Tae Takeda

Inhibition of in vitro growth of Shiga toxin-producing Escherichia coli O157:H7 by probiotic Lactobacillus strains due to production of lactic acid

The inhibiting characteristics of lactic acid bacteria on Shiga toxin-producing Escherichia coli (STEC) O157:H7 (three strains, clinically isolated) was investigated by using a batch fermentation system. The species such as Lactobacillus casei strain Shirota or L. acidophilus YIT 0070 exert growth inhibitory and bactericidal activities on STEC. The pH value and undissociated lactic acid (U-LA) concentration of the culture medium of STEC cocultured with L. casei or L. acidophilus dramatically lowered or increased, respectively [corrected], when compared with those of the control culture. The cytotoxic properties of U-LA on STEC strain 89020087 analyzed in vitro was divided into two phases, i.e., the bacteriostatic phase (between 3.2 to 62 mM) and the bactericidal phase (over 62 mM). These data suggest that the bactericidal effect of Lactobacillus on STEC depends on its lactic acid production and pH reductive effect.

Induction of Apoptosis in Normal Human Renal Tubular Epithelial Cells by Escherichia coli Shiga Toxins 1 and 2

The cytotoxicity of Shiga toxin (Stx) 1 and Stx2 produced by Escherichia coli to human renal cortical epithelial cells (HRCEC) in primary culture was investigated. HRCEC express CD24, the marker of renal distal tubules, as well as globotriaosyl ceramide/CD77, the receptor for Stxs. Binding of Stxs to HRCEC was confirmed by positive staining with specific antibodies to Stxs. Treatment of HRCEC with Stxs induced rapid cell death, which was reversed in the presence of neutralizing antibody specific for Stx. DNA fragmentation was found to be accompanied by Stx-mediated cell death in HRCEC, indicating that apoptosis was part of the process. These data and previous reports indicate that a variety of renal cell types, including tubular epithelial cells as well as glomerular capillary endothelial cells, may be targets for Stx-mediated apoptosis, which could contribute to the pathogenesis of hemolytic-uremic syndrome caused by Stx-producing E. coli infection.

Kinetic Analysis of Binding between Shiga Toxin and Receptor Glycolipid Gb3Cer by Surface Plasmon Resonance

Shiga toxin (Stx) binds to the receptor glycolipid Gb3Cer on the cell surface and is responsible for hemolytic uremic syndrome. Stx has two isoforms, Stx1 and Stx2, and in clinical settings Stx2 is known to cause more severe symptoms, although the differences between the mechanisms of action of Stx1 and Stx2 are as yet unknown. In this study, the binding modes of these two isoforms to the receptor were investigated with a surface plasmon resonance analyzer to compare differences by real time receptor binding analysis. A sensor chip having a lipophilically modified dextran matrix or quasicrystalline hydrophobic layer was used to immobilize an amphipathic lipid layer that mimics the plasma membrane surface. Dose responsiveness was observed with both isoforms when either the toxin concentration or the Gb3Cer concentration was increased. In addition, this assay was shown to be specific, because neither Stx1 nor Stx2 bound to GM3, but both bound weakly to Gb4Cer. It was also shown that a number of fitting models can be used to analyze the sensorgrams obtained with different concentrations of the toxins, and the “bivalent analyte” model was found to best fit the interaction between Stxs and Gb3Cer. This shows that the interaction between Stxs and Gb3Cer in the lipid bilayer has a multivalent effect. The presence of cholesterol in the lipid bilayer significantly enhanced the binding of Stxs to Gb3Cer, although kinetics were unaffected. The association and dissociation rate constants of Stx1 were larger than those of Stx2: Stx2 binds to the receptor more slowly than Stx1 but, once bound, is difficult to dissociate. The data described herein clearly demonstrate differences between the binding properties of Stx1 and Stx2 and may facilitate understanding of the differences in clinical manifestations caused by these toxins.

Virulence patterns of Vibrio cholerae non-01 strains isolated from hospitalised patients with acute diarrhoea in Calcutta, India

A collection of 28 strains of Vibrio cholerae non-O1 isolated during a 3-year period (1989-1991) from hospitalised patients with acute diarrhoea in Calcutta, India, were examined with regard to virulence-associated factors. Of the 28 isolates (each representing a case), 18 were isolated as the sole infecting agent; the remaining 10 were recovered as co-cultures from cases infected with V. cholerae O1. Of the strains isolated in this study, 82% could be serotyped, with serovars O5 (32.1%), O11 and O34 (14.3% each) predominant. Serovars O7, O14, O34, O39 and O97 were associated exclusively with sole infections. Two strains of V. cholerae non-O1 produced anti-cholera toxin IgG-absorbable cholera toxin (CT). Both CT-producing V. cholerae non-O1 strains hybridised with the DNA probe specific for the zonula occludens toxin (ZOT) but none of the remaining 26 strains hybridised with the ZOT probe. The majority of the strains were cytotoxic for CHO, HeLa and Vero cells, with end-point titres of 4-512. Fewer strains produced a cytotonic effect, with end-point titres of 2-16. Of the 28 strains of V. cholerae non-O1 examined, 75%, 75%, 25% and 14.3% produced haemolysin that was active against erythrocytes of rabbit, sheep (Eltor haemolysin), chicken and man, respectively. Strains that produced a haemolysin active against both rabbit and sheep erythrocytes were dominant (35.7%). Ten (35.7%) of the 28 strains examined showed cell-associated haemagglutinating activity on human blood. Of the 10 strains, nine were isolated as sole pathogen and only one strain was associated with mixed infection.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino acid sequence of a heat-stable enterotoxin isolated from enterotoxigenic Escherichia coli strain 18D

A heat‐stable enterotoxin produced by a strain of enterotoxigenic Escherichia coli 18D was purified by ion‐exchange and reversed‐phase high‐pressure liquid chromatography. The amino acid sequence of the purified toxin was determined by Edman‐degradation and a combination of fast atom bombardment mass spectrometry and carboxypeptidase digestion to be Asn‐Thr‐Phe‐Tyr‐Cys‐Cys‐Glu‐Leu‐Cys‐Cys‐Asn‐Pro‐Ala‐Cys‐Ala‐Gly‐Cys‐Tyr.

Essential structure for full enterotoxigenic activity of heat-stable enterotoxin produced by enterotoxigenic Escherichia coli.

Several analogues of heat‐stable enterotoxins (STh and STP) produced by enterotoxigenic Escherichia coli were synthesized. Peptides (STh[6–18] and STP[5–17]) consisting of 13 amino acid residues from the Cys residue near the N‐terminus to the Cys residue near the C‐terminus and linked by three disulfide bonds had the same biological and immunological properties as native STh and STP, respectively. The results indicated that the sequence with the 13 amino acid residues and three disulfide linkages is essential for full biological activity of ST.

The Detection of Shiga Toxins in the Kidney of a Patient with Hemolytic Uremic Syndrome

Infection of Shiga toxin (Stx)-producing Escherichia coli induces hemolytic uremic syndrome (HUS) in 10 to 15% of cases in infants and young children. Although the endothelial cell damage induced by Stx is widely believed to be a primary event of renal dysfunction in HUS, the precise mechanism remains to be elucidated. We were able to examine the kidney obtained at autopsy of a child who died after HUS associated with Stx-producing Escherichia coli O157:H7 infection, and immunohistochemistry indicated the deposition of Stx1 and Stx2 in a portion of the distal tubular epithelia. To our knowledge, this is the first report to show the presence of Stx in human tissue of a patient with HUS, and the results obtained in this study provide evidence that Stx indeed migrates into the kidney and binds to renal tubules during Stx-producing Escherichia coli infection.

Efficacy of Postinfection Treatment with Anti-Shiga Toxin (Stx) 2 Humanized Monoclonal Antibody TMA-15 in Mice Lethally Challenged with Stx-Producing Escherichia coli

Infection with Shiga toxin (Stx)-producing Escherichia coli (STEC) causes hemorrhagic colitis and hemolytic uremic syndrome. TMA-15 is a humanized monoclonal antibody against Stx2, a major pathogenic factor. In a mouse infection model that used B2F1, a virulent STEC strain, the efficacy of TMA-15 was assessed when it was administered after bacterial and toxin exposure. In this model, a time-course analysis of the serum Stx2 level showed that the toxin was detectable from 24 h after infection. In an evaluation of the time-dependent efficacy, treatment with TMA-15 up to 24 h after infection ameliorated the lethal challenge, although treatment at 48 h showed no efficacy. To determine the effective dose, escalating doses were administered at 24 h after infection. The number of mice that survived after doses of 0, 0.25, 0.5, 1.0, and 2.0 mg/kg were 0/20, 11/20, 17/20, 20/20, and 20/20, respectively. These findings suggest that TMA-15 shows potential for prevention of severe complications associated with STEC infection.

Globotriaosyl ceramide (CD77/Gb3) in the glycolipid-enriched membrane domain participates in B-cell receptor–mediated apoptosis by regulating Lyn kinase activity in human B cells

The role of CD77 expressed on a fraction of germinal center B cells, also known as glycosphyngolipid Gb3, and as a functional receptor for Shiga toxins (Stx) in B-cell receptor (BCR)-mediated apoptosis was investigated. Using Stx1-sensitive Burkitts lymphoma Ramos cells as an in vitro model of CD77(+) germinal center B cells, intracellular signaling events mediated by either Stx1 or anti-CD77 antibody were examined immunobiochemically and immunocytologically. We observed prompt activation of Lyn and Syk kinases leading to increased binding of these proteins to surface IgM (sIgM) in Ramos cells after Stx1 treatment. We also observed microscopic colocalization of CD77 and sIgM after stimulation with Stx1. Along with the synergism between the cross-linking of CD77 and that of sIgM in their effect on apoptosis induction, it was highly probable that CD77 cross-linking induces activation of the BCR signaling cascade. Analysis using sucrose density gradient centrifugation suggested that Stx1 binding to CD77 induced recruitment and activation of Lyn in the glycolipid-enriched membrane (GEM) fractions. Once activated, however, Lyn seemed to acquire an increased detergent solubility and moved outside of the GEM fractions. This study describes the participation of the GEM domain in BCR-signaling cascade and suggests a possible role of CD77 as a regulator of BCR-induced apoptosis in human B cells.

Clinical role for a superantigen in Yersinia pseudotuberculosis infection.

Yersinia pseudotuberculosis is an enteric pathogen that causes a variety of clinical symptoms in the human. Recently, we reported the production of a superantigen (Y. pseudotuberculosis-derived mitogen, YPM) by this organism and characterized the gene structure of ypm. To further study the potential pathogenic role of YPM in Y. pseudotuberculosis infection, we assayed IgG anti-YPM antibodies and T cell antigen receptor-Vbeta expression of the T cells in peripheral blood and in mesenteric lymph node in patients acutely infected with Y. pseudotuberculosis. 20 out of 33 patients (61%) had an elevated antibody titer compared with healthy controls (P = 0.0001). Patients with systemic symptoms such as lymphadenopathy, transient renal dysfunction, and arthritis had significantly higher titers of anti-YPM than patients with gastrointestinal tract symptoms alone. T cells bearing the Vbeta3 gene segment were significantly increased (P = 0.009) among acute phase patients compared with healthy children. During the convalescence phase of the illness, there was a reduction in the abnormal level of Vbeta3 T cells. Moreover, in the mesenteric lymph node, an elevated level of Vbeta3 T cells compared with peripheral blood and a sequence diversity in the junctional region of the T cell antigen receptor beta-chain containing Vbeta3 element was observed in one patient. Together, these findings suggest that YPM was produced in vivo and played an important role in the pathogenesis of Y. pseudotuberculosis infection.