Hemolysis induced by Bacillus cereus sphingomyelinase

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.