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Clifford A. Lingwood - One of the best experts on this subject based on the ideXlab platform.
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detergent resistant globotriaosyl ceramide may define Verotoxin glomeruli restricted hemolytic uremic syndrome pathology
Kidney International, 2009Co-Authors: Fahima Khan, Francois Proulx, Clifford A. LingwoodAbstract:Verotoxin binding to its receptor, globotriaosyl ceramide(Gb 3 ) mediates the glomerular pathology of hemolytic uremic syndrome, but Gb 3 is expressed in both tubular and glomerular cells. Gb 3 within detergent-resistant membranes, an index of glycolipid-cholesterol enriched lipid rafts, is required for in vitro cytotoxicity. We found that Verotoxin 1 and 2 binding to human adult renal glomeruli is detergent resistant, whereas the strong Verotoxin binding to renal tubules is detergent sensitive. Verotoxin binding to pediatric glomeruli was detergent resistant but binding to adult glomeruli was enhanced, remarkably for some samples, by detergent extraction. Detergent-sensitive glomerular components may provide age-related protection against Verotoxin glomerular binding. Mouse glomeruli remained Verotoxin unreactive after detergent extraction, whereas tubular binding was lost. Cholesterol extraction induced strong Verotoxin binding in poorly reactive adult glomeruli, suggesting cholesterol can mask Gb 3 in glomerular lipid rafts. Binding of the human immunodeficiency virus (HIV) adhesin, gp120 (another Gb 3 ligand) was detergent sensitive, tubule-restricted, and inhibited by Verotoxin B subunit pretreatment, and may relate to HIV nephropathy. Our study shows that differential membrane Gb 3 organization in glomeruli and tubules provides a basis for the age- and glomerular-restricted pathology of hemolytic uremic syndrome.
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differential intracellular transport and binding of Verotoxin 1 and Verotoxin 2 to globotriaosylceramide containing lipid assemblies
Journal of Cellular Physiology, 2008Co-Authors: Patty Tam, Radhia Mahfoud, Anita Nutikka, Aye Aye Khine, Beth Binnington, Paul Paroutis, Clifford A. LingwoodAbstract:Although Verotoxin-1 (VT1) and Verotoxin-2 (VT2) share a common receptor, globotriaosyl ceramide (Gb3), VT2 induces distinct animal pathology and is preferentially associated with human disease. Moreover VT2 cytotoxicity in vitro is less than VT1. We therefore investigated whether these toxins similarly traffic within cells via similar Gb3 assemblies. At 4°C, fluorescent-VT1 and VT2 bound both coincident and distinct punctate surface Gb3 microdomains. After 10 min at 37°C, similar distinct/coincident micropunctate intracellular localization was observed. Most internalized VT2, but not VT1, colocalized with transferrin. After 1 h, VT1 and VT2 coalesced during retrograde transport to the Golgi. During prolonged incubation (3–6 h), VT1, and VT2 (more slowly), exited the Golgi to reach the ER/nuclear envelope. At this time, VT2 induced a previously unreported, retrograde transport-dependent vacuolation. Cell surface and intracellular VT1 showed greater detergent resistance than VT2, suggesting differential ‘raft’ association. >90% 125I-VT1 cell surface bound, or added to detergent-resistant cell membrane extracts (DRM), was in the Gb3-containing sucrose gradient ‘insoluble’ fraction, whereas only 30% 125I-VT2 was similarly DRM-associated. VT1 bound more efficiently to Gb3/cholesterol DRMs generated in vitro. Only VT1 binding was inhibited by high cholesterol/Gb3 ratios. VT2 competed less effectively for 125I-VT1/Gb3 DRM-binding but only VT2-Gb3/cholesterol DRM-binding was augmented by sphingomyelin. Differential VT1/VT2 Gb3 raft-binding may mediate differential cell binding/intracellular trafficking and cytopathology. J. Cell. Physiol. 216: 750–763, 2008, © 2008 Wiley-Liss, Inc.
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membrane cytosolic translocation of Verotoxin a1 subunit in target cells
Microbiology, 2007Co-Authors: Patricia J Tam, Clifford A. LingwoodAbstract:In sensitive cells, Verotoxin 1 (VT1) utilizes a globotriaosylceramide receptor-dependent retrograde transport pathway from the cell surface to the Golgi/endoplasmic reticulum (ER). The VT1 A subunit (VTA) is an RNA glycanase. Although translocation of VTA from the ER to the cytosol is considered the route for protein synthesis inhibition, cell-based evidence is lacking. A dual-fluorescent-labelled VT1 holotoxin was constructed to simultaneously monitor VTA and VT1 B subunit (VTB) intracellular transport. By confocal microscopy, VTA/VTB subunits remained associated throughout the retrograde transport pathway without cytosolic staining. However, in [125I]VT1-treated cells, the selective cytosolic translocation (4 %) of the activated form of VTA, VTA1, was demonstrated for the first time by monitoring [125I]VTA1 release after plasma membrane permeabilization by streptolysin O (SLO). Lactacystin, a proteasome inhibitor, increased cytosolic VTA1 and enhanced VT1 cytotoxicity. VT1 ER arrival coincided with cytosolic VTA1 detection. Brefeldin A and 16 °C, conditions which inhibit VT1 retrograde transport to the Golgi/ER, prevented VTA1 cytosolic translocation; however, these treatments did not completely prevent VT1-induced protein synthesis inhibition. Thus, efficient cytosolic translocation of VTA1 requires transport to the Golgi/ER, but alternative minor escape pathways for protein synthesis inhibition may operate when transport to the Golgi/ER is prevented. Inhibition of protein synthesis was time and dose dependent, and not necessarily a valid index of subsequent cytopathology. Only protein synthesis inhibition following >3 h VT1 exposure correlated with eventual cell cytotoxicity. Extrapolation of translocated cytosolic VTA1 values indicates that about one molecule of translocated VTA1 per cell is sufficient to inhibit protein synthesis and kill a cell.
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a novel soluble analog of the hiv 1 fusion cofactor globotriaosylceramide gb3 eliminates the cholesterol requirement for high affinity gp120 gb3 interaction
Journal of Lipid Research, 2002Co-Authors: Radhia Mahfoud, Clifford A. Lingwood, Murugesapillai Mylvaganam, Jacques FantiniAbstract:We have analyzed the interaction of adamantyl Gb3 (adaGb3), a semi-synthetic soluble analog of Gb3, with HIV-1 surface envelope glycoprotein gp120. In this analog, which was orginally designed to inhibit Verotoxin binding to its glycolipid receptor, Gb3, the fatty acid chain is replaced with a rigid globular hydrocarbon frame (adamantane). Despite its solubility, adaGb3 forms monolayers at an air-water interface. Compression isotherms of such monolayers demonstrated that the adamantane substitution resulted in a larger minimum molecular area and a more rigid, less compressible film than Gb3. Insertion of gp120 into adaGb3 monolayers was exponential whereas the gp120/Gb3 interaction curve was sigmoidal with a lag phase of 40 min. Adding cholesterol into authentic Gb3 monolayers abrogated the lag phase and increased the initial rate of interaction with gp120. This effect of cholesterol was not observed with phosphatidylcholine or sphingomyelin. In addition, Verotoxin-bound adaGb3 or Gb3 plus cholesterol was recovered in fractions of comparable low density after ultracentrifugation through sucrose-density gradients in the presence of Triton X-100. The unique biological and physico-chemical properties of adaGb3 suggest that this analog may be a potent soluble mimic of Gb3, providing a novel concept for developing GSL-derived viral fusion inhibitors.
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effect of globotriaosyl ceramide fatty acid α hydroxylation on the binding by Verotoxin 1 and Verotoxin 2
Neurochemical Research, 2002Co-Authors: Beth Binnington, Anita Nutikka, Daniel Lingwood, Clifford A. LingwoodAbstract:Variation in the lipid moiety of the Verotoxin (VT) receptor glycosphingolipid, globotriaosyl ceramide (Gb3) can modulate toxin binding. The binding of VT1 and VT2 to C18 and C22 ahydroxy and nonhydroxy fatty acid isoforms of Gb3 were compared using a receptor ELISA and a 125l-labeled toxin/glycolipid microtitre plate direct binding assay. Increased binding to the hydroxylated species, particularly C22OH, was observed for both toxins. Increased RELISA binding at low glycolipid concentrations only, suggested the binding affinity is increased following Gb3 fatty acid hydroxylation. Nonlinear regression analysis of direct binding assay to these Gb3 isoforms confirmed the increased affinity of both toxins for the C22 hydroxylated Gb3. The capacity was also significantly increased. The increased binding of VTs for hydroxylated fatty acid Gb3 isoforms may be a factor in the selective renal pathology which can follow systemic verotoxemia, particularly in the mouse model. The more pronounced effect at lower glycolipid concentrations prompted investigation of VT1 binding affinity at different Gb3 concentrations. Unexpectedly, the VT1 Kd for Gb3 was found to decrease as an inverse function of the Gb3 concentration. This shows that glycolipids have “nonclassical” receptor properties.
Douglas L Marshall - One of the best experts on this subject based on the ideXlab platform.
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effect of trisodium phosphate adaptation on changes in membrane lipid composition Verotoxin secretion and acid resistance of escherichia coli o157 h7 in simulated gastric fluid
International Journal of Food Microbiology, 2006Co-Authors: Douglas L MarshallAbstract:Abstract Escherichia coli O157:H7 (HEC), E. coli O157:H7 rpoS mutant (HEC-RM), and nonpathogenic E. coli (NPEC) were step-wise adapted to trisodium phosphate (TSP) by incubation in broths of increasing concentration, from 0% to 0.6%, at 37 °C for 24 h. After incubation at each concentration, each population was examined for acid resistance (D value) in simulated gastric fluid of pH 1.5, cell envelope membrane lipid composition, and intracellular and extracellular Verotoxin concentrations. The ratio of cis-vaccenic acid (18:1ω7c) to palmitic acid (16:0) increased, indicating increased membrane fluidity with increasing TSP concentration up to 0.4%, but decreased at 0.6%. HEC and HEC-RM adapted at 0.4% TSP had the highest Verotoxin concentrations of 1805 and 1879 ng/ml, respectively. In addition, with HEC the ratio of extracellular to intracellular Verotoxin concentration decreased at higher TSP concentrations. In contrast, the ratio for HEC-RM increased at 0.4% TSP. HEC adapted to 0.4% TSP had the greatest survival in gastric fluid (58 min D value) among all treatments. For HEC, the increase in membrane fluidity was associated with increased acid resistance and extracellular Verotoxin concentration for cells adapted to 0.4% TSP. In contrast, the increase in membrane fluidity was associated with decreased acid resistance of TSP adapted HEC-RM although the extracellular Verotoxin concentration increased. Therefore, the deletion of the rpoS gene appeared to affect the changes in Verotoxin concentration and acid resistance of TSP adapted E. coli O157:H7.
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influence of acetic citric and lactic acids on escherichia coli o157 h7 membrane lipid composition Verotoxin secretion and acid resistance in simulated gastric fluid
Journal of Food Protection, 2005Co-Authors: Hyungyun Yuk, Douglas L MarshallAbstract:The effect of organic acid (acetic, citric, and lactic acids) adaptation at equivalent initial pH values (6.4 and 5.4) on changes in membrane lipid composition, Verotoxin concentration, and acid resistance in simulated gastric fluid (pH 1.5, 37°C) was determined for Escherichia coli O157:H7 ATCC 43895 (HEC) and an rpoS mutant of E. coli O157:H7 ATCC 43895 (RM, FRIK 816-3). For HEC, lactic acid–adapted (pH 5.4) cells had the greatest D-value (32.2 min) and acetic acid–adapted (pH 5.4) cells had the smallest D-value (16.6 min) in simulated gastric fluid. For RM, D-values of citric and acetic acid–adapted cells were similar to those for nonadapted cells grown at pH 7.3, but D-values increased from 13.1 to 27.9 min in lactic acid–adapted cells (from pH 7.3 to pH 5.4). For both strains, the ratio of cis-vaccenic to palmitic acids decreased for citric and lactic acid–adapted cells, but the ratio increased for acetic acid–adapted cells at pH 5.4. Organic acid–adapted cells produced less total Verotoxin than did ...
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adaptation of escherichia coli o157 h7 to ph alters membrane lipid composition Verotoxin secretion and resistance to simulated gastric fluid acid
Applied and Environmental Microbiology, 2004Co-Authors: Hyungyun Yuk, Douglas L MarshallAbstract:The influence of adaptation to pH (from pH 5.0 to 9.0) on membrane lipid composition, Verotoxin concentration, and resistance to acidic conditions in simulated gastric fluid (SGF) (pH 1.5, 37 degrees C) was determined for Escherichia coli O157:H7 (HEC, ATCC 43895), an rpoS-deficient mutant of ATCC 43895 (HEC-RM, FRIK 816-3), and nonpathogenic E. coli (NPEC, ATCC 25922). Regardless of the strain, D values (in SGF) of acid-adapted cells were higher than those of non-acid-adapted cells, with HEC adapted at pH 5.0 having the greatest D value, i.e., 25.6 min. Acid adaptation increased the amounts of palmitic acid (C16:0) and decreased cis-vaccenic acid (C18:1 omega 7c) in the membrane lipids of all strains. The ratio of cis-vaccenic acid to palmitic acid increased at acidic pH, causing a decrease in membrane fluidity. HEC adapted to pH 8.3 and HEC-RM adapted to pH 7.3 exhibited the greatest Verotoxin concentrations (2,470 and 1,460 ng/ml, respectively) at approximately 10(8) CFU/ml. In addition, the ratio of extracellular to intracellular Verotoxin concentration decreased at acidic pH, possibly due to the decrease of membrane fluidity. These results suggest that while the rpoS gene does not influence acid resistance in acid-adapted cells it does confer decreased membrane fluidity, which may increase acid resistance and decrease Verotoxin secretion.
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heat adaptation alters escherichia coli o157 h7 membrane lipid composition and Verotoxin production
Applied and Environmental Microbiology, 2003Co-Authors: Hyungyun Yuk, Douglas L MarshallAbstract:The influence of heat adaptation (growth at 42 and 45°C) on changes in membrane lipid composition and Verotoxin concentration of Escherichia coli O157:H7 (ATCC 43895), an rpoS mutant of ATCC 43895 (FRIK 816-3), a Verotoxin mutant E. coli O157:H7 (B6-914), and nonpathogenic E. coli (ATCC 25922) was investigated. D values (57°C) of heat-adapted cells were up to 3.9 min longer than those of control cells for all four strains. Heat adaptation increased the amounts of palmitic acid (16:0) and cis-vaccenic acid (18:1ω7c) in membrane lipids of ATCC 43895 and the rpoS mutant, whereas there was a reduction and no change in the amount of cis-vaccenic acid in nonpathogenic and Verotoxin mutant E. coli, respectively. The ratio of palmitic to cis-vaccenic acids decreased in ATCC 43895 and in the rpoS mutant, whereas the ratio increased in nonpathogenic E. coli and was not different in the Verotoxin mutant with elevated growth temperature. Total Verotoxin concentration decreased due to a reduction in intracellular Verotoxin amount in heat-adapted ATCC 43895 and rpoS mutant strains. However, extracellular Verotoxin concentration increased in heat-adapted cells. The rpoS gene did not influence membrane lipid composition changes although it did affect heat resistance. Results suggest that increased membrane fluidity may have caused increased Verotoxin secretion.
Hyungyun Yuk - One of the best experts on this subject based on the ideXlab platform.
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influence of acetic citric and lactic acids on escherichia coli o157 h7 membrane lipid composition Verotoxin secretion and acid resistance in simulated gastric fluid
Journal of Food Protection, 2005Co-Authors: Hyungyun Yuk, Douglas L MarshallAbstract:The effect of organic acid (acetic, citric, and lactic acids) adaptation at equivalent initial pH values (6.4 and 5.4) on changes in membrane lipid composition, Verotoxin concentration, and acid resistance in simulated gastric fluid (pH 1.5, 37°C) was determined for Escherichia coli O157:H7 ATCC 43895 (HEC) and an rpoS mutant of E. coli O157:H7 ATCC 43895 (RM, FRIK 816-3). For HEC, lactic acid–adapted (pH 5.4) cells had the greatest D-value (32.2 min) and acetic acid–adapted (pH 5.4) cells had the smallest D-value (16.6 min) in simulated gastric fluid. For RM, D-values of citric and acetic acid–adapted cells were similar to those for nonadapted cells grown at pH 7.3, but D-values increased from 13.1 to 27.9 min in lactic acid–adapted cells (from pH 7.3 to pH 5.4). For both strains, the ratio of cis-vaccenic to palmitic acids decreased for citric and lactic acid–adapted cells, but the ratio increased for acetic acid–adapted cells at pH 5.4. Organic acid–adapted cells produced less total Verotoxin than did ...
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adaptation of escherichia coli o157 h7 to ph alters membrane lipid composition Verotoxin secretion and resistance to simulated gastric fluid acid
Applied and Environmental Microbiology, 2004Co-Authors: Hyungyun Yuk, Douglas L MarshallAbstract:The influence of adaptation to pH (from pH 5.0 to 9.0) on membrane lipid composition, Verotoxin concentration, and resistance to acidic conditions in simulated gastric fluid (SGF) (pH 1.5, 37 degrees C) was determined for Escherichia coli O157:H7 (HEC, ATCC 43895), an rpoS-deficient mutant of ATCC 43895 (HEC-RM, FRIK 816-3), and nonpathogenic E. coli (NPEC, ATCC 25922). Regardless of the strain, D values (in SGF) of acid-adapted cells were higher than those of non-acid-adapted cells, with HEC adapted at pH 5.0 having the greatest D value, i.e., 25.6 min. Acid adaptation increased the amounts of palmitic acid (C16:0) and decreased cis-vaccenic acid (C18:1 omega 7c) in the membrane lipids of all strains. The ratio of cis-vaccenic acid to palmitic acid increased at acidic pH, causing a decrease in membrane fluidity. HEC adapted to pH 8.3 and HEC-RM adapted to pH 7.3 exhibited the greatest Verotoxin concentrations (2,470 and 1,460 ng/ml, respectively) at approximately 10(8) CFU/ml. In addition, the ratio of extracellular to intracellular Verotoxin concentration decreased at acidic pH, possibly due to the decrease of membrane fluidity. These results suggest that while the rpoS gene does not influence acid resistance in acid-adapted cells it does confer decreased membrane fluidity, which may increase acid resistance and decrease Verotoxin secretion.
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heat adaptation alters escherichia coli o157 h7 membrane lipid composition and Verotoxin production
Applied and Environmental Microbiology, 2003Co-Authors: Hyungyun Yuk, Douglas L MarshallAbstract:The influence of heat adaptation (growth at 42 and 45°C) on changes in membrane lipid composition and Verotoxin concentration of Escherichia coli O157:H7 (ATCC 43895), an rpoS mutant of ATCC 43895 (FRIK 816-3), a Verotoxin mutant E. coli O157:H7 (B6-914), and nonpathogenic E. coli (ATCC 25922) was investigated. D values (57°C) of heat-adapted cells were up to 3.9 min longer than those of control cells for all four strains. Heat adaptation increased the amounts of palmitic acid (16:0) and cis-vaccenic acid (18:1ω7c) in membrane lipids of ATCC 43895 and the rpoS mutant, whereas there was a reduction and no change in the amount of cis-vaccenic acid in nonpathogenic and Verotoxin mutant E. coli, respectively. The ratio of palmitic to cis-vaccenic acids decreased in ATCC 43895 and in the rpoS mutant, whereas the ratio increased in nonpathogenic E. coli and was not different in the Verotoxin mutant with elevated growth temperature. Total Verotoxin concentration decreased due to a reduction in intracellular Verotoxin amount in heat-adapted ATCC 43895 and rpoS mutant strains. However, extracellular Verotoxin concentration increased in heat-adapted cells. The rpoS gene did not influence membrane lipid composition changes although it did affect heat resistance. Results suggest that increased membrane fluidity may have caused increased Verotoxin secretion.
Michael P Doyle - One of the best experts on this subject based on the ideXlab platform.
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fate of enterohemorrhagic escherichia coli o157 h7 in bovine feces
Applied and Environmental Microbiology, 1996Co-Authors: G Wang, T Zhao, Michael P DoyleAbstract:Dairy cattle have been identified as a principal reservoir of Escherichia coli O157:H7. The fate of this pathogen in bovine feces at 5, 22, and 37 degrees C was determined. Two levels of inocula (10(3) and 10(5) CFU/g) of a mixture of five nalidixic acid-resistant E. coli O157:H7 strains were used. E. coli O157:H7 survived at 37 degrees C for 42 and 49 days with low and high inocula, respectively, and at 22 degrees C for 49 and 56 days with low and high inocula, respectively. Fecal samples at both temperatures had low moisture contents (about 10%) and water activities ( < 0.5) near the end of the study. E. coli O157:H7 at 5 degrees C survived for 63 to 70 days, with the moisture content (74%) of feces remaining high through the study. Chromosomal DNA fingerprinting of E. coli O157:H7 isolates surviving near the completion of the study revealed that the human isolate strain 932 was the only surviving strain at 22 or 37 degrees C. All five strains were isolated near the end of incubation from feces held at 5 degrees C. Isolates at each temperature were still capable of producing both Verotoxin 1 and Verotoxin 2. Results indicate that E. coli O157:H7 can survive in feces for a long period of time and retain its ability to produce Verotoxins. Hence, bovine feces are a potential vehicle for transmitting E. coli O157:H7 to cattle, food, and the environment. Appropriate handling of bovine feces is important to control the spread of this pathogen.
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Verotoxin glycolipid receptors determine the localization of microangiopathic process in rabbits given Verotoxin 1
Journal of Laboratory and Clinical Medicine, 1992Co-Authors: Carla Zoja, Michael P Doyle, Daniela Corna, Claudio Farina, Giuseppe Sacchi, Clifford Lingwood, Vikas V Padhye, Mauro Abbate, Giuseppe RemuzziAbstract:Infection with Verotoxin-producing Escherichia coli has been implicated in the cause of hemolytic-uremic syndrome. Cases of thrombotic thrombocytopenic purpura and Verotoxin infections have been also described. In this study we sought to determine the following: (1) whether Verotoxin induces microvascular lesions in the rabbit, (2) the organ distribution of such lesions, and (3) the distribution of Verotoxin glycolipid receptors in the various organs. Rabbits challenged with Verotoxin-1 purified from E. coli O157:H7 had anorexia, lethargia, and limb paralysis; renal function, however, was normal. Central nervous system lesions found included pericellular and perivascular edema, focal hemorrhage, vascular lesions, and severe alterations of Purkinje cells. Histologic changes were also seen in the colon, with mucosal and submucosal edema and hemorrhage, and in the lung, with interstitial fibrosis and focal lymphohistiocitic infiltration. No lesions were detected in kidney, heart, liver, and spleen. Screening of various tissues for the presence of the Verotoxin receptors revealed galabiosyl ceramide in the central nervous system and globotriosyl ceramide in the gastrointestinal tract, lung, and spleen. No receptors for Verotoxin were found in the heart, liver, and kidney. These results indicate that organ localization of the disease in rabbits is dependent on the distribution of Verotoxin receptors.
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detection and production of Verotoxin 1 of escherichia coli o157 h7 in food
Applied and Environmental Microbiology, 1991Co-Authors: R D Weeratna, Michael P DoyleAbstract:Verotoxin 1 (VT1) is a recognized virulence factor of Escherichia coli O157:H7, a cause of severe food-borne disease. The public health significance of preformed Verotoxin in food is unknown, and relatively little research has been done to determine the production of VT1 in food. The purposes of this study were to develop a sensitive method to detect VT1 in milk and in ground beef and to determine the conditions for VT1 production in these foods. A sandwich enzyme-linked immunosorbent assay in which we used VT1-specific monoclonal antibody 9C9F5 as the capture antibody and a rabbit polyclonal antibody raised against VT2 as the detection antibody was developed for the detection and quantification of VT1 in milk and in ground beef. The enzyme-linked immunosorbent assay was sensitive to a minimum of 0.5 ng of VT1 per ml of milk and 1.0 ng of VT1 per g of ground beef. The greatest amount of VT1 detected in milk (306 ng/ml) was detected in samples that were incubated at 37 degrees C with agitation (160 rpm) for 48 h. Very little toxin (1 ng/ml) was produced at 25 or 30 degrees C within 96 h. VT1 production was greater in ground beef than in milk; 452 ng of VT1 per g was produced in beef at 37 degrees C in 48 h. Relatively little VT1 was produced in beef within 96 h at 25 and 30 degrees C (2.1 and 9.8 ng of VT1 per g, respectively). Our results indicate that ground beef is a better medium for VT1 production than milk.(ABSTRACT TRUNCATED AT 250 WORDS)
Philip M Sherman - One of the best experts on this subject based on the ideXlab platform.
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Multiple seropathotypes of Verotoxin-producing Escherichia coli (VTEC) disrupt interferon-γ-induced tyrosine phosphorylation of signal transducer and activator of transcription (Stat)-1
Microbial Pathogenesis, 2007Co-Authors: Narveen Jandu, Songhai Shen, Mark E Wickham, Rohit Prajapati, Brett B Finlay, Mohamed A Karmali, Philip M ShermanAbstract:Abstract Verotoxin-producing Escherichia coli (VTEC) O157:H7 inhibits interferon-γ-stimulated tyrosine phosphorylation of signal transducer and activator of transcription (Stat)-1 in epithelial cells, independent of Verotoxins and the locus of enterocyte effacement pathogenicity island. Although E. coli O157:H7 is the major cause of disease in humans, non-O157:H7 VTEC also cause human disease. However, the virulence properties of non-O157:H7 VTEC are less well characterized. The aims of this study were to define the ability of VTEC strains of differing seropathotypes (classified as A–E) to inhibit interferon-γ stimulated Stat1-phosphorylation and to further characterize the bacterial-derived inhibitory factor. Confluent T84 and HEp-2 cells were infected with VTEC strains (MOI 100:1, 6 h, 37 °C), and then stimulated with interferon-γ (50 ng/mL) for 0.5 h at 37 °C. Whole-cell protein extracts of infected cells were collected and prepared for immunoblotting to detect tyrosine phosphorylation of Stat1. The effects of E. coli O55 strains, the evolutionary precursors of VTEC, on Stat1-tyrosine phosphorylation were also determined. The effects of isogenic mutants of O-islands 47 and 122 were tested to determine the role of genes encoded on these putative pathogenicity islands in mediating VTEC inhibition of the interferon-γ-Stat1 signaling cascade. To evaluate potential mechanism(s) of inhibition, VTEC O157:H7-infected cells were treated with pharmacological inhibitors, including, wortmannin and LY294002. Relative to uninfected cells, Stat1-tyrosine phosphorylation was significantly reduced after 6 h infection of both T84 and HEp-2 cells by VTEC strains of all five seropathotypes. E. coli O55 strains, but not enteropathogenic E. coli (EPEC), also caused inhibition of Stat1-tyrosine phosphorylation, suggesting that this effect was acquired early in the evolution of VTEC. Stat1-activation did not recover in epithelial cells infected with isogenic mutants of O-islands 47 and 122, indicating that the inhibitory factor was not contained in these genomic regions. Stat1-phosphorylation remained intact when VTEC-infected cells were treated with wortmannin (0–100 nM), but not by treatment with the more specific PI3-kinase inhibitor, LY294002. Inhibition of interferon-γ stimulated Stat1-tyrosine phosphorylation by VTEC of multiple seropathotypes indicates the presence of a common inhibitory factor that is independent of bacterial virulence in humans. The results of treatment with wortmannin suggest that the bacterial-derived inhibitory factor employs host cell signal transduction to mediate inhibition of Stat1-activation.
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Translocation of Verotoxin-1 across T84 monolayers: mechanism of bacterial toxin penetration of epithelium
American Journal of Physiology-gastrointestinal and Liver Physiology, 1997Co-Authors: Dana J. Philpott, Mary H Perdue, Cameron A. Ackerley, Amanda J Kiliaan, Mohamed A Karmali, Philip M ShermanAbstract:Verotoxin-producing Escherichia coli (VTEC) are pathogenic bacteria associated with diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome. Verotoxins (VTs) elaborated by these organisms prod...
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Multiple determinants of Verotoxin-producing Escherichia coli O157:H7 attachment-effacement.
Infection and Immunity, 1993Co-Authors: Marlene Dytoc, Rohini Soni, F Cockerill, J. C. S. De Azavedo, James Brunton, M Louie, Philip M ShermanAbstract:Abstract Verotoxin-producing Escherichia coli strains of the serotype O157:H7 belong to a class of gastrointestinal pathogens that adhere to epithelial cells in a characteristic pattern known as attaching and effacing. Recent insight into the nature of E. coli O157:H7 adhesion was provided by the cloning and sequencing of the chromosomal eaeA (for E. coli attaching and effacing) gene homolog (G. Beebakhee, M. Louie, J. De Azavedo, and J. Brunton, FEMS Microbiol. Lett. 91:63-68, 1992, and J. Yu and J. B. Kaper, Mol. Microbiol. 6:411-417, 1992) and isolation of a 60-MDa plasmid referred to as pO157 (I. Toth, M. L. Cohen, H. S. Rumschlag, L. W. Riley, E. H. White, J. H. Carr, W. W. Bond, and I. K. Wachsmuth, Infect. Immun. 58:1223-1231, 1990, and S. Tzipori, H. Karch, K. I. Wachsmuth, R. M. Robins-Browne, A. D. O'Brien, H. Lior, M. L. Cohen, J. Smithers, and M. M. Levine, Infect. Immun. 55:3117-3125, 1987) and an approximately 94-kDa outer membrane protein (94-kDa OMP; P. Sherman, F. Cockerill III, R. Soni, and J. Brunton, Infect. Immun. 59:890-899, 1991). In this study, we examined the gene products of both eaeA and pO157 in relation to the 94-kDa OMP and as candidate effectors for O157:H7 attachment-effacement. Peptide sequencing and immunoassay demonstrated that the C. coli O157:H7 eaeA gene product is distinct from the 94-kDa OMP. Using ultrastructural analyses, we found that both parent and pO157 plasmid-cured O157:H7 strains demonstrated attaching and effacing adhesion to host epithelial cells and reacted equally well to rabbit antiserum raised against the 94-kDa OMP. By both transmission electron microscopy and light microscopy, E. coli HB101 transformed separately with the cloned eaeA gene and the pO157 plasmid did not form attaching and effacing lesions on cultured epithelial cells in vitro and rabbit intestinal tissues in vivo. Since additional determinants may mediate the attaching and effacing phenotype, we examined transposon TnphoA mutants constructed from E. coli O157:H7 strain CL8. Two TnphoA mutants were found deficient in bacterial factors that are necessary for O157:H7 attachment-effacement and likely distinct from the eaeA gene product.
