Shiga Toxin (Stx) Type 1a Reduces the Oral Toxicity of Stx Type 2a.

BACKGROUND: Shiga toxin (Stx) is the primary virulence factor of Stx-producing Escherichia coli (STEC). STEC can produce Stx1a and/or Stx2a, which are antigenically distinct. However, Stx2a-producing STEC are associated with more severe disease than strains producing both Stx1a and Stx2a. METHODS AND RESULTS: To address the hypothesis that the reason for the association of Stx2a with more severe disease is because Stx2a crosses the intestinal barrier with greater efficiency that Stx1a, we covalently labeled Stx1a and Stx2a with Alexa Fluor 750 and determined the ex vivo fluorescent intensity of murine systemic organs after oral intoxication. Surprisingly, both Stxs exhibited similar dissemination patterns and accumulated in the kidneys. We next cointoxicated mice to determine whether Stx1a could impede Stx2a. Cointoxication resulted in increased survival and an extended mean time to death, compared with intoxication with Stx2a only. The survival benefit was dose dependent, with the greatest effect observed when 5 times more Stx1a than Stx2a was delivered, and was amplified when Stx1a was delivered 3 hours prior to Stx2a. Cointoxication with an Stx1a active site toxoid also reduced Stx2a toxicity. CONCLUSIONS: These studies suggest that Stx1a reduces Stx2a-mediated toxicity, a finding that may explain why STEC that produce only Stx2a are associated with more severe disease than strains producing Stx1a and Stx2a.

Shiga toxin 1, as DNA repair inhibitor, synergistically potentiates the activity of the anticancer drug, mafosfamide, on raji cells.

Shiga toxin 1 (Stx1), produced by pathogenic Escherichia coli, targets a restricted subset of human cells, which possess the receptor globotriaosylceramide (Gb3Cer/CD77), causing hemolytic uremic syndrome. In spite of the high toxicity, Stx1 has been proposed in the treatment of Gb3Cer/CD77-expressing lymphoma. Here, we demonstrate in a Burkitt lymphoma cell model expressing this receptor, namely Raji cells, that Stx1, at quasi-non-toxic concentrations (0.05-0.1 pM), inhibits the repair of mafosfamide-induced DNA alkylating lesions, synergistically potentiating the cytotoxic activity of the anticancer drug. Conversely, human promyelocytic leukemia cells HL-60, which do not express Gb3Cer/CD77, were spared by the toxin as previously demonstrated for CD34+ human progenitor cells, and hence, in this cancer model, no additive nor synergistic effects were observed with the combined Stx1/mafosfamide treatment. Our findings suggest that Stx1 could be used to improve the mafosfamide-mediated purging of Gb3Cer/CD77+ tumor cells before autologous bone marrow transplantation.

Characterization of monoclonal immunoglobulin a and g against shiga toxin binding subunits produced by intranasal immunization.

Immunoglobulin A (IgA) is considered to play a major role in protection of the mucosal surface. However, its immunological and biological properties have not been extensively studied because the production of IgA class monoclonal antibodies (mAbs) is difficult. We compared the properties of IgA and IgG mAbs against Shiga toxin B subunits (Stx1B). These mAbs were secreted from hybridomas that had been produced from mice after intranasal immunization with recombinant Stx1B and cholera toxin. The dose response curves for the binding of the IgA (clone G2G7) and IgG (clone D11C6) mAbs to immobilized Stx1B were similar, as revealed on ELISA. The majority of the IgA mAb formed dimers while the IgG mAb was monomeric, as judged by immunoblot analysis. The IgG mAb completely inhibited the binding of Stx1B to Burkitt's lymphoma cell line Ramos, while the inhibition by the IgA mAb was only partial. The IgG mAb was able to neutralize the cytotoxicity of Stx1 holotoxin towards Vero cells, whereas the IgA mAb was not. The binding affinity of each binding site was compared by means of surface plasmon resonance analysis involving a capture method, with which the binding of soluble Stx1B to immobilized mAb was detected. The association rate was similar but the dissociation rate was twofold faster in the case of the IgA mAb, resulting in twofold higher affinity of the IgG mAb. These results suggest that one can obtain high affinity IgA mAb but toxin neutralization is another challenge as to therapeutic antibodies of the IgA class.

Shiga-toxin-induced firm adhesion of human leukocytes to endothelium is in part mediated by heparan sulfate.

BACKGROUND: Shiga toxin (Stx) is the main pathogenic factor in the haemolytic-uraemic syndrome (HUS). Stx damages the renal endothelium, which leads to inflammation and coagulation. Endothelial heparan sulfate proteoglycans (HSPG), and heparan sulfate in particular, play an important role in the inflammatory process by acting as a ligand for l-selectin. Furthermore, leukocytes are able to interact with chemokines bound to HSPG (examples are IL-8, RANTES and MCP-1). This leads to an activation of integrins on leukocytes and results in more stable leukocyte-endothelial wall adhesion. In this study, we have evaluated the effect of a subtoxic dose of Stx1 and Stx2 on the HSPG and its role in adhesion of leukocytes. METHODS: Primary human umbilical venous endothelial cells (HUVEC) and primary human glomerular microvascular endothelial cells (GMVEC) were incubated for 24 h with a subtoxic dose of Stx1 or Stx2. Then, cells were treated with heparan sulfate-degrading enzyme heparitinase I or left untreated, followed by determination of binding leukocytes to endothelial cells in a parallel plate flow chamber. RESULTS: In both cell types, Stx increased the amount of firmly adherent leukocytes. After removal of endothelial heparan sulfate, the number of adhering leukocytes decreased. CONCLUSIONS: HSPG have a distinctive role in adhesion of leukocytes to endothelial cells stimulated by a subtoxic dose of Stx.

Efficient antiproliferative and antiangiogenic effects on human ovarian cancer growth by gene transfer of attenuated mutants of Shiga-like toxin I.

To evaluate the potential effect of anticancer and antiangiogenesis of Stx1(W203F) and Stx1(R170H), two attenuated mutants of Shiga-like toxin I (Stx1), in cancer gene therapy. Antiproliferative effects of these Stx1 mutants were tested in human ovarian carcinoma cell line SKOV3 and human umbilical vein endothelial cells (HUVECs) in vitro. Effect of these Stx1 mutants on inducing cell death and cell cycle arrest was analyzed in SKOV3 cells. Influence of these Stx1 mutants on endothelial cell function was analyzed in HUVECs. In vivo therapeutic effect of these Stx1 mutants on SKOV3 was explored using xenograft models in nude mice. These Stx1 mutants can inhibit the growth of SKOV3 or HUVECs and this effect can be abrogated by antibody specific for Stx1. They caused considerable cell death of SKOV3 cells in 24 h; neither caspase activity nor DNA fragmentation was observed, and necrosis is the major mode of cell death. These Stx1 mutants can induce cell cycle arrest of SKOV3 cells in G(2)-M or S phase depending on the dosage of gene transfer. Furthermore, they significantly decreased migration and capillary tube formation of HUVECs at low dose. In vivo study showed that Stx1(W203F) but not Stx1(R170H) significantly suppressed transplanted SKOV3 tumor growth in nude mice model. Interestingly, the microvessel densities of tumor treated with Stx1(W203F) and Stx1(R170H) were significantly reduced. This study suggests that genes encoding attenuated Stx1 can be selected as good candidates for the gene therapy of ovarian carcinoma because of their antiproliferative and antiangiogenic effects.

Renal prostacyclin biosynthesis in a baboon model of Shiga toxin mediated hemolytic uremic syndrome.

BACKGROUND/AIMS: Shiga toxin (Stx) and lipopolysaccharide (LPS) both participate in the pathogenesis of post-diarrheal (D+) hemolytic uremic syndrome (HUS), but little is known about factors that modulate the host response to these toxins. Prostacyclin (PGI(2)) is a potent renal vasodilator and inhibitor of platelet aggregation and adhesion. An inability to produce PGI(2) in response to endothelial cell injury could drive the pathogenic cascade. We therefore used a baboon model of HUS to measure PGI(2 )production following the administration of Stx and LPS. METHODS: Shiga toxin-1 (Stx-1), with and without LPS, was administered intravenously to baboons in various doses and schedules. 6-keto-PGF(1)alpha, the stable metabolite of PGI(2), was measured by ELISA in the plasma and urine. RESULTS: Plasma concentrations did not change significantly. Urine values increased significantly in some groups, but not in others, and HUS developed both in animals that did and did not exhibit a significant increase in urinary PGI(2) production. CONCLUSIONS: Renal PGI(2) biosynthesis appears to be affected by the dose and rate of Stx administration, and the timing of LPS infusion. PGI(2) does not protect our primate model from developing HUS.

Urinary concentrating defect in rats given Shiga toxin: elevation in urinary AQP2 level associated with polyuria.

Shiga toxin (Stx) plays a central role in the etiology of hemolytic uremic syndrome (HUS) associated with Stx-producing Escherichia coli infection. The deposition of Stx2 in the renal collecting duct epithelial cells of rats administered Stx2 intravenously has been demonstrated by immunohistochemistry, and these rats were shown to develop substantial morphological changes in the kidney tubules, associated with polyuria. Severe polyuria was observed as an early event with no other obvious sequelae after Stx administration, in parallel with elevated urinary level of aquaporin 2 (AQP2) water channel protein that was determined by a sandwich EIA assay. Immunoblotting revealed that Stx treatment markedly induced an elevation in urinary AQP2 level and reduction in AQP2 protein in the renal plasma membranes. Elevated urinary AQP2 level was a more sensitive marker to assess Stx-induced renal tubular damage than urinary beta2-microglobulin or N-acetyl-beta-D-glucosaminidase in rats. Stx2 caused more severe renal tubular impairment than Stx1. Change in urinary AQP2 level by Stx1 and Stx2 at non-lethal doses of 40 ng/kg and 10 ng/kg, respectively, was reversed at 7 days in association with recovery of urinary concentrating ability, suggesting that there is a causative link.

Cross-protection against challenge by intravenous Escherichia coli verocytotoxin 1 (VT1) in rabbits immunized with VT2 toxoid.

Rabbits challenged intravenously with Escherichia coli verocytotoxin (VT1, Shiga toxin 1, Stx1) die after developing diarrhea and paralysis, and this outcome can be prevented by pre-immunization with VT1 toxoid. In nonimmune rabbits, intravenously administered 125I-VT1 binds to the central nervous system and gastrointestinal tract, whereas in immunized animals, these organs are spared and the toxin localizes in the liver and spleen. In rabbits immunized with either VT1 or VT2 toxoids, both the homologous or heterologous toxins are prevented from binding to target organs. This has lead to the advancement of a hypothesis that cross-protection in vivo can be induced to both toxins by immunization with a toxoid even though these toxins do not exhibit cross-neutralization in vitro. It was shown that rabbits immunized with VT2 were fully protected from the intravenous administration of 10 LD50 and 50 LD50 of VT1, and this correlated directly with the protection from binding of this toxin to target organs. These findings have important implications on the design of the vaccination strategies to prevent human VT-mediated diseases and also validate the concept of testing for immunity to VT by monitoring the inhibition of binding of the 125I-VT to target organs in preference to performing LD50 assays.

Response to Shiga toxin-1, with and without lipopolysaccharide, in a primate model of hemolytic uremic syndrome.

Shiga toxin (Stx) and lipopolysaccharide (LPS) both participate in the pathogenesis of post-diarrheal hemolytic uremic syndrome (HUS), yet little is known about the factors that modulate the host response to these toxins. We have previously shown that the baboon develops HUS if 100 ng/kg of purified Stx-1 is administered rapidly as a single bolus, but not if it is given as four 25-ng/kg doses every 12 h. We therefore used this baboon model to study the response to small intravenous doses of Stx-1, with and without the co-administration of LPS. The co-administration of two 1-mg/kg doses of LPS (given at 0 and 24 h) and four 25-ng/kg doses of Stx-1 (given at 0, 12, 24, and 36 h) resulted in HUS, but the administration of either toxin separately did not. The development of HUS was associated with a rise in urinary, but not plasma concentrations of TNF, and a rise in both urinary and plasma concentrations of IL-6 and IL-8. We speculate that LPS is not required for disease expression in the human, but that it can augment the response to otherwise subtoxic amounts of Stx and this augmentation may be mediated by LPS-induced cytokine release.

Toxicity of Shiga toxin 1 in the central nervous system of rabbits.

The action of Shiga toxin (Stx) on the central nervous system was examined in rabbits. Intravenous Stx1 was 44 times more lethal than Stx2 and acted more rapidly than Stx2. However, Stx1 accumulated more slowly in the cerebrospinal fluid than did Stx2. Magnetic resonance imaging demonstrated a predominance of Stx1-dependent lesions in the spinal cord. Pretreatment of the animals with anti-Stx1 antiserum intravenously completely protected against both development of brain lesions and mortality.

Response to single and divided doses of Shiga toxin-1 in a primate model of hemolytic uremic syndrome.

Postdiarrheal hemolytic uremic syndrome is caused by Shiga toxin (Stx)-producing Escherichia coli. It was shown previously that the baboon, like the human, has glycolipid receptors for Stx in the gut and the kidney and that a single 50- to 200-ng/kg intravenous dose of purified Stx-1 results in thrombocytopenia, hemolytic anemia, and renal thrombotic microangiopathy. For further characterization of factors that modulate disease expression, the baboon's response to the intravenous administration of 100 ng/kg Stx-1 given either rapidly as a single bolus or slowly as four 25-ng/kg doses at 12-h intervals was compared. Animals that received the Stx-1 as a single dose developed thrombocytopenia, schistocytosis, and acute renal failure. Urinary but not plasma tumor necrosis factor-alpha concentrations rose significantly by 6 h and then declined rapidly. Urinary and plasma interleukin-6 concentrations rose later. Glomeruli showed reduced patency of capillary loops, fragmented red blood cells, fibrin and platelet microthrombi, necrosis and detachment of endothelial cells, and accumulation of flocculent material in subendothelial spaces. Damage to tubular epithelium and peritubular capillary endothelium also was seen. Animals that received four divided doses of Stx-1 developed no clinical or histologic features of hemolytic uremic syndrome. It is concluded that in the primate model, disease expression is modulated by the rate of Stx administration, and it is speculated that in the human, the rate of Stx absorption from the gut is one determinant of disease severity.

Nasal immunization with E. coli verotoxin 1 (VT1)-B subunit and a nontoxic mutant of cholera toxin elicits serum neutralizing antibodies.

Escherichia coli O157:H7 produces two forms of verotoxin (VT), VT1 and VT2, which cause hemorrhagic colitis with development, in some cases, of hemolytic uremic syndrome. These toxins consist of an enzymatically active A subunit and pentamers of B subunit responsible for their binding to host cells. We used the secretion-expression system of Bacillus brevis to produce recombinant VT1B and VT2B. The secreted B subunits were purified and sequenced to verify their structure. Receptor-binding showed that rVT1B but not rVT2B bound to Gb3-receptor. When mice were nasally immunized with rVT1B or rVT2B together with a nontoxic mutant of cholera toxin (mCT) or native cholera toxin (nCT) as adjuvants, serum IgG and mucosal IgA antibody responses to VT1B were induced. The VT1B-specific antibodies prevented VT1B binding to its Gb3 receptor. In contrast, poor serum and no mucosal VT2B-specific antibodies but brisk CTB-specific antibody responses were induced by nasal immunization with rVT2B in the presence of mCT or nCT. These results show that nasal immunization with rVTB and mCT as a nontoxic mucosal adjuvant is an effective regimen for the induction of VT1B but not VT2B antibody responses which inhibit VT1B binding to Gb3 receptor.