肺炎克雷伯菌对替加环素的新耐药机制研究

Published studies have demonstrated that tigecycline resistance mainly conferred by the overproduction of AcrAB efflux pump in Enterobacteriaceae. In our previous study, the mechanisms of 10 tigecycline-resitant Klebsiella pneumoniae have been explored, and the results revealed that the presented resistance mechanisms were absence in 6 of them, suggesting the presence of alternative resistance mechanisms in these isolates. This study aims to: 1) detect the difference of gene expression and mutational genes between tigecycline-resistant and -susceptible K. pneumoniae using RNA-seq and whole-genome sequencing, respectively, 2) find the change of the outer membrane proteins in tigecycline-resistant and -susceptible K. pneumoniae by proteomics and western blotting, 3) analyze the difference in the local regulatory sequence of the new tigecycline resistance gene, and then carry out gene clone, gene knockout, and site-directed mutagenesis to know the role of the local factors; the latter methods are used to confirm whether the common global transcriptional activators play a role in the tigecycline resistance mediating by the new gene, and finally identify the regulatory pathway of the novel resistance mechanism. Further study on the tigecycline resistance mechanisms will be helpful for clinical rational use of this agent, and provide clues for the development of new drugs.

现有研究提示，肠杆菌科细菌对替加环素的耐药主要由多药外排泵AcrAB的过表达引起。但申请人前期对10株替加环素耐药肺炎克雷伯菌的研究发现，有6株菌的耐药机制不能用现有机制解释，提示存在新的耐药机制。本研究拟：①通过基因转录组测序及全基因组测序，发现替加环素敏感与耐药肺炎克雷伯菌（包括临床及体外诱导耐药株）的差异表达基因及差异基因，再通过基因敲除或定点突变，确定新的替加环素耐药基因；②通过蛋白质组学和蛋白质印迹，发现替加环素耐药菌的差异表达外膜蛋白，采用敲除技术确定与耐药相关的外膜蛋白；③比较耐药与敏感菌株新耐药基因上游调控序列的差异，通过克隆、基因敲除/定点突变，了解新基因的局部调控通路；对常见整体调控子分别予以基因敲除或定点突变，确定调控新耐药基因的整体转录激活子，从而阐明新耐药基因的调控通路。通过对替加环素耐药机制的深入研究将为该药的临床合理应用提供支持，为新药的研发提供思路。

以往研究表明，肠杆菌科细菌对替加环素耐药主要由整体转录激活子RamA表达上调，导致多药外排泵AcrAB过表达而产生。本项目对替加环素不敏感的肺炎克雷伯菌的耐药机制开展研究。在临床分离菌中发现了不能用现有耐药机制解释的替加环素耐药肺炎克雷伯菌。通过基因敲除及回补、凝胶迁移实验等发现RamA蛋白可以与acrA和oqxB启动子的相互作用，RamA同时调控AcrAB及OqxAB 2个外排泵，首次阐明了RamA介导的肺炎克雷伯菌对替加环素耐药的外排泵调控新机制。在对临床治疗过程中收集的一对由替加环素敏感到耐药的肺炎克雷伯菌的研究中，首次发现外排泵表达抑制子 RamR 基因上游核糖体结合位点(RBS)丢失，导致外排泵（AcrAB）高表达而致肺炎克雷伯菌对替加环素的耐药。研究成果对于进一步明确肺炎克雷伯菌临床分离菌对替加环素的耐药机制、特别是外排泵的调控机制具有重要的意义。. 课题总体按原计划完成，到达预期目标。课题执行过程中，增加了对肺炎克雷伯菌替加环素体内耐药机制的研究内容。相关研究成果发表SCI论文10篇，2篇文章在投。获得授权国家发明专利1项。项目负责人在本领域大型国际会议ECCMID作特邀专题报告1次，研究成果作大会口头交流1次。课题组成员4名获得国家自然自然科学青年科学基金项目；培养博士研究生10名。项目负责人担任感染性疾病领域顶级杂志CID中国特刊主编，就中国当今耐药形势与挑战发表评述与综述，并组织我国的多学科专家，牵头制定广泛耐药(XDR)革兰阴性杆菌感染防治共识，指导临床耐药菌感染的抗菌治疗。