抗生素诱导鲍曼不动杆菌多重耐药AdeABC外排泵中核糖开关研究

The AdeABC efflux pump, which is regulated by the AdeRS two-component system, plays important role in multidrug-resistant (MDR) A.baumannii. Our recent work showed that the expression of adeC in A.baumannii was increased at the level of transcription after antibiotic induction. There was a similar riboswitch structure of hairpin between adeB and adeC. Based on our preliminary data and our recent publication of an aminoglycoside-binding riboswitch, we propose that adeC riboswitch can regulate the efflux pump genes in MDR A.baumannii.We will construct adeC riboswitch structural model and locate the antibiotic binding site by 1) the construction of the reporter gene expression system to verify the riboswitch function in bacteria, and 2) the interaction between the riboswitch and antibiotics. Based on our experimental data and the use of bioinformatic tools, the research in this application will lead to the identification of adeC riboswitch controling the expression of the AdeABC efflux pump genes or AdeRS. The overall objective is to understand the mechanism of inducible antibiotic resistance for developing new antibiotic targets and it will contribute to developing novel treatments for the infection diseases caused by MDR Acinetobacter baumannii.

由AdeRS调节的AdeABC外排泵在介导鲍曼不动杆菌多重耐药中发挥重要作用。本课题组成员研究证实核糖开关调控氨基糖甙类抗生素耐药基因表达，我们前期研究显示抗生素诱导AdeABC中adeC高表达，而adeB和adeC基因间存在核糖开关相关非翻译区及类似核糖开关的发夹结构，因此我们提出多重耐药鲍曼不动杆菌中adeC核糖开关调控AdeABC外排泵基因表达的假说。本课题拟通过载体构建、细菌体内功能实验，核糖开关与抗生素体外相互作用等方法，确定adeC核糖开关构象变化模型，明晰adeC核糖开关及寻找抗生素和adeC核糖开关结合位点。通过阐明adeC核糖开关调控AdeABC外排泵或AdeRS表达，揭示核糖开关在多重耐药外排泵机制中的作用，为发现新的药物作用靶点，研制新的抗菌药物及指定新的治疗策略奠定基础。

鲍曼不动杆菌的外排泵是介导细菌产生多重耐药的重要机制之一。课题组研究了鲍曼不动杆菌对碳青霉烯类、氨基糖苷类、喹诺酮类抗菌药物耐药机制及对消毒剂敏感性降低生物学特征，研究发现blaOXA-23、blaOXA-51、armA、gyrA和parC分别是碳青霉烯类、氨基糖苷类和喹诺酮类抗菌药物耐药的主要原因。课题组应用Real-time PCR鉴定了多重耐药菌株adeA、adeB、adeC、adeR、adeS基因存在与多重耐药产生的相关性，检测了标准株和临床株抗菌药物诱导前后adeABC外排泵基因的表达量变化；在鲍曼不动杆菌8种RND外排泵介导替加环素耐药表型的研究中表明adeB不仅介导替加环素耐药还在多重耐药的产生起到重要作用；运用BLAST、mfold及核糖开关预测软件对鲍曼不动杆菌adeR/adeA 、adeC转录产物前导序列和二级结构进行保守性进行了分析,发现adeR/adeA 5’端UTR和adeC前导序列核糖开关相关UTR具有高度的保守性且adeB-adeC、adeR-adeA间存在稳定非编码RNA发夹结构；运用基因敲除技术获得多重耐药鲍曼不动杆菌adeA、adeB、adeC、adeRS、adeR/adeA 5’端UTR和adeC 5’端核糖开关相关UTR 的基因敲除株，比较基因敲除前后菌株MIC值变化、生物被膜形成能力及外排泵基因adeA、adeB、adeC、adeR、adeS表达量，初步证明adeR/adeA 5’UTR和adeC 5’核糖开关相关UTR在调控鲍曼不动杆菌耐药性中起到重要作用。课题组还深入研究了鲍曼不动杆菌碳青霉烯类耐药机制、氨基糖苷类耐药机制、喹诺酮类耐药机制、消毒剂耐药机制、医院流行病学情况、生物被膜的形成、中药抗菌活性研究等，为医院鲍曼不动杆菌感染传播的控制提供新的方法和实验依据。课题组按计划初步完成了相关研究目标，并将继续深入adeR/adeA 5’UTR和adeC 5’核糖开关相关UTR 的非编码RNA对细菌耐药性调控机制研究。