鲍曼不动杆菌Omp34蛋白表达上调及OmpA蛋白表达下调与其致病性相关研究

Acinetobacter baumannii is an important gram-negative opportunistic pathogens, its pathogenesis has become an important scientific challenges in the current international field. Our group analyst the membrane proteins of carbapenem-sensitive Acinetobacter baumannii(CSAb)and carbapenem-resistant Acinetobacter baumannii(CRAb) after clinical imipenem treatment of one patient. The preliminary experiment identified an unknown 34KDa efflux protein upregulation. In addition, OmpA protein was deteced downregulation. Based on the above conclusions ，we speculate: in resistant strain, efflux protein upregulation enhanced its antimicrobial resistance; OmpA protein downregulation weakened its pathogenic, which is one of the self-protection mechanisms of confronting antibiotic pressure. On the basis of the above study, our group respectively knockout Omp34, OmpA and induce its expression through complementray. With C.elegans model and mouse infection model, using two-dimensional electrophoresis、Western blot and plasmid transfection, from the level of molecules、cells and the whole animal investigate the role of efflux pump Omp34 and OmpA protein in the pathogenic of Acinetobacter baumannii, laying the foundation of better clarifying its pathogenic mechanisms. This is of great significance to the discovery of new drug targets and control strategies of nosocomial infections.

鲍曼不动杆菌是一重要革兰阴性条件致病菌，其致病机制研究已成为目前国际该领域面临的重要科学难题。本课题组前期研究：收集同一病人经亚胺培南治疗前后碳青霉烯敏感菌和耐药菌，进行膜蛋白分析，在耐药菌中发现34KDa外排蛋白（Omp34）表达上调的同时，检测到OmpA蛋白表达下调。根据上述结果推测：在耐药菌中，外排蛋白高表达，增强其对抗菌药物的耐药；OmpA蛋白低表达，减弱其致病能力，是细菌对抗外界抗生素压力的一种自我保护机制。本课题组将在上述研究基础上，分别通过基因敲除Omp34与OmpA及其回补实验诱导其表达，采用秀丽隐杆线虫模型及小鼠感染模型，二维电泳、Western blot、质粒转染等手段，从分子、细胞及动物整体水平等多方面探讨Omp34蛋白与OmpA蛋白在鲍曼不动杆菌致病中的重要作用，为更好阐明鲍曼不动杆菌的致病机制奠定基础。这对发现新的药物作用靶点、有效控制院内感染具有重要意义。

鲍曼不动杆菌是重要的院内感染致病菌，碳青霉烯类耐药鲍曼不动杆菌日益增多，是临床抗感染治疗的一大难题。我们前期研究发现同一病人经亚胺培南治疗前后碳青霉烯敏感菌和耐药菌，进行膜蛋白分析，在耐药菌中发现34KDa外排蛋白（Omp34）表达上调的同时，检测到OmpA蛋白表达下调。我们分析鲍曼不动杆菌对常用抗菌药物的敏感性；采用多位点序列分析和eBURST方法对菌株进行同源性分析；PCR方法检测菌株耐药基因携带情况；并用AB–PBRT法对菌株进行质粒分型；通过双重同源重组技术构建ATCC 19606-△Omp34基因敲除株并用SDS-PAGE蛋白电泳验证，对野生株和敲除株进行药物敏感性试验。研究结果发现临床分离鲍曼不动杆菌对碳青霉烯类耐药率较高，仅对多粘菌素和替加环素等保持较高敏感性；多位点序列分析显示ST208型是主要克隆株；eBURST分析发现临床存在CRAB菌株播散流行（CC92）；blaoxa-23耐药基因表达在鲍曼不动杆菌碳青霉烯类耐药中发挥重要作用；Southern杂交证实blaoxa-23位于质粒上；CRAB菌株均检测到1-3个质粒复制酶基因，其中GR6 (repAci6)最常见。共检测到两种blaOXA-23所在转座子：Tn2006 和Tn2008，大部分是Tn2008。接合试验显示blaoxa-23耐药基因可通过Tn2008转座子上载高活动能力的质粒，导致CRAB传播。此外，研究显示Omp34外膜蛋白低表达仅仅是引起碳青霉烯类耐药的辅助因素。