Inhibitory activity of quinolones against DNA gyrase of Mycobacterium tuberculosis

Yoshikuni Onodera,*, Mayumi Tanaka and Kenichi Sato

New Product Research Laboratories I, Daiichi Pharmaceutical Co. Ltd, 16-13 Kitakasai 1-chome, Edogawa-ku, Tokyo 134-8630, Japan


    Abstract
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
The in vitro inhibitory activities of quinolones against Mycobacterium tuberculosis DNA gyrase were measured. The 50% inhibitory concentrations (IC50s) of sitafloxacin (DU-6859a), sparfloxacin, ciprofloxacin and levofloxacin against supercoiling activity of DNA gyrase were 1.67, 4.80, 12.2 and 13.9 mg/L, respectively, and correlated well with their MICs. Two altered proteins of GyrA containing Ala-90Val, or Ala-90Val and Asp-94Gly were also purified and the inhibitory activities of the quinolones ranged from 12 to >83 times weaker than those against the wild-type enzyme. These results suggest that mutations in the corresponding genes confer quinolone resistance.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The increasing incidence of tuberculosis and multi-drug-resistant tuberculosis poses serious problems around the world, and new pharmaceuticals are needed for control of this disease. Recently, several new quinolones were demonstrated to be active in vitro and in vivo against mycobacterial species, and to be effective in the treatment of infection caused by Mycobacterium tuberculosis, Mycobacterium leprae and Mycobacterium avium.14

Quinolones inhibit bacterial type II topoisomerase, DNA gyrase and topoisomerase IV. In 1998, the full genome sequence of M. tuberculosis was reported,5 but the topoisomerase IV gene was not identified in the data. Although it was not clear whether topoisomerase IV is present in M. tuberculosis, DNA gyrase would certainly be expected to play an important role in drug resistance. Genetic studies on M. tuberculosis revealed that single missense mutations in gyrA conferred low-level quinolone resistance, and bacteria with high-level resistance had two missense mutations in gyrA.6

To analyse the inhibitory activity of quinolones against DNA gyrase of M. tuberculosis in vitro and to examine whether targeted alterations in GyrA could confer quinolone resistance, we expressed and purified recombinant M. tuberculosis GyrA, GyrB and altered GyrA subunit proteins, reconstituted the DNA gyrase enzymic activity and measured the inhibitory activities of quinolones against these proteins.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacterial strains and antibacterial agents

The bacterial strain used in this study was the quinolone-susceptible strain M. tuberculosis H37Rv, which served as the wild-type strain of M. tuberculosis. Sitafloxacin, levofloxacin, ciprofloxacin and sparfloxacin were synthesized at New Product Research Laboratories I, Daiichi Pharmaceutical Co. Ltd (Tokyo, Japan).

Determination of MICs

Bacterial suspensions were prepared by diluting organisms grown in 7H9 medium (Difco Laboratories, Detroit, MI, USA) at 37°C for 7–10 days with 0.05% Tween 80 in saline to give a bacterial concentration of c. 106 cfu/mL. The bacterial suspension (5 µL) was spotted on to 7H11 agar plates (Difco Laboratories) containing 100 to 0.003 µg of drug per mL. The drugs were initially dissolved in 0.1 M NaOH, and the solution serially diluted two-fold with distilled water to the appropriate concentrations before it was added to the agar medium. The MICs of the drugs were determined 14 days after the start of cultivation at 37°C in a CO2 incubator (5% CO2, 95% humidified air). The MICs were determined as the minimum drug concentrations to completely inhibit the growth of organisms or to allow no more than five colonies per spot to grow.

Purification of genomic DNA

Five hundred microlitres (c. 108 cells) of M. tuberculosis H37Rv organisms in broth were centrifuged. The pellet was resuspended in TE (10 mM Tris–HCl pH 8.0, 1 mM EDTA) and the mixture was boiled for 20 min. Proteinase K (Sigma Chemical Co., St Louis, MO, USA) was added and the suspension was then incubated at 37°C for 1 h. After phenol, phenol–chloroform and chloroform extraction, genomic DNA was obtained by ethanol precipitation.

Construction of expression vectors

Two sets of oligonucleotide primers were designed for amplification of gyrA and gyrB genes and subsequent insertion into the pMAL-c2 fusion protein expression vector (New England Biolabs, Beverly, MA, USA). In each case, the sequence of the forward primer was chosen at the initiation codon. For reverse primers, an XhoI site or a PstI site was introduced for cloning purposes. For gyrA, the forward primer was 5'-ATGACAGACACGACGTTGCCGCCTG-3' (containing the 1–25 bp region of gyrA) and the reverse primer was 5'-CATCGTCGTCGCTCGAGCCTGATTAA-3' (2514–2539; inserted XhoI site underlined). Primers for the gyrB gene were 5'-ATGGGTAAAAACGAGGCCAGAAGATC-3' (1–26) and 5'-TGCATCTCCTGCAGGATGTCAACCG-3' (2229–2253; inserted PstI site underlined). PCR was carried out on genomic DNA from strain H37Rv by using the Expand High-Fidelity PCR System (Boehringer Mannheim, Indianapolis, IN, USA). Each gene was amplified for 25 cycles, for 0.5 min at 94°C for denaturation, 0.5 min at 63°C for annealing and 2 min at 72°C for polymerization. The resulting 2.5 kb gyrA and 2.2 kb gyrB fragments were digested with XhoI and PstI, respectively. The DNA fragments were ligated into the XmnI–SalI site, or the XmnI– PstI site of the pMAL-c2 expression vector, respectively, and used to transform Escherichia coli MC1061.

In vitro mutagenesis

Mutations were introduced in the gyrA gene of the pMAL-c2-GyrA expression vector by site-directed mutagenesis with Mutan-K (Takara, Tokyo, Japan) by using Pr-mut90 5'-CGCACGGCGACGTGTCGATCTACGA-3' or Pr-mut9094 5'-CGGCGACGTGTCGATCTACGGCAGCCTGG-3' (altered nucleotides underlined).

Purification of the enzymes

The GyrA, altered GyrA and GyrB proteins of DNA gyrase were purified separately as maltose-binding protein (MBP) fusion products. The E. coli MC1061/pMAL-c2 cells containing one of the above genes were incubated in Luria–Bertani broth until log-phase growth, and isopropyl-ß-D-thiogalactopyranoside was then added to the culture at a final concentration of 0.3 mM to induce protein synthesis. After 2 h incubation the cells were harvested and resuspended in TGED buffer [50 mM Tris–HCl pH 8.0, 10% glycerol, 1 mM EDTA, 1 mM dithiothreitol (DTT)] supplemented with 0.5 mg/mL lysozyme, and then incubated at 4°C for 30 min. The suspension was centrifuged at 100 000g for 40 min, and the supernatant was loaded on to an amylose resin column previously equilibrated with TGED buffer. The column was washed with 10 volumes of TGED buffer, and the fusion proteins were eluted with 10 mM maltose. The eluted fractions were dialysed twice against TGED buffer at 4°C for 6 h and concentrated by dialysis against 50 mM Tris–HCl pH 8.0, 50% glycerol, 1 mM EDTA, 1 mM DTT.

DNA supercoiling assay and determination of inhibitory activity of drugs

DNA supercoiling activity, reconstituted with purified M. tuberculosis GyrA and GyrB proteins, was assayed with relaxed pBR322 DNA as a substrate. The standard reaction mixture (20 µL) contained 20 mM Tris–HCl pH 8.0, 2 mM MgCl2, 50 mM KCl, 1 mM DTT, 1 mM ATP, 1 mM spermidine, 20 mg/L tRNA, 20 mg/L bovine serum albumin, 0.2 µg relaxed pBR322, and GyrA and GyrB proteins. The reaction mixture was incubated at 37°C for 1 h, and the reaction was terminated by addition of a dye mix and then analysed by electrophoresis in 0.8% agarose. One unit of supercoiling activity was defined as the amount of GyrA and GyrB proteins required to supercoil 50% of 0.2 µg of relaxed pBR322 plasmid DNA. The IC50 was defined as the drug concentration that reduced the enzymic activity observed with drug-free controls by 50%.


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 Materials and methods
 Results
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 References
 
Purification of DNA gyrase subunit proteins

The GyrA and GyrB proteins of DNA gyrase of M. tuberculosis H37Rv were purified separately as MBP fusion proteins, and a single band for each protein was observed by SDS–polyacrylamide gel electrophoresis (SDS–PAGE), at c. 130 and 110 kDa for MBP–GyrA and MBP–GyrB, respectively (FigureGo, a). A factor Xa recognition site had been introduced into the fusion proteins and, after factor Xa digestion, the GyrA and GyrB proteins migrated at approximately 90 and 70 kDa, respectively (FigureGo, a). Although no single protein had enzymic activity, GyrA and GyrB together showed supercoiling activity (FigureGo, b). Because these activities were not detected in the absence of ATP or Mg2+, these enzymes were ATP- and Mg2+-dependent (FigureGo, b). The optimum concentration range for the potassium cation was 40–200 mM, and that for the magnesium cation was >1 mM (data not shown).



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Figure. (a) SDS–PAGE of purified M. tuberculosis DNA gyrase. Lane 1, purified MBP–GyrA fusion protein; lane 2, purified MBP–GyrB fusion protein; lane 3, MBP–GyrA fusion protein after factor Xa cleavage; lane 4, MBP–GyrB fusion protein after factor Xa cleavage. Each protein sample was loaded on an SDS–7.5% polyacrylamide gel and the gel stained with Coomassie Brilliant Blue. Size of protein markers are indicated at the left. (b) Enzymic activities of purified DNA gyrase of M. tuberculosis H37Rv. Purified GyrA and GyrB proteins reconstitute an ATP-dependent DNA supercoiling activity. Relaxed plasmid pBR322 substrate was incubated with purified proteins in the absence or presence of 1 mM ATP. Reactions were stopped and the DNA was examined by electrophoresis in 0.8% agarose. Lane 1, relaxed DNA and GyrA protein with ATP; lane 2, relaxed DNA and GyrB protein with ATP; lane 3, relaxed DNA and both GyrA and GyrB with ATP; lane 4, relaxed DNA and GyrA and GyrB without ATP; lane 5, relaxed DNA and GyrA and GyrB without Mg2+. (c) Inhibitory activity of levofloxacin against the supercoiling activity of DNA gyrase from M. tuberculosis M37Rv. Relaxed pBR322 DNA was incubated with GyrA and GyrB proteins in the absence or presence of the drug. DNA was analysed as described in (b). Lanes 1–6: 0, 3.13, 6.25, 12.5, 25 and 50 mg/L levofloxacin, respectively.

 
Comparison of inhibitory activities of antibacterial agents against DNA gyrase

The quinolones inhibited the reconstituted enzyme activity in a concentration-dependent manner (FigureGo, c). In contrast, benzylpenicillin, which does not inhibit the enzyme, had no effect on the activity (data not shown). The IC50s of the quinolones were calculated by quantifying the bands corresponding to supercoiled DNA (TableGo). The IC50 values of the quinolones against the wild-type DNA gyrase correlated with their MIC values (r = 0.99). Among the quinolones tested, sitafloxacin showed the highest inhibitory activity against the enzyme.


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Table. Inhibitory activities of quinolones against DNA gyrase of M. tuberculosis
 
Inhibitory activities of quinolones against altered DNA gyrase

The altered GyrA proteins with a substitution of Val (GTG) for Ala90 (GCG), or a double substitution of Val (GTG) for Ala90 (GCG) and Gly (GGC) for Asp94 (GAC), were characterized. The IC50s of these enzymes reconstituted with altered GyrA of M. tuberculosis are also presented in the TableGo. The IC50s of the quinolones tested against DNA gyrase containing a single amino acid change were 12 to >83 times higher than those against the wild-type enzyme. These results suggest that mutations at the Ala90 codon conferred quinolone resistance. In addition, the IC50 of sitafloxacin against the enzyme that contained two amino acid changes was c. 6.5-fold higher than that against the enzyme with the single amino acid change. These results are consistent with the results of genetic studies of M. tuberculosis, in which two missense mutations in gyrA conferred high-level resistance.6


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We expressed and purified the M. tuberculosis H37Rv DNA gyrase subunit proteins. The GyrA and GyrB proteins together reconstituted supercoiling activity characteristic of a DNA gyrase. Sitafloxacin, levofloxacin, ciprofloxacin and sparfloxacin inhibited the enzyme and their IC50s against DNA gyrase correlated with their anti-M. tuberculosis activities. Among the quinolones tested, the inhibitory activity of sitafloxacin was especially high. These results are consistent with the good anti-tuberculosis activity reported for sitafloxacin in vitro.4

It was reported that the natural differences in the level of resistance to quinolones within the genus Mycobacterium are, at least in part, related to the amino acid residue at position 90 in the quinolone resistance determining region (QRDR) of GyrA (Ser83 of GyrA in E. coli).7 This was confirmed by biochemical studies in which the IC50 values of the purified DNA gyrase from M. avium and Mycobacterium smegmatis, which naturally have an alanine at position 83, were higher than those inhibiting the enzyme from Mycobacterium fortuitum bv. peregrinum, characterized by a serine residue at position 83 of GyrA.8 In this report, we purified an altered GyrA with a substitution of Val for Ala90, which was shown previously to be related to quinolone resistance by genetic studies on M. tuberculosis.6 The inhibitory activities of levofloxacin, ciprofloxacin and sparfloxacin against DNA gyrase that contained a single amino acid change in GyrA were >28 times weaker than those against the wild-type enzyme. These values are higher than those found in the previous genetic study, in which the MICs of quinolones against the in vitro quinolone-resistant mutant with a single missense mutation (Ala-90Val) were eight times higher than in the wild-type strain.6 These results indicate that while the mutation conferred quinolone resistance, an additional target could also contribute to quinolone resistance. The target might be topoisomerase IV, but as a topoisomerase IV gene was not identified in the M. tuberculosis genome sequence data,5 a different gene product factor may exist for topoisomerase IV activity.

We also purified GyrA with substitutions of Val for Ala90 and Gly for Asp94, and indicated that the addition of a second mutation caused even greater resistance to the drug. Moreover, as in other bacteria, mutations in the B subunit of DNA gyrase and appearance of efflux pumps were shown to be related to quinolone resistance.6,9,10 To understand the mechanism of quinolone resistance in mycobacteria, these factors should be combined and clarified by further study.


    Notes
 
* Corresponding author. Tel: +81-3-3680-0151; Fax: +81-3-5696-8344; E-mail: onode90j{at}daiichipharm.co.jp Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Alangaden, G. J. & Lerner, S. A. (1997). The clinical use of fluoroquinolones for the treatment of mycobacterial diseases. Clinical Infectious Disease 25, 1213–21.[ISI][Medline]

2 . Ji, B., Sow, S., Perani, E., Lienhardt, C., Diderot, V. & Grosset, J. (1998). Bactericidal activity of a single-dose combination of ofloxacin plus minocycline, with or without rifampin, against Mycobacterium leprae in mice and in lepromatous patients. Antimicrobial Agents and Chemotherapy 42, 1115–20.[Abstract/Free Full Text]

3 . Ji, B., Lounis, N., Truffort-Pernot, C. & Grosset, J. (1995). In-vitro and in-vivo activities of levofloxacin against Mycobacterium tuberculosis. Antimicrobial Agents and Chemotherapy 39, 1341–4.[Abstract]

4 . Tomioka, H., Sato, K., Akaki, T., Kajitani, H., Kawahara, S. & Sakatani, M. (1999). Comparative in vitro antimicrobial activities of the newly synthesized quinolone HSR-903, sitafloxacin (DU-6859a), gatifloxacin (AM-1155), and levofloxacin against Mycobacterium tuberculosis and Mycobacterium avium complex. Antimicrobial Agents and Chemotherapy 43, 3001–4.[Abstract/Free Full Text]

5 . Cole, S. T., Brosch, R., Parkhill, J., Garnier, T., Churcher, C., Harris, D. et al. (1998). Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 393, 537–44.[ISI][Medline]

6 . Kocagoz, T., Hackbarth, C. J., Unsal, I., Rosenberg, E. Y., Nikaido, H. & Chambers, H. F. (1996). Gyrase mutation in laboratory-selected, fluoroquinolone-resistant mutants of Mycobacterium tuberculosis H37Ra. Antimicrobial Agents and Chemotherapy 40, 1768–74.[Abstract]

7 . Guillemin, I., Jarlier, V. & Cambau, E. (1998). Correlation between quinolone susceptibility patterns and sequences in the A and B subunits of DNA gyrase in mycobacteria. Antimicrobial Agents and Chemotherapy 42, 2084–8.[Abstract/Free Full Text]

8 . Guillemin, I., Sougakoff, W., Cambau, E., Viravau, V. R., Moreau, N. & Jarlier, V. (1999). Purification and inhibition by quinolones of DNA gyrase from Mycobacterium avium, Mycobacterium smegmatis and Mycobacterium fortuitum bv. peregrinum. Microbiology 145, 2527–32.[Abstract/Free Full Text]

9 . Liu, J., Takiff, H. E. & Nikaido, H. (1996). Active efflux of fluoroquinolones in Mycobacterium smegmatis mediated by LfrA, a multidrug efflux pump. Journal of Bacteriology 178, 3791–5.[Abstract]

10 . Ainsa, J. A., Blokpoel, M. C., Otal, I., Young, D. B., De Smet, K. A. & Martin, C. (1998). Molecular cloning and characterization of Tap, a putative multidrug efflux pump present in Mycobacterium fortuitum and Mycobacterium tuberculosis. Journal of Bacteriology 180, 5836–43.[Abstract/Free Full Text]

Received 17 July 2000; returned 28 September 2000; revised 23 November 2000; accepted 7 December 2000