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 ␤-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.

Candidate vaginal microbicides with activity against Chlamydia trachomatis and Neisseriagonorrhoeae

“…This pathogen also requires iron for growth and has multiple mechanisms for acquiring iron, including high-affinity receptors for both transferrin and lactoferrin [14,32,33]. We attempted to determine whether natural and laboratory strains lacking lactoferrin or transferrin receptors were inhibited by the INPs and, if so, could the inhibition be reversed by the iron-rich form of either lactoferrin or transferrin.…”

Microbial Iron Acquisition: Marine and Terrestrial Siderophores

“…whereas the pathogen Actinobacillus (Haemophilus) pleuropneumoniae is able to bind and use pig, but not human, transferrin as an iron source 39,41. Two proteins, TbpA and TbpB work together as the functional transferrin receptor 42. TbpA serves as the outer membrane pore through which iron, removed from the host's transferrin, enters the bacterial cell periplasm.…”

Beyond the Crystal Structure: Insight into the Function and Vaccine Potential of TbpA Expressed by Neisseria gonorrhoeae

“…Tf-iron uptake assays were performed as described previously (32)(33)(34)(35). Briefly, apo-human Tf (Sigma) was saturated to 20% with 55 Fe (Perkin-Elmer).…”