Characterization of Salmonella Typhimurium DNA gyrase as a target of quinolones.
Quinolones exhibit good antibacterial activity against Salmonella spp. isolates and are often the choice of treatment for life-threatening salmonellosis due to multi-drug resistant strains. To assess the properties of quinolones, we performed an in vitro assay to study the antibacterial activities of quinolones against recombinant DNA gyrase. We expressed the S. Typhimurium DNA gyrase A (GyrA) and B (GyrB) subunits in Escherichia coli. GyrA and GyrB were obtained at high purity (>95%) by nickel-nitrilotriacetic acid agarose resin column chromatography as His-tagged 97-kDa and 89-kDa proteins, respectively. Both subunits were shown to reconstitute an ATP-dependent DNA supercoiling activity. Drug concentrations that suppressed DNA supercoiling by 50% (IC50 s) or generated DNA cleavage by 25% (CC25 s) demonstrated that quinolones highly active against S. Typhimurium DNA gyrase share a fluorine atom at C-6. The relationships between the minimum inhibitory concentrations (MICs), IC50 s and CC25 s were assessed by estimating a linear regression between two components. MICs measured against S. Typhimurium NBRC 13245 correlated better with IC50 s (R = 0.9988) than CC25 s (R = 0.9685). These findings suggest that the DNA supercoiling inhibition assay may be a useful screening test to identify quinolones with promising activity against S. Typhimurium. The quinolone structure-activity relationship demonstrated here shows that C-8, the C-7 ring, the C-6 fluorine, and N-1 cyclopropyl substituents are desirable structural features in targeting S. Typhimurium gyrase.
Division of Bioresources, Hokkaido University Research Center for Zoonosis Control, Sapporo, 001-0020, Japan.
Siriporn
S
Kongsoi
Central Research Laboratory, Kissei Pharmaceutical Co., Ltd, Nagano, Japan.
Kazumasa
K
Yokoyama
Department of Anatomy, Faculty of Veterinary Medicine, Kasetsart University, Bangkok, Thailand.
Apinun
A
Suprasert
Department of Microbiology, Faculty of Public Health, Mahidol University, Bangkok, Thailand.
Fuangfa
F
Utrarachkij
Division of Bioresources, Hokkaido University Research Center for Zoonosis Control, Sapporo, 001-0020, Japan.|Hokkaido University The Global station for Zoonosis Control, Sapporo, Japan.
Chie
C
Nakajima
Department of Microbiology, Faculty of Public Health, Mahidol University, Bangkok, Thailand.
Orasa
O
Suthienkul
Division of Bioresources, Hokkaido University Research Center for Zoonosis Control, Sapporo, 001-0020, Japan.|Hokkaido University The Global station for Zoonosis Control, Sapporo, Japan.
Yasuhiko
Y
Suzuki
D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000494:pharmacology*; D027081:DNA Gyrase / Q000378:metabolism*; D006801:Humans; D058990:Molecular Targeted Therapy; D015363:Quinolones / Q000737:chemistry / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000378:metabolism; D012480:Salmonella Infections / Q000188:drug therapy / Q000382:microbiology*; D012486:Salmonella typhimurium / Q000187:drug effects / Q000201:enzymology*; D059005:Topoisomerase II Inhibitors / Q000737:chemistry / Q000494:pharmacology*
D000900:Anti-Bacterial Agents; D015363:Quinolones; D011994:Recombinant Proteins; D059005:Topoisomerase II Inhibitors; D027081:DNA Gyrase
DNA gyrase; Salmonella Typhimurium; quinolones