Characterization of potential interactions between transferrin binding proteins in Neisseria gonorrhoeae

摘要

Neisseria gonorrhoeae requires iron for survival and establishment of infection in the human host. Pathogenic Neisseriae have evolved a repertoire of high-affinity iron acquisition systems to facilitate iron uptake in the human host. This requires specific outer membrane receptors and energy-harnessing cytoplasmic membrane proteins. The transferrin receptor proteins of Neisseria gonorrhoeae are necessary for iron uptake from transferrin in the host. The iron uptake system consists of two transferrin binding proteins, (Tbp) A and B. TbpA is an integral outer membrane, TonB-dependent transporter that forms the pore for iron internalization. TbpB is a surface exposed lipoprotein that makes the iron internalization process more efficient. TbpA is proposed to consist of two distinct domains: a β barrel and an N-terminal plug domain. Previous studies have shown that the EIEYE sequence in the TbpA plug domain plays an important role in iron internalization. We undertook a collaborative project to test the hypothesis that the conserved EIEYE sequence in the wild-type TbpA plug binds Fe3+ during the outer membrane iron transport process. CD spectra analysis and fluorescence emission titration spectra of purified recombinant wildtype and mutated plug proteins revealed that Fe3+ is sequestered by the wildtype TbpA plug protein, unlike the mutated plug protein. Modeling data with the wild-type plug predicts the EIEYE sequence is part of a flexible loop structure and acts as an Fe3+ binding site. Characterization of the Tbps constituting the gonococcal receptor is important to understanding how the gonococcus survives within its host. TbpA and TbpB act together to acquire iron from human transferrin. We hypothesize that the presence of TbpA impacts the exposure or conformation of TbpB. In this study, we have utilized photoactivable cross-linkers to assess the effect of TbpA on TbpB in live gonococcal cells and studied it in presence of ligand and TonB derived energy. We employed insertion mutants, in which TbpA and TbpB contained the hemagglutinin (HA) epitope tag, to probe for impact of TbpA on TbpB. Our results demonstrate that photo-cross-linking altered TbpB size and migration and was dependent on the presence of TbpA. HA epitope insertion mutants in surface exposed loops of TbpA and TbpB did not impact the mobility of cross-linked TbpB. Addition of human transferrin to the de-energized mutant caused a change in TbpB migration after cross-linking. This result indicates that when ligand is bound tightly and irreversibly to de-energized TbpA, the surface accessibility and perhaps conformation of TbpB is altered and TbpA does not interact with TbpB. Our findings were confirmed with recent structural studies of TbpA-TbpB-ligand triple complex, which illustrate that Tbps bind ligand through unique, non-overlapping binding sites such that TbpA and TbpB do not interact. TbpB is not an essential member of transferrin-iron acquisition pathway. It is surface exposed and tethered to the outer membrane by a lipid moiety. The role of TbpB has not been clearly outlined in the transferrin iron acquisition system. The last objective of this study was to look at the significance of two specific conserved regions of TbpB and its importance in transferrin iron utilization and TbpA-TbpB interaction. Using site-directed mutagenesis we created two mutants, in the first mutant the conserved lipobox of TbpB was replaced with a signal I peptidase cleavage site, and the second mutant contained a deletion of the conserved poly glycine residue stretch, immediately downstream of the lipobox. Our results indicate that lipobox is required for lipidation of TbpB, both the mutants were impaired for transferrin-iron utilization, and neither of the mutations altered TbpA-TbpB interaction. Overall, these studies help elucidate the functional importance of the specific regions in TbpA and TbpB in Neisseria gonorrhoeae, thereby adding to our understanding of the process of iron acquisition through the transferrin binding proteins.