Characterization Of Two Genes Associated With The Riemerella Anatipestifer Lipopolysaccharide Synthesis

Riemerella anatipestifer is an important pathogen that causes septicemia anserum exsudativa in ducks,geese and turkeys.Riemerella anatipestifer infection causes high morbidity and high mortality for ducks and is widely distributed around the world,leading to serious economic losses to the duck industry.Lipopolysaccharide（LPS）is considered to be a major virulence factor of Gram-negative bacteria,which is an important virulence factor involving in bacterial pathogenicity.In this study,we identified two LPS mutants by screening the RA random Tn4351 transpon mutant library,named RA1067 and RA1062.Biological characterization showed a distinct loss of ladder-like pattern in LPS significantly attenuated the virulence of both mutants in ducks,and animal experiments indicated inactivated mutants were effective in cross-protecting of the ducks from challenging with R.anatipestifer strains WJ4（serotype 1）,Yb2（serotype 2）and HXb2（serotype 10）.This study mainly contained two parts as described below:In this study,we obtained two RA LPS mutant strains（RA1067 and RA1062）by screening the random Tn4351 transposon library using anti-LPS MAb 8A9 in an indirect ELISA.The mutant strain RA1067 contained a single Tn4351 insertion in the chromosomal DNA was confirmed by Southern blot.Sequence analysis indicated M949_RS01915 gene was inactivated in the mutant strain RA1067.The LD₅₀ for the mutant strain RA1067 was 2.74×10¹⁰ CFU,which was more than 365 times attenuated virulence than that of the WT strain CH3（7.50×10⁷ CFU）.The bacterial loads in the blood of ducks infected with the mutant strain RA1067 were decreased significantly in comparison with those of the ducks infected with the WT strain CH3 at 48 h post infection.Moreover,RA1067 presented significantly increased sensitivity to normal duck serum in comparison with CH3 did in vitro.Furthermore,RA1067 displayed approximately 21.92-fold enhanced adherence ability and about25.18-fold increased invasion capacity in comparison with those of the WT strain CH3.Our study showed that ducks that were immunized with the mutant strain RA1067 were protected from challenge with R.anatipestifer strains WJ4（serotype 1）,Yb2（serotype 2）and HXb2（serotype 10）at 7/8,6/8 and7/8 respectively,suggesting that the RA1067 strain is an effective cross-protection vaccine candidate for protecting ducks against R.anatipestifer serotypes 1,2 and 10 strains infections in China.Strand-specific Illumina RNA-Seq analysis was used to investigate the differentially expressed genes between the WT strain CH3 and mutant strain RA1067.In total,9 genes were up-regulated and 10genes were down-regulated in the mutant strain RA1067 in comparison to the WT strain CH3,which were differentially expressed by over five-fold based on RNA-Seq analysis.Real-time qPCR further confirmed M949_RS01940 and M949_RS09405 genes were up-regulated over two-fold at transcriptional levels.Three genes of M949_RS07580,M949_RS10455 and M949_RS00790 were further verified to be down-regulated over two-fold at transcriptional levels.Down-regulated gene M949_RS07580 was annotated to encode polysaccharide biosynthesis protein CapD,which may be involved in LPS O-antigen biosynthesis.Our results demonstrated that the M949_RS01915 gene is involved in LPS O-antigen synthesis,bacterial virulence and gene regulation in R.anatipestifer.In this study,the mutant strain RA1062 was obtained based on the results of the previous indirect ELISA test.Sequence analysis indicates the M949_RS01035 gene encoding CPBP protease family protein is inactivated and is specifically existed in R.anatipestifer serotype 1 and serotype 10 strains.The results of Western blotting showed that the LPS O-antigen ladder pattern of RA1062 were significantly absent,compared to its wild-type strain CH3.The animal experiment result further confirmed the attenuated virulence of the mutant strain RA1062.Serum sensitivity assays showed that the mutant strain RA1062 was more sensitive to normal duck sera than that of the WT strain CH3.Furthermore,we prepared the inactivated RA1062 vaccine and evaluated the cross-protection of mutant RA1062 among RA serotype 1,2 and 10.The vaccinated ducks were100%,75.00%and 100%protected from challenge with WJ4,Yb2 and HXb2,respectively.Strand-specific Illumina RNA-Seq analysis was used to investigate the differentially expressed genes between the WT strain CH3 and mutant strain RA1062.In total,12 genes were up-regulated and 9 genes were downregulated in the mutant strain RA1062 in comparison to the WT strain CH3,which were differentially expressed by over five-fold based on RNA-Seq analysis.Real-time qPCR further confirmed M949_RS07300 and M949_RS04680genes were up-regulated over five-fold at transcriptional levels.Two genes of M949_RS07580 and M949_RS03025 were further verified to be down-regulated over five-fold at transcriptional levels.The coding products by up-regulated gene M949_RS07300 and M949_RS04680 and down-regulated gene M949_RS03025 were annotated as TonB-dependent receptor.Down-regulated gene M949_RS07580was annotated to encode polysaccharide biosynthesis protein CapD,which may be involved in LPS O-antigen biosynthesis.The above results indicated that M949_RS01035 gene was not only involved in the synthesis of lipopolysaccharide O-antigen and gene expression regulation in Riemerella anatipestifer,but also was associated with bacterial pathogenicity.