猪肺炎支原体LAP激活纤溶系统诱导宿主纤毛黏液屏障破坏机制研究

Destruction of the host cilium-mucous barrier is a typical feature of Mycoplasma hyopneumoniae (Mhp) infection. Activation of the host fibrinolytic system is the key step in the process, but the mechanism is still unknown. Leucine aminopeptidase (LAP) is a newly identified virulence factor of Mhp. The previous study found that it was located on the surface of Mhp cells. LAP not only mediates the adhesion of Mhp to the host, but also has plasminogen-binding activity. So it is supposed that LAP may play an important role in the process of cilium-mucous barrier destruction, which is mediated by fibrinolytic system during Mhp infection. In this project, we are going to determine the crystal structures of LAP and its complex with plasminogen. It will be helpful for understanding the interaction mechanism between LAP and plasminogen. On the other hand, we are going to block the functions of LAP and plasminogen respectively during Mhp infection, and to investigate the effect of LAP and plasminogen on the disruption of cilium-mucous barrier. This will help us to understand the mechanism of cilium-mucous barrier disruption, which is mediated by fibrinolytic system and induced by Mhp LAP. This project will help to understand the mechanism that how Mhp LAP induces the invasion to the host respiratory tract. It will also give insights for the development of new anti-mycoplasma drugs.

对宿主纤毛黏液屏障系统的破坏是猪肺炎支原体（Mycoplasma hyopneumoniae, Mhp）感染的典型特征，诱导宿主纤溶系统的激活是该过程的关键步骤，但目前机制尚不清楚。亮氨酸氨肽酶（LAP）是Mhp新鉴定的毒力因子，申请人前期研究发现，它位于Mhp细胞表面，不仅能介导Mhp对宿主的黏附，还具备纤溶酶原结合活性，提示LAP可能在Mhp激活宿主纤溶系统诱导纤毛黏液屏障破坏这一过程发挥重要作用。本项目，一方面结合互作分析，拟通过解析LAP及其与纤溶酶原复合物的晶体结构，从原子水平揭示LAP与纤溶酶原的相互作用机制；另一方面，通过本动物试验，分别阻LAP和纤溶酶原活性，观察Mhp攻毒后对纤毛黏液屏障破坏的影响，进一步明确LAP通过激活纤溶系统诱导纤毛黏液屏障的破坏作用及机制。本项目的开展，将有助于理解LAP诱导Mhp对宿主呼吸道的入侵机制，同时也为抗支原体药物新靶标的发现提供思路。

猪肺炎支原体(Mycoplasma hyopneumoniae, Mhp)是严重危害养猪业的重要病原，主要引起猪气喘病。发病猪饲料利用率低，免疫力低下，易于与其它病原混合感染，发生猪呼吸道疾病综合征(Porcine respiratory disease complex, PDRC)，造成巨大经济损失。.对宿主纤毛黏液屏障系统的破坏是猪肺炎支原体（Mycoplasma hyopneumoniae, Mhp）感染的典型特征，诱导宿主纤溶系统的激活是该过程的关键步骤，但目前机制尚不清楚。亮氨酸氨肽酶（LAP）是Mhp新鉴定的毒力因子，本研究发现，它位于Mhp细胞表面，能介导Mhp黏附猪气管上皮细胞，其抗体可以特异性地抑制猪肺炎支原体对猪气管上皮细胞的黏附，此外，Mhp LAP可以结合纤溶酶原，相互作用呈现剂量依赖型，提示LAP可能在Mhp激活宿主纤溶系统诱导纤毛黏液屏障破坏这一过程发挥重要作用。本项目解析了Mhp LAP的晶体结构，分辨率达到2.4Å。Mhp LAP是个六聚体，通过结构比对和序列比对发现Mhp LAP的N端结构域与其它氨肽酶差异显著，可能与其黏附功能相关。建立了猪纤溶酶原的纯化方法，验证其具备完整的功能，并通人工智能方法建模了其结构。通过对Mhp LAP和sPLG进行结构分析，模拟了二者的相互作用，推测Mhp LAP的N端功能域与纤溶酶原KR1之间通过正负电荷相吸的方式相互作用。通过多点突变和SPR测定，验证Mhp LAP的57位和115位对纤溶酶原的相互作为尤为重要。此外，用氨肽酶抑制剂Bestatin作用支原体，发现对猪肺炎支原体，猪鼻支原体，鸡毒支原体，山羊支原体均具有生长抑制作用，且呈现计量依赖性，提示氨肽酶可能是抗支原体药物的一个新型靶标。.烯醇化酶（Enolase，Eno）是纤溶酶原的一个保守受体，也是猪肺炎支原体的重要毒力因子。通过far-WB，SPR等实验发现Mhp Eno是纤溶酶原的另一重要受体。解析Mhp Eno 的晶体结构，分辨率达2.3埃。解析的结构发现Mhp Eno具备种属特异性区域，主要包括S3/H1 loop， H6/S6 loop，H7/H8 loop，H13区域，上面的带电氨基酸可能与纤溶酶原的结合有关。.综上所述，猪肺炎支原体表面黏附因子可以通过形成多重网络与纤溶酶原结合，激活纤溶系统，介导对宿主的纤毛黏液屏障的破坏。