The Analysis Of O-antigen Gene Cluster In Riemerella Anatipestifer CH-2 And The Functions Of G148₀871 Gene

Riemerella anatipestifer（RA）is a Gram-negative,flagella-free,spore-free and capsule-containing bacillus.Infectious serositis of duck caused by RA is a contagious disease that infects ducks,geese,turkeys and many other poultry and wild birds.Capsule that consists of polysaccharide or peptide covers the surface of bacterium.It can affect the physical and chemical properties of bacterial surface,and is related to the ability of bacterium to adapt to the environment and the change of bacterial virulence.In addition,lipopolysaccharide（LPS）is the main component of external leaflet of outer membrane in Gram-negative bacterium,involving in the integrity and stability of bacterial outer membrane.O-antigen is the main part of LPS immunogenicity,and Wzy protein is a polymerase that catalyzes the formation of capsular polysaccharide and O-antigen polysaccharide chains.But so far,the research on RA O-antigen oligosaccharide unit chemical structure and corresponding gene cluster has not been reported,and the gene encoding Wzy protein in RA is still obscure.In this study,combining the O-antigen oligosaccharide unit chemical structure of RA-CH-2 strain,the gene cluster associated with O-antigen was analyzed,and the sugar nucleotide synthesis genes,glycosyltransferase genes and O-antigen processing genes were predicted.The other genes located in this gene cluster were associated with biosynthesis of capsular polysaccharide.As the member of the O-antigen processing genes,the G148₀871 gene was selected for this study.Bio informatics analysis showed that G148₀871 gene might be involved in the biosynthesis of O-antigen and capsule of RA-CH-2 strain,and this gene might be a wzy-like gene.In order to validate this prediction,a G148₀871 gene-deleted mutant was successfully constructed by homologous recombination mediated by suicide plasmid.The biological characteristics of the mutant were compared with those of the wild-type strain.PCR and DNA sequencing results showed that the G148₀871 gene of the mutant RA-CH-2AG148₀871 was replaced by the spectinomycin（spec）gene.The result of Real-Time qPCR indicated that the transcriptional level of downstream genes which were adjacent to G148₀871 was not changed after mutating the G148₀871 gene.The results of biochemical characteristics,autoaggregation and growth curve showed that the mutant had no significant variation in the utilization of carbon source and nitrogen source compared with wild-type strain,but there was obvious autoaggregation,moreover,bacterial growth rate of mutant became faster than that of wild-type strain after culturing for 14 hours.The colony morphology of the mutant became rough compared with wild-type strain,which observed by visual inspection and crystal violet staining.The result of serum agglutination test showed that the mutant did not cause agglutinate reaction with the whole bacterial sera.No obvious O-antigen ladder was observed in silver-stained lipopolysaccharide extracted by thermal phenol-water,cell lysis and other methods.The capsule of the mutant was thinner than that of wild-type strain,which was obseverd under oil microscope after being stained by modified Indian ink staining.The result of observation under transmission electron microscopy was consistent with that of capsule staining.Consequently,the mutant was a capsule-deficient strain.It turned out that the survival rate of the mutant was lower than that of the wild-type strain in the dry environment,which was in accordance with strong hydrophilicity and anti-dry ability of capsular polysaccharide.The result of serum bactericidal test indicated that the survival rate of the mutant was closed to zero when serum concentration was greater than 12.5%,which was significantly decreased compared with that of wild-type strain,suggesting that the resistance of the mutant strain to complement was far less than that of the wild-type strain.The duckling LD50 of the wild-type strain was 2.79×107 CFU,while the LD50 of the mutant was 1.02×1012 CFU.It can be seen that the absence of this gene led to remarkable decline of RA virulence.The colonization determination result indicated that the colonization ability of the mutant in blood,liver and brain tissue of the ducklings was significantly lower than that of the wild-type strain at 6 h,12 h,24 h and 48 h post-infection.There were no obvious pathological changes observed in the histopathological slices of heart,liver,spleen and brain of ducklings infected with 5×10’CFU mutant strain at 36 h post-infection.However,this result was contrast with that of the wild-type strain.These results suggested that the G148₀871 gene was involved in the resistance and virulence of Riemerella anatipestifer.In summary,the chemical structure of O-antigen oligosaccharide unit of Riemerella anatipestifer was revealed for the first time,and the O-antigen gene cluster was analyzed subsequently.We speculated that this gene cluster was shared by the biosynthesis of O-antigen and capsular polysaccharide.The G148₀871 gene was deleted and the biological characteristics of the mutant were studied by different methods.It was proved that the gene was involved in the capsule biosynthesis,which could lay foundation for further studying the function and biosynthesis pathway of the capsular polysaccharide of Riemerella anatipestifer.Moreover,the deletion of this gene significantly reduced the resistance and virulence of Riemerella anatipestifer.which could provide a new direction for understanding the pathogenesis of Riemerella anatipestifer.