Design And Characterization Of A Novel Tool For The Antigenic Enrichment Of Actinobacillus Pleuropneumoniae Outer Membrane

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.