Wild-type Streptococcus pneumoniae pneumolysin induces autophagy in A549 cells through the PI3K-I/Akt/mTOR pathway

Abstract:

Objective：To study the Streptococcus pneumoniae（Sp）-induced autophagy in human pulmonary epithelial cells and its mechanism. Methods：Human pulmonary epithelial A549 cells and wild-type Sp strain were cultured by routine methods. The A549 cells were transfected with the eukaryotic expression vector green fluorescent protein-light chain 3（GFP-LC3） and were divided into experimental group（Sp，MOI=30∶1），negative control group（wortmannin[an autophagy inhibitor]，2 μmol/L，2 h），positive control group（rapamycin[an mTOR inhibitor]，1 mmol/L，10 h），and reference control group（co-treated with wortmannin and Sp）；each group was treated for 1 h，2 h，3 h，or 4 h. Each group was observed for the formation of autophagic spots by fluorescence microscopy，for the structure of autophagosomes by transmission electron microscopy（TEM），and for the expression of microtubule-associated protein light chain 3-Ⅰ/Ⅱ（LC3-Ⅰ/Ⅱ），phosphorylated UNC-51-like kinase1（P-ULK1），and sequestosome 1（P62） by Western blot. The A549 cells were transfected/co-transfected with red fluorescent pro－tein-tagged pneumolysin（RFP-PLY） or/and GFP-LC3. The expression levels of phosphoinositide 3-kinase-Ⅰ/Ⅲ（PI3K-Ⅰ/Ⅲ），protein kinase B（Akt），Beclin 1 protein（Beclin-1），and mammalian target of rapamycin（mTOR） were determined by Western blot. Results：After treatment of the A549 cells for 3 hours，compared with the negative control group，the Sp-infected groups had a significantly increased number of autophagic spots（t=41.313，P=0.001） and a significantly up-regulated expression level of LC3-Ⅱ（t=121.592，P=0.000），with typical structures of autophagosomes observed under the TEM. After transfection of the A549 cells by RFP-PLY for 3 hours，compared with the negative control group，the Sp-infected groups also showed obvious autophagy with significantly down-regulated expression of PI3K-I，Akt，and mTOR（tPI3K-I=16.544，PPI3K-I=0.004；tAkt=22.679，PAkt=0.002；tmTOR=19.503，PmTOR=0.003），but there were no significant differences between the above groups in the expression level of PI3K-Ⅲ or Beclin-1（tPI3K-Ⅲ=3.572，PPI3K-Ⅲ=0.070；tBeclin-1=0.799，PBeclin-1=0.508）. Conclusion：Wild-type Sp may induce autophagy in A549 cells through the PI3K-I/Akt/mTOR signaling pathway.