Membrane vesicles released by Actinobacillus pleuropneumoniae contain proteases and Apx toxins

Actinobacillus pleuropneumoniae serotype 1 releases vesicles containing proteases and Apx toxins into the culture medium. Vesicles were concentrated by ultracentrifugation and analyzed by electron microscopy and electrophoresis; their size ranged from 20 to 200 nm. A polyclonal antiserum raised against a purified high molecular mass secreted protease of serotype 1 recognized this protease on the surface of the vesicles by immunogold electron microscopy. Higher molecular mass polypeptides from vesicle extracts were recognized by the antiserum by Western immunoblot, indicating that the protease could form oligomers. However, these oligomers were not active against gelatin until secreted. Additionally, Apx toxins were also present in vesicles, and were recognized by Western immunoblot by an anti-serotype 1 toxins polyclonal serum. A. pleuropneumoniae antigens in vesicles were recognized by convalescent-phase pig sera from animals infected with serotype 1 or 5. The release of vesicles containing virulence factors could be a tissue damage mechanism in swine pleuropneumonia.