Immunogenicity of Actinobacillus pleuropneumoniae outer membrane proteins and enhancement of phagocytosis by antibodies to the proteins

Abstract

To determine the opsonic effect of antibodies to Actinobacillus (Haemophilus) pleuropneumoniae outer membrane proteins on phagocytosis by porcine polymorphonuclear leukocytes (PMN), we separated the integral outer membrane proteins (IOMPs) by Triton X-114 extraction. Four major IOMPs with molecular masses of 76, 50, 39, and 29 kDa were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These IOMPs were found to be essentially free of endotoxin in the Limulus amebocyte lysate assay. The 76-kDa protein exhibited a more intensely stained electrophoresis band when isolated from iron-restricted cultures, and a new band at 105 kDa was present in the whole-membrane fraction but not in the integral fraction, indicating that the 105-kDa iron-repressible protein is a peripheral membrane protein. The 76-, 50-, and 39-kDa proteins were shown to be surface exposed, since antibodies to these IOMPs could be absorbed out of convalescent-phase sera by whole cells. Percentages of phagocytosis by porcine PMN of A. pleuropneumoniae opsonized with convalescent-phase sera, convalescent-phase sera absorbed with IOMPs, or convalescent-phase sera absorbed with whole cells were 46.75, 21.81, and 7.96%, respectively. These results demonstrate that antibodies to IOMPs of A. pleuropneumoniae serve as important opsonins in phagocytosis by porcine PMN.