沙门菌spvB基因介导巨噬细胞迟发性焦亡及其加重炎症损伤的信号通路研究

The interaction between bacteria and their hosts has emerged as one of the most exciting areas of pathogen biology. The high incidence of Salmonella infection poses a considerable threat to public health globally. Salmonellae are facultative intracellular bacteria and mainly reproduce in macrophages during natural infections. Therefore, macrophages play an important role in Salmonellosis. It was reported that Salmonella plasmid virulence gene spvB could mediate delayed macrophage pyroptosis, while the exact mechanism remained to be elucidated. Our preliminary study showed that spvB could suppress autophagosome formation of macrophages and inhibit the activation of NLRC4 inflammasome, meanwhile, restrain both the degradation of IκBα and the activation of NF-κB at the early stage of infection. However, it could stimulate NLRP3 inflammasome later. These findings suggested that the crosstalk between autophagosome and inflammasome as well as IκBα/NF-κB pathway played vital roles in the delayed macrophage pyroptosis. In this research project, Salmonella-macrophage interaction mediated by spvB would be regarded as the breakthrough point, CRISPR/Cas9 gene editing technique, drug interventions, etc., are used in cells and animal infection models to illuminate the underlying molecular mechanisms of delayed macrophage pyroptosis induced by spvB via NLRC4/Beclin 1/ROS/NLRP3 and IκBα/NF-κB/pro-IL-1β/NLRP3 signal pathways. The project is aimed at revealing the unique contribution of spvB to pathogenesis of Salmonella and casting a new light on the crosstalk between autophagy and inflammation. It will give novel insights into bacterial virulence factors and has potential application in the development of new vaccines and drugs used for prevention and treatment of infectious diseases.

沙门菌是食源性致病菌代表，造成人类和动物多种疾病。巨噬细胞(MΦ)是其在体内寄居的主要场所，对MΦ的破坏直接影响感染进程。国外学者报道沙门菌质粒毒力基因spvB可致MΦ迟发性焦亡，但机制尚未阐明。本课题组首次发现spvB抑制MΦ自噬体形成，预研实验显示其在感染早期尚可抑制炎性体NLRC4活化、IκBα降解和NF-κB激活，后期则活化炎性体NLRP3。据此提出：自噬体与炎性体的相互作用及IκBα/NF-κB通路对spvB介导的MΦ迟发性焦亡作用关键。本项目拟结合CRISPR/Cas9基因编辑技术和药物干预等手段，从分子、细胞和整体水平，研究spvB通过NLRC4/Beclin1/ROS/NLRP3和IκBα/NF-κB/pro-IL-1β/NLRP3通路导致MΦ迟发性焦亡、加重炎症损伤的机理，旨在阐明spvB调控自噬与炎症反应的信号途径，揭示其致病新机制，为防治细菌感染开辟新思路并提供依据。

沙门菌经消化道传播，与人类健康和公共卫生关系极为密切。作为典型的兼性胞内菌，深入研究其与宿主细胞的互作，对形成新的抗感染策略具有重要意义。沙门菌质粒毒力基因spvB的编码蛋白（SpvB）在沙门菌毒力岛2编码的Ⅲ型分泌系统（T3SS-2）作用下分泌至宿主细胞。既往文献报道，SpvB影响细菌在细胞内的增殖和存活，导致巨噬细胞迟发性焦亡，但相关机制不明。本项目发现SpvB羧基端结构域介导的ADP核糖基转移酶活性所致细胞骨架解聚，可抑制沙门菌感染的巨噬细胞形成自噬体进而抑制自噬通量，抵抗细胞通过自噬清除细菌，促进沙门菌在巨噬细胞内存活，与其所致细胞迟发性焦亡、增强细菌在实验动物体内外的致病性密切相关；SpvB下调NF-κB信号通路的重要蛋白IKKβ的水平并抑制其磷酸化，进而抑制IκBα的降解和NF-κB通路的激活，使炎性体NLRP3及NLRC4活化的第一信号降低，抑制其活化，导致巨噬细胞焦亡的延迟。利用CRISPR/Cas9技术构建的敲除nlrp3基因的巨噬细胞感染模型，发现NLRP3是沙门菌spvB基因导致巨噬细胞迟发性焦亡的重要因素。此外尚发现spvB基因可下调巨噬细胞的铁输出蛋白FPN，使胞内铁含量增多，促进细菌增殖；spvB基因可协同spvC基因抑制巨噬细胞焦亡，促进沙门菌播散，加重全身感染。本项目围绕沙门菌spvB基因对巨噬细胞焦亡的影响及机制开展的系统研究，发现该基因介导细菌毒力具有多重效应，有助于深层了解细菌的致病机制，推进在宿主抗感染免疫应答领域获得新发现。