Reverse vaccinology-based identification of a novel surface lipoprotein that is an effective vaccine antigen against bovine infections caused by Pasteurella multocida.

Pasteurella multocida can infect a multitude of wild and domesticated animals, with infections in cattle resulting in hemorrhagic septicemia (HS) or contributing to bovine respiratory disease (BRD) complex. Current cattle vaccines against P. multocida consist of inactivated bacteria, which only offer limited and serogroup specific protection. Here, we describe a newly identified surface lipoprotein, PmSLP, that is present in nearly all annotated P. multocida strains isolated from cattle. Bovine associated variants span three of the four identified phylogenetic clusters, with PmSLP-1 and PmSLP-2 being restricted to BRD associated isolates and PmSLP-3 being restricted to isolates associated with HS. Recombinantly expressed, soluble PmSLP-1 (BRD-PmSLP) and PmSLP-3 (HS-PmSLP) vaccines were both able to provide full protection in a mouse sepsis model against the matched P. multocida strain, however no cross-protection and minimal serum IgG cross-reactivity was identified. Full protection against both challenge strains was achieved with a bivalent vaccine containing both BRD-PmSLP and HS-PmSLP, with serum IgG from immunized mice being highly reactive to both variants. Year-long stability studies with lyophilized antigen stored under various temperatures show no appreciable difference in biophysical properties or loss of efficacy in the mouse challenge model. PmSLP-1 and PmSLP-3 vaccines were each evaluated for immunogenicity in two independent cattle trials involving animals of different age ranges and breeds. In all four trials, vaccination with PmSLP resulted in an increase in antigen specific serum IgG over baseline. In a blinded cattle challenge study with a recently isolated HS strain, the matched HS-PmSLP vaccine showed strong efficacy (75-87.5% survival compared to 0% in the control group). Together, these data suggest that cattle vaccines composed of PmSLP antigens can be a practical and effective solution for preventing HS and BRD related P. multocida infections.

Structural and functional insights into iron acquisition from lactoferrin and transferrin in Gram-negative bacterial pathogens.

Iron is an essential element for various lifeforms but is largely insoluble due to the oxygenation of Earth's atmosphere and oceans during the Proterozoic era. Metazoans evolved iron transport glycoproteins, like transferrin (Tf) and lactoferrin (Lf), to keep iron in a non-toxic, usable form, while maintaining a low free iron concentration in the body that is unable to sustain bacterial growth. To survive on the mucosal surfaces of the human respiratory tract where it exclusively resides, the Gram-negative bacterial pathogen Moraxella catarrhalis utilizes surface receptors for acquiring iron directly from human Tf and Lf. The receptors are comprised of a surface lipoprotein to capture iron-loaded Tf or Lf and deliver it to a TonB-dependent transporter (TBDT) for removal of iron and transport across the outer membrane. The subsequent transport of iron into the cell is normally mediated by a periplasmic iron-binding protein and inner membrane transport complex, which has yet to be determined for Moraxella catarrhalis. We identified two potential periplasm to cytoplasm transport systems and performed structural and functional studies with the periplasmic binding proteins (FbpA and AfeA) to evaluate their role. Growth studies with strains deleted in the fbpA or afeA gene demonstrated that FbpA, but not AfeA, was required for growth on human Tf or Lf. The crystal structure of FbpA with bound iron in the open conformation was obtained, identifying three tyrosine ligands that were required for growth on Tf or Lf. Computational modeling of the YfeA homologue, AfeA, revealed conserved residues involved in metal binding.

Lactoferrin receptors in Gram-negative bacteria: an evolutionary perspective.

In this short review, we outline the major events that led to the development of iron acquisition systems in Gram-negative bacteria and mammals since the beginning of life on earth. Naturally, the interaction between these organisms led to the development of a wonderfully complex set of protein systems used for competition over a once prevalent (but no longer) biocatalytic cofactor. These events led to the appearance of the lactoferrin gene, which has since been exploited into adopting countless new functions, including the provision of highly bactericidal degradation products. In parallel to lactoferrin's evolution, evolving bacterial receptors have countered the bactericidal properties of this innate immunity protein.

Photo-Cross-Linking Mass Spectrometry and Integrative Modeling Enables Rapid Screening of Antigen Interactions Involving Bacterial Transferrin Receptors.

Structure-based approaches to the delineation of immunogens for vaccine development have a throughput requirement that is difficult to meet in practice with conventional methods of structure determination. Here we present a strategy for rapid and accurate structure generation in support of antigen engineering programs. The approach is developed around the modeling of interactions between host transferrin (Tf) and the bacterial vaccine target transferrin binding protein B (TbpB) from Gram-negative pathogens such as Neisseria meningitidis. Using an approach based solely on cross-linking mass spectrometry (XL-MS) data, monomeric structural models, and the Integrative Modeling Platform (IMP), we demonstrate that converged representations of the Tf:TbpB interactions can be returned that accurately reflect the binding interface and the relative orientation of the monomeric units, with the capacity to scale to the analysis of interactions from any number of additional strains. We show that a key element to accurate modeling involves the application of hetero-bifunctional cross-linkers incorporating fast-acting photoactivatable diazirines coupled with conventional amine-targeting N-hydroxysuccinimide esters, and we demonstrate that conventional homo-bifunctional reagents used in cross-linking kinetically trap dynamic states in the ensemble. Therefore, the application of both classes of cross-linker provides an opportunity to empirically detect protein dynamics during integrative structural modeling.

Lactoferrin binding protein B - a bi-functional bacterial receptor protein.

Lactoferrin binding protein B (LbpB) is a bi-lobed outer membrane-bound lipoprotein that comprises part of the lactoferrin (Lf) receptor complex in Neisseria meningitidis and other Gram-negative pathogens. Recent studies have demonstrated that LbpB plays a role in protecting the bacteria from cationic antimicrobial peptides due to large regions rich in anionic residues in the C-terminal lobe. Relative to its homolog, transferrin-binding protein B (TbpB), there currently is little evidence for its role in iron acquisition and relatively little structural and biophysical information on its interaction with Lf. In this study, a combination of crosslinking and deuterium exchange coupled to mass spectrometry, information-driven computational docking, bio-layer interferometry, and site-directed mutagenesis was used to probe LbpB:hLf complexes. The formation of a 1:1 complex of iron-loaded Lf and LbpB involves an interaction between the Lf C-lobe and LbpB N-lobe, comparable to TbpB, consistent with a potential role in iron acquisition. The Lf N-lobe is also capable of binding to negatively charged regions of the LbpB C-lobe and possibly other sites such that a variety of higher order complexes are formed. Our results are consistent with LbpB serving dual roles focused primarily on iron acquisition when exposed to limited levels of iron-loaded Lf on the mucosal surface and effectively binding apo Lf when exposed to high levels at sites of inflammation.

Are lactoferrin receptors in Gram-negative bacteria viable vaccine targets?

A number of important Gram-negative pathogens that reside exclusively in the upper respiratory or genitourinary tract of their mammalian host rely on surface receptors that specifically bind host transferrin and lactoferrin as a source of iron for growth. The transferrin receptors have been targeted for vaccine development due to their critical role in acquiring iron during invasive infection and for survival on the mucosal surface. In this study, we focus on the lactoferrin receptors, determining their prevalence in pathogenic bacteria and comparing their prevalence in commensal Neisseria to other surface antigens targeted for vaccines; addressing the issue of a reservoir for vaccine escape and impact of vaccination on the microbiome. Since the selective release of the surface lipoprotein lactoferrin binding protein B by the NalP protease in Neisseria meningitidis argues against its utility as a vaccine target, we evaluated the release of outer membrane vesicles, and transferrin and lactoferrin binding in N. meningitidis and Moraxella catarrhalis. The results indicate that the presence of NalP reduces the binding of transferrin and lactoferrin by cells and native outer membrane vesicles, suggesting that NalP may impact all lipoprotein targets, thus this should not exclude lactoferrin binding protein B as a target.

High Sensitivity Crosslink Detection Coupled With Integrative Structure Modeling in the Mass Spec Studio.

The Mass Spec Studio package was designed to support the extraction of hydrogen-deuterium exchange and covalent labeling data for a range of mass spectrometry (MS)-based workflows, to integrate with restraint-driven protein modeling activities. In this report, we present an extension of the underlying Studio framework and provide a plug-in for crosslink (XL) detection. To accommodate flexibility in XL methods and applications, while maintaining efficient data processing, the plug-in employs a peptide library reduction strategy via a presearch of the tandem-MS data. We demonstrate that prescoring linear unmodified peptide tags using a probabilistic approach substantially reduces search space by requiring both crosslinked peptides to generate sparse data attributable to their linear forms. The method demonstrates highly sensitive crosslink peptide identification with a low false positive rate. Integration with a Haddock plug-in provides a resource that can combine multiple sources of data for protein modeling activities. We generated a structural model of porcine transferrin bound to TbpB, a membrane-bound receptor essential for iron acquisition in Actinobacillus pleuropneumoniae Using mutational data and crosslinking restraints, we confirm the mechanism by which TbpB recognizes the iron-loaded form of transferrin, and note the requirement for disparate sources of restraint data for accurate model construction. The software plugin is freely available at www.msstudio.ca.

Active Transport of Phosphorylated Carbohydrates Promotes Intestinal Colonization and Transmission of a Bacterial Pathogen.

Efficient acquisition of extracellular nutrients is essential for bacterial pathogenesis, however the identities and mechanisms for transport of many of these substrates remain unclear. Here, we investigate the predicted iron-binding transporter AfuABC and its role in bacterial pathogenesis in vivo. By crystallographic, biophysical and in vivo approaches, we show that AfuABC is in fact a cyclic hexose/heptose-phosphate transporter with high selectivity and specificity for a set of ubiquitous metabolites (glucose-6-phosphate, fructose-6-phosphate and sedoheptulose-7-phosphate). AfuABC is conserved across a wide range of bacterial genera, including the enteric pathogens EHEC O157:H7 and its murine-specific relative Citrobacter rodentium, where it lies adjacent to genes implicated in sugar sensing and acquisition. C. rodentium ΔafuA was significantly impaired in an in vivo murine competitive assay as well as its ability to transmit infection from an afflicted to a naïve murine host. Sugar-phosphates were present in normal and infected intestinal mucus and stool samples, indicating that these metabolites are available within the intestinal lumen for enteric bacteria to import during infection. Our study shows that AfuABC-dependent uptake of sugar-phosphates plays a critical role during enteric bacterial infection and uncovers previously unrecognized roles for these metabolites as important contributors to successful pathogenesis.

Insights into the bacterial transferrin receptor: the structure of transferrin-binding protein B from Actinobacillus pleuropneumoniae.

Pathogenic bacteria from the Neisseriaceae and Pasteurellacea families acquire iron directly from the host iron-binding glycoprotein, transferrin (Tf), in a process mediated by surface receptor proteins that directly bind host Tf, extract the iron, and transport it across the outer membrane. The bacterial Tf receptor is comprised of a surface exposed lipoprotein, Tf-binding protein B (TbpB), and an integral outer-membrane protein, Tf-binding protein A (TbpA), both of which are essential for survival in the host. In this study, we report the 1.98 A resolution structure of TbpB from the porcine pathogen Actinobacillus pleuropneumoniae, providing insights into the mechanism of Tf binding and the role of TbpB. A model for the complex of TbpB bound to Tf is proposed. Mutation of a single surface-exposed Phe residue on TbpB within the predicted interface completely abolishes binding to Tf, suggesting that the TbpB N lobe comprises the sole high-affinity binding region for Tf.

A method for measuring binding constants using unpurified in vivo biotinylated ligands.

Obtaining accurate kinetics and steady-state binding constants for biomolecular interactions normally requires pure and homogeneous protein preparations. Furthermore, in many cases, one of the ligands must be labeled. Over the past decade, several technologies have been introduced that allow for the measurement of kinetics constants for multiple different interactions in parallel. One such technology is bio-layer interferometry (BLI), which has been used to develop systems that can measure up to 96 biomolecular interactions simultaneously. However, despite the ever-increasing throughput of the tools available for measuring protein-protein interactions, the preparation of pure protein still remains a bottleneck in the process of producing high-quality kinetics data. Here, we show that high-quality binding data can be obtained using soluble lysate fractions containing protein that has been biotinylated in vivo using BirA and then applied to BLI sensors without further purification. Furthermore, we show that BirA ligase does not necessarily need to be co-overexpressed with the protein of interest for biotinylation of the biotin acceptor peptide to occur, suggesting that the activity of endogenous BirA in Escherichia coli is sufficient for producing enough biotinylated protein for a binding experiment.

Nonbinding site-directed mutants of transferrin binding protein B exhibit enhanced immunogenicity and protective capabilities.

Host-adapted Gram-negative bacterial pathogens from the Pasteurellaceae, Neisseriaceae, and Moraxellaceae families normally reside in the upper respiratory or genitourinary tracts of their hosts and rely on utilizing iron from host transferrin (Tf) for growth and survival. The surface receptor proteins that mediate this critical iron acquisition pathway have been proposed as ideal vaccine targets due to the critical role that they play in survival and disease pathogenesis in vivo. In particular, the surface lipoprotein component of the receptor, Tf binding protein B (TbpB), had received considerable attention as a potential antigen for vaccines in humans and food production animals but this has not translated into the series of successful vaccine products originally envisioned. Preliminary immunization experiments suggesting that host Tf could interfere with development of the immune response prompted us to directly address this question with site-directed mutant proteins defective in binding Tf. Site-directed mutants with dramatically reduced binding of porcine transferrin and nearly identical structure to the native proteins were prepared. A mutant Haemophilus parasuis TbpB was shown to induce an enhanced B-cell and T-cell response in pigs relative to native TbpB and provide superior protection from infection than the native TbpB or a commercial vaccine product. The results indicate that binding of host transferrin modulates the development of the immune response against TbpBs and that strategies designed to reduce or eliminate binding can be used to generate superior antigens for vaccines.

Effectiveness of the standard and an alternative set of Streptococcus pneumoniae multi locus sequence typing primers.

BACKGROUND: Multi-locus sequence typing (MLST) is a portable, broadly applicable method for classifying bacterial isolates at an intra-species level. This methodology provides clinical and scientific investigators with a standardized means of monitoring evolution within bacterial populations. MLST uses the DNA sequences from a set of genes such that each unique combination of sequences defines an isolate's sequence type. In order to reliably determine the sequence of a typing gene, matching sequence reads for both strands of the gene must be obtained. This study assesses the ability of both the standard, and an alternative set of, Streptococcus pneumoniae MLST primers to completely sequence, in both directions, the required typing alleles. RESULTS: The results demonstrated that for five (aroE, recP, spi, xpt, ddl) of the seven S. pneumoniae typing alleles, the standard primers were unable to obtain the complete forward and reverse sequences. This is due to the standard primers annealing too closely to the target regions, and current sequencing technology failing to sequence the bases that are too close to the primer. The alternative primer set described here, which includes a combination of primers proposed by the CDC and several designed as part of this study, addresses this limitation by annealing to highly conserved segments further from the target region. This primer set was subsequently employed to sequence type 105 S. pneumoniae isolates collected by the Canadian Immunization Monitoring Program ACTive (IMPACT) over a period of 18 years. CONCLUSIONS: The inability of several of the standard S. pneumoniae MLST primers to fully sequence the required region was consistently observed and is the result of a shift in sequencing technology occurring after the original primers were designed. The results presented here introduce clear documentation describing this phenomenon into the literature, and provide additional guidance, through the introduction of a widely validated set of alternative primers, to research groups seeking to undertake S. pneumoniae MLST based studies.

The specificity of protection against cationic antimicrobial peptides by lactoferrin binding protein B.

A variety of Gram-negative pathogens possess host-specific lactoferrin (Lf) receptors that mediate the acquisition of iron from host Lf. The integral membrane protein component of the receptor, lactoferrin binding protein A specifically binds host Lf and is required for acquisition of iron from Lf. In contrast, the role of the bi-lobed surface lipoprotein, lactoferrin binding protein B (LbpB), in Lf binding and iron acquisition is uncertain. A common feature of LbpBs from most species is the presence of clusters of negatively charged amino acids in the protein's C-terminal lobe. Recently it has been shown that the negatively charged regions from the Neisseria meningitidis LbpB are responsible for protecting against an 11 amino acid cationic antimicrobial peptide (CAP), lactoferricin (Lfcin), derived from human Lf. In this study we investigated whether the LbpB confers resistance to other CAPs since N. meningitidis is likely to encounter other CAPs from the host. LbpB provided protection against the cathelicidin derived peptide, cathelicidin related antimicrobial peptide (mCRAMP), but did not confer protection against Tritrp 1 or LL37 under our experimental conditions. When tested against a range of rationally designed synthetic peptides, LbpB was shown to protect against IDR-1002 and IDR-0018 but not against HH-2 or HHC10.

A secreted bacterial protein protects bacteria from cationic antimicrobial peptides by entrapment in phase-separated droplets.

Mammalian hosts combat bacterial infections through the production of defensive cationic antimicrobial peptides (CAPs). These immune factors are capable of directly killing bacterial invaders; however, many pathogens have evolved resistance evasion mechanisms such as cell surface modification, CAP sequestration, degradation, or efflux. We have discovered that several pathogenic and commensal proteobacteria, including the urgent human threat Neisseria gonorrhoeae, secrete a protein (lactoferrin-binding protein B, LbpB) that contains a low-complexity anionic domain capable of inhibiting the antimicrobial activity of host CAPs. This study focuses on a cattle pathogen, Moraxella bovis, that expresses the largest anionic domain of the LbpB homologs. We used an exhaustive biophysical approach employing circular dichroism, biolayer interferometry, cross-linking mass spectrometry, microscopy, size-exclusion chromatography with multi-angle light scattering coupled to small-angle X-ray scattering (SEC-MALS-SAXS), and NMR to understand the mechanisms of LbpB-mediated protection against CAPs. We found that the anionic domain of this LbpB displays an α-helical secondary structure but lacks a rigid tertiary fold. The addition of antimicrobial peptides derived from lactoferrin (i.e. lactoferricin) to the anionic domain of LbpB or full-length LbpB results in the formation of phase-separated droplets of LbpB together with the antimicrobial peptides. The droplets displayed a low rate of diffusion, suggesting that CAPs become trapped inside and are no longer able to kill bacteria. Our data suggest that pathogens, like M. bovis, leverage anionic intrinsically disordered domains for the broad recognition and neutralization of antimicrobials via the formation of biomolecular condensates.

Developing a PmSLP3-based vaccine formulation that provides robust long-lasting protection against hemorrhagic septicemia-causing serogroup B and E strains of Pasteurella multocida in cattle.

Background: Pasteurella multocida is a bacterial pathogen that causes a variety of infections across diverse animal species, with one of the most devastating associated diseases being hemorrhagic septicemia. Outbreaks of hemorrhagic septicemia in cattle and buffaloes are marked by rapid progression and high mortality. These infections have particularly harmful socio-economic impacts on small holder farmers in Africa and Asia who are heavily reliant on a small number of animals kept as a means of subsistence for milk and draft power purposes. A novel vaccine target, PmSLP-3, has been identified on the surface of hemorrhagic septicemia-associated strains of P. multocida and was previously shown to elicit robust protection in cattle against lethal challenge with a serogroup B strain. Methods: Here, we further investigate the protective efficacy of this surface lipoprotein, including evaluating the immunogenicity and protection upon formulation with a variety of adjuvants in both mice and cattle. Results: PmSLP-3 formulated with Montanide ISA 61 elicited the highest level of serum and mucosal IgG, elicited long-lasting serum antibodies, and was fully protective against serogroup B challenge. Studies were then performed to identify the minimum number of doses required and the needed protein quantity to maintain protection. Duration studies were performed in cattle, demonstrating sustained serum IgG titres for 3 years after two doses of vaccine and full protection against lethal serogroup B challenge at 7 months after a single vaccine dose. Finally, a serogroup E challenge study was performed, demonstrating that PmSLP-3 vaccine can provide protection against challenge by the two serogroups responsible for hemorrhagic septicemia. Conclusion: Together, these data indicate that PmSLP-3 formulated with Montanide ISA 61 is an immunogenic and protective vaccine against hemorrhagic septicemia-causing P. multocida strains in cattle.

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

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.

Use of Epivolve phage display to generate a monoclonal antibody with opsonic activity directed against a subdominant epitope on extracellular loop 4 of Treponema pallidum BamA (TP0326).

Introduction: Syphilis, a sexually transmitted infection caused by the spirochete Treponema pallidum (Tp), is resurging globally. Tp's repertoire of outer membrane proteins (OMPs) includes BamA (ß-barrel assembly machinery subunit A/TP0326), a bipartite protein consisting of a 16-stranded ß-barrel with nine extracellular loops (ECLs) and five periplasmic POTRA (polypeptide transport-associated) domains. BamA ECL4 antisera promotes internalization of Tp by rabbit peritoneal macrophages. Methods: Three overlapping BamA ECL4 peptides and a two-stage, phage display strategy, termed "Epivolve" (for epitope evolution) were employed to generate single-chain variable fragments (scFvs). Additionally, antisera generated by immunizing mice and rabbits with BamA ECL4 displayed by a Pyrococcus furiosus thioredoxin scaffold (PfTrxBamA/ECL4). MAbs and antisera reactivities were evaluated by immunoblotting and ELISA. A comparison of murine and rabbit opsonophagocytosis assays was conducted to evaluate the functional ability of the Abs (e.g., opsonization) and validate the mouse assay. Sera from Tp-infected mice (MSS) and rabbits (IRS) were evaluated for ECL4-specific Abs using PfTrxBamA/ECL4 and overlapping ECL4 peptides in immunoblotting and ELISA assays. Results: Each of the five mAbs demonstrated reactivity by immunoblotting and ELISA to nanogram amounts of PfTrxBamA/ECL4. One mAb, containing a unique amino acid sequence in both the light and heavy chains, showed activity in the murine opsonophagocytosis assay. Mice and rabbits hyperimmunized with PfTrxBamA/ECL4 produced opsonic antisera that strongly recognized the ECL presented in a heterologous scaffold and overlapping ECL4 peptides, including S2. In contrast, Abs generated during Tp infection of mice and rabbits poorly recognized the peptides, indicating that S2 contains a subdominant epitope. Discussion: Epivolve produced mAbs target subdominant opsonic epitopes in BamA ECL4, a top syphilis vaccine candidate. The murine opsonophagocytosis assay can serve as an alternative model to investigate the opsonic potential of vaccinogens. Detailed characterization of BamA ECL4-specific Abs provided a means to dissect Ab responses elicited by Tp infection.

Anchor peptide of transferrin-binding protein B is required for interaction with transferrin-binding protein A.

Gram-negative bacterial pathogens belonging to the Pasteurellaceae, Moraxellaceae, and Neisseriaceae families rely on an iron acquisition system that acquires iron directly from host transferrin (Tf). The process is mediated by a surface receptor composed of transferrin-binding proteins A and B (TbpA and TbpB). TbpA is an integral outer membrane protein that functions as a gated channel for the passage of iron into the periplasm. TbpB is a surface-exposed lipoprotein that facilitates the iron uptake process. In this study, we demonstrate that the region encompassing amino acids 7-40 of Actinobacillus pleuropneumoniae TbpB is required for forming a complex with TbpA and that the formation of the complex requires the presence of porcine Tf. These results are consistent with a model in which TbpB is responsible for the initial capture of iron-loaded Tf and subsequently interacts with TbpA through the anchor peptide. We propose that TonB binding to TbpA initiates the formation of the TbpB-TbpA complex and transfer of Tf to TbpA.

Structural variations within the transferrin binding site on transferrin-binding protein B, TbpB.

Pathogenic bacteria acquire the essential element iron through specialized uptake pathways that are necessary in the iron-limiting environments of the host. Members of the Gram-negative Neisseriaceae and Pasteurellaceae families have adapted to acquire iron from the host iron binding glycoprotein, transferrin (Tf), through a receptor complex comprised of transferring-binding protein (Tbp) A and B. Because of the critical role they play in the host, these surface-exposed proteins are invariably present in clinical isolates and thus are considered prime vaccine targets. The specific interactions between TbpB and Tf are essential and ultimately might be exploited to create a broad-spectrum vaccine. In this study, we report the structure of TbpBs from two porcine pathogens, Actinobacillus pleuropneumoniae and suis. Paradoxically, despite a common Tf target, these swine related TbpBs show substantial sequence variation in their Tf-binding site. The TbpB structures, supported by docking simulations, surface plasmon resonance and hydrogen/deuterium exchange experiments with wild-type and mutant TbpBs, explain why there are structurally conserved elements within TbpB homologs despite major sequence variation that are required for binding Tf.

Conserved interaction between transferrin and transferrin-binding proteins from porcine pathogens.

Gram-negative porcine pathogens from the Pasteurellaceae family possess a surface receptor complex capable of acquiring iron from porcine transferrin (pTf). This receptor consists of transferrin-binding protein A (TbpA), a transmembrane iron transporter, and TbpB, a surface-exposed lipoprotein. Questions remain as to how the receptor complex engages pTf in such a way that iron is positioned for release, and whether divergent strains present distinct recognition sites on Tf. In this study, the TbpB-pTf interface was mapped using a combination of mass shift analysis and molecular docking simulations, localizing binding uniquely to the pTf C lobe for multiple divergent strains of Actinobacillus plueropneumoniae and suis. The interface was further characterized and validated with site-directed mutagenesis. Although targeting a common lobe, variants differ in preference for the two sublobes comprising the iron coordination site. Sublobes C1 and C2 participate in high affinity binding, but sublobe C1 contributes in a minor fashion to the overall affinity. Further, the TbpB-pTf complex does not release iron independent of other mediators, based on competitive iron binding studies. Together, our findings support a model whereby TbpB efficiently captures and presents iron-loaded pTf to other elements of the uptake pathway, even under low iron conditions.