沙门菌接合性质粒增强细菌突破肠血管屏障的机制及干预研究

Conjugative plasmid-mediated horizontal gene transfer is an important mechanism for pathogenic bacteria to establish infection in human by acquiring virulence factors and antibiotic resistance from exogenous source. As a typical facultative intracellular bacterium, Salmonella manages to adhesion, colonization and aggregation by outwitting break through the first line of defenses of gastrointestinal tract, which is mainly composed of intact gut-vascular barrier (GVB), normal flora barriers, phagocytosis, inflammatory response, and the second line of defenses mainly cellular-mediated immune response. pRST98, a chimeric plasmid originally isolated from Salmonella enterica serovar typhi, was involved in bacterial multidrug-resistance and virulence. In our preliminary study, it was shown that pRST98 could be easily transferred among different intestinal bacteria by conjugation both in vivo and in vitro. And this plasmid carries both Salmonella plasmid virulence genes (spv) and rck which is associated with biofilm formation. It is demonstrated that pRST98 enhances Salmonella adhesion and colonization, moreover, it promotes GVB dismantling induced by Salmonella which leads to a systemic immune response and severe infection. However, the exact mechanisms remain unknown. In this proposal we will dissect the underlying mechanism of GVB leakage in Salmonella infection by initiating the investigation of the role of pRST98 in commensal intestinal bacteria dysbiosis, biofilm formation and bacterial invasion into GVB. The completion of this project will provide novel insights into the development of new strategies in the prevention and treatment of Salmonella infection.

接合性质粒是细菌间遗传元件和信息传递最有效的方式和工具之一，对病原菌遗传变异、耐药性和致病性非常重要。沙门菌是典型的消化道传播兼性胞内菌，机体完整的肠血管屏障(gut-vascular barrier, GVB)、正常菌群微生态平衡和细胞免疫为主的应答是抵御感染的重要防线。项目组前期研究发现，沙门菌接合性质粒pRST98是具有介导多重耐药和毒力的“嵌合型”质粒，在体内外易在不同肠道菌间传递；pRST98上存在的沙门菌质粒毒力基因spv和与细菌生物膜形成相关的rck基因，能促进宿主菌在体内定植并穿透GVB导致系统感染，但确切机制不清楚。本课题以pRST98为切入点，用野生株、质粒消除株和经接合转移重新导入pRST98的接合子，建立细胞和小鼠感染模型，从肠道共生菌微生态失衡和细菌生物膜形成两方面，探讨其增强沙门菌突破GVB的作用机制及干预手段，为有效防治沙门菌等肠道病原菌感染提供新策略和靶点。

沙门菌在自然界广泛分布，是一种典型的经消化道传播病原菌，可引起人和动物多种疾病。沙门菌感染每年引起约1.8亿例腹泻病，导致约30万例腹泻相关死亡病例，在全球四大人类腹泻病病因中，沙门菌致死率居首。我国由沙门菌引起的食物中毒病例高达细菌性食物中毒病例总数的70-80%。沙门菌在抗生素压力下不断进化，并可通过接合转移等方式将耐药和毒力基因等在不同细菌间进行水平传递，导致新的耐药株和毒力株频现，是一个巨大的公共卫生安全隐患。.本课题组从上世纪八十年代暴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从spv功能性片段spvB破坏肠黏膜机械屏障、干扰宿主铁代谢，及spv影响宿主固有免疫和获得性免疫应答等多方面开展研究，系统深入地探索了沙门菌的致病作用及分子机制。.NLRP6是胞内模式识别受体NLRs家族成员，对维持肠道微生态稳定至关重要。本项目研究发现沙门菌感染可通过上调肠上皮细胞内NLRP6破坏细胞间连接，促进细菌突破肠黏膜屏障和肠外播散。spvB可介导肠上皮紧密连接蛋白表达失调和肠黏膜机械屏障受损，该过程与F肌动蛋白重排和PKC信号通路密切相关。铁是沙门菌繁殖和致病的必需营养物质，铁剥夺是机体营养免疫的重要组成部分，在抵抗沙门菌感染中十分重要。本项目发现SpvB效应蛋白可通过Hamp/FPN调节轴依赖和非依赖两种途径，影响宿主细胞内铁代谢和系统性铁代谢，以利于自身存活和繁殖。此外，沙门菌应对机体免疫应答时，spv基因可通过抑制自噬阻抑Ⅰ型干扰素反应和中性粒细胞趋化，亦可通过抑制Th1型细胞基因转录和细胞因子表达，导致Th1/Th2免疫应答向Th2偏移，从而加重感染。.相关成果发表论文7篇，培养研究生10名，授权专利1项。