Abstract
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.
Highlights
Enterohemorrhagic Escherichia coli (EHEC) are global causes of diarrhea and its severe extra-intestinal complication, hemolytic uremic syndrome (HUS) [1]
EHEC-Hly belongs to a large family of toxins, whose members typically kill target cells by inserting themselves into the cell membranes, which results in pore formation and cell lysis
We show that EHEC-Hly associated with outer membrane vesicles (OMVs) secreted by EHEC during growth does not lyse human microvascular endothelial and intestinal epithelial cells, which are the major targets in EHEC-mediated human diseases
Summary
Enterohemorrhagic Escherichia coli (EHEC) are global causes of diarrhea and its severe extra-intestinal complication, hemolytic uremic syndrome (HUS) [1]. 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 [1]. 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 [1,2], several other EHEC toxins can trigger or contribute to this pathology [3,4,5,6]. The contribution of EHEC-Hly to the pathogenesis of HUS is supported by the ability of the toxin to injure microvascular endothelial cells [6]. The pro-inflammatory potential of EHEC-Hly [9], its production by the vast majority of EHEC strains associated with HUS [13,14], and expression of the toxin during infection as demonstrated by the development of antiEHEC-Hly antibodies in most HUS patients [7] and by increased
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