| With the rapid development of the poultry farming industry,the morbidity and mortality caused by Avian pathogenic Escherichia coli(APEC)disease have increased,causing serious economic losses to the poultry farming industry.At the same time,Studies have shown that APEC,as a virulence gene pool for human enteric pathogenic Escherichia coli(Ex PEC),is considered to be closely related to human-derived intestinal pathogenic E.coli and poses a serious indirect threat to human health.Therefore,strengthening the prevention and control of APEC has important public health significance.Escherichia coli type III secretion system 2(ETT2)is a newly discovered virulence gene cluster in APEC that may cause the pathogenicity of APEC.In recent years,most of its research has remained on epidemiology.However,it is unclear whether ETT2 secretes effector proteins and its specific pathogenic ability.Based on the preliminary research in the laboratory,transcriptional regulators play a vital role in controlling the life of bacterial cells,and can affect the pathogenicity of APEC through various ways.But whether transcriptional regulators have the same function in ETT2 is unknown.This subject starts with the etr A gene in ETT2,analyzes its physicochemical properties and functions through bioinformatics,constructs the deleted and reverted strains of the etr A gene in APEC,evaluates its impact on the biological characteristics of APEC,and uses RNA-Seq technology to screen the expression of deleted strains Genes with significant differences explore the potential virulence factors of APEC and sort out its possible pathogenic mechanisms.Finally,the prokaryotic expression and verification of its protein are provided to provide a theoretical basis for the future search for downstream target genes,and to further study the effect of EtrA regulation of APEC on host pathogenicity.The main research contents and results of this study are summarized as follows:1.Bioinformatics analysis of EtrA and construction of gene-deleted and reverted strainsThe amino acid sequence of EtrA in Escherichia coli O157:H7 str.Sakai DNA(Gen Bank: BA000007.3)ETT2 was retrieved from the NCBI database for bioinformatics analysis.The results showed that EtrA is an acidic hydrophilic protein and there is no transmembrane structure in the molecule The signal peptide sequence contains 2phosphorylation sites,and the most composed of EtrA protein secondary structure is?-helix accounting for 50.00%.The prediction result of the tertiary structure is consistentwith the prediction of the secondary structure.According to the topological network prediction of its interacting proteins,EtrA belongs to the family of response regulator Lux R/Fix J operons and is a type of regulatory protein that plays an important role in the quorum sensing of Gram-negative bacteria.Through PCR detection,it was proved that the clinical isolate APEC40 strain contained the target gene etr A.The screening results of ampicillin and chloramphenicol showed that the two-step homologous recombination method of Red can be used to construct the APEC-etr A gene deletion strain,and at the same time,the p STV28 plasmid and the amplified etr A fragment containing the cleavage site were amplified using T4 The ligase was ligated and transferred into APEC40-?etr A to obtain the etr A gene reverted strain APEC40-C?etr A.2.Evaluation of the biological characteristics of etr A gene and verification of transcriptomics analysisIn this project,the differences between APEC40 wild strain,gene-deleted strain APEC40-?etr A and reverted strain APEC40-C?etr A in terms of motility and biofilm formation ability were first studied.The study found that there is no significant difference in the growth performance of the deleted and reverted strains in the LB medium compared with the wild strain(P>0.05);Deleted of etr A gene could reduce the motility of APEC40strain(P<0.01)and affected the formation of bacterial biofilm to a certain extent(P <0.001)compared with wild-type APEC40.The drug sensitivity test shows that the deletion of etr A causes the sensitivity to ampicillin to change from sensitive to intermediate.Based on the effect of etr A on biofilm and motility in previous studies,transcriptomics and fluorescent quantitative PCR were used to verify.The results showed that the two transcripts obtained a total of 176 differentially expressed genes,including 124up-regulated genes and 52 down-regulated genes.Transcriptomics GO function analysis.Differentially expressed genes mainly involve bacterial physiological metabolism,cellular processes,virulence,stress,binding and catalytic activity,and biological regulation.KEGG enrichment analysis showed that differentially expressed genes mainly Concentrate on environmental information processing,metabolism,membrane transport,and signal transduction pathways.The results of fluorescent quantitative PCR confirmed that the expression levels of the selected genes were consistent with the results of the transcriptome differential genes.From this,the reliability of the results of transcriptomics analysis can be determined.Based on the results of transcriptomics,the etr A gene not only affects the cellmovement and biofilm formation ability,but the differentially expressed genes are mainly involved in the biological processes of bacterial physiological metabolism,virulence,stress,and biological regulation.The biological characteristics were verified by phenotypic experiments.In the stress resistance experiment,wild-type APEC40 and APEC40-?etr A-deficient strains had significant differences under high osmotic pressure and acidic environment(P<0.001).There was no significant difference under the shock.The results of in vitro serum sensitivity test showed that when the serum content was above 70%,the sensitivity of the deleted strain APEC40-?etr A to serum was significantly higher than that of the wild strain APEC40(P <0.001).The results of hemolysis test showed that the deletion of etr A gene had no significant effect on wild strains.3.Prokaryotic expression and verification of avian E.coli ETT2-EtrA proteinTwo pairs of primers were designed respectively;the epitope enrichment region sequence of etr A gene was amplified by PCR and cloned into p ET32 a and p Creat S-II expression vectors,respectively.Through PCR and enzyme digestion analysis,recombinant plasmids containing the corresponding genes were obtained.The obtained recombinant plasmid was transformed into BL21(DE3),and induced expression by IPTG.After SDS-PAGE and Westem-Blot analysis,the two fusion proteins were 37.37 KD and33.4KD in size,respectively.The two fusion proteins obtained were named p ET32a-EtrA protein and p Creat S-II-EtrA,which were used for the later prediction of EtrA The regulatory mechanism and role of proteins provide a basis for research.In summary,this study analyzed EtrA as a transcriptional regulator through bioinformatics,and successfully constructed the etr A gene deletion and recovery strains.After the deletion of the etr A gene,there was no significant difference in the growth performance,hemolytic capacity and resistance to oxidative stress of the strain,Bacterial motility,acid resistance and anti-serum sensitivity are reduced,and biofilm formation ability and anti-osmotic pressure are significantly enhanced.RNA-Seq technology shows that the etr A gene affects the transcription level of bacterial metabolism,biofilm,outer membrane protein and stress resistance genes.These genes are all related to the pathogenicity of bacteria.It shows that etr A is involved in the regulation of APEC biophenotype and pathogenic effect,which may affect the pathogenesis of APEC by regulating the expression of bacterial metabolism,biofilm,outer membrane protein and stress resistance genes. |
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