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Shiga Toxin (shiga + toxin)
Selected AbstractsGlycochips from Polyanionic Glycopolymers as Tools for Detecting Shiga ToxinsCHEMBIOCHEM, Issue 17 2007Hirotaka Uzawa Dr. Abstract An alternating layer-by-layer adsorption methodology was applied to the assembly of glycochips by using synthetic polyanionic glycopolymers. Three glycochips carrying globobioside (Gb2), ,-lactoside (, -Lac), or , - D -mannoside (, -Man) residues were prepared, and used for the detection of Shiga toxins, Stx-1 and Stx-2, by using surface plasmon resonance (SPR). Using this method, we could confirm that both Stx-1 and Stx-2 show binding specificity for the Gb2 glycochip as well as a weak affinity for the , -Lac glycochip. The affinity constants of these toxins depended strongly on the sugar content of the Gb2 polymer used to prepare the glycochip. Greater affinity was observed for chips with a higher sugar content (up to 43,%) in the Gb2 glycopolymer. The maximal affinity constants of Stx-1 and Stx-2 (Ka=108,109,M,1) enabled highly sensitive and facile analysis (10 ng,mL,1, 30 min). When Gb2 glycopolymers were used as competitors, Stx-1 and Stx-2 behaved differently from one another in terms of their SPR response; this allowed us to perform discriminative analysis between the two toxins. [source] The Shiga toxin B-subunit targets antigen in vivo to dendritic cells and elicits anti-tumor immunityEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 5 2006Benoit Vingert Abstract The non-toxic B-subunit of Shiga toxin (STxB) interacts with the glycolipid Gb3, which is preferentially expressed on dendritic cells (DC) and B cells. After administration of STxB chemically coupled to OVA (STxB-OVA) in mice, we showed that the immunodominant OVA257,264 peptide restricted by Kb molecules is specifically presented by CD11c+CD8,, DC, some of them displaying a mature phenotype. Using mice carrying a transgene encoding a diphtheria toxin receptor (DTR) under the control of the murine CD11c promoter, which allows inducible ablation of DC, we showed that DC are required for efficient priming of CTL after STxB-OVA vaccination. Immunization of mice with STxB-OVA induced OVA-specific CD8+ T cells detected ex vivo; these cells were long lasting, since they could be detected even 91,days after the last immunization and were composed of both central and memory T cells. Vaccination of mice with STxB-OVA and STxB coupled to E7, a protein derived from HPV16, inhibited tumor growth in prophylactic and therapeutic experiments. This effect was mainly mediated by CD8+ T cells. STxB therefore appears to be a powerful carrier directly targeting DC in vivo, resulting in a strong and durable CTL response associated with tumor protection. [source] The A-subunit of surface-bound Shiga toxin stimulates clathrin-dependent uptake of the toxinFEBS JOURNAL, Issue 16 2005Maria L. Torgersen Shiga toxin can be internalized by clathrin-dependent endocytosis in different cell lines, although it binds specifically to the glycosphingolipid Gb3. It has been demonstrated previously that the toxin can induce recruitment of the toxin,receptor complex to clathrin-coated pits, but whether this process is concentration-dependent or which part of the toxin molecule is involved in this process, have so far been unresolved issues. In this article, we show that the rate of Shiga toxin uptake is dependent on the toxin concentration in several cell lines [HEp-2, HeLa, Vero and baby hamster kidney (BHK)], and that the increased rate observed at higher concentrations is strictly dependent on the presence of the A-subunit of cell surface-bound toxin. Surface-bound B-subunit has no stimulatory effect. Furthermore, this increase in toxin endocytosis is dependent on functional clathrin, as it did not occur in BHK cells after induction of antisense to clathrin heavy chain, thereby blocking clathrin-dependent endocytosis. By immunofluorescence, we show that there is an increased colocalization between Alexa-labeled Shiga toxin and Cy5-labeled transferrin in HeLa cells upon addition of unlabeled toxin. In conclusion, the data indicate that the Shiga toxin A-subunit of cell surface-bound toxin stimulates clathrin-dependent uptake of the toxin. Possible explanations for this phenomenon are discussed. [source] Rapid laboratory identification and characterization of verocytotoxigenic (Shiga toxin producing) Escherichia coli (VTEC/STEC)JOURNAL OF APPLIED MICROBIOLOGY, Issue 2 2003K.A. Bettelheim First page of article [source] Shiga toxin enhances functional tissue factor on human glomerular endothelial cells: implications for the pathophysiology of hemolytic uremic syndrome,JOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 4 2005E. NESTORIDI Summary.,Background:,The pathogenesis of Shiga toxin (Stx)-mediated childhood hemolytic uremic syndrome (HUS) is not fully delineated, although current evidence implicates a prothrombotic state. We hypothesized that the tissue factor (TF) pathway plays a major role in the pathophysiology of HUS. Materials and methods:,We measured cell surface TF activity in response to tumor necrosis factor-, (TNF-,) (20 ng mL,1, 2,144 h), Stx-1 (10,11 mol L,1, 4,144 h), or their combination (TNF-, 22 h and Stx-1 for the last 0.5,4 h of TNF-, incubation) on human glomerular (microvascular) endothelial cells (HGECs) and human umbilical vein (macrovascular) endothelial cells (HUVECs). Results and conclusions:,We observed that while TNF-, caused an increase in cell surface TF activity on both cell types, the combination of TNF-, and Stx-1 differentially affected HGECs. On these cells, TF activity was increased further by 2.67 ± 0.38-fold (n = 38, P < 0.001), consistent with our parallel observation that Stx-1 binds to HGECs but not to HUVECs. Anti-TF antibody abolished functional TF while anti-tissue factor pathway inhibitor antibody enhanced TF activity. Stx-1 alone did not induce TF activity on either cell type. Measurement of TF antigen levels and quantitative real-time polymerase chain reaction demonstrated that exposure to TNF-, markedly increased TF protein and TF mRNA for HGECs, but the exposure to the combination of TNF-, and Stx-1 did not increase further the amount of either TF protein or TF mRNA. We conclude that cytokine-activated HGECs, but not HUVECs, undergo a significant augmentation of cell surface TF activity following exposure to Stx, suggesting an important role for TF in the coagulopathy observed in HUS. [source] Production of Shiga toxin by Shiga toxin-expressing Escherichia coli (STEC) in broth media: from divergence to definitionLETTERS IN APPLIED MICROBIOLOGY, Issue 4 2007L.B. Rocha Abstract Aims:, To determine the suitability of eight different commercial broth media for Shiga toxin (Stx) production. Methods and Results:, Shiga toxin-producing Escherichia coli (STEC) strains producing Stx1 or Stx2 were grown at 37°C (250 rev min,1) for 24 h in brain heart infusion broth, E. coli broth, Evans medium, Luria-Bertani broth, Penassay broth, buffered-peptone water, syncase broth and trypticase soy broth. Toxin production was measured by enzyme-linked immunosorbent assay (ELISA) in polymyxin-treated cell pellets and/or supernatants of cultures, ELISA optical densities reached 1 when isolates were grown for 2,4 h in E. coli broth in the presence of antibiotic. Besides, a collection of STEC-expressing Stx strains was evaluated and the Stx production was assayed in the supernatants and in polymyxin-treated pellets of bacterial growth after 4 h of cultivation in E. coli broth in the presence of antibiotic. Conclusions:, The most suitable medium for Stx production was E. coli broth when the bacterial isolates were grown for 4 h in the presence of ciprofloxacin and the Stx production is detected in the supernatant. Significance and Impact of the Study:, This study presents the first comprehensive comparison of different broth media with regard to Stx production to establish optimal culture conditions for STEC detection in routine diagnostic laboratories. [source] On the structural diversity of Shiga toxin glycosphingolipid receptors in lymphoid and myeloid cells determined by nanoelectrospray ionization tandem mass spectrometryRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 15 2010Petra Hoffmann Shiga toxin (Stx, synonymous to verotoxin, VT) binds with high and low affinity to the globo-series neutral glycosphingolipids (GSLs), globotriaosylceramide (Gb3Cer or Gal,4Gal,4Glc,1Cer, also known as CD77) and globotetraosylceramide (Gb4Cer or GalNAc,3Gal,4Gal,4Glc,1Cer), respectively, which represent the targets of Stxs on many different cell types. B-cell-derived Raji cells and THP-1 cells of monocytic origin are widely used for the investigation of Stx-mediated cellular response, because Stx is known to cause cell death in both cell lines. Despite their functional importance, the Stx receptors of Raji and THP-1 cells have so far not been investigated. This prompted us to explore the structures of their GSL receptors in detail by means of nanoelectrospray ionization quadrupole time-of-flight mass spectrometry (nanoESI-QTOF-MS) with collision-induced dissociation (CID) in conjunction with Stx1 as well as anti-Gb3Cer and anti-Gb4Cer antibodies. Using the combination of a thin-layer chromatography (TLC) overlay assay and MS1 and MS2 analysis we identified Gb3Cer (d18:1, C24:1/C24:0) as the prevalent Stx1-receptor accompanied by less abundant Gb3Cer (d18:1, C16:0) in the neutral GSL fraction of Raji cells. The same Gb3Cer species but with almost equal proportions of the C24:1/C24:0 and C16:0 variants were found in THP-1 cells. In addition, unusual hydroxylated Gb3Cer (d18:1, C24:1/C24:0) and Gb3Cer (d18:1, C26:1) could be identified in trace quantities in both cell lines. As the most obvious difference between Raji and THP-1 cells we observed the expression of Gb4Cer in THP-1 cells, whereas Raji cells failed to express this elongation product of Gb3Cer. Both short- and long-chain fatty acid carrying Gb4Cer (d18:1, C16:0) and Gb4Cer (d18:1, C24:1/C24:0), respectively, were the prevalent Gb4Cer variants. This first report on the differential expression of Gb3Cer and Gb4Cer and their structural diversity in lymphoid and myeloid cell lines supports the hypothesis that such heterogeneities might play a functional role in the molecular assembly of GSLs in membrane organization and cellular signaling of Stx-susceptible cells. Copyright © 2010 John Wiley & Sons, Ltd. [source] Role of EHEC O157:H7 virulence factors in the activation of intestinal epithelial cell NF-,B and MAP kinase pathways and the upregulated expression of interleukin 8CELLULAR MICROBIOLOGY, Issue 10 2002M. Cecilia Berin Summary Enterohaemorrhagic Escherichia coli O157:H7 (EHEC) is a gastrointestinal pathogen that is generally non-invasive for intestinal epithelial cells, yet causes acute intestinal inflammation, diarrhoea, haemorrhagic colitis and haemolytic uraemic syndrome. To study signal transduction pathways activated in human intestinal epithelial cells by EHEC, we took advantage of EHEC O157:H7 and isogenic mutants deficient in the major EHEC virulence factors, intimin (eae,) and Shiga toxin (stx,). Infection with wild-type EHEC activated p38 and ERK MAP kinases and the nuclear translocation of the transcription factor NF-,B. Downstream, this was accompanied by increased expression of mRNA and protein for the neutrophil chemoattractant IL-8. Isogenic eae, and stx, mutants also activated p38 and ERK MAP kinases, and NF-,B and stimulated increases in IL-8 protein secretion similar to those of wild-type EHEC. Further, inhibition of either p38, ERK or NF-,B activation abrogated the IL-8 response induced by wild-type EHEC and the mutants. Epithelial cell MAP kinase and NF-,B pathways leading to IL-8 secretion were also activated by isolated EHEC H7 flagellin, which was active when added to either the apical or basolateral surface of polarized human intestinal epithelial cells. We conclude that EHEC interacting with intestinal epithelial cells activates intracellular signalling pathways and an epithelial cell proinflammatory response independent of either Shiga toxin or intimin, two of the major known virulence factors of EHEC. The activation of proinflammatory signals in human colon epithelial cells in response to this non-invasive pathogen appears to depend to a significant extent on H7 flagellin. [source] Glycochips from Polyanionic Glycopolymers as Tools for Detecting Shiga ToxinsCHEMBIOCHEM, Issue 17 2007Hirotaka Uzawa Dr. Abstract An alternating layer-by-layer adsorption methodology was applied to the assembly of glycochips by using synthetic polyanionic glycopolymers. Three glycochips carrying globobioside (Gb2), ,-lactoside (, -Lac), or , - D -mannoside (, -Man) residues were prepared, and used for the detection of Shiga toxins, Stx-1 and Stx-2, by using surface plasmon resonance (SPR). Using this method, we could confirm that both Stx-1 and Stx-2 show binding specificity for the Gb2 glycochip as well as a weak affinity for the , -Lac glycochip. The affinity constants of these toxins depended strongly on the sugar content of the Gb2 polymer used to prepare the glycochip. Greater affinity was observed for chips with a higher sugar content (up to 43,%) in the Gb2 glycopolymer. The maximal affinity constants of Stx-1 and Stx-2 (Ka=108,109,M,1) enabled highly sensitive and facile analysis (10 ng,mL,1, 30 min). When Gb2 glycopolymers were used as competitors, Stx-1 and Stx-2 behaved differently from one another in terms of their SPR response; this allowed us to perform discriminative analysis between the two toxins. [source] Human isolates of Aeromonas possess Shiga toxin genes (stx1 and stx2) highly similar to the most virulent gene variants of Escherichia coliCLINICAL MICROBIOLOGY AND INFECTION, Issue 10 2010A. Alperi Clin Microbiol Infect 2010; 16: 1563,1567 Abstract Strains producing Shiga toxins, encoded by stx1 and stx2 genes, can cause diarrhoea, haemorrhagic colitis and haemolytic uremic syndrome. PCR screening of 80 clinical Aeromonas strains showed that 19 were stx1-positive and only one was positive for both stx1 and stx2. PCR bands were very faint for some strains and negative results were obtained after subculturing. The obtained sequences of Aeromonas stx1 and stx2 genes were highly similar to those of the most virulent stx gene variants of Shiga toxin-producing Escherichia coli. These results may lead to a better understanding of the potential pathogenicity and virulence mechanisms of Aeromonas. [source] | ||||||||||||||||