Virulence Factor Cargo and Host Cell Interactions of Shiga Toxin-Producing Escherichia coli Outer Membrane Vesicles.

Outer membrane vesicles (OMVs), nanoparticles released by Shiga toxin-producing Escherichia coli (STEC), have been identified as novel efficient virulence tools of these pathogens. STEC O157 OMVs carry a cocktail of virulence factors including Shiga toxin 2a (Stx2a), cytolethal distending toxin V (CdtV), EHEC hemolysin, flagellin, and lipopolysaccharide. OMVs are taken up by human intestinal epithelial and microvascular endothelial cells, the major targets during STEC infection, and deliver the virulence factors into host cells.

Advanced Methods for MALDI-MS Imaging of the Chemical Communication in Microbial Communities.

Matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) is increasingly used to visualize the chemical communication between microorganisms. However, to fully exploit the potential of this label-free technique, crucial methodological advances are still needed. In particular, with current microbial MALDI-MSI methods chemical coverage is strongly limited to well ionizing compounds and a safe MSI-compatible inactivation of microbial viability and quenching of metabolism is not possible.

Determination of virulence and fitness genes associated with the , and integration sites of LEE-negative food-borne Shiga toxin-producing strains.

Background: In the current study, nine foodborne "Locus of Enterocyte Effacement" (LEE)-negative Shiga toxin-producing (STEC) strains were selected for whole genome sequencing and analysis for yet unknown genetic elements within the already known LEE integration sites , and . Foreign DNA ranging in size from 3.4 to 57 kbp was detected and further analyzed.

Enterohemorrhagic Escherichia coli O157 outer membrane vesicles induce interleukin 8 production in human intestinal epithelial cells by signaling via Toll-like receptors TLR4 and TLR5 and activation of the nuclear factor NF-κB.

Proinflammatory cytokines play important roles in the pathogenesis of diseases caused by enterohemorrhagic Escherichia coli (EHEC) O157, but the spectrum of bacterial components involved in the proinflammatory responses is not fully understood. Here, we investigated the abilities of outer membrane vesicles (OMVs), nanoparticles released by EHEC O157 during growth, to induce production of proinflammatory cytokines in human intestinal epithelial cells. OMVs from both EHEC O157:H7 and sorbitol-fermenting (SF) EHEC O157:H induced production of interleukin-8 (IL-8) in Caco-2, HCT-8, and HT-29 intestinal epithelial cell lines.

Sorbitol-Fermenting Enterohemorrhagic Escherichia coli O157:H Isolates from Czech Patients with Novel Plasmid Composition Not Previously Seen in German Isolates.

Sorbitol-fermenting (SF) enterohemorrhagic (EHEC) O157:H strains, first identified in Germany, have emerged as important pathogens throughout Europe. Besides chromosomally encoded Shiga toxin 2a (the major virulence factor), several putative virulence loci, including the , , and operons, encoding EHEC hemolysin, type II secretion system proteins, and Sfp fimbriae, respectively, are located on the 121-kb plasmid pSFO157 in German strains. Here we report novel SF EHEC O157:H strains isolated from patients in the Czech Republic.

Intrahost milieu modulates production of outer membrane vesicles, vesicle-associated Shiga toxin 2a and cytotoxicity in Escherichia coli O157:H7 and O104:H4.

Outer membrane vesicles (OMVs) are important virulence tools of enterohaemorrhagic Escherichia coli (EHEC), but other biological functions of these nanostructures are unknown. We tested the hypothesis that modulation of OMV production enables EHEC to resist the intrahost environment during infection by investigating if simulated human gastrointestinal conditions affect OMV production in EHEC O157:H7 and O104:H4. All the conditions tested including a low pH, simulated ileal and colonic media, presence of mucin, intestinal epithelial cell lysate or antimicrobial peptides, as well as iron limitation, significantly increased OMV production by these pathogens.

Antibiotic-Mediated Modulations of Outer Membrane Vesicles in Enterohemorrhagic Escherichia coli O104:H4 and O157:H7.

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.

Host cell interactions of outer membrane vesicle-associated virulence factors of enterohemorrhagic Escherichia coli O157: Intracellular delivery, trafficking and mechanisms of cell injury.

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.

On-Tissue Phospholipase C Digestion for Enhanced MALDI-MS Imaging of Neutral Glycosphingolipids.

Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) can be used to simultaneously visualize the lateral distribution of different lipid classes in tissue sections, but the applicability of the method to real-life samples is often limited by ion suppression effects. In particular, the presence of abundant phosphatidylcholines (PCs) can reduce the ion yields for all other lipid species in positive ion mode measurements. Here, we used on-tissue treatment with buffer-free phospholipase C (PLC) to near-quantitatively degrade PCs in fresh-frozen tissue sections.

Shiga toxin of enterohaemorrhagic Escherichia coli directly injures developing human erythrocytes.

Haemolytic anaemia is one of the characteristics of life-threatening extraintestinal complications in humans during infection with enterohaemorrhagic Escherichia coli (EHEC). Shiga toxins (Stxs) of EHEC preferentially damage microvascular endothelial cells of the kidney and the brain, whereby occluded small blood vessels may elicit anaemia through mechanical erythrocyte disruption. Here we show for the first time that Stx2a, the major virulence factor of EHEC, is also capable of direct targeting developing human erythrocytes.

Virulence from vesicles: Novel mechanisms of host cell injury by Escherichia coli O104:H4 outbreak strain.

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.

Hemolysin of enterohemorrhagic Escherichia coli: structure, transport, biological activity and putative role in virulence.

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.

Heteropathogenic virulence and phylogeny reveal phased pathogenic metamorphosis in Escherichia coli O2:H6.

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.

Enterohemorrhagic Escherichia coli hemolysin employs outer membrane vesicles to target mitochondria and cause endothelial and epithelial apoptosis.

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.

Lability of the pAA Virulence Plasmid in Escherichia coli O104:H4: Implications for Virulence in Humans.

Background: Escherichia coli O104:H4 that caused the large German outbreak in 2011 is a highly virulent hybrid of enterohemorrhagic (EHEC) and enteroaggregative (EAEC) E. coli. The strain displays "stacked-brick" aggregative adherence to human intestinal epithelial cells mediated by aggregative adherence fimbriae I (AAF/I) encoded on the pAA plasmid.

Association of Shiga toxin glycosphingolipid receptors with membrane microdomains of toxin-sensitive lymphoid and myeloid cells.

Glycosphingolipids (GSLs) of the globo-series constitute specific receptors for Shiga toxins (Stxs) released by certain types of pathogenic Escherichia coli strains. Stx-loaded leukocytes may act as transporter cells in the blood and transfer the toxin to endothelial target cells. Therefore, we performed a thorough investigation on the expression of globo-series GSLs in serum-free cultivated Raji and Jurkat cells, representing B- and T-lymphocyte descendants, respectively, as well as THP-1 and HL-60 cells of the monocyte and granulocyte lineage, respectively.

Facing glycosphingolipid-Shiga toxin interaction: dire straits for endothelial cells of the human vasculature.

The two major Shiga toxin (Stx) types, Stx1 and Stx2, produced by enterohemorrhagic Escherichia coli (EHEC) in particular injure renal and cerebral microvascular endothelial cells after transfer from the human intestine into the circulation. Stxs are AB(5) toxins composed of an enzymatically active A subunit and the pentameric B subunit, which preferentially binds to the glycosphingolipid globotriaosylceramide (Gb3Cer/CD77). This review summarizes the current knowledge on Stx-caused cellular injury and the structural diversity of Stx receptors as well as the initial molecular interaction of Stxs with the human endothelium of different vascular beds.

Uncommon membrane distribution of Shiga toxin glycosphingolipid receptors in toxin-sensitive human glomerular microvascular endothelial cells.

Membrane microdomain association of the glycosphingolipids (GSLs) globotriaosylceramide (Gb3Cer) and globotetraosylceramide (Gb4Cer), the highly and less effective receptors, respectively, for Shiga toxins (Stxs), is assumed as a functional requirement for Stx-mediated cytotoxicity. In a previous study, we demonstrated predominant localization of Stx receptors in cholesterol-enriched membrane microdomains of moderately Stx-sensitive human brain microvascular endothelial cells (HBMECs) by means of detergent-resistant membranes (DRMs). Here we report a different preferential distribution of Stx receptors in non-DRM fractions of human glomerular microvascular endothelial cells (GMVECs), the major targets of Stxs in the human kidney.

Effects of antibiotics on Shiga toxin 2 production and bacteriophage induction by epidemic Escherichia coli O104:H4 strain.

The role of antibiotics in treatment of enterohemorrhagic Escherichia coli (EHEC) infections is controversial because of concerns about triggering hemolytic-uremic syndrome (HUS) by increasing Shiga toxin (Stx) production. During the recent large EHEC O104:H4 outbreak, antibiotic therapy was indicated for some patients. We tested a diverse panel of antibiotics to which the outbreak strain is susceptible to interrogate the effects of subinhibitory antibiotic concentrations on induction of stx(2)-harboring bacteriophages, stx(2) transcription, and Stx2 production in this emerging pathogen.

Real-time multiplex PCR for detecting Shiga toxin 2-producing Escherichia coli O104:H4 in human stools.

A real-time multiplex PCR targeting stx(2), wzy(O104), and fliC(H4) of enterohemorrhagic Escherichia coli (EHEC) O104:H4 correctly determined the presence or absence of these genes in 253 EHEC isolates and enrichment cultures of stool samples from 132 patients. It is a rapid, sensitive, and specific tool for detecting EHEC O104:H4 in human stools.

Characterisation of the Escherichia coli strain associated with an outbreak of haemolytic uraemic syndrome in Germany, 2011: a microbiological study.

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.

Differential cytotoxic actions of Shiga toxin 1 and Shiga toxin 2 on microvascular and macrovascular endothelial 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.

Label-free quantitative cell division monitoring of endothelial cells by digital holographic microscopy.

Digital holographic microscopy (DHM) enables quantitative multifocus phase contrast imaging for nondestructive technical inspection and live cell analysis. Time-lapse investigations on human brain microvascular endothelial cells demonstrate the use of DHM for label-free dynamic quantitative monitoring of cell division of mother cells into daughter cells. Cytokinetic DHM analysis provides future applications in toxicology and cancer research.

Vacuolisation of human microvascular endothelial cells by enterohaemorrhagic Escherichia coli.

Enterohaemorrhagic Escherichia coli (EHEC) cause haemolytic uraemic syndrome (HUS), a thrombotic microangiopathy resulting from endothelial injury in the renal glomeruli and other organs. EHEC virulence factors that damage the microvascular endothelium play therefore major roles in the pathogenesis of HUS. We identified an EHEC strain that vacuolates and kills primary human glomerular microvascular endothelial cells (GMVECs) and a human brain microvascular endothelial cell (HBMEC) line.