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1
Matussek, Andreas, Joerg Lauber, Anna Bergau, Wiebke Hansen, Manfred Rohde, Kurt E. J. Dittmar, Matthias Gunzer, et al. "Molecular and functional analysis of Shiga toxin–induced response patterns in human vascular endothelial cells." Blood 102, no. 4 (August 15, 2003): 1323–32. http://dx.doi.org/10.1182/blood-2002-10-3301.
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Abstract Enterohemorrhagic Escherichia coli (EHEC) is the major cause of hemolyticuremic syndrome (HUS) characterized by microangiopathic hemolytic anemia, thrombocytopenia, and acute renal failure. EHEC produces one or more Shiga toxins (Stx1 and Stx2), and it was assumed that Stx's only relevant biologic activity was cell destruction through inhibition of protein synthesis. However, recent data indicate that in vivo the cytokine milieu may determine whether endothelial cells survive or undergo apoptosis/necrosis when exposed to Stxs. In this study, we analyzed the genome-wide expression patterns of human endothelial cells stimulated with subinhibitory concentrations of Stxs in order to characterize the genomic expression program involved in the vascular pathology of HUS. We found that Stxs elicited few, but reproducible, changes in gene expression. The majority of genes reported in this study encodes for chemokines and cytokines, which might contribute to the multifaceted inflammatory response of host endothelial cells observed in patients suffering from EHEC disease. In addition, our data provide for the first time molecular insights into the epidemiologically well-established higher pathogenicity of Stx2 over Stx1.
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Lentz, Erin K., Dinorah Leyva-Illades, Moo-Seung Lee, Rama P. Cherla, and Vernon L. Tesh. "Differential Response of the Human Renal Proximal Tubular Epithelial Cell Line HK-2 to Shiga Toxin Types 1 and 2." Infection and Immunity 79, no. 9 (June 27, 2011): 3527–40. http://dx.doi.org/10.1128/iai.05139-11.
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ABSTRACTShiga toxins (Stxs) are expressed by the enteric pathogensShigella dysenteriaeserotype 1 and certain serotypes ofEscherichia coli. Stx-producing bacteria cause bloody diarrhea with the potential to progress to acute renal failure. Stxs are potent protein synthesis inhibitors and are the primary virulence factors responsible for renal damage that may follow diarrheal disease. We explored the use of the immortalized human proximal tubule epithelial cell line HK-2 as anin vitromodel of Stx-induced renal damage. We showed that these cells express abundant membrane Gb3and are differentially susceptible to the cytotoxic action of Stxs, being more sensitive to Shiga toxin type 1 (Stx1) than to Stx2. At early time points (24 h), HK-2 cells were significantly more sensitive to Stxs than Vero cells; however, by 72 h, Vero cell monolayers were completely destroyed while some HK-2 cells survived toxin challenge, suggesting that a subpopulation of HK-2 cells are relatively toxin resistant. Fluorescently labeled Stx1 B subunits localized to both lysosomal and endoplasmic reticulum (ER) compartments in HK-2 cells, suggesting that differences in intracellular trafficking may play a role in susceptibility to Stx-mediated cytotoxicity. Although proinflammatory cytokines were not upregulated by toxin challenge, Stx2 selectively induced the expression of two chemokines, macrophage inflammatory protein-1α (MIP-1α) and MIP-1β. Stx1 and Stx2 differentially activated components of the ER stress response in HK-2 cells. Finally, we demonstrated significant poly(ADP-ribose) polymerase (PARP) cleavage after exposure to Stx1 or Stx2. However, procaspase 3 cleavage was undetectable, suggesting that HK-2 cells may undergo apoptosis in response to Stxs in a caspase 3-independent manner.
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Ogawa, Michinaga, Kensuke Shimizu, Koji Nomoto, Masatoshi Takahashi, Masaaki Watanuki, Ryuichiro Tanaka, Tetsuya Tanaka, Takashi Hamabata, Shinji Yamasaki, and Yoshifumi Takeda. "Protective Effect of Lactobacillus casei Strain Shirota on Shiga Toxin-Producing Escherichia coliO157:H7 Infection in Infant Rabbits." Infection and Immunity 69, no. 2 (February 1, 2001): 1101–8. http://dx.doi.org/10.1128/iai.69.2.1101-1108.2001.
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ABSTRACT We examined colonization patterns of Shiga toxin-producingEscherichia coli (STEC), concentrations of Shiga toxins (Stxs) and specific immunoglobulin A (lgA) against Stxs and STEC bacterial cell surface antigen in various portions of the gastrointestinal tract in an infant rabbit infection model. After inoculation of 3-day-old infant rabbits with STEC strain 89020087 at low doses (∼103 CFU/body), numbers of colonizing STEC bacteria and concentrations of Stxs in the intestine increased dramatically and the animals developed diarrhea within a couple of days after infection. Daily administration ofLactobacillus casei from the day of birth dramatically decreased the severity of diarrhea and lowered STEC colonization levels in the gastrointestinal tract 100-fold day 7 after infection. Both Stx1 and Stx2 concentrations in the intestines and histological damage to the intestinal mucus induced by STEC infection were decreased by the administration of L. casei. Examination of the concentrations of volatile fatty acids and pH of the intestinal contents revealed that the protective effect of L. caseiadministration against STEC infection was not due to fermented products such as lactic acid in the gastrointestinal tract. Administration ofL. casei increased levels of lgAs against Stx1, Stx2, and formalin-killed STEC cells in the colon approximately two-, four-, and threefold, respectively, compared with those of the untreated controls by day 7 after infection. These results suggest that administration ofL. casei strain Shirota enhances the local immune responses to STEC cells and Stxs and leads to elimination of STEC and thus decreases Stx concentrations in the intestines.
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Skinner, Craig, Guodong Zhang, Stephanie Patfield, and Xiaohua He. "AnIn VitroCombined Antibiotic-Antibody Treatment Eliminates Toxicity from Shiga Toxin-Producing Escherichia coli." Antimicrobial Agents and Chemotherapy 59, no. 9 (June 22, 2015): 5435–44. http://dx.doi.org/10.1128/aac.00763-15.
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ABSTRACTTreating Shiga toxin-producingEscherichia coli(STEC) gastrointestinal infections is difficult. The utility of antibiotics for STEC treatment is controversial, since antibiotic resistance among STEC isolates is widespread and certain antibiotics dramatically increase the expression of Shiga toxins (Stxs), which are some of the most important virulence factors in STEC. Stxs contribute to life-threatening hemolytic uremic syndrome (HUS), which develops in considerable proportions of patients with STEC infections. Understanding the antibiotic resistance profiles of STEC isolates and the Stx induction potential of promising antibiotics is essential for evaluating any antibiotic treatment of STEC. In this study, 42 O157:H7 or non-O157 STEC isolates (including the “big six” serotypes) were evaluated for their resistance against 22 antibiotics by using an antibiotic array. Tigecycline inhibited the growth of all of the tested STEC isolates and also inhibited the production of Stxs (Stx2 in particular). In combination with neutralizing antibodies to Stx1 and Stx2, the tigecycline-antibody treatment fully protected Vero cells from Stx toxicity, even when the STEC bacteria and the Vero cells were cultured together. The combination of an antibiotic such as tigecycline with neutralizing antibodies presents a promising strategy for future STEC treatments.
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Thorpe, Cheleste M., Wendy E. Smith, Bryan P. Hurley, and David W. K. Acheson. "Shiga Toxins Induce, Superinduce, and Stabilize a Variety of C-X-C Chemokine mRNAs in Intestinal Epithelial Cells, Resulting in Increased Chemokine Expression." Infection and Immunity 69, no. 10 (October 1, 2001): 6140–47. http://dx.doi.org/10.1128/iai.69.10.6140-6147.2001.
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ABSTRACT Exposure of humans to Shiga toxins (Stxs) is a risk factor for hemolytic-uremic syndrome (HUS). Because Stx-producingEscherichia coli (STEC) is a noninvasive enteric pathogen, the extent to which Stxs can cross the host intestinal epithelium may affect the risk of developing HUS. We have previously shown that Stxs can induce and superinduce IL-8 mRNA and protein in intestinal epithelial cells (IECs) in vitro via a ribotoxic stress response. We used cytokine expression arrays to determine the effect of Stx1 on various C-X-C chemokine genes in IECs. We observed that Stx1 induces multiple C-X-C chemokines at the mRNA level, including interleukin-8 (IL-8), GRO-α, GRO-β, GRO-γ, and ENA-78. Like that of IL-8, GRO-α and ENA-78 mRNAs are both induced and superinduced by Stx1. Furthermore, Stx1 induces both IL-8 and GRO-α protein in a dose-response fashion, despite an overall inhibition in host cell protein synthesis. Stx1 treatment stabilizes both IL-8 and GRO-α mRNA. We conclude that Stxs are able to increase mRNA and protein levels of multiple C-X-C chemokines in IECs, with increased mRNA stability at least one mechanism involved. We hypothesize that ribotoxic stress is a pathway by which Stxs can alter host signal transduction in IECs, resulting in the production of multiple chemokine mRNAs, leading to increased expression of specific proteins. Taken together, these data suggest that exposing IECs to Stxs may stimulate a proinflammatory response, resulting in influx of acute inflammatory cells and thus contributing to the intestinal tissue damage seen in STEC infection.
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Smith, Wendy E., Anne V. Kane, Sausan T. Campbell, David W. K. Acheson, Brent H. Cochran, and Cheleste M. Thorpe. "Shiga Toxin 1 Triggers a Ribotoxic Stress Response Leading to p38 and JNK Activation and Induction of Apoptosis in Intestinal Epithelial Cells." Infection and Immunity 71, no. 3 (March 2003): 1497–504. http://dx.doi.org/10.1128/iai.71.3.1497-1504.2003.
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ABSTRACT Shiga toxins made by Shiga toxin-producing Escherichia coli (STEC) are associated with hemolytic uremic syndrome. Shiga toxins (Stxs) may access the host systemic circulation by absorption across the intestinal epithelium. The effects of Stxs on this cell layer are not completely understood, although animal models of STEC infection suggest that, in the gut, Stxs may participate in both immune activation and apoptosis. Stxs have one enzymatically active A subunit associated with five identical B subunits. The A subunit inactivates ribosomes by cleaving a specific adenine from the 28S rRNA. We have previously shown that Stxs can induce multiple C-X-C chemokines in intestinal epithelial cells in vitro, including interleukin-8 (IL-8), and that Stx-induced IL-8 expression is linked to induction of c-Jun mRNA and p38 mitogen-activated protein (MAP) kinase pathway activity. We now report Stx1 induction of both primary response genes c-jun and c-fos and activation of the stress-activated protein kinases, JNK/SAPK and p38, in the intestinal epithelial cell line HCT-8. By 1 h of exposure to Stx1, mRNAs for c-jun and c-fos are induced, and both JNK and p38 are activated; activation of both kinases persisted up to 24 h. Stx1 enzymatic activity was required for kinase activation; a catalytically defective mutant toxin did not activate either. Stx1 treatment of HCT-8 cells resulted in cell death that was associated with caspase 3 cleavage and internucleosomal DNA fragmentation; this cytotoxicity also required Stx1 enzymatic activity. Blocking Stx1-induced p38 and JNK activation with the inhibitor SB202190 prevented cell death and diminished Stx1-associated caspase 3 cleavage. In summary, these data link the Stx1-induced ribotoxic stress response with both chemokine expression and apoptosis in the intestinal epithelial cell line HCT-8 and suggest that blocking host cell MAP kinases may prevent these Stx-associated events.
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Ishikawa, Satoshi, Kazuyoshi Kawahara, Yutaka Kagami, Yasunori Isshiki, Aki Kaneko, Hidenori Matsui, Nobuhiko Okada, and Hirofumi Danbara. "Protection against Shiga Toxin 1 Challenge by Immunization of Mice with Purified Mutant Shiga Toxin 1." Infection and Immunity 71, no. 6 (June 2003): 3235–39. http://dx.doi.org/10.1128/iai.71.6.3235-3239.2003.
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ABSTRACT Shiga toxin 1 (Stx1) of enterohemorrhagic Escherichia coli O157:H7 was cloned, and four mutant Stx1s were constructed by site-directed mutagenesis with PCR. The wild-type and mutant Stx1s with amino acid replacements at positions 167 and 170 of the A subunit were purified by one-step affinity chromatography with commercially available Globotriose Fractogel, and the mutant Stxs were used for the immunization of mice. The mutant toxins were nontoxic to Vero cells in vitro and to mice in vivo and induced the immunoglobulin G antibody against the wild-type Stx1, which neutralized the cytotoxicity of Stx1. The induced antibody titers depended on the mutation at position 170 of the A subunit. The mice immunized with the mutant Stx1s were protected against a challenge of approximately 100 times the 50% lethal dose of the wild-type Stx1, suggesting that the mutant toxins are good candidates for toxoid vaccines for infection by Stx1-producing E. coli.
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Leyva-Illades, Dinorah, Rama P. Cherla, Moo-Seung Lee, and Vernon L. Tesh. "Regulation of Cytokine and Chemokine Expression by the Ribotoxic Stress Response Elicited by Shiga Toxin Type 1 in Human Macrophage-Like THP-1 Cells." Infection and Immunity 80, no. 6 (March 19, 2012): 2109–20. http://dx.doi.org/10.1128/iai.06025-11.
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ABSTRACTShiga toxins (Stxs) are cytotoxins produced by the enteric pathogensShigella dysenteriaeserotype 1 and Shiga toxin-producingEscherichia coli(STEC). Stxs bind to a membrane glycolipid receptor, enter cells, and undergo retrograde transport to ultimately reach the cytosol, where the toxins exert their protein synthesis-inhibitory activity by depurination of a single adenine residue from the 28S rRNA component of eukaryotic ribosomes. The depurination reaction activates the ribotoxic stress response, leading to signaling via the mitogen-activated protein kinase (MAPK) pathways (Jun N-terminal protein kinase [JNK], p38, and extracellular signal-regulated kinase [ERK]) in human epithelial, endothelial, and myeloid cells. We previously showed that treatment of human macrophage-like THP-1 cells with Stxs resulted in increased cytokine and chemokine expression. In the present study, we show that individual inactivation of ERK, JNK, and p38 MAPKs using pharmacological inhibitors in the presence of Stx1 resulted in differential regulation of the cytokines tumor necrosis factor alpha and interleukin-1β (IL-1β) and chemokines IL-8, growth-regulated protein-β, macrophage inflammatory protein-1α (MIP-1α), and MIP-1β. THP-1 cells exposed to Stx1 upregulate the expression of select dual-specificity phosphatases (DUSPs), enzymes that dephosphorylate and inactivate MAPKs in mammalian cells. In this study, we confirmed DUSP1 protein production by THP-1 cells treated with Stx1. DUSP1 inhibition by triptolide showed that ERK and p38 phosphorylation is regulated by DUSP1, while JNK phosphorylation is not. Inhibition of p38 MAPK signaling blocked the ability of Stx1 to induce DUSP1 mRNA expression, suggesting that an autoregulatory signaling loop may be activated by Stxs. Thus, Stxs appear to be capable of eliciting signals which both activate and deactivate signaling for increased cytokine/chemokine production in human macrophage-like cells.
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Hurley, Bryan P., Cheleste M. Thorpe, and David W. K. Acheson. "Shiga Toxin Translocation across Intestinal Epithelial Cells Is Enhanced by Neutrophil Transmigration." Infection and Immunity 69, no. 10 (October 1, 2001): 6148–55. http://dx.doi.org/10.1128/iai.69.10.6148-6155.2001.
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ABSTRACT Shiga toxin-producing E. coli (STEC) is a food-borne pathogen that causes serious illness, including hemolytic-uremic syndrome (HUS). STEC colonizes the lower intestine and produces Shiga toxins (Stxs). Stxs appear to translocate across intestinal epithelia and affect sensitive endothelial cell beds at various sites. We have previously shown that Stxs cross polarized intestinal epithelial cells (IECs) via a transcellular route and remain biologically active. Since acute inflammatory infiltration of the gut and fecal leukocytes is seen in many STEC-infected patients and since polymorphonuclear leukocyte (PMN) transmigration across polarized IECs diminishes the IEC barrier function in vitro, we hypothesized that PMN transmigration may enhance Stx movement across IECs. We found that basolateral-to-apical transmigration of neutrophils significantly increased the movement of Stx1 and Stx2 across polarized T84 IECs in the opposite direction. The amount of Stx crossing the T84 barrier was proportional to the degree of neutrophil transmigration, and the increase in Stx translocation appears to be due to increases in paracellular permeability caused by migrating PMNs. STEC clinical isolates applied apically induced PMN transmigration across and interleukin-8 (IL-8) secretion from T84 cells. Of the 10 STEC strains tested, three STEC strains lackingeae and espB (eae- andespB-negative STEC strains) induced significantly more neutrophil transmigration and significantly greater IL-8 secretion thaneae- and espB-positive STEC or enteropathogenic E. coli. This study suggests that STEC interaction with intestinal epithelia induces neutrophil recruitment to the intestinal lumen, resulting in neutrophil extravasation across IECs, and that during this process Stxs may pass in greater amounts into underlying tissues, thereby increasing the risk of HUS.
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Leyva-Illades, Dinorah, Rama P. Cherla, Cristi L. Galindo, Ashok K. Chopra, and Vernon L. Tesh. "Global Transcriptional Response of Macrophage-Like THP-1 Cells to Shiga Toxin Type 1." Infection and Immunity 78, no. 6 (March 29, 2010): 2454–65. http://dx.doi.org/10.1128/iai.01341-09.
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ABSTRACT Shiga toxins (Stxs) are bacterial cytotoxins produced by the enteric pathogens Shigella dysenteriae serotype 1 and some serotypes of Escherichia coli that cause bacillary dysentery and hemorrhagic colitis, respectively. To date, approaches to studying the capacity of Stxs to alter gene expression in intoxicated cells have been limited to individual genes. However, it is known that many of the signaling pathways activated by Stxs regulate the expression of multiple genes in mammalian cells. To expand the scope of analysis of gene expression and to better understand the underlying mechanisms for the various effects of Stxs on host cell functions, we carried out comparative microarray analyses to characterize the global transcriptional response of human macrophage-like THP-1 cells to Shiga toxin type 1 (Stx1) and lipopolysaccharides. The data were analyzed by using a rigorous combinatorial approach with three separate statistical algorithms. A total of 36 genes met the criteria of upregulated expression in response to Stx1 treatment, with 14 genes uniquely upregulated by Stx1. Microarray data were validated by real-time reverse transcriptase PCR for genes encoding early growth response 1 (Egr-1) (transcriptional regulator), cyclooxygenase 2 (COX-2; inflammation), and dual specificity phosphatase 1 (DUSP1), DUSP5, and DUSP10 (regulation of mitogen-activated protein kinase signaling). Stx1-mediated signaling through extracellular signal-regulated kinase 1/2 and Egr-1 appears to be involved in the increased expression and production of the proinflammatory mediator tumor necrosis factor alpha. Activation of COX-2 is associated with the increased production of proinflammatory and vasoactive eicosanoids. However, the capacity of Stx1 to increase the expression of genes encoding phosphatases suggests that mechanisms to dampen the macrophage proinflammatory response may be built into host response to the toxins.
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Storck, Wiebke, Iris Meisen, Kathrin Gianmoena, Ina Pläger, Ivan U. Kouzel, Martina Bielaszewska, Jörg Haier, et al. "Shiga toxin glycosphingolipid receptor expression and toxin susceptibility of human pancreatic ductal adenocarcinomas of differing origin and differentiation." Biological Chemistry 393, no. 8 (August 1, 2012): 785–99. http://dx.doi.org/10.1515/hsz-2012-0165.
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Abstract Shiga toxins (Stxs) are composed of an enzymatically active A subunit (StxA) and a pentameric B subunit (StxB) that preferentially binds to the glycosphingolipid (GSL) globo\xadtriaosylceramide (Gb3Cer/CD77) and to a reduced extent to globotetraosylceramide (Gb4Cer). The identification of Gb3Cer as a tumor-associated GSL in human pancreatic cancer prompted us to investigate the expression of Gb3Cer and Gb4Cer in 15 human pancreatic ductal adenocarcinoma cell lines derived from primary tumors and liver, ascites, and lymph node metastases. Thin-layer chromatography overlay assays revealed the occurrence of Gb3Cer in all and of Gb4Cer in the majority of cell lines, which largely correlated with transcriptional expression analysis of Gb3Cer and Gb4Cer synthases. Prominent Gb3Cer and Gb4Cer lipoform heterogeneity was based on ceramides carrying predominantly C16:0 and C24:0/C24:1 fatty acids. Stx2-mediated cell injury ranged from extremely high sensitivity (CD50 of 0.94 pg/ml) to high refractiveness (CD50 of 5.8 μg/ml) and to virtual resistance portrayed by non-determinable CD50 values even at the highest Stx2 concentration (10 μg/ml) applied. Importantly, Stx2-mediated cytotoxicity did not correlate with Gb3Cer expression (the preferential Stx receptor), suggesting that the GSL receptor content does not primarily determine cell sensitivity and that other, yet to be delineated, cellular factors might influence the responsiveness of cancer cells.
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Harrison, Lisa M., Wilhelmina C. E. van Haaften, and Vernon L. Tesh. "Regulation of Proinflammatory Cytokine Expression by Shiga Toxin 1 and/or Lipopolysaccharides in the Human Monocytic Cell Line THP-1." Infection and Immunity 72, no. 5 (May 2004): 2618–27. http://dx.doi.org/10.1128/iai.72.5.2618-2627.2004.
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ABSTRACT Infection with Shiga toxin (Stx)-producing bacteria and the subsequent release of Stxs and endotoxins into the bloodstream may damage blood vessels in the colon, kidneys, and central nervous system, leading to bloody diarrhea, acute renal failure, and neurological complications. The proinflammatory cytokines tumor necrosis factor alpha (TNF-α) and interleukin-1β (IL-1β) may contribute to the pathogenesis of Stx-induced vascular lesions by up-regulating toxin receptor expression on endothelial cells. We previously showed that macrophages treated with purified Shiga toxin 1 (Stx1) or lipopolysaccharides (LPS) secrete TNF-α and IL-1β. Northern blot analysis revealed that treatment of the human monocytic cell line THP-1 with LPS induced a rapid and transient increase in steady-state TNF-α and IL-1β transcripts. In contrast, Stx1 induced slower but prolonged elevations in cytokine transcripts. The presence of both stimulants resulted in optimal cytokine mRNA induction in terms of kinetics and prolonged expression. Compared to LPS, Stx1 was a poor inducer of IL-1β protein expression, although levels of soluble IL-1β induced by all treatments continually increased over 72 h. IL-1β transcripts were not induced by Stx1 B-subunits. Using the transcriptional inhibitor actinomycin D, we determined that treatment with Stx1 or Stx1 plus LPS induced cytokine transcripts with increased stability compared to transcripts induced by LPS alone. For all treatments, IL-1β mRNA decay was slower than TNF-α. Collectively, our data suggest that Stxs affect cytokine expression, in part, at the posttranscriptional level by stabilizing mRNAs. Optimal TNF-α expression occurs when both Stxs and LPS are present.
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Lee, Sang-Yun, Rama P. Cherla, Isa Caliskan, and Vernon L. Tesh. "Shiga Toxin 1 Induces Apoptosis in the Human Myelogenous Leukemia Cell Line THP-1 by a Caspase-8-Dependent, Tumor Necrosis Factor Receptor-Independent Mechanism." Infection and Immunity 73, no. 8 (August 2005): 5115–26. http://dx.doi.org/10.1128/iai.73.8.5115-5126.2005.
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ABSTRACT Shiga toxins (Stxs) induce apoptosis in a variety of cell types. Here, we show that Stx1 induces apoptosis in the undifferentiated myelogenous leukemia cell line THP-1 in the absence of tumor necrosis factor alpha (TNF-α) or death receptor (TNF receptor or Fas) expression. Caspase-8 and -3 inhibitors blocked, and caspase-6 and -9 inhibitors partially blocked, Stx1-induced apoptosis. Stx1 induced the mitochondrial pathway of apoptosis, as activation of caspase-8 triggered the (i) cleavage of Bid, (ii) disruption of mitochondrial membrane potential, and (iii) release of cytochrome c into the cytoplasm. Caspase-8, -9, and -3 cleavage and functional activities began 4 h after toxin exposure and peaked after 8 h of treatment. Caspase-6 may also contribute to Stx1-induced apoptosis by directly acting on caspase-8. It appears that functional Stx1 holotoxins must be transported to the endoplasmic reticulum to initiate apoptotic signaling through the ribotoxic stress response. These data suggest that Stxs may activate monocyte apoptosis via a novel caspase-8-dependent, death receptor-independent mechanism.
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Debernardi, Justine, Catherine Pioche-Durieu, Eric Le Cam, Joëlle Wiels, and Aude Robert. "Verotoxin-1-Induced ER Stress Triggers Apoptotic or Survival Pathways in Burkitt Lymphoma Cells." Toxins 12, no. 5 (May 11, 2020): 316. http://dx.doi.org/10.3390/toxins12050316.
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Shiga toxins (Stxs) expressed by the enterohaemorrhagic Escherichia coli and enteric Shigella dysenteriae 1 pathogens are protein synthesis inhibitors. Stxs have been shown to induce apoptosis via the activation of extrinsic and intrinsic pathways in many cell types (epithelial, endothelial, and B cells) but the link between the protein synthesis inhibition and caspase activation is still unclear. Endoplasmic reticulum (ER) stress induced by the inhibition of protein synthesis may be this missing link. Here, we show that the treatment of Burkitt lymphoma (BL) cells with verotoxin-1 (VT-1 or Stx1) consistently induced the ER stress response by activation of IRE1 and ATF6—two ER stress sensors—followed by increased expression of the transcription factor C/REB homologous protein (CHOP). However, our data suggest that, although ER stress is systematically induced by VT-1 in BL cells, its role in cell death appears to be cell specific and can be the opposite: ER stress may enhance VT-1-induced apoptosis through CHOP or play a protective role through ER-phagy, depending on the cell line. Several engineered Stxs are currently under investigation as potential anti-cancer agents. Our results suggest that a better understanding of the signaling pathways induced by Stxs is needed before using them in the clinic.
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Ritchie, Jenny M., Patrick L. Wagner, David W. K. Acheson, and Matthew K. Waldor. "Comparison of Shiga Toxin Production by Hemolytic-Uremic Syndrome-Associated and Bovine-Associated Shiga Toxin-Producing Escherichia coli Isolates." Applied and Environmental Microbiology 69, no. 2 (February 2003): 1059–66. http://dx.doi.org/10.1128/aem.69.2.1059-1066.2003.
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ABSTRACT There is considerable diversity among Shiga toxin (Stx)-producing Escherichia coli (STEC) bacteria, and only a subset of these organisms are thought to be human pathogens. The characteristics that distinguish STEC bacteria that give rise to human disease are not well understood. Stxs, the principal virulence determinants of STEC, are thought to account for hemolytic-uremic syndrome (HUS), a severe clinical consequence of STEC infection. Stxs are typically bacteriophage encoded, and their production has been shown to be enhanced by prophage-inducing agents such as mitomycin C in a limited number of clinical STEC isolates. Low iron concentrations also enhance Stx production by some clinical isolates; however, little is known regarding whether and to what extent these stimuli regulate Stx production by STEC associated with cattle, the principal environmental reservoir of STEC. In this study, we investigated whether toxin production differed between HUS- and bovine-associated STEC strains. Basal production of Stx by HUS-associated STEC exceeded that of bovine-associated STEC. In addition, following mitomycin C treatment, Stx2 production by HUS-associated STEC was significantly greater than that by bovine-associated STEC. Unexpectedly, mitomycin C treatment had a minimal effect on Stx1 production by both HUS- and bovine-associated STEC. However, Stx1 production was induced by growth in low-iron medium, and induction was more marked for HUS-associated STEC than for bovine-associated STEC. These observations reveal that disease-associated and bovine-associated STEC bacteria differ in their basal and inducible Stx production characteristics.
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Betz, Josefine, Andreas Bauwens, Lisa Kunsmann, Martina Bielaszewska, Michael Mormann, Hans-Ulrich Humpf, Helge Karch, Alexander W. Friedrich, and Johannes Müthing. "Uncommon membrane distribution of Shiga toxin glycosphingolipid receptors in toxin-sensitive human glomerular microvascular endothelial cells." Biological Chemistry 393, no. 3 (March 1, 2012): 133–47. http://dx.doi.org/10.1515/hsz-2011-0288.
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Abstract Membrane microdomain association of the glycosphingolipids (GSLs) globotriaosylceramide (Gb3Cer) and globotetraosylceramide (Gb4Cer), the highly and less effective receptors, respectively, for Shiga toxins (Stxs), is assumed as a functional requirement for Stx-mediated cytotoxicity. In a previous study, we demonstrated predominant localization of Stx receptors in cholesterol-enriched membrane microdomains of moderately Stx-sensitive human brain microvascular endothelial cells (HBMECs) by means of detergent-resistant membranes (DRMs). Here we report a different preferential distribution of Stx receptors in non-DRM fractions of human glomerular microvascular endothelial cells (GMVECs), the major targets of Stxs in the human kidney. Full structural characterization of Stx receptors using electrospray ionization (ESI) mass spectrometry revealed Gb3Cer and Gb4Cer lipoforms with ceramide moieties mainly composed of C24:0/C24:1 or C16:0 fatty acid and sphingosine (d18:1) in GMVECs comparable to those previously found in HBMECs. Thin-layer chromatography immunostaining demonstrated an approximately 2-fold higher content of Gb3Cer and a 1.4-fold higher content of Gb4Cer in GMVECs than in HBMECs. However, this does not explain the remarkable higher cytotoxic action of Stx1 and Stx2 toward GMVECs as compared with HBMECs. Our finding opens new questions on the microdomain association of Stx receptors and the functional role of GSLs in the membrane assembly of GMVECs.
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Dong, Jing, Yong Zhang, Yutao Chen, Xiaodi Niu, Yu Zhang, Cheng Yang, Quan Wang, Xuemei Li, and Xuming Deng. "Baicalin Inhibits the Lethality of Shiga-Like Toxin 2 in Mice." Antimicrobial Agents and Chemotherapy 59, no. 11 (September 8, 2015): 7054–60. http://dx.doi.org/10.1128/aac.01416-15.
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ABSTRACTShiga-like toxins (Stxs), produced by pathogenicEscherichia coli, are a major virulence factor involved in severe diseases in human and animals. These toxins are ribosome-inactivating proteins, and treatment for diseases caused by them is not available. Therefore, there is an urgent need for agents capable of effectively targeting this lethal toxin. In this study, we identified baicalin, a flavonoid compound used in Chinese traditional medicine, as a compound against Shiga-like toxin 2 (Stx2). We found that baicalin significantly improves renal function and reduces Stx2-induced lethality in mice. Further experiments revealed that baicalin induces the formation of oligomers by the toxin by direct binding. We also identified the residues important for such interactions and analyzed their roles in binding baicalin by biophysical and biochemical analyses. Our results establish baicalin as a candidate compound for the development of therapeutics against diseases caused by Stxs.
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Lee, Moo-Seung, Rama P. Cherla, Dinorah Leyva-Illades, and Vernon L. Tesh. "Bcl-2 Regulates the Onset of Shiga Toxin 1-Induced Apoptosis in THP-1 Cells." Infection and Immunity 77, no. 12 (September 14, 2009): 5233–44. http://dx.doi.org/10.1128/iai.00665-09.
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ABSTRACT Shiga toxins (Stxs), which are proteins expressed by the enteric pathogens Shigella dysenteriae serotype 1 and some serotypes of Escherichia coli, are potent protein synthesis inhibitors. Stx-producing organisms cause bloody diarrhea with the potential to progress to acute renal failure and central nervous system complications. Studies using animal models of these diseases have shown that Stxs are major virulence factors, and purified toxins have been shown to be capable of killing many types of cells in vitro. We showed that Stx type 1 (Stx1) rapidly induced apoptosis in undifferentiated, monocytic THP-1 cells through a mechanism involving the endoplasmic reticulum (ER) stress response. Rapid apoptosis correlated with increased expression of C/EBP homologous protein (CHOP), TRAIL, and DR5, while expression of the antiapoptotic factor Bcl-2 was downregulated. Stx1 treatment of differentiated, macrophage-like THP-1 cells was associated with cytokine production and delayed apoptosis. The mechanisms contributing to cell maturation-dependent differences in responses to Stx1 are unknown. We show here that in macrophage-like cells, Stx1 activated the proximal ER stress sensors RNA-dependent protein kinase-like ER kinase and inositol-requiring ER signal kinase 1α but did not activate activating transcription factor 6. Proapoptotic signaling pathways mediated by CHOP and by Bax and Bak were activated by Stx1. However, the toxin also activated prosurvival signaling through increased expression, mitochondrial translocation, and alternative phosphorylation of Bcl-2.
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Nakao, Hiroshi, Nobutaka Kiyokawa, Junichiro Fujimoto, Shinji Yamasaki, and Tae Takeda. "Monoclonal Antibody to Shiga Toxin 2 Which Blocks Receptor Binding and Neutralizes Cytotoxicity." Infection and Immunity 67, no. 11 (November 1, 1999): 5717–22. http://dx.doi.org/10.1128/iai.67.11.5717-5722.1999.
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ABSTRACT A monoclonal antibody (MAb) was raised against Shiga toxin 2 (Stx2) of Escherichia coli O157:H7. MAb VTm1.1 belonged to the immunoglobulin G1 subclass and had a κ light chain, and it could neutralize the cytotoxic activity of Stx2 and variants derived from patient strains but not that of variants derived from animals. MAb VTm1.1 was shown to bind to the B subunit of these neutralized Stx2s by Western blotting. Comparison of B-subunit amino acid sequences and reactivities to these Stxs suggested six amino acids (Ser30, Ser53, Glu56, Gln65, Asn68, and Asp69) that were candidates for the MAb VTm1.1 epitope. Consequently, five Stx2 mutants (S30N, S53N, E56H, Q65K, and N68Ter) were prepared by site-directed mutagenesis to determine which residue is essential for the epitope. All of these mutants showed cytotoxicity almost equal to that of the wild-type Stx2. Of the five Stx2 mutants, only E56H could not be neutralized by MAb VTm1.1. Western blot analysis also showed that MAb VTm1.1 could not bind to the E56H B subunit. These results indicated that Glu56 is an important residue recognized by MAb VTm1.1. Immunofluorescence analysis further indicated that MAb VTm1.1 inhibits the binding of Stx2 to its receptors. MAb VTm1.1 could be a useful therapeutic agent for Shiga toxin-producingE. coli infection.
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Robert, Aude, and Joëlle Wiels. "Shiga Toxins as Antitumor Tools." Toxins 13, no. 10 (September 28, 2021): 690. http://dx.doi.org/10.3390/toxins13100690.
Full textAbstract:
Shiga toxins (Stxs), also known as Shiga-like toxins (SLT) or verotoxins (VT), constitute a family of structurally and functionally related cytotoxic proteins produced by the enteric pathogens Shigella dysenteriae type 1 and Stx-producing Escherichia coli (STEC). Infection with these bacteria causes bloody diarrhea and other pathological manifestations that can lead to HUS (hemolytic and uremic syndrome). At the cellular level, Stxs bind to the cellular receptor Gb3 and inhibit protein synthesis by removing an adenine from the 28S rRNA. This triggers multiple cellular signaling pathways, including the ribotoxic stress response (RSR), unfolded protein response (UPR), autophagy and apoptosis. Stxs cause several pathologies of major public health concern, but their specific targeting of host cells and efficient delivery to the cytosol could potentially be exploited for biomedical purposes. Moreover, high levels of expression have been reported for the Stxs receptor, Gb3/CD77, in Burkitt’s lymphoma (BL) cells and on various types of solid tumors. These properties have led to many attempts to develop Stxs as tools for biomedical applications, such as cancer treatment or imaging, and several engineered Stxs are currently being tested. We provide here an overview of these studies.
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Zhu, Hongchen, Takayuki Sonoyama, Misako Yamada, Wei Gao, Ryohei Tatsuno, Tomohiro Takatani, and Osamu Arakawa. "Co-Occurrence of Tetrodotoxin and Saxitoxins and Their Intra-Body Distribution in the Pufferfish Canthigaster valentini." Toxins 12, no. 7 (July 3, 2020): 436. http://dx.doi.org/10.3390/toxins12070436.
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Pufferfish of the family Tetraodontidae possess tetrodotoxin (TTX) and/or saxitoxins (STXs), but the toxin ratio differs, depending on the genus or species. In the present study, to clarify the distribution profile of TTX and STXs in Tetraodontidae, we investigated the composition and intra-body distribution of the toxins in Canthigaster valentini. C. valentini specimens (four male and six female) were collected from Amami-Oshima Island, Kagoshima Prefecture, Japan, and the toxins were extracted from the muscle, liver, intestine, gallbladder, gonads, and skin. Analysis of the extracts for TTX by liquid chromatography tandem mass spectrometry and of STXs by high-performance liquid chromatography with post-column fluorescence derivatization revealed TTX, as well as a large amount of STXs, with neoSTX as the main component and dicarbamoylSTX and STX itself as minor components, in the skin and ovary. The toxins were also detected in the other tissues, but in much lower amounts than in the skin and ovary. The TTX/STX ratio varied greatly, depending on the tissue, but TTX was the major toxin component in the whole body, and STXs accounted for 25% and 13% of the total toxin amount in males and females, respectively. Like the marine pufferfish of the genus Arothron, C. valentini should be considered a pufferfish with considerable amounts of both TTX and STXs present simultaneously.
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Dean, Karl J., Ryan P. Alexander, Robert G. Hatfield, Adam M. Lewis, Lewis N. Coates, Tom Collin, Mickael Teixeira Alves, et al. "The Common Sunstar Crossaster papposus—A Neurotoxic Starfish." Marine Drugs 19, no. 12 (December 7, 2021): 695. http://dx.doi.org/10.3390/md19120695.
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Saxitoxins (STXs) are a family of potent neurotoxins produced naturally by certain species of phytoplankton and cyanobacteria which are extremely toxic to mammalian nervous systems. The accumulation of STXs in bivalve molluscs can significantly impact animal and human health. Recent work conducted in the North Sea highlighted the widespread presence of various saxitoxins in a range of benthic organisms, with the common sunstar (Crossaster papposus) demonstrating high concentrations of saxitoxins. In this study, an extensive sampling program was undertaken across multiple seas surrounding the UK, with 146 starfish and 5 brittlestars of multiple species analysed for STXs. All the common sunstars analysed (n > 70) contained quantifiable levels of STXs, with the total concentrations ranging from 99 to 11,245 µg STX eq/kg. The common sunstars were statistically different in terms of toxin loading to all the other starfish species tested. Two distinct toxic profiles were observed in sunstars, a decarbomylsaxitoxin (dcSTX)-dominant profile which encompassed samples from most of the UK coast and an STX and gonyautoxin2 (GTX2) profile from the North Yorkshire coast of England. Compartmentalisation studies demonstrated that the female gonads exhibited the highest toxin concentrations of all the individual organs tested, with concentrations >40,000 µg STX eq/kg in one sample. All the sunstars, male or female, exhibited the presence of STXs in the skin, digestive glands and gonads. This study highlights that the common sunstar ubiquitously contains STXs, independent of the geographical location around the UK and often at concentrations many times higher than the current regulatory limits for STXs in molluscs; therefore, the common sunstar should be considered toxic hereafter.
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Sakiri, Ramesh, Belakere Ramegowda, and Vernon L. Tesh. "Shiga Toxin Type 1 Activates Tumor Necrosis Factor-α Gene Transcription and Nuclear Translocation of the Transcriptional Activators Nuclear Factor-κB and Activator Protein-1." Blood 92, no. 2 (July 15, 1998): 558–66. http://dx.doi.org/10.1182/blood.v92.2.558.
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Abstract Shiga toxins (Stxs) produced by Shigella dysenteriae 1 andEscherichia coli have been implicated in the pathogenesis of bloody diarrhea, acute renal failure, and neurologic abnormalities. The pathologic hallmark of Stx-mediated tissue damage is the development of vascular lesions in which endothelial cells are swollen and detached from underlying basement membranes. However, in vitro studies using human vascular endothelial cells demonstrated minimal Stx-induced cytopathic effects, unless the target cells were also incubated with the proinflammatory cytokines tumor necrosis factor-α (TNF-α) or interleukin-1β (IL-1β). These cytokines have been shown to upregulate the expression of the Stx-binding membrane glycolipid globotriaosylceramide (Gb3). We show here that purified Stx1 induces TNF secretion by a human monocytic cell line, THP-1, in a dose- and time-dependent manner. Treatment of cells with both lipopolysaccharides (LPS) and Stx1 results in augmented TNF production. Treatment with the nontoxic Gb3-binding subunit of Stx1 or with an anti-Gb3 monoclonal antibody did not trigger TNF production. Northern blot analyses show that Stx1 causes increased TNF-α production through transcriptional activation. Increased levels of TNF-α mRNA are preceded by the nuclear translocation of the transcriptional activators NF-κB and AP-1 and the loss of cytoplasmic IκB-α. These data are the first to show that, in addition to direct cytotoxicity, Stxs possess cellular signaling capabilities sufficient to induce the synthesis of cytokines that may be necessary for target cell sensitization and the development of vascular lesions.
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Sakiri, Ramesh, Belakere Ramegowda, and Vernon L. Tesh. "Shiga Toxin Type 1 Activates Tumor Necrosis Factor-α Gene Transcription and Nuclear Translocation of the Transcriptional Activators Nuclear Factor-κB and Activator Protein-1." Blood 92, no. 2 (July 15, 1998): 558–66. http://dx.doi.org/10.1182/blood.v92.2.558.414k35_558_566.
Full textAbstract:
Shiga toxins (Stxs) produced by Shigella dysenteriae 1 andEscherichia coli have been implicated in the pathogenesis of bloody diarrhea, acute renal failure, and neurologic abnormalities. The pathologic hallmark of Stx-mediated tissue damage is the development of vascular lesions in which endothelial cells are swollen and detached from underlying basement membranes. However, in vitro studies using human vascular endothelial cells demonstrated minimal Stx-induced cytopathic effects, unless the target cells were also incubated with the proinflammatory cytokines tumor necrosis factor-α (TNF-α) or interleukin-1β (IL-1β). These cytokines have been shown to upregulate the expression of the Stx-binding membrane glycolipid globotriaosylceramide (Gb3). We show here that purified Stx1 induces TNF secretion by a human monocytic cell line, THP-1, in a dose- and time-dependent manner. Treatment of cells with both lipopolysaccharides (LPS) and Stx1 results in augmented TNF production. Treatment with the nontoxic Gb3-binding subunit of Stx1 or with an anti-Gb3 monoclonal antibody did not trigger TNF production. Northern blot analyses show that Stx1 causes increased TNF-α production through transcriptional activation. Increased levels of TNF-α mRNA are preceded by the nuclear translocation of the transcriptional activators NF-κB and AP-1 and the loss of cytoplasmic IκB-α. These data are the first to show that, in addition to direct cytotoxicity, Stxs possess cellular signaling capabilities sufficient to induce the synthesis of cytokines that may be necessary for target cell sensitization and the development of vascular lesions.
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Guessous, Fadila, Marek Marcinkiewicz, Renata Polanowska-Grabowska, Sudawadee Kongkhum, Daniel Heatherly, Tom Obrig, and Adrian R. L. Gear. "Shiga Toxin 2 and Lipopolysaccharide Induce Human Microvascular Endothelial Cells To Release Chemokines and Factors That Stimulate Platelet Function." Infection and Immunity 73, no. 12 (December 2005): 8306–16. http://dx.doi.org/10.1128/iai.73.12.8306-8316.2005.
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ABSTRACT Shiga toxins (Stxs) produced by Shigella dysenteriae type 1 and enterohemorrhagic Escherichia coli are the most common cause of hemolytic-uremic syndrome (HUS). It is well established that vascular endothelial cells, mainly those located in the renal microvasculature, are targets for Stxs. The aim of the present research was to evaluate whether E. coli-derived Shiga toxin 2 (Stx2) incubated with human microvascular endothelial cells (HMEC-1) induces release of chemokines and other factors that might stimulate platelet function. HMEC-1 were exposed for 24 h in vitro to Stx2, lipopolysaccharide (LPS), or the Stx2-LPS combination, and chemokine production was assessed by immunoassay. More interleukin-8 was released than stromal cell-derived factor 1α (SDF-1α) or SDF-1β and RANTES. The Stx2-LPS combination potentiated chemokine release, but Stx2 alone caused more release of SDF-1α at 24 h than LPS or Stx2-LPS did. In the presence of low ADP levels, HMEC-1 supernatants activated platelet function assessed by classical aggregometry, single-particle counting, granule secretion, P-selectin exposure, and the formation of platelet-monocyte aggregates. Supernatants from HMEC-1 exposed only to Stx2 exhibited enhanced exposure of platelet P-selectin and platelet-THP-1 cell interactions. Blockade of platelet cyclooxygenase by indomethacin prevented functional activation. The chemokine RANTES enhanced platelet aggregation induced by SDF-1α, macrophage-derived chemokine, or thymus and activation-regulated chemokine in the presence of very low ADP levels. These data support the hypothesis that microvascular endothelial cells exposed to E. coli O157:H7-derived Stx2 and LPS release chemokines and other factors, which when combined with low levels of primary agonists, such as ADP, cause platelet activation and promote the renal thrombosis associated with HUS.
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Zhang, Yuchengmin, Hideto Tsutsui, Nobuhiro Yamawaki, Yasuhiro Morii, Gregory N. Nishihara, Shiro Itoi, Osamu Arakawa, and Tomohiro Takatani. "Geographic Variations in the Toxin Profile of the Xanthid Crab Zosimus aeneus in a Single Reef on Ishigaki Island, Okinawa, Japan." Marine Drugs 19, no. 12 (November 26, 2021): 670. http://dx.doi.org/10.3390/md19120670.
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Toxic crabs of the family Xanthidae contain saxitoxins (STXs) and/or tetrodotoxin (TTX), but the toxin ratio differs depending on their habitat. In the present study, to clarify within reef variations in the toxin profile of xanthid crabs, we collected specimens of the toxic xanthid crab Zosimus aeneus and their sampling location within a single reef (Yoshihara reef) on Ishigaki Island, Okinawa Prefecture, Japan, in 2018 and 2019. The STXs/TTX content within the appendages and viscera or stomach contents of each specimen was determined by instrumental analyses. Our findings revealed the existence of three zones in Yoshihara reef; one in which many individuals accumulate extremely high concentrations of STXs (northwestern part of the reef; NW zone), another in which individuals generally have small amounts of TTX but little STXs (central part of the reef; CTR zone), and a third in which individuals generally exhibit intermediate characteristics (southeastern part of the reef; SE zone). Furthermore, light microscopic observations of the stomach contents of crab specimens collected from the NW and CTR zones revealed that ascidian spicules of the genus Lissoclinum were dominant in the NW zone, whereas those of the genus Trididemnum were dominant in the CTR zone. Although the toxicity of these ascidians is unknown, Lissoclinum ascidians are considered good candidate source organisms of STXs harbored by toxic xanthid crabs.
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Menge, Christian. "The Role of Escherichia coli Shiga Toxins in STEC Colonization of Cattle." Toxins 12, no. 9 (September 21, 2020): 607. http://dx.doi.org/10.3390/toxins12090607.
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Many cattle are persistently colonized with Shiga toxin-producing Escherichia coli (STEC) and represent a major source of human infections with human-pathogenic STEC strains (syn. enterohemorrhagic E. coli (EHEC)). Intervention strategies most effectively protecting humans best aim at the limitation of bovine STEC shedding. Mechanisms enabling STEC to persist in cattle are only partialy understood. Cattle were long believed to resist the detrimental effects of Shiga toxins (Stxs), potent cytotoxins acting as principal virulence factors in the pathogenesis of human EHEC-associated diseases. However, work by different groups, summarized in this review, has provided substantial evidence that different types of target cells for Stxs exist in cattle. Peripheral and intestinal lymphocytes express the Stx receptor globotriaosylceramide (Gb3syn. CD77) in vitro and in vivo in an activation-dependent fashion with Stx-binding isoforms expressed predominantly at early stages of the activation process. Subpopulations of colonic epithelial cells and macrophage-like cells, residing in the bovine mucosa in proximity to STEC colonies, are also targeted by Stxs. STEC-inoculated calves are depressed in mounting appropriate cellular immune responses which can be overcome by vaccination of the animals against Stxs early in life before encountering STEC. Considering Stx target cells and the resulting effects of Stxs in cattle, which significantly differ from effects implicated in human disease, may open promising opportunities to improve existing yet insufficient measures to limit STEC carriage and shedding by the principal reservoir host.
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Wagner, Patrick L., Melody N. Neely, Xiaoping Zhang, David W. K. Acheson, Matthew K. Waldor, and David I. Friedman. "Role for a Phage Promoter in Shiga Toxin 2 Expression from a Pathogenic Escherichia coliStrain." Journal of Bacteriology 183, no. 6 (March 15, 2001): 2081–85. http://dx.doi.org/10.1128/jb.183.6.2081-2085.2001.
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ABSTRACT Shiga toxins (Stxs), encoded by the stxA andstxB genes, are important contributors to the virulence ofEscherichia coli O157:H7 and other Stx-producing E. coli (STEC) strains. The stxA and stxBgenes in STEC strains are located on the genomes of resident prophages of the λ family immediately downstream of the phage late promoters (p R′). The phage-encoded Q proteins modify RNA polymerase initiating transcription at the cognatep R′ promoter which creates transcription complexes that transcend a transcription terminator immediately downstream of p R′ as well as terminator kilobases distal to p R′. To test if this Q-directed processive transcription plays a role instx 2 AB expression, we constructed a mutant prophage in an O157:H7 clinical isolate from whichp R′ and part of Q were deleted but which has an intact pStx, the previously describedstx 2 AB-associated promoter. We report that production of significant levels of Stx2 in this O157:H7 isolate depends on the p R′ promoter. Since transcription initiating at p R′ ultimately requires activation of the phage lytic cascade, expression ofstx 2 AB in STEC depends primarily on prophage induction. By showing this central role for the prophage instx 2 AB expression, our findings contradict the prevailing assumption that phages serve merely as agents for virulence gene transfer.
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Lee, Moo-Seung, and Vernon Tesh. "Roles of Shiga Toxins in Immunopathology." Toxins 11, no. 4 (April 9, 2019): 212. http://dx.doi.org/10.3390/toxins11040212.
Full textAbstract:
Shigella species and Shiga toxin-producing Escherichia coli (STEC) are agents of bloody diarrhea that may progress to potentially lethal complications such as diarrhea-associated hemolytic uremic syndrome (D+HUS) and neurological disorders. The bacteria share the ability to produce virulence factors called Shiga toxins (Stxs). Research over the past two decades has identified Stxs as multifunctional toxins capable of inducing cell stress responses in addition to their canonical ribotoxic function inhibiting protein synthesis. Notably, Stxs are not only potent inducers of cell death, but also activate innate immune responses that may lead to inflammation, and these effects may increase the severity of organ injury in patients infected with Stx-producing bacteria. In the intestines, kidneys, and central nervous system, excessive or uncontrolled host innate and cellular immune responses triggered by Stxs may result in sensitization of cells to toxin mediated damage, leading to immunopathology and increased morbidity and mortality in animal models (including primates) and human patients. Here, we review studies describing Stx-induced innate immune responses that may be associated with tissue damage, inflammation, and complement activation. We speculate on how these processes may contribute to immunopathological responses to the toxins.
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Kim, Hansol, Hyunjun Park, Hui Wang, Hah Young Yoo, Jaeyeon Park, and Jang-Seu Ki. "Low Temperature and Cold Stress Significantly Increase Saxitoxins (STXs) and Expression of STX Biosynthesis Genes sxtA4 and sxtG in the Dinoflagellate Alexandrium catenella." Marine Drugs 19, no. 6 (May 21, 2021): 291. http://dx.doi.org/10.3390/md19060291.
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Toxic dinoflagellate Alexandrium spp. produce saxitoxins (STXs), whose biosynthesis pathway is affected by temperature. However, the link between the regulation of the relevant genes and STXs’ accumulation and temperature is insufficiently understood. In the present study, we evaluated the effects of temperature on cellular STXs and the expression of two core STX biosynthesis genes (sxtA4 and sxtG) in the toxic dinoflagellate Alexandrium catenella Alex03 isolated from Korean waters. We analyzed the growth rate, toxin profiles, and gene responses in cells exposed to different temperatures, including long-term adaptation (12, 16, and 20 °C) and cold and heat stresses. Temperature significantly affected the growth of A. catenella, with optimal growth (0.49 division/day) at 16 °C and the largest cell size (30.5 µm) at 12 °C. High concentration of STXs eq were detected in cells cultured at 16 °C (86.3 fmol/cell) and exposed to cold stress at 20→12 °C (96.6 fmol/cell) compared to those at 20 °C and exposed to heat stress. Quantitative real-time PCR (qRT-PCR) revealed significant gene expression changes of sxtA4 in cells cultured at 16 °C (1.8-fold) and cold shock at 20→16 °C (9.9-fold). In addition, sxtG was significantly induced in cells exposed to cold shocks (20→16 °C; 19.5-fold) and heat stress (12→20 °C; 25.6-fold). Principal component analysis (PCA) revealed that low temperature (12 and 16 °C) and cold stress were positively related with STXs’ production and gene expression levels. These results suggest that temperature may affect the toxicity and regulation of STX biosynthesis genes in dinoflagellates.
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Zhu, Hongchen, Towa Sakai, Yuji Nagashima, Hiroyuki Doi, Tomohiro Takatani, and Osamu Arakawa. "Tetrodotoxin/Saxitoxins Selectivity of the Euryhaline Freshwater Pufferfish Dichotomyctere fluviatilis." Toxins 13, no. 10 (October 16, 2021): 731. http://dx.doi.org/10.3390/toxins13100731.
Full textAbstract:
The present study evaluated differences in the tetrodotoxin (TTX)/saxitoxins (STXs) selectivity between marine and freshwater pufferfish by performing in vivo and in vitro experiments. In the in vivo experiment, artificially reared nontoxic euryhaline freshwater pufferfish Dichotomyctere fluviatilis were intrarectally administered a mixture of TTX (24 nmol/fish) and STX (20 nmol/fish). The amount of toxin in the intestine, liver, muscle, gonads, and skin was quantified at 24, 48, and 72 h. STX was detected in the intestine over a long period of time, with some (2.7–6.1% of the given dose) being absorbed into the body and temporarily located in the liver. Very little TTX was retained in the body. In the in vitro experiments, slices of intestine, liver, and skin tissue prepared from artificially reared nontoxic D. fluviatilis and the marine pufferfish Takifugu rubripes were incubated in buffer containing TTX and STXs (20 nmol/mL each) for up to 24 or 72 h, and the amount of toxin taken up in the tissue was quantified over time. In contrast to T. rubripes, the intestine, liver, and skin tissues of D. fluviatilis selectively took up only STXs. These findings indicate that the TTX/STXs selectivity differs between freshwater and marine pufferfish.
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Yoshida, Tomoaki, Tsuyoshi Sugiyama, Naoki Koide, Isamu Mori, and Takashi Yokochi. "Human microvascular endothelial cells resist Shiga toxins by IFN-γ treatment in vitro." Microbiology 149, no. 9 (September 1, 2003): 2609–14. http://dx.doi.org/10.1099/mic.0.26142-0.
Full textAbstract:
Shiga toxins (Stxs) produced by enterohaemorrhagic Escherichia coli or Shigella dysenteriae damage human endothelial cells predominantly in cooperation with pro-inflammatory cytokines, such as TNF-α. However, in this study, in vitro IFN-γ pre-treatment resulted in human lung microvascular endothelial cells becoming over 10 000-fold less sensitive to Stxs. In contrast, in their basal condition, they were extremely sensitive to Stxs. Interestingly, TNF-α addition to IFN-γ reverted the Stx-resistant phenotype, which corresponded with its well-established enhancing effect on Stx toxicity. Toxin binding to the cell was barely affected by IFN-γ. Also, the toxin uptake in the Stx-resistant phenotype was more than 100-fold greater than that of normal cells, when compared at Stx concentrations resulting in equivalent degrees of cell damage. Protein synthesis was inhibited by nearly 90 % in the Stx-resistant phenotype after 24 h toxin exposure. This indicated that the intracellular toxin was active as an N-glycosidase, while cells were still over 60 % viable, suggesting a possible unknown cytotoxic function of Stx. In conclusion, this study shows a unique effect of IFN-γ in the suppression of the toxicity of Stxs in a human microvascular endothelial cell model and the involvement of a novel mechanism in this suppression.
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Lee, Kyung-Soo, Yu-Jin Jeong, and Moo-Seung Lee. "Escherichia coli Shiga Toxins and Gut Microbiota Interactions." Toxins 13, no. 6 (June 11, 2021): 416. http://dx.doi.org/10.3390/toxins13060416.
Full textAbstract:
Escherichia coli (EHEC) and Shigella dysenteriae serotype 1 are enterohemorrhagic bacteria that induce hemorrhagic colitis. This, in turn, may result in potentially lethal complications, such as hemolytic uremic syndrome (HUS), which is characterized by thrombocytopenia, acute renal failure, and neurological abnormalities. Both species of bacteria produce Shiga toxins (Stxs), a phage-encoded exotoxin inhibiting protein synthesis in host cells that are primarily responsible for bacterial virulence. Although most studies have focused on the pathogenic roles of Stxs as harmful substances capable of inducing cell death and as proinflammatory factors that sensitize the host target organs to damage, less is known about the interface between the commensalism of bacterial communities and the pathogenicity of the toxins. The gut contains more species of bacteria than any other organ, providing pathogenic bacteria that colonize the gut with a greater number of opportunities to encounter other bacterial species. Notably, the presence in the intestines of pathogenic EHEC producing Stxs associated with severe illness may have compounding effects on the diversity of the indigenous bacteria and bacterial communities in the gut. The present review focuses on studies describing the roles of Stxs in the complex interactions between pathogenic Shiga toxin-producing E. coli, the resident microbiome, and host tissues. The determination of these interactions may provide insights into the unresolved issues regarding these pathogens.
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Thorpe, C. M., Bryan P. Hurley, Lisa L. Lincicome, Mary S. Jacewicz, Gerald T. Keusch, and David W. K. Acheson. "Shiga Toxins Stimulate Secretion of Interleukin-8 from Intestinal Epithelial Cells." Infection and Immunity 67, no. 11 (November 1, 1999): 5985–93. http://dx.doi.org/10.1128/iai.67.11.5985-5993.1999.
Full textAbstract:
ABSTRACT In the 1980s, Shiga toxin (Stx)-producing Escherichia coli O157:H7 (STEC) was identified as a cause of hemorrhagic colitis in the United States and was found to be associated with hemolytic uremic syndrome (HUS), a microangiopathic hemolytic anemia characterized by thrombocytopenia and renal failure. The precise way that Stxs cause hemorrhagic colitis and HUS is unclear. Stxs have been thought to cause disease by killing or irreversibly harming sensitive cells through a nonspecific blockade of mRNA translation, eventually resulting in cytotoxicity by preventing synthesis of critical molecules needed to maintain cell integrity. Because STEC is noninvasive, we have been exploring the host-toxin response at the level of the gastrointestinal mucosa, where STEC infection begins. We have found that Stx is capable of interleukin-8 (IL-8) superinduction in a human colonic epithelial cell line. Despite a general blockade of mRNA translation, Stx treatment results in increased IL-8 mRNA as well as increased synthesis and secretion of IL-8 protein. Our data suggest that an active Stx A subunit is required for this activity. Ricin, which has the same enzymatic activity and trafficking pathway as Stx, has similar effects. Exploration of the effects of other protein synthesis inhibitors (cycloheximide, anisomycin) suggests a mechanism of gene regulation that is distinct from a general translational blockade. Use of the specific p38/RK inhibitor SB202190 showed that blocking of this pathway results in decreased Stx-mediated IL-8 secretion. Furthermore, Stxs induced mRNA of the primary response gene c-jun, which was subsequently partially blocked by SB202190. These data suggest a novel model of how Stxs contribute to disease, namely that Stxs may alter regulation of host cell processes in sensitive cells via activation of at least one member of the mitogen-activated protein kinase family in the p38/RK cascade and induction of c-jun mRNA. Stx-induced increases in chemokine synthesis from intestinal epithelial cells could be important in augmenting the host mucosal inflammatory response to STEC infection.
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Guessous, Fadila, Renata Polanowska-Grabowska, Tiffany Keepers, Tom Obrig, Adrian Gear, and Marek Marcinkiewicz. "Shiga toxin 2 and lipopolysaccharide cause monocytic THP-1 cells to release factors which activate platelet function." Thrombosis and Haemostasis 94, no. 11 (2005): 1019–27. http://dx.doi.org/10.1160/th05-02-0115.
Full textAbstract:
SummaryPlatelet and monocyte activation may contribute to hemolytic anemia, thrombocytopenia and renal failure associated with the hemolytic uremic syndrome (HUS) caused by Escherichia coli O157:H7. Since Shiga toxins (Stxs) and lipopolysaccharide (LPS) from this bacterium are implicated in the pathogenesis of HUS, we examined whether stimulation of THP-1 human monocytic cells by Shiga toxin 2 (Stx2) and LPS can lead to the activation of platelet function. We now show that Stx2 caused THP-1 cells to release the chemokines IL-8, MDC, and RANTES and that the presence of LPS further stimulated this release. IL-8 was produced in greatest amount and was an effective co-agonist for inducing platelet aggregation. Primary human monocytes also released large amounts of IL-8 in response to LPS and Stx2. Factors released by THP-1 cells exposed to Stx2 and LPS activated platelet function as evidenced by increased aggregation, serotonin secretion, P-selectin exposure and by the formation of stable platelet-monocyte aggregates. Our data therefore show that monocytes exposed to E. coli-derived Stx2 and LPS release factors which activate platelet function.
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Hughes, Anna C., Stephanie Patfield, Reuven Rasooly, and Xiaohua He. "Validation of a Cell-Based Assay for Detection of Active Shiga Toxins Produced by Escherichia coli in Water." International Journal of Environmental Research and Public Health 17, no. 21 (October 28, 2020): 7901. http://dx.doi.org/10.3390/ijerph17217901.
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Shiga toxin-producing Escherichia coli (STEC) causes a wide spectrum of diseases, including hemorrhagic colitis and hemolytic uremic syndrome (HUS). Almost 5% of STEC infections result from waterborne exposures, yet there is no test listed in the EPA’s current Selected Analytical Methods for the detection of active Shiga toxins (Stxs) in water. In this study, a HeLa cell-based assay is validated for the detection of metabolically active Stxs produced by STEC in water, including tap, bottled, and pond water. Active Stxs are detected even when the number of Stx-producing bacteria is less than 0.4 CFU/mL and the assay performance is not affected by background flora or chlorine in the water. This assay is not only as simple and affordable as cell-free assays but also detects active holotoxins without the use of live animals. In addition, the assay is designed for use in multi-well formats, making it ideal for high-throughput screening of water samples and therefore useful for environmental public health surveillance programs to reduce human risk of infection with STEC.
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Shimizu, Takeshi, Satomi Kawakami, Toshio Sato, Terumi Sasaki, Masato Higashide, Takashi Hamabata, Toshiko Ohta, and Masatoshi Noda. "The Serine 31 Residue of the B Subunit of Shiga Toxin 2 Is Essential for Secretion in Enterohemorrhagic Escherichia coli." Infection and Immunity 75, no. 5 (February 26, 2007): 2189–200. http://dx.doi.org/10.1128/iai.01546-06.
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ABSTRACT Shiga toxins produced by enterohemorrhagic Escherichia coli (EHEC) include Shiga toxin 1 (Stx1) as well as Shiga toxin 2 (Stx2). Stx1 is cell associated, whereas Stx2 is localized to the culture supernatant. We have analyzed the secretion of Stx2 by generating histidine-tagged StxB (StxB-H). Although neither StxB1-H nor StxB2-H was secreted in StxB-H-overexpressed EHEC, StxB2-H-overexpressed EHEC showed inhibited Stx2 secretion. On the other hand, StxB1-H-overexpressed EHEC showed no alteration of Stx2 secretion. B-subunit chimeras of Stx1 and Stx2 were used to identify the specific residue of StxB2 that the Stx2 secretory system recognizes. Alteration of the serine 31 residue to an asparagine residue (S31N) in StxB2-H enabled the recovery of Stx2 secretion. On the other hand, alteration of the asparagine 32 residue to a serine residue (N32S) in StxB1-H caused the partial secretion of a point-mutated histidine-tagged B subunit in EHEC. Based on the evidence, it appeared possible that this residue might contain secretion-related information for Stx2 secretion. To investigate this hypothesis, we constructed an isogenic mutant EHEC (Stx1B subunit, N32S) strain and an isogenic mutant EHEC (Stx2B subunit, S31N) strain. Although the mutant Stx2 was cell associated in isogenic mutant EHEC, mutant Stx1 was not extracellular. However, when we used plasmids for the expression of the mutant holotoxins, the overexpressed mutant Stx1 was found in the supernatant fraction, and the overexpressed mutant Stx2 was found in the cell-associated fraction in mutant holotoxin gene-transformed EHEC. These results indicate that the serine 31 residue of the B subunit of Stx2 contains secretion-related information.
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Dwiyitno, D., G. R. Barokah, R. K. Rustiani, and S. Wibowo. "Distribution of saxitoxin producing algae in Jakarta Bay and the implication to saxitoxin concentration in green mussel." IOP Conference Series: Earth and Environmental Science 967, no. 1 (January 1, 2022): 012037. http://dx.doi.org/10.1088/1755-1315/967/1/012037.
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Abstract Harmful algae blooms (HABs) have been associated with an annual incidence in Jakarta Bay. The The present study aimed to investigate the concentration of dinoflagellates producing saxitoxins (STXs) in Cilincing and Kalibaru regions and the STXs concentration in mussel species from this coastal water. A sampling of phytoplankton, green mussel (Perna viridis), and environmental parameters were measured during the transition from wet to dry season. The water nutrients of the Cilincing region, mainly nitrite and ammonia in some green mussel aquaculture, have exceeded the recommended concentration to support the eutrophication/algae bloom. The N/P ratio at the study area was between 10 and 22. Plankton identification found STXs producing dinoflagellates, i.e., Alexandrium sp. and Gymnodinium sp. Other species of dinoflagellates and diatom showed predominantly to trigger algae Bloom, such as Skeletonema sp., Chaetoceros sp., Prorocentrum sp., Gonyaulax sp., Protoperidinium sp., and Nitzchia sp. Further analysis of saxitoxin in mussel samples from Cilincing and Kalibar showed STX concentrations of 10.15 μg/100 g and 21.24 μg/100 g, respectively, which is fairly below the official maximum limit (80 μg/100 g) as set by FAO/WHO or national standard.
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Basu, Indira, Witold A. Ferens, Diana M. Stone, and Carolyn J. Hovde. "Antiviral Activity of Shiga Toxin Requires Enzymatic Activity and Is Associated with Increased Permeability of the Target Cells." Infection and Immunity 71, no. 1 (January 2003): 327–34. http://dx.doi.org/10.1128/iai.71.1.327-334.2003.
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ABSTRACT This study expanded our earlier finding that Shiga toxin type 1 (Stx1) has activity against bovine leukemia virus (BLV) (W. A. Ferens and C. J. Hovde, Infect. Immun. 68:4462-4469, 2000). The Stx molecular motifs required for antiviral activity were identified, and a mechanism of Stx action on virally infected cells is suggested. Using inhibition of BLV-dependent spontaneous lymphocyte proliferation as a measure of antiviral activity, we showed that Stx2 had antiviral activity similar to that of Stx1. Enzymatic and antiviral activities of three StxA1 chain mutants deficient in enzymatic activity or aspects of receptor-mediated cytotoxicity were compared. Using protein synthesis inhibition to measure enzymatic activity, the mutant E167D was 300-fold less catalytically active than wild-type StxA1, was minimally active in antiviral assays, and did not inhibit synthesis of viral proteins. Two StxA1 mutants, A231D-G234E and StxA11 (enzymatically active but unable to kill cells via the classical receptor-mediated route), had undiminished antiviral activity. Although binding of radiolabeled StxA1 to bovine blood cells or to free virus was not detected, flow cytometric analysis showed that the number of BLV-expressing cells were specifically reduced in cultures treated with Stx. These unique and rare lymphocytes were highly permeable to 40- and 70-kDa fluorescent dextrans, indicating that direct absorption of toxins by virus-expressing cells is a potential mechanism of target cell intoxication. These results support the hypothesis that Stx-producing Escherichia coli colonization of the gastrointestinal tract may benefit ruminant hosts by the ability of Stxs to exert antiviral activity.
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Sy, Brandon M., Ruiting Lan, and Jai J. Tree. "Early termination of the Shiga toxin transcript generates a regulatory small RNA." Proceedings of the National Academy of Sciences 117, no. 40 (September 23, 2020): 25055–65. http://dx.doi.org/10.1073/pnas.2006730117.
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Enterohemorrhagic Escherichia coli is a significant human pathogen that causes disease ranging from hemorrhagic colitis to hemolytic uremic syndrome. The latter can lead to potentially fatal renal failure and is caused by the release of Shiga toxins that are encoded within lambdoid bacteriophages. The toxins are encoded within the late transcript of the phage and are regulated by antitermination of the PR′ late promoter during lytic induction of the phage. During lysogeny, the late transcript is prematurely terminated at tR′ immediately downstream of PR′, generating a short RNA that is a byproduct of antitermination regulation. We demonstrate that this short transcript binds the small RNA chaperone Hfq, and is processed into a stable 74-nt regulatory small RNA that we have termed StxS. StxS represses expression of Shiga toxin 1 under lysogenic conditions through direct interactions with the stx1AB transcript. StxS acts in trans to activate expression of the general stress response sigma factor, RpoS, through direct interactions with an activating seed sequence within the 5′ UTR. Activation of RpoS promotes high cell density growth under nutrient-limiting conditions. Many phages utilize antitermination to regulate the lytic/lysogenic switch and our results demonstrate that short RNAs generated as a byproduct of this regulation can acquire regulatory small RNA functions that modulate host fitness.
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Menge, Christian. "Molecular Biology of Escherichia coli Shiga Toxins’ Effects on Mammalian Cells." Toxins 12, no. 5 (May 23, 2020): 345. http://dx.doi.org/10.3390/toxins12050345.
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Shiga toxins (Stxs), syn. Vero(cyto)toxins, are potent bacterial exotoxins and the principal virulence factor of enterohemorrhagic Escherichia coli (EHEC), a subset of Shiga toxin-producing E. coli (STEC). EHEC strains, e.g., strains of serovars O157:H7 and O104:H4, may cause individual cases as well as large outbreaks of life-threatening diseases in humans. Stxs primarily exert a ribotoxic activity in the eukaryotic target cells of the mammalian host resulting in rapid protein synthesis inhibition and cell death. Damage of endothelial cells in the kidneys and the central nervous system by Stxs is central in the pathogenesis of hemolytic uremic syndrome (HUS) in humans and edema disease in pigs. Probably even more important, the toxins also are capable of modulating a plethora of essential cellular functions, which eventually disturb intercellular communication. The review aims at providing a comprehensive overview of the current knowledge of the time course and the consecutive steps of Stx/cell interactions at the molecular level. Intervention measures deduced from an in-depth understanding of this molecular interplay may foster our basic understanding of cellular biology and microbial pathogenesis and pave the way to the creation of host-directed active compounds to mitigate the pathological conditions of STEC infections in the mammalian body.
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Suhan, Michelle L., and Carolyn J. Hovde. "Disruption of an Internal Membrane-Spanning Region in Shiga Toxin 1 Reduces Cytotoxicity." Infection and Immunity 66, no. 11 (November 1, 1998): 5252–59. http://dx.doi.org/10.1128/iai.66.11.5252-5259.1998.
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ABSTRACT Shiga toxin type 1 (Stx1) belongs to the Shiga family of bipartite AB toxins that inactivate eukaryotic 60S ribosomes. The A subunit of Stxs are N-glycosidases that share structural and functional features in their catalytic center and in an internal hydrophobic region that shows strong transmembrane propensity. Both features are conserved in ricin and other ribosomal inactivating proteins. During eukaryotic cell intoxication, holotoxin likely moves retrograde from the Golgi apparatus to the endoplasmic reticulum. The hydrophobic region, spanning residues I224 through N241 in the Stx1 A subunit (Stx1A), was hypothesized to participate in toxin translocation across internal target cell membranes. The TMpred computer program was used to design a series of site-specific mutations in this hydrophobic region that disrupt transmembrane propensity to various degrees. Mutations were synthesized by PCR overlap extension and confirmed by DNA sequencing. Mutants StxAF226Y, A231D, G234E, and A231D-G234E and wild-type Stx1A were expressed in Escherichia coli SY327 and purified by dye-ligand affinity chromatography. All of the mutant toxins were similar to wild-type Stx1A in enzymatic activity, as determined by inhibition of cell-free protein synthesis, and in susceptibility to trypsin digestion. Purified mutant or wild-type Stx1A combined with Stx1B subunits in vitro to form a holotoxin, as determined by native polyacrylamide gel electrophoresis immunoblotting. StxA mutant A231D-G234E, predicted to abolish transmembrane propensity, was 225-fold less cytotoxic to cultured Vero cells than were the wild-type toxin and the other mutant toxins which retained some transmembrane potential. Furthermore, compared to wild-type Stx1A, A231D-G234E Stx1A was less able to interact with synthetic lipid vesicles, as determined by analysis of tryptophan fluorescence for each toxin in the presence of increasing concentrations of lipid membrane vesicles. These results provide evidence that this conserved internal hydrophobic motif contributes to Stx1 translocation in eukaryotic cells.
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Lee, Moo-Seung, Rama P. Cherla, Erin K. Lentz, Dinorah Leyva-Illades, and Vernon L. Tesh. "Signaling through C/EBP Homologous Protein and Death Receptor 5 and Calpain Activation Differentially Regulate THP-1 Cell Maturation-Dependent Apoptosis Induced by Shiga Toxin Type 1." Infection and Immunity 78, no. 8 (June 1, 2010): 3378–91. http://dx.doi.org/10.1128/iai.00342-10.
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ABSTRACT Shiga toxins (Stxs) induce apoptosis via activation of the intrinsic and extrinsic pathways in many cell types. Toxin-mediated activation of the endoplasmic reticulum (ER) stress response was shown to be instrumental in initiating apoptosis in THP-1 myeloid leukemia cells. THP-1 cells responded to Shiga toxin type 1 (Stx1) in a cell maturation-dependent manner, undergoing rapid apoptosis in the undifferentiated state but reduced and delayed apoptosis in differentiated cells. The onset of apoptosis was associated with calpain activation and changes in expression of C/EBP homologous protein (CHOP), Bcl-2 family members, and death receptor 5 (DR5). Ligation of DR5 by tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) activates the extrinsic pathway of apoptosis. We show here that expression of TRAIL and DR5 is increased by Stx1 treatment. Addition of exogenous TRAIL enhances, and anti-TRAIL antibodies inhibit, Stx1-induced apoptosis of THP-1 cells. Silencing of CHOP or DR5 expression selectively prevented caspase activation, loss of mitochondrial membrane potential, and Stx1-induced apoptosis of macrophage-like THP-1 cells. In contrast, the rapid kinetics of apoptosis induction in monocytic THP-1 cells correlated with rates of calpain cleavage. The results suggest that CHOP-DR5 signaling and calpain activation differentially contribute to cell maturation-dependent Stx1-induced apoptosis. Inhibition of these signaling pathways may protect cells from Stx cytotoxicity.
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Castro, Vinicius Silva, Rodrigo Ortega Polo, Eduardo Eustáquio de Souza Figueiredo, Emmanuel Wihkochombom Bumunange, Tim McAllister, Robin King, Carlos Adam Conte-Junior, and Kim Stanford. "Inconsistent PCR detection of Shiga toxin-producing Escherichia coli: Insights from whole genome sequence analyses." PLOS ONE 16, no. 9 (September 3, 2021): e0257168. http://dx.doi.org/10.1371/journal.pone.0257168.
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Shiga toxin-producing Escherichia coli (STEC) have been linked to food-borne disease outbreaks. As PCR is routinely used to screen foods for STEC, it is important that factors leading to inconsistent detection of STEC by PCR are understood. This study used whole genome sequencing (WGS) to investigate causes of inconsistent PCR detection of stx1, stx2, and serogroup-specific genes. Fifty strains isolated from Alberta feedlot cattle from three different studies were selected with inconsistent or consistent detection of stx and serogroup by PCR. All isolates were initially classified as STEC by PCR. Sequencing was performed using Illumina MiSeq® with sample library by Nextera XT. Virtual PCRs were performed using Geneious and bacteriophage content was determined using PHASTER. Sequencing coverage ranged from 47 to 102x, averaging 74x, with sequences deposited in the NCBI database. Eleven strains were confirmed by WGS as STEC having complete stxA and stxB subunits. However, truncated stx fragments occurred in twenty-two other isolates, some having multiple stx fragments in the genome. Isolates with complete stx by WGS had consistent stx1 and stx2 detection by PCR, although one also having a stx2 fragment had inconsistent stx2 PCR. For all STEC and 18/39 non-STEC, serogroups determined by PCR agreed with those determined by WGS. An additional three WGS serotypes were inconclusive and two isolates were Citrobacter spp. Results demonstrate that stx fragments associated with stx-carrying bacteriophages in the E. coli genome may contribute to inconsistent detection of stx1 and stx2 by PCR. Fourteen isolates had integrated stx bacteriophage but lacked complete or fragmentary stx possibly due to partial bacteriophage excision after sub-cultivation or other unclear mechanisms. The majority of STEC isolates (7/11) did not have identifiable bacteriophage DNA in the contig(s) where stx was located, likely increasing the stability of stx in the bacterial genome and its detection by PCR.
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Vinh, Shinohara, Yamada, Duc, Nakayama, Ozawa, Sato, Masuda, Honjoh, and Miyamoto. "Baicalein Inhibits Stx1 and 2 of EHE: Effects of Baicalein on the Cytotoxicity, Production, and Secretion of Shiga Toxins of Enterohaemorrhagic Escherichia coli." Toxins 11, no. 9 (August 29, 2019): 505. http://dx.doi.org/10.3390/toxins11090505.
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Shiga toxin-producing enterohaemorrhagic Escherichia coli (EHEC) O157:H7 is an important foodborne pathogen. Baicalein (5,6,7-trihydroxylflavone), a flavone isolated from the roots of Scutellaria baicalensis, is considered as a potential antibacterial agent to control foodborne pathogens. Among seven compounds selected by in silico screening of the natural compound database, baicalein inhibited the cytotoxicity of both Shiga toxins 1 and 2 (Stx1 and Stx2) against Vero cells after pretreatment at 0.13 mmol/L. In addition, baicalein reduced the susceptibility of Vero cells to both Stx1 and Stx2. Real-time qPCR showed that baicalein increased transcription of stx1 but not of stx2. However, baicalein had no effects on production or secretion of Stx1 or Stx2. Docking models suggested that baicalein formed a stable structure with StxB pentamer with low intramolecular energy. The results demonstrate that inhibitory activity of baicalein against the cytotoxicity of both Stx1 and Stx2 might be due to of the formation of a binding structure inside the pocket of the Stx1B and Stx2B pentamers.
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Liu, Yang, Songhai Tian, Hatim Thaker, and Min Dong. "Shiga Toxins: An Update on Host Factors and Biomedical Applications." Toxins 13, no. 3 (March 18, 2021): 222. http://dx.doi.org/10.3390/toxins13030222.
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Shiga toxins (Stxs) are classic bacterial toxins and major virulence factors of toxigenic Shigella dysenteriae and enterohemorrhagic Escherichia coli (EHEC). These toxins recognize a glycosphingolipid globotriaosylceramide (Gb3/CD77) as their receptor and inhibit protein synthesis in cells by cleaving 28S ribosomal RNA. They are the major cause of life-threatening complications such as hemolytic uremic syndrome (HUS), associated with severe cases of EHEC infection, which is the leading cause of acute kidney injury in children. The threat of Stxs is exacerbated by the lack of toxin inhibitors and effective treatment for HUS. Here, we briefly summarize the Stx structure, subtypes, in vitro and in vivo models, Gb3 expression and HUS, and then introduce recent studies using CRISPR-Cas9-mediated genome-wide screens to identify the host cell factors required for Stx action. We also summarize the latest progress in utilizing and engineering Stx components for biomedical applications.
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Watanabe, Ryuichi, Makoto Kanamori, Hidetsugu Yoshida, Yutaka Okumura, Hajime Uchida, Ryoji Matsushima, Hiroshi Oikawa, and Toshiyuki Suzuki. "Development of Ultra-Performance Liquid Chromatography with Post-Column Fluorescent Derivatization for the Rapid Detection of Saxitoxin Analogues and Analysis of Bivalve Monitoring Samples." Toxins 11, no. 10 (October 1, 2019): 573. http://dx.doi.org/10.3390/toxins11100573.
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Saxitoxin (STX) and its analogues produced by toxic dinoflagellates accumulate in bivalves, and routine monitoring of bivalves is important to prevent cases of human poisoning. In this study, we describe a rapid detection method for the analysis of STXs using ultra-performance liquid chromatography with post-column fluorescent detection and to investigate water depths and sampling points optimal for shellfish toxin monitoring. Cultured scallops (Mizuhopecten yessoensis) and mussels (Mytilus galloprovincialis) collected from various water depths and sampling points were used in this study. Irrespective of bivalve species, toxin concentrations in bivalves were lower at deeper water depths. The toxin concentrations of bivalves did not differ greatly when bivalves were collected from the same bay. Although the levels of contamination of bivalves with STXs can depend on various environmental and geographical factors, our findings are useful for formulating a sampling protocol for the prevention of harvesting contaminated shellfish.
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Osek, J. "Phenotypic and genotypic characterization of Escherichia coli O157 strains isolated from humans, cattle and pigs." Veterinární Medicína 49, No. 9 (March 29, 2012): 317–26. http://dx.doi.org/10.17221/5711-vetmed.
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A total of 90 Escherichia coli O157 isolates recovered from humans, cattle, and pigs, were examined for the presence of the H7 antigen, ability to produce Shiga toxins and enterohemolysin as well as for antimicrobial resistance and biochemical properties. Fourteen of the human strains (n = 23) and 21 of the bovine isolates (n = 29) were of the O157:H7 serotype as determined by agglutination and PCR methods. All E. coli O157 of porcine origin (n = 38) were H-negative. Based on the ability to produce Shiga toxins (Stxs), all human and cattle isolates and 11 strains recovered from swine were identified as Shiga toxin-producing E. coli (STEC). Among STEC, most human strains (18 isolates) were Stx1- and Stx2-positive whereas cattle strains were mostly Stx2-positive. Eleven porcine STEC produced either Stx1 (7 isolates) or Stx2 (4 strains) toxins; an additional 20 isolates recovered from these animals had the Stx2e toxin gene as previously determined by PCR. All human and cattle E. coli O157 produced enterohemolysin whereas only 4 strains recovered from pigs were ehly-positive. Moreover, the PCR identification of the lpf<sub>O113</sub> gene performed earlier revealed that this putative virulence marker was present in all porcine isolates, only in 5 strains of bovine origin but in none of E. coli O157 recovered from humans. All 90 E. coli O157 strains tested displayed 10 biochemical profiles that were different at least in one of the reaction tested. The most common atypical reaction observed among porcine O157 isolates was ability to ferment sorbitol (all strains) and production of β-glucuronidase (25 isolates). Moreover, none of the sorbitol-positive strains was able to produce indol. Four antimicrobial resistance profiles among 90 E. coli O157 strains tested were observed. Most of the isolates recovered from humans and all strains from cattle were resistant only to rifampicin whereas the porcine strains showed resistance to either 3 antimicrobials (4 isolates) or to 4 drugs tested (34 isolates). The phenotypic data shown in the present study, together with the previously published genotypic analyses of these strains, confirm earlier suggestions that the porcine E. coli O157 strains are mostly different from those of bovine and human O157 isolates and could therefore play less important role in human STEC O157 infections.
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Gao, Junjie, and Younghwan Pan. "Evaluating Influencing Factors of Tourists’ Experiences with Smart Tour Guide System: A Mixed Method Research." Sustainability 14, no. 23 (December 6, 2022): 16320. http://dx.doi.org/10.3390/su142316320.
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The issue that this study explores is evaluation of tourists’ experiences with smart tour guide system (STGS). To address this issue, the purpose of this study is to explore what factors influence tourists’ experiences with STGS in four Chinese smart tourism destinations (STDs). An exploratory sequential mixed method was used to collect the data. Tourists who had used STGS in four Chinese STDs participated in the research. In the first and second phase, Semi-structured interviews with 12 interviewees were conducted, and these interviewees put forward six factors affecting tourists’ experiences with STGS: approachability, visual, operability, function, offline service, and interactivity. The third phase involved an analysis of a survey of 248 participants who had used STGS in the Chinese four STDs to verify the influencing factors. By using the method of linear regression analysis, we found that approachability, visual, operability, function and offline service have a significant impact on tourists’ experiences with STGS while interactivity contributes little impact to tourists’ experiences with STGS. The findings will be useful for STDs to explore and promote STGS services, as well as enhancing tourists’ smart experiences with STTs.
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Schweppe, Christian, Helge Karch, Alexander Friedrich, and Johannes Müthing. "Shiga toxins, glycosphingolipid diversity, and endothelial cell injury." Thrombosis and Haemostasis 101, no. 02 (2009): 252–64. http://dx.doi.org/10.1160/th08-05-0317.
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SummaryShiga toxin (Stx)-producing Escherichia coli (STEC) cause an enteric illness that results in a spectrum of outcomes ranging from asymptomatic carriage to uncomplicated diarrhea, bloody diarrhea, and the postdiarrheal haemolytic uremic syndrome (HUS), which leads to renal and other organ microvascular thrombosis. Binding of Stx to the glycosphingolipid (GSL) globotriaosylceramide (Gb3Cer/CD77) on endothelial cells followed by receptor-mediated endocytosis is the linchpin in STEC-mediated disease. Only GSLs that associate strongly with lipid rafts appear to carry Stxs retrogradely from the plasma membrane through the Golgi apparatus to the endoplasmic reticulum where they are translocated to the cytosol and exert their toxic function. Thus, the biophysical features of the lipid moiety of GSL receptors may influence its incorporation into certain membrane domains and thereby affect toxin destination. Consequently, a detailed structural analysis of Stx-binding GSLs is required to illuminate the molecular causes that may underlie the different Stx susceptibilities of endothelial cells derived from various vascular beds. Solid phase overlay binding assays of thin-layer chromatography (TLC)-separated GSL preparations employing specific antibodies and/or Stxs in conjunction with anti-Stx-antibodies are commonly used for the identification of Stx-binding GSLs. Such GSL-profiling combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) represents a convenient strategy to structurally characterize Stx-receptors from any biological sources such as primary cells, cell lines, or organs. This approach may be helpful to gain insights into Stx-induced impairment of target cells that is suggested to originate at least partly from the structural heterogeneity of the cellular ligands of Stxs.