The Effect Of LpxL And LpxM Genes On Virulence Of Avian Pathogenic Escherichia Coli Belonging To O1 And O78 Serogroups

Avian pathogenic Escherichia coli(APEC)is the most important pathogen causing avian colibacillosis,which can cause a variety of poultry disease and form local or systemic typical infection symptoms,causing huge losses to the poultry industry.The structure of APEC antigens is complex and diverse,and O antigen is often used as the basis for APEC serotyping,and the dominant pathogenic serotypes such as O1,02,and O78 are the main types.Lipopolysaccharide(LPS)is the main component of the cell wall surface on E.coli,and it is also an important virulence factor for APEC,they are composed of discrete structural regions such as lipid A,core polysaccharides and O antigen side chains.The synthesis and transportation of LPS molecules are necessary for the survival of bacteria in their environment.Lipid A is closely related to pathogenicity and plays an important role in the endotoxin activity in LPS molecules.The structural integrity of lipid A plays an important role in the pathogenicity of APEC.Virulence genes involved in the synthesis of lipid A have become the focus of research at home and abroad.In this study,APEC E516 belonging to serogroup O1 and E522 belonging to serogroup O78 were employed as parental strains,aiming at the two late acyltransferases LpxL and LpxM in the lipid A synthesis process,the APEC E516,E522 lpxL and lpxM gene-deletion strains were constructed by ? phage Red homologous recombination technology,and their complementary strains were analyzed for their biological characteristics.Comparing its biological characteristics with that of wild strains,the influence of lpxL and lpxM genes on the virulence of APEC was further analyzed,which laid a foundation for in-depth study on the pathogenesis of APEC.The growth characteristics,electron microscopic observation,RT-PCR,HD-11 phagocytosis,intracellular viability and pathogenicity of APEC E516 and E522 strains and their lpxL and lpxM gene deletion and complement strains were studied to analyze the physiological and biochemical characteristics of the strains and the relationship between lpxL and lpxM gene pair and pathogenicity of different serotypes O1 E516 and O78 E522 strains.The growth curve measurement results show that the growth rate of each strain at 37?is higher than that at 42?,however,at 37? and 42?,the growth rate of all strains mentioned above in the minimum medium was equivalent.In the LB broth,E516?lpxL,E516?lpxL?lpxM,E522?lpxM,E522?lpxLAlpxM grew significantly slower than their parental strains.The morphological observation under a transmission electron microscopy showed that the outer membrane player and its appendages of gene-deletion strains E516?lpxL,E522?lpxM,E516?lpxL?lpxM and E522?lpxL?lpxM were significantly thinner than that of the wild-type strains,and the surface layer of the cells was even impaired.The surface structural integrity of the complementary strains was not fully restored compared with that of the parental strains.RT-PCR results showed that the homologous arms of the deleted gene togather with the FLP recognition target sites(FRT)left after knock out of chloramphenicol resistan gene(cat)were also normally transcripted,resulting in an"unexpected transcript".Serum complement bactericide test showed that in 12.50%and 25.00%specific pathogen free(SPF)chicken serum,the mortality rate of E516?lpxL,E516?lpxL?lpxM,E522?lpxM,E522?lpxL?lpxM was significantly higher than that of wild-type strains(P<0.05),indicating that lpxL and lpxM genes bore an anti-serum complement bactericide effect.The half lethal dose(LD50)of 1-day-old SPF suggested that the virulence of the gene-deletion strains was reduced by about 1000 times compared with the wild-type strains,and the virulence of the complementary strains had not been recovered to the level of the wild-type strains.The colonization and persistance experiment in 21-day-old SPF chicken showed that the ability of each gene-deletion strain to colonize and persist in each tested organ decreased significantly compared with that of the wild-type strain,and that of the complementary strains was partially restored,instead of complete restoring to the level of the wild-type strain.HD-11 cell phagocytosis and survival test results showed that the gene-deletion strains E516?lpxL,E516?lpxL?lpxM,E522?lpxM,E522?lpxL?lpxM had significantly reduced resistance to endocytosis and intracellular survival compared with wild-type strains,and survival of complementary strains ReE516?lpxL,ReE522?lpxM in HD-11 cells lay between wild-type and gene-deletion strains.Therefore,we conclude that lpxL and lpxM genes have important effects on pathogenicity and virulence of APEC O1 E516 and O78 E522 strains,but there are differences.The effect of lpxL gene on O1 E516 is greater than that of lpxM gene,and the effect of lpxM gene on O78 E522 is greater than that of IpxL gene.