sRNA18-AdeN-AdeIJK通路介导鲍曼不动杆菌对碳青霉烯类抗生素耐药的作用机制研究

The number of carbapenem-resistant Acinetobacter baumannii (CRAB) has been growing increasingly. The strains which do not produce carbapenemases bring big challenges in clinical infection control. The resistant mechanism of these CRAB strains is not known. Our previous work found that in these strains, the expressions of AdeIJK pump and its repressor AdeN were upregulated and downregulated, respectively. And AdeN was proofed to be able to induce carbapenem resistance in this kind of CRAB strains. Moreover, the amount of sRNA18, which regulated the expression of AdeN, was increased under the exposure of carbapenem. Therefore, we presume that sRNA18 promotes the overexpression of AdeIJK by inhibiting the expression of AdeN, thus results in carbapenem resistance in these strains. In this study, by using the methods of gene knock-out and knock-in, MIC, et al, the roles that AdeN and AdeIJK play in causing carbapenem resistance in A. baumannii will be investigated. By using the methods of molecular biology and bioinformatics, the upregulation of sRNA18 will be verified, and the hypothesis that sRNA18 could affect carbapenem resistance in A. baumannii through regulating the expressions of AdeN and AdeIJK will be confirmed. By using the methods of RNA footprinting, compensatory base-pair exchange, gel mobility shift assay and mRNA decay rate assay, the molecular mechanism that sRNA18 inhibits the expression of AdeN will be revealed. This study presents a new mechanism in CRAB, thus helps to develop new drugs and control infection.

不产碳青霉烯酶的耐碳青霉烯类鲍曼不动杆菌是控制临床感染的一大难题，其耐药机制尚不清楚。我们前期研究发现，主动外排泵AdeIJK过表达、其抑制性调控因子AdeN低表达参与这类细菌的耐药作用，并且发现抗生素压力下调控AdeN表达的sRNA18上调。故提出科学假说：sRNA18通过抑制AdeN的表达，造成AdeIJK过表达，导致鲍曼不动杆菌对碳青霉烯类抗生素耐药。本课题通过基因敲除与基因插入等技术，明确AdeN及AdeIJK在鲍曼不动杆菌对碳青霉烯类抗生素耐药中的作用；利用分子生物学和生物信息学等方法验证上游调控因子sRNA18的表达，并揭示sRNA18通过调控AdeN、AdeIJK的表达影响细菌耐药的机制；采用RNA足迹、mRNA衰退率检测等方法，阐明sRNA18抑制AdeN表达的分子机制。该研究提出了鲍曼不动杆菌对碳青霉烯类抗生素耐药的一种新的分子机制，为新药研发及耐药菌的控制提供新思路。

背景：不产碳青霉烯酶的耐碳青霉烯类鲍曼不动杆菌是控制临床感染的一大难题，其耐药机制尚不清楚。主动外排泵系统AdeN-AdeIJK参与这类细菌的耐药作用，但具体作用及调节机制尚不明确。.研究内容和结果：1. 对临床菌株进行检测发现，在美罗培南刺激下仅耐药菌的adeN和adeJ基因表达改变，其中所有adeN基因表达均不同程度下降，而adeJ基因表达均升高。在模式菌株中对adeN和adeJ进行基因敲除和回补，并检测其碳青霉烯类抗生素的MIC，发现adeN缺失后细菌对美罗培南耐药，恢复adeN的表达后MIC值降低，细菌对美罗培南恢复敏感性；adeJ缺失后细菌对美罗培南敏感，恢复adeJ的表达后MIC值升高，细菌对美罗培南恢复耐药性，证实AdeN-AdeIJK系统在鲍曼不动杆菌对碳青霉烯类耐药中的作用。2. RNA-Seq、Northern blotting及Real-time PCR等结果均证明可能参与调控adeN表达的sRNA18在药物刺激下表达升高。利用RACE技术明确sRNA18在鲍曼不动杆菌基因组中的位置后，对其进行基因敲除和回补，发现sRNA18的缺失不影响细菌的生长，但细菌的美罗培南MIC值降低，药物敏感性增加；恢复sRNA18表达后MIC值回复。同时发现sRNA18缺失后adeN的表达量升高，而adeJ的表达量下降，证实sRNA18通过调控AdeN-AdeIJK的表达介导细菌对碳青霉烯类抗生素耐药。3. 构建adeN 5’UTR-zeoR表达报告载体，并在adeN 5’UTR区及sRNA18结合区分别构建点突变，检测zeoR表达，发现结合位点突变后zeoR基因的表达发生改变，证明sRNA18与adeN 5’UTR区结合对后者进行表达调控。RAP检测提示Chaperoion蛋白参与sRNA18对adeN mRNA的协同调控。.结论：1. AdeN-AdeIJK系统参与鲍曼不动杆菌对碳青霉烯类抗生素耐药。2. sRNA18通过调控AdeN-AdeIJK的表达介导细菌对碳青霉烯类抗生素耐药。3. sRNA18与adeN 5’UTR区结合进行表达调控。