scholarly journals Oral Administration of Formaldehyde-Killed Recombinant Bacteria Expressing a Mimic of the Shiga Toxin Receptor Protects Mice from Fatal Challenge with Shiga-ToxigenicEscherichia coli

2001 ◽  
Vol 69 (3) ◽  
pp. 1389-1393 ◽  
Author(s):  
James C. Paton ◽  
Trisha J. Rogers ◽  
Renato Morona ◽  
Adrienne W. Paton
Keyword(s):  
Oral Administration ◽  
Shiga Toxin ◽  
High Efficiency ◽  
Gastrointestinal Disease ◽  
Protective Capacity ◽  
Recombinant Bacteria ◽  
Recombinant Bacterium ◽  
Life Threatening ◽  
Toxin Receptor ◽  
Uremic Syndrome

ABSTRACT Gastrointestinal disease caused by Shiga toxin-producingEscherichia 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.

scholarly journals Neutralization of Shiga Toxins Stx1, Stx2c, and Stx2e by Recombinant Bacteria Expressing Mimics of Globotriose and Globotetraose

2001 ◽  
Vol 69 (3) ◽  
pp. 1967-1970 ◽  
Author(s):  
Adrienne W. Paton ◽  
Renato Morona ◽  
James C. Paton
Keyword(s):  
High Efficiency ◽  
Gastrointestinal Disease ◽  
Recombinant Bacteria ◽  
Shiga Toxins ◽  
Edema Disease ◽  
E Coli ◽  
Recombinant Bacterium ◽  
Genes Encoding ◽  
Uremic Syndrome

ABSTRACT Strains of Escherichia coli producing Shiga toxins Stx1, Stx2, Stx2c, and Stx2d cause gastrointestinal disease and the hemolytic-uremic syndrome in humans. We have recently constructed a recombinant bacterium which displays globotriose (the receptor for these toxins) on its surface and adsorbs and neutralizes these Shiga toxins with very high efficiency. This agent has great potential for the treatment of humans with such infections. E. colistrains which cause edema disease in pigs produce a variant toxin, Stx2e, which has a different receptor specificity from that for the other members of the Stx family. We have now modified the globotriose-expressing bacterium such that it expresses globotetraose (the preferred receptor for Stx2e) by introducing additional genes encoding a N-acetylgalactosamine transferase and a UDP-N-acetylgalactosamine-4-epimerase. This bacterium had a reduced capacity to neutralize Stx1 and Stx2c in vitro, but remarkably, its capacity to bind Stx2e was similar to that of the globotriose-expressing construct; both constructs neutralized 98.4% of the cytotoxicity in lysates of E. coli JM109 expressing cloned stx 2e. These data suggest that either globotriose- or globotetraose-expressing constructs may be suitable for treatment and/or prevention of edema disease in pigs.

scholarly journals Pathogenesis and Diagnosis of Shiga Toxin-Producing Escherichia coli Infections

1998 ◽  
Vol 11 (3) ◽  
pp. 450-479 ◽  
Author(s):  
James C. Paton ◽  
Adrienne W. Paton
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Gastrointestinal Disease ◽  
Diagnostic Procedures ◽  
Host Cells ◽  
Food Borne ◽  
Large Numbers ◽  
Life Threatening ◽  
Uremic Syndrome ◽  
People Development

SUMMARY Since their initial recognition 20 years ago, Shiga toxin-producing Escherichia coli (STEC) strains have emerged as an important cause of serious human gastrointestinal disease, which may result in life-threatening complications such as hemolytic-uremic syndrome. Food-borne outbreaks of STEC disease appear to be increasing and, when mass-produced and mass-distributed foods are concerned, can involve large numbers of people. Development of therapeutic and preventative strategies to combat STEC disease requires a thorough understanding of the mechanisms by which STEC organisms colonize the human intestinal tract and cause local and systemic pathological changes. While our knowledge remains incomplete, recent studies have improved our understanding of these processes, particularly the complex interaction between Shiga toxins and host cells, which is central to the pathogenesis of STEC disease. In addition, several putative accessory virulence factors have been identified and partly characterized. The capacity to limit the scale and severity of STEC disease is also dependent upon rapid and sensitive diagnostic procedures for analysis of human samples and suspect vehicles. The increased application of advanced molecular technologies in clinical laboratories has significantly improved our capacity to diagnose STEC infection early in the course of disease and to detect low levels of environmental contamination. This, in turn, has created a potential window of opportunity for future therapeutic intervention.

scholarly journals Human Intestinal Tissue and Cultured Colonic Cells Contain Globotriaosylceramide Synthase mRNA and the Alternate Shiga Toxin Receptor Globotetraosylceramide

2010 ◽  
Vol 78 (11) ◽  
pp. 4488-4499 ◽  
Author(s):  
Steven D. Zumbrun ◽  
Leanne Hanson ◽  
James F. Sinclair ◽  
James Freedy ◽  
Angela R. Melton-Celsa ◽  
...  
Keyword(s):  
Shiga Toxin ◽  
Epithelial Cell Line ◽  
Intestinal Epithelial ◽  
Fresh Tissue ◽  
Tissue Sections ◽  
Human Intestinal Epithelial Cells ◽  
Life Threatening ◽  
Toxin Receptor ◽  
Uremic Syndrome ◽  
Colonic Cells

ABSTRACT Escherichia coli O157:H7 and other Shiga toxin (Stx)-producing E. coli (STEC) bacteria are not enteroinvasive but can cause hemorrhagic colitis. In some STEC-infected individuals, a life-threatening sequela of infection called the hemolytic uremic syndrome may develop that can lead to kidney failure. This syndrome is linked to the production of Stx by the infecting organism. For Stx to reach the kidney, the toxin must first penetrate the colonic epithelial barrier. However, the Stx receptor, globotriaosylceramide (Gb3), has been thought to be absent from human intestinal epithelial cells. Thus, the mechanisms by which the toxin associates with and traverses through the intestine en route to the kidneys have been puzzling aspects of STEC pathogenesis. In this study, we initially determined that both types of Stx made by STEC, Stx1 and Stx2, do in fact bind to colonic epithelia in fresh tissue sections and to a colonic epithelial cell line (HCT-8). We also discovered that globotetraosylceramide (Gb4), a lower-affinity toxin receptor derived from Gb3, is readily detectable on the surfaces of human colonic tissue sections and HCT-8 cells. Furthermore, we found that Gb3 is present on a fraction of HCT-8 cells, where it presumably functions to bind and internalize Stx1 and Stx2. In addition, we established by quantitative real-time PCR (qRT-PCR) that both fresh colonic epithelial sections and HCT-8 cells express Gb3 synthase mRNA. Taken together, our data suggest that Gb3 may be present in small quantities in human colonic epithelia, where it may compete for Stx binding with the more abundantly expressed glycosphingolipid Gb4.

scholarly journals Induction by Sphingomyelinase of Shiga Toxin Receptor and Shiga Toxin 2 Sensitivity in Human Microvascular Endothelial Cells

2003 ◽  
Vol 71 (2) ◽  
pp. 845-849 ◽  
Author(s):  
T. G. Obrig ◽  
R. M. Seaner ◽  
M. Bentz ◽  
C. A. Lingwood ◽  
B. Boyd ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Shiga Toxin ◽  
Microvascular Endothelial Cells ◽  
Cytotoxic Action ◽  
Ceramide Level ◽  
Shiga Toxins ◽  
Shiga Toxin 2 ◽  
Toxin Receptor ◽  
Uremic Syndrome ◽  
Microvascular Endothelial

ABSTRACT Shiga toxin-producing enterohemorrhagic Escherichia coli is the major cause of acute renal failure in young children. The interaction of Shiga toxins 1 and 2 (Stx1 and Stx2) with endothelial cells is an important step in the renal coagulation and thrombosis observed in hemolytic uremic syndrome. Previous studies have shown that bacterial lipopolysaccharide and host cytokines slowly sensitize endothelial cells to Shiga toxins. In the present study, bacterial neutral sphingomyelinase (SMase) rapidly (1 h) sensitized human dermal microvascular endothelial cells (HDMEC) to the cytotoxic action of Stx2. Exposure of endothelial cells to neutral SMase (0.067 U/ml) caused a rapid increase of intracellular ceramide that persisted for hours. Closely following the change in ceramide level was an increase in the expression of globotriaosylceramide (Gb3), the receptor for Stx2. A rapid increase was also observed in the mRNA for ceramide:glucosyltransferase (CGT), the first of three glycosyltransferase enzymes of the Gb3 biosynthetic pathway. The product of CGT (glucosylceramide) was also increased. In contrast, mRNA for the third enzyme of the pathway, Gb3 synthase, was constitutively produced and was not influenced by SMase treatment of HDMEC. These results describe a rapid response mechanism by which extracellular neutral SMase derived from either bacteria or eukaryotic cells may signal endothelial cells to become sensitive to Shiga toxins.

scholarly journals Sab, a Novel Autotransporter of Locus of Enterocyte Effacement-Negative Shiga-Toxigenic Escherichia coli O113:H21, Contributes to Adherence and Biofilm Formation

2009 ◽  
Vol 77 (8) ◽  
pp. 3234-3243 ◽  
Author(s):  
Sylvia Herold ◽  
James C. Paton ◽  
Adrienne W. Paton
Keyword(s):  
Escherichia Coli ◽  
Biofilm Formation ◽  
Gastrointestinal Disease ◽  
Pcr Analysis ◽  
Systemic Complications ◽  
E Coli ◽  
Acid Protein ◽  
Life Threatening ◽  
Uremic Syndrome ◽  
Enterocyte Effacement

ABSTRACT Shiga-toxigenic Escherichia coli (STEC) strains cause serious gastrointestinal disease, which can lead to potentially life-threatening systemic complications such as hemolytic-uremic syndrome. Although the production of Shiga toxin has been considered to be the main virulence trait of STEC for many years, the capacity to colonize the host intestinal epithelium is a crucial step in pathogenesis. In this study, we have characterized a novel megaplasmid-encoded outer membrane protein in locus of enterocyte effacement (LEE)-negative O113:H21 STEC strain 98NK2, termed Sab (for STEC autotransporter [AT] contributing to biofilm formation). The 4,296-bp sab gene encodes a 1,431-amino-acid protein with the features of members of the AT protein family. When expressed in E. coli JM109, Sab contributed to the diffuse adherence to human epithelial (HEp-2) cells and promoted biofilm formation on polystyrene surfaces. A 98NK2 sab deletion mutant was also defective in biofilm formation relative to its otherwise isogenic wild-type parent, and this was complemented by transformation with a sab-carrying plasmid. Interestingly, an unrelated O113:H21 STEC isolate that had a naturally occurring deletion in sab was similarly defective in biofilm formation. PCR analysis indicated that sab is present in LEE-negative STEC strains belonging to serotypes/groups O113:H21, O23, and O82:H8. These findings raise the possibility that Sab may contribute to colonization in a subset of LEE-negative STEC strains.

scholarly journals Upregulation of Shiga Toxin Receptor CD77/Gb3 and Interleukin-1β Expression in the Brain of EHEC Patients with Hemolytic Uremic Syndrome and Neurologic Symptoms

2014 ◽  
Vol 25 (2) ◽  
pp. 146-156 ◽  
Author(s):  
Christian Hagel ◽  
Susanne Krasemann ◽  
Judith Löffler ◽  
Klaus Püschel ◽  
Tim Magnus ◽  
...  
Keyword(s):  
Hemolytic Uremic Syndrome ◽  
Shiga Toxin ◽  
Interleukin 1Β ◽  
Neurologic Symptoms ◽  
Toxin Receptor ◽  
Uremic Syndrome ◽  
The Brain

scholarly journals A Novel Tail-Associated O91-Specific Polysaccharide Depolymerase from a Podophage Reveals Lytic Efficacy of Shiga Toxin-Producing Escherichia coli

2020 ◽  
Vol 86 (9) ◽  
Author(s):  
Yibao Chen ◽  
Xiangmin Li ◽  
Shuang Wang ◽  
Lingyu Guan ◽  
Xinxin Li ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Hemolytic Uremic Syndrome ◽  
Proinflammatory Cytokines ◽  
Shiga Toxin ◽  
Hemorrhagic Colitis ◽  
Content Type ◽  
Increased Risk ◽  
Specific Polysaccharide ◽  
Life Threatening ◽  
Uremic Syndrome

ABSTRACT Shiga toxin-producing Escherichia coli (STEC) strains are important zoonotic foodborne pathogens, causing diarrhea, hemorrhagic colitis, and life-threatening hemolytic uremic syndrome (HUS) in humans. However, antibiotic treatment of STEC infection is associated with an increased risk of HUS. Therefore, there is an urgent need for early and effective therapeutic strategies. Here, we isolated lytic T7-like STEC phage PHB19 and identified a novel O91-specific polysaccharide depolymerase (Dep6) in the C terminus of the PHB19 tailspike protein. Dep6 exhibited strong hydrolase activity across wide ranges of pH (pH 4 to 8) and temperature (20 to 60°C) and degraded polysaccharides on the surface of STEC strain HB10. In addition, both Dep6 and PHB19 degraded biofilms formed by STEC strain HB10. In a mouse STEC infection model, delayed Dep6 treatment (3 h postinfection) resulted in only 33% survival, compared with 83% survival when mice were treated simultaneously with infection. In comparison, pretreatment with Dep6 led to 100% survival compared with that of the control group. Surprisingly, a single PHB19 treatment resulted in 100% survival in all three treatment protocols. Moreover, a significant reduction in the levels of proinflammatory cytokines was observed at 24 h postinfection in Dep6- or PHB19-treated mice. These results demonstrated that Dep6 or PHB19 might be used as a potential therapeutic agent to prevent STEC infection. IMPORTANCE Shiga toxin-producing Escherichia coli (STEC) is an important foodborne pathogen worldwide. The Shiga-like toxin causes diarrhea, hemorrhagic colitis, and life-threatening hemolytic uremic syndrome (HUS) in humans. Although antibiotic therapy is still used for STEC infections, this approach may increase the risk of HUS. Phages or phage-derived depolymerases have been used to treat bacterial infections in animals and humans, as in the case of the “San Diego patient” treated with a phage cocktail. Here, we showed that phage PHB19 and its O91-specific polysaccharide depolymerase Dep6 degraded STEC biofilms and stripped the lipopolysaccharide (LPS) from STEC strain HB10, which was subsequently killed by serum complement in vitro. In a mouse model, PHB19 and Dep6 protected against STEC infection and caused a significant reduction in the levels of proinflammatory cytokines. This study reports the use of an O91-specific polysaccharide depolymerase for the treatment of STEC infection in mice.

scholarly journals Soluble Toll-Like Receptor 4 Impairs the Interaction of Shiga Toxin 2a with Human Serum Amyloid P Component

Toxins ◽  
2018 ◽  
Vol 10 (9) ◽  
pp. 379 ◽  
Author(s):  
Maurizio Brigotti ◽  
Valentina Arfilli ◽  
Domenica Carnicelli ◽  
Francesca Ricci ◽  
Pier Tazzari ◽  
...  
Keyword(s):  
Hemolytic Uremic Syndrome ◽  
Shiga Toxin ◽  
Flow Cytometric Analysis ◽  
Toll Like Receptor ◽  
Toll Like Receptor 4 ◽  
Amyloid P Component ◽  
Blood Constituents ◽  
Life Threatening ◽  
Uremic Syndrome ◽  
Amyloid P

Shiga toxin 2a (Stx2a) is the main virulence factor produced by pathogenic Escherichia coli strains (Stx-producing E. coli, STEC) responsible for hemorrhagic colitis and the life-threatening sequela hemolytic uremic syndrome in children. The toxin released in the intestine by STEC targets the globotriaosylceramide receptor (Gb3Cer) present on the endothelial cells of the brain and the kidney after a transient blood phase during which Stx2a interacts with blood components, such as neutrophils, which, conversely, recognize Stx through Toll-like receptor 4 (TLR4). Among non-cellular blood constituents, human amyloid P component (HuSAP) is considered a negative modulating factor that specifically binds Stx2a and impairs its toxic action. Here, we show that the soluble extracellular domain of TLR4 inhibits the binding of Stx2a to neutrophils, assessed by indirect flow cytometric analysis. Moreover, by using human sensitive Gb3Cer-expressing cells (Raji cells) we found that the complex Stx2a/soluble TLR4 escaped from capture by HuSAP allowing the toxin to target and damage human cells, as assayed by measuring translation inhibition, the typical Stx-induced functional impairment. Thus, soluble TLR4 stood out as a positive modulating factor for Stx2a. In the paper, these findings have been discussed in the context of the pathogenesis of hemolytic uremic syndrome.

scholarly journals Characterization of Shiga toxin-producing Escherichia coli in raw beef from informal and commercial abattoirs

PLoS ONE ◽  
2020 ◽  
Vol 15 (12) ◽  
pp. e0243828
Author(s):  
Kaarina N. Nehoya ◽  
Ndinomholo Hamatui ◽  
Renatus P. Shilangale ◽  
Harris Onywera ◽  
Jeya Kennedy ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Foodborne Pathogens ◽  
Shiga Toxin ◽  
Beef Products ◽  
Proper Training ◽  
Peptone Water ◽  
Life Threatening ◽  
Uremic Syndrome ◽  
Human Illness ◽  
Meat Samples

Shiga toxin-producing Escherichia coli are foodborne pathogens that are mostly associated with beef products and have been implicated in human illness. E.coli-associated illness range from asymptomatic conditions of mild diarrhoea to haemorrhagic colitis which can progress into life threatening haemolytic uremic syndrome (HUS). Beef from cattle are regarded as the main reservoir of Shiga toxin-producing E. coli (STEC) pathogen. The aim of this study was to assess the level and sources of contamination of raw beef with STEC, and determine the incidences of STEC strains in raw beef from informal and commercial abattoirs in Windhoek, Namibia. A total of 204 raw beef samples, 37 equipment and 29 hand swabs were collected and tested for STEC. The meat samples were first enriched with pre-warmed buffered peptone water, cultured on Tryptone Bile X-Glucuronide and CHROMagar STEC, and then sub-cultured on nutrient agar. The presence of E.coli in the samples was confirmed by using VITEK 2 E.coli identification cards and PCR. The overall prevalence of STEC in the meat samples from both the abattoirs was 41.66% raw beef samples; 5.40% equipment swabs; and none of the hand swabs was STEC positive. From the STEC positive meat samples 29.41% contained one of the major STEC strains. Moreover, 52% of the 25 samples that contained the major STECs were characterised by eae and stx1, 8% characterised by eae and stx2 while 40% were characterised by eae, stx1 and stx2 virulence genes. This study has revealed the necessity for proper training on meat safety (for meat handlers) as well as the development, implementation and maintenance of effective sanitary dressing procedures at abattoirs to eliminate beef contamination by STECs thereby ensuring the production of wholesome meat, and to prevent the occurrences of STEC infections.

scholarly journals Development of a homogeneous time-resolved FRET (HTRF) assay for the quantification of Shiga toxin 2 produced by E. coli

PeerJ ◽  
2021 ◽  
Vol 9 ◽  
pp. e11871
Author(s):  
Keiji Nakamura ◽  
Chikashi Tokuda ◽  
Hideyuki Arimitsu ◽  
Yoshiki Etoh ◽  
Mitsuhiro Hamasaki ◽  
...  
Keyword(s):  
Shiga Toxin ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Assay System ◽  
Time Resolved ◽  
Virulence Potential ◽  
Shiga Toxin 2 ◽  
Quantification Analysis ◽  
Life Threatening ◽  
Uremic Syndrome

Shiga toxin-producing Escherichia coli (STEC) is a major intestinal pathogen and causes serious gastrointestinal illness, which includes diarrhea, hemorrhagic colitis, and life-threatening hemolytic uremic syndrome. The major virulence factors of STEC are Shiga toxins (Stx1 and Stx2), which belong to the AB-type toxin family. Among several subtypes of Stx1 and Stx2, the production of Stx2a is thought to be a risk factor for severe STEC infections, but Stx2a production levels vary markedly between STEC strains, even strains with the same serotype. Therefore, quantitative analyses of Stx2 production by STEC strains are important to understand the virulence potential of specific lineages or sublineages. In this study, we developed a novel Stx2 quantification method by utilizing homogeneous time-resolved fluorescence resonance energy transfer (HTRF) technology. To determine suitable “sandwich” assay conditions, we tested 6 combinations of fluorescence-labeled monoclonal antibodies (mAbs) specific to Stx2 and compared the HTRF signal intensities obtained at various incubation times. Through this analysis, we selected the most suitable mAb pair, one recognizing the A subunit and the other recognizing the B subunit, thus together detecting Stx holotoxins. The optimal incubation time was also determined (18 h). Then, we optimized the concentrations of the two mAbs based on the range for linearity. The established HTRF assay detected 0.5 ng/ml of the highly purified recombinant Stx2a and Stx2e proteins and the working range was 1–64 ng/ml for both Stx2a and Stx2e. Through the quantification analysis of Stx proteins in STEC cell lysates, we confirmed that other Stx2 subtypes (Stx2b, Stx2c, Stx2d and Stx2g) can also be quantified at a certain level of accuracy, while this assay system does not detect Stx2f, which is highly divergent in sequence from other Stx2 subtypes, and Stx1. As the HTRF protocol we established is simple, this assay system should prove useful for the quantitative analysis of Stx2 production levels of a large number of STEC strains.

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