Date Published: December 12, 2013
Publisher: Public Library of Science
Author(s): Martina Bielaszewska, Christian Rüter, Lisa Kunsmann, Lilo Greune, Andreas Bauwens, Wenlan Zhang, Thorsten Kuczius, Kwang Sik Kim, Alexander Mellmann, M. Alexander Schmidt, Helge Karch, Steven R. Blanke.
Enterohemorrhagic Escherichia coli (EHEC) strains cause diarrhea and hemolytic uremic syndrome resulting from toxin-mediated microvascular endothelial injury. EHEC hemolysin (EHEC-Hly), a member of the RTX (repeats-in-toxin) family, is an EHEC virulence factor of increasingly recognized importance. The toxin exists as free EHEC-Hly and as EHEC-Hly associated with outer membrane vesicles (OMVs) released by EHEC during growth. Whereas the free toxin is lytic towards human endothelium, the biological effects of the OMV-associated EHEC-Hly on microvascular endothelial and intestinal epithelial cells, which are the major targets during EHEC infection, are unknown. Using microscopic, biochemical, flow cytometry and functional analyses of human brain microvascular endothelial cells (HBMEC) and Caco-2 cells we demonstrate that OMV-associated EHEC-Hly does not lyse the target cells but triggers their apoptosis. The OMV-associated toxin is internalized by HBMEC and Caco-2 cells via dynamin-dependent endocytosis of OMVs and trafficked with OMVs into endo-lysosomal compartments. Upon endosome acidification and subsequent pH drop, EHEC-Hly is separated from OMVs, escapes from the lysosomes, most probably via its pore-forming activity, and targets mitochondria. This results in decrease of the mitochondrial transmembrane potential and translocation of cytochrome c to the cytosol, indicating EHEC-Hly-mediated permeabilization of the mitochondrial membranes. Subsequent activation of caspase-9 and caspase-3 leads to apoptotic cell death as evidenced by DNA fragmentation and chromatin condensation in the intoxicated cells. The ability of OMV-associated EHEC-Hly to trigger the mitochondrial apoptotic pathway in human microvascular endothelial and intestinal epithelial cells indicates a novel mechanism of EHEC-Hly involvement in the pathogenesis of EHEC diseases. The OMV-mediated intracellular delivery represents a newly recognized mechanism for a bacterial toxin to enter host cells in order to target mitochondria.
Enterohemorrhagic Escherichia coli (EHEC) are global causes of diarrhea and its severe extra-intestinal complication, hemolytic uremic syndrome (HUS) . HUS, the most common cause of acute renal failure in children, is a thrombotic microangiopathy resulting from microvascular endothelial injury in the kidneys and the brain . EHEC produce a spectrum of virulence factors, which plausibly play a role in the pathogenesis of HUS. In addition to Shiga toxins (Stx), which are the major EHEC virulence factors involved in the microvascular endothelial injury , , several other EHEC toxins can trigger or contribute to this pathology -. The importance of the contribution of EHEC hemolysin (EHEC-Hly) , also designated EHEC toxin (Ehx)  is increasingly recognized , .
OMVs ubiquitously shed by Gram-negative bacteria are emerging as a new, highly sophisticated mechanism for secretion and a simultaneous, coordinated and direct delivery of bacterial virulence factors into host cells , , –. OMVs, which contain bacterial toxins, adhesins, invasins, and immunomodulatory compounds are considered as bacterial weapons that attack the host tissues and assist bacterial pathogens to establish their colonization niches, impair host cellular functions, and modulate host defense [reviewed in 39–41]. We now demonstrate that OMV-associated EHEC-Hly irreversibly injures human microvascular endothelial and intestinal epithelial cells, which are key players in the pathogenesis of EHEC-mediated diseases. Our data support the model (Figure 14) that the toxin is internalized via OMVs by dynamin-dependent endocytosis and trafficked together with its carriers to endo-lysosomal compartments (Figure 14, Step 1). During endosomal acidification EHEC-Hly is separated from OMVs (Step 2), escapes from the lysosomes, most probably via its pore-forming activity towards the lysosomal membrane (Step 3), and targets mitochondria (Step 4). The presence of EHEC-Hly in mitochondria leads to a reduction of the mitochondrial transmembrane potential and release of cytochrome c to the cytosol (Step 5). Subsequent activation of caspase-9 triggers the apoptotic cell death.