Study On The Completion Of Autophagy Fiux And The Intracellular Proliferation Induced By Mycoplasma Hyopneumoniae

Mycoplasmal pneumonia of swine is a respiratory infectious disease caused by M.hyopneumoniae infection that seriously harms pig industry.Our laboratory has proved that M.hyopneumoniae can enter into porcine alveolar macrophages and cause the accumulation of LC3-Ⅱand the induction of autophagy.The aim of this study is to investigate the integrity of autophagy flux induced by M.hyopneumoniae and the effect of autophagy on the growth of M.hyopneumoniae in cells.Meanwhile,we hope to preliminarily understand the host cell molecules that affect the integrity of autophagy flux induced by M.hyopneumoniae.1.Study on the optimal multiplicity of infection of 3D4/21 cells infected with M.hyopneumoniae AH strain and the optimal concentration of cells treated with autophagy modulatorsObjective:To determine the optimal multiplicity of infection of 3D4/21 cells infected with AH strain and the optimal concentration of 3D4/21 cells treated with autophagy modulator.Methods:Firstly,the growth curve of M.hyopneumoniae AH strain was determined.Then CCK-8 was used to determine the optimal infection number of AH strain infected 3D4/21 cells.Thirdly,3D4/21 cells were treated with different concentrations of rapamycin,3-Methyladenine,bafloromycin A1 and hydroxychloroquine to determine the optimal concentration and cytotoxicity of these autophagy regulators.Results:The growth curve of AH strain was Y=161.01X-18.633(X represented OD600nm,Y represented Ig CCU.)and the optimal multiplicity of infection of 3D4/21 was 200.The optimal working concentrations of rapamycin,3-Methyladenine,bafilomycin A1 and hydroxychloroquin were 1000 nmol/L,5mmol/L,200 nmol/L and 25μmol/L,respectively,and the cell viability was not affected by these autophagy modulators with the optimal working concentrations.Conclusion:The growth curve of AH strain,the optimal multiplicity of infection of3D4/21 cells infected with AH strain,the optimal working concentration of autophagy modulators and the toxicity to cells were determined,which laid a foundation for the subsequent experiments.2.Effect of M.hyopneumoniae infection on the completion of autophagy fiux in porcine alveolar macrophagesObjective:To determine the completion of autophagy flux in porcine alveolar macrophages infected by M.hyopneumoniae.Method:Thirty samples of porcine alveolar macrophages(PAM)were collected from the lungs with typical M.hyopneumoniae gross lesions of pigs,and 10 samples of PAM were collected from the lungs without any gross lesion of pigs.Western blot was used to detect the expression of Mhp366,LC3-Ⅱand p62,and indirect immunofluorescence was used to detect the co-localization of LC3 and Mhp366/LAMP2 in PAM.After infected with AH strain,the expression of p62 protein in 3D4/21 cells was detected by Western blot,the co-localization of GFP-LC3 and m RFP-LC3,GFP-LC3 and Lyso-Tracker Red in3D4/21 cells was observed by fluorescence microscope,the co-localization of LC3 and LAMP2 in 3D4/21 cells was detected by indirect immunofluorescence.Results:The expressions of LC3-Ⅱand p62 in M.hyopneumoniae-positive PAMs were significantly higher than in negative PAM.The expression of p62 in 3D4/21 cells was significantly increased after the infection of AH strain.The Mhp366 protein was colocalized with LC3 in M.hyopneumoniae-positive PAM.The colocalization number of LC3 with LAMP2 in porcine M.hyopneumoniae-negative PAMs was significantly higher than in positive PAMs.LC3 did not colocalize with LAMP2 in AH strain infected group,and the number of yellow fluorescent puncta(m RFP~+GFP~+)was significantly higher than red fluorescent puncta(m RFP~+GFP~-).The number of colocalization of GFP-LC3 with Lyso-tracker Red in EBSS group was significantly higher than AH strain-infected group.Conclusion:M.hyopneumoniae infection induces incomplete autophagy flux in PAM and 3D4/21 cells.3.The effect of autophagy on the intracellular proliferation of M.hyopneumoniaeObjective:To determine whether autophagy affects the intracellular proliferation of M.hyopneumoniae.Methods:3D4/21 cells were treated with 3-Methyladenine,and then infected with M.hyopneumoniae AH strain(MOI=2 or MOI=200).The expression of LC3-Ⅱ,p62 and P97 were evaluated by Western blot.The proliferation of M.hyopneumoniae AH strain in 3D4/21 cells were detected.3D4/21 cells infected with M.hyopneumoniae AH strain(MOI=2 or MOI=200),and then treated with rapamycin,bafilomycin A1 or hydroxychloroquin.The expression of LC3-Ⅱ,p62 and P97 were evaluated by Western blot.Results:After 3D4/21 cells were infected with AH strain(MOI=2 or MOI=200),the expression of LC3-Ⅱ,p62 and P97 and CCU of intracellular AH strain in 3-Methyladenine treated group were significantly lower than in 3-Methyladenine untreated group.After 3D4/21 cells were infected with AH strain of MOI=2,the expressions of LC3-Ⅱ,p62 and P97 in cells treated with rapamycin,bafilomycin A1 or hydroxychloroquin were increased,and CCU of intracellular AH strain was significantly increased.After 3D4/21 cells were infected with AH strain of MOI=200,compared with the untreated group,the expression of LC3-Ⅱ,p62 and P97 in cells treated with rapamycin or bafilomycin A1 did not change significantly,but the expression of LC3-Ⅱand p62 in cells treated with hydroxychloroquin were increased.Compared with the untreated group,CCU of AH strain in rapamycin,bafilomycin A1 or hydroxychloroquin treated group showed no significant difference.Conclusion:Autophagy promotes the intracellular proliferation of M.hyopneumoniae.4.Study on cleavage of the tethering factor connecting autophagosome with lysosome by M.hyopneumoniaeObjective:To determine whether M.hyopneumoniae can cleave the tethering factor connecting autophagosome with lysosome.Methods:The expression of P97,LC3-Ⅱ,STX17,SNAP29,PLEKHM1,ATG14,Rab7 and VAMP8 were evaluated by Western blot after the infection of 3D4/21 cells with AH strain.Results:Compared with the control group,the expression of STX17,SNAP29 and PLEKHM1 were decreased in AH strain infected group,while the expression of LC3-Ⅱ,ATG14,Rab7and VAMP8 were significantly increased.Conclusion:M.hyopneumoniae may destroy the STX17-SNAP29-VAMP8 and LC3-PLEKHM1-HOPS-Rab7 complexes by cleavages STX17,SNAP29 and PLEKHM1 to prevent the fusion of autophagosome and lysosome.