IDENTIFICATION OF HUMAN TRANSFERRIN-BINDING SITES WITHIN MENINGOCOCCAL TRANSFERRIN-BINDING PROTEIN-B

Transferrin-binding protein B (TbpB) from Neisseria meningitidis binds human transferrin (hTf) at the surface of the bacterial cell as part of the iron uptake process, To identify hTf binding sites within the m eningococcal TbpB, defined regions of the molecule were produced in Es cherichia coli by a translational fusion expression system and the abi lity of the recombinant proteins (rTbpB) to bind peroxidase-conjugated hTf was characterized by Western blot and dot blot assays, Both the N -terminal domain (amino acids [aa] 2 to 351) and the C-terminal domain (aa 352 to 691) were able to bind hTf, and by a peptide spot synthesi s approach, two and five hTf binding sites were identified in the N- a nd C-terminal domains, respectively. The hTf binding activity of three rTbpB deletion variants constructed within the central region (aa 346 to 543) highlighted the importance of a specific peptide (aa 377 to 3 94) in the ligand interaction, Taken together, the results indicated t hat the N- and C-terminal domains bound hTf approximately 10 and 1000 times less, respectively, than the full-length rTbpB (aa 2 to 691), wh ile the central region (aa 346 to 543) had a binding avidity in the sa me order of magnitude as the C-terminal domain, In contrast with the h Tf binding in the N-terminal domain, which was mediated by conformatio nal epitopes, linear determinants seemed to be involved in the hTf bin ding in the C-terminal domain, The host specificity for transferrin ap peared to be mediated by the N-terminal domain of the meningococcal rT bpB rather than the C-terminal domain, since we report that murine Tf binds to the C-terminal domain, Antisera raised to both N- and C-termi nal domains were bactericidal for the parent strain, indicating that b oth domains are accessible at the bacterial surface, We have thus iden tified hTf binding sites within each domain of the TbpB from N. mening itidis and propose that the N- and C-terminal domains together contrib ute to the efficient binding of TbpB to hTf with their respective affi nities and specificities for determinants of their ligand.