Apa在猪胸膜肺炎放线杆菌感染肺巨噬细胞中的作用与机制研究

Actinobacillus pleuropneumoniae （APP） is an important swine respiratory pathogen causing great economic losses worldwide. At present there is no ideal cross protective vaccine. Apa found by our team is a key virulence factor of APP triggering infection. And pulmonary macrophages (PAM) are the main damaged cells after APP's infection, so it is urgent and necessary to study deeply on function and mechanism of Apa in infection stage of swine pulmonary macrophages by APP strains for improvement of vaccine and drugs. With Apa as the object, based on our successfully constructed APP mutant missing Apa,this project aims to research function and mechanism of Apa in adhesion and phagocytose process, to explore APP survival mechanism in PAM, and to analyse signaling pathways of Apa mediating APP strains to induce PAM apoptosis when PAMs were infected by APPs, and to comfirm these results in animal model at last, through qPCR, transcriptome sequencing technology, iTRAQ technology. Thus revealing of the Apa role in adhesion, phagocytose, intracellular survival and inducing apoptosis process between bacteria and cell must provide much-needed theoretical basis and new targets for vaccine and drug research of swine contagious pleuropneumonia, and must promote the further research of other respiratory infections. This result is completely belongs to independent research, with independent intellectual property rights. And it has important scientific significance to the prevention and control of respiratory tract infection.

猪胸膜肺炎放线杆菌(APP)是世界性严重危害养猪业的呼吸道致病菌，目前尚无理想的交叉保护性商品疫苗。Apa是本课题组前期发现的该菌重要的致病因子，肺巨噬细胞（PAM）是该菌感染后的主要受损细胞，系统研究Apa在PAM感染受损过程中的作用及机制是疫苗和药物研究的重要基础。因此本项目以Apa为研究对象，利用课题组已构建的Apa缺失突变株，采用转录组测序技术、iTRAQ技术等，系统检测缺失株和野生株黏附PAM并致其凋亡过程中的参与基因和蛋白分子，比较其不同，筛选出与Apa密切相关的调节分子及信号通路，在细胞模型和实验动物体内一一验证，从而全面揭示Apa在细菌与细胞黏附互作、被吞噬后胞内存活、诱导细胞凋亡阶段的作用机制，为猪传染性胸膜肺炎疫苗与药物研究提供急需的理论基础和靶标，并推动其它呼吸道感染的深入研究。这一成果完全属于自主创新，拥有自主知识产权，对呼吸道感染的防控具有重要科学意义。

猪胸膜肺炎放线杆菌(APP)是世界性严重危害养猪业的呼吸道致病菌，三聚体自转运粘附素（Apa）是本课题组前期发现APP的重要致病因子，本项目利用原代猪肺泡巨噬细胞（PAM）感染模型，系统开展了胸膜肺炎放线杆菌野生菌株（APP 5b）、Apa承载区功能结构域缺失菌株（APP5b △Adh）与PAM互作研究。经APP全基因组表达谱芯片检测，发现Adh主要调控PAM 87个基因，其中包括46个上调基因和41个下调基因，上调基因主要涉及脂代谢、能量代谢、基因转录和加工等功能，下调基因则集中在氨基酸代谢、碳水化合物代谢等。Adh能够促进APP生物被膜形成，抑制宿主抗原加工与递呈等基因的表达，从而增强其致病性。首次发现Adh在猪肺巨噬细胞的互作分子OR5M11，Apa通过与OR5M11结合，介导APP菌株对细胞的粘附、侵入。当细菌被巨噬细胞吞噬后，Apa则通过显著诱导细胞凋亡使细菌逃避细胞的杀伤作用，其机制是Adh通过活化Fas和Bax等凋亡基因，激活caspase-8、9、3从而诱导PAMs的凋亡。Adh还能够激活p38信号通路介导PAM感染早期IL-8的高表达，从而实现对中性粒细胞的趋化。小鼠和仔猪感染模型证实Adh介导APP早期致病性，参与血液和肺组织中IL-8的分泌以及诱导肺组织中的PAM凋亡。表明Apa作为三聚体自转运粘附素，不仅能够介导APP菌株对肺泡巨噬细胞的粘附、在细胞内存活，而且能够诱导PAM凋亡、IL-8过度分泌、中性粒细胞趋化，加重肺炎损伤。该研究成果丰富了对细菌三聚体自转运黏附素的认识，为猪胸膜肺炎的防治提供了必要的理论基础。