Site-directed mutagenesis of transferrin binding protein B enhances immunogenicity and protective efficacy against Bibersteinia trehalosi

Bibersteinia trehalosi (B. trehalosi) is a gram-negative bacterial pathogen that causes respiratory diseases in goats. Its surface transferrin receptor, transferrin binding protein B(TbpB), is an ideal vaccine target as its expression is essential for colonization of the upper respiratory tract. Previous studies have shown that mutations that reduce affinity of TbpB toward transferrin can significantly enhance the immunogenicity of TbpB. In this study, amino acid residues that are key to the function of TbpB were determined using 3-D modelling of the protein structure. The key residues were mutated to alanine via site-directed mutagenesis, and a solid-phase binding assay was carried out to assess their binding activities to goat transferrin. Mice were immunized thrice with TbpB antigens. TbpB-specific antibody titers and bactericidal activity were evaluated. The mice were challenged with the pathogen after immunization, and histopathological analysis was performed after euthanasia. The results showed that the L156A mutant TbpB exhibited the highest serum antibody levels and maintained a 48.11 % bactericidal rate even after 12 h of co-incubation with B. trehalosi. Histopathological examination revealed that mice immunized with L156A TbpB mutant had the mildest pulmonary lesions. Altogether, these findings suggest that the leucine residue at position 156 of B. trehalose TbpB is crucial for transferrin binding and induced a stronger immune response and provided better protection.