Target optimization for peptide nucleic acid (PNA)-mediated antisense inhibition of the CmeABC multidrug efflux pump in Campylobacter jejuni.
Target optimization for peptide nucleic acid (PNA)-mediated antisense inhibition of the CmeABC multidrug efflux pump in Campylobacter jejuni.
复制标题
空肠弯曲杆菌中肽核酸 (PNA) 介导的 CmeABC 多药外排泵反义抑制的目标优化。
DOI：
10.1093/jac/dkt381
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发表时间：
2014
期刊：
影响因子：
--
通讯作者：
Jeon,Byeonghwa
中科院分区：
文献类型：
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作者：
Oh,Euna;Zhang,Qijing;Jeon,Byeonghwa
目的cmeabc是一种耐药-结节-细胞分裂（RND）型多药外排泵，对临床上重要的弯曲杆菌具有耐药性。本研究旨在确定反义肽核酸（PNA）抑制CmeABC的最佳靶点。方法设计18个PNAs结合到cmeabc的翻译起始区，跨越核糖体结合位点（RBS）和meabc基因的起始密码子。用不同浓度的CmeABC特异性PNAs （CmeABC-PNAs）处理空肠弯曲杆菌，并进行western blot检测CmeABC表达水平的变化。检测环丙沙星和红霉素的mic，评价CmeABC敲除对抗生素敏感性的影响。结果反义PNA显著影响CmeA和CmeB的表达，有趣的是，本研究中使用的任何CmeC-PNA均未改变CmeC的表达。CmeA-PNA靶向RBS ofcmeBand及其上游区域最有效地降低了CmeA的表达，而PNA结合RBS ofcmeBand及其下游区域时，CmeB的表达降低最为显著。CmeA-和CmeB-PNAs增加了c的易感性。空肠尼托环丙沙星和红霉素与免疫印迹法观察到的抑制水平成比例。结论该基因是反义PNA敲除CmeABC的最佳靶点。RBS是pna介导的CmeABC反义抑制的主要靶点。然而，其附近的区域也显著影响pna敲除CmeABC的有效性。
ObjectivesCmeABC is a resistance-nodulation-cell division (RND)-type multidrug efflux pump conferring resistance to clinically important antibiotics inCampylobacter. This study aimed to identify the optimal target sites for the inhibition of CmeABC with antisense peptide nucleic acid (PNA).MethodsEighteen PNAs were designed to bind to the translational initiation regions ofcmeABC, spanning the ribosome-binding site (RBS) and the start codon of thecmeABCgenes.Campylobacter jejuniwas treated with CmeABC-specific PNAs (CmeABC-PNAs) at various concentrations and subjected to western blotting to measure changes in the level of CmeABC expression. The MICs of ciprofloxacin and erythromycin were measured to evaluate the impact of CmeABC knockdown on antibiotic susceptibility.ResultsWhile antisense PNA significantly affected CmeA and CmeB expression, interestingly, CmeC expression was not altered by any of the CmeC-PNAs used in this study. A CmeA-PNA targeting the RBS ofcmeAand its upstream region reduced CmeA expression most efficiently, and CmeB expression was most significantly decreased by PNA binding to the RBS ofcmeBand its downstream region. CmeA- and CmeB-PNAs increased the susceptibility ofC. jejunito ciprofloxacin and erythromycin in proportion to the inhibition levels observed in western blotting.ConclusionsThecmeAgene is the best target to knockdown CmeABC with antisense PNA. The RBS is the major target for the PNA-mediated antisense inhibition of CmeABC. However, regions in its vicinity also significantly influence the effectiveness of the PNA-based knockdown of CmeABC.