Dataset Information

Design and Characterization of a Novel Tool for the Antigenic Enrichment of Actinobacillus pleuropneumoniae Outer Membrane.

ABSTRACT: Production and isolation of recombinant proteins are costly and work-intensive processes, especially in immunology when tens or hundreds of potential immunogens need to be purified for testing. Here we propose an alternative method for fast screening of immunogen candidates, based on genetic engineering of recombinant bacterial strains able to express and expose selected antigens on their outer membrane. In Actinobacillus pleuropneumoniae, a Gram-negative porcine pathogen responsible for extensive economic losses worldwide, we identified a conserved general secretion pathway (GSP) domain in the N-terminal part of the outer membrane protein ApfA (ApfA stem: ApfA_s). ApfA_s was used as an outer membrane anchor, to which potential immunogens can be attached. To enable confirmation of correct positioning, ApfA_s, was cloned in combination with the modified acyl carrier protein (ACP) fluorescent tag ACP mini (ACP_m) and the putative immunogen VacJ. The chimeric construct was inserted in the pMK-express vector, subsequently transformed into A. pleuropneumoniae for expression. Flow cytometry, fluorescence imaging and mass spectrometry analysis were employed to demonstrate that the outer membrane of the transformed strain was enriched with the chimeric ApfA_s-ACP_m-VacJ antigen. Our results confirmed correct positioning of the chimeric ApfA_s-ACP_m-VacJ antigen and supported this system's potential as platform technology enabling antigenic enrichment of the outer membrane of A. pleuropneumoniae.

SUBMITTER: Antenucci F

PROVIDER: S-EPMC7761619 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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Design and Characterization of a Novel Tool for the Antigenic Enrichment of <i>Actinobacillus pleuropneumoniae</i> Outer Membrane.

Antenucci Fabio F Ovsepian Armen A Wrobel Agnieszka A Winther-Larsen Hanne Cecilie HC Bojesen Anders Miki AM

Pathogens (Basel, Switzerland) 20201202 12

Production and isolation of recombinant proteins are costly and work-intensive processes, especially in immunology when tens or hundreds of potential immunogens need to be purified for testing. Here we propose an alternative method for fast screening of immunogen candidates, based on genetic engineering of recombinant bacterial strains able to express and expose selected antigens on their outer membrane. In <i>Actinobacillus pleuropneumoniae</i>, a Gram-negative porcine pathogen responsible for ...[more]

PMID: 33276526

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Project description:Outer membrane vesicles (OMVs) are spontaneously released by Gram-negative bacteria, including Actinobacillus pleuropneumoniae, which causes contagious pleuropneumonia in pigs and leads to considerable economic losses in the swine industry worldwide. A. pleuropneumoniae OMVs have previously been demonstrated to contain Apx toxins and proteases, as well as antigenic proteins. Nevertheless, comprehensive characterizations of their contents and interactions with host immune cells have not been made. Understanding the protein compositions and immunomodulating ability of A. pleuropneumoniae OMVs could help illuminate their biological functions and facilitate the development of OMV-based applications. In the current investigation, we comprehensively characterized the proteome of native A. pleuropneumoniae OMVs. Moreover, we qualitatively and quantitatively compared the OMV proteomes of a wild-type strain and three mutant strains, in which relevant genes were disrupted to increase OMV production and/or produce OMVs devoid of superantigen PalA. Furthermore, the interaction between A. pleuropneumoniae OMVs and porcine alveolar macrophages was also characterized. Our results indicate that native OMVs spontaneously released by A. pleuropneumoniae MIDG2331 appeared to dampen the innate immune responses by porcine alveolar macrophages stimulated by either inactivated or live parent cells. The findings suggest that OMVs may play a role in manipulating the porcine defense during the initial phases of the A. pleuropneumoniae infection. IMPORTANCE Owing to their built-in adjuvanticity and antigenicity, bacterial outer membrane vesicles (OMVs) are gaining increasing attention as potential vaccines for both human and animal use. OMVs released by Actinobacillus pleuropneumoniae, an important respiratory pathogen in pigs, have also been investigated for vaccine development. Our previous studies have shown that A. pleuropneumoniae secretes OMVs containing multiple immunogenic proteins. However, immunization of pigs with these vesicles was not able to relieve the pig lung lesions induced by the challenge with A. pleuropneumoniae, implying the elusive roles that A. pleuropneumoniae OMVs play in host-pathogen interaction. Here, we showed that A. pleuropneumoniae secretes OMVs whose yield and protein content can be altered by the deletion of the nlpI and palA genes. Furthermore, we demonstrate that A. pleuropneumoniae OMVs dampen the immune responses in porcine alveolar macrophages stimulated by A. pleuropneumoniae cells, suggesting a novel mechanism that A. pleuropneumoniae might use to evade host defense.

Dataset Information

Design and Characterization of a Novel Tool for the Antigenic Enrichment of Actinobacillus pleuropneumoniae Outer Membrane.

Publications

Design and Characterization of a Novel Tool for the Antigenic Enrichment of <i>Actinobacillus pleuropneumoniae</i> Outer Membrane.

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