CmeABC functions as a multidrug efflux system in Campylobacter jejuni
CmeABC functions as a multidrug efflux system in Campylobacter jejuni
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DOI：
10.1128/aac.46.7.2124-2131.2002
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发表时间：
2002-07-01
影响因子：
4.9
通讯作者：
Zhang, QJ
中科院分区：
文献类型：
--
作者：
Lin, J;Michel, LO;Zhang, QJ
空肠弯曲杆菌是一种引起人类胃肠炎的革兰氏阴性微生物，对抗生素的耐药性越来越强。然而，人们对该病原体的药物流出机制知之甚少。在这里，我们表征了由三基因操纵子（指定为 cmeABC）编码的外排泵，该外排泵有助于空肠弯曲菌 81-176 的多药耐药性。 CmeABC 与其他革兰氏阴性细菌中已知的三方多药外排泵具有显着的序列和结构同源性，它由周质融合蛋白 (CmeA)、属于耐药结节细胞分裂超家族 (CmeB) 的内膜外排转运蛋白和外膜蛋白 (CmeC) 组成。使用 CmeABC 特异性抗体进行的免疫印迹表明，cmeABC 在野生型 81-176 中表达；然而，在 cmeB 基因中插入转座子的同基因突变体 (9B6) 显示 CmeB 和 CmeC 的产生受损。与野生型 81-176 相比，9B6 对一系列抗生素、重金属、胆盐和其他抗菌剂的耐药性降低了 2 至 4,000 倍。积累测定表明，突变体9B6中比野生型81-176中积累了显着更多的溴化乙锭和环丙沙星。添加羰基氰化物间氯苯腙（一种外排泵抑制剂）使野生型81-176中环丙沙星的积累增加至突变体9B6的水平。 PCR和免疫印迹分析还表明，cmeABC广泛分布于各种空肠弯曲菌分离株中，并在野生型菌株中组成型表达。总之，这些发现正式证实了 CmeABC 作为三方多药外排泵发挥作用，有助于空肠弯曲菌对多种结构上不相关的抗菌药物产生内在耐药性。
Campylobacter jejuni, a gram-negative organism causing gastroenteritis in humans, is increasingly resistant to antibiotics. However, little is known about the drug efflux mechanisms in this pathogen. Here we characterized an efflux pump encoded by a three-gene operon (designated cmeABC) that contributes to multidrug resistance in C. jejuni 81-176. CmeABC shares significant sequence and structural homology with known tripartite multidrug efflux pumps in other gram-negative bacteria, and it consists of a periplasmic fusion protein (CmeA), an inner membrane efflux transporter belonging to the resistance-nodulation-cell division superfamily (CmeB), and an outer membrane protein (CmeC). Immunoblotting using CmeABC-specific antibodies demonstrated that cmeABC was expressed in wild-type 81-176; however, an isogenic mutant (9B6) with a transposon insertion in the cmeB gene showed impaired production of CmeB and CmeC. Compared to wild-type 81-176, 9B6 showed a 2- to 4,000-fold decrease in resistance to a range of antibiotics, heavy metals, bile salts, and other antimicrobial agents. Accumulation assays demonstrated that significantly more ethidium bromide and ciprofloxacin accumulated in mutant 9B6 than in wild-type 81-176. Addition of carbonyl cyanide m-chlorophenylhydrazone, an efflux pump inhibitor, increased the accumulation of ciprofloxacin in wild-type 81-176 to the level of mutant 9B6. PCR and immunoblotting analysis also showed that cmeABC was broadly distributed in various C. jejuni isolates and constitutively expressed in wild-type strains. Together, these findings formally establish that CmeABC functions as a tripartite multidrug efflux pump that contributes to the intrinsic resistance of C. jejuni to a broad range of structurally unrelated antimicrobial agents.