National Reference Laboratory for E. coli and Shigellae, National Institute of Public Health, Prague, Czech Republic.
Institute for Hygiene, University of Münster, Münster, Germany.
Methods Mol Biol. 2021;2291:177-205. doi: 10.1007/978-1-0716-1339-9_8.
Outer membrane vesicles (OMVs), nanoparticles released by Shiga toxin-producing Escherichia coli (STEC), have been identified as novel efficient virulence tools of these pathogens. STEC O157 OMVs carry a cocktail of virulence factors including Shiga toxin 2a (Stx2a), cytolethal distending toxin V (CdtV), EHEC hemolysin, flagellin, and lipopolysaccharide. OMVs are taken up by human intestinal epithelial and microvascular endothelial cells, the major targets during STEC infection, and deliver the virulence factors into host cells. There the toxins separate from OMVs and are trafficked via different pathways to their target compartments, i.e., the cytosol (Stx2a-A subunit), nucleus (CdtV-B subunit), and mitochondria (EHEC hemolysin). This leads to a toxin-specific host cell injury and ultimately apoptotic cell death. Besides their cytotoxic effects, STEC OMVs trigger an inflammatory response via their lipopolysaccharide and flagellin components. In this chapter, we describe methods for the isolation and purification of STEC OMVs, for the detection of OMV-associated virulence factors, and for the analysis of OMV interactions with host cells including OMV cellular uptake and intracellular trafficking of OMVs and OMV-delivered toxins.
外膜囊泡（OMVs）是产志贺毒素大肠杆菌（STEC）释放的纳米颗粒，已被鉴定为这些病原体新型有效的毒力工具。STEC O157 OMVs 携带包括志贺毒素 2a（Stx2a）、细胞毒素扩张毒素 V（CdtV）、EHEC 溶血素、鞭毛蛋白和脂多糖在内的多种毒力因子。OMVs 被人类肠道上皮细胞和微血管内皮细胞摄取，这些是 STEC 感染的主要靶细胞，并将毒力因子递送到宿主细胞内。在那里，毒素与 OMVs 分离，并通过不同的途径被运送到它们的靶位区，即细胞质（Stx2a-A 亚基）、细胞核（CdtV-B 亚基）和线粒体（EHEC 溶血素）。这导致毒素特异性的宿主细胞损伤，并最终导致细胞凋亡。除了细胞毒性作用外，STEC OMVs 还通过其脂多糖和鞭毛蛋白成分引发炎症反应。在本章中，我们描述了分离和纯化 STEC OMVs 的方法、检测与 OMV 相关的毒力因子的方法，以及分析 OMV 与宿主细胞相互作用的方法，包括 OMV 的细胞摄取和 OMV 及其携带的毒素的细胞内运输。
