猪肺炎支原体果糖二磷酸醛缩酶的致病机制研究

The fructose bisphosphate aldolase (FBA) of Mycoplasma hyopneumoniae（Mhp) function as a key enzyme of glucolysis and also considered as a membrane protein. In microorganisms, the FBA could be a virulence factor involved in the interaction between microbe and host by an unknown mechanism. At present, no genetic tools to manipulate Mhp genome was documented, which hampered further studies on molecular pathogenesis of Mhp. It was reported that, the expression of FBA differs between Mhp strain 168 (higher expression of FBA) and its attenuated strain 168L. Herein, we investigated the pathogenic mechanism of Mhp FBA. Firstly, the difference of glycolytic rate between Mhp strain 168 and its attenuated strain 168L were compared. Secondly, the agonist and the inhibitor of glycolysis, were used to control the glycolytic rate of both strains. Then the correlation between the glycolytic rate of the two strains and the apoptotic rate of swine trachea epithelial cell induced by the same strains was analyzed. Lastly, both the pathogenesis mechanisms and the membrane protein features will be identified. The monoclonal antibody of FBA will be used to block the FBA (cloning and expression) and FBA in both strains and the apoptotic rate in swine trachea epithelial cells induced by FBA will be detected before and after blocking with FBA-monoclonal antibody. Finally, the main regulator of apoptosis will be detected to revealing the pathogennic mechanisms of Mhp FBA. This study, will further deepen the understanding of Mhp pathophysiology and the molecular pathogenesis of Mhp which may help in designing therapeutic agents to control Mhp infection.

猪肺炎支原体（Mhp）果糖二磷酸醛缩酶（FBA）作为糖酵解关键酶和膜表面蛋白发挥功能。研究表明微生物中的FBA可作为毒力因子参与微生物与宿主间的相互作用，但具体分子机制还不清楚。在目前Mhp无法实现基因定点敲除的情况下，本项目拟利用的Mhp168强毒株（高表达FBA）与168L弱毒株表达FBA的差异，分别从糖酵解和膜蛋白两方面研究其致病机制。糖酵解方面，首先对比Mhp 168L株及168株糖酵解速率的差异，再利用糖酵解激动剂和抑制剂研究糖酵解对168L株和168株诱导猪气管上皮细胞凋亡的影响及分子机制；膜蛋白方面，以FBA单克隆抗体预作用下的FBA、168L株和168株诱导猪气管上皮细胞凋亡的变化探讨FBA的致病性。最后通过凋亡调节物的检测探讨FBA诱导猪气管上皮细胞凋亡的胞内通路，最终揭示FBA的致病机制。以上研究将加深对Mhp致病机制的了解，从而促进环境友好型Mhp防治技术的开发。

猪肺炎支原体(Mycoplasma hyopneumoniae, Mhp)果糖1,6-二磷酸醛缩酶（fructose 1,6-bisphosphate aldolase，FBA) 属于Ⅱ类醛缩酶，其主要功能是作为糖酵解途径的关键酶参与能量代谢，为猪肺炎支原体提供能量。在提供能量的同时产生一些自由基或其他物质，影响宿主细胞。同时，醛缩酶也可能作为猪肺炎支原体的一个毒力因子参与致病作用，但具体机制还不清楚。本项目揭示了猪肺炎支原体168L弱毒株及其亲本强毒株168株糖酵解速率的差异，发现强毒株168株的糖酵解速率比猪肺炎支原体168L弱毒株快，这一差异可能与强弱毒株醛缩酶表达量的差异有关。通过对比猪肺炎支原体168强毒株和弱毒株诱导猪气管上皮细胞凋亡的研究发现猪肺炎支原体168强毒株相比弱毒株更能诱导细胞的凋亡。进一步研究发现猪肺炎支原体诱导的细胞凋亡是通过线粒体途径诱导的。克隆表达猪肺炎支原体醛缩酶，通过Overlap PCR点突变扩增醛缩酶基因，成功构建了Mhp FBA重组菌，重组工程菌在IPTG诱导下成功获得表达融合蛋白猪肺炎支原体Ⅱ类果糖二磷酸醛缩酶（Mhp FBA），大小约为35 KDa，该重组蛋白具有正确的二级结构和酶的活性。通过流式细胞术和荧光显微镜观察显示猪肺炎支原体FBA蛋白定位在猪肺炎支原体膜上，且对猪气管上皮细胞具有黏附作用。进一步研究发现这种黏附作用可能与纤连蛋白有关。通过离体试验发现猪肺炎支原体FBA能够诱导猪气管上皮细胞凋亡，FBA多抗能都拮抗这种诱导凋亡的作用。进一步研究发现诱导的细胞凋亡可能与FBA作为糖酵解关键之一，促进能量代谢有关。通过细胞通路研究发现猪肺炎支原体FBA诱导的细胞凋亡与caspase依赖性途径有关。