Component Analysis And Pathogenicity Of Outer Membrane Vesicle Of Avian Pathogenic E.coli

Avian pathogenic Escherichia coli(APEC)as a gram negative bacteria,poultry has a high mortality and infection rate,which not only endangers the development of the poultry industry,but also with human urethral pathogenic Escherichia coli(UPEC)has a high homology,It has public health risk and endangers human health development.In the process of natural growth,stress and host infection,pathogens secrete nanosized Outer Membrane vesicles(OMV)composed of proteins and nucleic acids.Studies at the present stage have found that virulence factors and other substances can be secreted from the bacteria and have certain protective functions for these virulence factors.OMV can enter the host cells through the endocytosis of host cells and play intracellular regulatory functions to promote the infection of the source bacteria.Avian pathogenic Escherichia coli also secrets OMV during the growth process,but whether OMV is closely related to the pathogenicity of avian pathogenic Escherichia coli,and whether OMV components are different before and after APEC infection remains to be further studied.Therefore,avian pathogenic Escherichia coli(APEC clinical isolate AE17,O2serotype)was used as the research object.The proteomic analysis of APEC-OMV before and after co-culture of APEC and chicken embryo fibroblast DF-1,cell proteomic analysis of chicken trachea mucosal epithelial cells before and after infection with APEC-OMV,in vivo pathogenicity test of APEC-OMV on chickens and in vitro pathological changes observation were planned.The aim of this study was to analyze the proteomic differences before and after APEC-OMV infection of cells and the proteomic differences before and after APEC-OMV infection of host cells,and to preliminatively clarify the pathogenicity of APEC-OMV,so as to provide reference for further research on the pathogenesis of APEC-OMV in the later stage.The main research methods and results are as follows:1.Proteomic differences of APEC-OMV before and after APEC infection of DF-1 cellsOMV was extracted and purified from APEC by differential centrifugation and density gradient centrifugation.The shape and particle size distribution of APEC-OMV were analyzed by transmission electron microscopy and particle size distribution.The proteins and lipopolysaccharide components of APEC-OMV were detected by BCA protein concentration detection,SDS-PAGE protein electrophoresis,silver staining,and other methods.DNA content of APEC-OMV was detected by the Picogreen method.OMV was extracted after co-culture of APEC and DF-1 cells,and label-free omics analysis was performed to screen the differential proteins.A total of 462 significantly different proteins were screened,including 312 up-regulated proteins and 350 downregulated proteins.Through the analysis of protein data,it was found that the virulence factor differential proteins of APEC-OMV after co-culture with DF-1 cells mainly involved the outer membrane protein family,such as Omp A,Omp X,Omp T and Omp A showed significant changes.The regulation of LPS formation was mainly related to lipid A and the translation regulatory proteins related to LPS formation,such as Lpx D and Lpx A,which showed significant changes.GO functional analysis found that a total of 37 functions were annotated,which mainly involved cellular processes and metabolic processes.Cells and cell components,membranes and membrane components;Catalytic activity,binding,transcription activity,and other functions.KEGG pathway analysis showed that the differential proteins were mainly concentrated in carbon metabolism,followed by the ribosome,purine metabolism,pyrimidine metabolism,pyruvate metabolism,two-component system,oxidation metabolism,methane metabolism,and other pathways.2.Proteomic analysis of chicken tracheal mucosal epithelial cells before and after infection with APEC-OMVThe model of chicken tracheal mucosal epithelial cells infected by APEC-OMV was established,and the i-TRAQ proteomics technique was used to screen the cell differential proteins after the infection of chicken tracheal mucosal epithelial cells infected by APEC-OMV.A total of 659 significantly different proteins were screened,among which 330 proteins were up-regulated and 329 proteins were down-regulated.A total of 94 functions were annotated by GO functional analysis,which mainly involved monovalent inorganic cation transmembrane transporter activity,peptide metabolism,and cellular protein metabolism.Cell biosynthesis process,membrane intracellular nonmembrane bound organelles;Ribosome synthesis,hydrogen ion transmembrane transporter activity,etc.KEGG pathway analysis showed that the differential proteins were mainly concentrated in the ribosome,extracellular matrix,other types of O-glycan biosynthesis,oxidative phosphorylation,endoplasmic reticulum protein processing,and cell adhesion molecules.Through data analysis,it was found that APEC-OMV infection of chicken tracheal mucosal epithelium cells resulted in significant changes in related cell apoptosis and transmembrane transport proteins.The proteins associated with apoptosis were screened,including apoptotic family protein Caspase.Such as caspase-2,caspase-3,caspase-7,and caspase-8;Transmembrane transport of substances such as differential proteins involved in the CAM process,such as Integrin Alpha-5,Neogenin1,Cadherin-2,Syndecan-4,Syndecan,etc.Based on the results of differential protein analysis,some differential proteins were screened,and relevant quantitative fluorescence primer sequences were designed.Meanwhile,RNA was extracted from cell samples and m RNA was detected by real-time quantitative fluorescence PCR.The results showed that the trend of transcription level was consistent with proteomic data.3.Apoptosis of epithelial cells induced by APEC-OMV and its pathogenicity in chickensCCK8,MTT cytotoxicity and Annexin assay were used to detect the period of cell apoptosis and flow cytometry to detect the apoptotic rate of co-culture APEC-OMV and chicken trachea mucosal epithelial cells.The results showed that the cell growth rate decreased with the increase of the concentration of the outer membrane vesicles,and the outer membrane vesicles were more toxic to the cells with the increase of time.After coculture with Apec OMV for 4 h,the cell proliferation rate changed normally after 8 h.After co-culture for 12 h,the cell proliferation rate decreased.At 12 h,16 h,and 20 h,the cell proliferation rate decreased significantly with time.20?g APEC-OMV and chicken tracheal mucosal epithelial cells cell apoptosis was obvious,and the number of cell apoptosis increased with time.In the experiment of trachea injection of APEC-OMV into chickens,200 ?g/kg of APEC-OMV was used as the concentration of the attack.After 48 hours of injection of APEC-OMV into chickens,the extracted organ tissues were made into paraffin sections,and the results showed that the trachea,kidney,liver and heart of chickens had lesions,indicating that the isolated APEC-OMV had damage effect on animal tissues.Caused in vitro pathological changes of chicken.