空肠弯曲菌鞭毛相关新基因Cj0064c, Cj0371功能挖掘及其分子基础

Campylobacter jejuni is now recognised as one of the most common agents of reported zoonosis worldwide. However, current knowledge about the pathogenic mechanisms, virulence and pathogen-host interaction mechanisms of campylobacter is still limited. Flagellar mediated motility and pathogenicity play an important role in bacterial infection. However, the function and regulation mechanism of the flagellum synthesis genes remains unclear. We have discovered two new flagellum synthesis related virulence genes cj0064c and Cj0371 by in vivo-induced antigen technology from our previous study. In this project, we are aiming to analyse the biochemical and biological characteristics of cj0064c and / or Cj0371mutant, and study its function of controlling transcription level of the flagellar genes in the intracellular environment by quantitative fluorescent polymerase chain reaction (QF-PCR). By combining this with microarray technology, we will be able to detect the different expression level of the flagellar related genes, and analyse the mechanism. We will also employ Western Blot to discover the effect of C. jejuni secreted flagellins, and the interactions between these flagellins. Furthermore, the interaction mechanisms between flagellins and with host will also be studied by bacteria (and / or yeast) two-hybrid system. The launching of the project will be instrumental in clarifying the pathway of the C. jejuni flagellar biosynthesis cascade, revealing the coupling reactions between the flagellar pathogenic factors, and also to provide new candidates for research and development of live attenuated vaccines and subunit vaccines.

空肠弯曲菌是世界范围内引发兽医公共卫生和食品安全的主要人兽共患病病原菌之一，其致病机制仍未阐明。鞭毛介导的运动性和致病性在感染宿主和传染机制中发挥重要作用，是关键致病因子，但对于鞭毛合成的基因功能和调节机制仍不明晰。课题组通过体内诱导抗原技术筛选获得了与鞭毛合成相关的新基因，本研究拟通过构建新基因Cj0064c和Cj0371突变株，分析其生理生化和生物学特性；通过荧光定量PCR分析其对鞭毛主要结构基因、调节基因在胞内转录水平的作用，并联合应用微阵列技术检测相关基因的差异表达，分析其作用机制。同时通过蛋白免疫印迹分析鞭毛相关蛋白的影响，明确毒力因子藕联间的调节作用；通过细菌（或/和酵母）双杂交系统在蛋白水平分析鞭毛蛋白之间及与宿主之间的相互作用和调节机制。该项目的开展将有助于完善空肠弯曲菌鞭毛生物合成的级联通路，明晰与鞭毛相关致病因子间的偶联关系，同时为开发诊断和疫苗制剂提供新思路和新途径。

cj0064c和cj0371是本课题组前期通过体内诱导抗原技术筛选获得的可能与空肠弯曲菌鞭毛合成相关的新基因，本研究通过构建cj0064c和cj0371突变株，表达蛋白并制备其单克隆抗体，在生理、生化、分子以及毒力水平对这两个基因的功能进行了深入研究。结果显示：cj0064c与空肠弯曲菌鞭毛合成密切相关，该基因失活后空肠弯曲菌完全失去鞭毛结构以及运动力，在鞭毛组装过程中，其在级联通路的顶端通过介导早期鞭毛亚组分（FlhB）定位来发挥一种总调节子的作用，cj0064c失活后该菌毒力明显降低，可能与失去鞭毛有关；与之相比cj0371与鞭毛的直接关联性则不是很强，进一步研究发现该基因可能通过CheV蛋白对空肠弯曲菌趋化起着一种负调控作用并间接影响鞭毛通路，cj0371突变株的毒力相比较野生株有所上升，其可能是空肠弯曲菌的一种抗毒力因子。本课题解析并阐明了Cj0064c作为主调节子在鞭毛通路中所发挥的作用，并鉴定得到一个新趋化蛋白Cj0371，研究结果为解析空肠弯曲菌毒力机制提供了新借鉴。