ClpP蛋白酶在马链球菌兽疫亚种感染过程中的作用机制研究

Streptococcus equi ssp. zooepidemicus (SEZ) is responsible for a wide variety of infections in humans and many species. However, little was known about the pathogenesis of SEZ infection. In our previous project, we identified that clpP gene was up-regulated in necrotic porcine lung tissues, suggesting it related to their pathogenicity. Other studies have shown that ClpP protease participates in regulation of the growing of bacteria, biofilm formation and virulence and so on. But the regulation mechanism of ClpP in SEZ infection is still unkown. Thus, this study attempts to construct the clpP gene mutant and complementation strains by homologous recombination and analyze the pathogenicity; determine the regulation function of biofilm, growing and oxidative stress. The effectors of SEZ ClpP were identified by bioinformatics and comparative proteomics approach; and its effects on host cells of the levels of cytokines and apoptosis of SEZ were determined. The pathogenicity of effectors were also detected through animal experiments. This research will increase our knowledge about the regulation mechanism of ClpP in SEZ infection and contribute to the prevention and control of Streptococcosis in China.

马链球菌兽疫亚种（SEZ）是一种人兽共患病的病原，可感染人及多种动物并呈世界性分布。申请人前期研究发现clpP基因在SEZ感染的猪肺中呈现显著上调表达，提示该基因与SEZ的致病性相关。ClpP蛋白酶在多种细菌中被发现参与调控细菌生长发育、生物被膜形成、毒力等众多生理功能，但在SEZ感染过程中的作用机理及调控机制尚不清楚。本研究拟构建SEZ的clpP基因缺失株和互补株，利用小鼠和细胞模型研究其与毒力的关系，明确对生物被膜、生长和氧化应激等生物学特性的调控作用；通过生物信息学和比较蛋白组学技术筛选ClpP蛋白酶调控的效应蛋白；并分析效应蛋白对宿主细胞炎症因子水平和细胞凋亡的能力影响，以及通过动物致病试验评价效应蛋白对宿主致病性的作用。本项目旨在阐明ClpP蛋白酶在SEZ中的多种调控机制，为解析ClpP蛋白酶在SEZ感染中的致病机理提供理论依据，也为SEZ的防控提供新思路。

马链球菌兽疫亚种（SEZ)是猪链球菌病的重要病原之一，对养猪业造成严重的经济损失。在前期工作中，我们发现clpP基因可能与SEZ的致病性相关，但具体的功能和分子致病机制尚不清楚。本研究检测了SEZ野生株、clpP基因缺失株和互补株在感染小鼠血液、肝、肺和脾脏组织的活菌数，比较了各菌株引起的脏器病理变化情况；检测血清中的炎症因子IL-1β、IL-6、IL-8、TNFα水平；测定各菌株对生长曲线、生物被膜形成能力、氧化应激及对细胞炎症因子水平和信号通路的影响，证明了ClpP蛋白酶参与了SEZ的相关致病机制，并筛选出了ClpP蛋白酶调控的效应蛋白。进一步地，本项目在SEZ的防控方面完成了相关研究，进行了SEZ亚单位疫苗候选抗原的筛选。本项目研究成果初步阐明了ClpP蛋白酶的致病机理及调控机制，为SEZ所导致感染的防控提供新的思路。