新型分泌系统OMVs介导副猪嗜血杆菌LPS转运激活炎性体通路与致病的机制研究

Pigs infected by haemophilus parasuis (HPS)is characterized by fibrinous polyserositis, pericarditis, peritonitis, polyarthritis and meningitis. Recently, out membrane vesicles (OMVs) are paid more and mote attention as a novel secretion system. OMVs is considered as the crucial inducer for the inflammation and pathological process caused by lots of bacteria, but it is not known that the role of OMVs in HPS infection. In our previous research, we found that OMVs of HPS wild-type H45 strain up-regulated the production of cytokines in epithelial cells. According to the previous finding that LPS, which are rich in OMVs, plays an important role in activating the inflammasome, we will focus the function of OMVs on the transmembrane transport of LPS and the relation with inflammasome in this project. The HPS wild-type strains, strains lack of genes linked to LPS , suitable cell lines, inflammasome molecular and cell cytokines were targeted to explore the role OMVs of in the LPS transmembrane transport, the activation of inflammasome and the HPS pathogenic progress from multiple dimensions. In this project, we are aim to clear up the novel pathogenic mechanism of HPS and offer new ideas for designing or improving vaccines.

副猪嗜血杆菌（haemophilus parasruis,HPS）主要引发猪多发性浆膜炎等炎性反应。新型分泌系统---外膜囊泡（outer membrane vesicles,OMVs）作为多种细菌致炎致病的最关键因素之一，在HPS感染中的作用与机制研究尚为空白。课题组最新研究表明，HPS野毒株分泌的OMVs能强有力诱导上皮细胞分泌IL-8等促炎因子。鉴于NATURE的最新发现---脂多糖（LPS）可被OMVs跨膜传输至宿主细胞质内进而活化炎性体通路，导致炎性因子分泌和细胞焦亡，我们大胆推测OMV在HPS感染过程中发挥重要作用。本项目拟构建LPS相关基因缺失株，研究在HPS感染过程中OMVs介导的LPS胞内转运、炎性体信号通路活化、炎性因子分泌等随时空变化的动态规律，解析OMVs介导的LPS转运及致炎机制，进而为新药开发、新型疫苗设计提供新思路，为HPS的有效防控提供新的理论依据

本研究以副猪嗜血杆菌血清5型强毒株H45为对象，以常用小鼠巨噬细胞为感染模型，研究发现，H45的感染激活了NLRP3炎性体通路，并对已缺失脂寡糖类脂A合成过程中的酰基转移酶LpxM基因的ΔLpxM株进行生物学鉴定，使用ΔLpxM株感染小鼠巨噬细胞，炎性体的激活水平降低。此外成功构建了Caspase11基因沉默的小鼠巨噬细胞，并通过密度梯度离心法提取并纯化了副猪嗜血杆菌的OMVs，进行了电镜观察、质谱分析，使用提取的OMVs感染小鼠巨噬细胞发现，单独OMVs同样可激活炎性体通路，单独感染Caspase11基因沉默的小鼠巨噬细胞，炎性体激活水平显著降低。总的来说，本研究证实副猪嗜血杆菌感染引起炎性体的激活，其中副猪嗜血杆菌的脂寡糖起到重要作用。提取自副猪嗜血杆菌的OMVs单独即可引起炎性体的激活，OMVs在副猪嗜血杆菌致炎过程中起到重要作用，此外作为脂多糖胞内受体的Caspase11基因的沉默，显著降低了OMVs引起的炎性体的激活水平，进一步说明，在副猪嗜血杆菌感染过程中OMVs作为载体递送了其脂寡糖进入胞内，通过非经典炎性体通路激活了炎性体，加工并释放炎性因子IL1β和IL18，通过这两个炎性因子的进一步作用引起强烈炎症反应。为副猪嗜血杆菌杆菌引起的多发性浆膜炎等的治疗提供了新的方向和策略。