高致病2型猪链球菌89K毒力岛编码毒力蛋白SP1的结构和致病性研究

The major pathogenic host for streptococcus suis 2 (SS2) is swine. However,recently there have been outbreaks of SS2 epidemic strain cross-species infections in human that cause streptococcal toxic shock syndrome (STSS) with high mortality. The current study discloses that the high pathogenicity of the above-mentioned SS2 epidemic strains is due to an acquired 89k pathogenicity island (PAI), which encodes a type IV secretion system.. When certain effector molecules are released extracellularly by this type IV secretion system, they stimulate the excessive release of the host inflammatory factors, resulting in immunopathological damages and STSS. It was found that an SP1 protein encoded within the 89K PAI, the specific functions of which remain unknown, could be secreted into the medium, and showshas immunogenic and enzymatic activity. This is a strong indication that the SP1 protein is involved in the unusually high bacterial pathogenicity of SS2, and could be the STSS on-set factor researchers are trying to identify. Through screening experiments, several SP1-binding proteins have been isolated, including the peptidoglycan recognition protein (PGRP), which is an important component of the host non-specific immune defense system. It is likely that the binding of the SP1 protein to the PGRP leads to the disruption of the host's natural immunity, resulting in the high pathogenicity of the SS2 epidemic strains. The goal of the present project is to resolve the structure of the SP1 protein, illustrate the SP1 protein's relationships with the STSS and PGRP, which help to unreveal the mechanism of the high bacterial pathogenicity, especially the developmental process of STSS under the induction ofthe SS2 epidemic strains.

2型猪链球菌（SS2）主要感染宿主是猪，最近却发生跨种传播，在人群爆发并致被感染者发生病死率极高的链球菌中毒性休克综合征（STSS）。本项目研究发现，流行菌株的高致病性是因为获得一个89K毒力岛，岛编码的IV型分泌系统通过将菌体内的某种效应分子分泌到胞外，激发机体免疫系统，造成炎性因子过度释放和免疫病理损伤导致STSS。SP1是89K毒力岛编码的一个功能未知蛋白，实验发现 SP1可分泌到培养基中，具有抗原性和酶活性，强烈提示参与该菌的高致病性，可能就是我们在努力寻找的诱发STSS的效应分子。我们筛选到数种与SP1结合的蛋白，其中之一是肽聚糖结合蛋白(PGRP)，后者是宿主非特异性免疫防御的重要组分，SP1与之结合可能导致宿主的天然免疫力受到破坏，致使流行菌株具有高侵袭力。本项目的目标是解析SP1的结构，阐明SP1与STSS、PGRP的关系，为菌株的高致病性特别是STSS的发生求解。

我们首报SS2菌感染宿主发生重大改变，不仅在人群中爆发流行，而且使1/3患者发生病死率高达80%以上的中毒性休克（STSS），表明我国出现SS2高致病株。通过对该菌基因组测序发现高致病株基因组携有一独特的大小为89K的毒力岛（89KPAI），分析该毒力岛携载的基因除与毒力密切相关的二元调节系统，VI分泌系统（T4SS）外还有一潜在的毒力蛋白SP1。.通过酵母双杂交系统我们筛选出SP1能与保守的动物天然免疫蛋白PGRP相互作用并通过免疫共沉淀技术和多重截短SP1蛋白确定了这种相互作用及其与PGRP蛋白的潜在的结合区域。体外实验证明SP1能明显阻断天然免疫蛋白PGRP对细菌本身的抑杀作用。我们同时发现89KPAI携载的T4SS参与SP1蛋白的转运分泌，同时通过生物信息学分析发现一些共要的致病链球菌肺炎、无乳也含有类似的结构并通过实验证实，揭示了这类致病链球菌新的逃逸机体天然免疫抑菌的新机制。.我们从健康猪鼻咽部分离获得一株2型无毒猪链球菌，对全基因组测序发现该菌缺少 2型猪链球菌的主要毒力因子EF和SLY及毒力因子MRP发生较大变异。用该活菌作为疫苗免疫接种仔猪发现能特异性的免疫保护强毒株的感染。