Integrated Transcriptomic And Proteomic Analysis Of Swine Tracheal Mucosa Infected By Mycoplasma Hyopneumoniae And Its Pathogenesis

Mycoplasma hyopneumoniae is the etiological pathogen of Mycoplasma pneumonia of swine(MPS),which is characterized as a chronic respiratory disease.Economic losses related to M.hyopneumoniae are associated with decreased feed efficiency,reduced average daily gain,and increased medication costs.In an effort to mitigate these economic losses,swine veterinarians and producers utilize several control measures,including optimizing management and housing,vaccination,and strategic antimicrobial medication from the first isolation the pathogen in 1965.When control measures are insufficient,the research on the pathogenesis of M.hyopneumoniae is of great significance for the development of effective prevention and control measures.When M.hyopneumoniae infects pigs,the first step is to adhere with the cilia in swine trachea.It can help fight against physical,biochemical and immune clearance,causing tracheal cilia injury shedding,and gradually infect the lung,and finally leading to MPS.Till now,the studies on the pathogenic mechanism of M.hyopneumoniae mainly focus on adhesion-associated virulence factors.However,a comprehensive understanding on the interaction between the pathogen and the host is still insufficient.Therefore,the whole picture of swine tracheal mucosa infected with M.hyopneumoniae through the digital gene expression tag profiling(DGE)and isobaric tags for relative and absolute quantitation(iTRAQ)is illustrated.We found that the changes of swine tracheal mucosa infected by M hyopneumoniae mainly included cell death apoptosis,immunosuppression,and lipid metabolism.In the cell model,we found the lipid associated membrane proteins of M.hyopneumoniae and the recombinant protein P76 can induce apoptosis of immune cells.Forthermore,the interaction between M.hyopneumoniae infection and cholesterol was investigated in this study.1 Integration analysis of swine tracheal mucosal injury infected by M.hyopneumoniae using transcriptomic and proteomicSixteen 1 month old,healthy ternary hybrid pigs were divided randomly into 4 groups,4 pigs per group.8 pigs in control group were injected phosphate buffer solution(PBS)in tracheal,and 8 pigs in challenge group(Mhp)were injected with M.hyopneumoniae in tracheal.14 and 28 days post challenge(DPC),the lung,trachea and tracheal mucosa of 4 pigs in each group were collected,respectively.Chronic,non-productive coughing and asthma,the main clinical signs and macroscopic lesions,consisting of purple to grey areas of pulmonary consolidation,were observed in M.hyopneumoniae infected pigs.Histopathological analysis of the lungs showed interstitial pneumonia in M.hyopneumoniae infected pigs.Neither macroscopic nor microscopic lesions were observed in the control pigs.Scanning electron microscopy analysis showed the bronchial cilia breaking from the base in the infected pigs.The changes profiles of the tracheal mucosal tissues in the control group and the M.hyopneumoniae infection group were analyzed by digital gene expression tag profiles(DGE)and isobaric tags for relative and absolute quantitation(iTRAQ).Compared with the control group,425 genes and 281 proteins were differentially expressed in the M.hyopneumoniae infection group at 14 DPC,and 339 mRNAs and 148 proteins were differentially expressed at 28 DPC.The bio-functions,pathways and networks of differentially expressed genes and proteins were analyzed by Ingenuity pathway analysis software.The analysis results showed that the bio-functions,including infection,inflammation and death,were significantly activated by M.hyopneumoniae infection,while the bio-functions,including cell proliferation,differentiation,movement,adhesion and immunity,were significantly inhibited at 14 DPC.At 28 DPC,the bio-function of cell apoptosis,death,cell morphology,cell development were activated,and that bio-functions of cell number,movement,chemotaxis,adhesion,binding were inhibited.The affected cell type mainly included lymphocytes,mononuclear leukocytes,macrophages and other immune cells.The dysfunctional canonical pathways mainly focused on cholesterol metabolism pathway,apoptosis,inflammatory response,immune defection,protein modification at 14 DPC,and cholesterol metabolism,inflammatory response,apoptosis,signal transduction,dysfunction at 28 DPC.Furthermore,the infection caused significant changes in the network in cell death,development,inflammation,metabolism and immune at 14 DPC,while significant changes of development,metabolism,immunosuppression and other networks were found at 28 DPC.The results suggest that cholesterol metabolism,apoptosis and inflammation are invoved in the pathogenesis of M.hyopneumoniae infection.2 M.hyopneumoniae induce apoptosis of porcine alveolar macrophages 3D4/21 cell and mechanism involvedThe model of M.hyopneumoniae infected porcine alveolar macrophage(PAM)3D4/21 was established.The results showed that nitric oxide concentrations in the supernatants of 3D4/21 cells culture increased in a dose and time-dependent manner following M.hyopneumoniae infection(P<0.05).Meanwhile,the apoptotic rates of 3D4/21 cells infected with M.hyopneumoniae for 12 h and 24 h were significantly higher than those of the uninfected control group(P<0.05).This suggests that M.hyopneumoniae could induce apoptosis of 3D4/21 cells.It is reported that mycoplasma contains a lot of lipoproteins,which can adhere to the host cells and then induce cell damages.The lipid associated membrane lipoproteins(LAMPs)were extracted from M.hyopneumoniae by Trion-X 114.The assessment of MTT[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]assay showed that the half inhibitory concentration(IC50)of M.hyopneumoniae LAMPs for 3D4/21 cell was 0.34 mg/mL.After incubated with M.hyopneumoniae LAMPs for 12 h and 24 h,cells were stained with Annexin V-FITC and PI.The flow cytometry test results showed that cell apoptosis increased on a time-dependent manner(P<0.05).The contents of IL-6,TNF-?,superoxide and nitric oxide(NO)in cell and culture supernatant of 3D4/21 cells treated with M.hyopneumoniae LAMPs were significantly higher than those in the control group(P<0.05).Caspase 3 and caspase 8 in 3D4/21 were significantly activated by M.hyopneumoniae LAMPs.While 3D4/21 pretreated with antioxidant N-acetylcysteine(NAC)or total NOS inhibitor(L-NMMA)for 1 h,the cell apoptotic rate caused by M.hyopneumoniae LAMPs was significantly relieved.After exposure of 3D4/21 cell to M.hyopneumoniae LAMPs,Bax/Bcl-2 ratio was significantly increased(P<0.05),the phosphorylated ERK was increased at 1 h and gradually reverted at 3 h while phosphorylated p38 MAPK increased significantly from 3 h in 3D4/21 cells(P<0.01).Previous studies have shown that the expression of P76 protein in M.hyopneumoniae was significantly up-regulated after infection.E.coli was used to express recombinant P76 protein.We found that the recombinant P76 protein induced apoptosis in porcine alveolar macrophages via caspase 3 and caspase 8 activation by mitochondrial pathway.The results indicate that M.hyopneumoniae and its membrane lipoprotein and recombinant P76 protein could induce apoptosis in 3D4/21 cell,via activating caspase 8 and caspase 3,producting NO,superoxide and proinflammatory factors by p38 MAPK pathway.3 The role and mechanism of cholesterol in Mycoplasma hyopneumoniae infectionThe correlation analysis showed that the antibody in porcine serum was positively correlated with total cholesterol concentration(Tch)(P<0.01).The lung tissue with lesion caused by M.hyopneumoniae had higher cholesterol contents than that in not lesion lung tissue(P<0.05).The results showed that the level of cholesterol in lung tissues was correlated to some extent with M.hyopneumoniae infection.The effect of M.hyopneumoniae infection on cholesterol metabolism in host cells was studied on PAM cell model.Oil Red O staining and intracellular cholesterol assay were used to quantify cellular cholesterol levels.The results show that the total cholesterol content and cholesterol esters content in porcine alveolar macrophages treated with M.hyopneumoniae was significantly higher than those in control(P<0.05).Real-time reverse transcription polymerase chain reaction(RT-PCR)was used to measure messenger RNA(mRNA)expression of target genes.The mRNA expresion level of sterol regulatory element binding protein 2(SREBP2),famesyl ester X receptor(FXR)and low-density lipoprotein receptor(LDLR)were significantly up-regulated in 3D4/21 cells treated with M.hyopneumoniae(P<0.05),that of 3-Hydroxy-3-methylglutaryl coenzyme A reductase(HMGCR)was not changed;but that of apolipoprotein E(ApoE),type B scavenger receptor(SR-B1)and cholesterol 7? hydroxylase 1(CYP7?1)were significantly down-regulated(P<0.05).The above results suggest that M.hyopneumoniae infection increases the cholesterol content of porcine alveolar macrophages by affecting cholesterol transport and transformation.We investigated also the effect of cholesterol on PAM cell apoptosis induced by M.hyopneumoniae infection.Cholesterol supplement in infection system can promote the proliferation and adhesion to 3D4/21 cells of M.hyopneumoniae,and promote the increase of NO,caspase 3 activation and apoptosis in 3D4/21cells infected with M.hyopneumoniae(P<0.05).The mRNA expression levels of GR,TLR2,TLR4,IL-1?,IL-6 and TNF-? in the 3D4/21 cells co-treated with cholesterol and M.hyopneumoniae were significantly up-regulated(P<0.05)than those in 3D4/21 cells treated with M.hyopneumoniae.Cholesterol improved mRNA expression of genes associated with DNA and protein synthesis,glucose and lipid metabolism,partial proteins,phosphoenolpyruvate-phosphotransferase system and adhesion factors in M.hyopneumoniae(P<0.05).Cholesterol supplement in culture medium promoted M.hyopneumonia proliferation(P<0.05).The above results indicated that cholesterol promotes the M.hyopneumoniae adhesion to 3D4/21cells and increases apoptosis of 3D4/21 induced with M.hyopneumoniae via NO production,TNF-? expression and caspase 3 activation.In summary,the damages of swine respiratory tract mucosa caused by M.hyopneumoniae infection involve cell death,lipid metabolism and immune function classification,and the immune cells played an important role in the damages.The results showed that M.hyopneumoniae and its LAMPs and recombinant P76 protein could induce apoptosis of porcine alveolar macrophages.The NO,peroxides,inflammatory factors and caspase 3 are involved in this apoptosis process.M.hyopneumoniae infection can increase cholesterol content in porcine alveolar macrophage.The cholesterol can promote M.hyopneumoniae proliferation and adhesion,as well as apoptosis of infected porcine alveolar macrophages.This study provides a new insight in the understanding the pathogenesis of M.hyopneumoniae.