Outer membrane vesicles (OMVs) are important tools in bacterial virulence but their role in the pathogenesis of infections caused by enterohemorrhagic Escherichia coli (EHEC) O157, the leading cause of life-threatening hemolytic uremic syndrome, is poorly understood. Using proteomics, electron and confocal laser scanning microscopy, immunoblotting, and bioassays, we investigated OMVs secreted by EHEC O157 clinical isolates for virulence factors cargoes, interactions with pathogenetically relevant human cells, and mechanisms of cell injury. We demonstrate that O157 OMVs carry a cocktail of key virulence factors of EHEC O157 including Shiga toxin 2a (Stx2a), cytolethal distending toxin V (CdtV), EHEC hemolysin, and flagellin. The toxins are internalized by cells via dynamin-dependent endocytosis of OMVs and differentially separate from vesicles during intracellular trafficking. Stx2a and CdtV-B, the DNase-like CdtV subunit, separate from OMVs in early endosomes. Stx2a is trafficked, in association with its receptor globotriaosylceramide within detergent-resistant membranes, to the Golgi complex and the endoplasmic reticulum from where the catalytic Stx2a A1 fragment is translocated to the cytosol. CdtV-B is, after its retrograde transport to the endoplasmic reticulum, translocated to the nucleus to reach DNA. CdtV-A and CdtV-C subunits remain OMV-associated and are sorted with OMVs to lysosomes. EHEC hemolysin separates from OMVs in lysosomes and targets mitochondria. The OMV-delivered CdtV-B causes cellular DNA damage, which activates DNA damage responses leading to G2 cell cycle arrest. The arrested cells ultimately die of apoptosis induced by Stx2a and CdtV via caspase-9 activation. By demonstrating that naturally secreted EHEC O157 OMVs carry and deliver into cells a cocktail of biologically active virulence factors, thereby causing cell death, and by performing first comprehensive analysis of intracellular trafficking of OMVs and OMV-delivered virulence factors, we provide new insights into the pathogenesis of EHEC O157 infections. Our data have implications for considering O157 OMVs as vaccine candidates. Enterohemorrhagic Escherichia coli (EHEC) O157, the leading EHEC group causing diarrhea and the life-threatening hemolytic uremic syndrome in humans, produce several virulence factors which play distinct roles in the pathogenesis of these diseases. However, the mechanisms of their secretion and host cell injury are poorly understood. We show here that EHEC O157 strains isolated from patients shed nanostructures termed outer membrane vesicles (OMVs) which contain major EHEC O157 virulence factors including Shiga toxin 2a (Stx2a), cytolethal distending toxin V (CdtV), EHEC hemolysin, and flagellin. The OMVs are taken up by human intestinal epithelial and renal and brain microvascular endothelial cells, which are the major targets during EHEC O157 infections, and deliver the virulence factors intracellularly. Inside cells the virulence factors separate from OMVs and are transported via different pathways to their target compartments including the cytosol (Stx2a), nucleus (CdtV-B subunit), and mitochondria (EHEC hemolysin). Cells exposed to EHEC O157 OMVs develop G2 cell cycle arrest induced by CdtV-B-mediated DNA damage. This is followed by apoptotic cell death triggered by Stx2a and CdtV via caspase-9 activation. OMVs thus serve as novel tools of EHEC O157-mediated host injury and are quite likely involved in the pathogenesis of human diseases.

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. During the last 30 years, enterohemorrhagic Escherichia coli (EHEC) emerged as worldwide causes of diarrhea and hemolytic uremic syndrome, the most common cause of acute kidney failure in children. EHEC hemolysin (EHEC-Hly) is one of the toxins produced by EHEC during infection that afflict the human host. 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 ultimately cell lysis. Here 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. Instead, the OMV-associated EHEC-Hly uses the OMVs to enter the cells and acts intracellularly. The toxin separates from its carriers in lysosomes, translocates into mitochondria and triggers apoptotic death of the target cells via the mitochondrial pathway. EHEC-Hly is the first known bacterial toxin, which enters host cells via OMVs in order to attack mitochondria. The apoptotic potential of OMV-associated EHEC-Hly indicates a novel mechanism for this toxin to cause cell death during human EHEC infections.

Escherichia coli serogroup O26 consists of enterohemorrhagic E. coli (EHEC) and atypical enteropathogenic E. coli (aEPEC). The former produces Shiga toxins (Stx), major determinants of EHEC pathogenicity, encoded by bacteriophages; the latter is Stx negative. We have isolated EHEC O26 from patient stools early in illness and aEPEC O26 from stools later in illness, and vice versa. Intrapatient EHEC and aEPEC isolates had quite similar pulsed-field gel electrophoresis (PFGE) patterns, suggesting that they might have arisen by conversion between the EHEC and aEPEC pathotypes during infection. To test this hypothesis, we asked whether EHEC O26 can lose stx genes and whether aEPEC O26 can be lysogenized with Stx-encoding phages from EHEC O26 in vitro. The stx ₂ loss associated with the loss of Stx2-encoding phages occurred in 10% to 14% of colonies tested. Conversely, Stx2- and, to a lesser extent, Stx1-encoding bacteriophages from EHEC O26 lysogenized aEPEC O26 isolates, converting them to EHEC strains. In the lysogens and EHEC O26 donors, Stx2-converting bacteriophages integrated in yecE or wrbA . The loss and gain of Stx-converting bacteriophages diversifies PFGE patterns; this parallels findings of similar but not identical PFGE patterns in the intrapatient EHEC and aEPEC O26 isolates. EHEC O26 and aEPEC O26 thus exist as a dynamic system whose members undergo ephemeral interconversions via loss and gain of Stx-encoding phages to yield different pathotypes. The suggested occurrence of this process in the human intestine has diagnostic, clinical, epidemiological, and evolutionary implications.

Background. Some Escherichia coli produce Shiga toxin (Stx) in which cytotoxicity is increased (activated) by intestinal mucus and elastase (Stx2dact

The highly virulent Escherichia coli O104:H4 that caused the large 2011 outbreak of diarrhoea and haemolytic uraemic syndrome secretes blended virulence factors of enterohaemorrhagic and enteroaggregative E. coli, but their secretion pathways are unknown. We demonstrate that the outbreak strain releases a cocktail of virulence factors via outer membrane vesicles (OMVs) shed during growth. The OMVs contain Shiga toxin (Stx) 2a, the major virulence factor of the strain, Shigella enterotoxin 1, H4 flagellin and O104 lipopolysaccharide. The OMVs bind to and are internalised by human intestinal epithelial cells via dynamin-dependent and Stx2a-independent endocytosis, deliver the OMV-associated virulence factors intracellularly and induce caspase-9-mediated apoptosis and interleukin-8 secretion. Stx2a is the key OMV component responsible for the cytotoxicity, whereas flagellin and lipopolysaccharide are the major interleukin-8 inducers. The OMVs represent novel ways for the E. coli O104:H4 outbreak strain to deliver pathogenic cargoes and injure host cells.

Background. Enterohemorrhagic Escherichia coli (EHEC) O26 causes diarrhea and hemolytic uremic syndrome (HUS). Strains harboring the stx_1a gene prevail, but strains with stx_2a as the sole Shiga toxin–encoding gene are now emerging. The traits and virulence of the latter set of strains are unknown. We correlated stx genotypes of 272 EHEC O26 strains isolated in 7 European countries between 1996 and 2012 with disease phenotypes. We determined phylogeny, clonal structure, and plasmid gene profiles of the isolates and portray geographic and temporal distribution of the different subgroups. Methods. The stx genotypes and plasmid genes were identified using polymerase chain reaction, phylogeny was assigned using multilocus sequence typing, and clonal relatedness was established using pulsed-field gel electrophoresis. Results. Of the 272 EHEC O26 isolates, 107 (39.3%), 139 (51.1%), and 26 (9.6%) possessed stx_1a, stx_2a, or both genes, respectively. Strains harboring stx_2a only were significantly associated with HUS (odds ratio, 14.2; 95% confidence interval, 7.9–25.6; P < .001) compared to other stx genotypes. The stx_2a-harboring strains consist of 2 phylogenetically distinct groups defined by sequence type (ST) 21 and ST29. The ST29 strains are highly conserved and correspond by plasmid genes to the new virulent clone of EHEC O26 that emerged in Germany in the 1990s. This new clone occurred in 6 of the 7 countries and represented approximately 50% of all stx_2a-harboring EHEC O26 strains isolated between 1996 and 2012. Conclusions. A new highly virulent clone of EHEC O26 has emerged in Europe. Its reservoirs and sources warrant identification.