Impact of mutations in DNA gyrase genes on quinolone resistance in Campylobacter jejuni
Impact of mutations in DNA gyrase genes on quinolone resistance in Campylobacter jejuni
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DOI：
10.1002/dta.1937
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发表时间：
2016-10-01
影响因子：
2.9
通讯作者：
Suzuki, Yasuhiko
中科院分区：
文献类型：
--
作者：
Changkwanyeun, Ruchirada;Yamaguchi, Tomoyuki;Suzuki, Yasuhiko
在蛋白DNA旋转酶A亚单位(GyrA)的喹诺酮耐药决定区发现了对空肠弯曲菌具有喹诺酮耐药性的氨基酸替换，频率最高的是第86位，其次是第90位。本研究在大肠杆菌中表达野生型和突变型重组DNA旋转酶亚基，并用Ni-NTA琼脂糖柱层析进行纯化。可溶性97 kDa GyrA和87 kDa DNA旋转酶B亚基重建了依赖于ATP的DNA超螺旋活性。喹诺酮类药物抑制的超螺旋试验表明Thr86Ile、Thr86Ala、Thr86Lys、Asp90Asn和Asp90Tyr氨基酸替换在降低对喹诺酮类药物的敏感性方面起到了作用。Thr86Ile对所有被检测的喹诺酮类药物的显著作用表明，这种替代的优势与重复分离耐喹诺酮类空肠弯曲菌的情况一致。构效关系分析表明，喹诺酮类药物中8位取代基对于克服Thr86Ile的作用具有重要意义。西塔沙星(Sitafloxating，SIT)对包括环丙沙星耐药突变株在内的所有空肠弯曲菌具有最高的抑制活性，其R-1位有氟化环丙基环，R-8位有氯取代基，这是其他喹诺酮类药物所没有的。结果提示，SIT可作为治疗耐CIP空肠弯曲杆菌病的一种有前景的药物。版权所有(C)2016 John Wiley&Sons，Ltd.
Amino acid substitutions providing quinolone resistance to Campyloabcter jejuni have been found in the quinolone resistance-determining region of protein DNA gyrase subunit A (GyrA), with the highest frequency at position 86 followed by position 90. In this study, wild-type and mutant recombinant DNA gyrase subunits were expressed in Escherichia coli and purified using Ni-NTA agarose column chromatography. Soluble 97 kDa GyrA and 87 kDa DNA gyrase subunit B were shown to reconstitute ATP-dependent DNA supercoiling activity. A quinolone-inhibited supercoiling assay demonstrated the roles of Thr86Ile, Thr86Ala, Thr86Lys, Asp90Asn, and Asp90Tyr amino acid substitutions in reducing sensitivity to quinolones. The marked effect of Thr86Ile on all examined quinolones suggested the advantage of this substitution in concordance with recurring isolation of quinolone-resistant C. jejuni. An analysis of the structure-activity relationship showed the importance of the substituent at position 8 in quinolones to overcome the effect of Thr86Ile. Sitafloxacin (SIT), which has a fluorinate cyclopropyl ring at R-1 and a chloride substituent at R-8, a characteristic not found in other quinolones, showed the highest inhibitory activity against all mutant C. jejuni gyrases including ciprofloxacin-resistant mutants. The results suggest SIT as a promising drug for the treatment of campylobacteriosis caused by CIP-resistant C. jejuni. Copyright (C) 2016 John Wiley & Sons, Ltd.