Preliminary Study Of Autophagy In Lung Epithelial Cells Induced By M. Ovipneumoniae Infection

Background:Mycoplasma ovipneumoniae(MO)is a highly contagious disease that leads to Contagious ovine pleuropneumonia in sheep and goats. The pathogen is wide-spreading and can cause serious economic loss. Studies have showed that MO could attach and do harmful to Ovis aries lung cell lines.Thus, whether host cells adopted the same mechanism defence against MO is an intriguing topic.Autophagy is an evolutionally conserved cellular physiological mechanism in eukaryotes and important for the maintenance of cellular homeostasis. Autophagy phagocytosed and degras the cellular aging organelles andlong-lived proteins by fused with lysosome. The degradation products are recycled to maintain the function of cellular organelles and cellular homeostasis. In addition, autophagy has the important function to defense bacteria infections.Objectives:The objection of our research is to investigate whether MO could induce autophagy formation and what is the effect of autophagy on MO infection.Methods:Our research had been done in the lung epithelial cells of mice lines. Detect different stages(0h、6h、12h、24h) of MO infection induce autophagy in host cells, and uinfected cells for blank control.P62 or Atg7 gene was knocked down using RNA interference followed by MO infection,and the yield of MO was assessed by CCU.Results(1)The autophagy formation was confirmed by observation of autophagosome with transmission electron microscopy. MO could induced the formation of the double-membrane of autophagosome in 6h, induced the formation of autophagosome and autolysosome in 12h and 24h.(2)The distribution of LC3 detected by fluorescence microscopy shows that:LC3 gradually changed from dispersive distribution to aggregated distribution when the TC-1 cells are stimulated by MO of MOI 1:10 for 6 h. The fusion of Autophagy and lysosomes is obviously higher than the control group in 12h and 24h.(3)qPCR results showed that mRNA expression of autophagy-related genes LC3A/B, P62, Atg7 were upregulated following MO infection and Western blot results revealed a similar pattern compared with the results of the qPCR.(4)The CCU experimental suggested that TC-1 cells could decreased the replication of MO progeny.(5)Compared to mock cells,the amount of P62 or Atg7 proteins was decreased in TC-1 cells transfected with corresponding siRNA.Silencing the gene markedly boosted the replication of MO.Conclusion:To conclude,in this study we showed that MO infection could activate TC-1 cells autophagy and the highest level of autophagy occurs in 12 h. Autophagy involved in the clearance of MO by TC-1 cells.These results will contribute to further investigation of MO-host interactions and will be helpful for the new strategies for the control and treatment of MO infection.