猪链球菌2型逃避猪中性粒细胞胞外诱捕网杀菌机制的研究

Streptococcus suis serotype 2 (SS2) could inhibit swine neutrophils to form extracellular traps (NETs), which plays an essential role in clearance of bacteria. It would benefit the bacteria to evade NETs-mediate killing, which was one of the essential reasons that the bacteria could infect pigs and cause severe diseases. The present study would analyze the differentially expressed proteins of PGN (peptidoglycan, a pattern recognized molecular of Gram-positive bacteria)-induced swine neutrophils or mock treated neutrophils by label-free quantitative proteomics. And then we would identify the major swine signaling pathways for PGN-induced NETs with the activator and blocking reagent. On the basis of these results, the present study would identify the NETs formatting related signaling pathways which can be inhibited by SS2. Meanwhile, we would also identify the bacterial evasion-related factors by screening the recombinant SS2 surface proteins and secreted proteins pool. After that, the potential bacterial evasion-related factors would be confirmed and characterized by specific gene-deletion experiments and complementary experiments. The aim of this study was to clarify the mechanism how SS2 evade PGN-induced NETs formatting and subsequent host killing. It would contribute to the pathogenesis of SS2 diseases.

猪链球菌2型可抑制猪中性粒细胞形成胞外诱捕网（NETs），这无疑是该菌逃避NETs捕杀效应、成功建立感染并导致猪严重发病的重要机制之一。本研究拟通过Label-free比较蛋白质组学方法分析G+菌的模式分子PGN（肽聚糖）诱导猪中性粒细胞形成NETs时的差异蛋白，筛选并鉴定其激活的主要信号通路。在此基础上研究猪链球菌2型对信号通路激活NETs的抑制效应，从而鉴定猪链球菌抑制NETs形成的主要信号通路。同时基于构建的重组表达猪链球菌表面蛋白和分泌蛋白的“文库”，筛选可显著抑制猪NETs形成的关键因子。通过相应基因的缺失突变体和回复突变菌株，确认该基因在抑制猪NETs形成通路中的作用。力图阐明猪链球菌2型是通过表达哪些（个）蛋白来抑制哪条（些）信号途径，而抑制猪中性粒细胞形成NETs、从而逃避其杀菌效应的分子机制。为深入解析猪链球菌2型的免疫逃避机制奠定重要基础。

本项目首先通过动物感染试验首次发现了猪链球菌感染动物后可以诱导动物体内的感染部位形成NETs，并且证实诱导的NETs结构发挥着清除感染部位病原菌的抗感染功能。之后应用蛋白质组学方法分析了猪链球菌诱导NETs的主要差异蛋白，并以PMA作为阳性对照。发现猪链球菌和PMA一样通过PKC-NADPH-MPO途径激活NETs的形成。进一步对比低NETs和高NETs差异的蛋白质组学，发现高NETs水平时MMP-8、MMP-9和WDR5呈现显著变化，但低水平NETs形成时其变化受到明显抑制。并通过试验确认了NETs的大量形成需要MMP-8活性的抑制以及WDR5通过JMJD6和PAD4调控的histone的修饰，从而促进染色质解聚并释放。另外，猪链球菌的荚膜缺失株可以很好地诱导wdr5，并促使NETs的形成。这也是猪链球菌逃避NETs活化的重要机制。因此，本研究可以得出如下机制：在感染早期，猪链球菌由于菌量少虽然激活了PKC-ROS-NADPH-MPO途径，但却由于细菌荚膜的存在抑制了WDR5的诱导，也未能显著降解MMP-8等酶的活性和诱导WDR5调控的histone的修饰，从而不能很好促进染色质解聚并释放。这很可能是猪链球菌感染早期未能有效诱导NETs而逃避其介导杀菌效应的机制。.本研究按计划解析了NETs形成的信号通路以及猪链球菌逃避猪中性粒细胞胞外诱捕网大量激活而逃避其杀菌的机制，而且进一步证实缺失荚膜基因对于开发安全的弱毒疫苗的必要性。本研究已发表SCI 研究论文2篇，申请专利受理1 项。通过从事本研究，发表优秀学士学位论文1篇、校优秀硕士学位论文1篇。部分研究成果作为支撑材料获得国家科技进步二等奖一项。