43K-OMP在牛坏死杆菌黏附宿主靶细胞中的作用

Fusobacterium necrophorum (F. necrophorum) is a primary pathogen caused the necrotic infection of the various organs in cattle, and affects seriously the cows-rearing industry. The adhesion of bacteria to host cells is the first step in the establishment of infection. At present, adhesion-associated proteins of the F. necrophorum remained unknown. Outer membrane proteins (OMPs) have been reported as adhesion factors of host cells in gram-negative anaerobic bacilli, becoming a research focus. In our previous study, a 43 Ku of outer membrane protein (43K-OMP) was identified in the F. necrophorum strain H05. The 43K-OMP has a potential role in adhesion of F. necrophorum to host cells. To confirm the adhesion function of the 43K-OMP, the competition/inhibition experiments based on antibodies or natural 43K-OMP were carried out in primary cells from the targeted tissues for F. necrophorum; the receptor proteins that the 43K-OMP adheres host cells from bovine liver cells, hoof tissue cells, breast epithelial cells, and endometrial epithelial cells were identified by using co-immunoprecipitation (Co-IP), GST-pull down, over-expression/silencing of the targeted proteins, and expression of the targeted proteins in the non-host cell lines; and the preventive effect of the 43K-OMP recombinant protein as vaccine candidate on bovine F. necrophorum infection was evaluated through in vivo experiments in the moue model. These data will provide novel basis for understanding of pathogenic mechanism and antimicrobial drug design of bovine F. necrophorum.

牛坏死杆菌是引起牛多种器官坏死性感染的主要病原菌，给养牛业带来严重危害。病原菌对宿主细胞的黏附是建立感染的第一步。目前，牛坏死杆菌黏附宿主细胞的相关蛋白尚不明确。在前期研究中，我们在牛坏死杆菌中鉴定了1个43Ku的外膜蛋白（43K-OMP），其具有黏附宿主细胞的潜在作用。为了明确43K-OMP在牛坏死杆菌黏附宿主细胞中的作用，本研究拟通过抗体/天然蛋白的竞争抑制实验，在牛坏死杆菌感染宿主不同靶器官的原代细胞上，明确牛坏死杆菌43K-OMP的黏附作用；通过免疫共沉淀、GST-pull down、靶蛋白非宿主表达细胞系和靶基因沉默与过表达等方法和技术，鉴定牛坏死杆菌43K-OMP黏附宿主不同靶细胞的受体蛋白；以鼠为模型，通过体内实验，评价43K-OMP作为疫苗抗原对牛坏死杆菌感染的抑制作用。本研究成果将为牛坏死杆菌的致病机理及抗菌药物设计提供新的理论基础。

牛坏死杆菌可引起牛多种器官坏死性感染，给养牛业造成了巨大的经济损失。然而，牛坏死杆菌如何黏附入侵宿主细胞尚不明确。申请人在前期鉴定并推测43K-OMP在坏死杆菌黏附宿主细胞中可能发挥潜在作用。为明确43K-OMP在牛坏死杆菌黏附宿主细胞中的作用，本课题组在项目资助下，先后制备牛坏死杆菌43K-OMP的单克隆抗体和多克隆抗体，纯化重组蛋白和提取天然43K-OMP，并构建43K-OMP的真核表达载体，通过抗体阻断、蛋白竞争抑制等实验明确43K-OMP在牛坏死杆菌感染中的黏附作用，利用免疫共沉淀、蛋白质谱技术等进一步寻找牛坏死杆菌43K-OMP黏附宿主不同靶细胞的互作蛋白，同时在小鼠体内评价了43K-OMP对牛坏死杆菌感染的抑制作用。研究发现，43K-OMP可以黏附于BHK-21细胞、奶牛子宫内膜细胞和奶牛乳腺上皮细胞，预孵育43K-OMP抗体或天然43K-OMP均可降低黏附于BHK-21细胞、奶牛子宫内膜细胞和奶牛乳腺上皮细胞的坏死杆菌数量，提示43K-OMP在牛坏死杆菌黏附宿主靶细胞中发挥积极作用。利用CO-IP和蛋白质谱技术筛选宿主靶细胞上与43K-OMP作用的互作蛋白，共筛选出差异蛋白110个，通过蛋白的单细胞功能和亚细胞定位对质谱分析蛋白进行分析并验证，未发现与43K-OMP作用的互作蛋白，结合质谱分析的差异蛋白功能推测43K-OMP可能通过胶原蛋白或纤连蛋白介导牛坏死杆菌黏附宿主靶细胞。43K-OMP作为疫苗抗原，与坏死杆菌白细胞毒素截短表达蛋白、溶血素截短表达蛋白免疫小鼠后，可以产生高的抗体水平，诱发炎症细胞因子分泌增加，激发机体免疫反应，且对攻菌后的小鼠具有很好的免疫保护效果，抑制了牛坏死杆菌的感染。该研究为深入认识牛坏死杆菌的致病机制提供研究依据，也为牛坏死杆菌防治提供理论基础。