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A substantial disease burden in vertebrates is due to Gram-negative bacteria that exclusively inhabit the upper respiratory or genitourinary tracts of their hosts and rely on directly acquiring iron from the host iron-binding glycoproteins through surface receptor proteins. The receptors enable these bacteria to proliferate independently from their neighbors on the mucosal surface and during invasive infection of the host. The diversity in these receptors evolved over millions of years of evolution, which thus bodes well for long-lasting vaccine coverage. Experiments in food production animals provide proof of concept for the use of engineered antigens derived from the receptor proteins to prevent colonization and invasive infection in the natural host, strongly supporting development of these vaccines for use in humans. The presence of transferrin (Tf) receptors in Gram-negative bacteria that reside in the upper respiratory tract of mammals and birds indicate that they have existed for over 320 million years. Tf/lactoferrin receptors are essential for survival of Gram-negative bacterial pathogens (pathobionts) on the mucosal surface and during invasive infection and thus are ideal targets for vaccines. Structure-based antigen engineering can improve the immune response induced by Tf receptor proteins. Proof of concept experiments in pigs demonstrate that antigens targeting Tf receptors can prevent infection and colonization. [ABSTRACT FROM AUTHOR]

Lactoferrin has long been recognized for its antimicrobial properties, initially attributed primarily to iron sequestration. It has since become apparent that interaction between the host and bacteria is modulated by a complex series of interactions between lactoferrin and bacteria, lactoferrin and bacterial products, and lactoferrin and host cells. The primary focus of this review is the interaction between lactoferrin and bacteria, but interactions with the lactoferrin-derived cationic peptide lactoferricin will also be discussed. We will summarize what is currently known about the interaction between lactoferrin (or lactoferricin) and surface or secreted bacterial components, comment on the potential physiological relevance of the findings, and identify key questions that remain unanswered. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Pathogenic bacteria in the family Neisseriaceae express surface receptors to acquire iron from the mammalian iron-binding proteins. Transferrins and lactoferrins constitute a family of iron-binding proteins highly related in both sequence and structure, yet the bacterial receptors are able to distinguish between these proteins and uphold a strict binding specificity. In order to understand the molecular basis for this specificity, the interaction between human lactoferrin (hLf) and the lactoferrin-binding protein A (LbpA) from Moraxella catarrhalis was studied. A periplasmic expression system was designed for the heterologous expression of LbpA, which enabled the investigation of its binding activity in the absence of lactoferrin-binding protein B (LbpB). To facilitate delineation of the LbpA-binding regions of hLf, chimeric proteins composed of hLf and bovine transferrin were made. Binding studies performed with the chimeric proteins and recombinant LbpA identified two binding regions within the C-terminus of hLf. Furthermore, native LbpA from Moraxella and Neisseria spp. bound the identical spectrum of hybrid proteins as the recombinant receptor, demonstrating a conserved binding interaction with the C-lobe of hLf. [ABSTRACT FROM AUTHOR]

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 transferrinbinding 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. [ABSTRACT FROM AUTHOR]

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.La bactérie Moraxella bovis cause une conjonctivite infectieuse chez les bovins. Les cellules de M. bovis ayant une quantité limitée de fer, elles lient spécifiquement la lactoferrine et la transferrine bovines et utilisent ces protéines comme source de fer, afin d'assurer leur croissance. Une chromatographie d'affinité envers la transferrine bovine a permis d'obtenir deux protéines, facilement séparées par électrophorèse en gel de polyacrylamide en présence de dodécylsulfate de sodium (SDS-PAGE), tandis qu'une seule bande d'environ 100 kDa a été détectée après une chromatographie d'affinité envers la lactoferrine bovine. En utilisant une nouvelle stratégie de clonage, les régions comportant les gènes codant le récepteur de la lactoferrine et le récepteur de la transferrine ont été isolées et leurs séquences déterminées, ce qui a permis de démontrer que les deux régions comportent deux gènes : le gène tbpB ou lbpB suivi du gène tbpA ou lbpA. Les gènes lbp clonés ont été utilisés pour produire des mutants isogéniques déficients en protéines liant la lactoferrine A et/ou B et, par la suite, la croissance et la capacité de liaison des souches obtenues ont été mesurées. Les mutants isogéniques sont déficients dans l'utilisation de la lactoferrine pour leur croissance et leur pouvoir de lier la lactoferrine bovine est beaucoup diminué. La séquence des acides aminés prédite à partir du segment codant la protéine liant la lactoferrine bovine B révèle une homologie d'acides aminés interne, ce qui suggère que la protéine a deux lobes unis par un lobe C riche en acides aminés acides, mais elle n'a pas la tendance à l'agrégation des protéines liant la lactoferrine chez d'autres espèces.Mots clés : protéine membrane externe, fer protéine de liaison, lactoferrine, récepteur fer, transport, spécificité.[Traduit par Rédaction] [ABSTRACT FROM AUTHOR]

Reports on the characterization of the novel transferrin bTf receptor in bovine strains of Pasteurella multicoda. Prevalence and properties of the Tf receptor in the isolates; Cloning of the Tf receptor gene; Characterization of the chromosomal locus; Characterization of the Tf receptor gene.

Examines the relatedness of bacterial populations within taxonomic species by revealing and comparing their genome structures. Construction of genome maps by complete and incomplete endonuclease I-CeuI cleavages of genomic deoxyribonucleic acid; Similarities in the genome structure of Salmonella strain; Analysis of the P. haemolytica and multocida strains.

Pathogenic neisseriae have a repertoire of high-affinity iron uptake systems to facilitate acquisition of this essential element in the human host. They possess surface receptor proteins that directly bind the extracellular host iron-binding proteins transferrin and lactoferrin. Alternatively, they have siderophore receptors capable of scavenging iron when exogenous siderophores are present. Released intracellular haem iron present in the form of haemoglobin, haemoglobin–haptoglobin or free haem can be used directly as a source of iron for growth through direct binding by specific surface receptors. Although these receptors may vary in complexity and composition, the key protein involved in the transport of iron (as iron, haem or iron-siderophore) across the outer membrane is a TonB-dependent receptor with an overall structure presumably similar to that determined recently for Escherichia coli FhuA or FepA. The receptors are potentially ideal vaccine targets in view of their critical role in survival in the host. Preliminary pilot studies indicate that transferrin receptor-based vaccines may be protective in humans. [ABSTRACT FROM AUTHOR]

Presents a study which focused on the receptors, which facilitate iron acquisition from glycoprotein transferrin (Tf) and lactoferin (Lf), produced by the pathogenic members of the Neisseriaceae family. Details on Tf; Identification of several human pathogens; Methodology used in the study; Conclusions based on results.

The article presents Lactoferrin receptors in Gram-negative bacteria: an evolutionary perspective. Topics discussed include 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; and parallel to lactoferrin's evolution, evolving bacterial receptors have countered the bactericidal properties of this innate immunity protein.

Examines the development of a PCR-based method for amplification of segments of receptor protein genes. Information pertaining to Neisseriaceae and Pasteurellaceae; How the simple solidphase binding assays and affinity isolation protocols were used; Details on the development of genes.

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. [ABSTRACT FROM AUTHOR]

The article provides scientific information on lactoferrin, a member of a small family of nonheme iron binding proteins that includes transferrin. It discusses several of its functions including the regulation of inflammatory responses in vivo and inhibit inflammatory cytokine production. It also enumerates some of its benefits in human clinical trials and studies in molecular mechanisms.

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. Author summary: Surface lipoproteins have been used as components of subunit-based vaccines to combat gram negative bacterial infections. A few years ago, we discovered a gene encoding a predicted lipoprotein (PmSLP) in Pasteurella multocida adjacent to the gene encoding the Slam translocon and showed that it was translocated to the bacterial cell surface. Since Pasteurella multocida had been linked to some devastating cattle diseases, hemorrhagic septicemia (HS) and bovine respiratory disease (BRD), we sought to evaluate PmSLP as a potential vaccine antigen. Upon removal of the lipid anchor, the PmSLP protein was easy to purify and could be reconstituted after lyophilization for long term storage. We investigated whether this protein could provide protection against P. multocida infections by establishing a mouse infection model and demonstrated that PmSLP could provide protect against invasive disease. Finally, to illustrate that the antigen could provide protection in the organism that it naturally infects, we performed large animal vaccine experiments illustrating the protective properties of PmSLP in cattle. Thus, the surface protein PmSLP provides an exciting new protein antigen that could be a cost-effective vaccine to prevent BRD and HS in cattle. [ABSTRACT FROM AUTHOR]

Presents information on several conferences on lactoferrins, an iron-binding protein found in milk.

Bovine respiratory disease complex (BRDc) is a major cause of morbidity and mortality in beef cattle. There is recent evidence suggesting that the nasopharyngeal microbiota has a key role in respiratory health and disease susceptibility in cattle. However, there is a paucity of knowledge regarding evolution of the nasopharyngeal microbiota when cattle are most likely to develop BRDc ( i.e. , from weaning to 40 days after arrival at a feedlot). The objective was to describe the evolution of the nasopharyngeal microbiota of beef cattle from weaning to 40 days after arrival at a feedlot. Deep nasal swabs (DNS) from 30 Angus-cross steers were collected at weaning, on arrival at a feedlot, and at day 40 after arrival. The DNA was extracted from DNS and the hypervariable region V3 of the 16S rRNA gene was amplified and sequenced (Illumina MiSeq platform). Nasopharyngeal microbiota underwent a profound evolution from weaning to arrival at the feedlot and from arrival to day 40, with the abundance of 92 Operational Taxonomic Units (OTUs) significantly changing over time. Mycoplasma ( M. dispar and M. bovirhinis ) was the most abundant genus in the nasopharynx, accounting for 53% of the total bacterial population. Because an evolving bacterial community may be less capable of resisting colonization by pathogenic bacteria, the instability of the nasopharyngeal microbiota documented in this study might explain why cattle are most likely to be affected with BRDc during the first weeks after weaning and arrival at a feedlot. [ABSTRACT FROM AUTHOR]

Lactoferrin binding protein B (LbpB) is a bi-lobed membrane bound lipoprotein that is part of the lactoferrin receptor complex in a variety of Gram-negative pathogens. Despite high sequence diversity among LbpBs from various strains and species, a cluster of negatively charged amino acids is invariably present in the protein’s C-terminal lobe in all species except Moraxella bovis. The function of LbpB in iron acquisition has yet to be experimentally demonstrated, whereas in vitro studies have shown that LbpB confers protection against lactoferricin, a short cationic antimicrobial peptide released from the N- terminus of lactoferrin. In this study we demonstrate that the negatively charged regions can be removed from the Neisseria meningitidis LbpB without compromising stability, and this results in the inability of LbpB to protect against the bactericidal effects of lactoferricin. The release of LbpB from the cell surface by the autotransporter NalP reduces the protection against lactoferricin in the in vitro killing assay, attributed to removal of LbpB during washing steps, but is unlikely to have a similar impact in vivo. The protective effect of the negatively charged polysaccharide capsule in the killing assay was less than the protection conferred by LbpB, suggesting that LbpB plays a major role in protection against cationic antimicrobial peptides in vivo. The selective release of LbpB by NalP has been proposed to be a mechanism for evading the adaptive immune response, by reducing the antibody binding to the cell surface, but may also provide insights into the primary function of LbpB in vivo. Although TbpB and LbpB have been shown to be major targets of the human immune response, the selective release of LbpB suggests that unlike TbpB, LbpB may not be essential for iron acquisition, but important for protection against cationic antimicrobial peptides. [ABSTRACT FROM AUTHOR]

To study the periplasmic branch of iron (ferric ion) uptake systems in Gram-negative bacteria, genetic reconstitution experiments were initiated in Haemophilus influenzae involving exchange of the periplasmic iron-binding protein. The expression of many of the heterologous periplasmic ferric-binding proteins (FbpAs) was quite limited. Transformation experiments with the fbpA gene from Neisseria gonorrhoeae yielded two colony sizes with different phenotypic characteristics. The small colonies contained the intact N. gonorrhoeae fbpA gene and were deficient in utilization of transferrin iron. The large colonies contained hybrid H. influenzae/N, gonorrhoeae fbpA genes, were proficient in transferrin iron utilization and had enhanced levels of expression of FbpA. These hybrid genes included several that encoded the mature N. gonorrhoeae FbpA with the H. influenzae signal peptide. To more fully evaluate the effect of foreign signal peptides, a series of hybrid genes were prepared that exchanged the signal peptides from H. influenzae FbpA, N. gonorrhoeae FbpA and the TEM-1 β-Iactamase. The presence of the H. influenzae leader was required for functional expression of FbpAs and was shown to dramatically increase the level of β-Iactamase activity. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

• Multidrug resistant strains of Acinetobacter baumannii is a serious health issue. • Immunoprotectivity of Omp34 loops implanted in a loopless C-lobe TbpB were assessed. • Loop 3 of Omp34 induced protection against A.baumannii infection in the murine model. Acinetobacter baumannii is a common causative agent of nosocomial infections, with a mortality rate of 43% in infected patients. Due to the emergence of multidrug-resistant (MDR) strains, vaccine development has become necessary. Since the 34 kDa outer membrane protein Omp34 has been identified as a potential vaccine target, we implemented a hybrid antigen approach to target its extracellular loops. Using bioinformatic and structural analyses, we selected Loop 3 from Omp34 and displayed it on the loopless C-lobe (LCL) of TbpB of Neisseria meningitidis. The hybrid antigen and the LCL were produced and used to immunize mice for passive and active immunization and challenge experiments in which the reactivity of the sera was assessed by ELISAs, the bacterial load in the tissues measured and the survival of immunized mice compared. LCL was ineffective in immunization against A. baumannii thus the resulting immunity was due to the presence of Omp34 loop 3. It resulted in increased survival and a reduced bacterial load in the tissues compared to the control groups. The findings indicate that the immunogenicity of Omp34 loops can induce protection against A. baumannii infection , and it could probably be used as a vaccine candidate to control the pathogenesis of A. baumannii. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Actinobacillus pleuropneumoniae , Actinobacillus suis , and Haemophilus parasuis are bacterial pathogens from the upper respiratory tract that are responsible for a substantial burden of porcine disease. Although reduction of disease has been accomplished by intensive management practices, immunization remains an important strategy for disease prevention, particularly when intensive management practices are not feasible or suitable. An attractive target for vaccine development is the surface receptor involved in acquiring iron from host transferrin, since it is common to all three pathogenic species and has been shown to be essential for survival and disease causation. It has also recently been demonstrated that an engineered antigen derived from the lipoprotein component of the receptor, transferrin-binding protein B (TbpB), was more effective at preventing infection by H. parasuis than a commercial vaccine product. This study was initiated to explore the genetic and immunogenic diversity of the transferrin receptor system from these species. Nucleic acid sequences were obtained from a geographically and temporally diverse collection of isolates, consisting of 41 A. pleuropneumoniae strains, 30 H. parasuis strains, and 2 A. suis strains. Phylogenetic analyses demonstrated that the receptor protein sequences cluster independently of species, suggesting that there is genetic exchange between these species such that receptor-based vaccines should logically target all three species. To evaluate the cross-reactive response of TbpB-derived antigens, pigs were immunized with the intact TbpB, the TbpB N-lobe and the TbpB C-lobe from A. pleuropneumoniae strain H49 and the resulting sera were tested against a representative panel of TbpBs; demonstrating that the C-lobe induces a broadly cross-reactive response. Overall our results indicate that there is a common reservoir for transferrin receptor antigenic variation amongst these pathogens. While this could present a challenge to future vaccine development, our results suggest a rationally designed TbpB-based vaccine may provide protection against all three pathogens. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

• Acinetobacter baumannii infections are difficult-to-treat with high mortality. • The immunogenic loops from BauA and Omp34 were used as a hybrid antigen. • C-lobe of TbpB surface lipoprotein was used to display BauA and Omp34 loops. • The combination of both loops brought about 71.43% protection against A. baumannii. • Multivalent vaccine induces broadly reactive antibody responses against A. baumannii. The complexity of treating Acinetobacter baumannii infections with the newly developed resistant strains has led researchers to confront this pathogen by developing vaccines. In this study, we used two important virulence factors of A. baumannii to elicit immunity against the A. baumannii. The immunogenic loops were from Baumannii acinetobactin utilization A (BauA) and 34kD outer membrane protein (Omp34). C-lobe derivative of the TbpB surface lipoprotein was used to display the superficial epitopes of the TbpA receptor protein of Neisseria meningitidis. The resulting loopless C-lobe (LCL) with implanted nucleotide sequences of the immunogenic loops from BauA and Omp34 was used as a hybrid antigen. The hybrid antigens were expressed in the E. coli and were used to immunize mice. The mice were challenged with a clinical isolate of A. baumannii (ABI022). Immunization with the hybrid antigens of the BauA loop 7 (BauAL7P3), Omp34 loop 3 Omp34L3P1, and the combination of both loops (BauAL7P3Omp34L3P1) brought about 42.86%, 42.86%, and 71.43% protection against A. baumannii infection. Histopathological findings in the immunized mice showed bronchioles clear from inflammatory cells and normal texture of the spleen and liver. The findings support the use of a multivalent vaccine to induce broadly reactive antibody responses against heterologous A. baumannii strains. [ABSTRACT FROM AUTHOR]

The upper respiratory tract (URT) is a crucial site for host defense, as it is home to bacterial communities that both modulate host immune defense and serve as a reservoir of potential pathogens. Young children are at high risk of respiratory illness, yet the composition of their URT microbiota is not well understood. Microbial profiling of the respiratory tract has traditionally focused on culturing common respiratory pathogens, whereas recent culture-independent microbiome profiling can only report the relative abundance of bacterial populations. In the current study, we used both molecular profiling of the bacterial 16S rRNA gene and laboratory culture to examine the bacterial diversity from the oropharynx and nasopharynx of 51 healthy children with a median age of 1.1 years (range 1-4.5 years) along with 19 accompanying parents. The resulting profiles suggest that in young children the nasopharyngeal microbiota, much like the gastrointestinal tract microbiome, changes from an immature state, where it is colonized by a few dominant taxa, to a more diverse state as it matures to resemble the adult microbiota. Importantly, this difference in bacterial diversity between adults and children accompanies a change in bacterial load of three orders of magnitude. This indicates that the bacterial communities in the nasopharynx of young children have a fundamentally different structure from those in adults and suggests that maturation of this community occurs sometime during the first few years of life, a period that includes ages at which children are at the highest risk for respiratory disease. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Lactoferrin binding proteins A and B (LbpA and LbpB) compose the lactoferrin receptor of the obligate human pathogen Neisseria meningitidis. This receptor is thought to be important for colonization and initiation of invasive disease because of its role in acquiring host iron and providing protection from the cationic peptide, lactoferricin. By virtue of its function, the receptor is accessible to the host immune system and displays substantial sequence variation. In this study, we analyzed a broad collection of LbpAs (62) and LbpBs (101) to determine the distribution of sequence variation within each protein and to search for patterns between sequence similarity and strain typing. The sequence variation in LbpA was predominantly observed in 3 surface loops and, surprisingly, in the N-terminal region immediately upstream of the predicted TonB box. The analysis of LbpB revealed that the variability was distributed throughout the protein, particularly in the highly variable negatively charged regions in the C-lobe, but otherwise was greater in the N-lobe than the C-lobe. There was no readily identifiable correlation between the sequence variation within LbpA, LbpB, multi-locus sequence type, or serogroup. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 (TI), 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 hydro-gen/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. [ABSTRACT FROM AUTHOR]

Pathogenic bacteria in the Neisseriaceae possess a surface receptor mediating iron acquisition from human transferrin (hTf) that consists of a transmembrane iron transporter (TbpA) and a surface-exposed lipoprotein (TbpB). In this study, we used hydrogen/deuterium exchange coupled to mass spectrometry (H/DX-MS) to elucidate the effects on hTf by interaction with TbpB or derivatives of TbpB. An overall conserved interaction was observed between hTf and full-length or N-lobe TbpB from Neisseria meningitidis strains B16B6 or M982 that represent two distinct subtypes of TbpB. Changes were observed exclusively in the C-lobe of hTf and were caused by the interaction with the N-lobe of TbpB. Regions localized to the 'lip' of the C1 and C2 domains that flank the interdomain cleft represent sites of direct contact with TbpB whereas the peptides within the interdomain cleft that encompass iron binding ligands are inaccessible in the closed (holo) conformation. Although substantial domain separation upon binding TbpB cannot be excluded by the H/DX-MS data, the preferred model of interaction involves binding hTf C-lobe in the closed conformation. Alternate explanations are provided for the substantial protection from deuteration of the peptides encompassing iron binding ligands within the interdomain cleft but cannot be differentiated by the H/DX-MS data. [ABSTRACT FROM AUTHOR]

Summary: 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 Å 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. [Copyright &y& Elsevier]

Human lactoferrin (hLf) has been shown to interact with cells from the Caco-2 human small intestinal cell line. There currently is little information about the molecular details of its interaction. As a first step toward detailed characterization of this interaction, we used a series of Lf chimeras to analyze which part of Lf is responsible for the interaction with Caco-2 cells. Recombinant chimeric proteins consisting of segments of hLf and bovine transferrin (bTf) were produced in a baculovirus-insect cell system and purified by a combination of cation exchange chromatography and immobilized bTf antibody affinity chromatography. Each chimera was labeled with a green fluorescent dye to monitor its interaction with Caco-2 cells. Similarly, the intestinal Lf receptor (LfR), also known as intelectin, was probed with an anti-LfR antibody that was detected with a secondary antibody conjugated with a red-color fluorescent dye. The results demonstrated that chimeric proteins containing the N-lobe or the N1.1 subdomain of Lf bound as well as intact Lf to Caco-2 cells. Confocal microscopy analysis revealed that these proteins, along with the LfR, were internalized and targeted to the nucleus. These results indicate that the N1.1 subdomain of hLf is sufficient for binding, internalization, and targeting to the nucleus of Caco-2 cells. [ABSTRACT FROM AUTHOR]

The uptake of the element iron is vital for the survival of most organisms. Numerous pathogenic Gram-negative bacteria utilize a periplasm-to-cytosol ATP-binding cassette transport pathway to transport this essential atom in to the cell. In this study, we investigated the Yersinia enterocolitica (YfuA) and Serratia marcescens (SfuA) iron-binding periplasmic proteins. We have determined the 1.8-Å structures of iron-loaded (YfuA) and iron-free (SfuA) forms of this class of proteins. Although the sequence of these proteins varies considerably from the other members of the transferrin structural superfamily, they adopt the same three-dimensional fold. The iron-loaded YfuA structure illustrates the unique nature of this new class of proteins in that they are able to octahedrally coordinate the ferric ion in the absence of a bound anion. The iron-free SfuA structure contains a bound citrate anion in the iron-binding cleft that tethers the N- and C-terminal domains of the apo protein and stabilizes the partially open structure. [ABSTRACT FROM AUTHOR]

We have determined the 1.35- and 1.45-Å structures, respectively, of closed and open iron-loaded forms of Mannheimia haemolytica ferric ion-binding protein A. M. haemolytica is the causative agent in the economically important and fatal disease of cattle termed shipping fever. The periplasmic iron-binding protein of this gram-negative bacterium, which has homologous counterparts in many other pathogenic species, performs a key role in iron acquisition from mammalian host serum iron transport proteins and is essential for the survival of the pathogen within the host. The ferric (Fe3+) ion in the closed structure is bound by a novel asymmetric constellation of four ligands, including a synergistic carbonate anion. The open structure is ligated by three tyrosyl residues and a dynamically disordered solvent-exposed anion. Our results clearly implicate the synergistic anion as the primary mediator of global protein conformation and provide detailed insights into the molecular mechanisms of iron binding and release in the periplasm. [ABSTRACT FROM AUTHOR]

The periplasmic iron binding protein plays an essential role in the iron uptake pathway of Gram-negative pathogenic bacteria from the Pasteurellaceae and Neisseriaceae families and is critical for survival of these pathogens within the host. In this study, we report the crystal structures of two mutant forros of ferric ion-binding protein A (FbpA) from Haemophilus influenzae with bound multinuclear oxometal clusters. Crystals of site-directed mutants in the metal or anion binding ligands contain protein in the open conformation, and two mutant FbpAs, HgA and N175L, contain different cluster arrangements in the iron-binding pocket. The iron clusters are anchored by binding to the two tyrosine ligands (Tyr195 and Tyr196) positioned at the vertex of the iron-binding pocket but are not coordinated by the other metal binding ligands. Our results suggest that the metal clusters may have formed in situ, suggesting that the mutant FbpAs may serve as a simple model for protein-mediated mineralization. [ABSTRACT FROM AUTHOR]

Pasteurellosis caused by the Gram-negative pathogen Pasteurella haemolytica is a serious disease leading to death in cattle. To scavenge growth-limiting iron from the host, the pathogen utilizes the periplasmic ferric ion.binding protein A (PhFbpA) as a component of an ATP-binding cassette transport pathway. We report the 1.2-Å structure of the iron-free (apo) form of PhFbpA, which is a member of the transferrin structural superfamily. The protein structure adopts a closed conformation, allowing us to reliably assign putative iron-coordinating residues. Based on our analysis, PhFbpA utilizes a unique constellation of binding site residues and anions to octahedrally coordinate an iron atom. A surprising finding in the structure is the presence of two formate anions on opposite sides of the iron-binding pocket. The formate ions tether the N- and C-terminal domains of the protein and stabilize the closed structure, also providing clues as to probable candidates for synergistic anions in the iron-loaded state. PhFbpA represents a new class of bacterial iron-binding proteins. [ABSTRACT FROM AUTHOR]

Focuses on high resolution crystal structure of the iron-loaded form of the Haemophilus influenzae periplasmic ferric ion binding protein mutant H9Q. Production of the protein in the periplasm of Escherichia coli; Purification and conversion of the protein; Presence of EDTA located at the previously determined anion ternary binding site.

Traditional ELISA-based protein analysis has been predicated on the assumption that proteins bind randomly to the solid surface of the ELISA plate polymer (polystyrene or polyvinyl chloride). Random adherence to the plate ensures equal access to all faces of the protein, an important consideration when evaluating immunogenicity of polyclonal serum samples as well as when examining the cross-reactivity of immune serum against different antigenic variants of a protein. In this study we demonstrate that the soluble form of the surface lipoprotein transferrin binding protein B (TbpB) from three different bacterial pathogens (Neisseria meningitidis, Actinobacillus pleuropneumoniae, and Mannheimia haemolytica) bind the ELISA plate in a manner that consistently obscures the transferrin binding face of the proteins' N-lobe. In order to develop a non-biased ELISA where all faces of the protein are accessible, the strong interaction between biotin and avidin has been exploited by adding a biotin tag to these proteins during Escherichia coli- based cytoplasmic expression and utilizing streptavidin or neutravidin coated ELISA plates for protein capture and display. The use of avidin coated ELISA plates also allows for rapid purification of biotin-tagged proteins from crude E. coli lysates, removing the requirement of prior affinity purification of each protein to be included in the ELISA-based analyses. In proof of concept experiments we demonstrate the utility of this approach for evaluating immunogenicity and cross-reactivity of serum from mice and pigs immunized with TbpBs from human and porcine pathogens. [ABSTRACT FROM AUTHOR]

Neisseria meningitidis, the causative agent of bacterial meningitis, acquires the essential element iron from the host glycoprotein transferrin during infection through a surface transferrin receptor system composed of proteins TbpA and TbpB. Here we present the crystal structures of TbpB from N. meningitidis in its apo form and in complex with human transferrin. The structure reveals how TbpB sequesters and initiates iron release from human transferrin. [ABSTRACT FROM AUTHOR]