HP2082介导猪链球菌2型特异性结合纤维蛋白原促进脑膜炎形成的分子基础研究

Streptococcus suis serotype 2 (SS2) could the adhere to a variety of host plasma and matrix proteins, and promote bacteria adhesion and invasion, which plays an essential role in clearance of bacteria. It would benefit the bacteria to evade host cell killing which was one of the essential reasons that the bacteria could infect pigs and cause severe diseases. Our results in our Lab found that a SS2 surface protein called HP2082 binds fibrinogen, a major protein in human blood. This interaction enhances the attachment and virulence of SS2, and contributes to the development of meningitis, which play a important role in meningitis pathogensis of SS2. Although expression of HP2082 is associated with increased binding to fibrinogen, the molecular basis for this interaction is not well defined. Therefore, our study plans to character the binding domain of HP2082 and identify the binding site for HP2082 binding region based on bioinformatics and sequence analysis, ELISA assay, Western blotting and Far Western blotting, SPR、Fluorescent microscopy and so on. Then, we will determine the crystal structures of HP2082 binding region in complex with hFg. The key site of HP2082 binding region will be identified by site mutation. Finally, we will analyse the impact of the key site of HP2082 binding region on GBS adherence, virulence and the development of meningitis by Cell lines, infection assay and mouse model of meningitis. This study will also lay a theoretical foundation for pathogenic mechanism, the and development of new S. suis vaccines and drug targets of S. suis 2.

猪链球菌2型（SS2）与宿主血浆及基质蛋白互作，促进细菌粘附，是该菌逃避宿主免疫、成功建立感染并导致人和猪发病的重要机制之一。课题组前期结果发现，HP2082蛋白介导SS2结合人纤维蛋白原（hFg）增强细菌粘附和毒力，促进脑膜炎形成，提示HP2082在SS2感染致脑膜炎中扮演着重要作用。本项目拟通过生物信息学、ELISA、SPR、Western blotting、Far Western blotting等技术鉴定HP2082结合域及结合hFg具体肽链及区域；进一步通过蛋白结晶、点突变鉴定HP2082结合域关键位点；最后，通过构建HP2082结合域关键位点缺失突变株，利用细胞和动物感染分析HP2082结合域关键位点缺失对SS2粘附、毒力和脑膜炎形成的影响，阐明HP2082介导SS2特异性结合hFg促进脑膜炎形成的分子基础，为设计SS2新型疫苗和药物靶标提供新的思路。

猪链球菌2型是一种重要的人兽共患病病原菌，其毒力因子挖掘与致病性研究是药物靶标与疫病防控的研究基础。本研究利用细菌重组原理，通过温敏性质粒和抗性筛选得到hp2082基因缺失突变株，通过生物学特性研究、体外细胞实验及动物体内研究，发现蛋白HP2082在猪链球菌2型致病性乃至致脑膜炎中具有一定的促进作用。通过借助生物信息学分析了 HP2082结合域全长、N-端和C-端，并分段表达并纯化了相应Aα、Bβ 和 γ 多肽链重组蛋白；将纯化蛋白分别与纯化的 hFg 互作后确定了确定 HP2082 结合域（HP2082-BR）；通过RNA-seq、荧光PCR、Western blotting等技术，筛选并鉴定了HP2082结合域及结合hFg具体；进一步通过互作，发现△hp2082可以通过诱导提高HBMECs细胞间紧密连接蛋白表达，导致猪链球菌形成脑膜炎能力降低，说明HP2082蛋白是猪链球菌2型的毒力因子，并且在致脑膜炎中具有一定的作用。该研究初步阐明了猪链球菌引起脑膜炎的分子机制，为猪链球菌的药物靶标设计提供了素材。