Aldick Thomas, Bielaszewska Martina, Uhlin Bernt Eric, Humpf Hans-Ulrich, Wai Sun Nyunt, Karch Helge
Institute of Hygiene, University of Münster, 48149 Münster, Germany.
Mol Microbiol. 2009 Mar;71(6):1496-508. doi: 10.1111/j.1365-2958.2009.06618.x. Epub 2009 Feb 2.
Haemolysin from enterohaemorrhagic Escherichia coli (EHEC-Hly), a putative EHEC virulence factor, belongs to the RTX (repeat-in-toxin) family whose members rapidly inactivate themselves by self-aggregation. By investigating the status of EHEC-Hly secreted extracellularly, we found the toxin both in a free, soluble form and associated, with high tendency and independently of its acylation status, to outer membrane vesicles (OMVs) extruded by EHEC. We compared the interaction of both toxin forms with erythrocytes using scanning electron microscopy and binding assays. The OMV-associated toxin was substantially (80 times) more stable under physiological conditions than the free EHEC-Hly as demonstrated by prolonged haemolytic activity (half-life time 20 h versus 15 min). The haemolysis was preceded by calcium-dependent binding of OMVs carrying EHEC-Hly to erythrocytes; this binding was mediated by EHEC-Hly. We demonstrate that EHEC-Hly is a biologically active cargo in OMVs with dual roles: a cell-binding protein and a haemolysin. These paired functions produce a biologically potent form of the OMV-associated RTX toxin and augment its potential towards target cells. Our findings provide a general concept for stabilization of RTX toxins and open new insights into the biology of these important virulence factors.
肠出血性大肠杆菌（EHEC）溶血素（EHEC-Hly）是一种假定的EHEC毒力因子，属于RTX（毒素重复序列）家族，该家族成员会通过自我聚集迅速使自身失活。通过研究EHEC-Hly分泌到细胞外的状态，我们发现该毒素既以游离的可溶形式存在，又以高倾向性且独立于其酰化状态与EHEC挤出的外膜囊泡（OMV）相关联。我们使用扫描电子显微镜和结合试验比较了两种毒素形式与红细胞的相互作用。如延长的溶血活性所示（半衰期分别为20小时和15分钟），与OMV相关的毒素在生理条件下比游离的EHEC-Hly稳定得多（80倍）。溶血之前，携带EHEC-Hly的OMV会与红细胞发生钙依赖性结合；这种结合由EHEC-Hly介导。我们证明EHEC-Hly是OMV中具有双重作用的生物活性货物：一种细胞结合蛋白和一种溶血素。这些配对功能产生了一种生物活性形式的与OMV相关的RTX毒素，并增强了其对靶细胞的作用潜力。我们的发现为RTX毒素的稳定化提供了一个通用概念，并为这些重要毒力因子的生物学特性提供了新的见解。
