Identification of transferrin-binding domains in TbpB expressed by Neisseria gonorrhoeae.
The transferrin iron acquisition system of Neisseria gonorrhoeae is necessary for iron uptake from transferrin in the human host and requires the participation of two distinct proteins: TbpA and TbpB. TbpA is a TonB-dependent outer membrane transporter responsible for the transport of iron into the cell. TbpB is a lipid-modified protein, for which a precise role in receptor function has not yet been elucidated. These receptor complex proteins show promise as vaccine candidates; therefore, it is important to identify surface-exposed regions of the proteins required for wild-type functions. In this study we examined TbpB, which has been reported to be surface exposed in its entirety; however, this hypothesis has never been tested experimentally. We placed the hemagglutinin (HA) epitope into TbpB with the dual purpose of examining the surface exposure of particular epitopes as well as their impact on receptor function. Nine insertion mutants were created, placing the epitope downstream of the signal peptidase II cleavage site. We report that the HA epitope is surface accessible in all mutants, indicating that the full-length TbpB is completely surface exposed. By expressing the TbpB-HA fusion proteins in N. gonorrhoeae, we were able to examine the impact of each insertion on the function of TbpB and the transferrin acquisition process. We propose that TbpB is comprised of two transferrin-binding-competent lobes, both of which are critical for efficient iron uptake from human transferrin.
DeRocco
Amanda J
AJ
Department of Microbiology and Immunology, Virginia Commonwealth University Medical Center, Richmond, VA 23298-0678, USA.
Cornelissen
Cynthia Nau
CN
D000595:Amino Acid Sequence; D000939:Epitopes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D006388:Hemagglutinins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007501:Iron / Q000378:metabolism; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009344:Neisseria gonorrhoeae / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014168:Transferrin / Q000378:metabolism*; D033882:Transferrin-Binding Protein B / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D000939:Epitopes; D006388:Hemagglutinins; D011993:Recombinant Fusion Proteins; D014168:Transferrin; D033882:Transferrin-Binding Protein B; D007501:Iron