Construction Of The Tonb1and Tonb2Gene Deletion Mutants Of Riemerella Anatipestifer

Riemerella anatipestifer infection, which was also called infectious serositis, is primarily ahigh-contagious disease of domestic ducks, geese and turkeys. The disease occurs as an acute or chronicsepticemia characterized by fibrinous pericarditis, perihepatitis, airsacculitis, and meningitis. It’s one ofthe most serious diseases harming the duck-industry and had caused great economic loss. Currently, atleast21serotypes of R. anatipestifer have been identified. However, only very poor protection wasobserved among different serotypes of R. anatipestifer.Currently, the study of the pathogenicity of R. anatipestifer is just at starting stage. In this study, wehave constructed an E. coli-R. anatipestifer shuttle vector, which provides an important tool for studyingthe functions of genes of R. anatipestifer. On the other hand, TonB1and TonB2gene deletion mutantswere constructed. Our results suggested that TonB1and TonB2were involved in iron acquisition andrequired for the virulence of R. anatipestifer.1.Construction of an E. coli-R. anatipestifer shuttle vectorIn this study, an E. coli-R. anatipestifer shuttle vector pRES was successfully constructed based onthe plasmid pCP29. The vector contains the putative replication region of R. anatipestifer plasmidpRA7026for replication in R. anatipestifer and a ColE1ori for replication in Escherichia colirespectively. In addition, it contains multi-cloning site (MCS) and ompA promoter of R. anatipestifer forthe heterologous gene expression in R. anatipestifer. The stability assays showed that the vector pRESwas stable in R. anatipestifer.2. Construction of TonB1and TonB2gene deletion mutants of Riemerella anatipestifer CH3In this study, TonB1and TonB2genes were deleted by allelic exchange through recombinant suicidevector respectively, to generate the mutants CH3△TonB1and CH3△TonB2. Then TonB1ORF andTonB2ORF were ligated into pRES to generate the complemented plasmids pRES-TonB1andpRES-TonB2respectively. The two plasmids were introduced into the mutant CH3△TonB1and CH3△TonB2by conjugation respectively to generate the complemented strains of the mutants, and then thebiological characteristics of mutants CH3△TonB1and CH3△TonB2was studied. The results showedthat the growth of both mutant CH3△TonB1and CH3△TonB2was slower than that of wild type CH3.Although wild type CH3and CH3△TonB1and CH3△TonB2mutant did not grow in TSB containing200μM DIP, the addition of200μM of iron (III) chloride, holo-transferrin or hemoglobin to this culturerestored growth of wild type CH3and the complemented strains to the levels seen in TSB, but not forthe mutants. On the other hand, both TonB1and TonB2deletion in CH3strain could significantly reducethe biofilm formation and the adhesion and invasion capacities to Vero cells. Animal experimentsindicated that the median lethal dose (LD50) of mutants CH3△TonB1and CH3△TonB2to ducklingswas about450and90folds higher than that of wild type CH3. Additional analysis indicated thatbacterial loadings of blood in both CH3△TonB1and CH3△TonB2-infected ducklings were decreased significantly than those in CH3infected ducklings. Thus, our results showed that TonB1and TonB2were involved in iron acquisition and required for optimal bacterial virulence.