脂蛋白LppT在猪肺炎支原体感染与致病中的作用研究

Mycoplasma hyopneumoniae is the causative agent of porcine enzootic pneumonia (EP), which is responsible for major economic losses in the global swine industry. Relative control has been achieved through active vaccination programs, but this highly infectious organism is difficult to eradicate, and continues to be a major economic problem. Until now, the pathogenesis of M. hyopneumoniae is still unclear. To gain new insight into the components that contribute to virulence and the mechanisms by which M. hyopneumoniae causes disease, we had performed the first comparative genomics and proteomics analysis of virulent and its attenuated strains. The results indicated that the LppT membrane protein might be related to virulence. The aim of the present project is to study the role of the lipoprotein LppT in infection and pathogenisis of M. hyopneumoniae. The isogenic mutants of LppT would be constructed by using a replicable oriC plasmid through homologous recombination. Subsequently, the pathogenicity of mutant strains (LppT-) and wild-type strain are evaluated by in vitro and in vivo experiments, including the ability to bind ciliated epithelium cells, the ability to induce the apoptosis of lymphocytes and monocytes, the NF-κB/IL-6/IL-8/TNF-α secretion levels of porcine alveolar macrophage cells, and the damage degree to pigs. These results can be used to elucidate the correlation between the lipoprotein LppT and pathogenisis of M. hyopneumoniae, and provide theoretical basis for the development of new vaccines and drugs.

猪肺炎支原体（Mycoplasma hyopneumoniae，Mhp）是危害全球养猪业的重要病原，由于其感染率高、根除困难、造成的经济损失大，被喻为危害养猪业的“隐形杀手”。但迄今为止，人们对Mhp的毒力因子还知之甚少，阻碍了新型疫苗和药物的开发。申请人在前期研究中通过对Mhp强弱毒力菌株的比较基因组学和差异蛋白组学分析，发现脂蛋白LppT极可能是Mhp的潜在毒力因子。本项目拟利用已建立的猪肺炎支原体遗传操作系统构建LppT基因缺失突变株，比较野生型菌株和突变株（LppT-）对猪支气管纤毛上皮细胞黏附能力、诱导淋巴细胞和单核细胞凋亡、诱导猪肺泡巨噬细胞炎症反应以及对猪体致病力的差异，从体内和体外两方面深入研究脂蛋白LppT在Mhp感染与致病中的作用并揭示其作用机制，为阐明猪肺炎支原体的致病机制奠定基础，并为新型疫苗和药物的开发提供理论依据。

猪肺炎支原体（Mhp）是危害全球养猪业的重要病原菌之一，本课题以前期比较基因组学和差异蛋白质组学共同筛选出的潜在毒力因子LppT和LppB为研究对象，首先对其二级结构、亚细胞定位和自切割活性进行了解析，随后揭示了膜蛋白LppT和LppB在纤毛上皮细胞黏附、诱导肺泡巨噬细胞、淋巴细胞和单核细胞凋亡、诱导炎症等方面的功能，发现并证实了LppT和LppB在猪肺炎支原体感染与致病中的作用。已经发表研究论文5篇，其中SCI论文1篇；投稿BioMed Research International杂志1篇；参加国内学术会议3次，赴约翰霍普金斯大学医学院访学1年，培养研究生2名。毒力因子的发现对揭示猪肺炎支原体致病机制以及新型疫苗和药物的设计与开发具有重要的指导意义。