沙门菌毒力基因spv对免疫逃逸和动员肠道干细胞损伤修复功能的影响及干预研究

Salmonella infection poses great threat to the health of humans and animals in the world. As the typical facultative intracellular bacteria, Salmonella spread through gastrointestinal tract. The intact intestinal mucosa, phagocytes, antigen presenting cells and the cell immune response compose an important line of defense against Salmonella infection. Our preliminary studies showed that pRST98, a plasmid originally isolated from Salmonella enterica serovar typhi is transferable among enteric bacilli by conjugation, and involved in both bacterial multidrug-resistance and virulence. We found that this chimeric plasmid pRST98 carrying the Salmonella plasmid virulence gene (spv), inhibits the immune response mediated by dendritic cells (DC), promotes bacteria colonization and diffusion, and reduces colony formation of intestinal stem cells, eventually leading to serious intestinal tissue damage. However, the detailed mechanism still remains unknown. Based on our preliminary results, spv gene deletion strains and complementation strains were constructed to infect cells and mice to study the molecular mechanisms of spv effects on DC antigen recognition and intestinal stem cell repairing functions, and to elucidate the role of proteasome and mTOR in the intestinal tissue damage caused by Salmonella infection, with the research starting from the bacterial immune escape and the reduced repair ability of intestinal epithelia. The completion of this project will provide us new insight into the development of new strategies in the prevention and treatment of Salmonella infection via regulating immune escape and intestinal damage repair.

沙门菌感染严重危害人类和动物健康。作为一种典型的消化道传播兼性胞内菌，完整的肠黏膜上皮、吞噬细胞、抗原递呈细胞和以细胞免疫为主的适应性免疫应答是机体抵御沙门菌感染的重要防线。项目组前期研究发现，源自伤寒沙门菌的可接合性多重耐药质粒pRST98，具有介导细菌耐药性和显著增强宿主菌毒力的双重功能。这种"嵌合型"质粒上存在的沙门菌质粒毒力基因spv，能抑制树突状细胞（DC）免疫应答，促进宿主菌在体内定植和扩散，降低肠道干细胞克隆形成能力，导致严重肠道损伤，但确切机制不甚清楚。本项目在前期工作基础上，以免疫逃逸和损伤修复能力下降引起肠道损伤为切入点，用spv基因构建的重组沙门菌建立细胞模型和小鼠动物模型，系统研究spv影响DC抗原识别和肠道干细胞动员的分子机制；揭示干预蛋白酶体降解和mTOR等方法在spv导致肠道损伤中的作用；为通过靶向调控免疫逃逸和损伤修复能力从而防治沙门菌感染提供新策略和依据。

伤寒沙门菌所致的人类伤寒在发展中国家的发病率和死亡率仍居高不下。本课题组从上世纪八十年代爆发流行的临床多重耐药菌株中，分离到一个可接合传递的大质粒，命名为pRST98，并发现其具有其他致病性沙门菌毒力质粒上的高度保守序列spv，为本研究提供了具有我国特色的基因资源。本项目通过体内外模型探究spv对细菌免疫逃逸和肠道干细胞损伤修复功能的影响，以及spv对机体免疫应答的影响及机制，提出通过调控细胞自噬等来增强机体免疫功能，控制感染发生发展的新策略。.沙门菌经口进入机体首先接触肠上皮细胞，随后寄居于树突状细胞（DC）等抗原递呈细胞躲避免疫杀伤，进而播散、定植和侵染，引起持续炎症和严重肠道损伤。本项目发现spv可通过干扰DC的NLRP6表达，影响其识别和递呈抗原的能力，促进细菌扩散增殖，损伤肠黏膜；另一方面spv抑制肠道干细动员更新，肠道组织修复能力下降，损伤加重。作为固有免疫和适应性免疫的关键连接，DC识别细菌后激活固有免疫引起炎症反应和适应性免疫应答。斑马鱼感染模型结果显示，spv通过抑制中性粒细胞和巨噬细胞的趋化、聚集及吞噬杀伤，利于细菌逃逸固有免疫反应，并介导适应性免疫向Th2偏移，加重感染。对spv主要功能片段spvB基因的研究发现，spvB可能通过破坏细胞间连接促进宿主菌突破肠黏膜屏障从而加速感染进程。.自噬在抗感染中发挥天然免疫应答的作用，宿主细胞通过自噬清除细菌，细菌亦可通过某些机制逃避自噬，某些胞内菌还可通过诱导宿主细胞自噬促进自身存活。项目组发现spvB能通过解聚细胞骨架抑制自噬体形成，从而影响胞外诱捕网生成，逃逸机体免疫反应，加重炎症损伤。spv的重要功能片段spvC基因在感染早期可能通过ERK信号通路抑制宿主细胞自噬，促进胞内菌增殖；spvC亦可通过抑制自噬促进沙门菌在斑马鱼体内增殖和播散，加重感染和肠道病变。.相关成果发表论文8篇，培养研究生8名，授权专利1项。