Laboratoire de Recherche Moléculaire sur les Antibiotiques (LRMA), Université Pierre et Marie Curie (Paris VI), Faculté de Médecine Pitié-Salpêtrière, 91 Bd de lHôpital, 75634 Paris Cédex 13, France1
LRMA, Faculté de Médecine, Broussais-Hôtel Dieu, 75005 Paris, France2
Author for correspondence: Wladimir Sougakoff. Tel: +33 1 40 77 97 46. Fax: +33 1 45 82 75 77. e-mail: sougakof@ lmcp.jussieu.fr
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ABSTRACT |
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Keywords: Mycobacterium, DNA gyrase, quinolone inhibition assays, fluoroquinolone resistance
Abbreviations: QRDR, quinolone-resistance-determining regions
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INTRODUCTION |
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Mycobacteria are naturally less susceptible to quinolones than other bacteria (Wolfson & Hooper, 1989 ). Moreover, they are characterized by a wide range of quinolone susceptibility patterns (Leysen et al., 1989
; Yew et al., 1994
). Indeed, we have shown previously that several mycobacterial species, such as Mycobacterium fortuitum and M. peregrinum, are less susceptible to quinolones than Escherichia coli, but are more susceptible to these drugs than other mycobacterial species such as M. smegmatis and M. tuberculosis (Guillemin et al., 1995
, 1998
). By contrast, other species, such as M. avium, display a very high level of resistance to quinolones (Guillemin et al., 1998
). It is likely that the natural differences in the level of resistance to quinolones within the genus Mycobacterium is, at least in part, related to the primary structure of the DNA gyrase subunits A and B, and more specifically to the residue at position 83 in the QRDR GyrA (Cambau et al., 1994
; Guillemin et al., 1995
). However, such a hypothesis, based on genetic studies, has not been confirmed yet by biochemical experiments. In this report, we describe the purification and the biochemical properties of the DNA gyrases from M. avium, M. smegmatis and M. fortuitum bv peregrinum, which are representative of the various quinolone susceptibility patterns observed in mycobacteria. The inhibition by quinolones of the supercoiling activity of the purified enzymes has been investigated and compared to that observed for the DNA gyrase purified from E. coli.
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METHODS |
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Antibiotics.
The following antimicrobial agents were used: nalidixic acid, flumequine (Sigma), pefloxacin, sparfloxacin (Rhône-Poulenc Rorer), ofloxacin, levofloxacin (Roussel-Uclaf) and ciprofloxacin (Bayer Pharma).
DNA gyrase purification.
DNA gyrases were purified by affinity chromatography on novobiocin-Sepharose columns by the method of Bazile et al. (1992 ), with the following modifications. All purification steps were carried out at 4 °C. The bacterial pellets obtained from 6-litre cultures (approx. 20 g cells) of M. avium, M. smegmatis, M. fortuitum bv. peregrinum or E. coli, were suspended in 50 mM Tris/HCl (pH 7·5) containing 2 mM DTT and 1 mM PMSF. Bacteria were lysed in a cell homogenizer (B. Braun ScienceTec) for 3x30 s with glass beads (Sigma) of 212300 µm diameter for E. coli, and for 4x1 min with beads of 106 µm for M. avium, M. smegmatis and M. fortuitum bv. peregrinum.
The supernatant was recovered after 20 min centrifugation at 12000 g. To precipitate nucleic acids and ribosomes, KCl and magnesium acetate were added at final concentrations of 0·66 M and 5 mM, respectively. After 90 min ultracentrifugation at 53000 g, ammonium sulfate (Sigma) was added to the supernatant at a final saturation of 37%. After stirring for 1 h, the suspension was centrifuged for 20 min at 12000 g. The ammonium sulfate concentration in the supernatant was then adjusted to 55% for mycobacteria and 42% for E. coli. The precipitate was recovered after 20 min of centrifugation at 12000 g. The pellet was resuspended in 12 ml 25 mM HEPES, 1 mM EDTA, 6 mM ß-mercaptoethanol, 200 mM KCl, 10% (v/v) ethylene glycol (buffer A, pH 8), and was dialysed overnight against 1 litre of the same buffer.
After dialysis, proteins were loaded onto a 1x2 cm novobiocin-Sepharose column previously equilibrated in buffer A. The column was washed with buffer A until the A280 returned to the base line. The adsorbed proteins were eluted, first with buffer A containing 20 mM ATP, and then with buffer A containing 5 M urea. The protein content of each ATP and urea fraction was examined by SDS-PAGE. The eluates were finally dialysed overnight against buffer B (50 mM KH2PO4, 1 mM DTT, 0·2 mM EDTA, pH 7·6), and were concentrated by dialysis against the same buffer containing 50% (v/v) glycerol.
DNA supercoiling assay.
The DNA gyrase supercoiling activity was assessed by measuring the conversion of relaxed plasmid pBR322 DNA to the supercoiled form, as described previously (Bazile et al., 1992 ; Revel-Viravau et al., 1996
). The relaxed pBR322 DNA was prepared from the supercoiled form (Boehringer Mannheim) by treatment with prokaryotic topoisomerase I (Eurogentec), as described previously (Moreau et al., 1990
).
Supercoiling assays were carried out in 15 µl reaction mixtures containing the DNA gyrase assay buffer (20 mM HEPES, 25 mM KCl, 6 mM magnesium acetate, 2 mM spermine, 4 mM DTT, 3% ethylene glycol, v/v, 30 µg E. coli tRNA ml-1, 1 mM ATP) (pH 8·0), 150 ng relaxed pBR322, and 23 µl purified DNA gyrase. The mixture was incubated for 3 h at 30 °C for M. avium, for 30 min at 30 °C for M. smegmatis and M. fortuitum bv. peregrinum, and for 30 min at 37 °C for E. coli. The reaction was stopped by the addition of 50% glycerol containing 0·25% bromophenol blue, and the total reaction mixture was subjected to electrophoresis on 1% agarose gel in 1xTBE buffer (Tris/borate/EDTA, pH 8·3). After a run of 3 h at 90 V, the gel was stained with ethidium bromide (0·7 µg ml-1). Supercoiling activity was assessed by tracing the brightness of the bands corresponding to the supercoiled pBR322 DNA, using a Densylab densitometer (Bio-Rad). One unit (U) of enzyme activity was defined as the amount of DNA gyrase that converted 150 ng relaxed pBR322 to the supercoiled form in 30 min at 30 °C for M. smegmatis, M. fortuitum and M. avium, and at 37 °C for E. coli.
Inhibition by quinolones of DNA gyrase supercoiling activity.
Inhibition of the supercoiling activity of the purified DNA gyrases was performed using the method of Staudenbauer & Orr (1981 ), modified as described previously (Revel-Viravau et al., 1996
). In brief, a reaction mixture in the gyrase assay buffer (15 µl), containing 150 ng relaxed pBR322 DNA, 1 U purified DNA gyrase and a serial twofold dilution of the quinolone, was incubated as described above. The inhibitory effect of quinolones on DNA gyrase was assessed by determining the concentration of drug required to inhibit 50% of the supercoiling activity of the enzyme (IC50).
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RESULTS |
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DNA gyrase purification and supercoiling assays
The experimental conditions used to purify the mycobacterial DNA gyrases by chromatography on novobiocin-Sepharose columns were somewhat different from those used for E. coli. The DNA gyrase activity was recovered in the 3742% ammonium sulfate fraction for E. coli, whereas it was found in the 3755% fraction for mycobacteria. For every species, the supercoiling activity was detected both in the fractions eluted with ATP and urea (Fig. 1). However, the highest supercoiling activity was found in the fractions eluted with ATP for E. coli (data not shown) and in those eluted with urea for mycobacteria (Fig. 1
). The highest supercoiling activity for the purified DNA gyrases was reached after 3 h of incubation at 30 °C for M. avium, 30 min at 30 °C for M. smegmatis and M. fortuitum bv. peregrinum, and 30 min at 37 °C for E. coli. SDS-PAGE of the active fractions revealed, for each enzyme, two major bands corresponding to the A and B subunits (data not shown). The apparent molecular masses of the subunits and the specific activities of these DNA gyrases are presented in Table 2
.
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The IC50 values were far higher for the DNA gyrases from mycobacteria than for that from E. coli (see Table 1). The DNA gyrase from M. fortuitum bv. peregrinum displayed IC50 values two- to eightfold lower than those from M. smegmatis and M. avium. The enzymes from the latter two species displayed very similar IC50 values. A good correlation was found for the three purified DNA gyrases between the IC50 values and the corresponding MICs, as shown in Fig. 2
(correlation coefficient values, r: M. avium, 0·91; M. fortuitum, 0·98; M. smegmatis, 0·97).
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DISCUSSION |
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For M. smegmatis and M. fortuitum bv. peregrinum, the maximum supercoiling activity was obtained after an incubation time of 30 min, as found for E. coli. By contrast, 3 h was required to reach this maximum with the DNA gyrase from M. avium, which had a low specific activity (0·3x104 U mg-1) compared with the values found for the gyrases purified from the three other bacteria (23·4x104 U mg-1) (see Table 2). It must be noted here that the specific activity measured for the M. avium DNA gyrase is similar to the one reported previously for M. bovis BCG (Wu & Shahied, 1995
).
Site-directed mutagenesis experiments in E. coli have demonstrated that the substitution of Ser-83 with Ala can lead to a 10-fold increase in both MIC and IC50 values of quinolones (Hallet & Maxwell, 1991 ). The fact that the IC50 values found for the DNA gyrases from M. avium and M. smegmatis, which both naturally have an alanine at position 83, were significantly higher than those inhibiting the enzyme from M. fortuitum bv. peregrinum, characterized by a serine residue at this position, confirms the important role played by residue 83 of GyrA in the intrinsic quinolone resistance of mycobacteria (Cambau et al., 1994
; Revel et al., 1994
; Guillemin et al., 1995
). The recently determined three-dimensional structure of the E. coli DNA gyrase indicates that the serine residue at position 83 in the A subunit is located in a region where proteinDNA contacts occur (Morais Cabral et al., 1997
). Therefore, it is tempting to speculate that the natural presence of an alanine at position 83 in GyrA from M. avium and M. smegmatis could impair the binding of quinolones, probably by modifying the quinolone binding site in the DNADNA gyrase complex.
It is evident that the IC50 values found for the DNA gyrase from M. avium were similar to those found for M. smegmatis, while the MICs against the former species were markedly (30-fold) higher than those against the latter (see Table 1). This may indicate that the natural resistance of M. avium to quinolones is not solely related to the low susceptibility of its DNA gyrase to these drugs, but also to other factors such as the low permeability of the mycobacterial cell wall to hydrophilic compounds (Jarlier & Nikaido, 1994
). M. avium is assumed to have a particularly low permeability, although this has not been accurately measured so far (Rastogi et al., 1981
), which is likely to be an additional factor in its natural resistance. Finally, an efflux pump for quinolones, LfrA, has recently been described in ciprofloxacin-resistant mutants of M. smegmatis. The presence of such a pump has also been suggested in wild-type M. avium (Takiff et al., 1996
) and could be involved in the low intrinsic susceptibility of this species to quinolones, if it really is present and functional.
In conclusion, the data reported here highlight the important role played by the DNA gyrase in the natural low susceptibility of mycobacteria to quinolones. However, further investigations on the mode of entrance of the quinolones through the mycobacterial cell wall and the efflux pumps recently identified in mycobacteria will be necessary to fully understand why some mycobacterial species, such as M. avium, exhibit such high levels of resistance to these drugs.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 26 October 1998;
revised 8 April 1999;
accepted 18 May 1999.