双组份调控系统1094hk/rr参与高致病性猪链球菌2型诱导炎症反应机制研究

The highly pathogenic S. suis 2 strain ( 05ZY ) can trigger an excessive release and activation of several proin?ammatory cytokines, which may be responsible for its high pathogenicity. Our results in our Lab found that the expression of the two-component regulatory system 1094hk/rr was significantly up-regulated when strain 05ZY was interacted with human neutrophils and the mutant strain of deleting of 1094hk/rr could decrease the production of cytokine IL-1β and pathogenicity (virulence), indicating that 1094hk/rr may play an important role in inflammatory response. The present study plans to perform the comparative transcriptomic analysis of the 1094hk/rr deletion mutant and WT strains based on DNA microarray and proteomics. Together with ChIP-Seq、EMSA、SPR、 DNase I foot printing assay and so on, the virulence associated genes and sequences regulated by 1094hk/rr which can induce the production of IL-1β will be identified. Then using gene deletion and overexpression strain, combined with a series of pathway inhibitors, the signaling pathway activated by main genes will be identified in RAW264.2 model. So that the molecular mechanism of 1094hk/rr will be revealed, that is which genes were up-regulated to activate the cell signaling pathway and promote IL-1β production. This study will also lay a theoretical foundation for the development of new S. suis vaccines and anti-inflammatory drugs.

高致病猪链球菌2型（05ZY菌株）刺激机体导致炎症因子过度释放和激活可能是其强致病性的重要原因。课题组前期研究发现，05ZY与人中性粒细胞互作后1094hk/rr双组份调控系统显著上调表达，缺失1094hk/rr能显著降低炎症因子IL-1β产生和降低细菌致病性，提示1094hk/rr可能参与05ZY诱导炎症反应。本研究拟通过基因芯片和蛋白质组学，开展05ZY 及1094hk/rr敲除菌株的表达谱研究，结合ChIP-Seq、EMSA、SPR、DNase I足迹分析等技术，鉴定1094hk/rr调控的重要基因及序列，并从中鉴定可诱导IL-1β产生的关键基因。以RAW264.2巨噬细胞为模型，通过基因缺失和超表达，配合一系列通路抑制剂鉴定关键基因激活的信号通路。从而解析1094hk/rr是通过上调哪些基因表达激活细胞信号通路促使IL-1β产生的机制，为今后研制猪链球菌新型疫苗和抗炎药物提供基础。

猪链球菌作为一种重要的人兽共患病病原，不仅给全球养猪业造成巨大的经济损失，还严重危害公众健康。猪链球菌2型是世界范围内致病性最强，临床分离率最高的血清型。高致病猪链球菌2型（05ZY菌株）刺激机体导致炎症因子过度释放和激活可能是其强致病性的重要原因。课题组前期研究发现，05ZY与人中性粒细胞互作后1094hk/rr ( CiaRH )双组份调控系统显著上调表达，缺失1094hk/rr能显著降低炎症因子IL-1β产生和降低细菌致病性，提示1094hk/rr可能参与05ZY诱导炎症反应。.本项目以猪链球菌双组份调控系统CiaRH为研究对象，通过构建基因缺失突变株ΔciaRH，研究野生株与基因缺失突变株的致病性，突变株ΔciaRH较野生株在小鼠体内存活能力及毒力，感染仔猪的组织带菌量明显下降。结果表明：双组份CiaRH系统对影响猪链球菌2型的致病性起着重要作用。.本研究进一步通过基因芯片和蛋白质组学，开展05ZY 及1094hk/rr敲除菌株的表达谱研究，结合ChIP-Seq、EMSA、SPR、DNase I足迹分析等技术，鉴定1094hk/rr调控的重要基因SsPepO和SsPspC及序列，并从中鉴定可诱导IL-1β产生的关键基因。进一步构建SsPepO和SsPspC单基因缺失突变株ΔSsPepO、ΔSsPspC和双基因缺失突变株ΔSsPepO/ΔSsPspC；通过体外细胞和体内小鼠模型，评价了其对致病性及诱导炎症反应的影响。结果表明，ΔSsPepO/ΔSsPspC突变株毒力相比亲本菌，显著下降。确定了CiaRH调控的SsPepO和SsPspC蛋白能诱导MCP-1、IL-8炎症因子显著上调表达，初步阐明了双组份调控系统CiaRH参与猪链球菌2型诱导炎症反应的机制。为今后研制猪链球菌新型疫苗和抗炎药物提供基础。.项目发表相关研究论文9篇。获得大北农励志奖学金和国家奖学金。养毕业研究生6 人，其中博士2人，硕士4人；在读研究生2 人。