Preliminary Research Of Subcellular Localization And Cell Regulatory Pathway Of APEC Virulence Protein Hcp2

Avian pathogenic Escherichia coli(APEC)as a bacterial pathogen widely distributed around the world,not only poses a great threat to the poultry industry,but also brings serious impact to the prevention and control of animal food safety.The pathogenesis of APEC is complex that involves many pathogenic factors,which the secretion system plays an important role in the pathogenesis of APEC.Type VI secretion system(T6SS)as a bilayer transmembrane secretion system,participates in different biological processes of bacteria and plays an important role in the pathogenesis of bacteria.Hemolysin-coregulated protein(Hcp)participates in the assembly of the secretory tail tube in the T6 SS complex and acts as an effector protein alone.Hcp protein has a variety of functions,after knocking out the hcp gene,the ability of pathogenic bacteria to move,adhere and colonize will be affected.However,the regulatory mechanism of Hcp interaction with host cells during APEC infection remains unclear.In this study,the expression and purification of Hcp2 protein and the preparation of polyclonal antibody were complete by constructing p ET-28a-hcp2 recombinant plasmid.DF-1 cells were treated with a certain dose of purified virulence protein Hcp2,and through indirect immunofluorescence,subcellular localization and proteomics and other techniques to study whether the virulence proteins Hcp2 a and Hcp2 b can enter cells and their effects on the pathogenic effect of DF-1 cell;Moreover,the AE17 strain was resuspended by eluent containing purified Hcp2 protein and injected into chicks by trachea,the effects of overexpression of Hcp2 a and Hcp2 b on the transcriptome of tracheal mucosal epithelial cells in APEC infected hosts were studied.To provide a theoretical basis for further research on the pathogenic effect of T6 SS on APEC.The specific research contents and results are as follows:1.Prokaryotic expression of Hcp2 protein and preparation of polyclonal antibodyIn this study,two pairs of specific primers were designed based on the hcp2 a and hcp2 b gene sequences of avian pathogenic Escherichia coli AE17 strains for PCR amplification,and the restriction enzyme was linked to the p ET-28 a vector.After the recombinant vector was successfully constructed,it was transformed into E.coli BL21 competent cells.Add the inducer IPTG to induce the expression of the target protein,and analyze the protein expression form by SDS-PAGE.The results showed that the size of His-Hcp2 a protein was about 23 k Da,and the highest expression level was obtained at 16 ℃ and IPTG concentration of 0.25 mmol/L for 16 h;The protein size of His-Hcp2 b was about 18 k Da,and the expression of His-Hcp2 b was the best at 28 ℃ and IPTG concentration of 1mmol/L for 12 h.Both proteins were mainly expressed in the supernatant,The fusion proteins were purified by His-tag purification gels,respectively.The protein concentration was determined by BCA method,and the two purified fusion proteins were mixed with equal volume of Freund’s complete adjuvant as immunogens,and BALB/c mice were immunized to prepare murine polyclonal antibodies.The serum titer of Hcp2 a as measured by indirect ELISA was 1:408600 and the serum titer of Hcp2 b was 1:23400.Western Blot showed that the murine polyclonal antibody reacted well with the immunogen.2.Study on the subcellular localization of Hcp2 protein in DF-1 cellsThe DF-1 cells were treated with a certain dose of purified virulence effector protein Hcp2 for 48 h,and it was determined that the virulence effector protein Hcp2 could successfully enter the cells through the DF-1 cell membrane using indirect immunofluorescence and subcellular localization,and that the Hcp2 protein was clearly localized to the endoplasmic reticulum,where the Hcp2 a protein was also localized to the mitochondria.Proteomic sequencing found that Hcp2 a had 58 differentially expressed proteins,of which 25 were up-regulated and 33 were down-regulated.Mainly enriched in oxidative phosphorylation and p53 signaling pathway.Hcp2 b has 108 differentially expressed proteins,of which 60 are up-regulated and 48 are down-regulated.Mainly enriched in protein processing in the endoplasmic reticulum pathways and oxidative phosphorylation.Among them,ATP5 H and THBS1 proteins related to ATP supply and protein synthesis were both up-regulated.3.The effect of Hcp2 protein on the transcriptome of chick tracheal mucosaThe purified virulence proteins Hcp2 were added to avian pathogenic Escherichia coli AE17 strain to infect chicks by tracheal injection and 24 hours later,the tracheal mucosa epithelial cells of the chicks were collected for transcriptomic sequencing.The results showed that compared with the wild strain AE17,102 differentially expressed genes were found in the AE17 infection group with added virulence protein Hcp2 a,of which 53 genes were up-regulated and 49 genes were down-regulated.The AE17 infection group added with the virulence protein Hcp2 b obtained 85 differentially expressed genes,of which 46 genes were up-regulated and 39 genes were down-regulated,The GO function results showed that the differentially expressed genes of the AE17-infected group supplemented with the virulence effector protein Hcp2 a were mainly concentrated in GO function items such as redox process,oxygen transport,cytoplasm,biofilm and its membrane components;The differentially expressed genes of the AE17-infected group supplemented with the virulence effector protein Hcp2 b mainly focus on the functions of intracellular signal transduction,immune response,cytoplasm,biofilm and its membrane components;The results of KEGG analysis showed that the differentially expressed genes of the AE17-infected group supplemented with the virulence effector protein Hcp2 a were mainly enriched in NOD-like receptor signaling pathway,Toll-like receptor signaling pathway and phagosome and other pathways;The differentially expressed genes of the AE17-infected group supplemented with the virulence effector protein Hcp2 b were mainly enriched in Toll-like receptor signaling pathway,phagosome and cell adhesion molecules(CAMs).In conclusion,this research successfully constructed the prokaryotic expression plasmid of T6SS-2 virulence protein Hcp2 and completed the expression of the protein and the preparation of polyclonal antibody;The virulence protein Hcp2 can penetrate the DF-1cell membrane and localize in the endoplasmic reticulum,where Hcp2 a is also localized to the mitochondria.The results of proteomic sequencing showed that Hcp2 a protein can participate in oxidative phosphorylation signaling pathway in cells,and Hcp2 b protein can participate in protein processing signaling pathway and oxidative phosphorylation pathway in endoplasmic reticulum;The RNA-Seq results of chick tracheal mucosa epithelial cells showed that Hcp2 a and Hcp2 b proteins mainly affect the pathogenic mechanism of the host by regulating the Toll-like receptor signaling pathway and the regulation of immune-related pathways such as phagosomes.