| Avian Pathogenic Escherichia coli(APEC)belongs to Extra-intestinal pathogenic E.coli(ExPEC).In addition to a wide variety of serotypes,APEC also has a variety of virulence factors,transcription regulators,and secretion systems that participate in the pathogenesis of APEC,endangering the health of poultry and causing serious economic losses to the poultry industry.Escherichia coli Type ? Secretion System 2(ETT2)is a new type ? secretion system that affects pathogenic factors such as bacterial adhesion and motility.Yqe? is one of the five transcription factors of ETT2.It is homologous to MarT which affects virulence in SPI virulence island,and affects the secretion of LEE-encoded type ? secretion system together with ygeK,but its detailed functional role has not been reported in APEC.This study conducted a series of experiments to evaluate the effect of Yqe? on the pathogenic effect of APEC.Construct Yqe? gene-deleted and reverted strains to compare the biological characteristics differences between the mutant and original strains;use RNA-Seq to screen differential genes and use fluorescence quantitative verification to analyze differential gene function regulation pathways in conjunction with biological phenotype results;The pathogenesis of APEC,explore the characteristics of Yqe? against serum sterilization and cell adhesion,compare the distribution of missing and wild strains in animals,and specifically analyze the relationship between this gene and the pathogenicity of APEC.1.Construction of Avian pathogenic Escherichia coli Yqe? deleted and reverted strains and their effects on biological characteristics.Ampicillin and chloramphenicol-sensitive strains were selected from the clinical isolates stored in the laboratory,PCR screened the strain AE81 containing the Yqe? base strain,and the Yqe? gene deletion strain was successfully constructed using Red homologous recombination,and ligated using the low-copy plasmid pSTV-28 The Yqe? gene completes the replacement of the deleted strain.Biological characteristics evaluation of the obtained wild strains,deleted strains and reverted strains showed that there was no significant change in growth characteristics;the ability to exercise and biofilm formation decreased after the deletion of Yqe?,and the number of flagella of the deleted strains decreased by transmission electron microscope observation;The thickness of the membrane with the missing strain was reduced to a single layer,no multi-layer accumulation was formed,and the cellulose sticky structure between the cells disappeared;the sensitivity level of 18 kinds of gram-negative bacteria susceptibility cards was detected,but the sensitivity level was not changed.2.Screening and analysis of differentially expressed genes of Avian pathogenic Escherichia coli Yqe? deletion strain based on RNA-Seq.Transcriptomics sequencing of wild and deleted strains was performed to analyze the difference in gene expression of avian pathogenic E.coli from yqel.The results showed that Yqe? significantly affected 587 differential genes,including 391 up-regulations and 196 down-regulations.And screened for significant downregulation of eivE in the ETT2 gene cluster;KEGG pathway analysis involves 30 regulation including flagella,chemotaxis,biofilm,ABC transporter,antibiotic synthesis,metabolism in different environments,quorum sensing and two-component regulation Type,of which there are up to 51 differential genes for metabolic enrichment in different environments,and flagellar regulation pathways are the most multiples of differences;involved in 39 functions in GO function analysis,mainly enriching cellular processes and biological processes of metabolic processes,cells,cell groups Divide and cellular components of the membrane components,catalytic activity and molecular functions such as adhesives.Screening of flagella and biofilm-related regulatory genes with obvious changes in biological phenotype among the differential genes.Fluorescence quantitative results are consistent with the trend of transcriptome data,and the expression of flagella and biofilm genes in transcriptome sequencing was verified again.3.To detect the effect of Yqe? deletion on the pathogenic effect of Avian pathogenic Escherichia coli.Escherichia coli often causes sepsis,and hemolysis test and anti-blood sterilization test were carried out on the effect on blood components.The result of hemolysis showed that the loss of Yqe? did not change the characteristics of wild strains without hemolysis,but the antiserum sterilization test With the increase of serum concentration to 40%,the difference between the deleted strain and the wild strain over time is the largest,indicating that the presence of Yqe? can help the bacteria resist the bactericidal components in the blood of the host and achieve immune escape.The results of cell adhesion test showed that there was a difference in the ability of the missing and wild strains to adhere to the cells,which showed a weakening trend.Among the tissue load,the host tissue load was reduced,and the heart load was significantly different.The liver,spleen and The difference in lung load is very significant.Therefore,Yqe? is likely to play a positive role in regulating the pathogenesis of APEC.Combining the above three changes in biological characteristics,transcription levels and pathogenic effects,this study found that Yqe? can regulate the regulatory systems such as flagella,biofilm and substance transport required in multiple pathogenic processes of APEC,thereby maintaining bacterial pathogenicity The ability and self-protection ability in the host body confirmed that the transcription factor YqeI had an impact on the pathogenic role of APEC from multiple perspectives. |
PDF Full Text Request