Construction And Biological Characterization Of Avian Pathogenic Escherichia Coli Lipopolysaccharide Mutants

Avian colibacillosis is an infectious disease caused by Avian Pathogenic Escherichia coli (APEC) and featured with airsacculitis, synovitis, salpingitis, peritonitis. In addition to spontaneous infection of the pathogen, APEC can also be a secondary infection to other pathogens, resulting in more severe illness and greater losses in poultry industry. Lipopolysaccharide (LPS) is a major composition of the outer cell wall of Gram-negative bacteria which is not only the main source of endotoxin, but also an important indicator of the O antigen serotyping. More and more studies show that O antigen plays an important role in the pathogenic role of Gram-negative bacteria. The waal gene encodes O-antigen and lipid A ligase. the wzy gene, a polymerase of O-antigen, works together with wzze in O-antigen polymerization. These three genes coordinated with each other and played an important role in O-antigen biosynthesis. In this study, we constructed three LPS mutants by deletion of waal, wzy or wzze gene and investigated their roles on the bacterial phenotype and serotype. Moreover, the function of waal gene on APEC pathogenicity was investigated.In this study, three genes involved in O-antigen transportation have been cloned from DE17strain (APEC02serotype), and the corresponding mutant strains Awaa1、Awzy、Awzze were constructed using Red homologous recombination.Then, heat agglutination was used to detect the bacterial phenotypes. The mutant strains of Awaal and Awzy showed a rough phenotype characteristic of agglutination. Futhermore, serum agglutination showed that the Awaal mutant strain lost the sera reaction with standard O1、02、O78rabbit antiserum, indicating waal and wzy genes are associated with bacterial phenotype, and waal also plays a role on the bacterial serotype.To identify the function of waal gene, the LPS were extracted from the parent strain DE17, Awaal mutant strain and complementation strain CAwaal with hot phenol method to detect whether the waal gene affect the LPS structure. The sliver staining and western-blotting were used for the identification. The results demonstrated that the waal gene was involved in LPS biosynthesis, and inactivation of waal gene changed the O-antigen structure.To identify the role of waal on Characteristics of APEC, the growth curve was detected as normal method and the swimming medium were prepared to detect whether the waal gene affect the swimming motility. The results showed that the waal mutation decreased the swimming motility significantly. The adhesion and invasion abilities on DF-1cell, the bacterial lethal median dose (LD50), the environmental tolerance and biofilm formation of the parent strain, mutant strain and complementation strain were investigated to detect the effect of waal gene on APEC pathogenicity. The results showed that the adherence ability and the invasion ability were reduced by3.21-folds and4.42-folds respectively, compared with its parent strain DE17, the LD50were increased42.2-folds, the resistance to oxidative stress was over10-fold reduced, the resistance to alkali endurance showed8.13-fold reduced, the resistance to acid endurance for waal mutant remains no changes. The resistance to heat shock and biofilm formation were increased in waal inactivated strain, compared with its parent strain DE17.