The Effect Of APEC-OMV Virulence Factor OmpA On The Innate Immunity Of Chicken Tracheal Epithelial Cells

Avian pathogenic Escherichia coli(APEC),as a subgroup of extraintestinal pathogenic Escherichia coli(Ex PEC),can cause poultry of all ages to infect avian sepsis,chronic respiratory diseases,yolk peritonitis,etc.Affect weight gain and egg production rate.In addition,APEC has a high genetic homology with human urethral pathogenic Escherichia coli(UPEC)and human Ex PEC,which not only seriously endangers the development of poultry industry,but also poses a huge potential threat to human health.Therefore,the prevention and control of APEC is important public health significance.Outer membrane vesicles(OMV)are spherical double-layer lipid proteins secreted by gram-negative bacteria,which encapsulate the bioactive proteins,lipids,nucleic acids and virulence factors,etc.which are released after entering the host cell through endocytosis.The bioactive substances carried,then interact with intracellular proteins to increase the infectivity and destructive power of the source bacteria,and are considered to be new pathogens that do not depend on the source bacteria.These new discoveries of the pathogenicity of the pathogenic bacteria OMV make the research has become the latest hotspot in etiology research.Avian pathogenic Escherichia coli outer membrane vesicles(APEC-OMV)can infect host epithelial cells through multiple pathogenic mechanisms,induce the cells to release inflammatory mediators,and cause inflammation in the host,Although APEC-OMV plays a key role in the process of APEC infection,the key virulence factors in its components remain unclear.Therefore,this study obtained APEC-OMV component information through Label-free protein profiling technology,screened its key virulence factor OmpA,and then used APEC-OMV target cells—chicken tracheal mucosal epithelial cells as the entry point to establish an OmpA infection model to explore its location and apoptosis in cells and its influence on natural immune genes of cells provide references for revealing the pathogenic mechanism of APEC-OMV.The main research contents and results are as follows:(1)Proteomic analysis of outer membrane vesicles of avian pathogenic Escherichia coliThis study combines the ultrafiltration concentration method and density gradient centrifugation method to successfully extract APEC-OMV,and establishes the APEC-OMV extraction process;using Nanoparticles tracking analysis and transmission electron microscopy were used to identify the extracted APEC-OMV.The diameter and morphology of the extracted vesicles were consistent with those described in the literature.the component analysis was carried out by Label free protein profiling technology,and the main proteins identified were: inner membrane protein and outer membrane protein,periplasmic protein,cytoplasmic protein and extracellular protein.The GO function annotation analysis was performed on the proteins with expression abundance in the top 50.The proteins are mainly involved in the process of biology,cell composition,and molecular function.It is found that OmpA has the highest expression in APEC-OMV outer membrane protein.Combined with literature reports,it is speculated that OmpA is the key virulence factor of APEC-OMV.(2)Prokaryotic expression of APEC-OMV key virulence factor OmpA.Construction of APEC-OMV key virulence factor OmpA(APEC-OMV-OmpA)prokaryotic expression vector pET-28a-OmpA and p GEX-6p-1-OmpA,at an inducing concentration of At 0.5Mmol,temperature of 16?,and time of 16 hours,the recombinant protein pET-28a-OmpA with biological activity was induced to express and purified.After purification,the target band was single,and the recombinant protein pET-28a-OmpA was injected subcutaneously to immunize BALB /c mice,the immunization dose is 100?g/mouse,immunized once every two weeks,and boosted by intraperitoneal injection after three immunizations.One week later,the tail was cut off and blood was collected to extract serum.The antibody titer was detected by ELISA method,and the titer reached 1:25600.(3)APEC-OMV-OmpA induces apoptosis of chicken tracheal mucosal epithelial cells.The p CMV-Myc-OmpA eukaryotic recombinant expression vector was constructed and transiently transfected into chicken tracheal mucosal epithelial cells to over-express the OmpA gene in the cells.Confocal laser and Immunofluorescence technology detects the co-localization of OmpA in cells,and the results show that OmpA is mainly located in the cytoplasm.The Annexin ? detection method was used to detect the apoptosis of epithelial cells 24 h after p CMV-Myc-OmpA was transfected.The results showed that OmpA can induce early apoptosis of cells.(4)Effects of APEC-OMV-OmpA on innate immunity of chicken tracheal mucosal epithelial cellsThe transcription levels of innate immune genes were detected by q RT-PCR.The results showed that the expression levels of MDA5,STING,IRF7,NLRP3,IFN-?,IFN-? and TLR3 in innate immune genes were significantly up-regulated,while the expression levels of IL-1?,NF-?? and MAVS were down-regulated.In conclusion,this study successfully extracted APEC-OMV,and analyzed its protein composition information,and it is clear that OmpA is the key virulence factor of APEC-OMV.OmpA is mainly located in the cytoplasm of CTECs and can induce early apoptosis of CTECs,leading to significant up-regulation of MDA5,STING,IRF7,NLRP3,IFN-?,IFN-?,TLR3 expression levels,and IL-1?,NF-The expression levels of ?? and MAVS were down-regulated.This study enriches the biological functions of OmpA,and provides theoretical basis and data support for exploring the pathogenic mechanism of APEC-OMV.