Energy-dependent changes in the gonococcal transferrin receptor

The pathogenic Neisseria spp. are capable of iron utilization from host iron-binding proteins including transferrin and lactoferrin. Transferrin iron utilization is an energy-dependent, receptor-mediated event in which two identified transferrin-binding proteins participate. One of these proteins, TbpA, is homologous to the TonB-dependent family of outer membrane receptors that are required for high-affinity uptake of vitamin B₁₂ and ferric siderophores. The 'TonB box' is a conserved domain near the amino-terminus of these proteins that has been implicated in interaction with TonB. Interaction between a periplasmic domain of TonB and the TonB box allows energy transduction to occur from the cytoplasmic membrane to the energy-dependent receptor in the outer membrane. We created a TonB box mutant of gonococcal TbpA and demonstrated that its binding and protease accessibility characteristics were indistinguishable from those of gonococcal Ton system mutants. The protease exposure of the second transferrin-binding protein, TbpB, was affected by the energization of TbpA, consistent with an interaction between these proteins. TbpB expressed by the de-energized mutants was readily accessible to protease, similar to TbpB expressed in the absence of TbpA. The de-energized mutants exhibited a marked decrease in transferrin diffusion rate, suggesting that receptor energization was necessary for ligand release. We propose a model to explain the observed Ton-dependent changes in the binding parameters and exposures of TbpA and TbpB.     

Gonococcal transferrin-binding protein 1 is required for transferrin utilization and is homologous to TonB-dependent outer membrane receptors.

The pathogenic Neisseria species are capable of utilizing transferrin as their sole source of iron. A neisserial transferrin receptor has been identified and its characteristics defined; however, the biochemical identities of proteins which are required for transferrin receptor function have not yet been determined. We identified two iron-repressible transferrin-binding proteins in Neisseria gonorrhoeae, TBP1 and TBP2. Two approaches were taken to clone genes required for gonococcal transferrin receptor function. First, polyclonal antiserum raised against TBP1 was used to identify clones expressing TBP1 epitopes. Second, a wild-type gene copy was cloned that repaired the defect in a transferrin receptor function (trf) mutant. The clones obtained by these two approaches were shown to overlap by DNA sequencing. Transposon mutagenesis of both clones and recombination of mutagenized fragments into the gonococcal chromosome generated mutants that showed reduced binding of transferrin to whole cells and that were incapable of growth on transferrin. No TBP1 was produced in these mutants, but TBP2 expression was normal. The DNA sequence of the gene encoding gonococcal TBP1 (tbpA) predicted a protein sequence homologous to the Escherichia coli and Pseudomonas putida TonB-dependent outer membrane receptors. Thus, both the function and the predicted protein sequence of TBP1 were consistent with this protein serving as a transferrin receptor.     

Binding and surface exposure characteristics of the gonococcal transferrin receptor are dependent on both transferrin-binding proteins.

Neisseria gonorrhoeae is capable of iron utilization from human transferrin in a receptor-mediated event. Transferrin-binding protein 1 (Tbp1) and Tbp2 have been implicated in transferrin receptor function, but their specific roles in transferrin binding and transferrin iron utilization have not yet been defined. We utilized specific gonococcal mutants lacking Tbp1 or Tbp2 to assess the relative transferrin-binding properties of each protein independently of the other. The apparent affinities of the wild-type transferrin receptor and of Tbp1 and Tbp2 individually were much higher than previously estimated for the gonococcal receptor and similar to the estimates for the mammalian transferrin receptor. The binding parameters of both of the mutants were distinct from those of the parent, which expressed two transferrin-binding sites. Tbp2 discriminated between ferrated transferrin and apotransferrin, while Tbp1 did not. Results of transferrin-binding affinity purification, and protease accessibility experiments were consistent with the hypothesis that Tbp1 and Tbp2 interact in the wild-type strain, although both proteins were capable of binding to transferrin independently when separated in the mutants. The presence of Tbp1 partially protected Tbp2 from trypsin proteolysis, and Tbp2 also protected Tbp1 from trypsin exposure. Addition of transferrin to wild-type but not mutant cells protected Tbp1 from trypsin but increased the trypsin susceptibility of Tbp2. These observations indicate that Tbp1 and Tbp2 function together in the wild-type strain to evoke binding conformations that are distinct from those expressed by the mutants lacking either protein.     

A dynamic model of the meningococcal transferrin receptor.

Iron is an essential nutrient for all organisms and consequently, the ability to bind transferrin and sequester iron from his source constitutes a distinct advantage to a blood-borne bacterial pathogen. Levels of free iron are strictly limited in human serum, largely through the action of the iron-binding protein transferrin. The acquisition of trasferrin-iron is coincident with pathogenicity among Neisseria species and a limited number of other pathogens of human and veterinary significance. In Neisseria meningitidis, transferrin binding relies on two co-expressed, outer membrane proteins distinct in aspects of both structure and function. These proteins are independently and simultaneously capable of binding human transferrin and both are required for the optimal uptake of iron from this source. It has been established that transferrin-binding proteins (designated TbpA and TbpB) form a discrete, specific complex which may be composed of a transmembrane species (composed of the TbpA dimer) associated with a single surface-exposed lipoprotein (TbpB). This more exposed protein is capable of selectively binding iron-saturated transferrin and the receptor complex has ligand-binding properties which are distinct from either of its components. Previous in vivo analyses of N. gonorrhoeae, which utilizes a closely related transferrin-iron uptake system, indicated that this receptor exists in several conformations influenced in part by the presence (or absence) of transferrin.Here we propose a dynamic model of the meningococcal transferrin receptor which is fully consistent with the current data concerning this subject. We suggest that TbpB serves as the initial binding site for iron-saturated transferrin and brings this ligand close to the associated transmembrane dimer, enabling additional binding events and orientating transferrin over the dual TbpA pores. The antagonistic association of these receptor proteins with a single ligand molecule may also induce conformational change in transferrin, thereby favouring the release of iron. As, in vivo, transferrin may have iron in one or both lobes, this dynamic molecular arrangement would enable iron uptake from either iron-binding site. In addition, the predicted molecular dimensions of the putative TbpA dimer and hTf are fully consistent with these proposals. Given the diverse data used in the formulation of this model and the consistent characteristics of transferrin binding among several significant Gram-negative pathogens, we speculate that such receptor-ligand interactions may be, at least in part, conserved between species. Consequently, this model may be applicable to bacteria other than N. meningitidis.     

Peptide-peptide interactions between human transferrin and transferrin-binding protein B from Moraxella catarrhalis

Transferrin-binding protein B (TbpB) is one component of a bipartite receptor in several gram-negative bacterial species that binds host transferrin and mediates the uptake of iron for growth. Transferrin and TbpB are both bilobed proteins, and the interaction between these proteins seems to involve similar lobe-lobe interactions. Synthetic overlapping peptide libraries representing the N lobe of TbpB from Moraxella catarrhalis were prepared and probed with labeled human transferrin. Transferrin-binding peptides were localized to six different regions of the TbpB N lobe, and reciprocal experiments identified six different regions of the C lobe of transferrin that bound TbpB. Truncations of the N lobe of TbpB that sequentially removed each transferrin-binding determinant were used to probe an overlapping peptide library of the C lobe of human transferrin. The removal of each TbpB N-lobe transferrin-binding determinant resulted in a loss of reactivity with peptides from the synthetic peptide library representing the C lobe of transferrin. Thus, individual peptide-peptide interactions between ligand and receptor were identified. A structural model of human transferrin was used to map surface regions capable of binding to TbpB.     

Identification of sequences in human transferrin that bind to the bacterial receptor protein, transferrin-binding protein B

Alignment of amino-acid sequences from the N-terminal and C-terminal halves of transferrin-binding protein B revealed an underlying bilobed nature with several regions of identity. Based on this analysis, purified recombinant fusion proteins of maltose-binding protein (Mbp) with intact TbpB, its N-terminal half or C-terminal half from the human pathogens Neisseria meningitidis and Moraxella catarrhalis were produced. Solid-phase binding assays and affinity isolation assays demonstrated that the N-terminal and C-terminal halves of TbpB could bind independently to human transferrin (hTf). A solid-phase overlapping synthetic peptide library representing the amino-acid sequence of hTf was probed with soluble, labelled Mbp-TbpB fusions to localize TbpB-binding regions on hTf. An essentially identical series of peptides from domains within both lobes of hTf was recognized by intact TbpB from both organisms, demonstrating a conserved TbpB-hTf interaction. Both halves of TbpB from N. meningitidis bound the same series of peptides, which included peptides from equivalent regions on the two hTf lobes, indicating that TbpB interacts with each lobe of hTf in a similar manner. Mapping of the peptide-binding regions on a molecular model of hTf revealed a series of nearly adjacent surface regions that nearly encircled each lobe. Binding studies with chimeric hTf/bTf transferrins demonstrated that regions in the C-lobe of hTf were preferentially recognized by the N-terminal half of TbpB. Collectively, these results provide evidence that TbpB consists of two lobes, each with distinct yet homologous Tf-binding regions.     

Both the full-length and the N-terminal domain of the meningococcal transferrin-binding protein B discriminate between human iron-loaded and apo-transferrin

We have readdressed the ability of the transferrin-binding protein B (TbpB) from Neisseria meningitidis to discriminate between the iron-loaded and the iron-free human transferrin (hTf) by using the BIAcore technology, a powerful experimental technique for the observation of direct interactions between a receptor and its ligands, without the use of labels. Recombinant full-length TbpB from five N. meningitidis strains were produced and purified from Escherichia coli as fusion proteins. They showed a preference for the binding to iron-loaded hTf. As for the full-length molecule, we have demonstrated that the minimal N-terminal hTf binding domain of meningococcal TbpB from B16B6 and M982 strains was able to discriminate between both hTf forms.     

Comparative analysis of the transferrin and lactoferrin binding proteins in the family Neisseriaceae

Intact cells of several bacterial species were tested for their ability to bind human transferrin and lactoferrin by a solid-phase binding assay using horseradish peroxidase conjugated transferrin and lactoferrin. The ability to bind lactoferrin was detected in all isolates of Neisseria and Branhamella catarrhalis but not in isolates of Escherichia coli or Pseudomonas aeruginosa. Transferrin-binding activity was similarly detected in most isolates of Neisseria and Branhamella but not in E. coli or P. aeruginosa. The expression of transferrin- and lactoferrin-binding activity was induced by addition of ethylenediamine di-o-phenylacetic acid and reversed by excess FeCl3, indicating regulation by the level of available iron in the medium. The transferrin receptor was specific for human transferrin and the lactoferrin receptor had a high degree of specificity for human lactoferrin in all species tested. The transferrin- and lactoferrin-binding proteins were identified after affinity isolation using biotinylated human transferrin or lactoferrin and streptavidin-agarose. The lactoferrin-binding protein was identified as a 105-kilodalton protein in all species tested. Affinity isolation with biotinylated transferrin yielded two or more proteins in all species tested. A high molecular mass protein was observed in all isolates, and was of similar size (approximately 98 kilodaltons) in all species of Neisseria but was larger (105 kilodaltons) in B. catarrhalis.     

Identification and characterization of the transferrin receptor from Neisseria meningitidis

Expression of the meningococcal transferrin receptor, detected by assay with human transferrin conjugated to peroxidase, was regulated by the level of iron in the medium. The transferrin receptor was identified by SDS-PAGE and Western blot analysis, as a 71,000 molecular weight iron-regulated outer membrane protein in Neisseria meningitidis B16B6. Growth studies with iron-deficient cells and competition binding experiments demonstrated that the meningococcal receptor was species-specific for human transferrin. Reciprocal competitive binding experiments and limited proteolysis of intact cells indicated that the transferrin and lactoferrin receptors are distinct.     

Neisseria meningitidis transferrin-binding protein 1 expressed in Escherichia coli is surface exposed and binds human transferrin

Abstract A gene library of Neisseria meningitidis B15 P1.16 DNA was established in λ Zap II and clones containing DNA encoding transferrin binding protein 1 (TBP-1) identified following hybridisation with a 63-bp DNA probe based on the codon assignment for the first 21 N-terminal amino acids of TBP-1. Sequencing of the cloned DNA demonstrated that all of the intergenic DNA (i.e. upstream of bp-1 running through to the 3' end of the transferrin-binding protein 2 gene) and approx. 15% of tbp-1 had been cloned. The complete gene was generated using a polymerase chain reaction, with the primer for the 3' end being based on tbp-A of N. gonorrhoeae , and the approx. 2.9-kb DNA product cloned into pGem-3Z. The expressed protein (approx. 100 kDa) reacted with antiserum to an N-terminal peptide of TBP-1. In addition, the native product was surface-expressed by Escherichia coli and bound human transferrin.     

Receptors for transferrin in pathogenic bacteria are specific for the host's protein

Transferrin receptors detected by a solid-phase binding assay were shown to be specific for the host's transferrin in the representative bacterial pathogens Neisseria meningitidis (human), Pasteurella haemolytica (bovine), and Actinobacillus pleuropneumoniae (porcine). Consistent with the receptor specificity, iron-deficient bacteria were only capable of utilizing transferrin from the host as a source of iron for growth.     

Specificity of the lactoferrin and transferrin receptors in Neisseria gonorrhoeae

Lactoferrin (LF) and transferrin (TF) are postulated to be important physiological sources of iron for Neisseria gonorrhoeae. A dot binding assay involving the use of gonococcal total membranes derived from cells grown in iron-limited conditions demonstrated the presence of separate receptors for LF and TF. The ligand and functional specificities of these receptors were examined in competition-binding and growth experiments. The results indicate that the LF and TF receptors are highly specific for the human protein, suggesting that this property may be partially responsible for conferring the human host specificity of N. gonorrhoeae.     

Iron piracy: acquisition of transferrin-bound iron by bacterial pathogens

The mechanism of iron utilization from transferrin has been most extensively characterized in the pathogenic Neisseria species and Haemophilus species. Two transferrin-binding proteins, Tbp1 and Tbp2, have been identified in these pathogens and are thought to be components of the transferrin receptor. Tbp1 appears to be an integral, TonB-dependent outer membrane protein while Tbp2, a lipoprotein, may be peripherally associated with the outer membrane. The relative contribution of each of these proteins to transferrin binding and utilization is discussed and a model of iron uptake from transferrin is presented. Sequence comparisons of the genes encoding neisserial transferrin-binding proteins suggest that they are probably under positive selection for variation and may have resulted from inter-species genetic exchange.     

Protein H — a surface protein of Streptococcus pyogenes with separate binding sites for lgG and albumin

Protein H, a molecule expressed at the surface of some strains of Streptococcus pyogenes, has affinity for the constant (lgGFc) region of immunoglobulin (lg) G. In absorption experiments with human plasma, protein H–sepharose could absorb not only lgG but also albumin from plasma. The affinity constant for the reaction between albumin and protein H was 7.8 × 10⁹M⁻¹, which is higher than the affinity between lgG and protein H (K_a= 1.6 × 10⁹ M⁻¹). Fragments of protein H were generated with deletion plasmids and polymerase chain reaction (PCR) technology. Using these fragments in various protein–protein interaction assays, the binding of albumin was mapped to three repeats (C1–C3) in the C-terminal half of protein H. On the albumin molecule, the binding site for protein H was found to overlap the site for protein G, another albumin- and lgGFc-binding bacterial surface protein. Aiso lgGFc-binding could be mapped with the protein H fragments and the region was found N-terminally of the C repeats. A synthetic peptide (25 amino acid residues long) based on a sequence in this region was shown to inhibit the binding of protein H to immobilized lgG or lgGFc. This sequence was not found in previously described lgGFc-binding proteins. However, two other cell surface proteins of S. pyogenes exhibited highly homologous regions. The results identify lgGFc- and albumin binding regions of protein H and further define and emphasize the convergent evolution among bacterial surface proteins interacting with human plasma proteins.     

Utilization of transferrin-bound iron by Listeria monocytogenes

Abstract It has been demonstrated that under iron-restricted conditions, Listeria monocytogenes can utilize iron-loaded transferrin (Tf) from a range of species as its sole source of iron for growth. Human transferrin conjugated to horseradish-peroxidase (HRP-Tf) bound directly to whole cells of L. monocytogenes . This binding was blocked by apotransferrin indicating that the receptor can bind transferrin in either the iron-bound or iron-free form. Transferrin-binding was not host specific because both bovine and equine transferrin inhibited the binding of HRP-conjugated human transferrin. SDS-PAGE and Western blotting of bacterial surface extracts revealed the presence of a transferrin-binding protein of approximately 126 kDa.     

Friend erythroleukemia cell membrane transferrin receptors

We have compared the uptake of transferrin by murine Friend erythroleukemia cells with the uptake of transferrin by murine reticulocytes. Friend cells which had been induced to erythroid differentiation by dimethyl sulfoxide took up transferrin in a manner qualitatively and quantitatively similar to the uptake of transferrin by reticulocytes, while uninduced Friend cells took up only negligible amounts of transferrin. Specific transferrin-binding activity could be demonstrated in detergent extracts of membranes from induced cells and this activity was isolated from membrane extracts by the use of antibody to transferrin. The isolated membrane component(s) with transferrin-binding activity migrated electrophoretically as a single protein on sodium dodecyl sulfate gels and had similar properties to a transferrin-binding protein isolated previously from reticulocytes.     

Preparation and analysis of isogenic mutants in the transferrin receptor protein genes, tbpA and tbpB, from Neisseria meningitidis

Isogenic mutants were constructed in the tbpA and tbpB genes from Neisseria meningitidis strain B16B6, which code for the transferrin receptor proteins, Tbp1 and Tbp2. Insertion mutants of the tbpA and tbpB genes were obtained by shuttle mutagenesis and by in vitro cassette mutagenesis, respectively. The Isogenic mutants were verified by Southern blot and Western blot analysis. Isogenic mutants deficient in Tbp1 or Tbp2 demonstrated a reduced transferrin binding activity in intact cells and total membranes but were incapable of utilizing transferrin iron for growth. Tbp1 could be isolated by affinity methods from the mutant lacking Tbp2 but isolation of Tbp2 from the mutant lacking Tbp1 required the presence of exogenous Tbp1.     

Binding of bovine transferrin by Pasteurella multocida serotype B:2,5, a strain which causes haemorrhagic septicaemia in buffalo and cattle

Abstract Pasteurella multocida serotype B:2,5, which causes haemorrhagic septicaemia in buffalo and cattle, was examined for the presence of transferrin-binding proteins. An 82-kDa iron-regulated outer membrane protein was found which specifically binds bovine transferrin. In contrast, P. multocida serotype B:3,4, associated with haemorrhagic septicaemia in feral ruminants, did not express transferrin-binding proteins. These rsults might indicate a role for transferrin binding in the pathogenesis of haemorrhagic septicaemia in cattle and buffalo.     

FbpA — A bacterial transferrin with more to offer

Background

Gram negative bacteria require iron for growth and virulence. It has been shown that certain pathogenic bacteria such as Neisseria gonorrhoeae possess a periplasmic protein called ferric binding protein (FbpA), which is a node in the transport of iron from the cell exterior to the cytosol.

Scope of review

The relevant literature is reviewed which establishes the molecular mechanism of FbpA mediated iron transport across the periplasm to the inner membrane.

Major conclusions

Here we establish that FbpA may be considered a bacterial transferrin on structural and functional grounds. Data are presented which suggest a continuum whereby FbpA may be considered as a naked iron carrier, as well as a Fe–chelate carrier, and finally a member of the larger family of periplasmic binding proteins.

General significance

An investigation of the molecular mechanisms of action of FbpA as a member of the transferrin super family enhances our understanding of bacterial mechanisms for acquisition of the essential nutrient iron, as well as the modes of action of human transferrin, and may provide approaches to the control of pathogenic diseases. This article is part of a Special Issue entitled Transferrins: Molecular mechanisms of iron transport and disorders.     

The genes that encode the gonococcal transferrin binding proteins,TbpB and TbpA,are differentially regulated by MisR under iron‐replete and iron‐depleted conditions

Neisseria gonorrhoeae produces two transferrin binding proteins, TbpA and TbpB, which together enable efficient iron transport from human transferrin. We demonstrate that expression of the tbp genes is controlled by MisR, a response regulator in the two‐component regulatory system that also includes the sensor kinase MisS. The tbp genes were up‐regulated in the misR mutant under iron‐replete conditions but were conversely down‐regulated in the misR mutant under iron‐depleted conditions. The misR mutant was capable of transferrin‐iron uptake at only 50% of wild‐type levels, consistent with decreased tbp expression. We demonstrate that phosphorylated MisR specifically binds to the tbpBA promoter and that MisR interacts with five regions upstream of the tbpB start codon. These analyses confirm that MisR directly regulates tbpBA expression. The MisR binding sites in the gonococcus are only partially conserved in Neisseria meningitidis, which may explain why tbpBA was not MisR‐regulated in previous studies using this related pathogen. This is the first report of a trans‐acting protein factor other than Fur that can directly contribute to gonococcal tbpBA regulation.