副溶血弧菌ToxR对III型分泌系统和耐热性直接溶血素的表达调控研究

Vibrio parahaemolyticus expresses three major virulence factors called T3SS1, T3SS2, and TDH. The functions of the virulence regulator ToxR in V. parahaemolyticus are still poorly understood. In this study, the lethality against mice, cytotoxicity, enterotoxicity, and hemolytic activity will be systematically characterized for the wild-type, toxR mutant, and complemented mutant strain of pandemic V. parahaemolyticus. Subsequently, the regulation mechanisms of T3SS1, T3SS2α, and tdh2 genes by ToxR will be characterized by combined use of real-time quantitative RT-PCR, LacZ fusion reporter assay, western blot assay, primer extension analysis, gel mobility shift assay and DNase I footprinting. The final dataset will promote us to gain a deeper understanding of regulation of the lethality against mice, cytotoxicity, enterotoxicity, and hemolytic activity by ToxR in V. parahaemolyticus.

T3SS1、T3SS2α和TDH均是副溶血弧菌重要的毒力因子。ToxR是副溶血性弧菌的毒力调控因子，目前对其功能的认识还很肤浅。本研究在系统分析了野生株、toxR突变株和回补株的小鼠致死活性、细胞毒性、肠毒性、溶血活性等功能表型的基础上，选取T3SS1、T3SS2α和TDH相关基因为靶基因，运用实时定量RT-PCR、LacZ报告基因融合实验、Western blot实验、引物延伸实验、凝胶阻滞实验和DNA酶I足迹实验，深入研究ToxR对靶基因的精细分子调控机制；进而，将表型和分子实验数据结合起来，认识ToxR是如何通过调控T3SS1、T3SS2α和TDH的表达，实现对副溶血弧菌致病性的小鼠致死活性、细胞毒性、肠毒性和溶血活性的调控。

III型分泌系统（type III secretion system, T3SS）1、T3SS2和耐热性直接溶血素（TDH）是副溶血弧菌重要的毒力因子。ToxR是副溶血弧菌的毒力调控子，目前已知其对T3SS1和Vp-PAI（T3SS2和TDH）编码基因具有调控作用，但具体的机制未知。本研究以副溶血弧菌RIMD2210633为野生株，首先构建了toxR突变株（ΔtoxR）和回补株（C-toxR），进而探究了各菌株的毒力表型，结果发现ToxR对副溶血弧菌的小鼠致死毒性、肠毒性、细胞毒性和溶血活性等均具有调控作用。进一步研究发现：ToxR可以直接抑制T3SS1而直接激活Vp-PAI（T3SS2和tdh2）相关基因的转录；ToxR与CalR之间具有相互调控机制，ToxR直接激活calR而CalR直接抑制toxR和自身基因的转录，且CalR对T3SS1相关基因也具有直接的抑制作用；ToxR正调控副溶血弧菌的swimming、swarming和生物膜形成能力，但分子机制未知，后续研究中应着重探讨ToxR调控生物膜的分子机制。总之，本研究为我们理解ToxR的功能以及副溶血弧菌的致病机制提供一定的理论指导。