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

产气荚膜梭菌产生的Epsilon毒素和α毒素分别是家畜肠毒素血症和人类气性坏疽的主要毒素。为了阐明这两种毒素表现出毒性的分子机制，我们进行了这项研究。epsilon -毒素已被证明对神经元和肾细胞膜的脂筏具有高亲和力，并通过毒素的七聚体化渗透细胞膜。我们研究了膜脂环境如何影响毒素与脂筏的结合及其七聚化。MDCK细胞脂质层的主要脂质成分胆固醇的消耗和生物合成抑制抑制了毒素的七聚化，而伏马菌素B1处理对另一种鞘脂的生物合成抑制增加了MDCK细胞对epsilon-毒素的敏感性。用糖脂生物合成抑制剂PDMP观察到类似的结果。鞘磷脂是脂筏中具有代表性的鞘脂，当其生物合成受到抑制时，其敏感性降低。在MDCK细胞培养中外源性添加神经节苷脂G_<M1>可降低毒素的结合和七聚化，并降低MDCK细胞对毒素的敏感性。随着MDCK细胞培养时间的延长，神经节苷脂含量增加，但敏感性降低。经长时间培养的MDCK细胞经产气荚膜梭菌唾液酸酶处理后，其对毒素的敏感性增加。基于这些结果，我们得出结论，神经节苷脂含量的增加导致唾液酸的增加，从而抑制了epsilon毒素与脂筏的结合。我们还研究了α毒素诱导的筏和血小板聚集之间的关系，α毒素被认为是气性坏疽发病的主要原因。α -毒素具有磷脂酶C和鞘磷脂酶活性，对血小板脂筏具有较高的亲和力。它降解优先定位于脂筏中的鞘磷脂，生成神经酰胺。缺乏酶活性的突变α毒素优先结合脂筏，但不聚集血小板。我们还发现α -毒素聚集脂筏，这种能力被添加抗神经酰胺单克隆抗体抑制。这些结果使我们得出结论，由α毒素鞘磷脂酶活性产生的神经酰胺诱导脂筏聚集，从而触发参与血小板聚集的信号通路。少

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