Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna, Via San Giacomo 14, 40126 Bologna, Italy.
Institute of Hygiene and Medical Microbiology, Medical University of Innsbruck, A-6020 Innsbruck, Austria.
Toxins (Basel). 2021 Jan 26;13(2):94. doi: 10.3390/toxins13020094.
The pathogenesis of -induced hemolytic uremic syndrome (eHUS) caused by infections with pathogenic Shiga toxin (Stx) producing (STEC) is centered on bacterial (e.g., Stx) and host factors (circulating cells, complement system, serum proteins) whose interaction is crucial for the immediate outcome and for the development of this life-threatening sequela. Stx2a, associated to circulating cells (early toxemia) or extracellular vesicles (late toxemia) in blood, is considered the main pathogenic factor in the development of eHUS. Recently, it was found that the functional properties of Stx2a (binding to circulating cells and complement components) change according to modifications of the structure of the toxin, i.e., after a single cleavage of the A subunit resulting in two fragments, A1 and A2, linked by a disulfide bridge. Herein, we describe a method to be used for the detection of the cleaved form of Stx2a in the serum of STEC-infected or eHUS patients. The method is based on the detection of the boosted inhibitory activity of the cleaved toxin, upon treatment with reducing agents, on a rabbit cell-free translation system reconstituted with human ribosomes. The method overcomes the technical problem caused by the presence of inhibitors of translation in human serum that have been stalled by the addition of RNAase blockers and by treatment with immobilized protein G. This method, allowing the detection of Stx2a at concentrations similar to those found by ELISA in the blood of STEC-infected patients, could be a useful tool to study the contribution of the cleaved form of Stx2a in the pathogenesis of eHUS.
产志贺毒素（Stx）的致病性大肠杆菌（STEC）感染引起的 - 诱导的溶血性尿毒症综合征（eHUS）的发病机制集中在细菌（例如，Stx）和宿主因素（循环细胞、补体系统、血清蛋白）上，其相互作用对于即时结果和这种危及生命的后遗症的发展至关重要。与循环细胞（早期中毒）或血液中外泌体（晚期中毒）相关的 Stx2a 被认为是 eHUS 发展的主要致病因素。最近，人们发现 Stx2a 的功能特性（与循环细胞和补体成分结合）根据毒素结构的修饰而改变，即 A 亚基的单一切割导致两个片段 A1 和 A2 ，通过二硫键连接。在此，我们描述了一种用于检测 STEC 感染或 eHUS 患者血清中切割形式的 Stx2a 的方法。该方法基于检测经还原剂处理后切割毒素的增强抑制活性，在用人核糖体重建的兔无细胞翻译系统上进行。该方法克服了人血清中翻译抑制剂存在的技术问题，这些抑制剂通过添加 RNAase 阻滞剂和用固定化蛋白 G 处理而停滞不前。该方法允许以与 ELISA 在 STEC 感染患者血液中检测到的浓度相似的浓度检测 Stx2a，可作为研究切割形式的 Stx2a 在 eHUS 发病机制中的作用的有用工具。
