EtrA Regulation Of OmpW On APEC Biological Phenotype And Pathogenicity

Avian pathogenic Escherichia coli(APEC),as a common pathogen,often interacts with other pathogens,causing avian colibacillosis and even directly leading to the death of poultry.Therefore,it is of great significance to strengthen the research on the pathogenic mechanism of APEC for the development of aquaculture and the prevention and control of public health.Escherichia coli type Ⅲ secretion system 2(ETT2)is a type Ⅲ secretion system found in the genome of enterohemorrhagic Escherichia coli O157: H7 in 2001,which is involved in the secretion and transportation of bacterial toxins and other pathogenic processes.EtrA is one of the five transcriptional regulators of ETT2,which can inhibit the protein secretion of LEE pathogenicity island and participate in bacterial adhesion.Omp W is a small outer membrane protein(Outer membrane prorein,OMP)that is widely distributed in Gram-negative bacteria.These OMPs are involved in lipid metabolism,cell adhesion,membrane stability,and stress adaptation.Previous studies in the laboratory found that EtrA affects the transcription level of the gene ompW based on transcriptome.In this study,real-time quantitative PCR and β-galactosidase activity assay were used to explore the effect of etrA gene on the transcription level of ompW.Furthermore,Red homologous recombination system was used to construct missing strains of ?ompW and ?etrA?ompW.Combined with missing strains of ?etrA constructed in the laboratory,to evaluate the effects of etrA regulation of ompW on biological characteristics of APEC.Finally,in vitro and in vivo pathogenicity tests were used to evaluate their effects on the pathogenicity of APEC.The main research contents and results are summarized as follows:1.ETT2 Transcriptional Regulator EtrA Regulates the Transcriptional Level of Outer Membrane Protein Omp WIn order to explore whether ETT2 transcriptional regulator EtrA can regulate the transcription level of outer membrane protein ompW,the etrA-overexpression strain pET32a-EtrA was constructed,and the etrA transcription level of pET32a-EtrA was detected by real-time quantitative PCR.The results showed that the etrA transcription level of pET32a-EtrA was significantly increased(P<0.001),indicating that the etrA overexpression strain was successfully constructed.The transcription level of ompW in the deletion strain ?etrA and the overexpression strain pET32 aEtrA was detected,and the results showed that the transcription level of ompW in ?etrA was decreased(P<0.05),while that in pET32a-EtrA was increased(P<0.01).It is suggested that etrA has a regulatory effect on ompW.The Fusion expression vector Lac Z was further used to explore the relationship between the two,and Red homologous recombination technology was used to construct?etrA?lac Z and ?lac Z gene deletion strains,which were transformed into p RCL-ompW with ompW promoter,respectively.The β-galactosidase concentration of ?lac Z-pompW and ?etrA?lac Z-pompW was detected,and the results showed that the β-galactosidase concentration of?lac Z-pompW was higher than that of ?etrA?lac Z-pompW(P < 0.01),indicating that the transcription factor EtrA could act on the promoter region of ompW.It can regulate the transcription level of ompW.2.EtrA regulation of ompW on biological phenotype of avian pathogenic Escherichia coliRed homologous recombination technique was used to construct etrA,ompW gene single and double deletion strains.The results of growth curve showed that there was no significant difference in growth performance between wild strain AE40 and deletion strains ?etrA,?ompW and?etrA?ompW(P>0.05).The results of motility test showed that the motility of ?etrA,?ompW and ?etrA?ompW was significantly lower than that of wild strain AE40(P<0.01).The biofilm test results showed that the biofilm formation ability of ?etrA was significantly higher than that of the wild strain(P<0.01),while the biofilm formation ability of ?ompW was no significantly different from that of the wild strain(P>0.05),while the biofilm forming ability of ?etrA?ompW was significantly higher than that of wild strain(P<0.001).Compared with wild strain AE40,the survival rate of ?etrA,?ompW and ?etrA?ompW decreased significantly under acid stress(P<0.05);Compared with wild strain AE40,the survival rates of ?etrA,?ompW and ?etrA?ompW were significantly decreased under high osmotic stress(P<0.01),and the survival rates of etrA,ompW and ?etrA?ompW were significantly decreased under alkali stress(P<0.001),The survival rates of?etrA,?ompW and ?etrA?ompW were significantly decreased under 70 ℃ heat stress(P<0.05).The above results showed that the deletion of etrA and ompW genes changed some biological characteristics of APEC.3.EtrA regulation of ompW on pathogenicity of avian pathogenic Escherichia coliThe effect of etrA and ompW genes on pathogenicity of avian pathogenic Escherichia coli was investigated by in vitro and in vivo pathogenicity tests.In vitro serum bactericidal test results showed that the antiserum bactericidal ability of ?etrA,?ompW and ?etrA?ompW in serum of 100%was significantly lower than that of the wild strain(P<0.01),and the antiserum bactericidal ability in serum of 70%,50% or 10% was lower than that of the wild strain(P<0.05).There was no significant difference in antiserum bactericidal ability between the lost and wild strains in heat inactivated serum(P>0.05).Hemolytic test results showed that genes etrA and ompW had no relationship with bacterial hemolytic.The results of cell adhesion and invasion test showed that compared with wild strain AE40,the absence of etrA significantly enhanced the adhesion and invasion ability of APEC to chicken trachea mucosal epithelial cells(P<0.01),while ?ompW and?etrA?ompW significantly decreased the adhesion and invasion ability of APEC to chicken trachea mucosal epithelial cells(P<0.01).After cell infection,the transcription levels of TNF-α,IL-1β and IL-6 of ?etrA,?ompW and ?etrA?ompW were significantly higher than those of wild strain AE40(P<0.01).In vivo pathogenicity results showed that the deletion of etrA and ompW reduced the pathogenicity of avian pathogenic Escherichia coli to the host and the degree of damage to the host’s trachea,heart and liver.In conclusion,these results suggest that deletion of etrA and ompW reduces the pathogenicity of avian pathogenic E.coli.In summary,the overexpression strain pET32a-EtrA,the gene deletion strains ?etrA?lac Z and?lac Z,and the transcriptional fusions ?lac Z-pompW and ?etrA?lac Z-pompW with the reporter gene lac Z were successfully constructed.It was found that EtrA may positively regulate the transcription of ompW gene.?ompW and ?etrA?ompW deletion strains were successfully constructed,and compared with wild strain AE40,the motility,acid,alkali,salt and heat tolerance,serum bactericidal resistance and pathogenicity of the deletion strains were significantly reduced,but there was no significant difference in growth performance and hemolytic ability.The biofilm formation ability of ?etrA and ?etrA?ompW was significantly enhanced.The adhesion and invasion ability of ?etrA cells was significantly increased,while that of ?etrA?ompW cells was decreased.These results suggest that etrA and ompW are involved in the regulation of the biological phenotype and pathogenic process of APEC,and may affect the pathogenic mechanism of APEC.