Crystal structure of the N-lobe of lactoferrin binding protein B from Moraxella bovis1 1This paper is an invited article as a result of a presentation at the International Lactoferrin Conference held in Mazatlan, Mexico (May 2011), and has undergone the Journal’s usual peer review process.

Lactoferrin (Lf) is a bi-lobed, iron-binding protein found on mucosal surfaces and at sites of inflammation. Gram-negative pathogens from the Neisseriaceae and Moraxellaceae families are capable of using Lf as a source of iron for growth through a process mediated by a bacterial surface receptor that directly binds host Lf. This receptor consists of an integral outer membrane protein, lactoferrin binding protein A (LbpA), and a surface lipoprotein, lactoferrin binding protein B (LbpB). The N-lobe of the homologous transferrin binding protein B, TbpB, has been shown to facilitate transferrin binding in the process of iron acquisition. Currently there is little known about the role of LbpB in iron acquisition or how Lf interacts with the bacterial receptor proteins. No structural information on any LbpB or domain is available. In this study, we express and purify from Escherichia coli the full-length LbpB and the N-lobe of LbpB from the bovine pathogen Moraxella bovis for crystallization trials. We demonstrate that M. bovis LbpB binds to bovine but not human Lf. We also report the crystal structure of the N-terminal lobe of LbpB from M. bovis and compare it with the published structures of TbpB to speculate on the process of Lf mediated iron acquisition.

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The role of lactoferrin binding protein B in mediating protection against human lactoferricin1This article is part of a Special Issue entitled Lactoferrin and has undergone the Journal's usual peer review process.

Biochemistry and Cell Biology ◽

10.1139/o11-074 ◽

2012 ◽

Vol 90 (3) ◽

pp. 417-423 ◽

Cited By ~ 15

Author(s):

Ari Morgenthau ◽

Margaret Livingstone ◽

Paul Adamiak ◽

Anthony B. Schryvers

Keyword(s):

Peer Review Process ◽

Binding Protein ◽

Iron Acquisition ◽

Protein A ◽

Human Lactoferrin ◽

Cationic Antimicrobial Peptide ◽

Iron Source ◽

Iron Deficient ◽

Protein B

Bacteria that inhabit the mucosal surfaces of the respiratory and genitourinary tracts of mammals encounter an iron-deficient environment because of iron sequestration by the host iron-binding proteins transferrin and lactoferrin. Lactoferrin is also present in high concentrations at sites of inflammation where the cationic, antimicrobial peptide lactoferricin is produced by proteolysis of lactoferrin. Several Gram-negative pathogens express a lactoferrin receptor that enables the bacteria to use lactoferrin as an iron source. The receptor is composed of an integral membrane protein, lactoferrin binding protein A (LbpA), and a membrane-bound lipoprotein, lactoferrin binding protein B (LbpB). LbpA is essential for growth with lactoferrin as the sole iron source, whereas the role of LbpB in iron acquisition is not yet known. In this study, we demonstrate that LbpB from 2 different species is capable of providing protection against the killing activity of a human lactoferrin-derived peptide. We investigated the prevalence of lactoferrin receptors in bacteria and examined their sequence diversity. We propose that the protection against the cationic antimicrobial human lactoferrin-derived peptide is associated with clusters of negatively charged amino acids in the C-terminal lobe of LbpB that is a common feature of this protein.

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The structure of lactoferrin-binding protein B fromNeisseria meningitidissuggests roles in iron acquisition and neutralization of host defences

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x14019372 ◽

2014 ◽

Vol 70 (10) ◽

pp. 1312-1317 ◽

Cited By ~ 14

Author(s):

Cory L. Brooks ◽

Elena Arutyunova ◽

M. Joanne Lemieux

Keyword(s):

Binding Protein ◽

Iron Acquisition ◽

Protein A ◽

Transport Systems ◽

Cationic Peptide ◽

Moraxella Bovis ◽

Component Systems ◽

Two Component Systems ◽

Membrane Bound ◽

Protein B

Pathogens have evolved a range of mechanisms to acquire iron from the host during infection. Several Gram-negative pathogens including members of the generaNeisseriaandMoraxellahave evolved two-component systems that can extract iron from the host glycoproteins lactoferrin and transferrin. The homologous iron-transport systems consist of a membrane-bound transporter and an accessory lipoprotein. While the mechanism behind iron acquisition from transferrin is well understood, relatively little is known regarding how iron is extracted from lactoferrin. Here, the crystal structure of the N-terminal domain (N-lobe) of the accessory lipoprotein lactoferrin-binding protein B (LbpB) from the pathogenNeisseria meningitidisis reported. The structure is highly homologous to the previously determined structures of the accessory lipoprotein transferrin-binding protein B (TbpB) and LbpB from the bovine pathogenMoraxella bovis. Docking the LbpB structure with lactoferrin reveals extensive binding interactions with the N1 subdomain of lactoferrin. The nature of the interaction precludes apolactoferrin from binding LbpB, ensuring the specificity of iron-loaded lactoferrin. The specificity of LbpB safeguards proper delivery of iron-bound lactoferrin to the transporter lactoferrin-binding protein A (LbpA). The structure also reveals a possible secondary role for LbpB in protecting the bacteria from host defences. Following proteolytic digestion of lactoferrin, a cationic peptide derived from the N-terminus is released. This peptide, called lactoferricin, exhibits potent antimicrobial effects. The docked model of LbpB with lactoferrin reveals that LbpB interacts extensively with the N-terminal lactoferricin region. This may provide a venue for preventing the production of the peptide by proteolysis, or directly sequestering the peptide, protecting the bacteria from the toxic effects of lactoferricin.

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Transferrin binding protein B and Transferrin binding protein A 2 expand the transferrin recognition range of Histophilus somni

10.1101/730739 ◽

2019 ◽

Author(s):

Anastassia K. Pogoutse ◽

Trevor F. Moraes

Keyword(s):

Host Species ◽

Binding Protein ◽

Iron Acquisition ◽

Protein A ◽

Acquisition System ◽

Sheep And Goats ◽

Iron Acquisition System ◽

Histophilus Somni ◽

Bovine Transferrin ◽

Protein B

AbstractThe bacterial bipartite transferrin receptor is an iron acquisition system that is required for survival by several key human and animal pathogens. It consists of the TonB-dependent transporter Transferrin binding protein A (TbpA) and the surface lipoprotein Transferrin binding protein B (TbpB). Curiously, the Tbps are only found in host specific pathogens, and are themselves host specific, meaning that they will bind to the transferrin of their host species, but not to those of other animal species. While this phenomenon has long been established, neither the steps in the evolutionary process that led to this exquisite adaptation for the host, nor the steps that could alter it, are known. We sought to gain insight into these processes by studying Tbp specificity in Histophilus somni, a major pathogen of cattle. A past study showed that whole cells of H. somni specifically bind bovine transferrin, but not transferrin from sheep and goats, two bovids whose transferrins share 93% amino acid sequence identity with bovine transferrin. To our surprise, we found that H. somni can use sheep and goat transferrins as iron sources for growth, and that HsTbpB, but not HsTbpA, has detectable affinity for sheep and goat transferrins. Furthermore, a third transferrin binding protein, HsTbpA2, also showed affinity for sheep and goat transferrins. Our results show that H. somni TbpB and TbpA2 act to broaden the host transferrin recognition range of H. somni.ImportanceHost restricted pathogens infect a single host species or a narrow range of host species. Histophilus somni, a pathogen that incurs severe economic losses for the cattle industry, infects cattle, sheep, and goats, but not other mammals. The transferrin binding proteins, TbpA and TbpB, are thought to be a key iron acquisition system in H. somni, however, surprisingly, they were also shown to be cattle transferrin-specific. In our study we find that H. somni TbpB, and another little-studied Tbp, TbpA2, bind sheep and goat transferrins as well as bovine transferrin. Our results suggest that TbpA2 may have allowed for host range expansion, and provide a mechanism for how host specificity in Tbp containing pathogens can be altered.

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Steric and allosteric factors prevent simultaneous binding of transferrin-binding proteins A and B to transferrin

Biochemical Journal ◽

10.1042/bj20112133 ◽

2012 ◽

Vol 444 (2) ◽

pp. 189-197 ◽

Cited By ~ 4

Author(s):

Leslie P. Silva ◽

Rong-hua Yu ◽

Charles Calmettes ◽

Xue Yang ◽

Trevor F. Moraes ◽

...

Keyword(s):

Binding Site ◽

Binding Protein ◽

Iron Acquisition ◽

Protein A ◽

Structural Instability ◽

Gated Channel ◽

Surface Receptor ◽

Hydrogen Deuterium ◽

Study Support ◽

Transferrin Binding Proteins

The ability to acquire iron directly from host Tf (transferrin) is an adaptation common to important bacterial pathogens belonging to the Pasteurellaceae, Moraxellaceae and Neisseriaceae families. A surface receptor comprising an integral outer membrane protein, TbpA (Tf-binding protein A), and a surface-exposed lipoprotein, TbpB (Tf-binding protein B), mediates the iron acquisition process. TbpB is thought to extend from the cell surface for capture of Tf to initiate the process and deliver Tf to TbpA. TbpA functions as a gated channel for the passage of iron into the periplasm. In the present study we have mapped the effect of TbpA from Actinobacillus pleuropneumoniae on pTf (porcine Tf) using H/DX-MS (hydrogen/deuterium exchange coupled to MS) and compare it with a previously determined binding site for TbpB. The proposed TbpA footprint is adjacent to and potentially overlapping the TbpB-binding site, and induces a structural instability in the TbpB site. This suggests that simultaneous binding to pTf by both receptors would be hindered. We demonstrate that a recombinant TbpB lacking a portion of its anchor peptide is unable to form a stable ternary TbpA–pTf–TbpB complex. This truncated TbpB does not bind to a preformed Tf–TbpA complex, and TbpA removes pTf from a preformed Tf–TbpB complex. Thus the results of the present study support a model whereby TbpB ‘hands-off’ pTf to TbpA, which completes the iron removal and transport process.

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Analysis of the Immunological Responses to Transferrin and Lactoferrin Receptor Proteins fromMoraxella catarrhalis

Infection and Immunity ◽

10.1128/iai.67.8.3793-3799.1999 ◽

1999 ◽

Vol 67 (8) ◽

pp. 3793-3799 ◽

Cited By ~ 24

Author(s):

Rong-hua Yu ◽

Robert A. Bonnah ◽

Samuel Ainsworth ◽

Anthony B. Schryvers

Keyword(s):

Immune Response ◽

Solid Phase ◽

Binding Protein ◽

Iron Acquisition ◽

Polyacrylamide Gel Electrophoresis ◽

Protein A ◽

Sodium Dodecyl ◽

Receptor Proteins ◽

Convalescent Phase ◽

Protein B

ABSTRACT Moraxella catarrhalis expresses surface receptor proteins that specifically bind host transferrin (Tf) and lactoferrin (Lf) in the first step of the iron acquisition pathway. Acute- and convalescent-phase antisera from a series of patients with M. catarrhalis pulmonary infections were tested against Tf and Lf receptor proteins purified from the corresponding isolates. After the purified proteins had been separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting, we observed strong reactivity against Tf-binding protein B (TbpB; also called OMP1) and Lf-binding protein B (LbpB) but little or no reactivity against Tf-binding protein A (TbpA) or Lf-binding protein A (LbpA), using the convalescent-phase antisera. Considerable antigenic heterogeneity was observed when TbpBs and LbpBs isolated from different strains were tested with the convalescent-phase antisera. Comparison to the reactivity against electroblotted total cellular proteins revealed that the immune response against LbpB and TbpB constitutes a significant portion of the total detectable immune response to M. catarrhalis proteins. Preparations of affinity-isolated TbpA and LbpA reacted with convalescent-phase antisera in a solid-phase binding assay, but blocking with soluble TbpB, soluble LbpB, or extracts from an LbpA− mutant demonstrated that this reactivity was attributed to contaminants in the TbpA and LbpA preparations. These studies demonstrate the immunogenicity of M. catarrhalisTbpB and LbpB in humans and support their potential as vaccine candidates.

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The Crystal Structure ofHelicobacter pyloriCysteine-rich Protein B Reveals a Novel Fold for a Penicillin-binding Protein

Journal of Biological Chemistry ◽

10.1074/jbc.m108993200 ◽

2002 ◽

Vol 277 (12) ◽

pp. 10187-10193 ◽

Cited By ~ 30

Author(s):

Lucas Lüthy ◽

Markus G. Grütter ◽

Peer R. E. Mittl

Keyword(s):

Crystal Structure ◽

Binding Protein ◽

Penicillin Binding Protein ◽

Protein B

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Functional analysis of a murine monoclonal antibody against the repetitive region of the fibronectin-binding adhesins fibronectin-binding protein A and fibronectin-binding protein B from Staphylococcus aureus

FEBS Journal ◽

10.1111/j.1742-4658.2010.07835.x ◽

2010 ◽

Vol 277 (21) ◽

pp. 4490-4505 ◽

Cited By ~ 6

Author(s):

Giulio Provenza ◽

Maria Provenzano ◽

Livia Visai ◽

Fiona M. Burke ◽

Joan A. Geoghegan ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Monoclonal Antibody ◽

Functional Analysis ◽

Binding Protein ◽

Protein A ◽

Murine Monoclonal Antibody ◽

Repetitive Region ◽

Fibronectin Binding Protein ◽

Fibronectin Binding ◽

Protein B

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The bacterial receptor protein, transferrin-binding protein B, does not independently facilitate the release of metal ion from human transferrin

Biochemistry and Cell Biology ◽

10.1139/o03-057 ◽

2003 ◽

Vol 81 (4) ◽

pp. 275-283 ◽

Cited By ~ 6

Author(s):

Ulyana Nemish ◽

Rong-Hua Yu ◽

Leslie W Tari ◽

Karla Krewulak ◽

Anthony B Schryvers

Keyword(s):

Outer Membrane ◽

Metal Binding ◽

Metal Ion ◽

Binding Protein ◽

Protein A ◽

Nitrilotriacetic Acid ◽

Receptor Protein ◽

Binding Studies ◽

Metal Ion Removal ◽

Protein B

Pathogenic Gram-negative bacteria of the Pasteurellaceae and Neisseriaceae acquire iron for growth from host transferrin through the action of specific surface receptors. Iron is removed from transferrin by the receptor at the cell surface and is transported across the outer membrane to the periplasm. A periplasmic binding protein-dependent pathway subsequently transports iron into the cell. The transferrin receptor is composed of a largely surface-exposed lipoprotein, transferrin binding protein B, and a TonB-dependent integral outer membrane protein, transferrin binding protein A. To examine the role of transferrin binding protein B in the iron removal process, complexes of recombinant transferrin binding protein B and transferrin were prepared and compared with transferrin in metal-binding and -removal experiments. A polyhistidine-tagged form of recombinant transferrin binding protein B was able to purify a complex with transferrin that was largely monodisperse by dynamic light scattering analysis. Gallium was used instead of iron in the metal-binding studies, since it resulted in increased stability of recombinant transferrin binding protein B in the complex. Difference absorption spectra were used to monitor removal of gallium by nitrilotriacetic acid. Kinetic and equilibrium binding studies indicated that transferrin binds gallium more tightly in the presence of transferrin binding protein B. Thus, transferrin binding protein B does not facilitate metal ion removal and additional components are required for this process.Key words: iron, transport, outer membrane, lipoprotein, glycoprotein.

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Bacterial lactoferrin receptors: insights from characterizing theMoraxella bovisreceptors

Biochemistry and Cell Biology ◽

10.1139/o01-235 ◽

2002 ◽

Vol 80 (1) ◽

pp. 81-90 ◽

Cited By ~ 28

Author(s):

Rong-Hua Yu ◽

Anthony B Schryvers

Keyword(s):

Amino Acid ◽

Binding Protein ◽

Protein A ◽

Bovine Lactoferrin ◽

Predicted Amino Acid Sequence ◽

Amino Acid Homology ◽

Affinity Ligand ◽

Affinity Isolation ◽

Moraxella Bovis ◽

Isogenic Mutants

Moraxella bovis is the causative agent of infectious conjunctivitis in cattle. Moraxella bovis isolates were shown to specifically bind bovine lactoferrin (bLf) and bovine transferrin (bTf) and to use these proteins as a source of iron to support the growth of iron-limited cells. Affinity isolation experiments with immobilized bTf yielded two proteins readily resolved by SDS-PAGE analysis, whereas only a single band of approximately 100 kDa was detected when immobilized bLf was used as the affinity ligand. Using a novel cloning strategy, regions containing the genes encoding the lactoferrin (Lf) and transferrin (Tf) receptor proteins were isolated and sequenced, demonstrating that they both consisted of two genes, with the tbpB or lbpB gene preceding the tbpA or lbpA gene. The cloned lbp genes were used to generate isogenic mutants deficient in lactoferrin binding protein A and (or) B, and the resulting strains were tested in growth and binding assays. The isogenic mutants were deficient in their use of bLf for growth and had substantially diminished bLf binding capability. The predicted amino acid sequence from the segment encoding Lf binding protein B revealed an internal amino acid homology suggesting it is a bi-lobed protein, with a C-lobe enriched in acidic amino acids, but without the evident clustering observed in Lf-binding proteins from other species.Key words: outer membrane protein, iron-binding protein, lactoferrin, receptor, iron, transport, specificity.

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