猪链球2型双组份调控系统1910HK/RR调控机制研究

Streptococcus suis serotype 2 (SS2) is considered an important zoonotic disease pathogen which causing great economic loss to the pig industry worldwide. In recent years, research focused on the pathogenesis and prevention has been a hot spot. Bacterial two-component regulatory system (TCS) plays an important role in bacterium metabolism and regulation of virulence gene expression. Early study by our group demonstrated that deletion of the TCS 1910HK/RR in SS2 significantly reduced the virulence of SS2. However, the mechanisms still need further elucidation. This study intends to (1) select and validate differential expression genes between wild type strain and 1910rr/1911hk deletion strain by DNA microarray and real-time quantitative RT-PCR; (2) identify the genes and binding sequence of 1910HK/RR TCS regulation by ChIP-seq and electrophoretic mobility shift assay (EMSA); (3) further research some gene function of 1910 HK/RR TCS regulation by gene deletion or overexpression methods. Our aim is to systemically clarify how 1910rr/hk TCS regulates gene expression and further contributes to the virulence at the molecular level. The research would lay foundation for the development of prevention and control technology against SS2 and provide new sights of its pathogenesis.

猪2型链球菌病是由猪链球菌2型（SS2）引起的一种严重危害人畜健康的传染病。SS2致病机理研究是当前热点和难点。双组份调控系统（TCS）是致病菌中普遍存在的一种跨膜信号转导机制，在细菌代谢、调节毒力基因表达等方面发挥着重要作用。课题组前期研究结果发现，SS2国内流行株SC19双组份调控系统1910rr/hk 缺失后毒力显著降低，但作用机理尚不清楚。本项目拟进行研究（1）运用基因表达谱芯片筛选差异表达基因并通过荧光定量PCR验证；（2）通过染色质免疫共沉淀测序及凝胶迁移率试验鉴定1910rr/hk直接调控基因及DNA 序列；（3）利用基因缺失或过表达等方法研究受调控的部分功能基因。本项目旨在绘制SS2中1910rr/hk基因调控网络模型，阐明其调控机理，为SS2致病机理研究打下基础。

本项目通过构建基因缺失突变株的手段，运用了基因芯片来筛选差异表达基因并通过荧光定量PCR进行了验证，通过EMSA确定了1910HK/RR直接调控基因及DNA序列，最终阐明1910HK/RR是通过调控SS2荚膜合成相关基因来影响细菌致病性，为SS2 致病机理的研究奠定了理论基础。具体研究结果如下：.1．猪链球菌2型亲本株（WT）、基因缺失突变株（△1910hk/rr）和互补菌株（C△1910hk/rr）的体外生物学特性比较研究. 结果表明：Δ1910hk/rr的生长速率要低于WT和CΔ1910hk/rr；Δ1910hk/rr形态结构未发生改变。Δ1910hk/rr的细菌溶血活性并没有发生显著变化。双组分调控系统1910HK/RR缺失后使得SS2抵抗中性粒细胞（PMN）的杀伤能力降低。Δ1910hk/rr对HEp-2细胞的粘附和侵染能力降低。.2．双组份调控系统1910HK/RR缺失后对SS2毒力和致病性的影响. 分别用猪链球菌2型WT、Δ1910hk/rr和C△1910hk/rr感染5周龄左右断奶仔猪。结果为：感染后WT组存活率为16.7%（1/6），Δ1910hk/rr组存活率为83.3%（5/6）；肺、脑、血液组织载菌量比较分析发现，Δ1910hk/rr组也低于WT组。以上结果说明，双组分1910HK/RR缺失后SS2的致病性明显降低。.3．双组分调控系统1910HK/RR调控SS2致病性机制研究. 通过基因表达谱芯片片，发现在整个基因组2194个基因中，Δ1910hk/rr有219个基因转录水平发生了变化。其中105个基因下调表达1.5倍以上，114个基因上调1.5倍以上。有50个基因表达下调2倍以上，32个基因表达上调2倍以上。选取24下调表达的基因进行荧光定量PCR验证，证实了芯片结果的可靠性。克隆、表达反应调控蛋白1910RR，经EMSA证实，唾液酸合成相关基因启动子能与1910RR结合，初步证实了唾液酸合成相关基因受1910HK/RR的调控。阐明1910HK/RR通过调控唾液酸合成相关基因来影响SS2毒力和致病性的机制。项目发表相关SCI论文1篇。培养毕业研究生4名，在读硕士2名。. 关键词：猪链球菌2型；双组份调控系统1910HK/RR；调控；差异表达基因；毒力和致病性；