Regulation Of The Chaperone Protein FimC On The Biological Phenotype Of APEC And Its Downstream Target Protein Screening

Avian pathogenic Escherichia coli(APEC)can cause local and systemic infections,resulting in massive mortality in chicks and huge economic losses to the poultry industry around the world.Type I fimbriae are APEC adhesion-related virulence factors,which can assist bacteria to secrete adhesins on the surface of objects or hosts,including selfagglutination,to adhere and invade host cells and tissues.At the initial stage of APEC infection,after APEC colonizes host cells through type I fimbriae,it will further form a biofilm,resulting in persistent and chronic infection of APEC.Type I fimbriae are encoded by operons,mainly composed of subunits Fim A,Fim B,FimC,Fim D,Fim E,Fim F,Fim G and Fim H.Among them,FimC is a periplasmic chaperone(assembly factor)composed of two immunoglobulin-like domains,which helps the pilus subunits to fold correctly and ensures the correct assembly of type I fimbriae;however,FimC plays an important role in APEC initial adhesion and biofilm The role and regulatory mechanism in the formation process are still unclear.Therefore,it is particularly important to explore the molecular mechanism of fimbrial chaperone protein FimC on the adhesion and invasion of APEC in the initial infection.In order to study the function of the type I fimbrial chaperone protein FimC,a fimCdeficient strain was constructed,and the differential genes between the fimC-deficient strain and the wild strain were analyzed and compared by RNA-Seq technology,and the autotransporter gene agn43 was screened as a potential target gene.Biological characteristics and induced cell damage.The relationship between the fimbriae partner FimC and agn43 gene was analyzed,and the regulatory relationship between fimC and agn43 was revealed by fluorescence quantification.Further,it was found that fimC could directly bind to the promoter of agn43 by gel migration assay,and further provide a theoretical basis for the pathogenic mechanism of avian pathogenic Escherichia coli.The specific research is as follows:1.Functional verification of the fimC gene in APEC and screening of its downstream target genesIn this study,the fimC gene of type I fimbriae chaperone of avian pathogenic Escherichia coli APEC81 was used as the research object.The APEC81 deletion strain APEC81ΔfimC of fimC was successfully constructed by λ-Red homologous recombination technology,and the revertant strain APEC81CΔfimC was constructed by plasmid p STV28.Compared with the wild strain,APEC81ΔfimC had significantly fewer fimbriae;the molecular switch of type I fimbriae was in the off state;and there was a significant self-aggregation phenomenon mediated by the fimbrial subunit chaperone protein FimC.Transcriptome sequencing analysis of wild,fimC-deficient and revertant strains was performed,and verified by real-time PCR.The results showed that the fimC deletion resulted in a significant 8-fold increase in the transcription level of the autotransporter gene agn43.Therefore,agn43 was used as a potential target gene regulated by FimC for follow-up research.2.Differential effects of fimC gene and agn43 gene on APEC biological characteristics and induced cell damageIn order to explore whether fimC mediates agn43 to affect the biological phenotype of APEC and induce differences in cell damage,agn43 deletion strain APEC81Δagn43 and restorer strain APEC81CΔagn43 were successfully constructed in APEC81,and double gene deletion strains APEC81ΔfimCΔagn43 and Double revertant strain APEC81CΔfimCΔagn43.APEC81,APEC81ΔfimC,APEC81CΔfimC,APEC81Δagn43,APEC81CΔagn43,APEC81ΔfimCΔagn43,APEC81CΔfimCΔagn43 were measured for growth,biofilm formation,motility,self-agglutination,and cell adhesion and invasion assays.The results showed that compared with the wild strain,APEC81ΔfimC had significantly enhanced self-aggregation ability(p<0.0001),significantly weakened biofilm formation ability(P<0.0001),significantly enhanced exercise ability(p<0.0001),and had a significant effect on the adhesion of HD-11 cells.The ability was significantly weakened(p<0.0001),and the invasion ability was significantly enhanced(p<0.01).Compared with the deletion strain APEC81ΔfimC,the double deletion strain APEC81ΔfimCΔagn43 has significantly reduced self-agglutination ability(p<0.01),and compared with the wild strain and the deletion strain APEC81Δagn43(p<0.0001);The specific biofilm formation ability was significantly enhanced(p<0.0001),and the biofilm formation ability was significantly weakened compared with the wild strain and the deletion strain APEC81Δagn43(p<0.0001),the exercise capacity was significantly enhanced compared with the wild and deletion strain APEC81Δagn43(p<0.0001);for the adhesion ability of HD-11 cells,the double gene deletion strain APEC81ΔfimCΔagn43was comparable to the two single gene deletion strains(APEC81ΔfimC and APEC81Δagn43)The ratio was significantly attenuated(p<0.0001),while the invasion ability of HD-11 cells had no significant change.From the above results,it is speculated that there is a regulatory relationship between fimC and agn43.3.EMSA verifies that FimC binds to the promoter region of agn43 geneIn order to further verify the regulatory relationship between FimC and Ag43,the activity of the agn43 promoter was detected by β-galactosidase,and the promoter regions of the agn43 gene(agn43 I,agn43 II and agn43 III)were deleted in sections.Transcriptional activity.The results showed that the transcription level of Ag43 in the APEC81ΔfimCΔagn43 II double deletion strain decreased by 2 times,and the results indicated that agn43 II was the promoter of the agn43 gene.To further verify whether fimC inhibits the transcription level of agn43 by inhibiting the promoter activity of agn43.The expression vector p ET28a-fimC of FimC protein was constructed,and the induced expression and purification of FimC protein were carried out.The results of expression and purification were verified by WB.Through EMSA experiments,it was proved that FimC can bind to the promoter region of agn43.The above results indicate that the deletion of the fimbrial chaperone protein FimC can cause the self-aggregation of APEC,and the self-aggregation is mediated by the autotransporter Ag43.The results of real-time quantitative PCR and EMSA experiments showed that FimC can directly bind to the promoter region of agn43 and negatively regulate the promoter of agn43,thereby regulating the expression of agn43.This study analyzed the molecular mechanism of the type I fimbriae chaperone FimC negatively regulates Ag43 and thus affects APEC adhesion.