The Roles Of ETT2 And PhoP In The Biofilm Formation And Pathogenicity Of Avian Pathogenic Escherichia Coli

Avian colibacillosis is one of the bacterial infectious diseases that endanger the poultry industry.Due to its complex and variable serotype,it is difficult to effectively prevent and control,causing serious losses to the poultry industry.At the same time,APEC is the virulence gene pool of human parenteral pathogenic Escherichia coli(ExPEC).These virulence genes can enter the food chain through gene drift and pose a serious indirect threat to human health.Therefore,strengthen the prevention and prevention of APEC.Control has important public health implications.At present,although the pathogenic mechanism of avian pathogenic Escherichia coli has been reported at home and abroad,there are still many unknowns to explore and study.Biofilm is one of the pathogenic factors of APEC,and its formation can enhance the APEC anti-host immune system.And improve the viability in the environment,while it can also reduce the sensitivity to antibacterial drugs,thereby enhancing the pathogenicity of APEC.This poses a serious challenge to the prevention and control of APEC,and research related to the physiological characteristics and pathogenicity of biofilms is urgently needed.Virulence regulators mediate the expression of virulence-related genes through virulence regulation networks,which in turn affect the pathogenicity of bacteria.These virulence regulation networks involve bacterial secretion systems and binary regulatory systems.Escherichia coli type III secretion system 2(ETT2)acts as a secretory system similar to Salmonella SPI-1,and its appearance makes the pathogenesis of APEC more complicated.phoP acts as a transcriptional regulator of APEC,which regulates bacteria.A variety of life activities,however,the function and pathogenesis of ETT2 and phoP related to biofilm formation is unclear.In this paper,the research on the biofilm formation and pathogenicity of ETT2 and phoP in avian pathogenic Escherichia coli,which provides a theoretical basis for further understanding of the pathogenic mechanism of avian pathogenic Escherichia coli.Finally,it provides a new therapeutic approach for controlling APEC infection.The main research contents are as follows:1.Effect of ETT2 Deletion on APEC Biofilm Formation and PathogenicityTo analyze the distribution of ETT2 gene components in APEC clinical isolates,ECs3703 and ECs3737 were selected as ETT2 virulence island detection marker genes,and the integrity of ETT2 gene components in APEC strains containing ETT2 was detected.The results showed that 64.4%(47%)/73)APEC clinical isolates contain ETT2 virulence islands,of which the O1 serotype APEC strain containing ETT2 virulence island is 14.9%(7/47),and the 02 serotype APEC strain is 19.1%(9/47),078 The serotype APEC strain was 10.6%(5/47),and 55.4%(26/47)was an APEC strain that was not a "01/02/078" serotype.In addition,there is an 8.8-kb gene deletion in the APEC-ETT2 cluster eiv operon,which is truncated from eivJ(ecs3727)to eivF(ecs3734).On this basis,the deletion strain of ETT2 whole gene cluster was constructed.The experimental results showed that APEC biofilm formation ability was enhanced after ETT2 deletion,and resistance to amikacin and kanamycin was enhanced in heat and oxidation.Survival in the shock environment was significantly enhanced,while survival was significantly reduced under acid stress conditions,while deletion of ETT2 reduced flagellin synthesis protein(flgN/flgM/fliR)and flagella filament gene(flgB/flgC/flgF)The expression level of transcriptional level was down-regulated,and the expression level of type ? pili gene was up-regulated,resulting in decreased motility of APEC and enhanced adhesion to DF-1 cells.In addition,we found that the loss of ETT2 resulted in a change in the integrity of the outer membrane component of APEC,and the bacterial antisera bactericidal ability was enhanced.To find out whether ETT2 secretes effector proteins and whether it can affect the expression and secretion of other secretory systemic effector proteins,by analyzing the extracellular secreted proteins of ETT2 deletion strains and wild strains,the results showed that the expression of extracellular secreted proteins was decreased after ETT2 deletion.The composition contains a large number of flagellar-related protein expression down-regulation,and we have found through transcriptome data that the gene expression level of the virulence effector protein associated with T3SS has also changed.In addition,the two ETT2 groups of ecs3717 and ecs3718 The subgenes also undergo differential changes and are annotated as effector proteins in transcriptome data.These findings provide a reference for further understanding of the function of ETT2 and its pathogenesis in APEC.2.The role of ETT2 chaperone ygeG in the biofilm formation and pathogenicity of APECMolecular chaperones in T3SS are essential for the bacterial secretory effector protein to exert its pathogenicity,but the molecular chaperone function in ETT2 has not been confirmed.Here,we predicted and identified a new ETT2 chaperone ygeG through bioinformatics,and constructed a ygeG gene deletion strain and a restore strain.Our experimental results indicate that the loss of ygeG leads to APEC biofilm,motility,survival under oxidative stress conditions,cell adhesion and antiserum bactericidal effects have been enhanced to varying degrees,qRT-PCR results show that these life The transcriptional level of activity-related genes was significantly up-regulated.At the same time,the deletion of ygeG caused the transcription level of the stress-resistant gene yhbO/hdeB to be down-regulated,and the survival rate of APEC in heat shock,acid shock and hypertonic environment decreased.In addition,ygeG can affect the transcription level of the T3SS effector gene espL/espX.This study confirmed that the biological characteristics and virulence-related effects exhibited by APEC in APEC after deletion of ygeG are very similar to those after ETT2 deletion,and that ygeG deletion affects ETT2-encoded yqeH(ecs3703).The transcription level was up-regulated,and yqeH was analyzed as a transcriptional regulator.These results indicated that ygeG can negatively regulate the transcription level of the transcriptional regulator yqeH,and regulate the expression of downstream target genes through yqeH,ultimately contributing to the function of ETT2.3.The molecular mechanism of ETT2 transcriptional regulator yqeH regulating APEC biofilm formation and virulenceETT2 is present in most E.coli strains and plays a very important role in participating in bacterial pathogenicity.The role of regulatory factors in the pathogenesis of ETT2 is critical,but the function of the ETT2 regulatory factor has not been fully confirmed.Here,we show that yqeH,a new transcriptional regulator belonging to the LuxR family.We have carried out related work to study the role of ETT2-yqeH in APEC.We constructed the yqeH gene deletion strain and the restorer strain.The experimental results show that the yqeH deletion reduces the biofilm formation and motility of APEC,involving multiple biofilm formation.The transcription levels of related genes and flagella assembly genes were down-regulated.The P-galactosidase assay and EMSA confirmed that the target gene fliT regulated by yqeH is related to the motility of APEC,and the target gene lsrF regulated by yqeH is related to the formation of APEC biofilm.We also found that APEC40-yqeH affects the survival of bacteria in different environments by affecting biofilm formation and regulating the transcription of stress resistance genes.In addition,our study showed that the adhesion of APEC40-?yqeH strain to DF-1 cells was reduced because the deletion of yqeH reduced the transcription level of type ? pili.Similarly,we found that mutants of yqeH reduced transcription of the O-antigen capsule-forming protein gene,which may be responsible for serum resistance defects.It may provide a basis for analyzing the mechanism by which ETT2 is involved in bacterial sepsis.These findings indicate that the ETT2 regulatory factor yqeH is involved in the biofilm formation,movement and pathogenicity of APEC.4.Molecular mechanism of phoP regulating APEC biofilm formation and pathogenicityThis study is based on the phop-deficient strains that have been constructed in the previous laboratory,and based on the related work,it was found that the loss of phoP affects the synthesis of sugar chain-related genes in the lipopolysaccharide pathway,thereby affecting the hydrophobicity of the bacterial cell surface and itself.Agglutination.In addition,phoP regulates the assembly of bacteriophage proteins of bacterial flagella and curly pili,thereby affecting the formation of biofilms.In the end,phoP deficiency affects the multi-drug resistance of bacteria.In order to further analyze the biofilm formation and pathogenic mechanism of phoP in APEC,the differential gene tolC was screened by gene chip expression profiling data,and the specific sequence identification of phoP binding sequence was predicted by bioinformatics,and verified by EMSA.The results show that phoP can directly bind to the promoter of tolC.On this basis,the tolC deletion strain and the revertant strain were constructed.The crystal violet staining was used to quantify the biofilm formation and observe the change of rdar morphology.The results also proved that the loss of tolC reduced the biofilm formation,which further clarified the effect of phoP.The mechanism of action of bacterial biofilm formation.The loss of phoP reduces the pathogenicity of APEC to chicks,and the target gene tolC regulated by phoP plays an important role.We attacked the chicks and observed the pathological tissues and pathological sections.The results also showed that after the loss of tolC,the symptoms of the chicken heart,liver and spleen enlargement were alleviated,and the pathogenicity to the host cells decreased.qRT-PCR results found that tolC mutant strain affects the downregulation of the secA/secB/secE/secY transcription level of the T2SS secreting virulence effector protein element.Our results reveal the mechanism of phoP affecting APEC biofilm formation and pathogenicity of chicks,and provide a reference for comprehensive understanding of the pathogenic mechanism of avian pathogenic Escherichia coli.In conclusion,this paper starts with Escherichia coli type ? secretion system 2(ETT2)and the binary regulatory system transcriptional regulator phoP,and elucidates the mechanism of action of ETT2 and phoP in APEC biofilm formation and pathogenicity,namely ETT2 molecular chaperone ygeG Negative feedback regulates the transcriptional level of the transcriptional regulator yqeH,regulates the expression of downstream target genes through yqeH,and ultimately contributes to the biofilm formation and pathogenicity of ETT2 involved in APEC;the transcriptional regulator phoP affects the APEC by regulating the target gene tolC on biofilm formation and pathogenicity.These findings provide a theoretical reference for further revealing the pathogenesis of APEC.