Ciprofloxacin, meropenem, fosfomycin, and polymyxin B strongly increase production of outer membrane vesicles (OMVs) in
O104:H4 and O157:H7. Ciprofloxacin also upregulates OMV-associated Shiga toxin ...2a, the major virulence factor of these pathogens, whereas the other antibiotics increase OMV production without the toxin. These two effects might worsen the clinical outcome of infections caused by Shiga toxin-producing
Our data support the existing recommendations to avoid antibiotics for treatment of these infections.
Summary Background In an ongoing outbreak of haemolytic uraemic syndrome and bloody diarrhoea caused by a virulent Escherichia coli strain O104:H4 in Germany (with some cases elsewhere in Europe and ...North America), 810 cases of the syndrome and 39 deaths have occurred since the beginning of May, 2011. We analysed virulence profiles and relevant phenotypes of outbreak isolates recovered in our laboratory. Methods We analysed stool samples from 80 patients that had been submitted to the National Consulting Laboratory for Haemolytic Uraemic Syndrome in Münster, Germany, between May 23 and June 2, 2011. Isolates were screened with standard PCR for virulence genes of Shiga-toxin-producing E coli and a newly developed multiplex PCR for characteristic features of the outbreak strain ( rfbO104 , fliCH4 , stx2 , and terD ). Virulence profiles of the isolates were determined with PCR targeting typical virulence genes of Shiga-toxin-producing E coli and of other intestinal pathogenic E coli . We sequenced stx with Sanger sequencing and measured Shiga-toxin production, adherence to epithelial cells, and determined phylogeny and antimicrobial susceptibility. Findings All isolates were of the HUSEC041 clone (sequence type 678). All shared virulence profiles combining typical Shiga-toxin-producing E coli ( stx2 , iha, lpfO26 , lpfO113 ) and enteroaggregative E coli ( aggA, aggR, set1, pic, aap ) loci and expressed phenotypes that define Shiga-toxin-producing E coli and enteroaggregative E coli , including production of Shiga toxing 2 and aggregative adherence to epithelial cells. Isolates additionally displayed an extended-spectrum β-lactamase phenotype absent in HUSEC041. Interpretation Augmented adherence of the strain to intestinal epithelium might facilitate systemic absorption of Shiga toxin and could explain the high progression to haemolytic uraemic syndrome. This outbreak demonstrates that blended virulence profiles in enteric pathogens, introduced into susceptible populations, can have extreme consequences for infected people. Funding German Federal Ministry of Education and Research, Network Zoonoses.
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.
Abstract Enterohemorrhagic Escherichia coli (EHEC) cause diarrhea, bloody diarrhea and hemolytic-uremic syndrome (HUS), a thrombotic microangiopathy affecting the renal glomeruli, the intestine, and ...the brain. The pathogenesis of EHEC-mediated diseases is incompletely understood. In addition to Shiga toxins, the major virulence factors of EHEC, the contribution of EHEC hemolysin (EHEC-Hly), also designated EHEC toxin (Ehx), which is a member of the RTX (repeats-in-toxin) family, is increasingly recognized. The toxin and its activation and secretion machinery are encoded by the EHEC- hlyCABD operon, in which EHEC- hlyA is the structural gene for EHEC-Hly and the EHEC- hlyC product mediates post-translational activation of EHEC-Hly; the EHEC- hlyB - and EHEC- hlyD -encoded proteins form, together with genetically unlinked TolC, the type I secretion system that transports EHEC-Hly out of the bacterial cell. EHEC-Hly exists in two biologically active forms: as a free EHEC-Hly, and an EHEC-Hly associated with outer membrane vesicles (OMVs) that are released by EHEC during growth. The OMV-associated form results from a rapid binding of free EHEC-Hly to OMVs upon its extracellular secretion. The OMV association stabilizes EHEC-Hly and thus substantially prolongs its hemolytic activity compared to the free toxin. The two EHEC-Hly forms differ by their mechanism of toxicity toward human intestinal epithelial and microvascular endothelial cells, which are the major targets during EHEC infection. The free EHEC-Hly lyses human microvascular endothelial cells, presumably by pore formation in the cell membrane. In contrast, the OMV-associated EHEC-Hly does not lyse any of these cell types, but after its cellular internalization via OMVs it targets mitochondria and triggers caspase-9-mediated apoptosis. The proinflammatory potential of EHEC-Hly, in particular its ability to elicit secretion of interleukin-1β from human monocytes/macrophages, might be an additional mechanism of its putative contribution to the pathogenesis of EHEC-mediated diseases. Increasing understanding of molecular mechanisms underlying interaction of EHEC-Hly with target cells as well as the host cell responses to the toxin supports the involvement of EHEC-Hly in the pathogenesis of EHEC-mediated diseases and forms a basis for prevention of the EHEC-Hly-mediated injury during human infection.
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.
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.
Shiga toxin (Stx)-mediated injury to vascular endothelial cells in the kidneys, brain and other organs underlies the pathogenesis of haemolytic uraemic syndrome (HUS) caused by enterohaemorrhagic ...Escherichia coli (EHEC). We present a direct and comprehensive comparison of cellular injury induced by the two major Stx types, Stx1 and Stx2, in human brain microvascular endothelial cells (HBMECs) and EA.hy 926 macrovascular endothelial cells. Scanning electron microscopy of microcarrier-based cell cultures, digital holographic microscopy of living single cells, and quantitative apoptosis/necrosis assays demonstrate that Stx1 causes both necrosis and apoptosis, whereas Stx2 induces almost exclusively apoptosis in both cell lines. Moreover, microvascular and macrovascular endothelial cells have different susceptibilities to the toxins: EA.hy 926 cells are slightly, but significantly (∼ 10 times) more susceptible to Stx1, whereas HBMECs are strikingly (≥ 1,000 times) more susceptible to Stx2. These findings have implications in the pathogenesis of HUS, and suggest the existence of yet to be delineated Stx type-specific mechanisms of endothelial cell injury beyond inhibition of protein biosynthesis.
Extraintestinal pathogenic and intestinal pathogenic (diarrheagenic) Escherichia coli differ phylogenetically and by virulence profiles. Classic theory teaches simple linear descent in this species, ...where non‐pathogens acquire virulence traits and emerge as pathogens. However, diarrheagenic Shiga toxin‐producing E. coli (STEC) O2:H6 not only possess and express virulence factors associated with diarrheagenic and uropathogenic E. coli but also cause diarrhea and urinary tract infections. These organisms are phylogenetically positioned between members of an intestinal pathogenic group (STEC) and extraintestinal pathogenic E. coli. STEC O2:H6 is, therefore, a ‘heteropathogen,’ and the first such hybrid virulent E. coli identified. The phylogeny of these E. coli and the repertoire of virulence traits they possess compel consideration of an alternate view of pathogen emergence, whereby one pathogroup of E. coli undergoes phased metamorphosis into another. By understanding the evolutionary mechanisms of bacterial pathogens, rational strategies for counteracting their detrimental effects on humans can be developed.
Synopsis
Hybrid E. coli pathogens clinical significance was clearly shown during the deadly 2011 outbreak. STEC O2:H6 was diagnosed intestinal and uropathogenic in patients and is shown here as another heteropathogen that emerged via pathogroup conversion.
Shiga toxin‐producing Escherichia coli (STEC) O2:H6 were found to be phylogenetically positioned between intestinal pathogenic and uropathogenic E. coli, possess and express virulence factors of both STEC and uropathogenic E. coli, and cause diarrhea as well as urinary tract infections.
Based on their phylogeny, virulence features and pluripotential pathogenicity in intestinal and extraintestinal milieus, STEC O2:H6 are proposed to be ‘heteropathogens’ that occupy an evolutionary and pathogenic interface between intestinal and extraintestinal pathogenic E. coli, and are the first such hybrid virulent E. coli identified.
In contrast to the classic view that pathogens emerge from non‐pathogens via sequential, linear acquisition of virulence genes, STEC O2:H6 are portrayed as possessing heteropathogenic virulence potential.
The novel evolutionary concept of phased metamorphosis is compelled by our identification of genomes with dual virulence attributes.
Broader surveys combining backbone phylogeny and systematic virulence gene analyses are needed to determine if the pathogen emergence via transition is a generalized process.
Hybrid Escherichia coli pathogens clinical significance was clearly shown during the deadly 2011 outbreak. STEC O2:H6 was diagnosed intestinal and uropathogenic in patients and is shown here as another heteropathogen that emerged via pathogroup conversion.
PDF
