Study On The Biological Characteristics Of YbjX?pagp Mutant Strains And The Correlation With PhoP In Avian Pathogenic Escherichia Coli

The PhoP/Q two-component system is a signaling regulator system which is widely found in avian pathogenic Escherichia coli.The PhoP/Q two-component system regulates bacterial virulence and many cell activities by regulating the expression of endotoxin(LPS)-related genes,which may cause damage Intestinal tract,destruction of epithelial tissue,induction of the defense gene expression,and the expression of the response gene.However,the expression of the response genes havea great relationship with pathogenicity of APEC.The antimicrobial peptide gene(AMPR)modifies the outer membrane structure of bacteria by direct or indirect modification of LPS,enhancing the ability of bacteria to resist the penetration of host antimicrobial factors,thus helping to resist the resistance of bacterial peptides.In this study,we screened genes related to antimicrobial peptides ybjX and pagp on the basis of pre-gene chip,and to investigate the biological characteristics of ybjX and pagp gene deletion and the correlation with PhoP in Avian pathogenic Escherichia coli.The genes of ybjX and pagp in APEC O2 serotype were cloned by bioinformatics analysis and primers.The results of PCR amplification showed that ybjX and pagp were successfully cloned and the ybjX and pagp mutant strains were constructed by Red homologous recombination system.The results showed that ybjX and pagp deletion were successfully constructed.On the basis of this,we used pSTV-28 to constructe two complementation strains based on mutant strains.comparision AE17 with ybjX,pagp deletion strains of the biological characteristics.Based on the understanding of the effects of ybjX and pagp deletion on their biological characteristics,the regulation of ybjX,pagp by PhoP,and the resistance to cationic antimicrobial peptides were further studied.The concrete research contents and results are as follows:1.Construction of avian pathogenic Escherichia coli ybjX and pagp gene mutant strainsThe ybjX and pagp genes were amplified from an O2-sera-type avian pathogenic Escherichia coli AE17(sensitive to chloramphenicol and ampicillin)stored in the laboratory.There was no literature reported that ybjX and pagp genes shared a promoter.ybjX and pagp mutant strains were successfully constructed by using the method of Red homologous recombination.Low copy plasmid pSTV-28 is used to constructe complementation strains.The recombinant plasmid was transformed into a corresponding gene deletion strain by ligating the constructed vector into a corresponding gene deletion strain.The gene recovery strains CybjX,Cpagp were successfully constructed.2.Evaluation of biological characteristics of ybjX and pagp gene mutant strainsGrowth curve test,biochemical identification,motility assay,environmental tolerance test and biofilm formation ability were investigated.The study of ybjX mutant strain growth curve,complementation strain and wild strain growth in LB medium have no obvious change.pagp mutant strain have obvious change compared with the wild strain.The results of the study showed that the deletion of ybjX and pagp gene did not significantly reduce the mobility of AE17.Environmental tolerance test showed that ybjX mutant strain and pagp mutant strain respectively reduce tolerance to acid environments to2.2 times(p <0.05)and 4 times(p <0.01)than that of wild strain.The resistance of The tolerance of ybjX mutant strain and pagp mutant strain to alkali environment tolerance reduce to 1.5 times(p <0.05)and 2 times(p <0.05)than that of wild strain.(P <0.01).The resistance of ybjX mutant strain to thermal environment was 19.2% lower than that of wild plant,and the resistance of pagp mutant strain to thermal environment was reduced by10.4%?The results showed that the biofilm formation ability of the ybjX mutant strain is0.75 times that of wild strain(p <0.01)and was significantly lower than that of the wild strain.pagp deletion was 1.4 times that of wild strain(p <0.01)and was significantly higher than that of the wild strain.NPN test investigated the outer membrane permeability of two mutant strain.The results showed that the deletion of these two genes can enhance the permeability of APEC outer membrane to a certain extent.The antibiotic concentration experimental results keep consistent with the permeability of mutain strains.The higher permeability of the outer membrane showed higher sensitivity to polymyxin B and AVBD2.To explore the effect of two mutain strains of APEC pathogenicity,Test the ability of adhesion and invasion CEF cells,median lethal dose.The results showed that the adhesion and invasion of ybjX mutain strain to CEF reduced the bacterial virulence by 2.3 fold and 2fold respectively.The ability of adhesion and invasion of pagp deletion strain reduced the bacterial virulence by 1.3-fold(and the adhesion of the complementary strains was restored to the wild strain level).Half lethal dose test showed that ybjX and pagp deletion strains decreased the pathogenicity of chickens.3.Correlative analysis of ybjX and pagp genes and PhoP protein in avian pathogenicEscherichia coliThe recombinant protein of E.coli,BL21-pET32a/phoP was induced the expression of PhoP recombinant protein.The molecular weight was 42.9 KDa,conformed with its theoretical molecular weight.The cells were centrifuged and the PhoP protein was purified by supernatant of Ni-NTA affinity chromatography.The ybjX and pagp gene sequences were searched on Genbank.The binding sequence of PhoP protein and DNA was predicted by promoter prediction web.The electrophoretic mobility shift assay(EMSA)were applied to explore whether PhoP protein can bind to promoter of two genes.The results showed that ybjX,pagp is Indirectly regulated by phop protein.4.Effect of ybjX and pagp genes deletion on Antimicrobial peptidesIn this study,we compared the susceptibility of wild strains,ybjX and pagp deletion strains to polymyxin B and AVBD2.The diameter of the tumor show that the resistance of the two deletion strains to polymyxin B and AVBD2 was significantly reduced.The APEC single gene deletion strain was successfully constructed to determine the effect of ybjX and pagp gene deletion on CEF cells and chicks;The results prove that phop regulators could not directly control ybjX and pagp;ybjX and pagp deletion enhance the sensitivity of APEC to antimicrobial peptid respectively.The results showed that ybjX and pagp genes had an important effect on the pathogenicity of APEC and the resistance to antimicrobial peptides,which provide some experimental basis for further study the specific function of gens ybjX and pagp.