Membrane vesicles released by Actinobacillus pleuropneumoniae contain proteases and Apx toxins

Actinobacillus pleuropneumoniae serotype 1 releases vesicles containing pro teases and Apr toxins into the culture medium. Vesicles were concentrated b y ultracentrifugation and analyzed by electron microscopy and electrophores is; their size ranged from 20 to 200 nm. A polyclonal antiserum raised agai nst a purified high molecular mass secreted protease of serotype 1 recogniz ed this protease on the surface of the vesicles by immunogold electron micr oscopy. Higher molecular mass polypeptides from vesicle extracts were recog nized by the antiserum by Western immunoblot, indicating that the protease could form oligomers. However, these oligomers were not active against gela tin until secreted. Additionally, Apx toxins were also present in vesicles, and were recognized by Western immunoblot by an anti-serotype 1 toxins pol yclonal 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. (C) 2000 Published by Elsevier Science B.V, on behalf of the Federation of European Microbiological Societies.