PavA通过激活AMPK/mTOR通路诱导肺泡上皮细胞自噬在肺炎链球菌感染中的作用机制研究

Streptococcus pneumoniae is a natural resident of the upper and lower respiratory tracts of humans, as well as the major cause of community acquired pneumonia, pleurisy, otitis media, bacterial meningitis and septicemia. It has been shown that Streptococcus pneumoniae can invade epithelial and endothelial cells. One of the most important toxins release by streptococcus pneumoniae is pneumococcal adherence and virulence factor A (PavA) is displayed to the cell outer surface of Streptococcus pneumoniae and mediates pneumococcal binding to epithelial cells. PavA, which lacks a typical gram-positive signal sequence and cell surface anchorage motif, is essential for pneumococcal virulence. However, the mechanism of PavA in the interactive dialogue in the Streptococcus pneumoniae with human lung epithelial cells is still unknown...Our previous studies showed that streptococcus pneumoniae induces autophagy of alveolar epithelial cells in the infection of lung epithelial cells. Thus, we presume that PavA induces autophagy of alveolar epithelial cells in the infection of streptococcus pneumoniae via AMPK/mTOR pathway. Autophagy is an innate immune defense mechanism against intracellular Streptococcus pneumoniae. The mutant protein and the mutant strain were constructed. Cells low expression of AMPK was constructed by RNAi, high or low expression of AMPK or autophagy was constructed by a variety of methods. ..Thus, we aim to study autophagy in lung alveolar epithelial cells and to reveal the induction of autophagy by pneumococcal adherence and virulence factor A through activation of the AMPK/mTOR pathway. This observation can provide useful information for further understanding of the role of autophagy in respiratory pneumococcal infection and improve our knowledge of mucosal immunity against streptococcus pneumoniae...Therefore, this project intends to study the mechanism of autophagy defends alveolar epithelial cells against invading streptococcus pneumoniae mediated by PavA, to clarify the relationship between structure and function of PavA protein, to investigate the defense mechanism of autophagy in alveolar epithelial cells as the innate immune in the Streptococcus pneumoniae infections mediated by PavA.

肺炎链球菌粘附和毒力因子A(PavA)是肺炎链球菌早期感染过程中关键的毒力因子，PavA诱导的细胞和体液免疫能有效抵抗肺炎链球菌的感染，但PavA与人肺泡上皮细胞相互作用的分子机制研究尚属空白。本课题组前期研究发现肺炎链球菌可诱导肺泡上皮细胞自噬，进一步的试验又发现PavA可激活AMPK信号通路，故推测肺炎链球菌可通过PavA激活AMPK/mTOR信号通路参与肺泡上皮细胞自噬，在肺炎链球菌感染过程中发挥重要作用。本课题组已表达和纯化PavA及其突变蛋白，下一步拟利用CRISPR/Cas9技术构建PavA缺陷的突变菌株，利用多种方法分别构建AMPK和自噬表达上调及下调的细胞模型，来阐明肺炎链球菌感染肺泡上皮细胞过程中PavA蛋白发挥的作用，并全面证明肺炎链球菌通过AMPK/mTOR信号通路诱导肺泡上皮细胞自噬从而清除病原菌的分子机制，为研制针对PavA蛋白的疫苗和开发抑制性药物奠定研究基础。

肺炎链球菌粘附和毒力因子A(PavA)是肺炎链球菌早期感染过程中关键的毒力因子，PavA诱导的细胞和体液免疫能有效抵抗肺炎链球菌的感染，但PavA与人肺泡上皮细胞相互作用的分子机制研究尚属空白。.本项目主要阐明肺炎链球菌感染肺泡上皮细胞过程中PavA蛋白发挥的作用，并证明肺炎链球菌PavA通过AMPK/mTOR信号通路诱导肺泡上皮细胞自噬的分子机制，明确肺泡上皮细胞自噬体是否对PavA介导的肺炎链球菌感染具有保护作用。.本项目通过原核表达PavA及其突变蛋白，利用RNAi的方法分别构建Atg5和AMPK表达下调的细胞模型，来阐明肺炎链球菌感染肺泡上皮细胞过程中PavA蛋白发挥的作用，并证明肺炎链球菌通过AMPK/mTOR信号通路诱导肺泡上皮细胞自噬的分子机制。.本项目通过PavA蛋白与A549细胞的相互作用，证明了PavA可以激活AMPK信号通路，而mut-PavA并不能激活AMPK。AMPK可通过抑制mTOR通路诱导肺泡上皮细胞自噬。PavA作用于A549细胞后，细胞的凋亡和坏死增加，而PavA作用于Atg5基因沉默的A549细胞，细胞的凋亡和坏死有所减少，说明PavA蛋白可通过自噬途径引起细胞凋亡和坏死。另外，PavA作用于A549细胞后，ATG5、Beclin-1和AMPK蛋白的表达明显上升，而PavA作用于siAMPK的A549细胞后，ATG5、AMPK和Beclin-1的表达与对照组相比明显下降，这说明PavA可以通过激活AMPK介导自噬过程。.综上结论，肺炎链球菌PavA可以激活AMPK信号通路，AMPK可通过抑制mTOR通路诱导肺泡上皮细胞自噬，PavA通过AMPK/mTOR通路诱导肺泡上皮细胞自噬。.本研究全面证明肺炎链球菌通过AMPK/mTOR信号通路诱导肺泡上皮细胞自噬从而清除病原菌的分子机制，为研制针对PavA蛋白的疫苗和开发抑制性药物奠定研究基础。