RESUMO
Histophilus somni causes bovine pneumonia and septicemia, but protective immune responses are not well understood and immunodiagnostic methods are not well defined. We previously showed that antibody to a new virulence factor, IbpA, neutralizes cytotoxicity and immunization with a recombinant IbpA domain protects calves against experimental H. somni pneumonia. To further define immune responses to IbpA, we determined isotypic serum antibody responses to three IbpA domains (IbpA3, an N-terminal coiled coil region; IbpA5, a central region of 200 bp repeats and IbpA DR2, a C-terminal cytotoxic domain). ELISA was used to quantitate IgG1 or IgG2 antibodies to each of the IbpA subunits as well as H. somni whole cells (WCs) or culture supernatant (SUP). Calves experimentally infected with H. somni and monitored for up to 10 weeks had the least "0 time" (background) antibody levels to IbpA5, as well as the earliest and highest responses of greatest duration to the IbpA5 subunit. Responses of these calves were high to WC or SUP antigens but with higher "0 time" (background) antibody levels. We concluded that IbpA5 may be a useful immunodiagnostic antigen. Calves immunized with H. somni WC vaccine had antibody responses to WC antigens, but not to IbpA subunits before challenge. After challenge with H. somni, vaccinated calves had slight anamnestic responses to IbpA3 and IbpA5, but not to IbpA DR2. Since IbpA DR2 is a protective antigen, the data suggest the IbpA DR2 would be a useful addition to H. somni vaccines.
Assuntos
Formação de Anticorpos , Antígenos de Bactérias/imunologia , Doenças dos Bovinos/imunologia , Bovinos/imunologia , Infecções por Pasteurellaceae/veterinária , Pasteurellaceae/imunologia , Pneumonia Bacteriana/veterinária , Animais , Anticorpos Antibacterianos/sangue , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/metabolismo , Doenças dos Bovinos/microbiologia , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Imunoglobulina G/sangue , Masculino , Pasteurellaceae/patogenicidade , Infecções por Pasteurellaceae/imunologia , Infecções por Pasteurellaceae/microbiologia , Pneumonia Bacteriana/imunologia , Pneumonia Bacteriana/microbiologia , Subunidades Proteicas/imunologia , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/imunologia , Fatores de Virulência/imunologia , Fatores de Virulência/metabolismoRESUMO
Histophilus somni causes bovine pneumonia as well as septicemia and its sequelae but mechanisms of virulence and protective immunity are poorly understood. Since surface immunoglobulin binding proteins are virulence factors, we addressed their role as protective antigens in a mouse model of H. somni septicemia. Immunoglobulin binding protein A (IbpA), has homology to Bordetella pertussis filamentous hemagglutinin and other large bacterial exoproteins. IbpA is a major surface antigen encoded by the ibpA gene with many domains that may be important in pathogenesis and immune protection. Three IbpA recombinant protein subunits, IbpA3, IbpA5 and IbpADR2 were chosen for study because of putative functional domains and motifs. These recombinant GST fusion subunit proteins were compared with GST (negative control), formalin-killed H. somni (commercial vaccine control), live H. somni (to induce convalescent immunity) and H. somni culture supernatant (containing IbpA shed from the bacterial surface). In vaccination/challenge studies, both live H. somni (convalescent immunity) and supernatant protected equally but formalin-killed H. somni and GST did not protect against septicemia. The DR2 and A3 subunits protected moderately well and induced antibody responses against supernatant antigen and the homologous subunit in ELISA but not against whole cell antigens. Supernatant immunization protected better than the IbpA subunit antigens and induced high antibody activity against both whole cells and supernatant antigens. The results indicate that culture supernatant antigens or perhaps recombinant IbpA subunits may be useful in H. somni vaccines. These studies also provide insight into the contribution of IbpA domains to pathogenesis of H. somni septicemia.
Assuntos
Proteínas de Bactérias/imunologia , Vacinas Bacterianas/imunologia , Doenças dos Bovinos/prevenção & controle , Infecções por Pasteurellaceae/veterinária , Pasteurellaceae/imunologia , Sepse/veterinária , Animais , Anticorpos Antibacterianos/sangue , Proteínas de Bactérias/genética , Vacinas Bacterianas/genética , Bovinos , Ensaio de Imunoadsorção Enzimática , Feminino , Camundongos , Pasteurellaceae/genética , Infecções por Pasteurellaceae/prevenção & controle , Sepse/prevenção & controle , Índice de Gravidade de Doença , Análise de Sobrevida , Vacinas Atenuadas/imunologia , Vacinas de Produtos Inativados/imunologia , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologiaRESUMO
The role of bovine serum or plasma proteins in Haemophilus somnus virulence was investigated in a mouse model of septicemia. An increase in virulence was detected when the organism was pre-incubated for 5 min and inoculated with fetal calf serum. When purified bovine serum or plasma proteins were pre-incubated with H. somnus before inoculating into mice, transferrin was found to increase virulence. Bovine lactoferrin was also noted to increase virulence, but to a lesser extent and had a delayed time course when compared with transferrin. Using an ELISA assay, an increased amount of H. somnus whole cells and culture supernatant bound to bovine transferrin when the organism was grown in iron-restricted media. Lactoferrin also bound to H. somnus, but binding was not affected by growth in iron-restricted media and it was eliminated with 2M NaCl, which reversed charge mediated binding. Transferrin, but not lactoferrin, supported growth of H. somnus on iron-depleted agar based media using a disk assay. Therefore, lactoferrin increased virulence by an undetermined mechanism whereas transferrin increased virulence of H. somnus by binding to iron-regulated outer-membrane proteins (IROMPs) and providing iron to the pathogen.
Assuntos
Bacteriemia/microbiologia , Bovinos/microbiologia , Infecções por Haemophilus/microbiologia , Haemophilus somnus/patogenicidade , Transferrina/metabolismo , Animais , Bovinos/metabolismo , Meios de Cultura , Feminino , Haemophilus somnus/crescimento & desenvolvimento , Haemophilus somnus/metabolismo , Ferro/metabolismo , Lactoferrina/metabolismo , Camundongos , Camundongos Endogâmicos , Ligação Proteica , Transferrina/química , VirulênciaRESUMO
Bovine respiratory syncytial virus (BRSV) and Haemophilus somnus (H. somnus) co-infect to form a polymicrobial respiratory disease in calves. Both BRSV and H. somnus vaccinations have independently been shown to sometimes induce adverse IgE mediated responses. We hypothesized that combining these disease agents in vaccination would induce cytokine shifts resulting in greater IgE production and enhanced disease. Concurrent vaccination with subsequent infection with one or both pathogens in calves was conducted to evaluate the isotypic antibody responses, disease severity and cytokine response. BRSV-specific serum IgE levels were elevated for the most clinically diseased calves, while no difference was detected in the IgE levels to H. somnus among groups. The IFN-gamma message and H. somnus-specific IgG2 antibodies were significantly elevated in calves with the lowest clinical scores. Vaccination preferentially stimulated higher levels of IgG1 antibodies to BRSV, but in contrast higher levels of IgG2 antibodies to H. somnus. Concurrent vaccination induced IgE antibodies to BRSV, which were directly correlated with disease severity whereas vaccine induced IgG2 antibodies to H. somnus were inversely correlated with disease severity.
Assuntos
Doenças dos Bovinos/imunologia , Infecções por Haemophilus/veterinária , Vacinas Anti-Haemophilus/imunologia , Haemophilus somnus/imunologia , Infecções por Vírus Respiratório Sincicial/veterinária , Vacinas contra Vírus Sincicial Respiratório/imunologia , Vírus Sincicial Respiratório Bovino/imunologia , Animais , Anticorpos Antibacterianos/sangue , Anticorpos Antivirais/sangue , Bovinos , Doenças dos Bovinos/prevenção & controle , Citocinas/biossíntese , Ensaio de Imunoadsorção Enzimática , Infecções por Haemophilus/imunologia , Infecções por Haemophilus/fisiopatologia , Vacinas Anti-Haemophilus/administração & dosagem , Imunoglobulina E/sangue , Imunoglobulina G/sangue , Infecções por Vírus Respiratório Sincicial/imunologia , Infecções por Vírus Respiratório Sincicial/fisiopatologia , Vacinas contra Vírus Sincicial Respiratório/administração & dosagem , Vacinas de Produtos Inativados/administração & dosagem , Vacinas de Produtos Inativados/imunologiaRESUMO
DsbD and DsbB are two proteins that in Escherichia coli catalyze transmembrane electron flow in opposite directions, thereby allowing reversible oxidoreduction of periplasmic dithiol/disulfide-containing proteins. We have identified all recognizable homologues of these two proteins in the databases and have conducted structural and phylogenetic analyses of the two families. The larger DsbD family is more diverse in sequence, topology, function and organismal distribution than the smaller DsbB family. DsbB homologues are rarely found outside of the proteobacteria, although DsbD homologues are found in many bacterial kingdoms as well as archaea and plant chloroplasts. Few organisms with a fully sequenced genome and a DsbB homologue lack a DsbD homologue, and most of these DsbD homologues fall within two clusters in the DsbD tree, exhibiting phylogenetic relationships that are the same as those observed for the DsbB proteins. These observations suggest that a subset of the DsbD homologues evolved in parallel with the DsbB family to perform a single unified function involving reversible extracytoplasmic protein dithiol-disulfide bond interchange. DsbD family proteins are shown to have arisen by an internal gene duplication event, and this observation leads to prediction of the pathway taken for the evolutionary appearance of the different protein topological types found within this family.
Assuntos
Archaea/metabolismo , Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , Transporte de Elétrons , Proteínas de Membrana/metabolismo , Isomerases de Dissulfetos de Proteínas/metabolismo , Proteínas de Bactérias/classificação , Proteínas de Bactérias/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Potenciais da Membrana , Proteínas de Membrana/classificação , Proteínas de Membrana/genética , Isomerases de Dissulfetos de Proteínas/classificação , Isomerases de Dissulfetos de Proteínas/genética , Força Próton-MotrizRESUMO
The N-terminal domain of the human phagocyte flavocytochrome b558 NADPH oxidase, gp91phox, is believed to be a heme-containing voltage-gated H+ channel. The authors have conducted structural, sequence and phylogenetic analyses of the putative transmembrane channel/heme-binding domains of all homologous proteins in the NCBI GenBank database as of May 2001, as well as of the full-length proteins. Fifty-six homologues were identified, including 26 from animals, 19 from plants, seven from yeast, one from a slime mould and three from bacteria. Six well-defined sub-families were revealed by phylogenetic tree construction, two consisting of animal proteins, two of plant proteins, and one each of yeast and bacterial homologues, with the slime mould protein clustering loosely with one of the animal clusters. Signature sequences for the entire family as well as for the sub-families were determined. Most proteins have six putative TMSs, four of which may comprise the heme-binding H+ channel. The hydrophobic and amphipathic characteristics of each of the putative alpha-helical transmembrane segments were defined, and conserved residues that may be involved in heme binding, channel formation, and/or conformational changes were identified. The analyses lead to the suggestion that the oxidase domain became associated with the channel/heme-binding domain to form a single polypeptide chain early in evolutionary history, before eukaryotes diverged from prokaryotes, and that genetic transmission to present day organisms occurred primarily by vertical descent.