ETT2 Regulating The Serum Resistance Effect Of Avian Pathogenic Escherichia Coli.and The Pathogenic Mechanism Of Secretion Effector EspE3-lack E3

Avian pathogenic Escherichia coli(APEC)is a classic extraintestinal pathogenic Escherichia coli(ExPEC).It is mostly through fecal/mouth,causing inflammatory lesions and sepsis of various organs of poultry,resulting in poultry death.There is a variety of virulence secretion systems in APEC to promote its infection and pathogenic process in poultry.Escherichia coli type Ⅲ secretion system 2(ETT2)encoding many genes which can significantly enhance the pathogenicity of APEC,and its pathogenic mechanism has been widely studied.The transcriptional regulator encoded by ETT2 can regulate the biosynthesis of virulence factors such as flagella,pili and biofilm,and affect the pathogenic biological phenotype.Among them,the characteristics of ETT2 regulating APEC serum resistance are particularly obvious.Multiple transcriptional regulators encoded by ETT2 have a significant regulation effect on serum resistance.However,the reason for the effect of ETT2 gene cluster on APEC serum resistance has not been proved.At the same time,APEC-ETT2 can assist in the secretion of effector EspE3(Escherichia coli.secretion protein E3),which can enhance pathogenicity,but the mechanism of its pathogenic effect has not been explored.In order to reduce the risk of APEC infection and reduce the economic losses caused by poultry morbidity and mortality,it is of great significance to study the effect of APEC serum resistance mediated by ETT2 and the pathogenic mechanism mediated by ETT2 secretion effectors.Therefore,this study analyzed the serum resistance effect regulated by APEC-ETT2,and explored the pathogenic mechanism of ETT2 regulating secretion effector EspE3-lack E3(Escherichia coli.secretion protein E3-lack E3 domin).The research contents and results are as follows:1.Study on the effect of ETT2 regulating the serum resistance effect of avian pathogenic Escherichia coliSerum resistance is an important feature of APEC induced systemic infection,LPS and other bacterial extracellular polysaccharides can enhance bacterial resistance to serum sterilization mediated by complement.And the basis of previous research shows that many transcriptional regulators of ETT2(eivF-eivJ deletion subtype)have important influence to APEC serum resistance.Therefore,APEC40 coding ETT2(eivF-eivJ deletion subtype)was selected as the initial strain to study the serum resistance effect.In this study,the growth curves of ETT2 gene deleted strain(ΔETT2)and wild strain(APEC40)were determined.Then the two strains were cultured in serum,their growth curve and bacterial survival number in serum were recorded,and the effect of ETT2 gene cluster on APEC serum resistance was analyzed.After that,the time with the greatest difference in the survival number of the two strains in serum culture was selected for combined analysis of transcriptome and proteome sequencing.Finally,the biological phenotypes(LPS components and biofilm forming ability)related to the differential pathway of omics data were detected to evaluate the effect of ett2 on APEC antiserum.Studies have shown that there was no difference in growth ability betweenΔETT2 and APEC40.However,the growth ability of ΔETT2 in serum was stronger than that of APEC40,and the survival number of ΔETT2 cultured in serum was significantly higher than that of APEC40(P<0.05),which means ΔETT2 has stronger serum resistance effect.There were 79 differential genes and 49 differential proteins were identified by ETT2 and APEC40 under serum culture conditions.Combined with GO and KEGG analysis,ETT2 affected the down regulation of multiple genes in ATP binding cassette(ABC)transport system and quorum sensing system at gene transcription level and protein level.The two systems are involved in the formation of LPS and biofilm in APEC.And LPS production and biofilm formation ability of ΔETT2 were significantly higher than that of APEC40(P≤0.05),which was consistent with the possible phenotypic results of differential pathway.LPS can resist serum complement sterilization,and biofilm can help bacteria gather and resist adverse environment,so the effect of APEC serum resistance was enhanced.This study explored the influence of APEC-ETT2 on the characteristics of serum resistance,which provided research direction and research basis for the prevention of avian systemic infection caused by APEC.2.Study on the transcription level of espE3 regulated by ETT2 in avian pathogenic Escherichia coliE.coli type III secretory system(T3SS)often coexists with effectors in E.coli genome,and transcriptional regulators and chaperones usually regulate the transcription and secretion of effectors.ETT2 is involved in the secretion of APEC effector EspE3.Therefore,it is speculated that there may be correlation and transcriptional regulation between ETT2 and EspE3 in APEC clinical isolates.In this study,the transcription level of espE3-lack E3 was analyzed.APEC81,which encodes both ETT2 complete gene cluster and EspE3,was selected as the initial strain.The nucleic acid fragment of APEC81(AE81)-espE3(espE3-lack E3)was amplified and translated.The three-dimensional protein model was analyzed and amino acid sequence alignment was blast.After that,the ETT2 gene cluster,the transcription regulator gene yqeH encoded in ETT2,chaperone gene ygeG and espE3-lack E3 were detected by PCR in 107 APEC isolates;Deletion strain ΔyqeH was Construction,the expression of espE3-lack E3 was detected by fluorescence quantitative PCR inΔETT2、ΔyqeH、ΔygeG and wild strain AE81 at different growth stages,and the regulatory relationship between ETT2 and espE3-lack E3 was analyzed.Studies have shown that espE3-lack E3 does not predict the E3 ubiquitinase domain(E3),but has leucine repeat enrichment domain(LRR).This protein has 37%similarity with Salmonella E3 ubiquitinase effector SlrP and is a natural mutant of EspE3;Its gene espE3-lack E3 was associated with ETT2 gene cluster and ygeG in 107 APEC clinical isolates;yqeH and ygeG significantly positively regulated the transcription level of espE3-lack E3 in the logarithmic growth stage of APEC(P ≤0.05),and non significantly regulated the transcription level of espE3-lack E3 after the growth platform stage(P>0.05).This study provides a basis for the protein structure analysis and gene transcription regulation mechanism of espE3-lack E3.3.Study on the pathogenicity of avian pathogenic Escherichia coli secretion protein espE3-lack E3In the previous chapter of this study,it has been proved that espE3-lack E3 is a natural mutant of espE3,and the protein functional domain is different from espE3,which may lead to its failure to enhance the pathogenicity of APEC,so its pathogenicity was analyzed.In this study,the espE3-lack E3 deletion strain(ΔespE3-lack E3)and complementary strain(CespE3-lack E3)was constructed by Red homologous recombination method.After that,the pathogenic contribution of espE3-lack E3 to APEC was comprehensively analyzed and confirmed through biological characteristic test(growth curve detection,motility detection,biofilm detection,serum resistance ability detection,cell adhesion and invasion ability detection),cell infection test and animal in vivo infection test(chick survival rate and pathological tissue section detection).Studies have shown that espE3-lack E3 does not affect the growth ability(P>0.05),motility(P>0.05),biofilm formation(P>0.05)and serum resistance ability(P>0.05)of APEC;But significantly enhances the adhesion(P ≤0.05)and invasion ability(P≤0.05)of APEC to chicken tracheal mucosal cells;And significantly affect the host inflammatory response,resulting in IL-6(Interleukin-6),IL-8(Interleukin-8)and TNF-α(Tumor Necrosis Factor α)transcriptional levels changed significantly(P≤0.05);Significantly reduces the survival rate of 7-day-old chicks infected with APEC(P≤0.05),leads to more serious inflammatory lesions in chicks’ tissues and organs,and enhances APEC pathogenicity in trachea,lung,heart and liver.This study results showed that espE3-lack E3 had an important impact on pathogenicity in the process of APEC infection.4.Study on the pathogenic effect of EspE3-lack-E3 in chicken tracheal epithelial cells and screening of interaction proteinsIn the previous chapter of this study,it was confirmed that EspE3-lack E3 has an important impact on the pathogenicity of APEC.However,its pathogenic mechanism in the process of APEC infecting the host is not clear.Therefore,its secretion during infection was detected and its interaction proteins in host cells were screened.In this study,the prokaryotic expression vector of his-tag EspE3-lack E3 was constructed.After expressing the protein,its mouse polyclonal antibody was prepared to detect the EspE3-lack E3 protein secreted by APEC.After that,the antibody was used to verified by Western blotting and the secretion of EspE3-lack E3 was detected by indirect immunofluorescence.The GST-tag EspE3-lack E3 expression vector was constructed,and the host interaction protein of EspE3-lack E3 in chicken tracheal epithelial cells was screened by GST-pull down experiment and mass spectrometry.Studies have shown that soluble protein of EspE3-lack E3 was successfully expressed,and the polyclonal antibody of EspE3-lack E3 was successfully prepared.The antibody can detect the naturally secreted EspE3-lack E3 of APEC.ETT2 is involved in the secretion of EspE3-lack E3.After APEC infects chicken tracheal mucosal epithelial cells,EspE3-lack E3 can be secreted into cells.Finally,107 chicken tracheal epithelial cell interaction proteins such as eIF4H、SEM3 A、EPHB3、STAG2、SERPINF2、MCM5 and UBB were screened by GST-pull down.This study lays a foundation for exploring the pathogenic mechanism of APEC-ETT2 secretion virulence effector EspE3-lack E3,and provides a molecular target for the development of its interaction inhibitors.In conclusion,this study explored the regulation of serum resistance by APEC-ETT2 and the pathogenic mechanism of EspE3-lack E3.It was found that ETT2 regulates LPS and biofilm formation through ABC transport system and quorum sensing system,and mediates the serum resistance effect of APEC;ETT2 have relevance with EspE3-lack E3;The transcriptional regulator YqeH and chaperone YgeG encoded by ETT2 positively regulated the transcriptional level of EspE3-lack E3 in the logarithmic growth stage of APEC;APEC-ETT2 can assist EspE3-lack E3 in secreting into chicken tracheal epithelial cells;And 107 interacting proteins of chicken tracheal epithelial cells were screened by GST-pull down.The results provide a basis for the study of the pathogenic mechanism of APEC-ETT2.