Influence Of Two-component Regulatory System OmpR/envZ On The Pathogenicity Of APEC

Avian pathogenic Escherichia coli(APEC)causes avian colibacillosis,which mainly causes avian respiratory infections,pericarditis and sepsis.APEC has many virulence factors that play an important role in the pathogenesis.Mainly include adhesins,invaders,iron intake system,capsules,antiserum survival factors,two component regulatory system,toxins,etc.The omp R/envZ two component regulatory system is widely present in a variety of bacteria.It plays an important regulatory role in the process of bacterial response to osmotic changes in the environment,but its role in the pathogenesis of APEC has not been reported yet.In this study,we constructed the envZ single gene deleted and return strains and omp R single gene deleted and return strain of APEC O2 serotype,respectively,and then measured the growth curve,motility,biofilm formation ability and environment tolerance test of the gene deleted strains.By further comparing the transcription levels of virulence factors and the pathogenicity of chickens between wild strains and gene-deficient strains,the regulatory mechanism between the Omp R/Env Z two component regulatory system and APEC pathogenicity was revealed.The research content and results are as follows:1.The effect of envZ gene deletion on the biological characteristics and virulence of APECIn this experiment,Red homologous recombination technology was used to delete the envZ gene,and a single envZ gene deletion strain AE17?envZ was successfully constructed.The low copy plasmid p STV28 was used to successfully construct the envZ gene return strain AE17C-envZ.Through biological characteristics experiments,it was found that the deletion of the envZ gene did not affect its growth performance in LB liquid medium(p>0.05),but its motility ability was significantly enhanced(p<0.05);the biofilm formation ability was significantly reduced(p<0.05);Scanning electron microscope observation found that compared with wild strain AE17's biofilm was thicker and the connection between bacteria was tight.The biofilm of AE17?envZ strain was thinner,and the distribution between bacteria and bacteria was relatively loose,compared with wild strain The tolerance of the envZ deletion strain to the alkaline environment of p H=10 was significantly lower than that of the wild strain(p<0.01),and the resistance to the acidic environment of p H=5 was significantly lower than that of the wild strain(p<0.05);The resistance of the 50% serum environment was significantly reduced(p<0.05).The results of LD50 test showed that the pathogenicity of 7-day-old Roman chickens decreased by 7.50 times after the absence of envZ.2.Study on differentially expressed genes of envZ deletion strainsIn this experiment,RNA-Seq technology was used to explore the effect of envZ deletion on APEC gene levels.The results showed that 711 differentially expressed genes caused by envZ deletion were selected from transcriptome data,including 303up-regulation and 408 down-regulation.Differential genes expressed in flagellum,biofilms,outer membrane proteins,and Two-Component regulatory systems,etc.By annotating the differential genes into the GO database,a total of 269 differential genes were involved in the GO functional classification,and 1163 GO functional annotations were obtained,mainly involves biological processes such as fatty acid metabolism and molecular functions such as ion binding,etc.In terms of cellular components,significant GO function involves ATP binding(ABC)transporter complex Substances,outer membrane,etc.KEGG functional analysis found that there were 661 annotated differential genes involved in 78 signaling pathways.Mainly involves metabolic pathways,microbial metabolism in different environments,amino acid biosynthesis,two-component system,flagella assembly,biofilm formation and other pathways.3.The effect of ompR gene deletion on the biological characteristics and virulence of APECThe ompR gene was deleted using CRISPR-Cas9 editing technology,and the ompR single gene deletion strain AE17?omp R was successfully constructed and the low-copy plasmid p STV28 was used to successfully construct the omp R gene return strain AE17C-omp R.Then through a series of experiments to explore the impact of omp R gene deletion strain on APEC biological characteristics.It was found that the omp R gene deletion did not affect its growth in LB liquid medium(p>0.05),but the omp R gene deletion significantly reduced the AE17's motility ability(p<0.01).After transmission electron microscopy observed that omp R deletion reduced the number of flagella of wild strains.Compared with wild strains,omp R deletion significantly reduced the biofilm formation ability(p<0.01).Scanning electron microscopy of the biofilm revealed that the biofilm of the comparative wild strain AE17 was thicker and the connection between the bacteria and the bacteria was tight.The biofilm formed by the omp R deletion strain was thinner and the connections between the bacteria were relatively loose.Compared with wild strains,the tolerance of omp R deletion strains to alkaline environment at p H=10 was significantly reduced(p<0.01);tolerance to acidic environment at p H=5 was significantly reduced compared to wild strains(p<0.05);Tolerance to 50% serum environment is extremely significantly lower than that of wild strains(p<0.01).The result of the LD50 test showed that the pathogenicity of omp R deletion strains in chickens decreased by 74.98 times.Fluorescent quantitative PCR detected the expression levels of related virulence genes and found that flh D?fli N?ycg R?dks A?glg C?iss and tsh virulence genes were all down regulated to varying degrees,while lux S virulence genes have been upregulated.In summary,this study successfully constructed the deleted and reverted strains of the envZ and omp R genes.Deletion of envZ has declined the biofilm formation ability,environmental tolerance,and chicken pathogenicity,But its swimming ability is enhanced,And through transcriptome data analysis of envZ-deleted strains may cause the above-mentioned situation.After omp R is deleted,the Swimming ability,biofilm formation ability,alkali resistance,acid resistance and serum sterilization resistance and pathogenicity of chickens are all reduced to varying degrees.Fluorescence quantitative PCR detected the down-regulation of flh D,fli N,ycg R,dks A,glg C,iss,and tsh transcription levels,while the lux S virulence gene transcription levels were up-regulated.The results show that the envZ and omp R genes have an important influence on the pathogenicity of APEC,which lays a certain research foundation for the follow-up research on the mechanism of APEC resistance host innate immune defense.