Screening Of Downstream Target Proteins Of Apoptosis Of Chicken Trachea Epithelium Cells Induced By The Key Virulence Factor OmpA Of APEC-OMV

Avian pathogenic Escherichia coli(APEC),a typical extraintestinal pathogen,has a wide host spectrum and seriously endangers the development of the poultry industry.As a potential class of human-animal symbionts,APEC may pose a threat to human health.Therefore,it is potential public health significance to carry out research on the pathogenic mechanisms of APEC.Outer membrane vesicle(OMV)is a kind of spherical vesicle naturally secreted by Gram-negative bacteria with a bilayer proteolipid structure.As an extremely important virulence vector of pathogens,OMV can carry key virulence factors into infected host cells through endocytosis and improve the pathogenicity and destruction of source bacteria.Whether APEC-OMV is a kind of virulence vectors of APEC still needs further study.Whether there is random packaging during infection of host cells.Whether some of the key virulence factors still have virulence effects after weaning from OMV packaging.In order to explore the above issues,this study focused on APEC-OMV,with the goal of screening its key virulence factors and elucidating the molecular mechanism of host cell apoptosis induced by key virulence factors.OMVs from APEC clinical isolate AE17 before and after co-culture with host chicken trachea epithelium cells(CTECs)were analyzed for proteomic differences to screen large differential proteins.Out membrane protein A(Omp A)was used as an entry point to construct omp A gene deletion and overexpression strains and establish a cell model of APEC-OMV infection of CTECs.Thus,the regulation of APEC-OMV formation and the main peak of particle size by Omp A and its effect on the mechanism of host cell apoptosis were explored.CTECs were respectively transfected with eukaryotic plasmid pCMV-Myc and eukaryotic expression vector pCMV-Myc-Omp A for co-immunoprecipitation(Co-IP)and mass spectrometry analysis.Thus,the effect of Omp A on the regulatory pathways of CTECs was explored.Through the development of this study,the key virulence factors of APEC-OMV were screened and the downstream target proteins of its induced lesions in CTECs were identified.Main study contents and results are as follows:1.Based on proteomic screening of differential proteins of OMVs before and after co-culture of APEC with CTECsTwo kinds of OMV were respectively extracted and purified from the culture medium of APEC bacterial solution supernatant and APEC bacterial solution after 2 h of co-culture with CTECs.Mass spectrometric detection and bioinformatics analysis of two kinds of APEC-OMVs using label free quantitative method.It was found that there were 605 differential proteins in OMV extracted after co-culture of APEC bacterial solution with CTECs compared with OMV extracted from APEC bacterial solution.These differential proteins are mainly involved in various signaling pathways,for example energy production and transformation,cell cycle control and cell division,nuclear peptide transport and metabolism,carbohydrate transport and metabolism and so on.According to GO functional annotation and KEGG pathway analysis,these differential proteins are enriched in many signaling pathways which are carbon metabolism,ribosome,oxidative phosphorylation and so on.These results suggest that APEC may affect proteins or enzymes in the respiratory chain or ribosome at the translational level after co-culture with CTECs thereby regulating the protein composition of OMV and having some regulatory effects on the pathogenic effects of OMV.2.Function of virulence gene omp A in apoptosis of CTECs induced by APEC-OMVThe authors’ group of the paper previously found that Omp A is the most abundantly expressed outer membrane protein in APEC-OMV,which can lead to significant changes in the expression levels of some innate immune genes in CTECs.Furthermore,it is speculated that Omp A may be a key virulence factor of APEC-OMV.In this study,clinical strain AE17 of APEC used as a wild strain,omp A gene deletion strain was constructed using CRISPR/Cas 9 system.Then the unload strain and omp A gene overexpression strain was successfully constructed using expression plasmid pET-28 a.The different kinds of OMV were respectively extracted from the original,deletion,unloaded and overexpression strains.Transmission electron microscopy and nanoparticle analysis revealed that the number of OMV produced by the deletion strain was significantly reduced and the main peak of particle size were both decreased compared with OMV produced by the original strain,while the number of OMV produced by the overexpression strain and the main peak of particle size both increased.Annexin V-FITC/PI double staining by fluorescence-activated cell sorter and fluorescence microscopy revealed that the number of apoptotic cells in the early and late stages were increased after OMV infection of CTECs with primitive,deletion,unloaded and overexpressing strains compared with normal cultured cells.Compared with the original strain OMV,the induction of apoptosis in CTECs was reduced by the deletion strain OMV and the number of apoptosis was relatively the highest after infection of CTECs with the overexpression strain OMV.Ultrastructural pathological sections of CTECs infected with four kinds of OMV from APEC were observed by transmission electron microscopy.The results revealed that CTECs after OMV infection with the overexpression strain presented significant lesions,such as fading of the mitochondrial matrix,disappearance of cristae or even transformation into vesicular structures and so on.The above experimental results further clarified that omp A gene had a positive regulatory effect on the number and particle size main peak of APEC-OMV production and promoted APEC-OMV induced apoptosis in CTECs.3.Screening of downstream target protein for apoptosis of CTECs by virulence factor Omp AThe eukaryotic expression plasmid pCMV-Myc and the recombinant plasmid pCMV-Myc-Omp A were respectively transfected into CTECs for 24 h.It was found that the virulence factor Omp A could promote the increase in the number of early and late apoptotic cells in CTECs,and the apoptotic changes were more significant compared with the control group.Ultrastructural pathological sections were observed by transmission electron microscopy.The results showed that compared with the blank control group,the pathological changes of CTECs transfected with virulence factor Omp A were significant,such as larger and round mitochondria with shorter and less cristae or even disappeared,some mitochondria had been transformed into vesicular structures and so on.Fifteen differentially proteins were found by Co-IP and mass spectrometry analysis in CTECs transfected with the virulence factor Omp A,which are mainly involved in biological functions as protein binding and isomerase activity and cellular composition processes as T cell activation and cell-cell adhesion mediator activity.There are six proteins that may interact with Omp A based on String database analysis,namely PGK1(3-phosphoglycerate kinase),PKM2(pyruvate kinase),EEF1A1(elongation factor 1-alpha 1),ACTG1(actin cytoplasmic type 5),FSCN1(actin bundle protein-1),and PABPC1(polyadenylate binding protein-1).The results showed that the virulence factor Omp A had a pathogenic effect on host cell CTECs.In this study,we found differences in the composition of OMV proteins extracted and purified after 2 h co-culture of APEC with host cell CTECs.The virulence factor Omp A was screened for successful construction of omp A gene deletion and overexpression strains.The omp A gene was found to positively regulate the formation of APEC-OMV and promote APEC-OMV-induced apoptosis in CTECs.Meanwhile,we preliminarily screened the downstream target proteins of apoptosis of CTECs induced by the virulence factor Omp A in combination with Co-IP and mass spectrometry analysis,which provided a new way for further in-depth study of the pathogenic mechanism of APEC-OMV.