The Effect Of ETT2-YgeG On The Pathogenicity Of APEC And The Screening Of Candidate Interacting Proteins

Avian pathogenic Escherichia coli(APEC)is a typical extra-intestinal pathogen with a broad host spectrum in poultry.The potential reservoir of extraintestinal pathogenic Escherichia coli virulence genes,due to the complex and diverse serotypes and the high mutation rate of virulence gene clusters,there are still many difficulties in its control.Therefore,research on the pathogenic mechanism of APEC is of great significance to the development of poultry industry and public health and safety.Escherichia coli Type III Secretion System 2(ETT2)exists in most E.coli genomes and is involved in a series of bacterial pathogenic processes.During this process,chaperones can regulate the secretion of effector proteins and play a crucial role in their pathogenicity.ygeG is a potential chaperone of ETT2.At present,only preliminary studies have been carried out in E.coli with partial deletion of the ETT2 gene cluster,and it has been found that it has a certain effect on the pathogenicity of bacteria.So far,there has been no research on the regulatory function of ygeG in strains containing the complete ETT2 gene cluster,and the prediction of its structure and function lacks certain confirmation,so the research on this gene is essential.In this study,Red homologous recombination technology was used to construct ETT2 potential chaperone protein gene ygeG deletion strains and revertant strains,and the biological characteristics were analyzed.Through cell and animal experiments,the effect of ygeG on APEC in infected host cells and chick colonization was explored.Function;Build a YgeG prokaryotic expression vector,express the protein and verify,and screen ygeG interacting proteins by GST pull-down combined with mass spectrometry.The main research contents and results are summarized as follows:1.Construction of avian pathogenic Escherichia coli ygeG deletion strains and revertant strains and their effects on biological characteristicsTaking the strain APEC81 containing the complete ETT2 gene cluster preserved in the laboratory as the research object,the APEC81-ΔygeG deletion strain was constructed by Red homologous recombination technology,and the APEC81-CΔygeG revertant strain was constructed by using the low-copy plasmid p STV28.The effects of ygeG on the biological characteristics of APEC were analyzed through experiments such as growth characteristics,motility,biofilm formation ability,and environmental tolerance.The growth curve measurement results showed that the growth characteristics of the deletion strain APEC81-ΔygeG had no significant change(P>0.05);the biofilm test results showed that the biofilm formation ability of the deletion strain APEC81-ΔygeG was significantly reduced(P<0.01);the motility test results showed that,the exercise capacity of the deletion strain APEC81-ΔygeG was significantly increased(P<0.001);the results of the environmental tolerance test showed that the ability of the deletion strain APEC81-ΔygeG to tolerate acid and oxidative stress was extremely significantly reduced(P<0.001),and the ability of the deletion strain APEC81-ΔygeG was extremely significantly reduced(P<0.001),osmotic pressure and tolerance to heat shock were significantly decreased(P<0.01).The above experimental results indicate that ygeG is involved in regulating the pathogenicity-related biological phenotypes of APEC.2.The effect of ETT2 chaperone gene ygeG on the pathogenic effect of avian pathogenic Escherichia coliThe effect of ygeG on the pathogenic effect of APEC was explored through bacterial hemolysis test,antiserum bactericidal test,cell adhesion and invasion test,detection of inflammatory factors and chick pathological tissue sections.The results of bacterial hemolysis test showed that the hemolytic state of APEC81-ΔygeG in the deletion strain had no significant change compared with that of the wild strain.When the concentration of APEC81-ΔygeG was 30% and 100%,the bactericidal effect of antiserum was extremely significant(P<0.001);the cell adhesion and invasion test results showed that the adhesion ability of the deletion strain APEC81-ΔygeG to chicken tracheal mucosa epithelial cells was significantly decreased(P<0.001).The invasion ability was significantly increased(P<0.01).The results of the cytokine assay showed that the transcription levels of TNF-α and IL-1β were extremely significantly increased(P<0.001),IL-6 was significantly increased(P<0.01)and IFN-γ was increased(P<0.05)after APEC81-ΔygeG was infected.After the wild strain APEC81,the deletion strain APEC81-ΔygeG and the revertant strain APEC81-CΔygeG were infected with chicks,the results of histopathological sections showed that,compared with the control group,the three strains had different degrees of pathological changes after infection.,the damaged trachea mucosa was shed,the myocardial fibers were partially broken into corrugations and the fiber gaps were enlarged,the hepatocytes were enlarged and inflammatory cells were aggregated,and there were certain pathological characteristics in the APEC81-ΔygeG and APEC81-CΔygeG infection groups,but the same Compared with the APEC81 infection group,the degree was milder.The above experimental results further indicated that ygeG was involved in regulating the pathogenic effect of APEC.3.ygeG prokaryotic protein expression and preliminary screening of interacting proteinsThe prokaryotic expression vector p GEX-6P-1-ygeG was constructed and transformed into E.coli expression strain BL21(DE3)for IPTG-induced expression and its expression conditions were optimized.The ygeG recombinant protein has a size of about 44 ku,is highly expressed in Escherichia coli BL21(DE3),mainly exists in the form of soluble protein,and has good immunogenicity and reactogenicity.The results of SDS-PAGE and Western-blot identification showed that the optimal conditions were as follows: the final concentration of IPTG was 0.25 mmol/L,and the cells were induced at 16 ℃ for 16 h.Using purified ygeG as the bait protein and APEC81 whole bacterial protein as the prey protein,1115 proteins with potential interaction with ygeG were preliminarily screened by GST pull-down combined with mass spectrometry.The interacting protein library is of great significance for analyzing the function of the chaperone YgeG.To sum up,the deletion strain APEC81-ΔygeG and the revertant strain APEC81-CΔygeG were constructed in this experiment.After ygeG deletion,the growth performance and hemolytic ability of APEC81 did not change significantly,and the biofilm formation ability,environmental tolerance ability,and antiserum bactericidal ability did not change significantly.ygeG affects the transcription levels of genes such as biofilm,flagella,and cell inflammatory factors,and affects the pathological changes of chick trachea,heart,liver and other tissues degree,indicating that ygeG is involved in the regulation of APEC biological phenotype and pathogenic effects.The ygeG protein was expressed and purified,and the ygeG potential interacting protein library was successfully obtained by GST pull-down combined with mass spectrometry.This study clarified the biological function of ygeG gene,initially explored its possible interacting proteins,and provided research basis and support for the later search for its target and ETT2 secreted protein.