Department of Global Epidemiology, Hokkaido University Research Center for Zoonosis Control, Sapporo 001-0020, Japan.
Antimicrob Agents Chemother. 2011 Aug;55(8):3661-7. doi: 10.1128/AAC.00042-11. Epub 2011 Jun 6.
Amino acid substitutions conferring resistance to quinolones in Mycobacterium tuberculosis have generally been found within the quinolone resistance-determining regions (QRDRs) in the A subunit of DNA gyrase (GyrA) rather than the B subunit of DNA gyrase (GyrB). To clarify the contribution of an amino acid substitution, E540V, in GyrB to quinolone resistance in M. tuberculosis, we expressed recombinant DNA gyrases in Escherichia coli and characterized them in vitro. Wild-type and GyrB-E540V DNA gyrases were reconstituted in vitro by mixing recombinant GyrA and GyrB. Correlation between the amino acid substitution and quinolone resistance was assessed by the ATP-dependent DNA supercoiling assay, quinolone-inhibited supercoiling assay, and DNA cleavage assay. The 50% inhibitory concentrations of eight quinolones against DNA gyrases bearing the E540V amino acid substitution in GyrB were 2.5- to 36-fold higher than those against the wild-type enzyme. Similarly, the 25% maximum DNA cleavage concentrations were 1.5- to 14-fold higher for the E540V gyrase than for the wild-type enzyme. We further demonstrated that the E540V amino acid substitution influenced the interaction between DNA gyrase and the substituent(s) at R-7, R-8, or both in quinolone structures. This is the first detailed study of the contribution of the E540V amino acid substitution in GyrB to quinolone resistance in M. tuberculosis.
结核分枝杆菌中对喹诺酮类药物产生耐药性的氨基酸取代通常发生在 DNA 回旋酶 A 亚基（GyrA）而非 DNA 回旋酶 B 亚基（GyrB）的喹诺酮类药物耐药决定区（QRDRs）内。为了阐明 GyrB 中氨基酸取代 E540V 对结核分枝杆菌中喹诺酮类药物耐药性的贡献，我们在大肠杆菌中表达了重组 DNA 回旋酶，并在体外对其进行了表征。通过混合重组 GyrA 和 GyrB，在体外重新构成野生型和 GyrB-E540V DNA 回旋酶。通过 ATP 依赖性 DNA 超螺旋化测定、喹诺酮抑制超螺旋化测定和 DNA 切割测定评估氨基酸取代与喹诺酮类药物耐药性之间的相关性。与野生型酶相比，携带 GyrB 中 E540V 氨基酸取代的 DNA 回旋酶对八种喹诺酮类药物的 50%抑制浓度高出 2.5 至 36 倍。同样，E540V 回旋酶的 25%最大 DNA 切割浓度比野生型酶高 1.5 至 14 倍。我们进一步证明，E540V 氨基酸取代影响了 DNA 回旋酶与喹诺酮结构中 R-7、R-8 或两者的取代基之间的相互作用。这是首次详细研究 GyrB 中 E540V 氨基酸取代对结核分枝杆菌中喹诺酮类药物耐药性的贡献。
