Analysis of microdomain-association of Shiga toxin glycosphingolipid receptors in primary human endothelial cells

感染产生志贺毒素（Stx）的大肠杆菌（STEC）可导致一系列后果，从无症状携带到无并发症的腹泻、血性腹泻和溶血性尿毒症综合征（HUS）。Stxs被认为引起微血管内皮损伤，这是出血性结肠炎和溶血性尿毒综合征的主要组织病理事件和病理生理过程。Stxs被细分为两个家族，Stx1和Stx2，以及几个变体（Stx1c, Stx1d, Stx2c, Stx2d, Stx2e, Stx2f）。在大多数情况下，这些毒素的受体结合特性尚不清楚。虽然我们已经表明，产肠毒素大肠杆菌感染的临床结果在很大程度上取决于感染菌株产生的Stx型，但这种分化的原因尚不清楚。为了确定这些差异是否与不同的受体结合能力相关，我们将纯化Stx家族成员，并使用纳米电喷雾电离四极杆飞行时间质谱（nanoESI-QTOF MS）鉴定其受体，我们最近将其应用于Stx1的高亲和力和低亲和力结合配体的详细结构表征的定义。接下来，我们将研究这些受体在内皮细胞上是否有不同的定性和定量分布。由于globotriaosyl神经酰胺（Gb3Cer）的脂肪酸可以影响所使用的内噬途径，并且由于Stx的有效逆行运输需要筏定位，因此我们将对来自人内皮细胞的脂筏（分离为耐洗涤剂膜、根尖/基底外侧膜制剂和小泡）进行表征。我们将特别研究鞘糖脂（GSLs）和GSL/胆固醇相关膜蛋白的组成。这种表征将在stx细胞结合相互作用的背景下进行。通过确定不同stx与其细胞靶点相互作用的精确机制，我们希望为stc介导的疾病制定战略性的预防和治疗措施。

Infection with Shiga toxin (Stx) producing Escherichia coli (STEC) results in a spectrum of outcomes ranging from asymptomatic carriage to uncomplicated diarrhea, bloody diarrhea, and the hemolytic-uremic syndrome (HUS). Stxs are believed to cause microvascular endothelial injury which is the primary histopathological event and pathophysiological process underlying hemorrhagic colitis and HUS. Stxs have been subdivided into two families, Stx1 and Stx2, and several variants (Stx1c, Stx1d, Stx2c, Stx2d, Stx2e, Stx2f). In most instances, there are as yet unknown receptor binding characteristics of these toxins. Although we have shown that the clinical outcome of an STEC infection depends, in large part, on the Stx type produced by the infecting strain, the reason for this differentiation is not well understood. To determine if these differences correlate with different receptorbinding capacities, we will purify the members of the Stx families and identify their receptors using nanoelectrospray ionization quadrupole time-of-flight mass spectrometry (nanoESI-QTOF MS), which we have recently applied to the definition of the detailed structural characterization of high- and low-affinity binding ligands of Stx1. Next, we will investigate if these receptors have different qualitative and quantitative distributions on endothelial cells. Because the fatty acid of globotriaosylceramide (Gb3Cer) can influence the endocytic pathway used, and because raft localization is required for efficient retrograde transport of Stx, we will characterize lipid rafts (isolated as detergent-resistant membranes, apical/basolateral membrane preparations, and caveolae) from human endothelial cells. We will specifically examine the composition of glycosphingolipids (GSLs) and GSL/cholesterol associated membrane proteins. This characterization will be performed in the context of Stx-cell binding interactions. By defining precise mechanisms by which different Stxs interact with their cellular targets, we hope to develop strategic preventive and therapeutic measures for STEC-mediated diseases.