嗅觉受体OR5M11介导猪胸膜肺炎放线杆菌感染肺泡巨噬细胞的机制研究

Porcine Alveolar Macrophages (PAMs) are important target cells of Actinobacillus pleuropneumoniae (APP) infection. Adhesion is the primary step of APP infection and associate with trimetric autotransporter adhesion Adh. We first found and confirmed that PAMs surface Olfactory Receptor OR5M11 is an interactive protein of Adh. However, the mechanism of OR5M11 mediated PAMs activation by Adh is still poorly understood. Therefore, we will use APP wild type (APP 5b WT) and Adh deficiency APP (5b ΔAdh) as the model strains, OR5M11 as the research object, combine with eukaryotic cell surface transient overexpression, antibody block and lentivirus-CRISPR/Cas9 gene editing technology to overexpress the expression of OR5M11 or knock-out OR5M11 gene to systemic study the intrinsic molecular mechanisms of Adh mediated adhesion, invasion and activation of APP to PAMs and increased the PAMs apoptosis and secretion of inflammatory cytokines IL-8 by binding to OR5M11 in vitro. Finally to verify those results on piglets model one by one in vivo, and reveal the molecular mechanism of Olfactory Receptor OR5M11 mediated APP infection of PAMs. This will provide a novel idea for understanding the molecular mechanisms of selective infection of host cells by APP.

猪肺泡巨噬细胞（PAMs）是猪胸膜肺炎放线杆菌（APP）感染的重要靶细胞，黏附是APP感染PAMs的首要步骤，且该过程与三聚体自转运粘附素功能域Adh密切相关，PAMs表面嗅觉受体OR5M11是本课题组首次发现并初步证实为Adh的互作蛋白，而OR5M11介导Adh活化PAMs的机制目前尚不清楚。因此，本项目拟以APP野生菌（APP 5b WT）和Adh基因缺失菌（5b ∆Adh）为模式菌株，OR5M11为研究对象，采用真核细胞表面过表达、抗体封闭和慢病毒-CRISPR/Cas9基因敲除等相关技术，在细胞水平上系统研究Adh通过结合OR5M11促进APP黏附和侵袭PAMs，激活PAMs并诱导其产生细胞凋亡和分泌细胞因子IL-8的内在信号通路，最后在仔猪动物模型上进行验证，从而揭示嗅觉受体OR5M11介导APP感染肺泡巨噬细胞的分子机制，这将为APP宿主细胞选择性感染机制的研究提供新思路。

猪胸膜肺炎放线杆菌（APP）是典型经猪呼吸道感染并侵害肺脏的细菌，导致肺脏严重不可逆损伤，猪肺泡巨噬细胞（PAMs）是APP感染的重要靶细胞，黏附是APP感染PAMs的首要步骤，且该过程与三聚体自转运粘附素功能域Adh密切相关，PAMs表面嗅觉受体OR5M11是本课题组首次发现并初步证实为Adh的互作蛋白，因此，阐明Adh如何通过OR5M11活化PAMs的分子机制显得尤为重要。本项目通过嗅觉受体OR5M11过表达和缺失，证实了三聚体粘附素Adh在肺泡巨噬细胞表面的受体为OR5M11；较为全面揭示Adh通过嗅觉受体OR5M11活化PAMs 的关键信号通路及分子机制，发现Adh可以通过OR5M11激活NLRP3炎性小体的活化，进一步加重APP对肺脏的早期病理性损伤；采用TMT标记技术，较为系统的研究了APP野生菌和Adh基因缺失菌感染后PAMs蛋白质组学的差异，筛选并分析Adh参与的APP感染PAMs关键靶蛋白。另一方面，针对Adh筛选了特异性抗APP的多肽类抗菌药物MPX，并且完成了体外和体内抗APP分子机制的研究。该研究成果丰富了对细菌三聚体自转运黏附素的认识，为APP宿主细胞选择性感染机制的研究提供新思路，也为猪胸膜肺炎的防治提供了必要的理论基础。