A study on the mechanism underlying the action of Clostridium perfringens alpha- and epsilon-toxins to membrane lipid rafts

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Epsilon- and alpha-toxins produced by Clostridium perfringens are major toxins most responsible for enterotoxeamia in domestic animals and human gas gangrene, respectively. In an attempt to elucidate the molecular mechanism by which the two toxins exhibit their toxicity, we have undertaken this research. Epsilon-Toxin has been shown to exhibit high affinity to lipid rafts of neuronal and kidney cell membranes and to permealize the membranes through heptamerization of the toxin. We examined how a membrane lipid environment affects the binding of the toxin to lipid rafts and its heptamerization. The depletion and bio synthesis inhibition of cholesterol, a major lipid component of MDCK cell lipid rafts, inhibited the heptamerization of the toxin, while the bio synthesis inhibition of sphingolipid, another one, by the treatment with fumonisin B1 increased the sensitivity of MDCK cells against epsilon-toxin. Similar results were observed with PDMP, an inhibitor of glycosphigolipid bio synth … More esis. When the bio synthesis of sphingomyelin, a representative sphingolipid of lipid rafts, was inhibited, their sensitivity was decreased. The exogenous addition of ganglioside G_<M1> to MDCK cell cultures caused decreases in the binding and heptamerization of the toxin and also in the sensitivity of MDCK cells to the toxin. Prolongation of culture time of MDCK cells caused an increase in ganglioside contents but did a decrease in their sensitivity. When MDCK cells, which had been cultured for a prolonged time, was treated with C. perfringens sialidase, their sensitivity to the toxin was increased. Based on these results, we concluded that an increase in ganglioside contents leading to an increase in sialic acid results in the inhibition of epsilon-toxin-binding to lipid rafts. We have also examined relationship between rafts and platelet aggregation induced by alpha-toxin, which is regarded as being most responsible for the pathogenesis of gas gangrene. Alpha-Toxin, which possesses both phospholipase C and sphingomyelinase activities showed high affinity to the lipid rafts of platelets. It degraded sphingomyelin locating preferentially in lipid rafts, generating ceramide. A mutant alpha-toxin lacking enzymatic activity bound preferentially to lipid rafts but did not aggregate platelets. We also showed that alpha-toxin aggregates lipid rafts and this ability was inhibited by the addition of anti-ceramide monoclonal antibody. These results led us to conclude that ceramide created by sphingomyelinase activity of alpha-toxin induces clustering of lipid rafts, leading to triggering of signal pathways involved in platelet aggregation. Less