| Avian pathogenic Escherichia coli(APEC)is a kind of extraintestinal pathogenic Escherichia coli.It is often infected by respiratory tract colonization,causing gasbag inflammation,peritonitis,salpingitis,synovitis,septicemia and cellulitis in poultry.Because of its complex serotypes and numerous pathogenic factors,its prevention and treatment is still a major problem.Therefore,it is of great significance to strengthen the research on the pathogenicity of avian pathogenic Escherichia coli for the development of breeding industry and public health safety.ETT2 is an important virulence island of Escherichia coli,which is involved in the secretion and transportation of bacterial toxins.It has been reported that ETT2 deletion strain has great influence on the motility,biofilm formation and serum viability of APEC.Serum resistance is one of the most important virulence factors in APEC,which is closely related to its pathogenicity.ygeP gene is located at the end of ETT2 invasion gene cluster.So far,there is no research on the biological characteristics and pathogenicity of ygeP.In this study,we used CRISPR/Cas9 gene editing technology to construct the ygeP deletion strain of ETT2 gene of avian Pathogenic E.coli,and constructed the revertant strain to evaluate its effects on the biological characteristics of APEC.The pathogenicity of the deletion strain AE81 was studied by serum bactericidal test and tissue bacterial load.RT-PCR was used to screen the significant differential expression genes of serum resistance of the deletion strain AE81?ygeP and to screen the potential targets regulated by ygeP.Finally,Red homologous recombination technology was used to construct differential gene deletion strains,and the serum resistance of the deletion strains was verified to evaluate the relationship between ygeP and differential genes in the regulation mechanism of serum resistance.This study laid a foundation for further study on the function of ETT2 and pathogenic mechanism of avian pathogenic Escherichia coli.The main research contents and results are summarized as follows:1.Construction and characterization of ygeP deletion and revertant strains of avian pathogenic Escherichia coliIn this study,ygeP deletion strain was constructed on AE81 wild strain by CRISPR/Cas9 gene editing technology,and ygeP revertant strain was constructed by p STV28 plasmid.The biological characteristics of AE81 wild strain,AE81?ygeP deletion strain and AE81C?ygeP replying strain were evaluated.The results of growth curve showed that the growth curves of AE81 wild strain,AE81?ygeP deletion strain and AE81C?ygeP revertant strain were consistent,which indicated that ygeP did not affect their growth;the results of drug sensitivity test showed that AE81 wild strain,AE81?ygeP deletion strain and AE81C?ygeP revertant strain were sensitive to 14 antibiotics,but the sensitivity of the three strains to antibiotics was not significant The results of motility test showed that compared with AE81,the motility of AE81?ygeP deletion strain was significantly decreased(P<0.05),and the revertant strain was restored;the transcription expression of flagellum synthesis gene was detected by fluorescence quantitative PCR,and the results showed that the flagellum synthesis genes Fli I,fli J,flg B,flg E of ygeP deletion strain were significantly decreased(P<0.05),The results of biofilm test showed that the biofilm forming ability of AE81?ygeP deletion strain was significantly higher than that of AE81 wild strain(P<0.05),and there was no significant difference between the revertant strain and AE81 wild strain.There was no significant change in gar P and gho T(P>0.05),which was consistent with the results of biological phenotype.The results of stress resistance test showed that compared with AE81 wild strain,ygeP deficient strain had significantly higher acid tolerance(P<0.05),lower alkali tolerance(P<0.05),lower heat tolerance(P< 0.05),higher hypertonic tolerance(P<0.05)and higher oxidative stress(P< 0.05).2.The effect of ygeP deficiency on pathogenicity of avian pathogenic Escherichia coliThe pathogenicity of AE81 wild strain,AE81?ygeP deletion strain and AE81C?ygeP revertant strain were analyzed by hemolysis test,in vivo distribution test,serum bactericidal test and cell adhesion test.Hemolysis test showed that ygeP deletion did not affect the hemolysis of APEC;in vivo bacterial load test showed that the bacterial colonization of AE81?ygeP deletion strain in heart tissue was significantly higher than that of AE81 wild strain(P<0.05),and the revertant strain recovered;in lung tissue,the bacterial colonization of AE81?ygeP deletion strain was significantly higher than that of AE81 wild strain(P<0.001),and the revertant strain recovered;There was no significant difference in the distribution of AE81 wild strain,AE81?ygeP deletion strain and AE81C?ygeP in liver and spleen tissues.The results of serum bactericidal test showed that the serum viability of AE81?ygeP deletion strain in 20% serum group and 50% serum group was significantly higher than that of AE81 wild strain(P<0.05),and the revertant strain recovered;there was no significant difference in the serum viability of AE81 wild strain,AE81?ygeP deletion strain and AE81C?ygeP deletion strain in 10% serum group and 100% serum group(P>0.05).The serum viability of AE81?ygeP deletion strains in50% serum + 50% complement group,50% serum + 20% complement group and 20%serum + 20% complement group was significantly higher than that of AE81 wild strain(P<0.001),and the serum viability of AE81?ygeP deletion strains in 10% serum + 10%complement group,50% serum + 10% complement group and 20% serum + 20%complement group was significantly higher than that of AE81 wild strain(P<0.05)The results showed that compared with AE81 wild strain,the transcriptional levels of rcaA,rff D,wza and lpx A genes in AE81?ygeP deletion strain were significantly up-regulated(P<0.01);the transcriptional levels of rcs B,wcaA and nik B genes in AE81?ygeP deletion strain were significantly down regulated(P<0.01),while the transcriptional levels of tsa,mur E and tus genes were not significantly down regulated.The results of chicken embryo fibroblast adhesion test showed that the adhesion ability of AE81?ygeP deficient strain was significantly enhanced(P<0.05).3.Preliminary study on the role of ygeP in serum sterilizationThe lacZ reporter vector of AE81 wild strain and AE81?ygeP deletion strain was constructed,and the ?-galactosidase activity of wcaA,rffd,wza,lpx A gene promoters of different genes was detected.The results showed that the ?-galactosidase activity of wcaA,rffd,wza,lpx A gene promoters of AE81?ygeP deletion strain was significantly higher than that of AE81 wild strain(P<0.05).wcaA single deficient strain and AE81?ygeP? wcaA double deficient strain were successfully constructed by Red homologous recombination.The serum bactericidal test of AE81 wild strain,AE81?ygeP deletion strain,AE81-? wcaA deletion strain and AE81?ygeP? wcaA deletion strain was carried out.The results showed that the serum viability of AE81?ygeP? wcaA deletion strain was significantly higher than that of AE81 wild strain and AE81?ygeP deletion strain under the condition of 50% serum+ 50% antibody interaction at 37 ? for 2 h(P<0.05),and there was no significant difference between AE81?wcaA deletion strain and AE81 wild strain(P>0.05).To sum up,we successfully constructed ygeP deletion strain and revertant strain.After the deletion of ygeP,the movement ability of AE81 wild strain was significantly reduced,and the biofilm formation ability,serum survival ability,bacterial load in heart and lung tissue,cell adhesion ability were significantly enhanced,which affected the bacterial stress resistance system.There was no significant difference in growth performance and drug sensitivity.ygeP affected the expression of serum resistance related genes Transcription level.The ?-galactosidase activities of wcaA,rffd,wza and lpx A gene promoters of AE81?ygeP deletion strain were significantly higher than those of AE81 wild strain.The serum viability of AE81?ygeP? wcaA deletion strain was significantly higher than that of AE81 wild strain and AE81?ygeP deletion strain.These results suggest that ygeP is involved in regulating the phenotype and pathogenicity of APEC,and ygeP may affect the serum resistance by regulating the extracellular polysaccharide forming gene wcaA. |
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