Shiga toxin is the main virulence factor of non-invasive enterohemorrhagic Escherichia coli strains capable of causing hemolytic uremic syndrome. Our group has previously shown that the toxin can reach the kidney within microvesicles where it is taken up by renal cells and the vesicles release their cargo intracellularly, leading to toxin-mediated inhibition of protein synthesis and cell death. The aim of this study was to examine if recipient cells must express the globotriaosylceramide (Gb3) toxin receptor for this to occur, or if Gb3-negative cells are also susceptible after uptake of Gb3-positive and toxin-positive microvesicles. To this end we generated Gb3-positive A4GALT-transfected CHO cells, and a vector control lacking Gb3 (CHO-control cells), and decreased Gb3 synthesis in native HeLa cells by exposing them to the glycosylceramide synthase inhibitor PPMP. We used these cells, and human intestinal DLD-1 cells lacking Gb3, and exposed them to Shiga toxin 2-bearing Gb3-positive microvesicles derived from human blood cells. Results showed that only recipient cells that possessed endogenous Gb3 (CHO-Gb3 transfected and native HeLa cells) exhibited cellular injury, reduced cell metabolism and protein synthesis, after uptake of toxin-positive microvesicles. In Gb3-positive cells the toxin introduced via vesicles followed the retrograde pathway and was inhibited by the retrograde transport blocker Retro-2.1. CHO-control cells, HeLa cells treated with PPMP and DLD-1 cells remained unaffected by toxin-positive microvesicles. We conclude that Shiga toxin-containing microvesicles can be taken up by Gb3-negative cells but the recipient cell must express endogenous Gb3 for the cell to be susceptible to the toxin.

中文翻译：

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仅当细胞表达毒素受体时，带有志贺毒素的微泡才会对受体细胞产生细胞毒性作用。

志贺毒素是能够引起溶血性尿毒症综合征的非侵入性肠出血性大肠杆菌菌株的主要毒力因子。我们的小组先前已经表明，毒素可以在微囊泡内到达肾脏，在那里它被肾细胞吸收，并且囊泡在细胞内释放它们的货物，导致毒素介导的蛋白质合成抑制和细胞死亡。本研究的目的是检查受体细胞是否必须表达球三酰神经酰胺 (Gb3) 毒素受体才能发生这种情况，或者 Gb3 阴性细胞在摄取 Gb3 阳性和毒素阳性微泡后是否也易感。为此，我们产生了 Gb3 阳性 A4GALT 转染的 CHO 细胞，以及缺乏 Gb3 的载体对照（CHO 对照细胞），并通过将天然 HeLa 细胞暴露于糖基神经酰胺合酶抑制剂 PPMP 来减少天然 HeLa 细胞中 Gb3 的合成。我们使用这些细胞和缺乏 Gb3 的人类肠道 DLD-1 细胞，并将它们暴露于来自人类血细胞的带有志贺毒素 2 的 Gb3 阳性微泡。结果表明，在摄取毒素阳性微泡后，只有具有内源性 Gb3 的受体细胞（CHO-Gb3 转染和天然 HeLa 细胞）表现出细胞损伤、细胞代谢和蛋白质合成减少。在 Gb3 阳性细胞中，通过囊泡引入的毒素遵循逆行途径，并被逆行转运阻滞剂 Retro-2.1 抑制。CHO 对照细胞、用 PPMP 处理的 HeLa 细胞和 DLD-1 细胞不受毒素阳性微泡的影响。