In vitro production of a secreted hemolytic
cytolysin of Actinobacillus pleuropneumoniae has been reported to be dependent on the presence of
calcium in
culture media. This is not the case with Escherichia coli
hemolysins, however, where
calcium has been shown to be required only for activation and binding to target cells. Because the
cytolysins of A. pleuropneumoniae have structural and functional similarities to those of hemolytic E. coli, we sought to reexamine the role that
calcium plays in the secretion and activity of A. pleuropneumoniae
cytolysins. A. pleuropneumoniae hemolytic strain S4074 secreted two major
proteins into culture supernatants independent of the presence of
calcium in growth medium. These
proteins were identified with murine
monoclonal antibodies as the 105-kDa
cytolysin I and the 103-kDa
cytolysin II. It was found that both
cytolysins required
calcium for binding to erythrocyte membranes. Culture fluids from bacteria grown with
calcium lysed porcine erythrocytes even after free
calcium in the fluid was removed prior to the hemolytic assay. When bacteria were grown in medium depleted of
calcium, no lysis of erythrocytes was detected unless
calcium was added to assay
buffers. Culture supernatants from A. pleuropneumoniae nonhemolytic strain 1421 grown with or without
calcium contained two predominant
proteins, which were identified with mouse
monoclonal antibodies as the 103-kDa
cytolysin II and the 120-kDa
cytolysin III. Binding to erythrocytes (without
hemolysis) by
cytolysin II was dependent on
calcium.
Cytolysin III did not bind to erythrocytes. These results indicate that the ability of strain S4074 to lyse swine erythrocytes (and the inability of strain 1421 to do so) was directly correlated with the presence of
cytolysin I.
Cytolysins I, II, and III bound to swine neutrophils and purified neutrophil membranes only in the presence of
calcium. When
calcium was depleted,
cytolysin I of strain S4074 had a reduced binding and cytolysis II and III of strain 1421 did not bind at all. The data suggest that regardless of the target cell involved,
calcium plays an integral role in the function but not the production of A. pleuropneumoniae
cytolysins.