河弧菌VI型分泌系统致病机制研究及功能性效应蛋白-免疫分子的筛检

Vibrio fluvialis is an emerging foodborne pathogen and has been implicated in both outbreaks and sporadic cases of acute diarrhea with clinical symptoms similar to those of Vibrio cholerae, as well as various extra-intestinal infections. Despite increasing public health concern about V. fluvialis, knowledge about its virulence factors, pathogenesis and environmental fitness is still limited. Bacterial pathogenicity is closely related to its secretion system, quorum sensing (QS) system and environmental survival. Based on our previous genome sequencing analysis of a clinical isolate of V. fluvialis, we studied QS regulatory cascades and the regulation on the virulence, and characterized the genetic contents, functions, and the environmental conditions that control the expression of VflT6SS2, one of the two predicted type VI secretion systems (T6SSs) in V. fluvialis. In this study, we intend to further investigate the physiological function of T6SS in the virulence, pathogenesis and environmental survival of V. fluvialis, regulation mechanism and the crosstalk with other regulation systems. Research will be launched from the following aspects: to determine the roles of VflT6SS2 component genes in the pathogenesis and interbacterial virulence or competition of the bugs; to find out the environmental stimuli or host factors which regulate the VflT6SS2 expression; to explore the potential effectors and immunity effectors of the VflT6SS2, and the function and action mechanism of these molecules; to understand the crosstalk between VflT6SS2 and other vital virulence regulation systems, such as QS system, and important global regulators; to investigate the function, induction condition and regulatory mechanism of the other predicted T6SS cluster in V. fluvialis, VflT6SS1. The information obtained will greatly enhance our understanding of the pathogenic mechanism and environmental survival of the emerging foodborne pathogen and benefit the precaution and treatment of V. fluvialis infection.

河弧菌(Vibrio fluvialis) 是一种新发的食源性致病菌, 主要引起与甲类传染病霍乱相似的胃肠炎症状，曾造成大的暴发流行。目前，对于河弧菌毒力、致病机制和环境适应存活等仍缺乏了解。细菌的致病性与分泌系统和密度感应系统及环境生存密切相关。我们在一株河弧菌临床分离株基因组序列分析的基础上，探讨了其密度感应系统的功能和对毒力的调控，初步研究了VI型分泌系统(T6SS)基因簇VflT6SS2的组成及诱导表达分泌。本项目拟进一步研究河弧菌T6SS的生物学功能，探索VflT6SS2组成基因的作用，寻找效应蛋白及相应的免疫分子，探索效应蛋白作用机制；分析影响VflT6SS2表达的环境、宿主因素及与密度感应系统等重要毒力调节系统和全局性调控子的调控网络关系；探索另一个T6SS基因簇VflT6SS1的功能和表达调控机制，从而扩大我们对河弧菌致病性和环境生存等的认识。

河弧菌是一种新发的食源性致病菌，主要引起与甲类传染病霍乱相似的急性胃肠炎感染病例。关于河弧菌毒力、致病机制和环境适应存活等仍缺乏了解。本项目从与病原体致病和环境适应性生存密切相关的VI 型分泌系统入手，研究河弧菌VflT6SS2的生物学功能，影响其表达的环境宿主因素及与其他毒力调节系统和调控子的调控关系，毒性效应蛋白和免疫分子及作用机制；主要的研究内容和成果包括：.首次阐明河弧菌CqsA/LuxS-HapR QS调节VflT6SS2的分子机制。系统证明HapR通过接合VflT6SS2核心基因簇启动子区的两个Motif 1类型的HapR结合位点和hcp启动子区的一个Motif 2类型的HapR结合位点直接转录激活VflT6SS2的表达和其介导的菌间杀伤功能。.首次阐明宿主整合因子IHF调节河弧菌VflT6SS2参与环境适应性生存的机制。在VflT6SS2核心基因簇启动子区和hcp启动子区分别鉴定到两个和一个保守的IHF结合基序，并证明IHF通过直接结合于相应的位点转录水平正性调控VflT6SS2的表达和杀菌活性。.首次阐明霍乱弧菌和河弧菌T6SS附属基因簇的转录组织结构和调控因子。明确了原认为一个操纵子结构的附属基因簇实际转录成hcp(tssD)单顺反子和由vgrG(tssI)及其下游4个ORF组成的多顺反子两个转录单元，证明调控因子IHF和HapR在转录水平直接正性调控vgrG (tssI)操纵子。.阐明了河弧菌VflT6SS2核心基因簇上游包含两个ORF的一个小基因簇的结构特征、功能及其对河弧菌 VflT6SS2 功能的影响。明确AL536_RS29530和AL536_RS29525为共转录的操纵子结构，ORF有17bp的重叠，AL536_RS29525编码蛋白具有TPR样（TPR-like helical domain）的螺旋结构域，AL536_RS29530基因编码PAAR结构域蛋白，分别缺失该两基因均导致VflT6SS2的Hcp效应蛋白表达和分泌水平的明显降低和杀菌能力的缺陷，说明两者编码产物是河弧菌 VflT6SS2的组成部分，为其功能所需。.研究了河弧菌VflT6SS2核心基因簇C末端tssI2-tsiI2基因的功能，明确其编码产物为T6SS的效应子-免疫分子蛋白对，缺失tssI2虽不影响Hcp效应蛋白表达和分泌水平，但显著影响VflT6SS2介导的菌间