Identification of discrete domains within gonococcal transferrin-binding protein A that are necessary for ligand binding and iron uptake functions

The availability of free iron in vivo is strictly limited, in part by the i ron-binding protein transferrin. The pathogenic Neisseria spp. can sequeste r iron from this protein, dependent upon two iron-repressible, transferrin- binding proteins (TbpA and TbpB). TbpA is a TonB-dependent, integral, outer membrane protein that may form a beta -barrel exposing multiple surface lo ops, some of which are likely to contain ligand-binding motifs. In this stu dy we propose a topological model of gonococcal TbpA and then test some of the hypotheses set forth by the model by individually deleting three putati ve loops (designated loops 4, 5, and 8). Each mutant TbpA could be expresse d without toxicity and was surface exposed as assessed by immunoblotting, t ransferrin binding, and protease accessibility. Deletion of loop 4 or loop 5 abolished transferrin binding to whole cells in solid- and liquid-phase a ssays, while deletion of loop 8 decreased the affinity of the receptor for transferrin without affecting the copy number. Strains expressing any of th e three mutated TbpAs were incapable of growth on transferrin as a sole iro n source. These data implicate putative loops 4 and 5 as critical determina nts for receptor function and transferrin-iron uptake by gonococcal TbpA. T he phenotype of the Delta L8TbpA mutant suggests that high-affinity ligand interaction is required for transferrin-iron internalization.