Iron, an essential nutrient for most microorganisms, is sequestered in the host by iron-binding proteins, such as lactoferrin and transferrin. Neisseria gonorrhoeae utilizes transferrin as an iron source and its iron acquisition system is composed of two transferrin binding proteins: TbpA and TbpB. TbpA is a TonB-dependent, outer membrane transporter and TbpB is a bilobed, surface exposed lipoprotein. TbpB can distinguish between apo- and holo-transferrin which is involved in increasing the efficiency of iron uptake through the Tbps. It is anchored in the outer leaflet of the outer membrane by its lipid moiety. We aimed to identify the mechanism of TbpB export to the cell surface. No conclusions could be made from our results but we identified a protein that could potentially be involved in lipoprotein transport. TbpB is a bilobed protein with controversy over which lobe is involved in transferrin binding. In this study, we constructed a C-lobe deletion of TbpB to determine the role of the C-lobe in TbpB function. Results presented here showed deletion of the C-lobe caused degradation of TbpB and the minimal protein expressed was unable to bind transferrin both in vitro and in vivo. We were also able to demonstrate the TbpB C-lobe deletion is able to support limited transferrin-mediated growth, indicating some function of TbpB is retained. These results confirmed that both lobes are necessary for wild-type function of TbpB.

The transferrin iron acquisition system of Neisseria consists of two dissimilar proteins, transferrin binding protein A and B (TbpA and TbpB). TbpA and TbpB both specifically and independently bind human transferrin (Tf). TbpA is a TonB-dependent transporter, expression of which is necessary for Tf iron acquisition. In contrast, the lipoprotein TbpB is not necessary for iron internalization; however it makes this process more efficient. The role of TbpB in the transferrin iron acquisition system has not been completely elucidated. It has been suggested that TbpB is entirely surface exposed and tethered to the outer membrane by its lipid moiety. We inserted the hemagluttinin antigen (HA) epitope into TbpB in an effort to examine surface accessible and functional domains of the lipoprotein. We determined that TbpB was entirely surface exposed from just beyond the mature N-terminus. It was previously reported that the N- and C-terminus of TbpB independently bind Tf. HA epitope analysis defined both the N-terminal and C-terminal binding domains. TbpB was previously reported to play an important role in the release of Tf from the receptor. We established that TbpB exhibited a biphasic dissociation pattern; a C-terminal rapid release followed by a slower N-terminal release. These results suggested that the C-terminus plays a role in ligand turnover of the wild-type receptor. Little is known about the transport of TbpB to the outer membrane. In an attempt to identify the signals/mechanisms required for TbpB localization, the signal sequence of the protein was altered. In the absence of lipid modification, TbpB remained associated with the cell, localized to the periplasm. We also noted that internal cysteine residues were not critical for TbpB localization. Our results suggested that TbpB was transported by a lipoprotein-specific mechanism. Additionally, we demonstrated the major outer membrane secretin, PilQ, was not necessary for proper localization of TbpB. The mechanism responsible for this process remains elusive. This body of work represents the first comprehensive study of TbpB topology and function, utilizing the lipoprotein expressed in its native membrane. These results may translate to other, similar lipoprotein receptors of the pathogenic Neisseria, helping to shed light on these poorly understood proteins.