Identification of human transferrin-binding sites within meningococcal transferrin-binding protein B

Abstract

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 meningococcal TbpB, defined regions of the molecule were produced in Escherichia coli by a translational fusion expression system and the ability 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 synthesis approach, two and five hTf binding sites were identified in the N-and 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 394) in the ligand interaction. Taken together, the results indicated that the N-and C-terminal domains bound hTf approximately 10 and 1000 times less, respectively, than the full-length rTbpB (aa 2 to 691), while the central region (aa 346 to 543) had a binding avidity in the same order of magnitude as the C-terminal domain. In contrast with the hTf binding in the N-terminal domain, which was mediated by conformational epitopes, linear determinants seemed to be involved in the hTf binding in the C-terminal domain. The host specificity for transferrin appeared to be mediated by the N-terminal domain of the meningococcal rTbpB 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-terminal domains were bactericidal for the parent strain, indicating that both domains are accessible at the bacterial surface. We have thus identified hTf binding sites within each domain of the TbpB from N. meningitidis and propose that the Nand C-terminal domains together contribute to the efficient binding of TbpB to hTf with their respective affinities and specificities for determinants of their ligand.

Iron Transport Systems in Neisseria meningitidis

“…As mentioned previously, TbpB is a bilobed protein and both halves are capable of binding transferrin (28,126,166). The interactions of each lobe of TbpB with transferrin appear to be identical, since experiments demonstrate that the same peptide derivatives of transferrin are recognized by both binding domains (129).…”

“…The crystal structure of TbpB has not yet been determined, but it is predicted to be a bilobed protein like its transferrin substrate since it contains two transferrin-binding domains and has internal amino acid repeat regions (94,126). A high-affinity transferrin-binding domain which remains stable after exposure to heat and sodium dodecyl sulfate is located in the amino-terminal domain, and a second transferrin-binding site is located in the carboxyl-terminal domain (45,126,166). A unique feature of LbpB, not shared by TbpB, is the presence of two long stretches of acidic amino acid residues.…”

Host Specificity of Bacterial Pathogens

“…One strategy for iron acquisition is to strip iron from the human iron-binding protein transferrin using transferrin-binding protein B (TbpB) (reviewed in Schryvers and Stojiljkovic 1999). This process is host-specific, because TbpB of N. meningitidis binds human transferrin but not murine transferrin (Renauld-Mongenie et al 1997). N. meningitidis can persist at least 2 d in intraperitoneally challenged transgenic mice expressing human transferrin, whereas bacteria are rapidly cleared from human transferrin transgenic mice challenged with a N. meningitidis tbpB mutant or from wild-type mice challenged with wild-type N. meningitidis (Zarantonelli et al 2007).…”

Peptide-Peptide Interactions between Human Transferrin and Transferrin-Binding Protein B from Moraxella catarrhalis

“…Prior studies with overlapping peptides libraries of TbpB proteins from A. pleuropneumoniae (38) and N. meningitidis (33) led to the identification of a smaller number of Tf-binding peptides. Three Tf-binding peptides were identified in the A. pleuropneumoniae TbpB N lobe, and seven peptides were identified as positive in a library representing the intact TbpB from N. meningitidis, two from the N lobe and five from the C lobe.…”

“…TbpB truncations were generated by PCR-based cloning into the pMal expression system (New England BioLabs) as described previously (33). Essentially, amplified tbpB gene segments were subcloned into the pMal-c2 expression vector to generate in-frame fusions with the malE gene.…”