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

由产气荚膜梭菌产生的埃帕霉素和α-毒素分别是导致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。已显示ε-毒素对神经元和肾细胞膜的脂筏表现出高亲和力，并通过毒素的七聚化渗透膜。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。胆固醇是MDCK细胞脂筏的主要脂质成分，胆固醇的耗竭和生物合成的抑制可抑制毒素的七聚化，而鞘脂的生物合成的抑制可增加MDCK细胞对ε毒素的敏感性。用鞘糖脂生物合成抑制剂PdR观察到类似的结果。 ...更多信息 是的。当脂筏中代表性的鞘脂鞘磷脂的生物合成受到抑制时，它们的敏感性降低。外源性神经节苷脂G_1<M1>可降低MDCK细胞对毒素的结合和七聚化，并降低MDCK细胞对毒素的敏感性。MDCK细胞培养时间延长，神经节苷脂含量增加，但敏感性降低。长期培养的MDCK细胞经C.产气荚膜杆菌唾液酸酶，它们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，导致ε毒素与脂筏结合的抑制。我们还研究了筏与α毒素诱导的血小板聚集的关系，α毒素被认为是气性坏疽发病机制的主要原因。具有磷脂酶C和鞘磷脂酶活性的α-毒素对血小板的脂筏表现出高亲和力。它优先降解位于脂筏中的鞘磷脂，产生神经酰胺。缺乏酶活性的突变体α-毒素优先结合脂筏，但不聚集血小板。我们还发现，α-毒素聚集脂筏和这种能力被抑制的抗神经酰胺单克隆抗体的加入。这些结果使我们得出结论，神经酰胺产生的鞘磷脂酶活性的α-毒素诱导聚集的脂筏，导致触发的信号通路参与血小板聚集。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