globotriaosylceramide and Escherichia-coli-Infections

Topics: Animals; CRISPR-Cas Systems; Escherichia coli Infections; Hemolytic-Uremic Syndrome; Host-Pathogen Interactions; Humans; Immunotoxins; Models, Molecular; Neoplasms; Protein Conformation; Shiga Toxins; Shiga-Toxigenic Escherichia coli; Structure-Activity Relationship; Trihexosylceramides

Many cattle are persistently colonized with Shiga toxin-producing

Topics: Animals; Bacterial Shedding; Bacterial Zoonoses; Cattle; Cattle Diseases; Disease Vectors; Escherichia coli Infections; Host-Pathogen Interactions; Shiga Toxins; Shiga-Toxigenic Escherichia coli; Trihexosylceramides; Virulence

The leading cause of hemolytic uremic syndrome (HUS) in children is Shiga toxin-producing Escherichia coli (STEC) infection, which has a major outbreak potential. Since the early 2010s, STEC epidemiology is characterized by a decline of the historically predominant O157 serogroup and the emergence of non-O157 STEC, especially O26 and O80 in France. STEC contamination occurs through the ingestion of contaminated food or water, person-to-person transmission, or contact with ruminants or their contaminated environment. The main symptom is diarrhea, which is bloody in about 60% of patients and occurs after a median incubation period of three days. Shiga toxins released by STEC induce a cascade of thrombogenic and inflammatory changes of microvascular endothelial cells. HUS is observed in 5-15% of STEC infection cases, defined by the triad of mechanical hemolytic anemia, thrombocytopenia, and acute renal injury. The diagnosis of STEC infection relies on biological screening for Shiga toxins and STEC in stools and serology. Treatment of STEC-HUS is mainly symptomatic, as no specific drug has proved effective. The effect of antibiotics in STEC infection and STEC-HUS remains debated; however, some bacteriostatic antibiotics might have a beneficial effect. Proofs of evidence of a benefit from complement blockade therapy in STEC-HUS are also lacking. Clinical and bacteriological STEC-HUS surveillance needs to be continued. Ongoing prospective studies will document the role of bacteriostatic antibiotics in STEC infection and STEC-HUS, and of complement blockade therapy in STEC-HUS.

Topics: Adult; Animals; Anti-Bacterial Agents; Antibodies, Monoclonal, Humanized; Blood Transfusion; Child, Preschool; Combined Modality Therapy; Complement Pathway, Alternative; Contraindications, Drug; Diarrhea; Disease Outbreaks; Endothelium, Vascular; Environmental Exposure; Escherichia coli Infections; Feces; France; Hemolytic-Uremic Syndrome; Humans; Infant; Plasma Exchange; Prognosis; Shiga Toxin; Shiga-Toxigenic Escherichia coli; Trihexosylceramides; Zoonoses

Topics: Brain; Endothelial Cells; Enterohemorrhagic Escherichia coli; Escherichia coli Infections; Globosides; Hemolytic-Uremic Syndrome; Host-Pathogen Interactions; Humans; Kidney; Primary Cell Culture; Shiga Toxin 1; Shiga Toxin 2; Trihexosylceramides

Infection with Shiga toxin (Stx)-producing Escherichia coli (STEC), including O157:H7, causes bloody diarrhea and hemorrhagic colitis in humans, occasionally resulting in fatal systemic complications, such as neurological damage and hemolytic-uremic syndrome. Because Stx is a major virulence factor of the infectious disease, a series of Shiga toxin neutralizers with various structural characteristics has been developed as promising therapeutic agents. Most of these agents function to bind to the toxin directly and inhibit the binding to its receptor present on the target cells. Other neutralizers do not inhibit receptor binding but induce aberrant intracellular transport of the toxin, resulting in effective detoxification. Such a novel type of Stx neutralizer provides a new therapeutic strategy against STEC infections. Here, recent progress of the development of Stx neutralizers is reviewed.

Topics: Animals; Anti-Bacterial Agents; Binding Sites; Combinatorial Chemistry Techniques; Drug Design; Endoplasmic Reticulum; Escherichia coli Infections; Escherichia coli O157; Globosides; Hemolytic-Uremic Syndrome; Humans; Macrophages, Peritoneal; Mice; Peptides; Polymers; Rabbits; Serum Amyloid P-Component; Shiga Toxin 1; Shiga Toxin 2; Silanes; Trihexosylceramides; Trisaccharides; Virulence Factors

After ingestion via contaminated food or water, enterohaemorrhagic E. coli colonises the intestinal mucosa and produces Shiga toxins (Stx). No Stx-specific secretion system has been described so far, and it is assumed that Stx are released into the gut lumen after bacterial lysis. Human intestinal epithelium does not express the Stx receptor Gb3 or other Stx binding sites, and it remains unknown how Stx cross the intestinal epithelial barrier and gain access to the systemic circulation. This review summarises current knowledge about the influence of the intestinal environment on Stx production and release, Stx interaction with intestinal epithelial cells and intracellular uptake, and toxin translocation into underlying tissues. Furthermore, it highlights gaps in understanding that need to be addressed by future research.

Topics: Animals; Bacterial Outer Membrane Proteins; Bacteriophages; Binding Sites; Enterohemorrhagic Escherichia coli; Escherichia coli Infections; Host-Pathogen Interactions; Humans; Intestinal Mucosa; Protein Transport; Shiga Toxin 1; Shiga Toxin 2; Trihexosylceramides

Shiga toxin (Stx)-producing Escherichia coli (STEC) cause an enteric illness that results in a spectrum of outcomes ranging from asymptomatic carriage to uncomplicated diarrhea, bloody diarrhea, and the postdiarrheal haemolytic uremic syndrome (HUS), which leads to renal and other organ microvascular thrombosis. Binding of Stx to the glycosphingolipid (GSL) globotriaosylceramide (Gb3Cer/CD77) on endothelial cells followed by receptor-mediated endocytosis is the linchpin in STEC-mediated disease. Only GSLs that associate strongly with lipid rafts appear to carry Stxs retrogradely from the plasma membrane through the Golgi apparatus to the endoplasmic reticulum where they are translocated to the cytosol and exert their toxic function. Thus, the biophysical features of the lipid moiety of GSL receptors may influence its incorporation into certain membrane domains and thereby affect toxin destination. Consequently, a detailed structural analysis of Stx-binding GSLs is required to illuminate the molecular causes that may underlie the different Stx susceptibilities of endothelial cells derived from various vascular beds. Solid phase overlay binding assays of thin-layer chromatography (TLC)-separated GSL preparations employing specific antibodies and/or Stxs in conjunction with anti-Stx-antibodies are commonly used for the identification of Stx-binding GSLs. Such GSL-profiling combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) represents a convenient strategy to structurally characterize Stx-receptors from any biological sources such as primary cells, cell lines, or organs. This approach may be helpful to gain insights into Stx-induced impairment of target cells that is suggested to originate at least partly from the structural heterogeneity of the cellular ligands of Stxs.

Topics: Amino Acid Sequence; Animals; Chromatography, Thin Layer; Endocytosis; Endothelium, Vascular; Escherichia coli Infections; Hemolytic-Uremic Syndrome; Humans; Ligands; Membrane Microdomains; Molecular Sequence Data; Peptide Mapping; Protein Conformation; Protein Subunits; Protein Transport; Proteomics; Shiga Toxins; Shiga-Toxigenic Escherichia coli; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Structure-Activity Relationship; Trihexosylceramides

Topics: Amino Acid Motifs; Animals; Biological Transport; Drug Design; Endoplasmic Reticulum; Escherichia coli Infections; Escherichia coli O157; Golgi Apparatus; Humans; Oligosaccharides; Peptide Library; Shiga Toxin; Trihexosylceramides

Eukaryotic cell surface glycolipids can act as both the primary interface between bacteria and their host and secondly as a targeting mechanism for bacterial virulence factors. The former is characterized by redundancy in adhesin-receptor interactions and the latter by a higher affinity, more restrictive glycolipid binding specificity for targeting. Interactions of verotoxin with its glycolipid receptor globotriaosylceramide and Helicobacter pylori binding to a variety of different glycolipids, which can be environmentally regulated, provide examples of these differing modes of glycolipid receptor function. Verotoxins are involved in endothelial targeting in the microangiopathies of hemorrhagic colitis and hemolytic uremic syndrome (HUS). The highly restricted binding specificity and crystal structure of the verotoxin B subunit have allowed theoretical modeling of the Gb3 binding site of the verotoxin B subunit pentamer which provides an approach to intervention. Studies of the role of glycolipid function in verotoxin-induced disease have concentrated on the distribution of Gb3 and its ability to mediate the internalization of the toxin within the target cell. The distribution of Gb3 within the renal glomerulus plays a central role in defining the age-related etiology of HUS following gastrointestinal infection with VT producing Escherichia coli. H. pylori, on the other hand, instigates a less distinct but more complex disseminated gastric inflammation. Studies on the role of glycolipid receptors in H. pylori infection have been bogged down in establishing the importance of each binding specificity defined. In addition, the physiological condition of the organism within the various binding assays has not been extensively considered, such that spurious non-physiological interactions may have been elucidated. The identification and cloning of a Le(b) binding adhesin and the identification of cell surface hsp70 as a mediator of sulfoglycolipid binding under stress conditions may now allow a more molecular approach to define the role of glycolipid recognition in this infection.

Topics: Adhesins, Bacterial; Age Factors; Animals; Bacterial Toxins; Binding Sites; Escherichia coli Infections; Gastrointestinal Diseases; Helicobacter Infections; Helicobacter pylori; Hemolytic-Uremic Syndrome; Humans; Hydrogen-Ion Concentration; Kidney Glomerulus; Receptors, Cell Surface; Shiga Toxin 1; Stomach; Stress, Physiological; Trihexosylceramides

The hemolytic uremic syndrome (HUS) is the end result of a variety of etiologic agents that can induce endothelial cell injury and thrombotic microangiopathy (TMA) mostly within the kidney. The typical, post-diarrheal verocytotoxin associated HUS (D + HUS) is the major cause of acute renal failure in children worldwide. In the course of HUS treatment, fluid overload is usually the result of overhydration in the context of oliguria or anuria which cause edema, hypertension, worsening of neurologic signs and cardiac failure. Appropriate and timely use of dialysis has dramatically reduced complications of renal failure and extra-renal complications are now the main causes of mortality and morbidity in D + HUS. The reasons for treatment by infusion of fresh frozen plasma and/or plasmapheresis for D + HUS are theoretical and their therapeutic effects are inconclusive. We believe that plasma administration for regular D + HUS has no value and is potentially harmful. Until new strategies become available in clinical practice, the general consensus for the moment is that careful supportive management with patience is still the most appropriate form of D + HUS therapy.

Topics: Escherichia coli Infections; Escherichia coli O157; Hemolytic-Uremic Syndrome; Humans; Immunoglobulins, Intravenous; Oligosaccharides; Plasma; Plasmapheresis; Renal Dialysis; Trihexosylceramides

Hemolytic uremic syndrome (HUS) is caused by endothelial cell damages. Ninety percent of children with HUS have verotoxin-producing E.coli infection. Verotoxin binds to glycolipid receptors globotriaosyl ceramide (Gb3), and the difference of Gb3 expression level in each organ would lead to specific organ involvement. The receptors are expressed in human renal cortex and medulla. The expression level of Gb3 in normal human brain has not been characterized completely. However involvement of central nervous system is a severe complication of HUS. Spreading of microvascular thrombosis caused by combined effects of lipopolysaccharide, cytokine, enhanced shear stress, and verotoxin would play a major role in the development of central nervous dysfunction.

Topics: Bacterial Toxins; Central Nervous System Diseases; Escherichia coli Infections; Escherichia coli O157; Gastrointestinal Hemorrhage; Hemolytic-Uremic Syndrome; Humans; Purpura, Thrombotic Thrombocytopenic; Shiga Toxin 1; Trihexosylceramides

Topics: Adsorption; Amino Acid Sequence; Bacterial Toxins; Clinical Trials as Topic; Escherichia coli Infections; Escherichia coli O157; Humans; Molecular Sequence Data; Oligosaccharides; Shiga Toxin 1; Silicon Dioxide; Trihexosylceramides

Shiga toxins (Stx) are virulence factors produced by selected bacteria pathogenic for humans. These multicomponent protein complexes are among the more potent toxins known. As inhibitors of eukaryotic protein synthesis, these toxins selectively inactivate ribosomes in an enzymatic manner. Specificity of cell targeting is determined by the high-affinity binding of Stx to its receptor, a glycosphingolipid (Gb3) located in the plasma membrane or some eukaryotic cells. Elaborated by food-borne E. coli O157:H7 bacteria, isotypes of Stx (Stx1 & Stx2) are required for the ensuing vascular changes in humans, including hemorrhagic colitis and renal hemolytic uremic syndrome. Experimental therapeutic intervention of Stx-associated disease includes the Stx receptor immobilized on biologically inert particles designed for oral presentation.

Topics: Animals; Endocytosis; Escherichia coli Infections; Escherichia coli O157; Humans; Receptors, Cell Surface; Shiga Toxins; Trihexosylceramides; Vascular Diseases

E. coli cause greater than 90% of urinary tract infections (UTI) in childhood. The capacity to adhere to urinary tract epithelial cells characterizes E. coli strains that cause acute pyelonephritis. Galactose alpha 1-4Galactose beta is the minimal receptor for adhering uropathogenic E. coli. Gal alpha 1-4Gal beta-binding bacteria caused significantly higher body temperature, C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR), pyuria, and lower renal concentrating capacity than E. coli lacking this specificity. The binding bacteria thus appeared to be more potent inducers of acute inflammation. Since inflammation may lead to tissue damage, we examined the relationship of infection with Gal alpha 1-4Gal beta-positive bacteria to renal scarring. The frequency of renal scarring was 5% in boys with Gal alpha 1-4Gal beta-positive and 40% in boys with Gal alpha 1-4Gal beta-negative E. coli. Analysis of binding capacity with the help of a newly developed latex agglutination assay can thus be used as an effective predictor of risk for renal scarring.

Topics: Bacterial Adhesion; Child; Child, Preschool; Cicatrix; Escherichia coli Infections; Female; Humans; Male; Pyelonephritis; Trihexosylceramides; Urinary Tract Infections

Encephalopathy associated with hemolytic uremic syndrome is produced by enterohemorrhagic E. coli (EHEC) infection, which releases the virulence factors Shiga toxin (Stx) and lipopolysaccharide (LPS). Neurological compromise is a poor prognosis and mortality factor of the disease, and the thalamus is one of the brain areas most frequently affected. We have previously demonstrated the effectiveness of anti-inflammatory drugs to ameliorate the deleterious effects of these toxins. However, the thalamic production of cytokines involved in pro-inflammatory processes has not yet been acknowledged. The aim of this work attempts to determine whether systemic sublethal Stx2a or co-administration of Stx2a with LPS are able to rise a proinflammatory profile accompanying alterations of the neurovascular unit in anterior and lateral ventral nuclei of the thalamus (VA-VL) and motor behavior in mice. After 4 days of treatment, Stx2a affected the lectin-bound microvasculature distribution while increasing the expression of GFAP in reactive astrocytes and producing aberrant NeuN distribution in degenerative neurons. In addition, increased swimming latency was observed in a motor behavioral test. All these alterations were heightened when Stx2a was co-administered with LPS. The expression of pro-inflammatory cytokines TNFα, INF-γ and IL-2 was detected in VA-VL. All these effects were concomitant with increased expression of the Stx receptor globotriaosylceramide (Gb3), which hints at receptor involvement in the neuroinflammatory process as a key finding of this study. In conclusion, Stx2a to Gb3 may be determinant in triggering a neuroinflammatory event, which may resemble clinical outcomes and should thus be considered in the development of preventive strategies.

Topics: Animals; Cytokines; Escherichia coli; Escherichia coli Infections; Lipopolysaccharides; Mice; Shiga Toxin; Shiga Toxin 2; Thalamus; Trihexosylceramides

Topics: Animals; Cell Survival; Chlorocebus aethiops; Epithelial Cells; Escherichia coli Infections; Globosides; Hemolytic-Uremic Syndrome; Humans; Kidney Cortex; Membrane Microdomains; Primary Cell Culture; Protein Binding; Shiga Toxin 1; Shiga Toxin 2; Shiga-Toxigenic Escherichia coli; Trihexosylceramides; Vero Cells

Shiga toxin (Stx) produced by enterohemorrhagic

Topics: Animals; Brain Diseases; Cytokines; Disease Models, Animal; Enterohemorrhagic Escherichia coli; Escherichia coli Infections; Heat-Shock Response; Hemolytic-Uremic Syndrome; Inflammation; Lipopolysaccharides; Macrophages; Microglia; Rats; Rats, Wistar; Shiga Toxin 2; Trihexosylceramides

Hemolytic uremic syndrome (HUS) and acute encephalopathy caused by enterohemorrhagic Escherichia coli infection occur commonly in children, whereas adult-onset disease is rare. Here we report the case of a 24-year-old woman who developed acute encephalopathy and recovered without sequelae. She initially developed abdominal pain and diarrhea. On day 6, O-157 Shiga toxin was detected in her stool and she developed HUS. On day 11, acute encephalopathy developed and she required artificial ventilation. She was treated with steroid pulse therapy and plasma exchange (PE) and then discharged on day 53 without any sequelae. Globotriaosylceramide, a Shiga toxin receptor, is more frequently present on the cellular membranes of women than on those of men. Therefore, it is conceivable that adult women are at a higher risk of developing acute encephalopathy than men. Steroid pulse therapy and PE may effectively treat acute encephalopathy by reducing inflammatory cytokine levels in the blood; therefore, these treatments should be proactively considered.

Topics: Acute Disease; Antigens, Tumor-Associated, Carbohydrate; Brain Diseases; Enterohemorrhagic Escherichia coli; Escherichia coli Infections; Female; Hemolytic-Uremic Syndrome; Humans; Methylprednisolone; Plasma Exchange; Prednisolone; Pulse Therapy, Drug; Risk; Shiga Toxin; Treatment Outcome; Trihexosylceramides; Young Adult

Shiga-toxin-producing Escherichia coli (STEC) infections usually cause haemolytic uraemic syndrome (HUS) equally in male and female children. This study investigated the localization of globotriaosylceramide (Gb3) in human brain and kidney tissues removed from forensic autopsy cases in Japan. A fatal case was used as a positive control in an outbreak of diarrhoeal disease caused by STEC O157:H7 in a kindergarten in Urawa in 1990. Positive immunodetection of Gb3 was significantly more frequent in female than in male distal and collecting renal tubules. To correlate this finding with a clinical outcome, a retrospective analysis of the predictors of renal failure in the 162 patients of two outbreaks in Japan was performed: one in Tochigi in 2002 and the other in Kagawa Prefecture in 2005. This study concludes renal failure, including HUS, was significantly associated with female sex, and the odds ratio was 4·06 compared to male patients in the two outbreaks. From 2006 to 2009 in Japan, the risk factor of HUS associated with STEC infection was analysed. The number of males and females and the proportion of females who developed HUS were calculated by age and year from 2006 to 2009. In 2006, 2007 and 2009 in adults aged >20 years, adult women were significantly more at risk of developing HUS in Japan.

Topics: Adolescent; Adult; Age Factors; Aged; Aged, 80 and over; Brain; Child; Child, Preschool; Diarrhea; Disease Outbreaks; Escherichia coli Infections; Female; Hemolytic-Uremic Syndrome; Humans; Infant; Infant, Newborn; Japan; Kidney; Male; Middle Aged; Renal Insufficiency; Retrospective Studies; Risk Factors; Sex Factors; Shiga-Toxigenic Escherichia coli; Trihexosylceramides; Young Adult

This study investigated the efficacy of selenium (Se) in reducing Escherichia coli O157:H7 verotoxin production and toxin gene expression. Additionally, the effect of Se on globotriaosylceramide (Gb3) receptor in human lymphoma cells was determined.. The effect of Se on verotoxin synthesis was determined by standard ELISA, whereas its effect on Gb3 receptor was determined by flow cytometry and real-time quantitative PCR.. Se reduced extracellular and intracellular verotoxin concentration by 40-60% and 80-90%, respectively (p < 0.05), and downregulated verotoxin genes (p < 0.05). Se reduced Gb3 receptor synthesis in lymphoma cells, and real-time quantitative PCR data revealed a significant downregulation of LacCer synthase gene (GalT2) involved in Gb3 synthesis. Further studies are warranted to validate these results in an appropriate animal model.

Topics: Escherichia coli Infections; Escherichia coli O157; Humans; Receptors, Cell Surface; Selenium; Shiga Toxins; Trihexosylceramides

In 2011, a large outbreak of Shiga toxin-producing enterohemorrhagic Escherichia coli (EHEC) infections occurred in northern Germany, which mainly affected adults. Out of 3842 patients, 104 experienced a complicated course comprising hemolytic uremic syndrome and neurological complications, including cognitive impairment, aphasia, seizures and coma. T2 hyperintensities on magnet resonance imaging (MRI) bilateral in the thalami and in the dorsal pons were found suggestive of a metabolic toxic effect. Five of the 104 patients died because of toxic heart failure. In the present study, the post-mortem neuropathological findings of the five EHEC patients are described. Histological investigation of 13 brain regions (frontal, temporal, occipital cortex, corpora mammillaria, thalamus, frontal operculum, corona radiata, gyrus angularis, pons, medulla oblongata, cerebellar vermis and cerebellar hemisphere) showed no thrombosis, ischemic changes or fresh infarctions. Further, no changes were found in electron microscopy. In comparison with five age-matched controls, slightly increased activation of microglia and a higher neuronal expression of interleukin-1β and of Shiga toxin receptor CD77/globotriaosylceramide 3 was observed. The findings were confirmed by Western blot analyses. It is suggested that CD77/globotriaosylceramide upregulation may be a consequence to Shiga toxin exposure, whereas increased interleukin-1β expression may point to activation of inflammatory cascades.

Topics: Aged; Aged, 80 and over; Brain; Cognition Disorders; Encephalitis; Enterohemorrhagic Escherichia coli; Escherichia coli Infections; Female; Hemolytic-Uremic Syndrome; Humans; Interleukin-1beta; Male; Trihexosylceramides; Up-Regulation

Shiga toxin (Stx)-producing Escherichia coli (STEC) cause hemolytic uremic syndrome (HUS). This study investigated whether Stx2 induces hemolysis and whether complement is involved in the hemolytic process. RBCs and/or RBC-derived microvesicles from patients with STEC-HUS (n = 25) were investigated for the presence of C3 and C9 by flow cytometry. Patients exhibited increased C3 deposition on RBCs compared with controls (p < 0.001), as well as high levels of C3- and C9-bearing RBC-derived microvesicles during the acute phase, which decreased after recovery. Stx2 bound to P1 (k) and P2 (k) phenotype RBCs, expressing high levels of the P(k) Ag (globotriaosylceramide), the known Stx receptor. Stx2 induced the release of hemoglobin and lactate dehydrogenase in whole blood, indicating hemolysis. Stx2-induced hemolysis was not demonstrated in the absence of plasma and was inhibited by heat inactivation, as well as by the terminal complement pathway Ab eculizumab, the purinergic P2 receptor antagonist suramin, and EDTA. In the presence of whole blood or plasma/serum, Stx2 induced the release of RBC-derived microvesicles coated with C5b-9, a process that was inhibited by EDTA, in the absence of factor B, and by purinergic P2 receptor antagonists. Thus, complement-coated RBC-derived microvesicles are elevated in HUS patients and induced in vitro by incubation of RBCs with Stx2, which also induced hemolysis. The role of complement in Stx2-mediated hemolysis was demonstrated by its occurrence only in the presence of plasma and its abrogation by heat inactivation, EDTA, and eculizumab. Complement activation on RBCs could play a role in the hemolytic process occurring during STEC-HUS.

Topics: Adult; Aged; Antibodies, Monoclonal, Humanized; Child; Child, Preschool; Coated Vesicles; Complement Activation; Complement C3; Complement C9; Complement Membrane Attack Complex; Edetic Acid; Erythrocytes; Escherichia coli Infections; Escherichia coli O157; Female; Gene Expression; Hemolysis; Hemolytic-Uremic Syndrome; Humans; Infant; L-Lactate Dehydrogenase; Male; Middle Aged; Purinergic P2 Receptor Antagonists; Receptors, Purinergic P2; Shiga Toxin; Suramin; Trihexosylceramides

Shiga toxin (Stx)-producing Escherichia coli (STEC) infections in cattle are asymptomatic; however, Stx impairs the initiation of an adaptive immune response by targeting bovine peripheral and intraepithelial lymphocytes. As presumptive bovine mucosal macrophages (Mø) are also sensitive to Stx, STEC may even exert immune modulatory effects by acting on steps preceding lymphocyte activation at the Mø level. We therefore studied the expression of the Stx receptor (CD77), cellular phenotype and functions after incubation of primary bovine monocyte-derived Mø with purified Stx1. A significant portion of bovine Mø expressed CD77 on their surface, with the recombinant B-subunit of Stx1 binding to >50% of the cells. Stx1 down-regulated significantly surface expression of CD14, CD172a and co-stimulatory molecules CD80 and CD86 within 4 h of incubation, while MHC-II expression remained unaffected. Furthermore, incubation of Mø with Stx1 increased significantly numbers of transcripts for IL-4, IL-6, IL-10, IFN-γ, TNF-α, IL-8 and GRO-α but not for IL-12, TGF-β, MCP-1 and RANTES. In the course of bovine STEC infections, Stx1 appears to induce in Mø a mixed response pattern reminiscent of regulatory Mø, which may amplify the direct suppressive effect of the toxin on lymphocytes.

Topics: Animals; Cattle; Cells, Cultured; Cytokines; Escherichia coli; Escherichia coli Infections; Lipopolysaccharide Receptors; Lymphocytes; Macrophages; Recombinant Proteins; Shiga Toxin 1; Trihexosylceramides

The outbreak of hemolytic-uremic syndrome and diarrhea caused by Shiga toxin-producing Escherichia coli O104:H4 in Germany during May to July 2011 involved severe and characteristic neurologic manifestations with a strong female preponderance. Owing to these observations, we designed a series of experimental studies to evaluate the underlying mechanism of action of this clinical picture.. A magnetic resonance imaging and electroencephalographic study of patients was performed to evaluate the clinical picture in detail. Thereafter, combinations of different experimental settings, including electrophysiological and histological analyses, as well as calcium imaging in brain slices of rats, were conducted.. We report on 7 female patients with neurologic symptoms and signs including bilateral thalamic lesions and encephalopathic changes indicative of a predominant involvement of the thalamus. Experimental studies in rats revealed an enhanced expression of the Shiga toxin receptor globotriaosylceramide on thalamic neurons in female rats as compared to other brain regions in the same rats and to male animals. Incubation of brain slices with Shiga toxin 2 evoked a strong membrane depolarization and intracellular calcium accumulation in neurons, associated with neuronal apoptosis, predominantly in the thalamic area.. These findings suggest that the direct cytotoxic effect of Shiga toxin 2 in the thalamus might contribute to the pathophysiology of neuronal complications in hemolytic-uremic syndrome.

Topics: Adult; Aged; Animals; Animals, Newborn; Apoptosis; Astrocytes; Calcium; Electroencephalography; Escherichia coli Infections; Female; Gene Expression Regulation; Humans; In Vitro Techniques; Magnetic Resonance Imaging; Membrane Potentials; Nervous System Diseases; Neurons; Patch-Clamp Techniques; Rats; Rats, Long-Evans; RNA, Messenger; Sex Characteristics; Shiga Toxin 2; Thalamus; Trihexosylceramides; Young Adult

Hemolytic uremic syndrome (HUS) caused by intestinal Shiga toxin-producing Escherichia coli infections is a worldwide health problem, as dramatically exemplified by the German outbreak occurred in summer 2011 and by a constant burden of cases in children. Shiga toxins (Stx) play a pivotal role in HUS by triggering endothelial damage in kidney and brain through globotriaosylceramide (Gb3Cer) receptor targeting. Moreover, Stx interact with human neutrophils, as experimentally demonstrated in vitro and as observed in patients with HUS. A neutrophil-protective role on endothelial damage (sequestration of circulating toxins) and a causative role in toxin delivery from the gut to the kidney (piggyback transport) have been suggested in different studies. However, the receptor that recognizes Stx in human neutrophils, which do not express Gb3Cer, has not been identified. In this study, by competition and functional experiments with appropriate agonists and antagonists (LPS, anti-TLR4 Abs, respectively), we have identified TLR4 as the receptor that specifically recognizes Stx1 and Stx2 in human neutrophils. Accordingly, these treatments displaced both toxin variants from neutrophils and, upon challenge with Stx1 or Stx2, neutrophils displayed the same pattern of cytokine expression as in response to LPS (assessed by quantitative RT-PCR, ELISA, or multiplexed Luminex-based immunoassays). Moreover, data were supported by adequate controls excluding any potential interference of contaminating LPS in Stx-binding and activation of neutrophils. The identification of the Stx-receptor on neutrophils provides additional elements to foster the understanding of the pathophysiology of HUS and could have an important effect on the development of therapeutic strategies.

Topics: Antibodies, Monoclonal; Cytokines; Escherichia coli; Escherichia coli Infections; Hemolytic-Uremic Syndrome; Humans; Lipopolysaccharides; Neutrophils; Shiga Toxin 1; Shiga Toxin 2; Toll-Like Receptor 4; Trihexosylceramides

Diarrhea-associated hemolytic uremic syndrome (D(+)HUS) is caused by the ingestion of Escherichia coli that produce Shiga toxin (Stx), which is composed of a cytotoxic A subunit and pentameric B subunits that bind globotriaosylceramide on susceptible cells. Stx occurs in 2 types, Stx1 and Stx2. B subunits of either type stimulate von Willebrand factor (VWF) secretion from human umbilical vein endothelial cells (HUVECs), and Stx2B can cause thrombotic microangiopathy in Adamts13(-/-) mice. We have now determined that Stx1B and Stx2B activate different signaling pathways in HUVECs. VWF secretion induced by Stx1B is associated with a transient rise in intracellular Ca(2+) level that is blocked by chelation with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester, removal of extracellular Ca(2+), the phospholipase C inhibitor U73122, the protein kinase inhibitor staurosporine, or small interfering RNA knockdown of protein kinase Cα. In contrast, Stx2B-induced VWF secretion is associated with activation of protein kinase A (PKA) and is blocked by the PKA inhibitor H89 or small interfering RNA knockdown of PKA. Stx2B does not increase cAMP levels and may activate PKA by a cAMP-independent mechanism. The activation of distinct signaling pathways may be relevant to understanding why E coli that express Stx2 are more likely to cause D(+)HUS than are E coli expressing only Stx1.

Topics: Animals; Calcium; Cyclic AMP-Dependent Protein Kinases; Diarrhea; Egtazic Acid; Endothelial Cells; Escherichia coli; Escherichia coli Infections; Gene Silencing; Hemolytic-Uremic Syndrome; Humans; Mice; Protein Binding; Protein Kinase C-alpha; Protein Kinase Inhibitors; RNA, Small Interfering; Shiga Toxin 1; Shiga Toxin 2; Signal Transduction; Trihexosylceramides; Umbilical Veins; von Willebrand Factor

Neurological damage caused by intoxication with Shiga toxin (Stx) from enterohemorrhagic Escherichia coli is the most unrepairable and untreatable outcome of Hemolytic Uremic Syndrome, and occurs in 30% of affected infants. In this work intracerebroventricular administration of Stx2 in rat brains significantly increased the expression of its receptor globotriaosylceramide (Gb(3)) in neuronal populations from striatum, hippocampus and cortex. Stx2 was immunodetected in neurons that expressed Gb(3) after intracerebroventricular administration of the toxin. Confocal immunofluorescence of microtubule-associated protein 2 showed aberrant dendrites in neurons expressing increased Gb(3). The pro-apoptotic Bax protein was concomitantly immunodetected in neurons and other cell populations from the same described areas including the hypothalamus. Confocal immunofluorescence showed that Gb(3) colocalized also with glial fibrillary acidic protein only in reactive astrocytic processes, and not in vehicle-treated normal ones. Rats showed weight variation and motor deficits as compared to controls. We thus suggest that Stx2 induces the expression of Gb(3) in neurons and triggers neuronal dysfunctions.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Biomarkers; Brain; Chlorocebus aethiops; Dendrites; Escherichia coli Infections; Glial Fibrillary Acidic Protein; Immunohistochemistry; Injections, Intraventricular; Male; Microscopy, Confocal; Microtubule-Associated Proteins; Nerve Degeneration; Neurotoxicity Syndromes; Rats; Rats, Sprague-Dawley; Shiga Toxin 2; Trihexosylceramides; Up-Regulation; Vero Cells

Shiga toxin 1 and 2 production is a cardinal virulence trait of enterohemorrhagic Escherichia coli infection that causes a spectrum of intestinal and systemic pathology. However, intestinal sites of enterohemorrhagic E. coli colonization during the human infection and how the Shiga toxins are taken up and cross the globotriaosylceramide (Gb3) receptor-negative intestinal epithelial cells remain largely uncharacterized. We used samples of human intestinal tissue from patients with E. coli O157:H7 infection to detect the intestinal sites of bacterial colonization and characterize the distribution of Shiga toxins. We further used a model of largely Gb3-negative T84 intestinal epithelial monolayers treated with B-subunit of Shiga toxin 1 to determine the mechanisms of non-receptor-mediated toxin uptake. We now report that E. coli O157:H7 were found at the apical surface of epithelial cells only in the ileocecal valve area and that both toxins were present in large amounts inside surface and crypt epithelial cells in all tested intestinal samples. Our in vitro data suggest that macropinocytosis mediated through Src activation significantly increases toxin endocytosis by intestinal epithelial cells and also stimulates toxin transcellular transcytosis. We conclude that Shiga toxin is taken up by human intestinal epithelial cells during E. coli O157:H7 infection regardless of the presence of bacterial colonies. Macropinocytosis might be responsible for toxin uptake by Gb3-free intestinal epithelial cells and transcytosis. These observations provide new insights into the understanding of Shiga toxin contribution to enterohemorrhagic E. coli-related intestinal and systemic diseases.

Topics: Actins; Animals; cdc42 GTP-Binding Protein; Cell Line; Cell Membrane; Chlorpromazine; Clathrin; Disease Models, Animal; Endocytosis; Enzyme Activation; Epithelial Cells; Escherichia coli Infections; Escherichia coli O157; Ethylmaleimide; Humans; Intestinal Mucosa; Intestines; Male; N-Ethylmaleimide-Sensitive Proteins; Pinocytosis; Rabbits; Recombinant Proteins; Shiga Toxin 1; Shiga Toxin 2; src-Family Kinases; Tetradecanoylphorbol Acetate; Time Factors; Trihexosylceramides

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.

Topics: Cytokines; Enteritis; Enterohemorrhagic Escherichia coli; Epithelial Cells; Escherichia coli Infections; Host-Pathogen Interactions; HT29 Cells; Humans; I-kappa B Proteins; Inflammation Mediators; Interleukin-1beta; Interleukin-8; Intestinal Mucosa; NF-KappaB Inhibitor alpha; Protein Transport; Shiga Toxins; Trihexosylceramides; Tumor Necrosis Factor-alpha

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.

Topics: Animals; Cattle; Cell Survival; Cells, Cultured; Chemokine CCL5; Chemokine CXCL1; Colon; Epithelial Cells; Escherichia coli Infections; Flow Cytometry; Fluorescent Antibody Technique; Gene Expression; Interleukin-8; Intestinal Mucosa; Mesoderm; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Shiga Toxin 1; Shiga-Toxigenic Escherichia coli; Transcription, Genetic; Transforming Growth Factor beta; Trihexosylceramides

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.

Topics: Animals; Bacteriophages; Caco-2 Cells; Cecum; Disease Models, Animal; DNA, Bacterial; Escherichia coli Infections; Escherichia coli O157; Feces; Female; Hemolytic-Uremic Syndrome; Humans; Kidney; Male; Molecular Sequence Data; Rabbits; Sequence Analysis; Shiga Toxin; Time Factors; Trihexosylceramides

Granulocytes play a pivotal role in the pathogenesis of Shiga toxin (Stx)-producing Escherichia coli (STEC) related diseases in humans. Granulocytes are attracted and activated by Stxs in the enteric mucosa and are believed to thereby contribute to the intestinal inflammation. Mature ruminants, the main reservoir hosts of STEC, do not develop pathological changes that can be attributed to the Stxs. To prove whether the latter phenomenon correlates with the inability of the Stxs to affect granulocytes of ruminants, we investigated the ability of Stx1 to bind to granulocytes of cattle and sheep and analysed the effects of Stx1 on viability, phagocytosis, and oxidative burst activity. Bovine granulocytes from blood and milk did not express Stx1-binding sites even after activation of the cells and also were resistant to Stx1. In contrast to bovine granulocytes, granulocytes of sheep constitutively expressed Stx1-receptors of the Gb(3)/CD77 type ex vivo and bound the recombinant B-subunit of Stx1 (rStxB1). Stx1 holotoxin induced apoptosis in ovine granulocytes after prolonged incubation (18h) but Stx1 only slightly altered the phagocytosis and oxidative burst activities. The rStxB1 had no effect on granulocytes of either species. While arguing in favour of our initial hypothesis, that granulocytes of both, cattle and sheep are not activated by Stxs, the results of our study are the first evidences for differences in the cellular distribution of Stx-receptors in species equally regarded as STEC carriers.

Topics: Animals; Binding Sites; Cattle; Cattle Diseases; Cell Survival; Escherichia coli; Escherichia coli Infections; Female; Flow Cytometry; Granulocytes; Immunophenotyping; Intestinal Diseases; Milk; Phagocytosis; Respiratory Burst; Sheep; Sheep Diseases; Shiga Toxin 1; Trihexosylceramides

Fabry disease is an X-linked recessive disorder in which affected persons lack alpha-galactosidase A (alpha -GalA), which leads to excess glycosphingolipids in tissues, mainly globotriaosylceramide (Gb3). Gb3 is the cellular receptor for Shiga toxin (Stx), the primary virulence factor of enterohemorrhagic Escherichia coli. alpha-GalA-knockout mice were significantly protected against lethal intraperitoneal doses of Stx2 or oral doses of Stx2-expressing bacteria, compared with wild-type (wt) control mice. Kidneys of moribund wt mice revealed tubular necrosis, but no histopathologic changes were observed in Gb3-overexpressing mice. Reducing Gb3 levels in alpha-GalA-knockout mice by the intravenous injection of recombinant human alpha-GalA restored the susceptibility of knockout mice to lethal doses of Stx2. These results suggest that excess amounts of Gb3 in alpha-GalA-deficient mice may impair toxin delivery to susceptible tissues.

Topics: Administration, Oral; Animals; Escherichia coli Infections; Escherichia coli O157; Fabry Disease; Female; Injections, Intraperitoneal; Kidney; Male; Mice; Mice, Knockout; Shiga Toxins; Trihexosylceramides

Kidney lesions similar to those in humans with hemolytic-uremic syndrome were observed histologically in 82 of 122 piglets inoculated intragastrically with Shiga-toxigenic Escherichia coli but not in 29 controls. The locations of lesions matched locations where Stx-2 binding and Gb3 (globotriasylceramide receptors for Stx) were identified immunohistochemically.

Topics: Animals; Animals, Newborn; Escherichia coli; Escherichia coli Infections; Hemolytic-Uremic Syndrome; Humans; Immunohistochemistry; Kidney; Retrospective Studies; Shiga Toxin 2; Swine; Trihexosylceramides

Topics: Animals; Anti-Bacterial Agents; Clinical Trials as Topic; Escherichia coli Infections; Escherichia coli O157; Hemolytic-Uremic Syndrome; Humans; Mice; Molecular Mimicry; Polymers; Receptors, Cell Surface; Trihexosylceramides

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.

Topics: Acrylamide; Animals; Brain Chemistry; Carbohydrate Sequence; Disease Models, Animal; Dose-Response Relationship, Drug; Escherichia coli Infections; Escherichia coli O157; Female; Hemolytic-Uremic Syndrome; Mice; Mice, Inbred C57BL; Molecular Sequence Data; Polymers; Protein Binding; Receptors, Cell Surface; Shiga Toxin 1; Shiga Toxin 2; Shiga Toxins; Trihexosylceramides; Trisaccharides

We have previously constructed a recombinant bacterium expressing a modified lipopolysaccharide (LPS) mimicking the Shiga toxin receptor, which binds toxin with high avidity. This involved cloning Neisseria galactosyl transferase genes (lgtC and lgtE) in pK184 in a derivative of Escherichia coli R1 (CWG308). Such constructs have considerable potential for prevention of disease caused by Shiga toxin-producing E. coli (STEC). However, neither the E. coli host strain nor the expression plasmid is suitable for human use, because the former is derived from a clinical isolate and the latter contains a kanamycin-resistance gene. We have constructed, as a prelude to human trials, a nonpathogenic E. coli K-12 C600 derivative with deletions in waaO and waaB, such that it has the same LPS core structure as CWG308. We also deleted the thyA gene from this strain, rendering it thymine dependent. The kanamycin-resistance gene was also deleted from pK184 and was replaced with Salmonella typhimurium thyA. Neisseria lgtCE was then cloned into this plasmid and transformed into C600 Delta waaOB Delta thyA. The plasmid was stably maintained, and the construct produced a modified LPS and neutralized Stx1 and Stx2c. Moreover, mice challenged with an otherwise fatal dose of STEC were completely protected by oral administration of the novel construct.

Topics: Animals; Carbohydrate Sequence; Escherichia coli; Escherichia coli Infections; Galactosyltransferases; Genetic Vectors; Humans; Lipopolysaccharides; Mice; Molecular Sequence Data; Plasmids; Probiotics; Shiga Toxin 1; Shiga Toxin 2; Thymidylate Synthase; Trihexosylceramides

Enterohemorrhagic Escherichia coli (EHEC) is the major cause of hemolyticuremic syndrome (HUS) characterized by microangiopathic hemolytic anemia, thrombocytopenia, and acute renal failure. EHEC produces one or more Shiga toxins (Stx1 and Stx2), and it was assumed that Stx's only relevant biologic activity was cell destruction through inhibition of protein synthesis. However, recent data indicate that in vivo the cytokine milieu may determine whether endothelial cells survive or undergo apoptosis/necrosis when exposed to Stxs. In this study, we analyzed the genome-wide expression patterns of human endothelial cells stimulated with subinhibitory concentrations of Stxs in order to characterize the genomic expression program involved in the vascular pathology of HUS. We found that Stxs elicited few, but reproducible, changes in gene expression. The majority of genes reported in this study encodes for chemokines and cytokines, which might contribute to the multifaceted inflammatory response of host endothelial cells observed in patients suffering from EHEC disease. In addition, our data provide for the first time molecular insights into the epidemiologically well-established higher pathogenicity of Stx2 over Stx1.

Topics: Apoptosis; Chemokines; Cytokines; DNA Primers; Endothelium, Vascular; Enzyme-Linked Immunosorbent Assay; Escherichia coli; Escherichia coli Infections; Flow Cytometry; Gene Expression Profiling; Gene Expression Regulation; Hemolytic-Uremic Syndrome; Humans; Immunohistochemistry; Receptors, Cell Surface; Shiga Toxins; Trihexosylceramides; Umbilical Veins

Oral infection with enterohemorrhagic Escherichia coli (EHEC) may cause severe enteritis, followed in up to 10% of cases by an extraintestinal complication, the hemolytic uremic syndrome (HUS). HUS is characterized by a triad of symptoms: anemia, thrombocytopenia, and acute renalfailure due to thrombotic microangiopathy. EHEC produces several virulence factors, among which a family of phage-encoded cytotoxins, called Shiga toxin 1 and Shiga toxin 2, seems to be most important. However, since an appropriate animal model is not available, pathogenicity of these emerging enteric pathogens is still poorly understood. Germ-free gnotobiotic piglets infected orally with an O1577:H7 or an O26:H11 EHEC wild-type isolate, both producing Shiga toxin 2, developed intestinal and extraintestinal manifestations of EHEC disease, including thrombotic microangiopathy in the kidneys, the morphologic hallmark of HUS in humans. Thus, gnotobiotic piglets are suitable to further study the pathophysiology of EHEC-induced HUS. It can be expected that data obtainedfrom this animal model will improve our current standard of knowledge about this emerging infectious disease.

Topics: Animals; Child, Preschool; Digestive System; Disease Models, Animal; Endothelium, Vascular; Escherichia coli Infections; Escherichia coli O157; Female; Germ-Free Life; Hemolytic-Uremic Syndrome; Humans; Immunohistochemistry; Kidney; Microcirculation; Purpura, Thrombotic Thrombocytopenic; Shiga Toxin 2; Swine; Swine Diseases; Trihexosylceramides

Bacterial adherence to mucosal cells is a key virulence trait of pathogenic bacteria. The type 1 fimbriae and the P-fimbriae of Escherichia coli have both been described to be important for the establishment of urinary tract infections. While P-fimbriae recognize kidney glycosphingolipids carrying the Galalpha4Gal determinant, type 1 fimbriae bind to the urothelial mannosylated glycoproteins uroplakin Ia and Ib. The F1C fimbriae are one additional type of fimbria correlated with uropathogenicity. Although it was identified 20 years ago its receptor has remained unidentified. Here we report that F1C-fimbriated bacteria selectively interact with two minor glycosphingolipids isolated from rat, canine, and human urinary tract. Binding-active compounds were isolated and characterized as galactosylceramide, and globotriaosylceramide, both with phytosphingosine and hydroxy fatty acids. Comparison with reference glycosphingolipids revealed that the receptor specificity is dependent on the ceramide composition. Galactosylceramide was present in the bladder, urethers, and kidney while globotriaosylceramide was present only in the kidney. Using a functional assay, we demonstrate that binding of F1C-fimbriated Escherichia coli to renal cells induces interleukin-8 production, thus suggesting a role for F1C-mediated attachment in mucosal defense against bacterial infections.

Topics: Animals; Bacterial Adhesion; Chromatography, Thin Layer; Dogs; Escherichia coli; Escherichia coli Infections; Galactosylceramides; Glycosphingolipids; Humans; Interleukin-8; Magnetic Resonance Spectroscopy; Mucous Membrane; Rats; Receptors, Immunologic; Sphingosine; Trihexosylceramides; Urinary Tract; Urinary Tract Infections

The relationship between differential susceptibility to hemolytic-uremic syndrome (HUS) and levels of globotriaosylceramide (Gb3) in serum was studied in patients infected with verotoxin-producing Escherichia coli (VTEC). The serum Gb3 levels in patients with HUS were lower than these in diarrheal patients without subsequent HUS or in patients without clinical symptoms, indicating that individuals with a lower content of serum Gb3 show a higher incidence of HUS following VTEC infection.

Topics: Adolescent; Adult; Aged; Child; Child, Preschool; Diarrhea; Disease Susceptibility; Escherichia coli; Escherichia coli Infections; Female; Hemolytic-Uremic Syndrome; Humans; Male; Middle Aged; Shiga Toxins; Trihexosylceramides

Gastrointestinal disease caused by Shiga toxin-producing Escherichia coli (STEC) is frequently complicated by life-threatening toxin-induced systemic sequelae, including the hemolytic uremic syndrome. We previously constructed a recombinant bacterium displaying a Shiga toxin receptor mimic on its surface which neutralized Shiga toxins with very high efficiency. Moreover, oral administration of the live bacterium completely protected mice from challenge with virulent STEC. In this study, we investigated the protective capacity of formaldehyde-killed receptor mimic bacteria, as these are likely to be safer for administration to humans. The killed bacteria completely protected STEC-challenged mice when administered three times daily; incomplete protection was achieved using two doses per day. Commencement of therapy could be delayed for up to 48 h after challenge without diminishing protection, depending on the virulence of the challenge strain. Thus, administration of this agent early in the course of human STEC disease may prevent progression to life-threatening complications.

Topics: Administration, Oral; Animals; Escherichia coli; Escherichia coli Infections; Escherichia coli Vaccines; Formaldehyde; Hemolytic-Uremic Syndrome; Male; Mice; Mice, Inbred BALB C; Molecular Mimicry; Receptors, Cell Surface; Shiga Toxin; Trihexosylceramides; Vaccines, Inactivated; Vaccines, Synthetic

In a previous study, we showed that infection with Shiga toxin (Stx)-producing Escherichia coli O157:H7 (strain Sm(r)N-9) caused neurologic symptoms in malnourished mice with positive immunoreactions of Stx2 in brain tissues. The present study explores the mechanism of how Stx injures the vascular endothelium to enter the central nervous system in mice. Oral infection with strain Sm(r)N-9 elicited a tumor necrosis factor alpha (TNF-alpha) response in the blood as early as 2 days after infection, while Stx was first detected at 3 days postinfection. In the brain, TNF-alpha was detected at day 3, and its quantity was increased over the next 3 days. Frozen sections of the brains from moribound mice contained high numbers of apoptotic cells. Glycolipids recognized by an anti-Gb3 monoclonal antibody were extracted from the brain, and purified Stx2 was able to bind to the glycolipids. In human umbilical vascular endothelial cells (HUVEC) cultured with fluorescein-labeled Stx2 (100 ng/ml), TNF-alpha (20 U/ml) significantly facilitated the intracellular compartmentalization of fluorescence during 24 h of incubation, suggesting the enhanced intracellular processing of Stx2. Consequently, higher levels of apoptosis in HUVEC were found at 48 h. Short-term exposure of HUVEC to Stx2 abrogated their apoptotic response to subsequent incubation with TNF-alpha alone or TNF-alpha and Stx2. In contrast, primary exposure of HUVEC to TNF-alpha followed by exposure to Stx2 alone or TNF-alpha and Stx2 induced apoptosis at the same level as obtained after 48-h incubation with these two agents. These results suggest that the rapid production of circulating TNF-alpha after infection induces a state of competence in vascular endothelial cells to undergo apoptosis, which would be finally achieved by subsequent elevation of Stx in the blood. In this synergistic action, target cells must be first exposed to TNF-alpha. Such cell injury may be a prerequisite to brain damage after infection with Stx-producing E. coli O157:H7.

Topics: Animals; Apoptosis; Bacterial Toxins; Brain; Cytokines; Endothelium, Vascular; Escherichia coli Infections; Escherichia coli O157; Female; Humans; Mice; Mice, Inbred C57BL; Shiga Toxins; Trihexosylceramides; Tumor Necrosis Factor-alpha

Acute renal failure is one of the hallmarks of the hemolytic uremic syndrome (HUS). Infection with a verocytotoxin (VT)- or Shiga-like toxin (SLT)-producing Escherichia coli has been strongly implicated in the etiology of the epidemic form of HUS. The functional receptor for these closely related toxins appears to be a glycosphingolipid, globotriaosylceramide (Gb3). Endothelial damage in the glomeruli and arterioles of the kidney induced by VT is believed to play a crucial role in the pathogenesis of HUS. However, little information is available regarding the effects of VT on mesangial cells, which also play an important role in glomerular function. In this study, the effects of VT on human mesangial cells in vitro were investigated. Mesangial cells were enriched by collecting hillock-shaped outgrowths derived from adult human glomeruli and subsequently purified by elimination of contaminating epithelial cells by immunoseparation with ulex europaeus lectin-I (UEA-I)-coated dynabeads. The obtained and subcultured mesangial cell populations were >98% pure. Their mesangial nature was established by the presence of a-smooth muscle cell actin in highly confluent cultures and the absence of cytokeratin or platelet/endothelial cell adhesion molecule-1. Mesangial cells bound VT to bands of Gb3 and a closely related glycolipid, which is similar to a glycolipid involved in the VT-dependent cytokine production in monocytes. VT did not induce the release of cytokines or chemokines in mesangial cells. In VT-susceptible cells, binding of VT to Gb3 causes cell death by the inhibition of protein synthesis. Although protein synthesis was inhibited in mesangial cells, all cells remained viable, both under basal and tumor necrosis factor-alpha-stimulated conditions. However, the marked reduction in protein synthesis may impair a proper response of the cells in conditions of increased demand of newly synthesized proteins. Furthermore, VT markedly inhibited DNA synthesis and proliferation of mesangial cells. The inhibition of mitogenesis was also found with the B-subunit of VT-1 alone, albeit to a lesser extent, without a significant effect on protein synthesis. Because the inhibition of protein synthesis involves the A-subunit, this suggests that two distinct mechanisms contribute to the effects of VT on protein synthesis and mitogenesis. Intracellular routing of VT (A- and B-subunits) may vary between cell types and result in differential effects on human mesangial cells wh

Topics: Acute Kidney Injury; Adult; Bacterial Toxins; Cell Division; Cell Survival; Cells, Cultured; DNA Replication; Escherichia coli Infections; Glomerular Mesangium; Glycolipids; Growth Inhibitors; Hemolytic-Uremic Syndrome; Humans; Muscle, Smooth, Vascular; Protein Biosynthesis; Protein Synthesis Inhibitors; Shiga Toxin 1; Trihexosylceramides; Tumor Necrosis Factor-alpha; Up-Regulation

Gastrointestinal infection with verotoxin-producing Escherichia coli (VTEC) has been strongly implicated in the etiology of the hemolytic uremic syndrome (HUS), the leading cause of pediatric acute renal failure. The binding of fluorescein-conjugated VT1 overlaid on to frozen human renal sections has been examined. Sections from biopsies of infants aged < 2 years were compared with those from adult autopsies. VT primarily stained distal convoluted tubules, particularly those adjacent to glomeruli, and collecting ducts. VT-binding was detected within the infant glomerulus but not the adult. Binding of the toxin was removed when the section was pretreated with alpha-galactosidase, confirming the receptor-binding specificity for globotriaosyl ceramide (gal alpha 1-4gal beta 1-4 glucosylceramide), the glycolipid receptor for VT. These studies may suggest that differential localization of this glycolipid in the pediatric renal glomerulus is a risk factor for the development of HUS following infection with VTEC.

Topics: Adult; Age Factors; alpha-Galactosidase; Bacterial Toxins; Carbohydrate Sequence; Child; Child, Preschool; Escherichia coli Infections; Gastrointestinal Diseases; Glycolipids; Hemolytic-Uremic Syndrome; Hepatorenal Syndrome; Histocytochemistry; Humans; In Vitro Techniques; Infant; Kidney; Molecular Sequence Data; Nephrosis, Lipoid; Receptors, Cell Surface; Shiga Toxin 1; Trihexosylceramides

Fresh and transformed human B lineage cells were found to be sensitive to the cytotoxic action of Shiga-like toxin (SLT), a bacterial cytotoxin. The toxin was specifically bound by the glycolipids globotriosylceramide and galabiosylceramide expressed on the surface of sensitive cells. Mutant Daudi cells selected for resistance to SLT cytotoxicity (SLTR20) were deficient in SLT-binding glycolipids and failed to bind SLT to their surface, suggesting a role for these glycolipids in the mediation of SLT cytotoxicity. Of a number of normal and transformed lymphoid and myeloid cells screened for SLT sensitivity, only B lymphoid cells were susceptible to SLT action. Moreover, B lymphoid cells were the only cells expressing the SLT binding glycolipids. In vitro B cell activation studies with Epstein-Barr virus and pokeweed mitogen both indicated that the vast majority of SLT-sensitive B cells belong to the IgG and IgA committed subset, whereas most IgM and IgM/D producing cells were resistant to SLT toxicity. The selective elimination of IgG and IgA committed cells may explain the production of only IgM class anti-SLT antibodies in Shigella-infected humans leading to the failure of long-term immunity to dysenteric disease.

Topics: Antibodies, Bacterial; B-Lymphocytes; Bacterial Toxins; Burkitt Lymphoma; Cells, Cultured; Colitis; Cytotoxins; Escherichia coli; Escherichia coli Infections; Gangliosides; Glycolipids; Hemolytic-Uremic Syndrome; Humans; Lymphocyte Activation; Receptors, Cell Surface; Shiga Toxin 1; Trihexosylceramides; Tumor Cells, Cultured

Infection with verotoxin producing Escherichia coli has been strongly implicated in the etiology of the hemolytic uremic syndrome (HUS). We have previously shown that this toxin specifically binds to a glycolipid receptor-globotriosyl ceramide (Gb3). We have therefore quantitated the level of this glycolipid by HPLC in human renal cortex and medulla as a function of age. We have also measured the binding of verotoxin to Gb3 isolated from each renal tissue sample. Gb3 was a major component of the glycolipid fraction of all renal samples analyzed. The levels were found to be higher in the cortex than medulla, correlating with the clinical incidence of renal lesions in HUS, but reduced in the kidneys of infants as compared to adults. Verotoxin binding was directly proportional to the renal Gb3 content. Thus, human renal tissue is a rich source of the verotoxin receptor glycolipid. However, changes in receptor concentration cannot explain the age-related incidence of HUS.

Topics: Aging; Bacterial Toxins; Chromatography, High Pressure Liquid; Cytotoxins; Escherichia coli Infections; Globosides; Glycolipids; Glycosphingolipids; Hemolytic-Uremic Syndrome; Humans; Kidney Cortex; Kidney Medulla; Receptors, Cell Surface; Receptors, Immunologic; Shiga Toxin 1; Trihexosylceramides