RESUMEN
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.
Asunto(s)
Bacillus cereus/enzimología , Membrana Eritrocítica/química , Hemólisis , Microdominios de Membrana/química , Esfingomielina Fosfodiesterasa/química , Esfingosina/análogos & derivados , Animales , Bacillus cereus/genética , Toxinas Bacterianas/química , Proteínas de Unión al Calcio/química , Ceramidas/química , Fluidez de la Membrana , Toxina del Pertussis/química , Ovinos , Esfingomielina Fosfodiesterasa/genética , Esfingosina/química , Fosfolipasas de Tipo C/químicaRESUMEN
Clostridium perfringens alpha-toxin, an important agent of gas gangrene with inflammatory myopathies, possesses lethal, hemolytic, and necrotic activities. Here, we show that alpha-toxin-induced lethality in mice was inhibited by i.v. preadministration of erythromycin (ERM). Administration of ERM resulted in a drastic reduction in the release of tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, and IL-6 and systemic hemolysis induced by alpha-toxin, whereas the administration of kitasamycin did not. Furthermore, the lethality and systemic hemolysis caused by alpha-toxin were blocked by the preinjection of anti-TNF-alpha, but not the anti-IL-1beta- or anti-IL-6-antibody. In addition, TNF-alpha-deficient mice were resistant to alpha-toxin, indicating that TNF-alpha plays an important role in the lethality. ERM inhibited the toxin-induced release of TNF-alpha from neutrophils and phosphorylation of toropomyosin-related kinase receptor A (TrkA) and extracellular-regulated kinase (ERK) 1/2. Furthermore, K252a, a TrkA inhibitor, and PD98059 (2'-amino-3'-methoxyflavone), an ERK1/2 inhibitor, inhibited the toxin-induced release of TNF-alpha from neutrophils. The observation shows that the toxin-induced release of TNF-alpha is dependent on the activation of ERK/mitogen-activated protein kinase signal transduction via TrkA in neutrophils and that ERM specifically blocks the toxin-induced events through the activation of neutrophils.