EHEC hemolysin: Molecular and cellular mechanisms of host injury

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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.