Bacillus cereus
sphingomyelinase (Bc-SMase) induces
hemolysis of sheep erythrocytes which contain large amounts of
sphingomyelin. We investigated the mechanism of this
hemolysis in comparison to that induced by
Clostridium perfringens alpha-toxin.
Pertussis toxin, a Gi-specific inhibitor, N-oleoylethernolamine, a
ceramidase inhibitor, and
dihydrosphingosine, a
sphingosine kinase inhibitor, did not inhibit the
hemolysis by Bc-SMase, but did inhibit that by alpha-toxin. Bc-SMase broadly bound to whole membranes, and alpha-toxin specifically bound to the
detergent-resistant membrane fractions,
lipid rafts. The level of
ceramide production induced by Bc-SMase in sheep erythrocytes was 6- to 15-fold that induced by alpha-toxin, when the extent of the
hemolysis by Bc-SMase was the same as that by the toxin. However, the level of
ceramide production induced by Bc-SMase in SM-
liposomes was equal to that triggered by the toxin, when the carboxyl
fluorescein-release from
liposomes induced by Bc-SMase was the same as that induced by alpha-toxin. Confocal
laser microscopy showed that treatment of the cells with Bc-SMase resulted in the formation of
ceramide-rich domains. A photobleaching analysis suggested that treatment of the cells with Bc-SMase leads to a reduction in membrane fluidity. These results show that Bc-SMase-induced
hemolysis of sheep erythrocytes is related to the formation of interface between
ceramide-rich domains and
ceramide-poor domains through production of
ceramide from SM.