Infections of the human intestinal tract with enterohemorrhagic Escherichia coli (EHEC) result in massive extraintestinal complications due to translocation of EHEC-released Shiga toxins (Stxs) from the gut into the circulation. Stx-mediated damage of the cerebral microvasculature raises serious brain dysfunction being the most frequent cause of acute mortality in patients suffering from severe EHEC infections. Stx2a and Stx2e are associated with heavy and mild course of infection, respectively. Stx2a preferentially binds to globotriaosylceramide (Gb3Cer, Galα1-4Galβ1-4Glcβ1-1Cer), while Stx2e prefers globotetraosylceramide (Gb4Cer, GalNAcβ1-3Galα1-4Galβ1-4Glcβ1-1Cer). Both glycosphingolipids (GSLs) were detected in detergent-resistant membranes (DRMs) of primary human brain microvascular endothelial cells (pHBMECs) resembling microdomains of the plasma membrane. In this study, we show that Gb3Cer and Gb4Cer of pHBMECs with saturated C16:0, C22:0, and C24:0 fatty acids dominated in DRMs, corresponding to the liquid-ordered membrane phase, whereas lipoforms carrying unsaturated C24:1 and C24:2 fatty acids prevailed in the non-DRM fractions, which correspond to the liquid-disordered membrane phase. Similarly, a shift of the phospholipids from saturated lipoforms in the DRM to unsaturated species in the non-DRM fractions was observed. Real-time biomolecular interaction analysis using affinity-purified Stx2a and Stx2e, recorded with a surface acoustic wave (SAW) biosensor, evidenced high binding strength of both toxins toward DRMs and failure in interaction with non-DRMs. These results support the hypothesis of preferential binding of Stxs toward microdomains harboring GSL receptors carrying saturated fatty acids in their lipid anchors. Collectively, unraveling the precise mechanisms of Stx-microdomain interaction may help to develop antiadhesive compounds to combat Stx-mediated cellular injury.

中文翻译：

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志贺毒素与人脑微血管内皮细胞膜微区的实时相互作用分析。

肠道出血性大肠杆菌（EHEC）感染人肠道会导致大量肠外并发症，这是因为EHEC释放的志贺毒素（Stxs）从肠道转移到循环系统中。Stx介导的脑微血管系统损害引起严重的脑功能障碍，是严重的EHEC感染患者急性死亡的最常见原因。Stx2a和Stx2e分别与重度和轻度感染过程相关。Stx2a优先结合球果糖基神经酰胺（Gb3Cer，Galα1-4Galβ1-4Glcβ1-1Cer），而Stx2e优选球果糖基神经酰胺（Gb4Cer，GalNAcβ1-3Galα1-4Galβ1-4Glcβ1-1Cer）。两种糖鞘脂（GSL）在类似于质膜微区的原代人脑微血管内皮细胞（pHBMECs）的耐去污剂膜（DRM）中检测到。在这项研究中，我们表明在DRM中，具有饱和C16：0，C22：0和C24：0脂肪酸的pHBMECs的Gb3Cer和Gb4Cer占主导地位，对应于液序膜相，而携带不饱和C24：1和C24的脂质形式非DRM馏分中主要存在2：2脂肪酸，这对应于液体无序的膜相。类似地，观察到磷脂从DRM中的饱和脂质形式转移到非DRM部分中的不饱和物质。使用亲和纯化的Stx2a和Stx2e进行实时生物分子相互作用分析，并通过表面声波（SAW）生物传感器进行记录，证明了两种毒素对DRM的高结合强度以及与非DRM相互作用的失败。这些结果支持以下假设：Stxs优先结合微区，该微区包含在脂质锚中携带饱和脂肪酸的GSL受体。集体地，揭开Stx微域相互作用的确切机制可能有助于开发抗粘附化合物，以对抗Stx介导的细胞损伤。