Construction Of GldN And TbdR3 Gene Deletion Mutants Of Riemerella Anatipestifer And Analysis Of Their Biological Characteristics

Riemerella anatipestifer(RA)is an important pathogenic bacterium of ducks,geese and turkeys,which causes serious economic losses to duck industry.In order to control Riemerella ducklings effectively,we studied its biological mechanism at the molecular level.IX secretion system(T9SS),also known as Por secretion system,has been identified in recent years and is only found in Bacteroides.All 8 R.anatipestifer strains that have published full genome sequences on GenBank carry 13 genes encoding the core component of T9 SS,suggesting that the bacteria have a complete IX secretion system.Therefore,T9 SS is the only known secretion system of R.anatipestifer,and its role in the interaction between the bacteria and the host,and in the pathogenesis deserves special attention.In this study,we will construct Yb2?gldN gene deletion strain to analyze its related function of R.anatipestifer T9 SS.In this study,the upstream and downstream homologous arms of gldN gene were linked to suicide plasmid 313,and the gldN gene deletion mutant Yb2?gldN was constructed by conjugation transduction.Then the gldN gene ORF of wild strain Yb2 was linked to the shuttle expression plasmid pRES1 of E.coli-R.anatipestifer,and the recombinant plasmid pRES1-gldN was introduced into Yb2?gldN by conjugation transduction.The complement strain cYb2?gldN was obtained.Then some biological characteristics of wild strain Yb2,the deletion mutant Yb2?gldN and complement strain cYb2?gldN were compared.The results showed that there was no significantly difference in the growth rate in TSB between Yb2?gldN and wild strain Yb2.Protease hydrolysis test showed that no transparent hydrolysis zones was found around Yb2?gldN colonies after 24 hours incubation,while appeared around the wild strain Yb2 and complement strain colonies,with similar size of transparent hydrolysis zones.Compared with those of wild strain Yb2,the adhesion and invasion ability of deletion mutant Yb2?gldN to Vero cells decreased significantly.In addition,median lethal dose(LD50)of the deletion mutant Yb2?gldN to 8-day-old ducklings was 54 times higher than that of wild strain Yb2,and the bacterial load in blood,liver and brain tissues of mutant Yb2?gldN-infected ducklings was significantly lower than that of wild strain Yb2,while the complement strain could recover the pathogenicity and bacterial load to a certain extent.These results suggest that gldN gene and T9 SS is related to the pathogenicity of R.anatipestifer.In addition,our previous studies showed that the 375 # transposon insertion mutant of CH3 strain could not agglutinate serotype 1 positive serum,and could not lethal ducklings.The results of reverse PCR showed that the transposon Tn4351 inserted into TonB-dependent outer membrane protein receptor(tbdR3)on the genome of CH3.In this study,a deletion mutant of this gene will be constructed to identify whether the protein encoded by the gene is an agglutination-related antigen of R.anatipestifer.BLAST and PCR were used to analyze which strain carry the tbdR3.The results showed that serotype 1 strains CH3,NJ-2 and CQ3,serotype 10 strain HXb2,and serotype undetermined strain JY-6 carried tbdR3,while serotype 1 strains WJ4,YL4 and YXb12 did not carry tbdR3 e.It indicated that tbdR3 was not a specific and conservative gene of serotype 1 R.anatipestifer.Using the same method,we have constructed tbdR3 deletion mutants CH3?tbdR3,NJ2?tbdR3 and CQ3?tbdR3 of R.anatipestifer serotype 1 strain CH3,NJ-2 and CQ3,and their corresponding complemented strains cCH3?tbdR3,cNJ2?tbdR3 and cCQ3?tbdR3 respectively.The results of the biological characteristics of the deletion mutants showed that there was no significant difference in the growth rates between the deletion mutants CH3?tbdR3,CQ3?tbdR3,NJ2?tbdR3 and the corresponding wild strains.In addition,all three deletion mutants CH3?tbdR3,NJ2?tbdR3 and CQ3?tbdR3 could agglutinate rabbit anti-R.anatipestifer serotype 1 serum,just as the wild type strains CH3,NJ-2 and CQ3.Moreover,adhesion and invasion to Vero cells of deletion mutants CH3?tbdR3 and CQ3?tbdR3 was higher than those of wild type CH3 and CQ3,while wild type NJ-2 and mutant NJ2?tbdR3 had weak ability to adhere and invade Vero cells.Our results also showed that wild type CH3,mutant CH3?tbdR3,and the complemented strain cCH3?tbdR3 could not kill ducklings.This study will be helpful to elucidate the molecular pathogenesis and function of related genes of R.anatipestifer.