Molecular biological analysis of the exotoxin derived from periodontopathic bacteria on peridontal medicine

伴生放线杆菌产生一种毒素，称为细胞致死性膨胀毒素(CDT)，它会导致宿主细胞DNA损伤，从而诱导DNA损伤检查点通路。CDT由三个亚基组成，即CDtA、CDtB和CDtC。CDT B是CDT的活性亚基，发挥核酸酶的作用，损伤核DNA，触发细胞周期停滞。在本研究中，我们证实了导致细胞周期停滞的唯一毒素蛋白组合是所有三个重组CDT(RCDT)蛋白亚基的组合。此外，为了证明rCDT的毒性，有必要让CDtA和CDtC在CDtB之前访问细胞。CDtA和CDtC的共存是这些亚基与细胞结合的必要条件。用葡萄糖神经酰胺合成抑制剂1-phenyl-2-palmitoylamino-3-morpholino-1-propanol处理的细胞对重组CDT诱导的细胞毒性有抵抗作用。此外，缺乏鞘脂生物合成的LY-B细胞也表现出对rCDT诱导的细胞毒作用的抵抗。为了评估每个亚基与葡萄糖神经酰胺的结合，我们进行了薄层色谱免疫染色。结果表明，各亚基均与GM1、GM2、GM3、Gb3、Gb4发生反应。与含有GM3的脂质体孵育的rCDT混合物显示部分降低的毒性。这些结果表明GM3可以作为CDT受体发挥作用。

Actinobacillus actinomycetemcomitans produces a toxin, called cytolethal distending toxin (CDT), which causes host cell DNA damage leading to the induction of DNA damage checkpoint pathways. CDT consists of three subunits, CdtA, CdtB, and CdtC CdtB is the active subunit of CDT and exerts its effect as a nuclease that damages nuclear DNA, triggering cell cycle arrest. In the present study, we confirmed that the only combination of toxin proteins causing cell cycle arrest was that of all three recombinant CDT (rCDT) protein subunits. Furthermore, in order for rCDT to demonstrate toxicity, it was necessary for CdtA and CdtC to access the cell before CdtB. The coexistence of CdtA and CdtC was necessary for these subunits to bind to the cell. Cells treated with the glucosylceramide synthesis inhibitor 1-phenyl-2-palmitoylamino-3-morpholino-1-propanol showed resistance to the cytotoxicity induced by rCDT. Furthermore, LY-B cells, which are deficient in the biosynthesis of sphingolipid, also showed resistance to the cytotoxicity induced by rCDT. To evaluate the binding of each subunit for glucosylceramides, we performed thin-layer chromatography immunostaining. The results indicated that each subunit reacted with the GM1, GM2, GM3, Gb3, and Gb4. The rCDT mixture incubated with liposomes containing GM3 displayed partially reduced toxicity. These results indicate that GM3 can act as a CDT receptor.