EHEC hemolysin: Molecular and cellular mechanisms of host injury

肠出血性大肠杆菌溶血素（EHEC-Hly）是一种致孔RTX毒素，是一种重要的肠出血性大肠杆菌毒力因子。该毒素以游离的EHEC-Hly形式存在，其作为溶细胞素，并且EHEC-Hly与EHEC在生长期间释放的外膜囊泡（OMV）相关。通过研究OMV相关的EHEC-Hly对人微血管内皮细胞和肠上皮细胞的生物学效应，这是EHEC感染过程中的主要靶点，我们在第一个项目期间证明了OMV相关的EHEC-Hly不会裂解细胞，但会引发细胞凋亡。OMV相关毒素通过OMV被靶细胞内化，并与OMV一起运输到溶酶体中。在此，EHEC-Hly与OMV分离，从溶酶体逃逸并靶向线粒体，这通过内在的半胱天冬酶-9依赖性途径导致凋亡性细胞死亡。OMV相关的EHEC-Hly的细胞凋亡潜力证明了毒素参与EHEC介导的疾病的发病机制的新机制，并将OMV相关的EHEC-Hly鉴定为毒素的病理生理学高效形式。因此，第二个项目期的目的是更深入地了解含EHEC-Hly的OMV与靶细胞的相互作用以及OMV递送的EHEC-Hly与其主要靶细胞器（即线粒体）的相互作用。 具体来说，我们将确定OMV配体和细胞受体参与细胞结合和内化的EHEC-Hly-窝藏OMV。我们将确定EHEC-Hly转运到线粒体的机制，并确定参与EHEC-Hly线粒体输入的细胞器蛋白。我们将分析由OMV递送的EHEC-Hly在肠上皮和微血管内皮细胞中引起的线粒体的形态学变化。最后，我们将分析含EHEC-Hly的OMV破坏肠上皮屏障完整性并穿过该边界的能力。我们将阐明EHEC-Hly和其他主要生物活性OMV组分（包括OmpA和LPS）对肠屏障作用的贡献。阐明含EHEC-Hly的OMV在致病相关宿主细胞中引起的事件及其潜在机制将有助于更好地理解EHEC介导的疾病的发病机制。

The hemolysin of enterohemorrhagic Escherichia coli (EHEC-Hly), a pore-forming RTX toxin, is an EHEC virulence factor of increasing importance. The toxin exists as a free EHEC-Hly, which acts as cytolysin, and EHEC-Hly associated with outer membrane vesicles (OMVs) released by EHEC during growth. By investigating biological effects of OMV-associated EHEC-Hly on human microvascular endothelial and intestinal epithelial cells, which are the major targets during EHEC infection, we demonstrated during the first project period that OMV-associated EHEC-Hly does not lyse the cells, but triggers their apoptosis. The OMV-associated toxin is internalized by target cells via OMVs and trafficked with OMVs into lysosomes. Here EHEC-Hly separates from OMVs, escapes from the lysosomes and targets mitochondria, which leads to apoptotic cell death via the intrinsic caspase-9-dependent pathway. The apoptotic potential of OMV-associated EHEC-Hly demonstrates a novel mechanism of the toxin involvement in the pathogenesis of EHEC-mediated diseases and identifies the OMV-associated EHEC-Hly as a pathophysiologically highly efficient form of the toxin. The aim of the second project period is therefore to gain deeper insights into the interactions of EHEC-Hly-containing OMVs with the target cells and of OMV-delivered EHEC-Hly with its major target organelle, i.e. mitochondria. Specifically, we will identify OMV ligands and cellular receptors involved in cellular binding and internalization of EHEC-Hly-harboring OMVs. We will determine the mechanism involved in transport of EHEC-Hly to mitochondria and identify organelle proteins involved in EHEC-Hly mitochondrial import. We will analyze morphological changes of mitochondria elicited by OMV-delivered EHEC-Hly in intestinal epithelial and microvascular endothelial cells. Finally, we will analyze the ability of EHEC-Hly-containing OMVs to disrupt the integrity of the intestinal epithelial barrier and to cross this border. We will elucidate contributions of EHEC-Hly and of the other major biologically active OMV components including OmpA and LPS to the effects on the intestinal barrier. Clarifying the events elicited by EHEC-Hly-containing OMVs in pathogenetically relevant host cells and their underlying mechanisms will contribute to better understanding of the pathogenesis of EHEC-mediated diseases.