Analysis Of Pathogenic Characteristics Of APEC Phylogroup F Isolates And The Mechanism Of Dksa-mediated APEC Infection

Avian pathogenic E.coli（APEC）belongs to a pathogenic subtype of extra-intestinal pathogenic E.coli（Ex PEC）.The E.coli can be divided into eight system groups（A,B1,B2,C,D,E,F,and Clade I）by phylogenetically grouping.Studies have pointed out that most of the B2 poultry-derived Escherichia coli belong to pathogenic APEC,rather than symbiotic and weakly virulent E.coli.The B2 group APEC has strong pathogenicity to poultry,and also has the potential for zoonotic diseases.The first part of this study is to explore the molecular epidemiological characteristics of avian pathogenic Escherichia coli,mainly to study group F avian Escherichia coli.We analyze the virulence genotype and pathogenic phenotype of F group of poultry Escherichia coli.Studies have found that most F group of poultry-derived Escherichia coli have pathogenicity,similar to the high pathogenicity APEC of group B2,and even have zoonotic potential.The second part explores the molecular pathogenesis of APEC.We construct the transcription factor Dks A gene deletion strain and complementary strain,and study the effect of Dks A gene deletion on the pathogenic phenotype of APEC virulent strain.The lack of Dks A leads to a significant decrease in the virulence of APEC strains,and there are also significant differences in the transcriptome and proteome results of the deletion strain and the wild strain.We have thoroughly explored the molecular regulation mechanism of Dks A and found that Dks A is a key virulence regulator of APEC.Dks A mediates APEC to infect macrophages through small RNA post-transcriptional regulatory pathways.We mainly explored the pathogenic molecular mechanism and molecular epidemiological characteristics of APEC from these two aspects.The third part evaluates the immune protection effect of FY26ΔDks A as a candidate strain of attenuated vaccine.1.Study on pathogenicity and zoonotic potential of F group avian Escherichia coliThe F group of Escherichia coli is classified by MLST.These F group avian E.coli belong to 29 STs.The ST group containing more than 8 strains is called the dominant ST.There are 13 dominant STs（ST59,ST62,ST115,ST117,ST135,ST354,ST362,ST393,ST405,ST457,ST501,ST648 and ST1158）.The genotype and phenotype analysis of F group Escherichia coli show that most F group of poultry-derived Escherichia coli have similar genotype and virulence characteristics to APEC virulent strains.F Group Escherichia coli can produce biofilms and is generally motility;75.4%of the strains show complement tolerance.In contrast,strains containing Col V/BM large plasmids have significantly higher complement tolerance than negative strains,and strains with a virulence gene score that show a VF score≥15 have a higher complement tolerance ratio than strains with VF<15.F group avian Escherichia coli causes a high proportion of avian colibacillosis,and the mortality rate of Col V/BM plasmid-positive strains in the chick model is higher than that of Col V/BM-negative strains.We screen 38 dominant ST group strains and use animal models of sepsis,meningitis and urinary tract infection to assess the zoonotic risk of these 38 strains of poultry-derived F group Escherichia coli.The average disease score and mortality of mice and chicks of these 38 strains within 3dpi are close to the positive control DE205B.All 38 strains can cause bloodstream infections in rats and induce sepsis within 24 hours.The proliferation level of the virulent strains of group F in the urethra and bladder of mice are significantly higher than that of the negative control MG1655.These results indicate that F group of poultry-derived Escherichia coli is similar to human Ex PEC in pathogenicity and has zoonotic potential.In summary,F group of poultry-derived Escherichia coli is generally pathogenic and is a typical avian pathogenic Escherichia coli（APEC）.Some F group strains have zoonotic potential.2.Pathogenic phenotype changes and molecular regulation mechanism of Dks A gene deletion strainDks A is an important transcription factor.Most transcription factors can regulate the transcription function of RNA polymerase（RNAP）by binding to the promoter or the region near the promoter.Animal experiments show that the in vivo competitiveness of the deletion strain FY26ΔDks A is significantly lower than that of the wild strain FY26.Compared with the wild strain FY26,the bacterial load of FY26ΔDks A in the lung and blood of infected chicks is greatly reduced.In the chick model,the LD₅₀ of the deletion strain FY26ΔDks A and the wild strain FY26 are 2.5×10⁷ CFU/bird and 2.0×10⁵ CFU/bird,respectively;in the mouse model,the LD₅₀ of the deletion strain FY26ΔDks A and the wild strain FY26 are 6.3×10⁶ CFU/head and 3.2×10⁵ CFU/head,respectively.The results show that the virulence of the deletion strain FY26ΔDks A is significantly lower than that of the wild strain.The wild strains,deletion strain,and complementary strains are cultured overnight in LB liquid medium at 37°C,and the OD₆₀₀ values are measured to draw their growth curves.The results show that there are no significant differences between the strains.The results of cell adhesion and intracellular survival test show that compared with the wild strain FY26,the adhesion ability and intracellular survival ability of the deletion strain FY26ΔDks A are significantly reduced.Transcriptome sequencing of the wild strain FY26and the deletion strain FY26ΔDks A by RNA-seq reveal that the transcriptional expression profiles of the wild strain FY26 and the deletion strain FY26ΔDks A are significantly different.We screen s RNAs whose transcription levels are more obviously regulated by Dks A,such as s RNA057,s RNA091,s RNA051,s RNA167,s RNA029 and Dsr A,and verify them by Northern blot,the results are consistent with the transcriptome sequencing results.3.Evaluation of immune effect of Dks A gene deletion strain as candidate strain of attenuated vaccineCapsular staining and transmission electron microscopic observation of the wild strain FY26,the deletion strain FY26ΔDks A and the complementary strain FY26CΔDks A show that the capsule of the deletion strain is significantly thicker than that of the wild strain and the complementary strain,which is consistent with the transcriptome data.The deletion strain FY26ΔDks A has weaker pathogenicity,but the thickening of the capsule is more obvious.This characteristic indicates that it has the potential of attenuated vaccine application.To evaluate the immune effect and serum titer of FY26ΔDks A.The results show that regardless of the vaccine dose of 1×10⁶ CFU/head or 5×10⁵ CFU/head,the immune protection rate after the first immunization is greater than 90%,and after the second immunization,the immune protection rate is 100%.The FY26ΔDks A has a high level of serum titer.The results of the colonization clearance test in vivo show that FY26ΔDks A can be gradually cleared in the body over time.In summary,FY26ΔDks A attenuated vaccine candidate strain has a good protective effect against infections of homologous strains,and at the same time has high safety.The protection of the attenuated vaccine candidate strain against infection of different serotype strains needs to be evaluated.