title: Identification of TbpA residues required for transferrin-iron utilization by Neisseria gonorrhoeae.
abstract: Neisseria gonorrhoeae requires iron for survival in the human host and therefore expresses high-affinity receptors for iron acquisition from host iron-binding proteins. The gonococcal transferrin-iron uptake system is composed of two transferrin binding proteins, TbpA and TbpB. TbpA is a TonB-dependent, outer membrane transporter critical for iron acquisition, while TbpB is a surface-exposed lipoprotein that increases the efficiency of iron uptake. The precise mechanism by which TbpA mediates iron acquisition has not been elucidated; however, the process is distinct from those of characterized siderophore transporters. Similar to these TonB-dependent transporters, TbpA is proposed to have two distinct domains, a beta-barrel and a plug domain. We hypothesize that the TbpA plug coordinates iron and therefore potentially functions in multiple steps of transferrin-mediated iron acquisition. To test this hypothesis, we targeted a conserved motif within the TbpA plug domain and generated single, double, and triple alanine substitution mutants. Mutagenized TbpAs were expressed on the gonococcal cell surface and maintained wild-type transferrin binding affinity. Single alanine substitution mutants internalized iron at wild-type levels, while the double and triple mutants showed a significant decrease in iron uptake. Moreover, the triple alanine substitution mutant was unable to grow on transferrin as a sole iron source; however, expression of TbpB compensated for this defect. These data indicate that the conserved motif between residues 120 and 122 of the TbpA plug domain is critical for transferrin-iron utilization, suggesting that this region plays a role in iron acquisition that is shared by both TbpA and TbpB.
authors.affiliation: Department of Microbiology and Immunology, Virginia Commonwealth University Medical Center, Richmond, VA 23298-0678, USA.
authors.forename: Jennifer M
authors.initials: JM
authors.lastname: Noto
authors.forename: Cynthia Nau
authors.initials: CN
authors.lastname: Cornelissen
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D007501:Iron / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009344:Neisseria gonorrhoeae / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary / Q000235:genetics; D016415:Sequence Alignment; D014168:Transferrin / Q000378:metabolism*; D033881:Transferrin-Binding Protein A / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D014168:Transferrin; D033881:Transferrin-Binding Protein A; D007501:Iron