of cytochrome P450.
Microsomes were isolated from C. albicans as described previously (Vanden Bossche et al., 1986 ). In summary, C. albicans cells, grown in semianaerobic conditions, were harvested by centrifugation, then washed and resuspended in ice-cold 0·65 M mannitol. All subsequent steps were done at 4 °C. PMSF was added to a concentration of 1 mM. Then the suspension was immediately homogenized in a Bead-Beater as described above. The homogenate was differentially centrifuged for 5 min at 1500 g, 20 min at 10000 g and 1 h at 100000 g. The pellet thus obtained was resuspended in 0·05 M potassium phosphate buffer containing 0·01 M EDTA (pH 7·4) and the suspension was recentrifuged for 1 h at 100000 g (Vanden Bossche et al., 1986
). The final pellet was resuspended in 0·1 M potassium phosphate buffer (pH 7·4). The P-450 content and the effects of azoles on the interaction of CO with the reduced haem iron of P-450 were measured as described previously (Vanden Bossche et al., 1986
).
PCR amplification and sequence analysis of the C. albicans ERG11 gene.
To amplify the ERG11 gene encoding the cytochrome P450 14-demethylase we used 5'-TAATACGACTCACTATAGGGAAGATCATAACTCAATATGGCTATTGTTG-3' composed of a 19 bp T7 sequence and nucleotides 131161 from the Lai & Kirsch (1989
; accession number X13296) sequence, including the first four codons of the ORF, as sense primer and 5'-ATTTAGGTGACACTATAGGAAAGTTGCCGTTTTATTAAAACATAC-3' composed of the SP6 sequence (18 bp) and the 17241750 region of the published sequence surrounding the stop codon of the ORF as antisense primer. Heat-activatable AmpliTaq Gold (0·5 units; Perkin Elmer) was used with 2·5 mM MgCl2. DNA from all C. albicans strains was prepared by the Qiagen DNA extraction method according to the procedures of the manufacturer with zymolyase (60 U; 5000 U g-1; Arthrobacter luteus; Seikagaku Kogyo) used as the cell-wall-degrading enzyme. The PCR parameters were 10 min at 94 °C to activate the polymerase and then 30 cycles of 1 min annealing at 60 °C, 2 min elongation at 72 °C and 1 min denaturation at 92 °C. After the reaction, the 1657 bp PCR product was purified with a Qiagen PCR cleanup kit and a sample was separated on a 1% agarose gel in 0·5x TBE with Boehringer molecular mass standard VI. The 1657 bp amplification products from the different isolates were sequenced on both strands using the PCR primers and internal primers every 300 bp as follows: (name, sequence, nucleotide numbering according to Lai & Kirsch,1989
; accession number X13296, direction) Ca ERG11-01, TTAGGTCCAAAAGGTC, 432, sense; Ca ERG11-02, CATGACCTTTTGGACC, 451, antisense; Ca ERG11-03, GACCGTTCATTTGCTC, 787, sense; Ca ERG11-04, GAGCAAATGAACGGTC, 802, antisense; Ca ERG11-05, ATTCTTATGGGTGGTC, 1057, sense; Ca ERG11-06, GCAGAAGTAGAAGCAG, 1097, antisense; Ca ERG11-07, TCTCCAGGTTATGCTC, 1360, sense; Ca ERG11-08, CCCATCTAGTTGGATC, 1439, antisense). Primers were designed by visual inspection of the sequence for stretches of 1618 nucleotides of normal composition (4060% GC, no palindromes, no homopolymeric stretches). Primers were ordered from Eurogentec (Seraing) and were synthesized according to the ß-cyanoethylphosphoramidite method. Sequencing reactions were performed with the ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction Kit, used according to the instructions of the manufacturer (Perkin-Elmer), except that half of the volume of terminator mix was replaced by HalfTerm (GenPak). Sequencing reactions were run on an Applied Biosystems 377 XL DNA sequencer (Perkin-Elmer).
Sequences were assembled from the individual runs into single contig sequences by means of Sequencher software (Gene Codes Corporation). Ambiguity positions were scored by setting the threshold as low as 30% (i.e. secondary peaks at 30% of primary peak results in ambiguity call) and by inspecting all of the ambiguity calls on all available readings.
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RESULTS |
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Erg11p activity and sensitivity determination
In recent papers, Lamb et al. (1997) and Sanglard et al. (1998)
described elegant strategies to test for alterations in affinity of variant cytochrome P450 14-demethylases for azole derivatives. Both used heterologous expression in a S. cerevisiae host using a strong GAL promoter to overexpress the Candida Erg11p. In the Lamb et al. (1997)
system, the recombinant protein was purified and analysed with biochemical assays, whereas in the Sanglard et al. (1998b)
method, affinity changes were directly correlated with changes in MIC values obtained with the engineered Saccharomyces strains. In this study we used biochemical assays with subcellular extracts prepared from the different isolates to look for the effects of different azole concentrations on either the incorporation of [2-14C]mevalonic acid into sterols or on the CO-complex formation of reduced microsomal cytochrome P450 fractions. Because two strains used in the Sanglard et al. (1998b)
paper, C26 and C40, were included in this study, the results obtained with the different strategies could be compared.
The results obtained with the subcellular sterol biosynthesis method are summarized in Table 1. In the case of J913004/I and 6406/8, addition of PMSF to the homogenization buffer was found to be essential for enzyme activity, whereas no improvement was observed for the other isolates tested. For consistency reasons, PMSF was added to all preparations. According to the IC50 values obtained for itraconazole, only strain NCPF 3363, previously reported to contain a mutated cytochrome P450 (Vanden Bossche et al., 1990
), was less sensitive, having an IC50 value of 92 nM. All other isolates gave IC50 values in the range 1440 nM and as such can be regarded as itraconazole-sensitive. However, differences were seen at concentrations above the IC50 value, as shown in Table 1
. Indeed at 100 nM, a near complete inhibition was seen with the susceptible reference strains ATCC 28516 and ATCC 44858, and with isolates B59626, B59630 and J913004/I. A leaky ergosterol biosynthesis inhibition (1424% of control) at this concentration was seen with extracts from 6406/8, C26 and C40, suggestive of a decreased affinity for itraconazole. For fluconazole, more pronounced differences were observed. IC50 values ranged more than 100-fold, from 41 nM for the most sensitive isolate up to 4880 nM for the C26 isolate. As well as in this C26 strain, reductions in affinity were seen for three other isolates, NCPF 3363, J913004/I and C40. Fluconazole was as active as itraconazole with regard to inhibition of subcellular sterol biosynthesis for both reference ATCC strains, whereas for all other azole-resistant strains tested, a 2·4- to 244-fold difference was observed in favour of itraconazole, suggesting that fluconazole binding is more sensitive to the amino acid variations present in these isolates. Incomplete inhibition of ergosterol biosynthesis was more pronounced with fluconazole because even at 1000 nM, isolates NCPF 3363, J913004/1, 6406/8, C26 and C40 still produced 1678% of the amounts found in control conditions.
Prevention of CO-complex formation in the reduced microsomal cytochrome P450 preparations is another assay that can be used to test the affinity of the protein for an azole (Vanden Bossche et al., 1987 ; Yoshida, 1988
). With this method, a constant content of 0·1 nmol cytochrome P450 ml-1 is used, enabling direct comparison of susceptibility between different isolates. Fluconazole was consistently found to be less active compared to itraconazole, even in the sensitive reference strains ATCC 28516 and ATCC 44858. Itraconazole was able to prevent COcytochrome complex formation in all isolates from which an active extract could be isolated. For strains J913004/I and 6406/8 we consistently failed to obtain a fraction suitable for analysis. The addition of a protease inhibitor such as PMSF, or a protease inhibitor mix (leupeptin, PMSF, 4-amidino-PMSF) did not sufficiently improve the yield of active cytochrome P450. For the other strains, as seen in the sterol biosynthesis assays, differences were far more pronounced for fluconazole: IC50 values ranged almost 35-fold from 140 to 5030 nM. Again, microsomes isolated from C26 and C40 showed the largest shifts, although the C40 microsomal fraction was the least sensitive in this assay. The cytochrome P450 isolated from NCPF 3363 was as sensitive as that isolated from susceptible strains.
Computer-aided sequence analysis of identified mutations
In Table 2 the published sequence variations of C. albicans ERG11 alleles are summarized next to the available growth sensitivity data. In total 53 full sequences were obtained, resulting in 98 variations relative to the Lai & Kirsch (1989)
sequence at 29 different locations, in addition to the PCR-induced T315A mutation reported by Lamb et al. (1997)
. This is a mean of 1·85 mutations per sequence. Because not all authors mention the silent nucleotide mutations found, a precise number of nucleotide changes cannot be given. For the seven strains sequenced in this study, 59 silent mutations were found (16 positions) compared to 18 amino acid changes (12 different), which suggests a three-fold higher frequency of silent mutations. To visualize the position and the frequency of the amino acids substitutions reported, a graphical representation was made (Fig. 1
). At the top of the figure, the aligned sequences of the gene products of C. albicans ERG11 and that of the Pseudomonas putida CYP101 gene (P450cam) and their helical secondary structures are visualized. The alignment is taken from the model of Boscott & Grant (1994)
. Gaps are represented as thin lines and predicted
-helices are represented by boxes, for which the starting position and length are indicated. The letter code for the helices is indicated above the boxes. The mutations are represented by bars, for which the length is proportional to the frequency of occurrence. Different filling patterns are used to categorize the mutation: mutations found both in azole-sensitive and -resistant strains are represented by open bars and are probably not important for azole affinity. These substitutions could reflect allelic or strain variation. A filled bar is used for the mutations experimentally demonstrated to be important for the affinity of an azole for cytochrome P450. Non-characterized mutations are shown represented by hatched bars. The PCR-induced mutation is represented by an arrow. From Fig. 1
it is clear that the majority of the mutations were found in three regions: region 1 from amino acids 105 to 165, region 2 from 266 to 287 and region 3 from 405 to 488. This last region also contains C470, the fifth ligand of the haem. Three of the four mutations found with the highest frequency, D116E, K128T and E266D, were also found in azole-sensitive strains; only G464S has been seen exclusively in resistant strains. No spontaneous mutation was found in the I helix, which is highly conserved over the 14-demethylase cytochrome P450 family.
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DISCUSSION |
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Three different methods are available to investigate the affinity of cytochrome P450 for an azole inhibitor and each method has its strengths and limitations. The CO-binding assay is very easy to perform and allows immediate comparison between different isolates because a standard amount of cytochrome P450 is used. A drawback of this method is that preparations must be made from strains growing in high glucose and with oxygen limitation. Moreover, the preparation could contain non-14-demethylase cytochrome P450 and the long preparation time needed to obtain the washed microsomal preparation increases exposure to proteolysis. The subcellular ergosterol bioassay requires less preparation and as such is less sensitive to proteolysis. With both of these methods the enzymes are in their natural Candida membrane environment. This is in contrast with the third method: heterologous expression in S. cerevisiae. This heterologous expression method allows measurements of single allele products but because of differences in preferred codon usage between Candida and Saccharomyces could also introduce contributions from silent mutations. The use of a strong GAL promoter could also introduce unnaturally high concentrations of the cytochrome. Indeed Lamb et al. (1997) found concentrations of 2·5 nmol microsomal protein mg-1, at least 25 times the basal level. This huge overexpression of membrane-bound protein forces cells to hyperproliferate their membranes, which often form multilamellar membrane structures around the nucleus (karmella) (Vergeres et al., 1993
; Supply et al., 1993
). It is therefore not surprising that the results obtained with the three methods do not match perfectly. The C26 Erg11p variant has a lower affinity for fluconazole according to all three methods. In contrast, the eightfold decrease in affinity for itraconazole suggested by the heterologous expression system was not confirmed by CO binding or by a decrease in IC50 in the subcellular assay. The incomplete enzyme inhibition at 100 nM itraconazole observed in the subcellular assay could, however, be of importance because for growth inhibition in C. albicans a depletion of ergosterol is needed. If a leaky inhibition is sufficient for resistance, then four out of nine isolates (NCPF 3363, 6406/8, C26 and C40) could be regarded as itraconazole-refractory and in the case of fluconazole isolate J913004/1 is also resistant. These criteria suggest that the E165Y mutation found in the laboratory mutant interferes with both itraconazole and fluconazole binding. To demonstrate this, the E165Y mutation should be introduced by mutagenesis; if one envisages heterologous expression in a Saccharomyces host, one should moreover prevent the unintended S263L mutation resulting from the different translation of the CUG codon in Candida versus Saccharomyces. Three other refractory alleles contain the Y132H substitution, corroborating its importance. As suggested by Sanglard et al. (1998b)
a concomitant substitution of S405F or R467K further decreases affinity, especially for fluconazole. The smaller, hydrophilic fluconazole molecule has fewer stabilization sites in the active pocket compared to the lipophilic itraconazole molecule which may explain this difference in activity (Vanden Bossche & Koymans, 1997
). Moreover, because the CO-binding assay did not reveal a diminished sensitivity for the NCPF 3363 isolate (having a homozygotic Y132H substitution), it is likely that a concomitant mutation is necessary for maximal effect. In this isolate, the originally reported shift in CO-binding sensitivity (Smith et al., 1986
), comparable to that found in the C26 and C40 isolates for fluconazole, could not be reproduced on later occasions; this could be due to loss of a second mutation on one or on both alleles. Unfortunately, the isolate possessing the original phenotype could not be retrieved to investigate this hypothesis.
The sequence available for Candida guilliermondii Erg11p also contained a phenylalanine aligning with S405 from C. albicans. Because different C. guilliermondii isolates tend to vary substantially in their fluconazole susceptibility (Odds, 1992 ), it would be interesting to know the MIC of fluconazole for the isolate used for sequencing and to verify whether the presence of that phenylalanine correlates with higher fluconazole MIC values. From the substitutions identified in the J913004/I isolate, D116E and K128T probably do not contribute to the decrease in fluconazole activity because these substitutions were also found in multiple azole-sensitive strains. In fact, it is likely that all substitutions found in azole-sensitive strains, and presumably also a selection of the non-characterized substitutions found only in resistant strains so far, reflect strain variation. From the substitutions found in the N-terminal and central region of the protein, only Y132H was associated with resistance. In these regions a higher proportion of the substitutions was also found in azole-sensitive strains when compared to the C-terminal region. No spontaneous substitutions have been found so far in the very conserved central I helix. A reason for this could be that substitutions in this region induce loss of function of the enzyme that would result in slower growth rates and therefore would be disadvantagous for survival. Such a mutation, G310D, has been described in S. cerevisiae (Ishida et al., 1988
). The C-terminal part contains three substitutions associated with resistance, and from the remaining substitutions only one was found in azole-sensitive strains. This region therefore looks to be of greater importance for azole resistance. Further study is needed to verify to what extent V452A and G464S mutations contribute to resistance. The fact that G464S was found by several investigators in azole-resistant strains makes this a prioritized substitution for investigation. The V452A substitution, as well as the G448E, F449L and G450E mutations are situated near the end of the central I helix and the J helix, an area of the protein that could be important for the docking of cytochrome P450 reductase. Indeed it is hypothesized that this region, which is absent in the Cyp101 sequence, and as such also in the model, is creating the recognition and docking site for the reductase. It has to be stressed that the three-dimensional model used is based on a soluble isozyme of bacterial origin, with different substrate specificity. For this reason, only approximate locations of the mutations can be given. The Mycobacterium tuberculosis CYP51-like gene, identified by in silico analysis of the full genome, could become a better model because this soluble enzyme could 14-demethylate dihydrolanosterol but crystallization and structure determination has not been achieved yet (Aoyama et al., 1998
; Bellamine et al., 1998
). The availability of multiple sequences of Erg11p from Candida krusei, a species with a 14
-demethylase less susceptible to azoles (Marichal et al., 1995
; Orozco et al., 1998
) could provide additional comparative material to identify important regions for binding of azoles to the cytochrome P450.
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ACKNOWLEDGEMENTS |
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Received 15 March 1999;
revised 28 June 1999;
accepted 10 June 1999.