Functional Study Of The WzxC Gene Of R. Anatipestifer CH-1 Strain

Riemerella anatipestifer (R. anatipestifer) infection is one of the most economically important pathogens of domestic ducks, which usually appeared in 2-7 week-old ducklings with morbidity and mortality as high as 75%. R. anatipestifer always causes fibrinous pericarditis, perihepatitis, air sacculitis, caseous salpingitis and meningitis in ducks, which manifested drowsiness, diarrhea and ataxia. Polysaccharide, especially the capsular polysaccharide (CPS), was an important factor in bacteria for the virulence and resistance of external environment pressure, however the synthesis of CPS in R. anatipestifer is unknown. The Wzx protein is an oligosaccharide lipase participating in the two biosynthetic pathways of CPS and LPS. Bioinformatics analysis showed that the WzxC protein encoded by B739-1128 gene posssibly participates in the biosynthetic pathway of CPS in R. anatipestifer CH-1 strain. In order to testify this conjecture, we have done the researches below.First, we used the suicide vector pYE4278 to construct RA-CH-1 ?wzxC mutant strain with homologous recombination, followed by detecting the downstream B739-1129 gene of wzxC and wzxL gene with RT-PCR and real-time fluorescent quantitative PCR separately. The result showed the deletion of the wzxC gene hadn't brought any changes with the two strains.Then, comparative analysis of the parent and mutants strain was processed, including the phenotype characteristic, capsule staining, biochemical characteristics and growth curve. It indicated the deletion of wzxC has caused the lose of CPS and the decrease of bacteria amount in the stable phase. Then, the resistance of the two strains to drugs, desiccation, oxidation, the sterilization effect by complement in the serum have been detected as well as the virulence to SPF chick embryos and the adhesion and invasion ability to vero cell. It has determined primarily in experimental that the deletion of wzxC gene has caused the decrease of desiccation resistance, oxidation resistance, complement sterilization tolerance. The ELD50 to SPF chicken embryo of the mutant strain was reduced by 4.38 orders of magnitude compared with the parent strain, while the adhesion and invasion ability were increased 3.05 and 2.26 orders of magnitude. These have revealed preliminarily wzxC was involved in the biosynthesis pathway of CPS, and the CPS might inhibit the adhesion and invasion ability in RA-CH-1.Finally, we extracted LPS and CPS in both strains, and measured the content of polysaccharide in CPS with the method of sulfuric acid-phenol. The results showed that when the wzxC gene was deleted, the content of CPS was reduced by 21.23%, and medium- and long-chain of CPS had been lost, while no obvious change had been found for LPS in both strains. These have proved directly wzxC gene participates in the synthesis pathway of CPS and might have litter relationship of LPS.After all, we have testified the WzxC protein encoded by B739-1128 gene participates in the biosynthesis pathway of CPS in R. anatipestifer CH-1 with bioinformatics analysis and laboratory method, which provides a new basis for the research of synthesis pathway of CPS and LPS in R. anatipestifer and more material for the study of Wzx, especially for its function and substrate specificity. Meanwhile, the RA-CH-1 ?wzxC mutant strain we have constructed in this study has lower virulence and higher adhesion invasion ability compared with the parent strain, which provides a direction and thought for the research of attenuated R. anatipestifer vaccine strain.