小RNA Qrr5调控副溶血性弧菌毒力因子T3SS1表达的机制研究

Vibrio parahaemolyticus is the leading cause of foodborne outbreaks associated with aquatic products. The pathogenic mechanism of V. parahaemolyticus is always a key issue for researchers. Type III secretion system 1 (T3SS1) is considered as a key factor for the cytotoxicity of V. parahaemolyticus. Previous studies have showed that the expression of T3SS1 during infection of host might be regulated by sRNA Qrr5, while the regulatory mechanism remains unclear. In this project, homologous recombination technology will be conducted to generate a qrr5 deletion strain from a wild type pathogenicity strain of V. parahaemolyticus. After validation of the impact of qrr5 deletion on cytotoxicity and T3SS1 expression, RNA-seq and weighted gene co-expression network analysis will be carried out to identify the target gene of qrr5 during infection of host. The RNA:RNA EMSA will be applied to test for the interaction of Qrr5 and target gene, and target gene will be knocked out to validate its role in cytotoxicity. Finally, the regulatory network of Qrr5 and its target gene on T3SS1 expression will be constructed. This project is aimed to illustrate the regulatory mechanism of sRNA during pathogenic processes of V. parahaemolyticus, which will provide the theoretical basis for pathogenicity of V. parahaemolyticus, and will be meaningful to the strategy for preventing and controlling of this pathogen.

副溶血性弧菌是水产品中首要的食源性致病菌，其致病机理一直是对该菌研究中的重点问题。III型分泌系统1（T3SS1）是引起该菌细胞毒性的重要毒力因子，前期研究表明T3SS1在该菌侵染宿主过程中的表达可能受小RNA Qrr5的调控，但是具体的调控机制仍未阐明。本项目拟利用同源重组技术构建副溶血性弧菌致病性菌株的qrr5基因的缺失株，在明确qrr5缺失对细胞毒性和T3SS1表达影响的基础上，通过RNA-seq和权重基因共表达网络分析挖掘Qrr5在该菌侵染宿主细胞过程中作用的靶基因。通过RNA:RNA EMSA实验验证Qrr5与靶基因的互作，并利用基因敲除方法确证Qrr5的靶基因对细胞毒性的作用，最后阐明Qrr5及其靶基因对T3SS1的表达调控网络。本项目旨在揭示小RNA在副溶血性弧菌致病过程中的基因调控机制，为解析副溶血性弧菌的危害形成机制提供理论依据，对制定该菌的危害控制策略有重要意义。

副溶血性弧菌是水产品中首要的食源性致病菌，其致病机理一直是该菌研究中的重点问题。III型分泌系统1（T3SS1）是引起该菌细胞毒性的重要毒力因子，前期研究表明T3SS1在该菌侵染宿主过程中的表达受小RNA Qrr5的调控，但是具体的调控机制仍未阐明。本项目利用同源重组技术构建副溶血性弧菌致病性菌株的qrr5基因的缺失株，在明确qrr5缺失对细胞毒性和T3SS1表达影响的基础上，通过转录组测序和权重基因共表达网络分析挖掘Qrr5在该菌侵染宿主细胞过程中作用的潜在靶基因。同时，进一步通过蛋白组检测挖掘受Qrr5调控的蛋白，并构建了调控蛋白的下游互作网络。最后，基于转录组和蛋白组阐明Qrr5及其靶基因对T3SS1的表达调控网络。本项目揭示了小RNA在副溶血性弧菌致病过程中的基因调控机制，为解析副溶血性弧菌的危害形成机制提供理论依据，对制定该菌的危害控制策略有重要意义。