Construction Of TbdR1Deletion Mutant Strain Fariemerella Anatipestifer CH3

Riemerella anatipestifer (RA) infection, also called duck infectious serositis, is a contagious disease of domestic ducks, geese, turkeys and some other domestic and wild birds. RA infection had caused serious economic losses in the duck industry worldwide. However, little is known about the molecular basis of its pathogenesis and the virulence factors involved. To date, four full R. anatipestifer genomic sequences have been submitted to the GenBank database and31TonB-dependent outer membrane receptors, which may play critical roles in host-bacteria interactions, were predicted for R. anatipestifer strain GSM15868. In previous study, TonB-dependent receptor (TbdR1) was found to be a cross immunogenic antigen among R. anatipestifer serotypes1,2and10, but the biological functions of TbdR1in R. anatipestifer remain unclear.In this study, tbdR1gene distribution and the amino acids homology from different R. anatipestifer strains were first analyzed. The results showed that all the tested strains carried tbdRl gene and the TbdRl from different R. anatipestifer strains shared98.2%-100%homology. The highly conserved tbdRl gene suggested that the protein may have important biological functions.The tbdRl gene of the R. anatipestifer CH3strain was deleted by allelic exchange through a recombinant suicide plasmid pDS132-LSR, which replaced a2726-bp internal fragment of the gene with a1119-bp spectinomycin resistance cassette. The mutant strain was designated CH3ΔtbdR1. Then a complemented strain cCH3AtbdRl of the mutant CH3ΔtbdR1was constructed by transferring a recombinant E coli-R. anatipestifer shuttle plasmid, in which an intact tbdRl gene and R. anatipestifer ompA promoter region were cloning into the E. coli-R. anatipestifer shuttle vector pRES, into the mutant.To determine whether TbdR1plays a role in iron acquisition of the R. anatipestifer strain CH3, strain CH3AtbdRl growth in TSB supplemented with2’-2’-dipyridyl (DIP), an iron chelator, was measured. The results showed that the growth of the CH3ΔtbdR1mutant and wild type CH3was almost nonexistent in TSB supplemented with50μmol/L DIP and200μmol/L DIP respectively. Furthermore, the addition of200μmol/L of iron (Ⅲ) chloride to this culture restored growth of wild type CH3and the complemented strain cCE3ΔtbdR1under TSB containing200μmol/L DIP to the levels seen in TSB. Therefore, The results revealed that TbdRl was involved in iron acquisition of R. anatipestifer strain CH3. Iron utilization assay showed that the growth defect of CH3under200μmol/LDIP could be rescued by1μmol/L of hemin, demonstrating that R. anatipestifer CH3can utilize hemin as the sole iron source. On the other hand, our results showed that tbdRl deletion significantly reduced biofilm formation and adhesion to and invasion of Vero cells.Animal experiments indicated that the median lethal dose of the CH3ΔtbdR1mutant in ducklings was about45-fold higher than that of the wild-type CH3strain. Additional analysis indicated that bacterial loads in blood, liver, and brain tissues in CH3ΔtbdR1-infected ducklings were decreased significantly compared to those in wild-type CH3-infected ducklings. Thus, the results in this study demonstrated that TbdRl was involved in hemin iron acquisition and necessary for optimal bacterial virulence.