Role Of GldK Gene In Riemerella Anatipestifer CH-1

Riemerella anatipestifer(R.anatipestifer)is an important pathogen of ducks and other birds,with a worldwide distribution.The major clinical symptoms of infected ducks and other birds are pericarditis,perihepatitis and air sacculitis.In this paper,we studied the gldK gene,results are as follows:1.The bioinformatic analysis of gldK geneThrough our analysis,we found gldK gene was a major component of the type IX secretion system,and gldK gene was also present in RA-CH-1 genome.Blast analysis indicated that gldK gene showed high conservation among different strains of R.anatipestifer,and GldK protein has a typical lipoprotein signal peptide.2.Construction of gldK gene mutant strain in RA-CH-1Left flanking sequence of gldK,SpcR cassette and the right flanking sequence of gldK were ligated using the overlap PCR method.The fusion DNA fragments were recombined on the suicide plasmid pYA4278.The recombinant suicide plasmid was transformed in the donor strain E.coli X7312?pir finally.Then suicide plasmid in donor strain E.coli X7312 was transformed into RA-CH-1 recipient strain by conjugational transfer method.The transconjugants were selected on blood agar plates containing Cfx antibiotics,and combine to the identification of PCR,Finally,single-gene mutant strain RA-CH-1 AgldK was constructed.3.Construction of gldK gene complementation strainThe gldK gene and E.coli-R.antipestifer shuttle plsmid were ligated using Enzyme digestion-Ligase-PCR method.Then recombinant plasmid was transformed in the donor strain E.coli S17-1?pir,the recombinant plasmid in donor strain E.coli S17-1 was transformed into RA-CH-1?gldK recipient strain by conjugational transfer method.The transconjugants were selected on blood agar plates containing corresponding antibiotics,and combine to the identification of PCR,Finally,gldK gene complementation strain RA-CH-1C?gldK was constructed.4.The function research of gldKThe three-day old Cherry Valley ducks were challenged with the same dose(1010CFU)of RA-CH-1,RA-CH-1 ?gldK,RA-CH-1C?gldK.Then observed for a week,results indicated that the survival rate of ducks infected with RA-CH-1 was 20%,infected with RA-CH-1 ?gldK was 80%,infected with RA-CH-1C?gldK was 40%,so it showed gldK had an effect on pathogenicity of R.anatipestifer.The LD50 for RA-CH-1?gldK was 2.25×1011 CFU,which was 1.5×103 times higher than that of wild-type strain RA-CH-1(1.44×108 CFU).The complement strain RA-CH-1C?gldK was 1.24x 109 CFU,further proved the role of gldK in virulence in R.anatipestifer.To further investigate the effect of gldK gene on systemic infection in vivo,the bacterial loading in blood,liver,brain of infected ducks was quantitative,results all indicated the bacterial loading of gldK mutant strain was significantly decreased than that of RA-CH-1 wild type.At 36 h post infected,we collected heart,brain,spleen,liver tissue samples to make pathology slices.When stained with hematoxylin and eosin,we can clearly see the difference between gldK mutant strain and RA-CH-1 wild type strain.Both brain tissues were no visible damage.However,we can found just liver had edema,other tissues were no visible changes in gldK mutant strain,while for RA-CH-1 wild-type strain,we found liver cord disorder,a large area of vacuole-like change,myocardial necrosis,numerous neutrophils infiltrate in spleen,the histological changes of complement strain was similar with RA-CH-1 wild type strain.So we come to a conclusion that gldK did have an effect on the pathogenicity of R.anatipestifer.In summary:gldK gene of Riemerella anatipestifer is a non-essential gene,and also a virulence gene,deletion of gldK gene of Riemerella anatipestifer,it can still grow and reproduce,but reduced the pathogenicity to ducks.