Pathogenic Effects Of Etk/Etp And Wzc/Wzb Tyrosine Phosphorylation Systems On Avian Pathogenic Escherichia Coli And Their Regulatory Mechanisms

Colibacillosis caused by avian pathogenic Escherichia coli(APEC)is a bacterial disease that poses a serious threat to the development of the poultry industry.Tyrosine phosphorylation is widespread in bacteria,with tyrosine kinases and phosphatases being the main components of the tyrosine phosphorylation system,involved in bacterial life and pathogenesis.Currently,two groups of tyrosine kinases and phosphatases,Etk and Etp and Wzc and Wzb,have been identified in Escherichia coli to regulate the synthesis of polysaccharide in the bacterial capsule and extracellular polysaccharide synthesis,but whether they are involved in regulating other biological functions and pathogenic effects is unclear,and the mechanism of infection with host cells is unanswered.Based on this,this study aims to investigate the pathogenic mechanisms of avian pathogenic Escherichia coli,starting from these two groups of bacterial tyrosine kinases and phosphatases.Firstly,the mutant and complemented strains of kinase and phosphatase were constructed respectively to analyze the effects of these genes on the biological function and pathogenicity of APEC,and compare the contribution of these two groups of tyrosine kinases and phosphatases to the pathogenicity of APEC.Tyrosine mutant strains of the kinases Etk and Wzc were constructed to simulate dephosphorylated and persistent phosphorylation states and to search for key phosphorylation sites.Further,comparison of the effects of tyrosine kinases Etk and Wzc on extracellular proteins of APEC based on iTRAQ quantitative proteomics,analysis of differential proteins and signaling pathways,and screening of their potential substrate proteins.Finally,we used a chicken tracheal mucosal epithelial cell infection model to analyze the effects of kinases Etk and Wzc on host cells,and used cellular transcriptomics to explore the differential genes and signaling pathways of APEC-infected chicken tracheal mucosal epithelial cells,and the phosphorylation of NF-κB inflammatory signaling pathway was explored.This study provides an important basis for the in-depth analysis of bacterial tyrosine kinase and phosphatase functions and their regulatory mechanisms,and provides new directions for the prevention and control of colibacillosis and the screening of novel antibacterial drugs.The main research contents and results of this thesis are as follows:1.Pathogenic effects of tyrosine kinase Etk and phosphatase Etp on avian pathogenic Escherichia coliCompared to wild strain AE81,the deletion of etk and etp genes significantly reduced AE81’s motility(P < 0.01),biofilm formation(P < 0.001),resistance to serum bactericide and environmental tolerance.The number and length of the flagella of the Δetk and Δetp mutant strains were reduced and the adherent material between bacteria was reduced,and only a single biofilm was formed on the surface of the medium by electron microscopy.The flagellar flg B,flg E,flg M,fli C and fli H genes and the biofilm csg D,mcb R,mlr A,fim H and fim A genes were reduced to varying degrees in the mutant strains Δetk and Δetp.Compared with AE81,the Δetk and Δetp mutant strains had decreased adhesion ability to DF-1 cells(P< 0.01),significantly reduced phagocytosis against HD11 cells(P < 0.001),reduced pathogenicity in chicks and significantly reduced load in heart,liver,spleen and lung tissues.A point mutant strain with 12 tyrosine sites was constructed by bioinformatically predicting the kinase Etk phosphorylation site,with sites Y56 and Y574 affecting APEC biological function.In this study,we comprehensively analyzed the effects of kinase Etk and phosphatase Etp on the biological function and pathogenicity of APEC,indicating that Etk and Etp positively regulate the pathogenic process of APEC.2.Pathogenic effects of tyrosine kinase Wzc and phosphatase Wzb on avian pathogenic Escherichia coliIn this study,both kinase wzc and phosphatase wzb gene mutant and complemented strains were constructed to compare the contribution of the two kinase and phosphatase groups to the pathogenic effect of APEC.Compared with AE81,the deletion of wzc gene significantly decreased the biofilm formation ability of AE81(P < 0.001),reduced the survival ability under hypertonic conditions(P < 0.05)and enhanced the survival ability under acidic and hydrogen peroxide conditions(P < 0.01).Deletion of wzb gene significantly decreased the motility of AE81(P < 0.05)and enhanced the ability to survive under different environmental conditions.The mutant strains Δwzc and Δwzb reduced resistance to serum bactericidal activity,adhesion to DF-1 cells and phagocytosis of HD11 cells.The ability ofΔwzc to colonize the heart,liver,spleen and lung tissue of chicks was enhanced,while that of Δwzb was reduced in heart,liver,spleen and lung tissues.Ten Wzc tyrosine phosphorylation site mutants were constructed,in which four tyrosine sites,Y205,Y569,Y705 and Y710,affected APEC biological properties.Based on the above two studies,the regulatory effects of two important tyrosine phosphorylation systems on APEC pathogenicity were systematically analyzed,proving that tyrosine phosphorylation is widely involved in APEC pathogenicity.3.iTRAQ-based proteomic analysis of the extracellular protein profiles of APEC kinases Etk and WzcIn this study,we quantified the differentially expressed proteins of APEC extracellular secretion based on iTRAQ quantitative analysis.The results showed that a total of 1370 differential expressed proteins were quantified in Δetk mutant strain,of which 363 proteins were up-regulated and 1007 proteins were down-regulated.By GO and KEGG enrichment analysis,the differential proteins were mainly enriched in the biosynthesis of secondary metabolites,antibiotic biosynthesis,carbon metabolism,ABC transport,the ribosome,twocomponent system and quorum sensing pathways.A total of 493 differential proteins were quantified in Δwzc proteome,of which 237 were up-regulated and 256 were down-regulated.The differential proteins were mainly enriched for microbial metabolism in diverse environments,two-component system,ribosome,flagellar assembly,oxidative phosphorylation,biofilm formation-E.coli,bacterial chemotaxis and bacterial secretion system.The flagellin(flg D,fli C and flg M),response regulator omp R,secretory system proteins(esp Y,sec G and sec D)and heat shock protein dna K genes,which were significantly different and associated with the above phenotypes,were selected for fluorescent quantitative PCR and their up-and down-regulation trends were consistent with the results of proteomics.Based on proteomics analysis,this study found that kinases Etk and Wzc affect the expression of bacterial metabolic and virulence proteins provides potential targets for subsequent validation of kinase substrate proteins.4.Study on the pathogenic effects of kinases Etk and Wzc on chicken tracheal mucosal epithelial cellsThe pathogenic effects of tyrosine kinases Etk and Wzc on host cells were investigated in a chicken tracheal mucosal epithelial cell infection model.Compared with AE81,deletion of tyrosine kinase and phosphatase genes reduced the virulence effect on CTECs(P < 0.05).The mutant strain Δetk infected CTECs cells with a total of 191 differentially expressed host genes,of which 168 were significantly up-regulated and 23 were down-regulated.The differentially expressed genes were mainly enriched in IL-17 signaling pathway,cytokine and cytokine receptor interaction,TNF signaling pathway,Toll-like receptor signaling pathway and NF-κB signaling pathway.After infection with Δwzc,a total of 386 differentially expressed genes were detected,among which 263 were significantly upregulated and 123 were down-regulated.The differentially expressed genes were mainly enriched in signaling pathways such as ribosome,oxidative phosphorylation,RNA degradation and thiamine metabolism.The deletion of wzc gene mainly causes changes in cellular substance synthesis and metabolism-related pathways,and the deletion of etk gene affected changes in cellular immune inflammatory signaling pathways,suggesting that the two play different roles in cell biology.By flow cytometry,Annexin V-FITC/PI double staining and WB assay,it was found that deletion of kinase etk and wzc genes resulted in reduced apoptosis and decreased Caspase-3 expression in CTECs.Compared with AE81,the mutant strain Δetk enhanced p65 protein phosphorylation(P < 0.01),and Δwzc inhibited IκBα and p65 protein phosphorylation(P < 0.01).These results indicate that tyrosine kinases Etk and Wzc are actively involved in the pathogenesis of host cells.In summary,the study was conducted to investigate the effects of avian pathogenic E.coli tyrosine kinases and phosphatases on the biological functions and pathogenicity of APEC,and found that they are involved in regulating APEC pathogenicity by affecting bacterial flagella,biofilm,environmental tolerance,resistance to serum bactericidal,cell adhesion and anti-phagocytosis processes,and screening for some tyrosine phosphorylation sites and potential substrates.The protein kinases Etk and Wzc can cause damage and apoptosis in chicken tracheal mucosa epithelial cells and are involved in the phosphorylation process of NF-κB signaling pathway,providing new directions for subsequent research on the pathogenesis of avian pathogenic E.coli and the development of related drug targets.