Toxins

2024

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13 pages, 3483 KB

Open AccessArticle

In Silico Analysis of Shiga Toxin-Producing Escherichia coli O157:H7 Strains from Presumptive Super- and Low-Shedder Cattle

by Emmanuel W. Bumunang, Vinicius S. Castro, Trevor Alexander, Rahat Zaheer, Tim A. McAllister, Le Luo Guan and Kim Stanford

Toxins 2024, 16(2), 86; https://doi.org/10.3390/toxins16020086 - 5 Feb 2024

Cited by 1 | Viewed by 2671

Abstract

Cattle are the primary reservoir for STEC O157, with some shedding >10⁴ CFU/g in feces, a phenomenon known as super-shedding (SS). The mechanism(s) responsible for SS are not understood but have been attributed to the environment, host, and pathogen. This study aimed [...] Read more.

Cattle are the primary reservoir for STEC O157, with some shedding >10⁴ CFU/g in feces, a phenomenon known as super-shedding (SS). The mechanism(s) responsible for SS are not understood but have been attributed to the environment, host, and pathogen. This study aimed to compare genetic characteristics of STEC O157 strains from cattle in the same commercial feedlot pens with SS or low-shedding (LS) status. Strains from SS (n = 35) and LS (n = 28) collected from 11 pens in three feedlots were analyzed for virulence genes, Shiga toxin-carrying bacteriophage insertion sites, and phylogenetic relationships. In silico analysis showed limited variation regarding virulence gene profiles. Stx-encoding prophage insertion sites mrlA and wrbA for stx1a and stx2a, respectively, were all occupied, but two isolates had fragments of the stx-carrying phage in mrlA and wrbA loci without stx1a and stx2a. All strains screened for lineage-specific polymorphism assay (LSPA-6) were 111111, lineage I. Of the isolates, 61 and 2 were clades 1 and 8, respectively. Phylogenetic analysis revealed that pens with more than one SS had multiple distantly related clusters of SS and LS isolates. Although virulence genes and lineage were largely similar within and across feedlots, multiple genetic origins of strains within a single feedlot pen illustrate challenges for on-farm control of STEC. Full article

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2023

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13 pages, 3491 KB

Open AccessArticle

Characteristics of Shiga Toxin-Producing Escherichia coli Circulating in Asymptomatic Food Handlers

by Xinxia Sui, Xi Yang, Ming Luo, Hua Wang, Qian Liu, Hui Sun, Yujuan Jin, Yannong Wu, Xiangning Bai and Yanwen Xiong

Toxins 2023, 15(11), 640; https://doi.org/10.3390/toxins15110640 - 2 Nov 2023

Cited by 7 | Viewed by 3636

Abstract

Shiga toxin-producing Escherichia coli (STEC) is a foodborne zoonotic pathogen that causes diarrhea, hemorrhagic colitis (HC), and hemolytic uremic syndrome (HUS) worldwide. Since the infection can be asymptomatic, the circulation of STEC in some asymptomatic carriers, especially in healthy-food-related professionals, is not yet [...] Read more.

Shiga toxin-producing Escherichia coli (STEC) is a foodborne zoonotic pathogen that causes diarrhea, hemorrhagic colitis (HC), and hemolytic uremic syndrome (HUS) worldwide. Since the infection can be asymptomatic, the circulation of STEC in some asymptomatic carriers, especially in healthy-food-related professionals, is not yet well understood. In this study, a total of 3987 anal swab samples from asymptomatic food handlers were collected, and ten swabs recovered STEC strains (0.251%). Of the ten STEC isolates, seven serotypes and eight sequence types (ST) were determined using whole genome sequencing (WGS). Two stx1 subtypes (stx1a and stx1c) and four stx2 subtypes (stx2a, stx2b, stx2d, and stx2e) were detected. Seven different insertion sites were found in fourteen Stx prophages, and the dmsB and yfhL were the newly identified insertion sites. The ten strains showed the variable Stx transcription levels after the mitomycin C induction. The whole-genome phylogeny indicated that the strains from the asymptomatic food handlers were genetically distant from the strains of HUS patients. The STEC isolates circulating in asymptomatic carriers might pose a low potential to cause disease. Full article

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15 pages, 4108 KB

Open AccessArticle

Shiga Toxin (Stx) Type 1a and Stx2a Translocate through a Three-Layer Intestinal Model

by Rebecca A. Bova, Andrew C. Lamont, Theodore J. Picou, Vincent B. Ho, Kristin H. Gilchrist and Angela R. Melton-Celsa

Toxins 2023, 15(3), 207; https://doi.org/10.3390/toxins15030207 - 9 Mar 2023

Cited by 2 | Viewed by 3171

Abstract

Shiga toxins (Stxs) produced by ingested E. coli can induce hemolytic uremic syndrome after crossing the intact intestinal barrier, entering the bloodstream, and targeting endothelial cells in the kidney. The method(s) by which the toxins reach the bloodstream are not fully defined. Here, [...] Read more.

Shiga toxins (Stxs) produced by ingested E. coli can induce hemolytic uremic syndrome after crossing the intact intestinal barrier, entering the bloodstream, and targeting endothelial cells in the kidney. The method(s) by which the toxins reach the bloodstream are not fully defined. Here, we used two polarized cell models to evaluate Stx translocation: (i) a single-layer primary colonic epithelial cell model and (ii) a three-cell-layer model with colonic epithelial cells, myofibroblasts, and colonic endothelial cells. We traced the movement of Stx types 1a and 2a across the barrier models by measuring the toxicity of apical and basolateral media on Vero cells. We found that Stx1a and Stx2a crossed both models in either direction. However, approximately 10-fold more Stx translocated in the three-layer model as compared to the single-layer model. Overall, the percentage of toxin that translocated was about 0.01% in the epithelial-cell-only model but up to 0.09% in the three-cell-layer model. In both models, approximately 3- to 4-fold more Stx2a translocated than Stx1a. Infection of the three-cell-layer model with Stx-producing Escherichia coli (STEC) strains showed that serotype O157:H7 STEC reduced barrier function in the model and that the damage was not dependent on the presence of the eae gene. Infection of the three-layer model with O26:H11 STEC strain TW08571 (Stx1a+ and Stx2a+), however, allowed translocation of modest amounts of Stx without reducing barrier function. Deletion of stx2a from TW08571 or the use of anti-Stx1 antibody prevented translocation of toxin. Our results suggest that single-cell models may underestimate the amount of Stx translocation and that the more biomimetic three-layer model is suited for Stx translocation inhibitor studies. Full article

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2022

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28 pages, 1825 KB

Open AccessReview

Diagnosis and Treatment for Shiga Toxin-Producing Escherichia coli Associated Hemolytic Uremic Syndrome

by Yang Liu, Hatim Thaker, Chunyan Wang, Zhonggao Xu and Min Dong

Toxins 2023, 15(1), 10; https://doi.org/10.3390/toxins15010010 - 23 Dec 2022

Cited by 43 | Viewed by 23909

Abstract

Shiga toxin-producing Escherichia coli (STEC)-associated hemolytic uremic syndrome (STEC-HUS) is a clinical syndrome involving hemolytic anemia (with fragmented red blood cells), low levels of platelets in the blood (thrombocytopenia), and acute kidney injury (AKI). It is the major infectious cause of AKI in [...] Read more.

Shiga toxin-producing Escherichia coli (STEC)-associated hemolytic uremic syndrome (STEC-HUS) is a clinical syndrome involving hemolytic anemia (with fragmented red blood cells), low levels of platelets in the blood (thrombocytopenia), and acute kidney injury (AKI). It is the major infectious cause of AKI in children. In severe cases, neurological complications and even death may occur. Treating STEC-HUS is challenging, as patients often already have organ injuries when they seek medical treatment. Early diagnosis is of great significance for improving prognosis and reducing mortality and sequelae. In this review, we first briefly summarize the diagnostics for STEC-HUS, including history taking, clinical manifestations, fecal and serological detection methods for STEC, and complement activation monitoring. We also summarize preventive and therapeutic strategies for STEC-HUS, such as vaccines, volume expansion, renal replacement therapy (RRT), antibiotics, plasma exchange, antibodies and inhibitors that interfere with receptor binding, and the intracellular trafficking of the Shiga toxin. Full article

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2018

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10 pages, 2196 KB

Open AccessArticle

Human Recombinant Fab Fragment Neutralizes Shiga Toxin Type 2 Cytotoxic Effects in vitro and in vivo

by Daniela Luz, Maria Marta Amaral, Flavia Sacerdoti, Alan Mauro Bernal, Wagner Quintilio, Ana Maria Moro, Marina Sandra Palermo, Cristina Ibarra and Roxane Maria Fontes Piazza

Toxins 2018, 10(12), 508; https://doi.org/10.3390/toxins10120508 - 2 Dec 2018

Cited by 11 | Viewed by 4308

Abstract

Shiga toxin (Stx) producing Escherichia coli (STEC) is responsible for causing hemolytic uremic syndrome (HUS), a life-threatening thrombotic microangiopathy characterized by thrombocytopenia, hemolytic anemia, and acute renal failure after bacterially induced hemorrhagic diarrhea. Until now, there has been neither an effective treatment nor [...] Read more.

Shiga toxin (Stx) producing Escherichia coli (STEC) is responsible for causing hemolytic uremic syndrome (HUS), a life-threatening thrombotic microangiopathy characterized by thrombocytopenia, hemolytic anemia, and acute renal failure after bacterially induced hemorrhagic diarrhea. Until now, there has been neither an effective treatment nor method of prevention for the deleterious effects caused by Stx intoxication. Antibodies are well recognized as affinity components of therapeutic drugs; thus, a previously obtained recombinant human FabC11:Stx2 fragment was used to neutralize Stx2 in vitro in a Vero cell viability assay. Herein, we demonstrated that this fragment neutralized, in a dose-dependent manner, the cytotoxic effects of Stx2 on human glomerular endothelial cells, on human proximal tubular epithelial cells, and prevented the morphological alterations induced by Stx2. FabC11:Stx2 protected mice from a lethal dose of Stx2 by toxin-antibody pre-incubation. Altogether, our results show the ability of a new encouraging molecule to prevent Stx-intoxication symptoms during STEC infection. Full article

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2017

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27 pages, 4094 KB

Open AccessArticle

Shiga Toxin Glycosphingolipid Receptors in Human Caco-2 and HCT-8 Colon Epithelial Cell Lines

by Ivan U. Kouzel, Gottfried Pohlentz, Julia S. Schmitz, Daniel Steil, Hans-Ulrich Humpf, Helge Karch and Johannes Müthing

Toxins 2017, 9(11), 338; https://doi.org/10.3390/toxins9110338 - 25 Oct 2017

Cited by 31 | Viewed by 7459

Abstract

Shiga toxins (Stxs) released by enterohemorrhagic Escherichia coli (EHEC) into the human colon are the causative agents for fatal outcome of EHEC infections. Colon epithelial Caco-2 and HCT-8 cells are widely used for investigating Stx-mediated intestinal cytotoxicity. Only limited data are available regarding [...] Read more.

Shiga toxins (Stxs) released by enterohemorrhagic Escherichia coli (EHEC) into the human colon are the causative agents for fatal outcome of EHEC infections. Colon epithelial Caco-2 and HCT-8 cells are widely used for investigating Stx-mediated intestinal cytotoxicity. Only limited data are available regarding precise structures of their Stx receptor glycosphingolipids (GSLs) globotriaosylceramide (Gb3Cer) and globotetraosylceramide (Gb4Cer), and lipid raft association. In this study we identified Gb3Cer and Gb4Cer lipoforms of serum-free cultivated Caco-2 and HCT-8 cells, chiefly harboring ceramide moieties composed of sphingosine (d18:1) and C16:0, C22:0 or C24:0/C24:1 fatty acid. The most significant difference between the two cell lines was the prevalence of Gb3Cer with C16 fatty acid in HCT-8 and Gb4Cer with C22–C24 fatty acids in Caco-2 cells. Lipid compositional analysis of detergent-resistant membranes (DRMs), which were used as lipid raft-equivalents, indicated slightly higher relative content of Stx receptor Gb3Cer in DRMs of HCT-8 cells when compared to Caco-2 cells. Cytotoxicity assays revealed substantial sensitivity towards Stx2a for both cell lines, evidencing little higher susceptibility of Caco-2 cells versus HCT-8 cells. Collectively, Caco-2 and HCT-8 cells express a plethora of different receptor lipoforms and are susceptible towards Stx2a exhibiting somewhat lower sensitivity when compared to Vero cells. Full article

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18 pages, 6522 KB

Open AccessArticle

Soluble CD40 Ligand and Oxidative Response Are Reciprocally Stimulated during Shiga Toxin-Associated Hemolytic Uremic Syndrome

by Maria J. Abrey Recalde, Romina S. Alvarez, Fabiana Alberto, Maria P. Mejias, Maria V. Ramos, Romina J. Fernandez Brando, Andrea C. Bruballa, Ramon A. Exeni, Laura Alconcher, Cristina A. Ibarra, María M. Amaral and Marina S. Palermo

Toxins 2017, 9(11), 331; https://doi.org/10.3390/toxins9110331 - 25 Oct 2017

Cited by 6 | Viewed by 5418

Abstract

Shiga toxin (Stx), produced by Escherichia coli, is the main pathogenic factor of diarrhea-associated hemolytic uremic syndrome (HUS), which is characterized by the obstruction of renal microvasculature by platelet-fibrin thrombi. It is well known that the oxidative imbalance generated by Stx induces [...] Read more.

Shiga toxin (Stx), produced by Escherichia coli, is the main pathogenic factor of diarrhea-associated hemolytic uremic syndrome (HUS), which is characterized by the obstruction of renal microvasculature by platelet-fibrin thrombi. It is well known that the oxidative imbalance generated by Stx induces platelet activation, contributing to thrombus formation. Moreover, activated platelets release soluble CD40 ligand (sCD40L), which in turn contributes to oxidative imbalance, triggering the release of reactive oxidative species (ROS) on various cellular types. The aim of this work was to determine if the interaction between the oxidative response and platelet-derived sCD40L, as consequence of Stx-induced endothelium damage, participates in the pathogenic mechanism during HUS. Activated human glomerular endothelial cells (HGEC) by Stx2 induced platelets to adhere to them. Although platelet adhesion did not contribute to endothelial damage, high levels of sCD40L were released to the medium. The release of sCD40L by activated platelets was inhibited by antioxidant treatment. Furthermore, we found increased levels of sCD40L in plasma from HUS patients, which were also able to trigger the respiratory burst in monocytes in a sCD40L-dependent manner. Thus, we concluded that platelet-derived sCD40L and the oxidative response are reciprocally stimulated during Stx2-associated HUS. This process may contribute to the evolution of glomerular occlusion and the microangiopathic lesions. Full article

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14 pages, 5431 KB

Open AccessArticle

Ouabain Protects Human Renal Cells against the Cytotoxic Effects of Shiga Toxin Type 2 and Subtilase Cytotoxin

by María M. Amaral, Magalí C. Girard, Romina S. Álvarez, Adrienne W. Paton, James C. Paton, Horacio A. Repetto, Flavia Sacerdoti and Cristina A. Ibarra

Toxins 2017, 9(7), 226; https://doi.org/10.3390/toxins9070226 - 18 Jul 2017

Cited by 6 | Viewed by 5351

Abstract

Hemolytic uremic syndrome (HUS) is one of the most common causes of acute renal failure in children. The majority of cases are associated with Shiga toxin (Stx)-producing Escherichia coli (STEC). In Argentina, HUS is endemic and presents the highest incidence rate in the [...] Read more.

Hemolytic uremic syndrome (HUS) is one of the most common causes of acute renal failure in children. The majority of cases are associated with Shiga toxin (Stx)-producing Escherichia coli (STEC). In Argentina, HUS is endemic and presents the highest incidence rate in the world. STEC strains expressing Stx type 2 (Stx2) are responsible for the most severe cases of this pathology. Subtilase cytotoxin (SubAB) is another STEC virulence factor that may contribute to HUS pathogenesis. To date, neither a licensed vaccine nor effective therapy for HUS is available for humans. Considering that Ouabain (OUA) may prevent the apoptosis process, in this study we evaluated if OUA is able to avoid the damage caused by Stx2 and SubAB on human glomerular endothelial cells (HGEC) and the human proximal tubule epithelial cell (HK-2) line. HGEC and HK-2 were pretreated with OUA and then incubated with the toxins. OUA protected the HGEC viability from Stx2 and SubAB cytotoxic effects, and also prevented the HK-2 viability from Stx2 effects. The protective action of OUA on HGEC and HK-2 was associated with a decrease in apoptosis and an increase in cell proliferation. Our data provide evidence that OUA could be considered as a therapeutic strategy to avoid the renal damage that precedes HUS. Full article

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18 pages, 1221 KB

Open AccessArticle

Efficacy of Urtoxazumab (TMA-15 Humanized Monoclonal Antibody Specific for Shiga Toxin 2) Against Post-Diarrheal Neurological Sequelae Caused by Escherichia coli O157:H7 Infection in the Neonatal Gnotobiotic Piglet Model

by Rodney A. Moxley, David H. Francis, Mizuho Tamura, David B. Marx, Kristina Santiago-Mateo and Mojun Zhao

Toxins 2017, 9(2), 49; https://doi.org/10.3390/toxins9020049 - 26 Jan 2017

Cited by 18 | Viewed by 6039

Abstract

Enterohemorrhagic Escherichia coli (EHEC) is the most common cause of hemorrhagic colitis and hemolytic uremic syndrome in human patients, with brain damage and dysfunction the main cause of acute death. We evaluated the efficacy of urtoxazumab (TMA-15, Teijin Pharma Limited), a humanized monoclonal [...] Read more.

Enterohemorrhagic Escherichia coli (EHEC) is the most common cause of hemorrhagic colitis and hemolytic uremic syndrome in human patients, with brain damage and dysfunction the main cause of acute death. We evaluated the efficacy of urtoxazumab (TMA-15, Teijin Pharma Limited), a humanized monoclonal antibody against Shiga toxin (Stx) 2 for the prevention of brain damage, dysfunction, and death in a piglet EHEC infection model. Forty-five neonatal gnotobiotic piglets were inoculated orally with 3 × 10⁹ colony-forming units of EHEC O157:H7 strain EDL933 (Stx1⁺, Stx2⁺) when 22–24 h old. At 24 h post-inoculation, piglets were intraperitoneally administered placebo or TMA-15 (0.3, 1.0 or 3.0 mg/kg body weight). Compared to placebo (n = 10), TMA-15 (n = 35) yielded a significantly greater probability of survival, length of survival, and weight gain (p <0.05). The efficacy of TMA-15 against brain lesions and death was 62.9% (p = 0.0004) and 71.4% (p = 0.0004), respectively. These results suggest that TMA-15 may potentially prevent or reduce vascular necrosis and infarction of the brain attributable to Stx2 in human patients acutely infected with EHEC. However, we do not infer that TMA-15 treatment will completely protect human patients infected with EHEC O157:H7 strains that produce both Stx1 and Stx2. Full article

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2016

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24 pages, 5714 KB

Open AccessArticle

A Topographical Atlas of Shiga Toxin 2e Receptor Distribution in the Tissues of Weaned Piglets

by Daniel Steil, Robert Bonse, Iris Meisen, Gottfried Pohlentz, German Vallejo, Helge Karch and Johannes Müthing

Toxins 2016, 8(12), 357; https://doi.org/10.3390/toxins8120357 - 30 Nov 2016

Cited by 16 | Viewed by 5425

Abstract

Shiga toxin (Stx) 2e of Stx-producing Escherichia coli (STEC) is the primary virulence factor in the development of pig edema disease shortly after weaning. Stx2e binds to the globo-series glycosphingolipids (GSLs) globotriaosylceramide (Gb3Cer, Galα1-4Galβ1-4Glcβ1-1Cer) and globotetraosylceramide (Gb4Cer, GalNAcβ1-3Galα1-4Galβ1-4Glcβ1-1Cer), the latter acting as the [...] Read more.

Shiga toxin (Stx) 2e of Stx-producing Escherichia coli (STEC) is the primary virulence factor in the development of pig edema disease shortly after weaning. Stx2e binds to the globo-series glycosphingolipids (GSLs) globotriaosylceramide (Gb3Cer, Galα1-4Galβ1-4Glcβ1-1Cer) and globotetraosylceramide (Gb4Cer, GalNAcβ1-3Galα1-4Galβ1-4Glcβ1-1Cer), the latter acting as the preferential Stx2e receptor. We determined Stx receptor profiles of 25 different tissues of a male and a female weaned piglet using immunochemical solid phase binding assays combined with mass spectrometry. All probed tissues harbored GSL receptors, ranging from high (category I) over moderate (category II) to low content (category III). Examples of Gb4Cer expression in category I tissues are small intestinal ileum, kidney pelvis and whole blood, followed by colon, small intestinal duodenum and jejunum belonging to category II, and kidney cortex, cerebrum and cerebellum as members of category III organs holding true for both genders. Dominant Gb3Cer and Gb4Cer lipoforms were those with ceramides carrying constant sphingosine (d18:1) and a variable C16:0, C22:0 or C24:1/C24:0 fatty acid. From the mapping data, we created a topographical atlas for Stx2e receptors in piglet tissues and organs, which might be helpful to further investigations on the molecular and cellular mechanisms that underlie infections of Stx2e-producing STEC in pigs and their zoonotic potential for humans. Full article

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13 pages, 1800 KB

Open AccessFeature PaperArticle

Mechanisms that Determine the Differential Stability of Stx⁺ and Stx⁻ Lysogens

by Michael P. Colon, Dolonchapa Chakraborty, Yonatan Pevzner and Gerald B. Koudelka

Toxins 2016, 8(4), 96; https://doi.org/10.3390/toxins8040096 - 31 Mar 2016

Cited by 15 | Viewed by 7681

Abstract

Phages 933W, BAA2326, 434, and λ are evolutionarily-related temperate lambdoid phages that infect Escherichia coli. Although these are highly-similar phages, BAA2326 and 933W naturally encode Shiga toxin 2 (Stx⁺), but phage 434 and λ do not (Stx⁻). Previous [...] Read more.

Phages 933W, BAA2326, 434, and λ are evolutionarily-related temperate lambdoid phages that infect Escherichia coli. Although these are highly-similar phages, BAA2326 and 933W naturally encode Shiga toxin 2 (Stx⁺), but phage 434 and λ do not (Stx⁻). Previous reports suggest that the 933W Stx⁺ prophage forms less stable lysogens in E. coli than does the Stx⁻ prophages λ, P22, and 434. The higher spontaneous induction frequency of the Stx⁺ prophage may be correlated with both virulence and dispersion of the Stx2-encoding phage. Here, we examined the hypothesis that lysogen instability is a common feature of Stx⁺ prophages. We found in both the absence and presence of prophage inducers (DNA damaging agents, salts), the Stx⁺ prophages induce at higher frequencies than do Stx⁻ prophages. The observed instability of Stx⁺ prophages does not appear to be the result of any differences in phage development properties between Stx⁺ and Stx⁻ phages. Our results indicate that differential stability of Stx⁺ and Stx⁻ prophages results from both RecA-dependent and RecA-independent effects on the intracellular concentration of the respective cI repressors. Full article

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23 pages, 1448 KB

Open AccessFeature PaperReview

Shiga Toxins as Multi-Functional Proteins: Induction of Host Cellular Stress Responses, Role in Pathogenesis and Therapeutic Applications

by Moo-Seung Lee, Sunwoo Koo, Dae Gwin Jeong and Vernon L. Tesh

Toxins 2016, 8(3), 77; https://doi.org/10.3390/toxins8030077 - 17 Mar 2016

Cited by 90 | Viewed by 17605

Abstract

Shiga toxins (Stxs) produced by Shiga toxin-producing bacteria Shigella dysenteriae serotype 1 and select serotypes of Escherichia coli are primary virulence factors in the pathogenesis of hemorrhagic colitis progressing to potentially fatal systemic complications, such as hemolytic uremic syndrome and central nervous system [...] Read more.

Shiga toxins (Stxs) produced by Shiga toxin-producing bacteria Shigella dysenteriae serotype 1 and select serotypes of Escherichia coli are primary virulence factors in the pathogenesis of hemorrhagic colitis progressing to potentially fatal systemic complications, such as hemolytic uremic syndrome and central nervous system abnormalities. Current therapeutic options to treat patients infected with toxin-producing bacteria are limited. The structures of Stxs, toxin-receptor binding, intracellular transport and the mode of action of the toxins have been well defined. However, in the last decade, numerous studies have demonstrated that in addition to being potent protein synthesis inhibitors, Stxs are also multifunctional proteins capable of activating multiple cell stress signaling pathways, which may result in apoptosis, autophagy or activation of the innate immune response. Here, we briefly present the current understanding of Stx-activated signaling pathways and provide a concise review of therapeutic applications to target tumors by engineering the toxins. Full article

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10 pages, 896 KB

Open AccessFeature PaperArticle

Vitamin B₁₂ Uptake by the Gut Commensal Bacteria Bacteroides thetaiotaomicron Limits the Production of Shiga Toxin by Enterohemorrhagic Escherichia coli

by Charlotte Cordonnier, Guillaume Le Bihan, Jean-Guillaume Emond-Rheault, Annie Garrivier, Josée Harel and Grégory Jubelin

Toxins 2016, 8(1), 14; https://doi.org/10.3390/toxins8010014 - 5 Jan 2016

Cited by 48 | Viewed by 8767

Abstract

Enterohemorrhagic Escherichia coli (EHEC) are foodborne pathogens responsible for the development of bloody diarrhea and renal failure in humans. Many environmental factors have been shown to regulate the production of Shiga toxin 2 (Stx2), the main virulence factor of EHEC. Among them, soluble [...] Read more.

Enterohemorrhagic Escherichia coli (EHEC) are foodborne pathogens responsible for the development of bloody diarrhea and renal failure in humans. Many environmental factors have been shown to regulate the production of Shiga toxin 2 (Stx2), the main virulence factor of EHEC. Among them, soluble factors produced by human gut microbiota and in particular, by the predominant species Bacteroides thetaiotaomicron (B. thetaiotaomicron), inhibit Stx2 gene expression. In this study, we investigated the molecular mechanisms underlying the B. thetaiotaomicron-dependent inhibition of Stx2 production by EHEC. We determined that Stx2-regulating molecules are resistant to heat treatment but do not correspond to propionate and acetate, two short-chain fatty acids produced by B. thetaiotaomicron. Moreover, screening of a B. thetaiotaomicron mutant library identified seven mutants that do not inhibit Stx2 synthesis by EHEC. One mutant has impaired production of BtuB, an outer membrane receptor for vitamin B₁₂. Together with restoration of Stx2 level after vitamin B₁₂ supplementation, these data highlight vitamin B₁₂ as a molecule produced by gut microbiota that modulates production of a key virulence factor of EHEC and consequently may affect the outcome of an infection. Full article

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2015

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13 pages, 275 KB

Open AccessArticle

The Effects of Shiga Toxin 1, 2 and Their Subunits on Cytokine and Chemokine Expression by Human Macrophage-Like THP-1 Cells

by Jeremy R. Brandelli, Thomas P. Griener, Austin Laing, George Mulvey and Glen D. Armstrong

Toxins 2015, 7(10), 4054-4066; https://doi.org/10.3390/toxins7104054 - 9 Oct 2015

Cited by 14 | Viewed by 6352

Abstract

Infection by Shiga toxin (Stx)-producing enterohemorrhagic Escherichia coli (EHEC) results in severe diarrhea, hemorrhagic colitis, and, occasionally, hemolytic-uremic syndrome (HUS). HUS is associated with an increase in pro-inflammatory cytokines and chemokines, many of which are produced by macrophages in the kidneys, indicating that [...] Read more.

Infection by Shiga toxin (Stx)-producing enterohemorrhagic Escherichia coli (EHEC) results in severe diarrhea, hemorrhagic colitis, and, occasionally, hemolytic-uremic syndrome (HUS). HUS is associated with an increase in pro-inflammatory cytokines and chemokines, many of which are produced by macrophages in the kidneys, indicating that localized host innate immunity likely plays a role in renal pathogenesis. EHEC serotypes may express one or two classes of serologically defined but structurally and functionally-related Shiga toxins called Stx1 and Stx2. Of these, Stx2 appears to be linked to higher rates of HUS than Stx1. To investigate a possible reason for this, we exposed human macrophage-like THP-1 cells to Stx1 or Stx2 and then used the Luminex multiplex system to assess cytokine/chemokine concentrations in culture supernatant solutions. This analysis revealed that, relative to Stx1, Stx2 significantly caused increased expression of GRO, G-CSF, IL-1β, IL-8 and TNFα in macrophage-like THP-1 cells. This was determined to not be due to a difference in cytotoxicity since both Stx1 and Stx2 displayed similar cytotoxic activities on macrophage-like THP-1 cells. These observations indicate that, in vitro, Stx2 can provoke a greater pro-inflammatory response than Stx1 in macrophages and provides a possible partial explanation for higher rates of HUS in patients infected with EHEC strains expressing Stx2. To begin to determine a mechanism for Shiga toxin-mediated cytokine production, we exposed macrophage-like THP-1 cells to Stx1 or Stx2 A and B subunits. Luminex analysis of cytokines in cell culture supernatant solutions demonstrated that neither subunit alone induced a cytokine response in THP-1 cells. Full article

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13 pages, 1383 KB

Open AccessArticle

UV-Sensitivity of Shiga Toxin-Converting Bacteriophage Virions Φ24_B, 933W, P22, P27 and P32

by Sylwia Bloch, Bożena Nejman-Faleńczyk, Gracja Topka, Aleksandra Dydecka, Katarzyna Licznerska, Magdalena Narajczyk, Agnieszka Necel, Alicja Węgrzyn and Grzegorz Węgrzyn

Toxins 2015, 7(9), 3727-3739; https://doi.org/10.3390/toxins7093727 - 21 Sep 2015

Cited by 12 | Viewed by 5948

Abstract

Shiga toxin-converting bacteriophages (Stx phages) are present as prophages in Shiga toxin-producing Escherichia coli (STEC) strains. Theses phages can be transmitted to previously non-pathogenic E. coli cells making them potential producers of Shiga toxins, as they bear genes for these toxins in their [...] Read more.

Shiga toxin-converting bacteriophages (Stx phages) are present as prophages in Shiga toxin-producing Escherichia coli (STEC) strains. Theses phages can be transmitted to previously non-pathogenic E. coli cells making them potential producers of Shiga toxins, as they bear genes for these toxins in their genomes. Therefore, sensitivity of Stx phage virions to various conditions is important in both natural processes of spreading of these viruses and potential prophylactic control of appearance of novel pathogenic E. coli strains. In this report we provide evidence that virions of Stx phages are significantly more sensitive to UV irradiation than bacteriophage λ. Following UV irradiation of Stx virions at the dose of 50 J/m², their infectivity dropped by 1–3 log₁₀, depending on the kind of phage. Under these conditions, a considerable release of phage DNA from virions was observed, and electron microscopy analyses indicated a large proportion of partially damaged virions. Infection of E. coli cells with UV-irradiated Stx phages resulted in significantly decreased levels of expression of N and cro genes, crucial for lytic development. We conclude that inactivation of Stx virions caused by relatively low dose of UV light is due to damage of capsids that prevents effective infection of the host cells. Full article

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15 pages, 763 KB

Open AccessArticle

Reduced Toxicity of Shiga Toxin (Stx) Type 2c in Mice Compared to Stx2d Is Associated with Instability of Stx2c Holotoxin

by Joshua C. Bunger, Angela R. Melton-Celsa, Ernest L. Maynard and Alison D. O'Brien

Toxins 2015, 7(6), 2306-2320; https://doi.org/10.3390/toxins7062306 - 23 Jun 2015

Cited by 9 | Viewed by 5652

Abstract

Shiga toxin (Stx) is an AB₅ ribotoxin made by Stx-producing Escherichia coli (STEC). These organisms cause diarrhea, hemorrhagic colitis and the hemolytic uremic syndrome. STEC make two types of Stxs, Stx1 and/or Stx2. Stx2 has one prototype (a) and six subtypes (b–g), [...] Read more.

Shiga toxin (Stx) is an AB₅ ribotoxin made by Stx-producing Escherichia coli (STEC). These organisms cause diarrhea, hemorrhagic colitis and the hemolytic uremic syndrome. STEC make two types of Stxs, Stx1 and/or Stx2. Stx2 has one prototype (a) and six subtypes (b–g), but only STEC that make Stx2a, and/or Stx2c, or Stx2d are associated with severe disease. However, Stx2c is about 10-fold less toxic than Stx2d in vivo despite only two amino acid differences in the A subunit at positions 291 and 297. We made mutations at these two sites to create intermediate toxins between Stx2c and Stx2d, and determined the 50% cytotoxic dose on Vero cells before and after heat treatment, and the 50% lethal dose in mice of the toxins. We found that serine 291 was associated with increased toxicity in vivo and that either amino acid change from that in Stx2c to that in Stx2d increased heat stability. We also assessed the secondary structure of Stx2c and Stx2d by circular dichroism (CD) spectroscopy. The CD studies suggest that Stx2c has a less-ordered secondary structure than Stx2d. We conclude that both amino acids at positions 291 and 297 in Stx2c contribute to its decreased stability and in vivo toxicity compared to Stx2d. Full article

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11 pages, 697 KB

Open AccessArticle

Detection of Shiga Toxins by Lateral Flow Assay

by Kathryn H. Ching, Xiaohua He, Larry H. Stanker, Alice V. Lin, Jeffery A. McGarvey and Robert Hnasko

Toxins 2015, 7(4), 1163-1173; https://doi.org/10.3390/toxins7041163 - 3 Apr 2015

Cited by 27 | Viewed by 8312

Abstract

Shiga toxin-producing Escherichia coli (STEC) produce shiga toxins (Stxs) that can cause human disease and death. The contamination of food products with STEC represents a food safety problem that necessitates rapid and effective detection strategies to mitigate risk. In this manuscript, we report [...] Read more.

Shiga toxin-producing Escherichia coli (STEC) produce shiga toxins (Stxs) that can cause human disease and death. The contamination of food products with STEC represents a food safety problem that necessitates rapid and effective detection strategies to mitigate risk. In this manuscript, we report the development of a colorimetric lateral flow assay (LFA) for the rapid detection of Stxs in <10 min using a pair of monoclonal antibodies that bind epitopes common to Stx1 and six Stx2 variants. This LFA provides a rapid and sensitive test for the detection of Stxs directly from STEC culture supernatants or at risk food samples with a 0.1 ng/mL limit of detection (LOD) for Stx2a. This Stx LFA is applicable for use in the rapid evaluation of Stx production from cultured E. coli strains or as a tool to augment current methods as part of food safety testing. Full article

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2011

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24 pages, 525 KB

Open AccessArticle

Impact of the Nature and Size of the Polymeric Backbone on the Ability of Heterobifunctional Ligands to Mediate Shiga Toxin and Serum Amyloid P Component Ternary Complex Formation

by Pavel I. Kitov, Eugenia Paszkiewicz, Joanna M. Sadowska, Zhicheng Deng, Marya Ahmed, Ravin Narain, Thomas P. Griener, George L. Mulvey, Glen D. Armstrong and David R. Bundle

Toxins 2011, 3(9), 1065-1088; https://doi.org/10.3390/toxins3091065 - 25 Aug 2011

Cited by 14 | Viewed by 9692

Abstract

Inhibition of AB₅-type bacterial toxins can be achieved by heterobifunctional ligands (BAITs) that mediate assembly of supramolecular complexes involving the toxin’s pentameric cell membrane-binding subunit and an endogenous protein, serum amyloid P component, of the innate immune system. Effective in vivo [...] Read more.

Inhibition of AB₅-type bacterial toxins can be achieved by heterobifunctional ligands (BAITs) that mediate assembly of supramolecular complexes involving the toxin’s pentameric cell membrane-binding subunit and an endogenous protein, serum amyloid P component, of the innate immune system. Effective in vivo protection from Shiga toxin Type 1 (Stx1) is achieved by polymer-bound, heterobifunctional inhibitors-adaptors (PolyBAITs), which exhibit prolonged half-life in circulation and by mediating formation of face-to-face SAP-AB₅ complexes, block receptor recognition sites and redirect toxins to the spleen and liver for degradation. Direct correlation between solid-phase activity and protective dose of PolyBAITs both in the cytotoxicity assay and in vivo indicate that the mechanism of protection from intoxication is inhibition of toxin binding to the host cell membrane. The polymeric scaffold influences the activity not only by clustering active binding fragments but also by sterically interfering with the supramolecular complex assembly. Thus, inhibitors based on N-(2-hydroxypropyl) methacrylamide (HPMA) show significantly lower activity than polyacrylamide-based analogs. The detrimental steric effect can partially be alleviated by extending the length of the spacer, which separates pendant ligand from the backbone, as well as extending the spacer, which spans the distance between binding moieties within each heterobifunctional ligand. Herein we report that polymer size and payload of the active ligand had moderate effects on the inhibitor’s activity. Full article

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6 pages, 149 KB

Open AccessShort Note

Loss of vtx Genes after the First Subcultivation Step of Verocytotoxigenic Escherichia coli O157 and Non-O157 during Isolation from Naturally Contaminated Fecal Samples

by Maria-Adelheid Joris, Karen Verstraete, Koen De Reu and Lieven De Zutter

Toxins 2011, 3(6), 672-677; https://doi.org/10.3390/toxins3060672 - 20 Jun 2011

Cited by 33 | Viewed by 7009

Abstract

Verocytotoxins VT1 and VT2, produced by Verocytotoxigenic Escherichia coli (VTEC), are encoded on temperate bacteriophages. Several studies reported the loss of the vtx genes after multiple subcultivation steps or long preservation. The objective of this study was to determine if the loss of [...] Read more.

Verocytotoxins VT1 and VT2, produced by Verocytotoxigenic Escherichia coli (VTEC), are encoded on temperate bacteriophages. Several studies reported the loss of the vtx genes after multiple subcultivation steps or long preservation. The objective of this study was to determine if the loss of the verocytotoxin genes can already occur during the first subcultivation step. Consequently, the stability of the vtx genes were tested in 40 isolates originating from 40 vtx-positive fecal samples after the first subcultivation step following the isolation procedure. The loss occurred in 12 out of 40 strains tested and was rather rare among the O157 strains compared to the non-O157 strains. This is the first study demonstrating that the loss of the verocytotoxin genes can already occur after the first subcultivation step. This may lead to an underestimation of VTEC positive samples. Full article

7 pages, 219 KB

Open AccessBrief Report

Detection of stx₁ and stx₂ Genes in Pennsylvanian White-Tailed Deer

by Whitney M. Kistler, Surafel Mulugeta and Steven A. Mauro

Toxins 2011, 3(6), 640-646; https://doi.org/10.3390/toxins3060640 - 16 Jun 2011

Cited by 8 | Viewed by 7394

Abstract

Shiga toxin-producing E. coli carrying the stx₁ and/or stx₂ genes can cause multi-symptomatic illness in humans. A variety of terrestrial and aquatic environmental reservoirs of stx have been described. Culture based detection of microbes in deer species have found a low [...] Read more.

Shiga toxin-producing E. coli carrying the stx₁ and/or stx₂ genes can cause multi-symptomatic illness in humans. A variety of terrestrial and aquatic environmental reservoirs of stx have been described. Culture based detection of microbes in deer species have found a low percentage of samples that have tested positive for Stx-producing microbes, suggesting that while deer may contain these microbes, their overall abundance in deer is low. In this study, quantitative PCR (qPCR) was utilized to test for the presence of stx genes in white-tailed deer fecal matter in western Pennsylvania. In this culture independent screening, nearly half of the samples tested positive for the stx₂ gene, with a bias towards samples that were concentrated with stx₂. This study, while limited in scope, suggests that deer may be a greater reservoir for stx than was previously thought. Full article

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14 pages, 257 KB

Open AccessReview

Shiga Toxin Interaction with Human Intestinal Epithelium

by Stephanie Schüller

Toxins 2011, 3(6), 626-639; https://doi.org/10.3390/toxins3060626 - 14 Jun 2011

Cited by 96 | Viewed by 21031

Abstract

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. [...] Read more.

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. Full article

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18 pages, 336 KB

Open AccessReview

Shiga Toxin: Expression, Distribution, and Its Role in the Environment

by Steven A. Mauro and Gerald B. Koudelka

Toxins 2011, 3(6), 608-625; https://doi.org/10.3390/toxins3060608 - 14 Jun 2011

Cited by 64 | Viewed by 12169

Abstract

In this review, we highlight recent work that has increased our understanding of the production and distribution of Shiga toxin in the environment. Specifically, we review studies that offer an expanded view of environmental reservoirs for Shiga toxin producing microbes in terrestrial and [...] Read more.

In this review, we highlight recent work that has increased our understanding of the production and distribution of Shiga toxin in the environment. Specifically, we review studies that offer an expanded view of environmental reservoirs for Shiga toxin producing microbes in terrestrial and aquatic ecosystems. We then relate the abundance of Shiga toxin in the environment to work that demonstrates that the genetic mechanisms underlying the production of Shiga toxin genes are modified and embellished beyond the classical microbial gene regulatory paradigms in a manner that apparently “fine tunes” the trigger to modulate the amount of toxin produced. Last, we highlight several recent studies examining microbe/protist interactions that postulate an answer to the outstanding question of why microbes might harbor and express Shiga toxin genes in the environment. Full article

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