A Preliminary Study On Autophagy Induced By Mycoplasma Hyopneumoniae

Mycoplasmal pneumonia of swine is a chronic respiratory disease of swine caused by Mycoplasma hyopneumoniae.It characterized by widespread epidemic,high morbidity and low mortality.Through long-term research,our group found that M.hyopneumoniae infection mainly stimulates humoral immunity,and due to the capacity of antibacterial in host is limited,and antibiotics and vaccines cannot completely eliminate or kill M.hyopneumoniae,resulting in continuous latent infection of M.hyopneumoniae.Therefore,we speculate that M.hyopneumoniae can also exist in host cells as a traditional extracellular bacterium.Autophagy is a degradation process that depends on lysosomes and plays an important role in maintaining the stability of the intracellular environment.On the one hand,autophagy can clear the pathogens that enter host cells.On the other hand,some pathogenic microorganisms can use autophagy to live in host cells and to escape the killing of immune cells.Therefore,we speculated that M.hyopneumoniae could survive or even proliferate in host cells by autophagy.In order to explore the autophagy response induced by M.hyopneumoniae infection and the relationship between proliferation of M.hyopneumoniae and autophagy,this study will investigate the relationship between M.hyopneumoniae and autophagy from the following aspects:1.Identifying the existence of M.hyopneumonaie in cellsObjective: Identifying the existence of M.hyopneumonaie in cells.Methods: Firstly,we detected whether M.hyopneumoniae can be resided in pig lung cells.Thirty lung tissues with typical enzootic pneumonia lesions and 30 healthy lung tissues were collected for detection of M.hyopneumonaie by nested PCR.The PCR positive lung tissues and PCR negative lung tissues were cultured for detection of M.hyopneumonaie in the cells by indirect immunofluorescence.Secondly,it was determined whether M.hyopneumonaie could infect the porcine cell line.The porcine kidney cell line PK15 was used as the cell line in our study used to study the interaction between M.hyopneumonaie and cells.After determining the growth curve of M.hyopneumonaie AH strain and the optimal multiplicity of infection PK15 cells,AH strain infects PK15 cells with the optimal multiplicity of infection to determine whether AH strain can enter PK15 cells.Results: M.hyopneumonaie was detected in 28 lung tissues with typical MPS lesions,9 of which were successfully cultured and detected in lung cells.The optimal multiplicity of infection of PK15 cells infected by AH strain was 500.Immunofluorescence test showed that AH could enter PK15 cells.Conclusion: M.hyopneumonaie can enter the host cells after infection.2.Research on autophagy induced by M.hyopneumoniae infectionObjective: Confirming that M.hyopneumoniae could induce autophagy.Methods: First,indirect immunofluorescence was used to detect the number of LC3 spots in pathological lung cells with MPS typical lesions and in normal lung cells without M.hyopneumoniae infection.Then the autophagosomes in PK15 cells infected by M.hyopneumoniae AH strain was detected by transmission electron microscopy.The expressions of autophagy markers LC3-?,Atg5 and Beclin 1 in PK15 cells infected with M.hyopneumoniae were detected by Western blot,and the co-localization of M.hyopneumoniae and LC3 in PK15 cells was detected by indirect immunofluorescence.Results: The number of LC3 spots in pathological lung cells was significantly higher than that in normal lung cells.Transmission electron microscopy showed that,compared with the uninfected group,there are a large number of autophagosomes in PK15 cells infected with M.hyopneumoniae.In PK15 cells,M.hyopneumoniae and LC3 proteins were co-localized.Compared with the uninfected group,the number of LC3 spots in PK15 cells infected with M.hyopneumoniae increased significantly.Conclusion: M.hyopneumoniae infection can induce autophagy in porcine lung tissue cells and PK15 cells.3.M.hyopneumoniae infection can induce incomplete autophagy fluxObjective: To determine whether M.hyopneumoniae infection can induce complete autophagy flux and the key signal molecules that affect autophagy flux.Methods: After infection with M.hyopneumoniae AH strain,the expression of p62,a marker of autophagy flux was detected by Western blot.Also,the expressions of LC3-? and p62 in AH strain-infected PK15 cells was detected by Western blot after treatment with autophagy inhibitor 3-methyladenine(3-MA).Results: At 4 h,8 h and 12 h after M.hyopneumoniae AH strain infection,the expression of p62 was significantly higher than that of the control group,and increased with the extension of infection time,indicating that complete autophagy flux was not formed after infection of PK15 cells by M.hyopneumoniae AH strain.When treated with 3-MA,the protein levels of p62 and LC3-II in the cells decreased significantly at 4 h,8 h and 12 h after infection,indicating that the autophagy flux was also inhibited after the inhibition of autophagy induction,thus inhibiting the expression of p62.Conclusion: Incomplete autophagy flux was induced after M.hyopneumoniae infection.4.Autophagy promotes the proliferation of M.hyopneumoniae in PK15 cellsObjective: To determine the effect of autophagy on the proliferation of M.hyopneumoniae in cells.Methods: After infection with M.hyopneumoniae AH strain,the expression of P97,a structural protein of M.hyopneumoniae was detected by Western blot;the color changing unit(CCU)of M.hyopneumoniae in cells,treated with and withou inhibitor 3-MA were determined.Results: At 4 h after M.hyopneumoniae AH strain infection,the expression of P97 in cells treated with 3-MA was significantly lower than that of without 3-MA group.At 4 h,8 h,and 12 h after M.hyopneumoniae AH strain infection,the content of M.hyopneumoniae in cells treated with 3-MA was significantly lower than that of without 3-MA group.Conclusion: Autophagy promotes the proliferation of M.hyopneumoniae in cells.In summary,M.hyopneumoniae can enter cells after infecting cells and induce the occurrence of autophagy,but autophagosomes cannot fuse with lysosomes to form autophagolysosomes to degrade M.hyopneumoniae,thereby enabling the proliferation of M.hyopneumoniae in cells.