红斑丹毒丝菌毒力相关因子的发掘及其影响黏附、侵入的分子机理研究

Nowadays, swine erysipelas is an intractable re-emerging problem following by a sharp increase in cases in China. Erysipelothrix rhusiopathiae is the causative agent of swine erysipelas, which is infectious to a wide variety of animals, as well as human. It poses great threats to the swine industry. At present, virulence factors reported are limited, far from explaining its pathogenic mechanism. Cell wall proteins of the pathogenic bacteria is a biomacromolecule contacts directly with the host, participating in the course of adhesion and invasion. Until now, the role of its cell wall components to pathogenisis has not been systematic explored. This application will focus on the cell wall protein of Erysipelothrix rhusiopathiae. We will obtain candidate proteins related to virulence by analyzing the differentially expressed proteins between the virulent strain and avirulent strain through shotgun mass spectrometry. We will identify the proteins involved in adhesion and invasion of Erysipelothrix rhusiopathiae by protein interaction proteomics technology. The interacted proteins of the virulence associated factors from the host cell surface will be found by pull-down technology. The interaction will be testified by yeast two-hybrid technology and antibody blocking assay. Through deciphering the molecular mechanism of the interaction between virulence factors and its host, the adhesion and invasion of Erysipelothrix rhusiopathiae will be revealed. Completion of these studies will not only lay a fundation for the pathogenisis, but also provides new diagnostic marks and drug targets for the preventing and controlling of swine erysipelas.

当前我国猪丹毒病例急剧增多，已成为"老病新发"的棘手问题。其病原红斑丹毒丝菌能导致人和多种动物发病，是危害养猪业的一种重要细菌病原。当前已报道的少量毒力因子研究，远远不足以解释其致病机制。病原菌胞壁蛋白作为与宿主直接接触的生物大分子，参与黏附、侵入的致病过程。迄今为止，该病原胞壁组分参与致病的作用尚未被系统地发掘。本研究以红斑丹毒丝菌胞壁蛋白为研究对象，拟通过shotgun质谱分析强、弱菌株胞壁差异表达的蛋白，鉴定潜在的毒力相关蛋白；并结合蛋白质互作组学技术筛选出与黏附、侵入相关的毒力因子；利用pull-down技术垂钓与其互作的宿主细胞表面蛋白；采用酵母双杂交技术、抗体封闭试验等进行互作验证；通过进一步研究毒力因子与宿主蛋白相互作用的分子细节，最终揭示红斑丹毒丝菌黏附、侵入宿主细胞的分子机制。为阐明红斑丹毒丝菌的致病机理奠定基础。本研究将为猪丹毒的防控提供新的候选诊断标识和药物靶点。

当前我国猪丹毒"老病新发"，是危害养猪业的一种重要细菌病，且关于猪丹毒致病机制的研究尚浅。本研究以红斑丹毒丝菌胞壁蛋白为研究对象，开展了猪丹毒致病机制相关研究，取得成果如下：1.利用iTRAQ 结合LC-MS/MS的方法鉴定了临床分离的强弱毒菌株差异表达的细胞壁相关蛋白，共鉴定到125个差异表达蛋白，其中强毒菌株中高丰度蛋白为70个，低丰度蛋白为55个。试验证明sbp是红斑丹毒丝菌新的毒力因子。2.完成了红斑丹毒丝菌强毒株SE38与弱毒株G4T10基因组测序工作，对其GC含量，特异性基因进行比较分析。针对红斑丹毒丝菌强弱毒中的差异基因SpaA进行研究，发现缺失了部分SpaA片段的强毒株毒力下降20%~40%。说明SpaA在致病过程中起重要作用。3. 通过对红斑丹毒丝菌转录组学分析，发现红斑丹毒丝菌侵袭猪体心脏组织主要引起：炎性反应和免疫反应，新陈代谢，蛋白磷酸化和去磷酸化等通路的变化。4. 成功克隆并表达了红斑丹毒丝状杆菌蛋白SpaA、CbpB和GAPDH，经流式细胞术检测发现这三个蛋白均在红斑丹毒丝菌表面展示，是重要的候选毒力因子；发现红斑丹毒丝菌具有招募和利用宿主分子（纤连蛋白和纤溶酶原）的作用，这些宿主分子被招募后可以发挥粘附因子、蛋白水解酶及致炎因子等方面的作用。本项目通过研究毒力因子与宿主蛋白相互作用的分子细节，最终揭示红斑丹毒丝菌黏附、侵入宿主细胞的分子机制，为阐明红斑丹毒丝菌的致病机理奠定基础和猪丹毒的防控提供新的候选诊断标识和药物靶点。已发表SCI研究论文9篇；中文期刊1篇；获国家授权发明专利3项；培养毕业研究生4名。