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1.
Methods Mol Biol ; 2414: 115-140, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34784035

RESUMO

Metal ion transporters in the outer membrane of gram-negative bacteria that are responsible for acquiring iron and zinc are attractive vaccine targets due to their essential function. The core function is mediated by an integral outer membrane TonB-dependent transporter (TBDT) that mediates the transport of the metal ion across the outer membrane. Some TBDTs also have a surface lipoprotein (SLP) that assists in the efficient capture of the metal ion-containing host protein from which the metal ion is extracted. The challenges in producing the integral outer membrane protein for a commercial subunit vaccine prompted us to develop a hybrid antigen strategy in which surface loops of the TBDT are displayed on the lipoprotein, which can readily be produced as a soluble protein. The focus of this chapter will be on the methods for production of hybrid antigens and evaluating the immune response they elicit.


Assuntos
Bactérias Gram-Negativas , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bactérias Gram-Negativas/metabolismo , Proteínas de Membrana Transportadoras
2.
Front Microbiol ; 12: 714815, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34630348

RESUMO

Moraxella catarrhalis is a Gram-negative bacterium that is responsible for a substantial proportion of upper respiratory infections in children and lower respiratory infections in the elderly. Moraxella catarrhalis resides exclusively on the mucosal surfaces of the upper respiratory tract of humans and is capable of directly acquiring iron for growth from the host glycoproteins human transferrin (hTf) and human lactoferrin (hLf). The iron-bound form of these glycoproteins is initially captured by the surface lipoproteins Tf or Lf binding protein B (TbpB or LbpB) and delivered to the integral outer membrane TonB-dependent transport (TBDT) proteins, Tf binding protein A (TbpA) or Lf binding protein A (LbpA). The extraction of iron involves conformational changes in Lf and Tf to facilitate iron removal followed by its transport across the outer membrane by a well characterized process for TBDTs. Surprisingly the disruption of the gene encoding another TBDT, CopB, results in a reduction in the ability to grow on human Tf or Lf. The possibility that this could have been due to an artifact of mutant construction that resulted in the inhibition of TonB-mediated process was eliminated by a complete deletion of the CopB gene. A systematic evaluation of the impact on growth under various conditions by deletions of the genes encoding TbpA, LbpA, and CopB as well as mutations of the iron liganding residues and TonB box region of CopB was implemented. The results indicate that although CopB is capable of effectively acquiring iron from the growth medium, it does not directly acquire iron from Tf or Lf. We propose that the indirect effect on iron transport from Tf and Lf by CopB could possibly be explained by the association of TBDTs at gaps in the peptidoglycan layer that may enhance the efficiency of the process. This concept is supported by previous studies demonstrating an indirect effect on growth of Tf and Lf by deletion of the peptidoglycan binding outer membrane lipoprotein RmpM in Neisseria that also reduced the formation of larger complexes of TBDTs.

3.
J Immunol Methods ; 493: 113037, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33722512

RESUMO

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.


Assuntos
Actinobacillus pleuropneumoniae/química , Ensaio de Imunoadsorção Enzimática , Mannheimia haemolytica/química , Neisseria meningitidis/química , Proteína B de Ligação a Transferrina/imunologia , Actinobacillus pleuropneumoniae/imunologia , Avidina/química , Avidina/imunologia , Biotina/química , Biotina/imunologia , Mannheimia haemolytica/imunologia , Neisseria meningitidis/imunologia , Poliestirenos/química , Cloreto de Polivinila/química , Proteína B de Ligação a Transferrina/química
4.
Biochem Cell Biol ; 99(1): 102-108, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33464172

RESUMO

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.


Assuntos
Proteínas de Bactérias/metabolismo , Bactérias Gram-Negativas/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Humanos , Lactoferrina/genética , Lactoferrina/metabolismo
5.
Front Immunol ; 11: 158, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32117294

RESUMO

Acinetobacter baumannii is an important human pathogen causing substantial mortality in hospitalized patients for which treatment with antibiotics has become problematic due to growing antibiotic resistance. In an attempt to develop alternative strategies for dealing with these serious infections surface antigens are being considered as targets for vaccines or immunotherapy. The surface receptor proteins required for zinc acquisition in Gram-negative bacterial pathogens have been proposed as vaccine targets due to their crucial role for growth in the human host. In this study we selected the putative ZnuD outer membrane receptor from A. baumannii as a target for vaccine development. Due to challenges in production of an integral outer membrane protein for vaccine production, we adopted a recently described hybrid antigen approach in which surface epitopes from the Neisseria meningitidis TbpA receptor protein were displayed on a derivative of the C-lobe of the surface lipoprotein TbpB, named the loopless C-lobe (LCL). A structural model for ZnuD was generated and four surface loops were selected for hybrid antigen production by computational approaches. Hybrid antigens were designed displaying the four selected loops (2, 5, 7, and 11) individually or together in a single hybrid antigen. The hybrid antigens along with ZnuD and the LCL scaffold were produced in the E. coli cytoplasm either as soluble antigens or as inclusion bodies, that were used to generate soluble antigens upon refolding. Mice were immunized with the hybrid antigens, ZnuD or LCL and then used in an A. baumannii sepsis model to evaluate their ability to protect against infection. As expected, the LCL scaffold did not induce a protective immune response, enabling us to attribute observed protection to the displayed loops. Immunization with the refolded ZnuD protein protected 63% of the mice while immunization with hybrid antigens displaying individual loops achieved between 25 and 50% protection. Notably, the mice immunized with the hybrid antigen displaying the four loops were completely protected from infection.


Assuntos
Infecções por Acinetobacter/imunologia , Acinetobacter baumannii/imunologia , Antígenos de Bactérias/imunologia , Vacinas Bacterianas/imunologia , Infecções por Acinetobacter/prevenção & controle , Animais , Proteínas da Membrana Bacteriana Externa/imunologia , Camundongos , Engenharia de Proteínas/métodos
6.
Front Immunol ; 10: 247, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30837995

RESUMO

The surface transferrin receptor proteins from Neisseria gonorrhoeae have been recognized as ideal vaccine targets due to their critical role in survival in the human male genitourinary tract. Recombinant forms of the surface lipoprotein component of the receptor, transferrin binding protein B (TbpB), can be readily produced at high levels in the Escherichia coli cytoplasm and is suitable for commercial vaccine production. In contrast, the integral outer membrane protein, transferrin binding protein A (TbpA), is produced at relatively low levels in the outer membrane and requires detergents for solubilization and stabilization, processes not favorable for commercial applications. Capitalizing on the core ß-barrel structural feature common to the lipoprotein and integral outer membrane protein we engineered the lipoprotein as a scaffold for displaying conserved surface epitopes from TbpA. A stable version of the C-terminal domain of TbpB was prepared by replacing four larger exposed variable loops with short linking peptide regions. Four surface regions from the plug and barrel domains of Neisseria TbpA were transplanted onto this TbpB C-lobe scaffold, generating stable hybrid antigens. Antisera generated in mice and rabbits against the hybrid antigens recognized TbpA at the surface of Neisseria meningitidis and inhibited transferrin-dependent growth at levels comparable or better than antisera directed against the native TbpA protein. Two of the engineered hybrid antigens each elicited a TbpA-specific bactericidal antibody response comparable to that induced by TbpA. A hybrid antigen generated using a foreign scaffold (TbpB from the pig pathogen Haemophilus parasuis) displaying neisserial TbpA loop 10 was evaluated in a model of lower genital tract colonization by N. gonorrhoeae and a model of invasive infection by N. meningitidis. The loop 10 hybrid antigen was as effective as full length TbpA in eliminating N. gonorrhoeae from the lower genital tract of female mice and was protective against the low dose invasive infection by N. meningitidis. These results demonstrate that TbpB or its derivatives can serve as an effective scaffold for displaying surface epitopes of integral outer membrane antigens and these antigens can elicit protection against bacterial challenge.


Assuntos
Neisseria gonorrhoeae/imunologia , Neisseria meningitidis/imunologia , Ligação Proteica/imunologia , Proteína A de Ligação a Transferrina/imunologia , Proteína B de Ligação a Transferrina/imunologia , Transferrina/imunologia , Sequência de Aminoácidos , Animais , Proteínas da Membrana Bacteriana Externa/imunologia , Sítios de Ligação/imunologia , Feminino , Gonorreia/imunologia , Ferro/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Coelhos , Alinhamento de Sequência , Suínos
7.
J Proteome Res ; 18(3): 934-946, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30582701

RESUMO

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.


Assuntos
Proteínas de Bactérias/imunologia , Reagentes para Ligações Cruzadas/química , Espectrometria de Massas/métodos , Receptores da Transferrina/imunologia , Proteínas de Bactérias/metabolismo , Vacinas Bacterianas/imunologia , Reagentes para Ligações Cruzadas/efeitos da radiação , Bactérias Gram-Negativas , Modelos Moleculares , Neisseria meningitidis , Receptores da Transferrina/metabolismo , Proteína B de Ligação a Transferrina/imunologia , Proteína B de Ligação a Transferrina/metabolismo
8.
Biometals ; 31(3): 381-398, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29767396

RESUMO

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.


Assuntos
Vacinas Bacterianas/imunologia , Moraxella catarrhalis/imunologia , Neisseria meningitidis/imunologia , Receptores de Superfície Celular/imunologia , Testes de Sensibilidade Microbiana , Moraxella catarrhalis/química , Neisseria meningitidis/química
9.
PLoS Pathog ; 13(3): e1006244, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-28257520

RESUMO

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.


Assuntos
Proteína B de Ligação a Transferrina/química , Proteína B de Ligação a Transferrina/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Interferometria , Ferro/metabolismo , Espectrometria de Massas , Modelos Moleculares , Simulação de Acoplamento Molecular , Mutagênese Sítio-Dirigida , Neisseria meningitidis/química , Neisseria meningitidis/metabolismo , Ligação Proteica
10.
Mol Cell Proteomics ; 15(9): 3071-80, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27412762

RESUMO

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.


Assuntos
Actinobacillus pleuropneumoniae/metabolismo , Reagentes para Ligações Cruzadas/química , Peptídeos/análise , Proteína B de Ligação a Transferrina/metabolismo , Transferrina/metabolismo , Actinobacillus pleuropneumoniae/química , Actinobacillus pleuropneumoniae/genética , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Modelos Moleculares , Mutação , Peptídeos/química , Ligação Proteica , Conformação Proteica , Software , Suínos , Espectrometria de Massas em Tandem , Transferrina/química , Proteína B de Ligação a Transferrina/química , Proteína B de Ligação a Transferrina/genética
11.
Vet Microbiol ; 187: 75-81, 2016 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-27066712

RESUMO

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 40days after arrival at a feedlot). The objective was to describe the evolution of the nasopharyngeal microbiota of beef cattle from weaning to 40days 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.


Assuntos
Biodiversidade , Microbiota/fisiologia , Nasofaringe/microbiologia , Animais , Bactérias/genética , Bactérias/isolamento & purificação , Complexo Respiratório Bovino/microbiologia , Bovinos , Suscetibilidade a Doenças/microbiologia , Masculino , Mycoplasma/genética , Mycoplasma/isolamento & purificação , RNA Ribossômico 16S/genética , Desmame
12.
Anal Biochem ; 501: 35-43, 2016 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-26898305

RESUMO

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.


Assuntos
Técnicas Biossensoriais/métodos , Carbono-Nitrogênio Ligases/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Interferometria/métodos , Mapeamento de Interação de Proteínas/métodos , Proteínas Repressoras/metabolismo , Proteínas de Bactérias/metabolismo , Biotinilação , Humanos , Ligantes , Ligases/metabolismo , Neisseria meningitidis/metabolismo , Ligação Proteica , Mapas de Interação de Proteínas , Transferrina/metabolismo , Proteína B de Ligação a Transferrina/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo
13.
PLoS Pathog ; 11(8): e1005107, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26295949

RESUMO

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.


Assuntos
Metabolismo dos Carboidratos/fisiologia , Infecções por Enterobacteriaceae/metabolismo , Infecções por Enterobacteriaceae/transmissão , Intestinos/microbiologia , Animais , Transporte Biológico Ativo/fisiologia , Calorimetria , Cromatografia Líquida , Citrobacter rodentium , Mucosa Intestinal/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Mutagênese Sítio-Dirigida , Fosforilação , Filogenia , Espectrometria de Massas em Tandem
14.
Vaccine ; 33(42): 5700-5707, 2015 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-26263196

RESUMO

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.


Assuntos
Actinobacillus pleuropneumoniae/metabolismo , Actinobacillus suis/metabolismo , Proteínas de Bactérias/imunologia , Haemophilus parasuis/metabolismo , Receptores da Transferrina/imunologia , Proteína B de Ligação a Transferrina/imunologia , Actinobacillus pleuropneumoniae/genética , Actinobacillus suis/genética , Animais , Variação Antigênica , Proteínas de Bactérias/genética , Reações Cruzadas , Haemophilus parasuis/genética , Masculino , Simulação de Acoplamento Molecular , Filogenia , Receptores da Transferrina/genética , Suínos , Proteína B de Ligação a Transferrina/genética
16.
Microbiologyopen ; 4(3): 491-504, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25800619

RESUMO

Neisseria meningitidis inhabits the human upper respiratory tract and is an important cause of sepsis and meningitis. A surface receptor comprised of transferrin-binding proteins A and B (TbpA and TbpB), is responsible for acquiring iron from host transferrin. Sequence and immunological diversity divides TbpBs into two distinct lineages; isotype I and isotype II. Two representative isotype I and II strains, B16B6 and M982, differ in their dependence on TbpB for in vitro growth on exogenous transferrin. The crystal structure of TbpB and a structural model for TbpA from the representative isotype I N. meningitidis strain B16B6 were obtained. The structures were integrated with a comprehensive analysis of the sequence diversity of these proteins to probe for potential functional differences. A distinct isotype I TbpA was identified that co-varied with TbpB and lacked sequence in the region for the loop 3 α-helix that is proposed to be involved in iron removal from transferrin. The tightly associated isotype I TbpBs had a distinct anchor peptide region, a distinct, smaller linker region between the lobes and lacked the large loops in the isotype II C-lobe. Sequences of the intact TbpB, the TbpB N-lobe, the TbpB C-lobe, and TbpA were subjected to phylogenetic analyses. The phylogenetic clustering of TbpA and the TbpB C-lobe were similar with two main branches comprising the isotype 1 and isotype 2 TbpBs, possibly suggesting an association between TbpA and the TbpB C-lobe. The intact TbpB and TbpB N-lobe had 4 main branches, one consisting of the isotype 1 TbpBs. One isotype 2 TbpB cluster appeared to consist of isotype 1 N-lobe sequences and isotype 2 C-lobe sequences, indicating the swapping of N-lobes and C-lobes. Our findings should inform future studies on the interaction between TbpB and TbpA and the process of iron acquisition.


Assuntos
Proteínas de Bactérias , Variação Genética , Neisseria meningitidis/genética , Receptores da Transferrina/química , Receptores da Transferrina/genética , Sequência de Aminoácidos , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Neisseria meningitidis/classificação , Filogenia , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Alinhamento de Sequência
17.
ISME J ; 9(5): 1246-59, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25575312

RESUMO

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.


Assuntos
Infecções Bacterianas/microbiologia , Trato Gastrointestinal/microbiologia , Microbiota/imunologia , Nasofaringe/microbiologia , Orofaringe/microbiologia , Adulto , Fatores Etários , Infecções Bacterianas/diagnóstico , Carga Bacteriana , Criança , Pré-Escolar , DNA Bacteriano/genética , Voluntários Saudáveis , Humanos , Lactente , Filogenia , RNA Ribossômico 16S/genética , Streptococcus pneumoniae
18.
Infect Immun ; 83(3): 1030-8, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25547790

RESUMO

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.


Assuntos
Anticorpos Antibacterianos/biossíntese , Infecções por Haemophilus/prevenção & controle , Vacinas Anti-Haemophilus/imunologia , Haemophilus parasuis/imunologia , Imunoglobulina M/biossíntese , Proteína B de Ligação a Transferrina/imunologia , Animais , Linfócitos B/citologia , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Expressão Gênica , Infecções por Haemophilus/imunologia , Infecções por Haemophilus/microbiologia , Vacinas Anti-Haemophilus/administração & dosagem , Vacinas Anti-Haemophilus/genética , Haemophilus parasuis/química , Haemophilus parasuis/efeitos dos fármacos , Imunidade Celular , Imunidade Humoral , Ferro/metabolismo , Mutagênese Sítio-Dirigida , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Suínos , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Transferrina/genética , Transferrina/metabolismo , Proteína B de Ligação a Transferrina/administração & dosagem , Proteína B de Ligação a Transferrina/genética , Vacinação
19.
Biometals ; 27(5): 923-33, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25038734

RESUMO

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.


Assuntos
Peptídeos Catiônicos Antimicrobianos/antagonistas & inibidores , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/metabolismo , Lactoferrina/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Transporte/química , Proteínas de Transporte/genética , Catelicidinas/antagonistas & inibidores , Especificidade de Hospedeiro , Humanos , Ferro/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Neisseria meningitidis/genética , Neisseria meningitidis/metabolismo , Neisseria meningitidis/patogenicidade , Conformação Proteica , Receptores de Superfície Celular/metabolismo , Serina Endopeptidases/metabolismo
20.
BMC Microbiol ; 14: 143, 2014 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-24889110

RESUMO

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.


Assuntos
Primers do DNA/genética , Tipagem de Sequências Multilocus/métodos , Streptococcus pneumoniae/classificação , Streptococcus pneumoniae/genética , Adolescente , Canadá , Criança , Pré-Escolar , Humanos , Lactente , Recém-Nascido , Infecções Estreptocócicas/microbiologia , Streptococcus pneumoniae/isolamento & purificação
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