RHH超家族转录因子对耐药质粒复制的调控机制研究

A number of resistance genes are located on plasmids, conferring antimicrobial resistance in bacteria. In previous investigations of our group it was found that the overproduction of KPC carbapenemases caused by the increasing copy number of blaKPC-carrying plasmids significantly decreased the susceptibility of KPC-Kp to ceftazidime/avibactam. Proteomics analysis revealed a new member of RHH superfamily of transcription factors, which was designated CopC, was involved in regulation of resistance plasmid replication. With blaKPC-carrying plasmids as the model resistance plasmids, this study aims to: 1) analyze the correlation between the prevalence of copC in clinical isolates with their susceptibility to antibiotics and the sequence types, respectively; 2) illustrate the DNA target of CopC in plasmid replicon and the mode of interaction. To solve the crystal structure of CopC and its DNA target complex, and to screen the cofactors involved in CopC’s regulation and to determine their biological functions to elucidate the regulation pathway of CopC in resistance plasmid replication; 3) investigate the coordinated regulation of resistance plasmid replication between CopC and the replicon encoded local regulator, CopB, to better address the regulation network of RHH transcription factors. This research focuses on the interaction between chromosome and resistance plasmids to investigate the mechanisms of the development of antimicrobial resistance and the related regulation pathway, posing theoretical and practical significance.

许多耐药基因由质粒携带，介导细菌耐药。本课题组前期研究发现，携带blaKPC耐药基因质粒拷贝数升高导致过度产生碳青霉烯酶KPC，是造成肺炎克雷伯菌对阿维巴坦/头孢他啶合剂敏感性下降的主要原因。蛋白组学分析提示，染色体编码的新型RHH超家族调控因子CopC（本课题组暂命名）参与耐药质粒复制的调控。本项目以KPC耐药质粒为模式质粒：①通过流行病学分析copC基因与各类抗菌药的耐药性及菌株ST分型的关系。②明确CopC直接结合靶核酸序列及其作用模式，解析CopC-靶序列复合物晶体结构，确定相关的辅助蛋白及其功能，从而阐明以CopC 为核心的耐药质粒复制的调控通路。③探讨CopC与质粒复制子自身编码的局部调控子CopB蛋白对耐药质粒复制的协同调控作用，完善RHH超家族转录因子对耐药质粒复制的调控网络。本项目从染色体与耐药质粒的相互作用出发，探讨细菌耐药的形成及调控机制，具理论及实用价值。

本研究发现并鉴定了一个新型乙酰基转移酶类II型毒素-抗毒素（Toxin-Antitoxin systems, TA）系统，并将其命名为KtaA-KtaT（原称CopC-GANT），KtaT通过乙酰化修饰tRNA发挥毒素作用，而ktaA过表达可以解除KtaT带来的生长抑制。ktaA-ktaT基因敲除株生物膜形成能力明显增强，考虑生物膜形成与细菌耐药及临床感染反复发作发挥重要作用，故调整研究方向为新型TA系统参与肺炎克雷伯菌中生物膜形成新型调控通路研究。在此基础上深入研究：通过基因敲除及转录组学测序描绘生物膜形成新调控通路网络：KtaA-KtaT负调控III型菌毛基因、pgaA及EPS，以调控生物膜形成；通过流行病学分析发现肺炎克雷伯菌CG258克隆复合群为携带KtaA-KtaT及KPC碳青霉烯酶的优势克隆；采用迁移凝胶电泳实验明确其结合靶核苷酸序列为CGTAATGACATCTGCGTTACG；进而通过解析KtaA-KtaT复合物结构，发现4分子KtaA与2分子KtaT形成异源六聚体，KtaA通过影响KtaT二聚体形成发挥其拮抗作用，为抗菌药物研发提供结构基础。.发表SCI论文12篇。获得授权国家发明专利1项。培养博士研究生13名，硕士研究生6名。课题组成员2名获得国家自然自然科学青年科学基金项目；1人获得上海市浦江人才，1人获扬帆计划资助。