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Cloning, sequencing, expression, and protective capacity of the oma87 gene encoding the Pasteurella multocida 87-kilodalton outer membrane antigen.

ABSTRACT: Membrane proteins of Pasteurella multocida have been shown previously to elicit protective immunity. We have identified an 87-kDa outer membrane antigen, Oma87, which is present in all 16 serotypes of P. multocida. The gene encoding this protein was cloned and sequenced and found to have significant similarity to the D15 protective surface antigen of Haemophilus influenzae. Oma87 was localized to the outer membrane of the cell, and proteinase K treatment suggested that the protein is surface exposed. Native and recombinant Oma87 were strongly immunostained by convalescent-phase antiserum, indicating that the protein is expressed in vivo. Specific Oma87 antiserum protected mice against homologous, lethal P. multocida challenge. These results suggest that Oma87 is a protective outer membrane antigen of P. multocida.

SUBMITTER: Ruffolo CG

PROVIDER: S-EPMC174202 | biostudies-other | 1996 Aug

REPOSITORIES: biostudies-other

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Cloning, sequencing, expression, and protective capacity of the oma87 gene encoding the Pasteurella multocida 87-kilodalton outer membrane antigen.

Ruffolo C G CG Adler B B

Infection and immunity 19960801 8

Membrane proteins of Pasteurella multocida have been shown previously to elicit protective immunity. We have identified an 87-kDa outer membrane antigen, Oma87, which is present in all 16 serotypes of P. multocida. The gene encoding this protein was cloned and sequenced and found to have significant similarity to the D15 protective surface antigen of Haemophilus influenzae. Oma87 was localized to the outer membrane of the cell, and proteinase K treatment suggested that the protein is surface exp ...[more]

PMID: 8757848

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Project description:In our continued effort to search for a Streptococcus suis protein(s) that can serve as a vaccine candidate or a diagnostic reagent, we constructed and screened a gene library with a polyclonal antibody raised against the whole-cell protein of S. suis type 2. A clone that reacted with the antibody was identified and characterized. Analysis revealed that the gene encoding the protein is localized within a 2.0-kbp EcoRI DNA fragment. The nucleotide sequence contained an open reading frame that encoded a polypeptide of 445 amino acid residues with a calculated molecular mass of 46.4 kDa. By in vitro protein synthesis and Western blot experiments, the protein exhibited an electrophoretic mobility of approximately 38 kDa. At the amino acid level the deduced primary sequence shared homology with sequences of unknown function from Streptococcus pneumoniae (89%), Streptococcus mutans (86%), Lactococcus lactis (80%), Listeria monocytogenes (74%), and Clostridium perfringens (64%). Except for strains of serotypes 20, 26, 32, and 33, Southern hybridization analysis revealed the presence of the gene in strains of other S. suis serotypes and demonstrated restriction fragment length differences caused by a point mutation in the EcoRI recognition sequence. We confirmed expression of the 38-kDa protein in the hybridization-positive isolates using specific antiserum against the purified protein. The recombinant protein was reactive with serum from pigs experimentally infected with virulent strains of S. suis type 2, suggesting that the protein is immunogenic and may serve as an antigen of diagnostic importance for the detection of most S. suis infections. Pigs immunized with the recombinant 38-kDa protein mounted antibody responses to the protein and were completely protected against challenge with a strain of a homologous serotype, the wild-type virulent strain of S. suis type 2, suggesting that it may be a good candidate for the development of a vaccine that can be used as protection against S. suis infection. Analysis of the cellular fractions of the bacterium by Western blotting revealed that the protein was present in the surface and cell wall extracts. The functional role of the protein with respect to pathogenesis and whether antibodies against the antigen confer protective immunity against diseases caused by strains of other pathogenic S. suis capsular types remains to be determined.

Project description:Pasteurella multocida is an important bacterial pathogen that can cause diseases in both animals and humans. Its elevated morbidity and mortality rates in animals result in substantial economic repercussions within the livestock industry. The prevention of diseases caused by P. multocida through immunization is impeded by the absence of a safe and effective vaccine. Outer membrane vesicles (OMVs) secreted from the outer membrane of Gram-negative bacteria are spherical vesicular structures that encompass an array of periplasmic components in conjunction with a diverse assortment of lipids and proteins. These vesicles can induce antibacterial immune responses within the host. P. multocida has been shown to produce OMVs. Nonetheless, the precise characteristics and immunomodulatory functions of P. multocida OMVs have not been fully elucidated. In this study, OMVs were isolated from P. multocida using an ultrafiltration concentration technique, and their morphology, protein constitution, and immunomodulatory properties in RAW264.7 cells were studied. Transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA) revealed that the OMVs exhibited typical spherical and bilayered lipid vesicular architecture, exhibiting an average diameter of approximately 147.5 nm. The yield of OMVs was 2.6 × 1011 particles/mL. Proteomic analysis revealed a high abundance of membrane-associated proteins within P. multocida OMVs, with the capability to instigate the host's immune response. Furthermore, OMVs stimulated the proliferation and cellular uptake of macrophages and triggered the secretion of cytokines, such as TNF-ɑ, IL-1β, IL-6, IL-10, and TGF-β1. Consequently, our results indicated that OMVs from P. multocida could directly interact with macrophages and regulate their immune function in vitro. These results supported the prospective applicability of P. multocida OMVs as a platform in the context of vaccine development. KEY POINTS: • Preparation and characterization of P. multocida OMVs. • P. multocida OMVs possess a range of antigens and lipoproteins associated with the activation of the immune system. • P. multocida OMVs can activate the proliferation, internalization, and cytokine secretion of macrophages in vitro.

Dataset Information

Cloning, sequencing, expression, and protective capacity of the oma87 gene encoding the Pasteurella multocida 87-kilodalton outer membrane antigen.

Publications

Cloning, sequencing, expression, and protective capacity of the oma87 gene encoding the Pasteurella multocida 87-kilodalton outer membrane antigen.

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