肺炎克雷伯菌高毒力株分泌蛋白Hcp1调控PMN凋亡机制及其在脓毒症继发迁徙性感染中的作用

Hypervirulent clinical variant of Klebsiella pneumoniae (hvKP) has been documented as the major pathogenic cause of pyogenic liver abscess, which often complicated by sepsis and metastatic infection and posed a serious health threat to human beings. Delayed neutrophil apoptosis is widely accepted to be associated with the progression and prognosis of sepsis. Our previous study demonstrated that the Hcp1 protein secreted by hvKP was a important effector of T6SS and could inhibit the expression and activation of caspase-3 to cause the delayed netrophil apotosis. However, which signaling pathway the Hcp1 protein use to inhibit the expression and activation of caspase-3, and how to affect the sepsis and the secondary metastatic infection and the prognosis by this process remain unclear. In this project, based on our previous study, we establish the murine model of sepsis secondary metastatic infection, choose the Hcp1 protein as the breakthrough point, set the delayed neutrophil apoptosis as the main line, and capture the important aspects of the caspase-3 apoptosis singaling pathway, in order to further demonstrate the mechanism of delayed neutrophil apoptosis regulated by the Hcp1 protein from the molecular, cellular and animal level and give us a further understanding for the effect of Hcp1 protein in septic metastatic infection and prognosis in sepsis. Hope, the research is expected to provide new insights into the exploration of new targets for the development of preventive or therapeutic drugs and/or vaccines against hvKP infection.

肺炎克雷伯菌高毒力株（hvKP）是原发性肝脓肿的重要病原菌，常可并发脓毒症及迁徙性感染，严重威胁人类健康。中性粒细胞（PMN）凋亡延迟是影响脓毒症进展及预后的关键。课题组前期研究发现，hvKP可通过VI型分泌系统特异分泌Hcp1蛋白，以抑制PMN内Caspase-3的表达及活化，引发凋亡延迟。但Hcp1蛋白通过何种途径抑制Caspase-3的表达及活化，这一过程对hvKP脓毒症继发迁徙性感染及预后有何影响目前还不清楚。本项目拟在前期基础上，通过建立小鼠脓毒症继发迁徙性感染模型，利用Hcp1蛋白为切入点，以PMN凋亡延迟为主线，抓住Caspase-3凋亡通路的重要环节，分别在分子、细胞和动物水平探讨解析Hcp1蛋白调控PMN凋亡的机制，阐明Hcp1蛋白对hvKP脓毒症继发迁徙性感染过程及转归的影响，为寻找新型干预hvKP感染的药物靶标提供新思路。

近年来高毒力肺炎克雷伯菌严重危险人类健康，其极易并发脓毒血症及迁徙性感染并严重影响患者预后。本研究拟通过比较基因组学、基因敲除和回补，结合细胞和动物模型研究，阐明分泌蛋白Hcp1在高毒力肺炎克雷伯菌诱导中性粒细胞凋亡延迟中的作用及信号通路，并进一步探讨其对耐药形成的影响。研究首次发现bcl-2/bax-Caspase-3-NFκB信号通路在高毒力肺炎克雷伯菌诱导中性粒细胞凋亡延迟中发挥重要作用，还可显著增强中性粒细胞呼吸爆发功能，促进炎症因子的释放。首次发现了江西地区CR-hvKP菌以产KPC酶为主，主要为ST11型，CR-hvKP菌株的质粒多态性显著高于CS-hvKP菌株，Souther blot显示毒力基因与碳青霉烯酶基因的质粒播散是江西地区CR-hvKP菌株的主要耐药及致病机制。首次基因组学分析发现江西地区部分CR-hvKP菌株可能形成机制有四种：1）cKP菌株通过获取毒力质粒而形成CR-hvKP菌株，其主要流行在ST11型菌株中。2）hvKP菌株通过获取碳青霉烯酶质粒而形成CR-hvKP菌株，其主要流行于ST23、ST65等hvKP菌株中。3）通过碳青霉烯酶质粒和毒力质粒共同聚集于肺炎克雷伯菌而形成CR-hvKP菌株，此机制还促使了融合了毒力和耐药为一体的质粒在肺炎克雷伯菌中流行，提升了其耐药及毒力传播的高效性。4）首次发现毒力质粒可以在肺炎克雷伯菌中传递，表明毒力质粒的传播可增加CR-hvKP菌株的形成的可能性和广泛性。以上研究明确了质粒播散在CR-hvKP菌株形成中发挥着重要的作用，为控制CR-hvKP菌的产生和流行奠定理论基础。本项目研究期间，协助培养博士研究生2名。发表相关论文20篇，其中SCI7篇。