PavA Induces Autophagy Of Alveolar Epithelial Cells In The Infection Of Streptococcus Pneumoniae Via AMPK/mTOR Pathway

BackgroundStreptococcus 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.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.ObjectivesThis study aims 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.In this study,PavA and its truncated protein were expressed in prokaryotic cells,and the down-regulated cell models of Atg5 and AMPK were constructed by RNAi method to elucidate the role of PavA protein in the process of pneumococcal infiltration of alveolar epithelial cells,as well as to understand the molecular mechanism of alveolar epithelial cell autophagy by AMPK/mTOR signaling pathway to remove pathogensMethodsCells low expression of AMPK was constructed by RNAi,high or low expression of AMPK or autophagy was constructed by a variety of methods.In order to observe whether PavA protein and truncated-PavA protein could activate the AMPK signal pathway of alveolar epithelial cells and whether PavA activates AMPK signal pathway,We expressed the PavA protein and truncated-PavA protein in prokaryotic cells Alveolar epithelial cells were treated with AMPK agonist AICAR and inhibitor CompoundC,then the levels of gene and protein of autophagy-associated protein were detected by reverse transcription and Western blot.We successfully established alveolar epithelial cell line which was transfected with siAMPK virus to understand the effect of AMPK on the autophagy signal pathway,and whether AMPK induced autophagy of alveolar epithelial cells by inhibiting mTOR pathway.We established the low autophagic alveolar epithelial cell lines via RNAi technique to observe the expression of Beclin-1 and LC3 and the replication level in the process of different autophagic level alveolar epithelial cells,as well as to understand the role of autophagy in resisting PavA-mediated Streptococcus pneumonia infection.And to investigate whether alveolar epithelial autophagy has a protective effect on PavA-mediated Streptococcus pneumoniThe recombinant plasmid pLVX-shRNA2-AMPK transfected the human a infection.alveolar type ? Atg5-deficient epithelial cell line successfully as WesternBlot demonstrated.And this plasmid significantly reduced the expression of Atg5 in A549 cells.ResultsIn this research,the PavA and truncated-PavA gene were synthesized and put into pGEX-4T-1 vector in which was induced by IPTG for expression.The expressed fusion protein carries a GST tag for further affinity purification.And the fusion protein also carries a thrombin-specific cleavage site in order to separate the target protein from the fusion protein.The recombinant plasmid pLVX-shRNA2-Atg5 transfected the human alveolar type ? Atg5-deficient epithelial cell line successfully as WesternBlot demonstrated.This plasmid significantly reduced the expression of Atg5 in A549 cells.The recombinant plasmid pLVX-shRNA2-AMPK transfected the human alveolar type ? Atg5-deficient epithelial cell line successfully as WesternBlot demonstrated.This plasmid significantly reduced the expression of Atg5 in A549 cells.This study demonstrated the interaction between PavA protein and A549 cells and showed that PavA can activates AMPK signaling pathway while truncated-PavA cannot.ConclusionsOur studies showed that streptococcus pneumoniae induces autophagy of alveolar epithelial cells in the infection of lung epithelial cells.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.