Stx Receptor Research Articles

EnterohemorrhagicEscherichia colisuppresses inflammatory response to cytokines and its own toxin

Infection with the enteric pathogen enterohemorrhagic Escherichia coli (EHEC) causes a variety of symptoms ranging from nonbloody diarrhea to more severe sequelae including hemorrhagic colitis, altered sensorium and seizures, and even life-threatening complications, such as hemolytic uremic syndrome and thrombotic thrombocytopenic purpura. The more severe consequences of EHEC infection are attributable to the production of Shiga toxin (Stx) and its subsequent effects on the vasculature, which expresses high levels of the Stx receptor, Gb3. Interestingly, the intestinal epithelium does not express Gb3. Despite the lack of Gb3 receptor expression, intestinal epithelial cells translocate Stx. The effect of Stx on intestinal epithelial cells is controversial with some studies demonstrating induction of inflammation and others not. This may be difficult to resolve because EHEC expresses both proinflammatory molecules, such as flagellin, and factor(s) that dampen the inflammatory response of epithelial cells. The goal of our study was to define the effect of Stx on the inflammatory response of intestinal epithelial cells and to determine whether infection by EHEC modulates this response. Here we show that Stx is a potent inducer of the inflammatory response in intestinal epithelial cells and confirm that EHEC attenuates the induction of IL-8 by host-derived proinflammatory cytokines. More importantly, however, we show that infection with EHEC attenuates the inflammatory response by intestinal epithelial cells to its own toxin. We speculate that the ability of EHEC to dampen epithelial cell inflammatory responses to Stx and cytokines facilitates intestinal colonization.

551 Shiga Toxin 1 Uptake By Intestinal Epithelial Cells Is CDC-42 Mediated and Clathrin-Independent

Shiga toxins (Stx) 1 and 2 are major contributors to foodborne disease caused by enterohemorrhagic E.coli (EHEC). Human intestinal epithelial cells (IEC) do not normally express Stx receptor (the glycosphingolipid Gb3), leaving the mechanism of Stx uptake poorly understood. Inhibition of clathrin-mediated endocytosis significantly increases Stx1 uptake in Gb3-free IEC. We thus now test the hypothesis that cell division cycle 42 (Cdc-42) dependent fluid phase endocytosis might be involved in clathrin-independent Stx1 uptake by IEC In Vitro and In Vivo. Methods: In Vivo: We compared pathologies in New Zealand white rabbits fed with either a) an experimental RDEC-H19A strain engineered to express Stx1, since it produces intestinal pathology quite similar to that noted in human EHEC or b) a control RDEC-1 E. coli strain that adheres to rabbit mucosa to provide an attaching and effacing lesion or c) PBS, to provide a negative control for effects of bacterial infection. Immunofluorescence of frozen sections from cecal tissue of rabbits studied at times up to 7 days after infection assessed a) uptake of Stx1 and b) presence of activated Cdc-42 in IEC. In Vitro: The apical uptake of the recombinant B-subunit of Stx1 labeled with Alexa 680 fluorescent dye (Stx1B) was assessed in T84 IEC that were: a) treated with agents known to inhibit clathrin-mediated endocytosis, b) depleted of Cdc42 by shRNA or c) untreated controls using biochemical and immunofluorescence methods. Results: 1) Cecal tissues from rabbits treated with either RDEC-1 or RDEC-H19A bacteria, but not those from PBS-treated animals, displayed Cdc42 activation in subsets of epithelial cells at sites near and those distant from attached bacteria. 2) T84 cells incubated with the inhibitors of clathrin mediated endocytosis chlorpromazine or low potassium increased Stx1B uptake in a time-dependent fashion. This stimulation was virtually eliminated, however, in T84 cells in which Cdc-42 was downregulated using shRNA. 3) The specific blocker of Cdc-42 function and nucleotide exchange, Pirl-1, also prevented stimulation of Stx1B uptake in concentration-dependent way in cells treated with inhibitors of clathrin-mediated endocytosis. 4) This clathrin independent Cdc-42 mediated Stx1B uptake was also dynamin dependent. Dynasore, a specific inhibitor of dynamin GTPase activity, inhibited the Stx1B uptake in cells with inactivated clathrin induced endocytic pathway. Conclusions: 1) Basal Stx1uptake by Gb3 negative T84 cells is regulated by Cdc-42. 2) Cdc-42 dependent clathrin independent pathway is likely to be involved in IEC uptake of Stx1 during EHEC infections.

Epithelial and Mesenchymal Cells in the Bovine Colonic Mucosa Differ in Their Responsiveness toEscherichia coliShiga Toxin 1

Bovine colonic crypt cells express CD77 molecules that potentially act as receptors for Shiga toxins (Stx). The implication of this finding for the intestinal colonization of cattle by human pathogenic Stx-producing Escherichia coli (STEC) remains undefined. We used flow cytometric and real-time PCR analyses of primary cultures of colonic crypt cells to evaluate cell viability, CD77 expression, and gene transcription in the presence and absence of purified Stx1. A subset of cultured epithelial cells had Stx receptors which were located mainly intracellularly, with a perinuclear distribution, and were resistant to Stx1-induced apoptosis and Stx1 effects on chemokine expression patterns. In contrast, a population of vimentin-positive cells, i.e., mesenchymal/nonepithelial cells that had high numbers of Stx receptors on their surface, was depleted from the cultures by Stx1. In situ, CD77(+) cells were located in the lamina propria of the bovine colon by using immunofluorescence staining. A newly established vimentin-positive crypt cell line with high CD77 expression resisted the cytolethal effect of Stx1 but responded to Stx1 with a significant increase in interleukin-8 (IL-8), GRO-alpha, MCP-1, and RANTES mRNA. Combined stimulation with lipopolysaccharide and Stx1 increased IL-10 mRNA. Our results show that bovine colonic crypt cells of epithelial origin are resistant to both the cytotoxic and modulatory effects of Stx1. In contrast, some mucosal mesenchymal cells, preliminarily characterized as mucosal macrophages, are Stx1-responsive cells that may participate in the interaction of STEC with the bovine intestinal mucosa.

Latrunculin B facilitates Shiga toxin 1 transcellular transcytosis across T84 intestinal epithelial cells

Shiga toxins (Stx), released into the intestinal lumen by enterohemorrhagic Escherichia coli (EHEC), are major virulence factors responsible for gastrointestinal and systemic illnesses. These pathologies are believed to be due to the action of the toxins on endothelial cells, which express the Stx receptor, the glycosphingolipid Gb3. To reach the endothelial cells, Stx must translocate across the intestinal epithelial monolayer. This process is poorly understood. We investigated Stx1 movement across the intestinal epithelial T84 cell model and the role of actin turnover in this transcytosis. We showed that changes in the actin cytoskeleton due to latrunculin B, but not cytochalasin D or jasplakinolide, significantly facilitate toxin transcytosis across T84 monolayers. This trafficking is transcellular and completely inhibited by tannic acid, a cell impermeable plasma membrane fixative. This indicates that actin turnover could play an important role in Stx1 transcellular transcytosis across intestinal epithelium in vitro. Since EHEC attachment to epithelial cells causes an actin rearrangement, this finding may be highly relevant to Stx-induced disease.

Neuronal apoptosis and inflammatory responses in the central nervous system of a rabbit treated with Shiga toxin-2

BackgroundShiga toxins (Stxs) are the major agents responsible for hemorrhagic colitis and hemolytic-uremic syndrome (HUS) during infections caused by Stx-producing Escherichia coli (STEC) such as serotype O157:H7. Central nervous system (CNS) involvement is an important determinant of mortality in diarrhea associated-HUS. It has been suggested that vascular endothelial injuries caused by Stxs play a crucial role in the development of the disease. The current study investigates the relationship between the cytotoxic effects of Stxs and inflammatory responses in a rabbit brain treated with Stx2.MethodsIn a rabbit model treated with purified Stx2 or PBS(-), we examined the expression of the Stx receptor globotriaosylceramide (Gb3)/CD77 in the CNS and microglial activation using immunohistochemistry. The relationship between inflammatory responses and neuronal cell death was analyzed by the following methods: real time quantitative reverse transcriptase (RT)-polymerase chain reaction (PCR) to determine the expression levels of pro-inflammatory cytokines, and the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) method to detect apoptotic changes.ResultsGb3/CD77 expression was detected in endothelial cells but not in neurons or glial cells. In the spinal cord gray matter, significant levels of Gb3/CD77 expression were observed. Severe endothelial injury and microvascular thrombosis resulted in extensive necrotic infarction, which led to acute neuronal damage. Conversely, in the brain, Stx receptor expression was much lower. The observed neuropathology was less severe. However, neuronal apoptosis was observed at the onset of neurological symptoms, and the number of apoptotic cells significantly increased in the brain at a later stage, several days after onset. Microglial activation was observed, and tumor necrosis factor (TNF)-α and interleukin (IL)-1β mRNA in the CNS parenchyma was significantly up-regulated. There was significant overexpression of TNF-α transcripts in the brain.ConclusionThis study indicates that Stx2 may not directly damage neural cells, but rather inflammatory responses occur in the brain parenchyma in response to primary injury by Stx2 in vascular endothelial cells expressing Gb3/CD77. These findings suggest that neuroinflammation may play a critical role in neurodegenerative processes during STEC infection and that anti-inflammatory intervention may have therapeutic potential.

Interactions of Shiga-like toxin with human peripheral blood monocytes

The cytotoxic effect of Shiga-like toxin (Stx; produced by certain Escherichia coli strains) plays a central role in typical hemolytic uremic syndrome (HUS). It damages the renal endothelium by inhibiting the cellular protein synthesis. Also, the monocyte has a specific receptor for Stx but is not sensitive for the cytotoxic effect. In this work, monocytes were studied as a potential transporter for Stx to the renal endothelium. Coincubation of isolated human monocytes loaded with Stx and target cells (vero cells and human umbilical vascular endothelial cells) were performed. Transfer was determined by measuring the protein synthesis of target cells and by flow cytometry. Furthermore, the effect of a temperature shift on loaded monocytes was investigated. Stx-loaded monocytes reduced the protein synthesis of target cells. After adding an antibody against Stx, incomplete recovery occurred. Also, adding only the supernatant of coincubation was followed by protein synthesis inhibition. Stx detached from its receptor on the monocyte after a change in temperature, and no release was detected without this temperature shift. Although the monocyte plays an important role in the pathogenesis of HUS, it has no role in the transfer of Stx.

Renal Injury Is a Consistent Finding in Dutch Belted Rabbits Experimentally Infected with EnterohemorrhagicEscherichia coli

Enterohemorrhagic Escherichia coli (EHEC) produces Shiga toxin (Stx) and causes renal disease in humans. Dutch Belted (DB) rabbits naturally infected with EHEC O153 develop hemolytic-uremic syndrome-like disease. The aims of this study were to experimentally reproduce O153-induced renal disease in DB rabbits and investigate bacterial and host factors involved in pathogenesis. The pathogenicity of E. coli O157:H7 was also investigated in rabbits. The stx1AB region of O153 was sequenced. By use of liquid chromatography-tandem mass spectrometry, we identified homologs of the Stx receptor, globotriaosylceramide (Gb3), in rabbit kidney extracts. Infected rabbits developed clinical signs and intestinal and kidney lesions. Renal pathological changes consisted of intimal swelling, perivascular edema, erythrocyte fragmentation, capillary thickening, luminal constriction, leukocytic infiltration, mesangial deposits, and changes in Bowman's capsule and space. Sequence analysis of a approximately 7-kb region of the O153 chromosome indicated homology to the Stx1-producing bacteriophage H19B. Our findings indicate that DB rabbits are suitable for the study of the renal manifestations of EHEC infection in humans.

Role of Shiga toxin versus H7 flagellin in enterohaemorrhagic Escherichia coli signalling of human colon epithelium in vivo

Enterohaemorrhagic Escherichia coli O157:H7 (EHEC) is a clinically important foodborne pathogen that colonizes human colon epithelium and induces acute colonic inflammation, but does not invade the epithelial cells. Whereas Shiga toxin (Stx) and bacterial flagellin have been studied for their ability to upregulate the production of proinflammatory chemokines by cultured human colon cancer cell lines, the relevance of studies in colon cancer cell lines to the production of proinflammatory signals by normal epithelial cells in EHEC-infected human colon is not known. We show herein that Stx does not bind to human colon epithelium in vivo. Moreover, globotriaosylceramide (Gb3/CD77) synthase, the enzyme required for synthesis of the Gb3/CD77 receptor for Stx, was not expressed by normal or inflamed human colon epithelium in vivo. In contrast, Toll-like receptor (TLR) 5, the receptor for bacterial flagellin, was expressed by normal human colon epithelium and by colon epithelium in human intestinal xenografts. EHEC H7 flagellin instilled in the lumen of human colon xenografts that contain an intact human epithelium upregulated the expression of epithelial cell proinflammatory chemokines, which was accompanied by a subepithelial influx of neutrophils. Isogenic mutants of EHEC that lacked flagellin did not significantly upregulate prototypic neutrophil and dendritic cell chemoattractants by model human colon epithelia, irrespective of Stx production. We conclude that EHEC H7 flagellin and not Stx is the major EHEC factor that directly upregulates proinflammatory chemokine production by human colon epithelium in vivo.

Lipopolysaccharide Upregulates Renal Shiga Toxin Receptors in a Primate Model of Hemolytic Uremic Syndrome

Background: Although Shiga toxin (Stx) mediates classical hemolytic uremic syndrome (HUS), it is not fully understood why only some subjects exposed to Stx-expressing Escherichia coli develop HUS. We have previously shown in a baboon model of Stx-mediated HUS that coadministration of lipopolysaccharide (LPS) results in an augmented host response to otherwise subtoxic Stx1 doses. We used this model to test the hypothesis that LPS upregulates renal Stx receptor (Gb₃) expression. Methods: Juvenile baboons were treated with either Stx1 (100 ng/kg), LPS (1 mg/kg as two divided doses 24 h apart), or a sham injection of saline, and sacrificed and immediately autopsied at 72 h. Renal cortical tissue Gb₃ content was quantitated by lipid extraction and thin-layer chromatography, and Stx1 and Gb₃/CD77 immunostaining was assessed by quantitative immunofluorescent microscopy. Results: Compared to saline-in jected controls, LPS administration resulted in a 2.2-fold increase in renal cortical Gb₃ by chromatography (p < 0.01), a 2.5-fold increase in Stx1 staining (p = 0.003) and a 1.7-fold increase in CD77 immunostaining (p = 0.004). Stx treatment did not significantly alter either Stx or CD77 immunostaining. Conclusion: These observations suggest that LPS primes the host for Stx-mediated renal injury by upregulating renal Gb₃ expression.

VerotoxigenicEscherichia colifrom animals, humans and foods: who's who?

Verocytotoxigenic (shigatoxigenic) and enterohaemorrhagic Escherichia co/i, VTEC (STEC) and EHEC, produce a toxin active on Vero cells in vitro. VTEC and EHEC have been isolated from humans and different animal species, mainly ruminants and pigs. The verocytotoxins, also named shiga toxins (Stx), are active in vivo on the endothelial cells of the blood vessels of the gastro-intestinal mucosa, the kidneys, the brain, and other tissues of humans and piglets, leading to fluid leakage or haemorrhages. Conversely, their role in diseases of young ruminants remains unclear. Adult ruminants can also act as asymptomatic carriers of VTEC and EHEC strains similar to those causing diseases in humans. And they are incriminated as an important source of direct or indirect contamination of humans by the most famous EHEC strain belonging to the O157:H7 serotype, through faecal contamination of either foods of animal origin, or other foodstuffs (fruit, vegetables, etc.), or the environment. But dozens of non-O157 human and ruminant VTEC and EHEC strains with similar general and virulence-associated properties, have been described, whose epidemiology is much less well understood. The purpose of this review manuscript is to describe and compare the properties of human, ruminant and food VTEC and EHEC strains.

Identification of the Optimal Structure Required for a Shiga Toxin Neutralizer with Oriented Carbohydrates to Function in the Circulation

Shiga toxin (Stx) is a major virulence factor of Stx-producing Escherichia coli. Recently, we developed a therapeutic Stx neutralizer with 6 trisaccharides of globotriaosyl ceramide, a receptor for Stx, in its dendrimer structure (referred to as "SUPER TWIG [1]6") to function in the circulation. Here, we determined the optimal structure of SUPER TWIG for it to function in the circulation and identified a SUPER TWIG with 18 trisaccharides, SUPER TWIG (2)18, as another potent Stx neutralizer. SUPER TWIGs (1)6 and (2)18 shared a structural similarity, a dumbbell shape in which 2 clusters of trisaccharides were connected via a linkage with a hydrophobic chain. The dumbbell shape was found to be required for formation of a complex with Stx that enables efficient uptake and degradation of Stx by macrophages and, consequently, for potent Stx-neutralizing activity in the circulation. We also determined the binding site of the SUPER TWIGs on Stx.

Hemolytic Uremic Syndrome

Hemolytic uremic syndrome (HUS) is a thrombotic microangiopathy defined by thrombocytopenia, nonimmune microangiopathic hemolytic anemia, and acute renal failure. The most frequent form is associated with infections by Shiga-like toxin-producing bacteria (STEC-HUS). Rarer cases are triggered by neuraminidase-producing Streptococcus pneumoniae (pneumococcal-HUS). The designation of aHUS is used to refer to those cases in which an infection by Shiga-like toxin-producing bacteria or S. pneumoniae can be excluded. Studies performed in the last two decades have documented that hyperactivation of the complement system is the pathogenetic effector mechanism leading to the endothelial damage and the microvascular thrombosis in aHUS. Recent data suggested the involvement of the complement system in the pathogenesis of STEC-HUS and pneumococcal-HUS as well. Clinical signs and symptoms may overlap among the different forms of HUS; however, pneumococcal-HUS and aHUS have a worse prognosis compared with STEC-HUS. Early diagnosis and identification of underlying pathogenetic mechanism allows instating specific support measures and therapies. In clinical trials in patients with aHUS, complement inhibition by eculizumab administration leads to a rapid and sustained normalization of hematological parameters with improvement in long-term renal function. This review summarizes current concepts about the epidemiological findings, the pathological and clinical aspects of STEC-HUS, pneumococcal-HUS, and aHUS, and their diagnosis and management.

Decrease of thrombomodulin contributes to the procoagulant state of endothelium in haemolytic uraemic syndrome

Sirs, The observation that Shiga-like toxin 2 (stx2) appears to reduce the surface expression of thrombomodulin (TM) on human glomerular endothelial cells is intriguing, and might be relevant in the pathogenesis of haemolytic uraemic syndrome (HUS) if it is confirmed [1]. Fern ndez et al. [1] pre-stimulated the cells with tumour necrosis factor-a (TNF) to increase surface expression of the stx receptor, Gb3, and thus increase the sensitivity of the cells to the pleomorphic effects of the toxin. These effects include protein synthesis inhibition, stress responses and cell death, depending on the dose of toxin, the position of the cells in the cell cycle and the specific responses of the cell type. Surface-bound TM was quantified using an in situ cell ELISA method. For the groups of cells pre-stimulated with TNF there is a broad correlation between the reduction in measured TM and the reduction in protein synthesis. However, when a different protein synthesis inhibitor, cycloheximide, was used alone at a concentration that gave a modest reduction in protein synthesis, there was no reduction in TM. The authors concluded that stx2 specifically downregulates the expression of TM. However, this cannot be deduced from the data as they are presented. What the reader does not know is whether the same number of endothelial cells remains viable under the conditions of the experiment. Cell killing by different protein synthesis inhibitors is not consistently proportional to the level of inhibition of protein synthesis achieved. If there was more endothelial detachment or cell death with stx2, the apparent reduction in TM might simply represent the smaller number of endothelial cells left at the end of the incubation period. What one actually wants to know is whether the TM expression per cell is reduced. The authors could very easily go some way towards addressing this by vital staining and counting the cells at the end of the experiment to show that the monolayer of endothelial cells was in fact intact. It might be even better to use fluorescence-activated cell sorting, or to test for and quantify cell necrosis and apoptosis. Without this, the suggested down-regulation of TM by stx2 and its role in the pathogenesis of HUS remain somewhat speculative.

Inhibition of p38 Mitogen‐Activated Protein Kinase Ameliorates Cytokine Up‐Regulated Shigatoxin‐1 Toxicity in Human Brain Microvascular Endothelial Cells

Brain injury in hemolytic-uremic syndrome (HUS) may be enhanced by inflammatory cytokine up-regulation of endothelial cell sensitivity to shigatoxin (Stx). The present study investigated whether inflammatory cytokine up-regulation of Stx toxicity could be ameliorated by inhibiting candidate signal transduction pathways. Exposure of human brain endothelial cells (HBECs) to tumor necrosis factor (TNF) greatly increased Stx-1 and Stx-2 cytotoxicity; this was reduced by inhibition of p38 mitogen-activated protein kinase (MAPK), but not c-Jun kinase. SB203580, a specific inhibitor of p38 MAPK, reduced TNF-stimulated Stx cytotoxicity in HBECs, TNF-stimulated (125)Stx-1 binding to intact HBECs, the cellular content of Gb3 (galactose alpha 1,4, galactose ss 1,4, glucose-ceramide) (the Stx receptor), and TNF-stimulated Gb3 synthase and glucosylceramide synthase activities but did not affect lactosylceramide synthase activities or mRNA content. Thus, inhibition of p38 MAPK substantially reduces inflammatory cytokine up-regulation of Stx-receptor synthesis and cell-surface expression, thereby decreasing Stx cytotoxicity. Inhibition of p38 MAPK may be of therapeutic benefit in HUS.

Chemokine Expression in the Monocytic Cell Line THP-1 in Response to Purified Shiga Toxin 1 and/or Lipopolysaccharides

Infections with Shiga toxin (Stx)-producing bacteria are associated with bloody diarrhea and postdiarrheal sequelae, including hemolytic uremic syndrome and central nervous system (CNS) abnormalities. Stx-induced intestinal, renal, and CNS vascular lesions may involve a localized production of proinflammatory cytokines in target organs, as tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) up-regulate Stx receptor globotriaosylceramide (Gb(3)) expression on vascular endothelial cells. However, leukocyte recruitment to injured sites may also exacerbate vascular damage. A cytokine macroarray analysis of transcripts derived from macrophage-like THP-1 cells treated with Stx1, lipopolysaccharides (LPS), or both demonstrated a consistent up-regulation of TNF-alpha, IL-1beta, and four genes encoding the chemokines interleukin-8 (IL-8), macrophage inflammatory protein-1alpha (MIP-1alpha), MIP-1beta, and growth-related oncogene beta (GRO-beta). Real-time PCR analysis verified the macroarray results. Northern blot analyses after the addition of the transcriptional inhibitor actinomycin D revealed increased IL-8 mRNA stability in THP-1 cells treated with Stx1 or Stx1 plus LPS. Finally, enzyme-linked immunosorbent assay data for Stx1- plus LPS-treated cells demonstrated a poor correlation between IL-8, MIP-1alpha, MIP-1beta, and GRO-beta mRNA levels and protein production, indicating a posttranscriptional regulatory effect. Our data suggest that in response to Stx1 and LPS, macrophages may be a source of chemokines that promote tissue damage through leukocyte recruitment and activation.

Shiga toxin binds to activated platelets

Hemolytic uremic syndrome (HUS) is associated with acute renal failure in children and can be caused by Shiga toxin (Stx)-producing Escherichia coli. Thrombocytopenia and formation of renal thrombi are characteristic of HUS, suggesting that platelet activation is involved in its pathogenesis. However, whether Shiga toxin directly activates platelets is controversial. The present study evaluates if potential platelet sensitization during isolation by different procedures influences platelet interaction with Shiga toxin. Platelets isolated from sodium citrate anticoagulated blood were exposed during washing to EDTA and higher g forces than platelets prepared from acid-citrate-dextrose (ACD) plasma. Platelet binding of Stx was significantly higher in EDTA-washed preparations relative to ACD-derived platelets. Binding of Stx was also increased with ACD-derived platelets when activated with thrombin (1 U mL-1) and exposure of the Gb3 Stx receptor was detected only on platelets subjected to EDTA, higher g forces or thrombin. EDTA-exposed platelets lost their normal discoid shape and were larger. P-selectin (CD62P) exposure was significantly increased in EDTA-washed preparations relative to ACD-derived platelets, suggesting platelet activation. Taken together, these results suggest that direct binding of Stx occurs only on 'activated' platelets rather than on resting platelets. The ability of Stx to interact with previously activated platelets may be an important element in understanding the pathogenesis of HUS.

Oral therapeutic agents with highly clustered globotriose for treatment of Shiga toxigenic Escherichia coli infections.

Shiga toxin (Stx) is a major virulence factor in infection with Stx-producing Escherichia coli (STEC). We developed a series of linear polymers of acrylamide, each with a different density of trisaccharide of globotriaosylceramide (Gb3), which is a receptor for Stx, and identified Gb3 polymers with highly clustered trisaccharides as Stx adsorbents functioning in the gut. The Gb3 polymers specifically bound to both Stx1 and Stx2 with high affinity and markedly inhibited the cytotoxic activities of these toxins. Oral administration of the Gb3 polymers protected mice after administration of a fatal dose of E. coli O157:H7, even when the polymers were administered after the infection had been established. In these mice, the serum level of Stx was markedly reduced and fatal brain damage was substantially suppressed, which suggests that the Gb3 polymers entrap Stx in the gut and prevent its entrance into the circulation. These results indicate that the Gb3 polymers can be used as oral therapeutic agents that function in the gut against STEC infections.

Forssman synthetase expression results in diminished shiga toxin susceptibility: a role for glycolipids in determining host-microbe interactions.

Forssman glycolipid (FG), the product of Forssman synthetase (FS), is widely expressed among nonprimate mammalian species. Here, we describe a molecular and genetic relationship between FG expression and Shiga toxin (Stx) susceptibility. We have isolated the FS cDNA from human, canine, and murine cells. Whereas the murine and canine FS genes express a functional enzyme, the human FS cDNA was found to express a protein that lacks FS activity, despite a high degree of sequence identity with the enzymatically active murine and canine FS genes. In order to examine the relationship between FG expression and Stx susceptibility, Vero cells were transfected with the three FS orthologues or a vector control. Complementation with the human FS cDNA had no effect on Stx susceptibility, whereas stable expression of the canine and murine FS resulted in markedly decreased susceptibility to toxin. Among individual cells, an inverse correlation between FG expression and Stx binding was demonstrated. Moreover, only strongly FG-reactive cells were capable of growing in the presence of Stx. These cells were found to have high levels of FG expression and a correspondingly diminished GbO(3) content. We conclude that expression of a functionally active FS modifies Stx receptor glycolipids to FG and results in markedly decreased susceptibility to toxin. We speculate that inactivation of the FS gene during primate evolution may account, at least in part, for the marked susceptibility of human cells to Stx.

Decrease of thrombomodulin contributes to the procoagulant state of endothelium in hemolytic uremic syndrome.

The typical form of hemolytic uremic syndrome (D+HUS) is a thrombotic microangiopathy that causes acute renal failure in children. The etiology of this disease is a toxin called Shiga-like toxin (Stx), present in certain strains of gram-negative bacteria. Vascular endothelial cell (EC) injury appears to be central in the pathogenesis of D+HUS. Thrombomodulin (TM) is a glycoprotein present in EC with anti-thrombogenic properties. The objective of this study was to investigate the effects of Stx on the surface expression of TM in EC using an in vitro culture of human glomerular microvascular endothelial cells. We also evaluated other inflammatory mediators [tumor necrosis factor-alpha (TNF-alpha) and lipopolysaccharide], which are known to increase Stx receptor expression and are potentially involved in the pathogenesis of D+HUS. Stx2 induced a significant decrease of TM expression in this cell type after pre-incubation with TNF-alpha. This decrease could not be attributed to the inhibition of protein synthesis only, as cycloheximide, another inhibitor of protein synthesis, did not affect TM surface expression. These results suggest that the Stx2-induced decrease of TM expression in glomerular EC might contribute to the local procoagulant state present in D+HUS.

Sex steroids do not affect shigatoxin cytotoxicity on human renal tubular or glomerular cells.

BackgroundThe greater susceptibility of children to renal injury in post-diarrheal hemolytic-uremic syndrome (HUS) may be related, at least in part, to heightened renal cell sensitivity to the cytotoxic effect of Shiga toxin (Stx), the putative mediator of kidney damage in HUS. We hypothesized that sexual maturation, which coincides with a falling incidence of HUS, may induce a relatively Stx-resistant state in the renal cells.MethodsCultured human glomerular endothelial (HGEN), human glomerular visceral epithelial (HGEC) and human proximal tubule (HPT) cells were exposed to Stx-1 after pre-incubation with progesterone, β-estradiol or testosterone followed by determination of cytotoxicity.ResultsUnder basal conditions, Stx-1 potently and dose-dependently killed HPT and HGEC, but had relatively little effect on HGEN. Pre-incubation for 1, 2 or 7 days with physiologic or pharmacologic concentrations of progesterone, β-estradiol or testosterone had no effect on Stx-1 cytotoxicity dose-response on any cell type. In addition, no steroid altered Gb3 expression (Stx receptor) by any cell type at any time point.ConclusionThese data do not support the notion that hormonal changes associated with puberty induce an Stx-resistant state within kidney cells.