Abstract
Neisseria meningitidis, a causative agent of bacterial meningitis and septicemia, obtains transferrin-bound iron by expressing two outer membrane-located transferrin-binding proteins, TbpA and TbpB. A novel system was developed to investigate the interaction between Tbps and human transferrin. Copurified TbpA-TbpB, recombined TbpA-TbpB, and individual TbpA and TbpB were reconstituted into liposomes and fused onto an HPA chip (BIAcore). All preparations formed stable monolayers, which, with the exception of TbpB, could be regenerated by removing bound transferrin. The ligand binding properties of these monolayers were characterized with surface plasmon resonance and shown to be specific for human transferrin. Kinetic data for diferric human transferrin binding showed that recombined TbpA-TbpB had K(a) and K(d) values similar to those of copurified TbpA-TbpB. Individual TbpA and TbpB also displayed K(a) values similar to those of copurified TbpA-TbpB, but their K(d) values were one order of magnitude higher. Chemical cross-linking studies revealed that TbpA and TbpB, in the absence of human transferrin, formed large complexes with TbpA as the predominant species. Upon human transferrin binding, a complex was formed with a molecular mass corresponding to that of a TbpB-human transferrin heterodimer as well as a higher-molecular-mass complex of this heterodimer cross-linked to TbpA. This indicates that TbpA and TbpB form a functional meningococcal receptor complex in which there is cooperativity in the human transferrin binding kinetics. However, iron loss from the diferric human transferrin-TbpA-TbpB complex was not greater than that from human transferrin alone, suggesting that additional meningococcal transport components are involved in the process of iron removal.