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Topical Collection "Clostridium difficile/Clostridium sordellii and Clostridium perfringens Toxins"

A topical collection in Toxins (ISSN 2072-6651). This collection belongs to the section "Bacterial Toxins".

Editors

Collection Editor
Prof. Dr. Harald Genth

Institute for Toxicology, Hannover Medical School, Germany
E-Mail
Phone: +49-511-532-9168
Fax: +49-511-532-2879
Interests: Modulation of cellular functions by bacterial toxins
Collection Editor
Prof. Michel R. Popoff

Bacteries Anaerobies et Toxines, Institut Pasteur, 28 rue du Docteur Roux, Paris 75724, France
E-Mail
Phone: 33 1 45688307
Fax: +33 1 40613123
Interests: bacterial protein toxins; clostridial toxins; pore-forming toxins; cellular uptake of bacterial toxins; Rho-GTPases; interactions of clostridial toxins with the actin cytoskeleton; botulinum neurotoxins; passage of the neurotoxins through epithelial barrier; regulation of clostridial toxin synthesis

Topical Collection Information

Dear Colleagues,

Clostridium difficile/Clostridium sordellii and Clostridium perfringens are the most common toxigenic clostridia involved in human and animal diseases. The main C. difficile and C. sordellii toxins belong to the large clostridial glucosylating toxin family, which are intracellularly active toxins through inactivation of Rho/Ras-GTPases, whereas C. perfringens toxins are mainly active on cell membrane via a pore-forming activity. In the few last decades, a great effort has been done to better understand the molecular mode of action of these toxins notably by unraveling the toxin structures and their interaction with receptor and/or substrates, as well as by identifying the cell signaling cascade(s) triggered by the toxins and leading to cell perturbation or death. Indeed, a better comprehension of toxin activity is the basis of the development of efficient inhibitors. Moreover, genetic analysis of the toxigenic clostridia shed light on diversity and evolution of toxin variants, and on possible mechanisms of genetic transfers between clostridia and also from other bacteria. Although C. difficile/C. sordellii and C. perfringens are potent toxins responsible for severe pathologies, they represent potential therapeutic tools, for example to kill specific cell populations or to transport therapeutic compounds in specific cells.

Prof. Dr. Michel R. Popoff
Prof. Dr. Harald Genth
Collection Editors

Manuscript Submission Information

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Keywords

  • Clostridium difficile toxins
  • Clostridium sordellii toxins
  • Clostridium perfringens toxins
  • large clostridial glucosylating toxins
  • pore-forming toxins
  • actin cytoskeleton
  • Rho-GTPases
  • Ras-GTPases
  • apoptosis
  • cell necrosis
  • toxin gene evolution

Published Papers (26 papers)

2019

Jump to: 2018, 2017, 2016, 2015

Open AccessArticle
Clostridium perfringens Delta-Toxin Damages the Mouse Small Intestine
Received: 27 March 2019 / Revised: 8 April 2019 / Accepted: 17 April 2019 / Published: 22 April 2019
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Abstract
Clostridium perfringens strains B and C cause fatal intestinal diseases in animals. The secreted pore-forming toxin delta-toxin is one of the virulence factors of the strains, but the mechanism of intestinal pathogenesis is unclear. Here, we investigated the effects of delta-toxin on the [...] Read more.
Clostridium perfringens strains B and C cause fatal intestinal diseases in animals. The secreted pore-forming toxin delta-toxin is one of the virulence factors of the strains, but the mechanism of intestinal pathogenesis is unclear. Here, we investigated the effects of delta-toxin on the mouse ileal loop. Delta-toxin caused fluid accumulation and intestinal permeability to fluorescein isothiocyanate (FITC)-dextran in the mouse ileal loop in a dose- and time-dependent manner. Treatment with delta-toxin induced significant histological damage and shortening of villi. Delta-toxin activates a disintegrin and metalloprotease (ADAM) 10, leading to the cleavage of E-cadherin, the epithelial adherens junction protein, in human intestinal epithelial Caco-2 cells. In this study, E-cadherin immunostaining in mouse intestinal epithelial cells was almost undetectable 1 h after toxin treatment. ADAM10 inhibitor (GI254023X) blocked the toxin-induced fluid accumulation and E-cadherin loss in the mouse ileal loop. Delta-toxin stimulated the shedding of intestinal epithelial cells. The shedding cells showed the accumulation of E-cadherin in intracellular vesicles and the increased expression of active caspase-3. Our findings demonstrate that delta-toxin causes intestinal epithelial cell damage through the loss of E-cadherin cleaved by ADAM10. Full article
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Open AccessArticle
Application of an Endothelial Cell Culture Assay for the Detection of Neutralizing Anti-Clostridium Perfringens Beta-Toxin Antibodies in a Porcine Vaccination Trial
Received: 4 March 2019 / Revised: 8 April 2019 / Accepted: 9 April 2019 / Published: 15 April 2019
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Abstract
Background: Beta-toxin (CPB) is the major virulence factor of Clostridium perfringens type C, causing hemorrhagic enteritis in newborn pigs but also other animals and humans. Vaccines containing inactivated CPB are known to induce protective antibody titers in sow colostrum and neutralization of the [...] Read more.
Background: Beta-toxin (CPB) is the major virulence factor of Clostridium perfringens type C, causing hemorrhagic enteritis in newborn pigs but also other animals and humans. Vaccines containing inactivated CPB are known to induce protective antibody titers in sow colostrum and neutralization of the CPB activity is thought to be essential for protective immunity in newborn piglets. However, no method is available to quantify the neutralizing effect of vaccine-induced antibody titers in pigs. (2) Methods: We developed a novel assay for the quantification of neutralizing anti-CPB antibodies. Sera and colostrum of sows immunized with a commercial C. perfringens type A and C vaccine was used to determine neutralizing effects on CPB induced cytotoxicity in endothelial cells. Antibody titers of sows and their piglets were determined and compared to results obtained by an ELISA. (3) Results: Vaccinated sows developed neutralizing antibodies against CPB in serum and colostrum. Multiparous sows developed higher serum and colostrum antibody titers after booster vaccinations than uniparous sows. The antibody titers of sows and those of their piglets correlated highly. Piglets from vaccinated sows were protected against intraperitoneal challenge with C. perfringens type C supernatant. (4) Conclusions: The test based on primary porcine endothelial cells quantifies neutralizing antibody activity in serum and colostrum of vaccinated sows and could be used to reduce and refine animal experimentation during vaccine development. Full article
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Open AccessArticle
Targeted Mass Spectrometry Analysis of Clostridium perfringens Toxins
Received: 1 March 2019 / Revised: 18 March 2019 / Accepted: 21 March 2019 / Published: 23 March 2019
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Abstract
Targeted proteomics recently proved to be a technique for the detection and absolute quantification of proteins not easily accessible to classical bottom-up approaches. Due to this, it has been considered as a high fidelity tool to detect potential warfare agents in wide spread [...] Read more.
Targeted proteomics recently proved to be a technique for the detection and absolute quantification of proteins not easily accessible to classical bottom-up approaches. Due to this, it has been considered as a high fidelity tool to detect potential warfare agents in wide spread kinds of biological and environmental matrices. Clostridium perfringens toxins are considered to be potential biological weapons, especially the epsilon toxin which belongs to a group of the most powerful bacterial toxins. Here, the development of a target mass spectrometry method for the detection of C. perfringens protein toxins (alpha, beta, beta2, epsilon, iota) is described. A high-resolution mass spectrometer with a quadrupole-Orbitrap system operating in target acquisition mode (parallel reaction monitoring) was utilized. Because of the lack of commercial protein toxin standards recombinant toxins were prepared within Escherichia coli. The analysis was performed using proteotypic peptides as the target compounds together with their isotopically labeled synthetic analogues as internal standards. Calibration curves were calculated for each peptide in concentrations ranging from 0.635 to 1101 fmol/μL. Limits of detection and quantification were determined for each peptide in blank matrices. Full article
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Open AccessFeature PaperArticle
Two Clostridium perfringens Type E Isolates in France
Received: 12 February 2019 / Revised: 22 February 2019 / Accepted: 23 February 2019 / Published: 1 March 2019
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Abstract
Clostridium perfringens type E is a less frequently isolated C. perfringens type and has not previously been reported in France. We have characterized two recent type E isolates, C. perfringens 508.17 from the intestinal content of a calf that died of enterotoxemia, and [...] Read more.
Clostridium perfringens type E is a less frequently isolated C. perfringens type and has not previously been reported in France. We have characterized two recent type E isolates, C. perfringens 508.17 from the intestinal content of a calf that died of enterotoxemia, and 515.17 from the stool of a 60-year-old woman, subsequent to food poisoning, which contained the plasmid pCPPB-1 with variant iota toxin and C. perfringens enterotoxin genes. Full article
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Open AccessArticle
Treatment and Prevention of Recurrent Clostridium difficile Infection with Functionalized Bovine Antibody-Enriched Whey in a Hamster Primary Infection Model
Received: 19 December 2018 / Revised: 29 January 2019 / Accepted: 1 February 2019 / Published: 6 February 2019
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Abstract
Toxin-induced Clostridium difficile infection (CDI) is a major disease characterized by severe diarrhea and high morbidity rates. The aim with this study was to develop an alternative drug for the treatment of CDI. Cows were repeatedly immunized to establish specific immunoglobulin G and [...] Read more.
Toxin-induced Clostridium difficile infection (CDI) is a major disease characterized by severe diarrhea and high morbidity rates. The aim with this study was to develop an alternative drug for the treatment of CDI. Cows were repeatedly immunized to establish specific immunoglobulin G and A titers against toxins A (TcdA) and B (TcdB) and against C. difficile cells in mature milk or colostrum. The effect of three different concentrations of anti-C. difficile whey protein isolates (anti-CD-WPI) and the standard of care antibiotic vancomycin were investigated in an animal model of CD infected hamsters (6 groups, with 10 hamsters each). WPI obtained from the milk of exactly the same cows pre-immunization and a vehicle group served as negative controls. The survival of hamsters receiving anti-CD-WPI was 50, 80 and 100% compared to 10 and 0% for the control groups, respectively. Vancomycin suppressed the growth of C. difficile and thus protected the hamsters at the time of administration, but 90% of these hamsters nevertheless died shortly after discontinuation of treatment. In contrast, the surviving hamsters of the anti-CD-WPI groups survived the entire study period, although they were treated for only 75 h. The specific antibodies not only inactivated the toxins for initial suppression of CDI, but also provoked the inhibition of C. difficile growth after discontinuation, thus preventing recurrence. Oral administration of anti-CD-WPI is a functional therapy of CDI in infected hamsters for both primary treatment and prevention of recurrence. Thus, anti-CD-WPI could address the urgent unmet medical need for treating and preventing recurrent CDI in humans. Full article
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2018

Jump to: 2019, 2017, 2016, 2015

Open AccessFeature PaperArticle
Clostridium perfringens Enterotoxin: The Toxin Forms Highly Cation-Selective Channels in Lipid Bilayers
Received: 30 July 2018 / Revised: 14 August 2018 / Accepted: 14 August 2018 / Published: 22 August 2018
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Abstract
One of the numerous toxins produced by Clostridium perfringens is Clostridium perfringens enterotoxin (CPE), a polypeptide with a molecular mass of 35.5 kDa exhibiting three different domains. Domain one is responsible for receptor binding, domain two is involved in hexamer formation and domain [...] Read more.
One of the numerous toxins produced by Clostridium perfringens is Clostridium perfringens enterotoxin (CPE), a polypeptide with a molecular mass of 35.5 kDa exhibiting three different domains. Domain one is responsible for receptor binding, domain two is involved in hexamer formation and domain three has to do with channel formation in membranes. CPE is the major virulence factor of this bacterium and acts on the claudin-receptor containing tight junctions between epithelial cells resulting in various gastrointestinal diseases. The activity of CPE on Vero cells was demonstrated by the entry of propidium iodide (PI) in the cells. The entry of propidium iodide caused by CPE was well correlated with the loss of cell viability monitored by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test. CPE formed ion-permeable channels in artificial lipid bilayer membranes with a single-channel conductance of 620 pS in 1 M KCl. The single-channel conductance was not a linear function of the bulk aqueous salt concentration indicating that point-negative charges at the CPE channel controlled ion transport. This resulted in the high cation selectivity of the CPE channels, which suggested that anions are presumably not permeable through the CPE channels. The possible role of cation transport by CPE channels in disease caused by C. perfringens is discussed. Full article
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Open AccessArticle
The Binary Toxin CDT of Clostridium difficile as a Tool for Intracellular Delivery of Bacterial Glucosyltransferase Domains
Received: 9 April 2018 / Revised: 28 May 2018 / Accepted: 30 May 2018 / Published: 1 June 2018
Cited by 2 | PDF Full-text (2278 KB) | HTML Full-text | XML Full-text | Supplementary Files
Abstract
Binary toxins are produced by several pathogenic bacteria. Examples are the C2 toxin from Clostridium botulinum, the iota toxin from Clostridium perfringens, and the CDT from Clostridium difficile. All these binary toxins have ADP-ribosyltransferases (ADPRT) as their enzymatically active component that [...] Read more.
Binary toxins are produced by several pathogenic bacteria. Examples are the C2 toxin from Clostridium botulinum, the iota toxin from Clostridium perfringens, and the CDT from Clostridium difficile. All these binary toxins have ADP-ribosyltransferases (ADPRT) as their enzymatically active component that modify monomeric actin in their target cells. The binary C2 toxin was intensively described as a tool for intracellular delivery of allogenic ADPRTs. Here, we firstly describe the binary toxin CDT from C. difficile as an effective tool for heterologous intracellular delivery. Even 60 kDa glucosyltransferase domains of large clostridial glucosyltransferases can be delivered into cells. The glucosyltransferase domains of five tested large clostridial glucosyltransferases were successfully introduced into cells as chimeric fusions to the CDTa adapter domain (CDTaN). Cell uptake was demonstrated by the analysis of cell morphology, cytoskeleton staining, and intracellular substrate glucosylation. The fusion toxins were functional only when the adapter domain of CDTa was N-terminally located, according to its native orientation. Thus, like other binary toxins, the CDTaN/b system can be used for standardized delivery systems not only for bacterial ADPRTs but also for a variety of bacterial glucosyltransferase domains. Full article
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Open AccessReview
Mechanisms of Action and Cell Death Associated with Clostridium perfringens Toxins
Received: 27 April 2018 / Revised: 18 May 2018 / Accepted: 19 May 2018 / Published: 22 May 2018
Cited by 6 | PDF Full-text (2262 KB) | HTML Full-text | XML Full-text
Abstract
Clostridium perfringens uses its large arsenal of protein toxins to produce histotoxic, neurologic and intestinal infections in humans and animals. The major toxins involved in diseases are alpha (CPA), beta (CPB), epsilon (ETX), iota (ITX), enterotoxin (CPE), and necrotic B-like (NetB) toxins. CPA [...] Read more.
Clostridium perfringens uses its large arsenal of protein toxins to produce histotoxic, neurologic and intestinal infections in humans and animals. The major toxins involved in diseases are alpha (CPA), beta (CPB), epsilon (ETX), iota (ITX), enterotoxin (CPE), and necrotic B-like (NetB) toxins. CPA is the main virulence factor involved in gas gangrene in humans, whereas its role in animal diseases is limited and controversial. CPB is responsible for necrotizing enteritis and enterotoxemia, mostly in neonatal individuals of many animal species, including humans. ETX is the main toxin involved in enterotoxemia of sheep and goats. ITX has been implicated in cases of enteritis in rabbits and other animal species; however, its specific role in causing disease has not been proved. CPE is responsible for human food-poisoning and non-foodborne C. perfringens-mediated diarrhea. NetB is the cause of necrotic enteritis in chickens. In most cases, host–toxin interaction starts on the plasma membrane of target cells via specific receptors, resulting in the activation of intracellular pathways with a variety of effects, commonly including cell death. In general, the molecular mechanisms of cell death associated with C. perfringens toxins involve features of apoptosis, necrosis and/or necroptosis. Full article
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2017

Jump to: 2019, 2018, 2016, 2015

Open AccessEditor’s ChoiceArticle
Effect of Clostridium perfringens β-Toxin on Platelets
Toxins 2017, 9(10), 336; https://doi.org/10.3390/toxins9100336
Received: 2 October 2017 / Revised: 19 October 2017 / Accepted: 20 October 2017 / Published: 24 October 2017
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Abstract
Clostridium perfringens β-toxin (CPB) is the major virulence factor of C. perfringens type C causing a hemorrhagic enteritis in animals and humans. In experimentally infected pigs, endothelial binding of CPB was shown to be associated with early vascular lesions and hemorrhage but [...] Read more.
Clostridium perfringens β-toxin (CPB) is the major virulence factor of C. perfringens type C causing a hemorrhagic enteritis in animals and humans. In experimentally infected pigs, endothelial binding of CPB was shown to be associated with early vascular lesions and hemorrhage but without obvious thrombosis of affected vessels, suggesting altered hemostasis in the early phase of the disease. The objective of the present study was to investigate the effect of CPB on platelets, with respect to primary hemostasis. Our results demonstrate that CPB binds to porcine and human platelets and forms oligomers resulting in a time- and dose-dependent cell death. Platelets showed rapid ultrastructural changes, significantly decreased aggregation and could no longer be activated by thrombin. This indicates that CPB affects the physiological function of platelets and counteracts primary hemostasis. Our results add platelets to the list of target cells of CPB and extend the current hypothesis of its role in the pathogenesis of C. perfringens type C enteritis. Full article
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2016

Jump to: 2019, 2018, 2017, 2015

Open AccessCorrection
Correction: Chen, S., et al. Identification of an Essential Region for Translocation of Clostridium difficile Toxin B. Toxins 2016, 8, 241
Toxins 2016, 8(12), 352; https://doi.org/10.3390/toxins8120352
Received: 29 November 2016 / Revised: 30 November 2016 / Accepted: 1 December 2016 / Published: 2 December 2016
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Open AccessReview
Recombinant Alpha, Beta, and Epsilon Toxins of Clostridium perfringens: Production Strategies and Applications as Veterinary Vaccines
Toxins 2016, 8(11), 340; https://doi.org/10.3390/toxins8110340
Received: 20 October 2016 / Revised: 10 November 2016 / Accepted: 14 November 2016 / Published: 21 November 2016
Cited by 6 | PDF Full-text (2635 KB) | HTML Full-text | XML Full-text
Abstract
Clostridium perfringens is a spore-forming, commensal, ubiquitous bacterium that is present in the gastrointestinal tract of healthy humans and animals. This bacterium produces up to 18 toxins. The species is classified into five toxinotypes (A–E) according to the toxins that the bacterium produces: [...] Read more.
Clostridium perfringens is a spore-forming, commensal, ubiquitous bacterium that is present in the gastrointestinal tract of healthy humans and animals. This bacterium produces up to 18 toxins. The species is classified into five toxinotypes (A–E) according to the toxins that the bacterium produces: alpha, beta, epsilon, or iota. Each of these toxinotypes is associated with myriad different, frequently fatal, illnesses that affect a range of farm animals and humans. Alpha, beta, and epsilon toxins are the main causes of disease. Vaccinations that generate neutralizing antibodies are the most common prophylactic measures that are currently in use. These vaccines consist of toxoids that are obtained from C. perfringens cultures. Recombinant vaccines offer several advantages over conventional toxoids, especially in terms of the production process. As such, they are steadily gaining ground as a promising vaccination solution. This review discusses the main strategies that are currently used to produce recombinant vaccines containing alpha, beta, and epsilon toxins of C. perfringens, as well as the potential application of these molecules as vaccines for mammalian livestock animals. Full article
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Open AccessArticle
Identification of an Essential Region for Translocation of Clostridium difficile Toxin B
Received: 19 July 2016 / Revised: 8 August 2016 / Accepted: 8 August 2016 / Published: 15 August 2016
Cited by 4 | PDF Full-text (3202 KB) | HTML Full-text | XML Full-text
Abstract
Clostridium difficile toxin A (TcdA) and toxin B (TcdB) are the major virulence factors involved in C. difficile-associated diarrhea and pseudomembranous colitis. TcdA and TcdB both contain at least four distinct domains: the glucosyltransferase domain, cysteine protease domain, receptor binding domain, and [...] Read more.
Clostridium difficile toxin A (TcdA) and toxin B (TcdB) are the major virulence factors involved in C. difficile-associated diarrhea and pseudomembranous colitis. TcdA and TcdB both contain at least four distinct domains: the glucosyltransferase domain, cysteine protease domain, receptor binding domain, and translocation domain. Few studies have investigated the translocation domain and its mechanism of action. Recently, it was demonstrated that a segment of 97 amino acids (AA 1756–1852, designated D97) within the translocation domain of TcdB is essential for the in vitro and in vivo toxicity of TcdB. However, the mechanism by which D97 regulates the action of TcdB in host cells and the important amino acids within this region are unknown. In this study, we discovered that a smaller fragment, amino acids 1756–1780, located in the N-terminus of the D97 fragment, is essential for translocation of the effector glucosyltransferase domain into the host cytosol. A sequence of 25AA within D97 is predicted to form an alpha helical structure and is the critical part of D97. The deletion mutant TcdB∆1756–1780 showed similar glucosyltransferase and cysteine protease activity, cellular binding, and pore formation to wild type TcdB, but it failed to induce the glucosylation of Rho GTPase Rac1 of host cells. Moreover, we found that TcdB∆1756–1780 was rapidly degraded in the endosome of target cells, and therefore its intact glucosyltransferase domain was unable to translocate efficiently into host cytosol. Our finding provides an insight into the molecular mechanisms of action of TcdB in the intoxication of host cells. Full article
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Open AccessReview
Regulation of Toxin Production in Clostridium perfringens
Received: 20 March 2016 / Accepted: 24 June 2016 / Published: 5 July 2016
Cited by 9 | PDF Full-text (541 KB) | HTML Full-text | XML Full-text
Abstract
The Gram-positive anaerobic bacterium Clostridium perfringens is widely distributed in nature, especially in soil and the gastrointestinal tracts of humans and animals. C. perfringens causes gas gangrene and food poisoning, and it produces extracellular enzymes and toxins that are thought to act synergistically [...] Read more.
The Gram-positive anaerobic bacterium Clostridium perfringens is widely distributed in nature, especially in soil and the gastrointestinal tracts of humans and animals. C. perfringens causes gas gangrene and food poisoning, and it produces extracellular enzymes and toxins that are thought to act synergistically and contribute to its pathogenesis. A complicated regulatory network of toxin genes has been reported that includes a two-component system for regulatory RNA and cell-cell communication. It is necessary to clarify the global regulatory system of these genes in order to understand and treat the virulence of C. perfringens. We summarize the existing knowledge about the regulatory mechanisms here. Full article
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Open AccessArticle
The Sialidase NanS Enhances Non-TcsL Mediated Cytotoxicity of Clostridium sordellii
Received: 24 March 2016 / Accepted: 7 June 2016 / Published: 17 June 2016
Cited by 5 | PDF Full-text (1272 KB) | HTML Full-text | XML Full-text | Supplementary Files
Abstract
The clostridia produce an arsenal of toxins to facilitate their survival within the host environment. TcsL is one of two major toxins produced by Clostridium sordellii, a human and animal pathogen, and is essential for disease pathogenesis of this bacterium. C. sordellii [...] Read more.
The clostridia produce an arsenal of toxins to facilitate their survival within the host environment. TcsL is one of two major toxins produced by Clostridium sordellii, a human and animal pathogen, and is essential for disease pathogenesis of this bacterium. C. sordellii produces many other toxins, but the role that they play in disease is not known, although previous work has suggested that the sialidase enzyme NanS may be involved in the characteristic leukemoid reaction that occurs during severe disease. In this study we investigated the role of NanS in C. sordellii disease pathogenesis. We constructed a nanS mutant and showed that NanS is the only sialidase produced from C. sordellii strain ATCC9714 since sialidase activity could not be detected from the nanS mutant. Complementation with the wild-type gene restored sialidase production to the nanS mutant strain. Cytotoxicity assays using sialidase-enriched culture supernatants applied to gut (Caco2), vaginal (VK2), and cervical cell lines (End1/E6E7 and Ect1/E6E7) showed that NanS was not cytotoxic to these cells. However, the cytotoxic capacity of a toxin-enriched supernatant to the vaginal and cervical cell lines was substantially enhanced in the presence of NanS. TcsL was not the mediator of the observed cytotoxicity since supernatants harvested from a TcsL-deficient strain displayed similar cytotoxicity levels to TcsL-containing supernatants. This study suggests that NanS works synergistically with an unknown toxin or toxins to exacerbate C. sordellii-mediated tissue damage in the host. Full article
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Open AccessReview
The Regulatory Networks That Control Clostridium difficile Toxin Synthesis
Received: 2 April 2016 / Revised: 3 May 2016 / Accepted: 5 May 2016 / Published: 14 May 2016
Cited by 32 | PDF Full-text (1094 KB) | HTML Full-text | XML Full-text
Abstract
The pathogenic clostridia cause many human and animal diseases, which typically arise as a consequence of the production of potent exotoxins. Among the enterotoxic clostridia, Clostridium difficile is the main causative agent of nosocomial intestinal infections in adults with a compromised gut microbiota [...] Read more.
The pathogenic clostridia cause many human and animal diseases, which typically arise as a consequence of the production of potent exotoxins. Among the enterotoxic clostridia, Clostridium difficile is the main causative agent of nosocomial intestinal infections in adults with a compromised gut microbiota caused by antibiotic treatment. The symptoms of C. difficile infection are essentially caused by the production of two exotoxins: TcdA and TcdB. Moreover, for severe forms of disease, the spectrum of diseases caused by C. difficile has also been correlated to the levels of toxins that are produced during host infection. This observation strengthened the idea that the regulation of toxin synthesis is an important part of C. difficile pathogenesis. This review summarizes our current knowledge about the regulators and sigma factors that have been reported to control toxin gene expression in response to several environmental signals and stresses, including the availability of certain carbon sources and amino acids, or to signaling molecules, such as the autoinducing peptides of quorum sensing systems. The overlapping regulation of key metabolic pathways and toxin synthesis strongly suggests that toxin production is a complex response that is triggered by bacteria in response to particular states of nutrient availability during infection. Full article
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Open AccessFeature PaperReview
Clostridium difficile Toxins A and B: Insights into Pathogenic Properties and Extraintestinal Effects
Received: 25 February 2016 / Revised: 22 April 2016 / Accepted: 25 April 2016 / Published: 3 May 2016
Cited by 26 | PDF Full-text (3816 KB) | HTML Full-text | XML Full-text
Abstract
Clostridium difficile infection (CDI) has significant clinical impact especially on the elderly and/or immunocompromised patients. The pathogenicity of Clostridium difficile is mainly mediated by two exotoxins: toxin A (TcdA) and toxin B (TcdB). These toxins primarily disrupt the cytoskeletal structure and the tight [...] Read more.
Clostridium difficile infection (CDI) has significant clinical impact especially on the elderly and/or immunocompromised patients. The pathogenicity of Clostridium difficile is mainly mediated by two exotoxins: toxin A (TcdA) and toxin B (TcdB). These toxins primarily disrupt the cytoskeletal structure and the tight junctions of target cells causing cell rounding and ultimately cell death. Detectable C. difficile toxemia is strongly associated with fulminant disease. However, besides the well-known intestinal damage, recent animal and in vitro studies have suggested a more far-reaching role for these toxins activity including cardiac, renal, and neurologic impairment. The creation of C. difficile strains with mutations in the genes encoding toxin A and B indicate that toxin B plays a major role in overall CDI pathogenesis. Novel insights, such as the role of a regulator protein (TcdE) on toxin production and binding interactions between albumin and C. difficile toxins, have recently been discovered and will be described. Our review focuses on the toxin-mediated pathogenic processes of CDI with an emphasis on recent studies. Full article
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Open AccessFeature PaperArticle
Metal Ion Activation of Clostridium sordellii Lethal Toxin and Clostridium difficile Toxin B
Received: 21 March 2016 / Revised: 5 April 2016 / Accepted: 5 April 2016 / Published: 13 April 2016
Cited by 2 | PDF Full-text (1544 KB) | HTML Full-text | XML Full-text
Abstract
Lethal Toxin from Clostridium sordellii (TcsL) and Toxin B from Clostridium difficile (TcdB) belong to the family of the “Large clostridial glycosylating toxins.” These toxins mono-O-glucosylate low molecular weight GTPases of the Rho and Ras families by exploiting UDP-glucose as a hexose donor. [...] Read more.
Lethal Toxin from Clostridium sordellii (TcsL) and Toxin B from Clostridium difficile (TcdB) belong to the family of the “Large clostridial glycosylating toxins.” These toxins mono-O-glucosylate low molecular weight GTPases of the Rho and Ras families by exploiting UDP-glucose as a hexose donor. TcsL is casually involved in the toxic shock syndrome and the gas gangrene. TcdB—together with Toxin A (TcdA)—is causative for the pseudomembranous colitis (PMC). Here, we present evidence for the in vitro metal ion activation of the glucosyltransferase and the UDP-glucose hydrolysis activity of TcsL and TcdB. The following rating is found for activation by divalent metal ions: Mn2+ > Co2+ > Mg2+ >> Ca2+, Cu2+, Zn2+. TcsL and TcdB thus require divalent metal ions providing an octahedral coordination sphere. The EC50 values for TcsL were estimated at about 28 µM for Mn2+ and 180 µM for Mg2+. TcsL and TcdB further require co-stimulation by monovalent K+ (not by Na+). Finally, prebound divalent metal ions were dispensible for the cytopathic effects of TcsL and TcdB, leading to the conclusion that TcsL and TcdB recruit intracellular metal ions for activation of the glucosyltransferase activity. With regard to the intracellular metal ion concentrations, TcsL and TcdB are most likely activated by K+ and Mg2+ (rather than Mn2+) in mammalian target cells. Full article
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Open AccessArticle
EGA Protects Mammalian Cells from Clostridium difficile CDT, Clostridium perfringens Iota Toxin and Clostridium botulinum C2 Toxin
Received: 25 January 2016 / Revised: 22 March 2016 / Accepted: 24 March 2016 / Published: 1 April 2016
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Abstract
The pathogenic bacteria Clostridium difficile, Clostridium perfringens and Clostridium botulinum produce the binary actin ADP-ribosylating toxins CDT, iota and C2, respectively. These toxins are composed of a transport component (B) and a separate enzyme component (A). When both components assemble on the [...] Read more.
The pathogenic bacteria Clostridium difficile, Clostridium perfringens and Clostridium botulinum produce the binary actin ADP-ribosylating toxins CDT, iota and C2, respectively. These toxins are composed of a transport component (B) and a separate enzyme component (A). When both components assemble on the surface of mammalian target cells, the B components mediate the entry of the A components via endosomes into the cytosol. Here, the A components ADP-ribosylate G-actin, resulting in depolymerization of F-actin, cell-rounding and eventually death. In the present study, we demonstrate that 4-bromobenzaldehyde N-(2,6-dimethylphenyl)semicarbazone (EGA), a compound that protects cells from multiple toxins and viruses, also protects different mammalian epithelial cells from all three binary actin ADP-ribosylating toxins. In contrast, EGA did not inhibit the intoxication of cells with Clostridium difficile toxins A and B, indicating a possible different entry route for this toxin. EGA does not affect either the binding of the C2 toxin to the cells surface or the enzyme activity of the A components of CDT, iota and C2, suggesting that this compound interferes with cellular uptake of the toxins. Moreover, for C2 toxin, we demonstrated that EGA inhibits the pH-dependent transport of the A component across cell membranes. EGA is not cytotoxic, and therefore, we propose it as a lead compound for the development of novel pharmacological inhibitors against clostridial binary actin ADP-ribosylating toxins. Full article
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Open AccessArticle
The Tip of the Four N-Terminal α-Helices of Clostridium sordellii Lethal Toxin Contains the Interaction Site with Membrane Phosphatidylserine Facilitating Small GTPases Glucosylation
Received: 5 February 2016 / Revised: 1 March 2016 / Accepted: 10 March 2016 / Published: 25 March 2016
Cited by 5 | PDF Full-text (4390 KB) | HTML Full-text | XML Full-text | Supplementary Files
Abstract
Clostridium sordellii lethal toxin (TcsL) is a powerful virulence factor responsible for severe toxic shock in man and animals. TcsL belongs to the large clostridial glucosylating toxin (LCGT) family which inactivates small GTPases by glucosylation with uridine-diphosphate (UDP)-glucose as a cofactor. Notably, TcsL [...] Read more.
Clostridium sordellii lethal toxin (TcsL) is a powerful virulence factor responsible for severe toxic shock in man and animals. TcsL belongs to the large clostridial glucosylating toxin (LCGT) family which inactivates small GTPases by glucosylation with uridine-diphosphate (UDP)-glucose as a cofactor. Notably, TcsL modifies Rac and Ras GTPases, leading to drastic alteration of the actin cytoskeleton and cell viability. TcsL enters cells via receptor-mediated endocytosis and delivers the N-terminal glucosylating domain (TcsL-cat) into the cytosol. TcsL-cat was found to preferentially bind to phosphatidylserine (PS)-containing membranes and to increase the glucosylation of Rac anchored to the lipid membrane. We have previously reported that the N-terminal four helical bundle structure (1–93 domain) recognizes a broad range of lipids, but that TcsL-cat specifically binds to PS and phosphatidic acid. Here, we show using mutagenesis that the PS binding site is localized on the tip of the four-helix bundle which is rich in positively-charged amino acids. Residues Y14, V15, F17, and R18 on loop 1, between helices 1 and 2, in coordination with R68 from loop 3, between helices 3 and 4, form a pocket which accommodates L-serine. The functional PS-binding site is required for TcsL-cat binding to the plasma membrane and subsequent cytotoxicity. TcsL-cat binding to PS facilitates a high enzymatic activity towards membrane-anchored Ras by about three orders of magnitude as compared to Ras in solution. The PS-binding site is conserved in LCGTs, which likely retain a common mechanism of binding to the membrane for their full activity towards membrane-bound GTPases. Full article
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Open AccessFeature PaperReview
Clostridium perfringens Enterotoxin: Action, Genetics, and Translational Applications
Received: 23 February 2016 / Revised: 4 March 2016 / Accepted: 8 March 2016 / Published: 16 March 2016
Cited by 40 | PDF Full-text (2264 KB) | HTML Full-text | XML Full-text
Abstract
Clostridium perfringens enterotoxin (CPE) is responsible for causing the gastrointestinal symptoms of several C. perfringens food- and nonfood-borne human gastrointestinal diseases. The enterotoxin gene (cpe) is located on either the chromosome (for most C. perfringens type A food poisoning strains) or [...] Read more.
Clostridium perfringens enterotoxin (CPE) is responsible for causing the gastrointestinal symptoms of several C. perfringens food- and nonfood-borne human gastrointestinal diseases. The enterotoxin gene (cpe) is located on either the chromosome (for most C. perfringens type A food poisoning strains) or large conjugative plasmids (for the remaining type A food poisoning and most, if not all, other CPE-producing strains). In all CPE-positive strains, the cpe gene is strongly associated with insertion sequences that may help to assist its mobilization and spread. During disease, CPE is produced when C. perfringens sporulates in the intestines, a process involving several sporulation-specific alternative sigma factors. The action of CPE starts with its binding to claudin receptors to form a small complex; those small complexes then oligomerize to create a hexameric prepore on the membrane surface. Beta hairpin loops from the CPE molecules in the prepore assemble into a beta barrel that inserts into the membrane to form an active pore that enhances calcium influx, causing cell death. This cell death results in intestinal damage that causes fluid and electrolyte loss. CPE is now being explored for translational applications including cancer therapy/diagnosis, drug delivery, and vaccination. Full article
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Open AccessReview
Perfringolysin O Theta Toxin as a Tool to Monitor the Distribution and Inhomogeneity of Cholesterol in Cellular Membranes
Received: 4 February 2016 / Revised: 26 February 2016 / Accepted: 26 February 2016 / Published: 8 March 2016
Cited by 12 | PDF Full-text (1288 KB) | HTML Full-text | XML Full-text | Correction
Abstract
Cholesterol is an essential structural component of cellular membranes in eukaryotes. Cholesterol in the exofacial leaflet of the plasma membrane is thought to form membrane nanodomains with sphingolipids and specific proteins. Additionally, cholesterol is found in the intracellular membranes of endosomes and has [...] Read more.
Cholesterol is an essential structural component of cellular membranes in eukaryotes. Cholesterol in the exofacial leaflet of the plasma membrane is thought to form membrane nanodomains with sphingolipids and specific proteins. Additionally, cholesterol is found in the intracellular membranes of endosomes and has crucial functions in membrane trafficking. Furthermore, cellular cholesterol homeostasis and regulation of de novo synthesis rely on transport via both vesicular and non-vesicular pathways. Thus, the ability to visualize and detect intracellular cholesterol, especially in the plasma membrane, is critical to understanding the complex biology associated with cholesterol and the nanodomains. Perfringolysin O (PFO) theta toxin is one of the toxins secreted by the anaerobic bacteria Clostridium perfringens and this toxin forms pores in the plasma membrane that causes cell lysis. It is well understood that PFO recognizes and binds to cholesterol in the exofacial leaflets of the plasma membrane, and domain 4 of PFO (D4) is sufficient for the binding of cholesterol. Recent studies have taken advantage of this high-affinity cholesterol-binding domain to create a variety of cholesterol biosensors by using a non-toxic PFO or the D4 in isolation. This review highlights the characteristics and usefulness of, and the principal findings related to, these PFO-derived cholesterol biosensors. Full article
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Open AccessReview
Reactive Oxygen Species as Additional Determinants for Cytotoxicity of Clostridium difficile Toxins A and B
Received: 7 December 2015 / Revised: 7 January 2016 / Accepted: 13 January 2016 / Published: 18 January 2016
Cited by 9 | PDF Full-text (493 KB) | HTML Full-text | XML Full-text
Abstract
Clostridium difficile infections can induce mild to severe diarrhoea and the often associated characteristic pseudomembranous colitis. Two protein toxins, the large glucosyltransferases TcdA and TcdB, are the main pathogenicity factors that can induce all clinical symptoms in animal models. The classical molecular mode [...] Read more.
Clostridium difficile infections can induce mild to severe diarrhoea and the often associated characteristic pseudomembranous colitis. Two protein toxins, the large glucosyltransferases TcdA and TcdB, are the main pathogenicity factors that can induce all clinical symptoms in animal models. The classical molecular mode of action of these homologous toxins is the inhibition of Rho GTPases by mono-glucosylation. Rho-inhibition leads to breakdown of the actin cytoskeleton, induces stress-activated and pro-inflammatory signaling and eventually results in apoptosis of the affected cells. An increasing number of reports, however, have documented further qualities of TcdA and TcdB, including the production of reactive oxygen species (ROS) by target cells. This review summarizes observations dealing with the production of ROS induced by TcdA and TcdB, dissects pathways that contribute to this phenomenon and speculates about ROS in mediating pathogenesis. In conclusion, ROS have to be considered as a discrete, glucosyltransferase-independent quality of at least TcdB, triggered by different mechanisms. Full article
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2015

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Open AccessReview
Membrane-Binding Mechanism of Clostridium perfringens Alpha-Toxin
Toxins 2015, 7(12), 5268-5275; https://doi.org/10.3390/toxins7124880
Received: 27 October 2015 / Revised: 17 November 2015 / Accepted: 30 November 2015 / Published: 3 December 2015
Cited by 11 | PDF Full-text (1142 KB) | HTML Full-text | XML Full-text
Abstract
Clostridium perfringens alpha-toxin is a key mediator of gas gangrene, which is a life-threatening infection that manifests as fever, pain, edema, myonecrosis, and gas production. Alpha-toxin possesses phospholipase C and sphingomyelinase activities. The toxin is composed of an N-terminal domain (1–250 aa, [...] Read more.
Clostridium perfringens alpha-toxin is a key mediator of gas gangrene, which is a life-threatening infection that manifests as fever, pain, edema, myonecrosis, and gas production. Alpha-toxin possesses phospholipase C and sphingomyelinase activities. The toxin is composed of an N-terminal domain (1–250 aa, N-domain), which is the catalytic site, and a C-terminal domain (251–370 aa, C-domain), which is the membrane-binding site. Immunization of mice with the C-domain of alpha-toxin prevents the gas gangrene caused by C. perfringens, whereas immunization with the N-domain has no effect. The central loop domain (55–93 aa), especially H….SW84Y85….G, plays an important role in the interaction with ganglioside GM1a. The toxin binds to lipid rafts in the presence of a GM1a/TrkA complex, and metabolites from phosphatidylcholine to diacylglycerol through the enzymatic activity of alpha-toxin itself. These membrane dynamics leads to the activation of endogenous PLCγ-1 via TrkA. In addition, treatment with alpha-toxin leads to the formation of diacylglycerol at membrane rafts in ganglioside-deficient DonQ cells; this in turn triggers endocytosis and cell death. This article summarizes the current the membrane-binding mechanism of alpha-toxin in detail. Full article
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Open AccessReview
The Role of Rho GTPases in Toxicity of Clostridium difficile Toxins
Toxins 2015, 7(12), 5254-5267; https://doi.org/10.3390/toxins7124874
Received: 24 September 2015 / Revised: 18 November 2015 / Accepted: 18 November 2015 / Published: 2 December 2015
Cited by 22 | PDF Full-text (1061 KB) | HTML Full-text | XML Full-text
Abstract
Clostridium difficile (C. difficile) is the main cause of antibiotic-associated diarrhea prevailing in hospital settings. In the past decade, the morbidity and mortality of C. difficile infection (CDI) has increased significantly due to the emergence of hypervirulent strains. Toxin A (TcdA) [...] Read more.
Clostridium difficile (C. difficile) is the main cause of antibiotic-associated diarrhea prevailing in hospital settings. In the past decade, the morbidity and mortality of C. difficile infection (CDI) has increased significantly due to the emergence of hypervirulent strains. Toxin A (TcdA) and toxin B (TcdB), the two exotoxins of C. difficile, are the major virulence factors of CDI. The common mode of action of TcdA and TcdB is elicited by specific glucosylation of Rho-GTPase proteins in the host cytosol using UDP-glucose as a co-substrate, resulting in the inactivation of Rho proteins. Rho proteins are the key members in many biological processes and signaling pathways, inactivation of which leads to cytopathic and cytotoxic effects and immune responses of the host cells. It is supposed that Rho GTPases play an important role in the toxicity of C. difficile toxins. This review focuses on recent progresses in the understanding of functional consequences of Rho GTPases glucosylation induced by C. difficile toxins and the role of Rho GTPases in the toxicity of TcdA and TcdB. Full article
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Open AccessArticle
Veal Calves Produce Less Antibodies against C. Perfringens Alpha Toxin Compared to Beef Calves
Toxins 2015, 7(7), 2586-2597; https://doi.org/10.3390/toxins7072586
Received: 28 May 2015 / Revised: 30 June 2015 / Accepted: 7 July 2015 / Published: 10 July 2015
Cited by 3 | PDF Full-text (344 KB) | HTML Full-text | XML Full-text | Supplementary Files
Abstract
Enterotoxaemia is a disease with a high associated mortality rate, affecting beef and veal calves worldwide, caused by C. perfringens alpha toxin and perfringolysin. A longitudinal study was conducted to determine the dynamics of antibodies against these toxins in 528 calves on 4 [...] Read more.
Enterotoxaemia is a disease with a high associated mortality rate, affecting beef and veal calves worldwide, caused by C. perfringens alpha toxin and perfringolysin. A longitudinal study was conducted to determine the dynamics of antibodies against these toxins in 528 calves on 4 beef and 15 veal farms. The second study aimed to determine the effect of solid feed intake on the production of antibodies against alpha toxin and perfringolysin. The control group only received milk replacer, whereas in the test group solid feed was provided. Maternal antibodies for alpha toxin were present in 45% of the veal calves and 66% of the beef calves. In beef calves a fluent transition from maternal to active immunity was observed for alpha toxin, whereas almost no veal calves developed active immunity. Perfringolysin antibodies significantly declined both in veal and beef calves. In the second study all calves were seropositive for alpha toxin throughout the experiment and solid feed intake did not alter the dynamics of alpha and perfringolysin antibodies. In conclusion, the present study showed that veal calves on a traditional milk replacer diet had significantly lower alpha toxin antibodies compared to beef calves in the risk period for enterotoxaemia, whereas no differences were noticed for perfringolysin. Full article
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Open AccessReview
Perfringolysin O: The Underrated Clostridium perfringens Toxin?
Toxins 2015, 7(5), 1702-1721; https://doi.org/10.3390/toxins7051702
Received: 9 April 2015 / Accepted: 8 May 2015 / Published: 14 May 2015
Cited by 14 | PDF Full-text (697 KB) | HTML Full-text | XML Full-text
Abstract
The anaerobic bacterium Clostridium perfringens expresses multiple toxins that promote disease development in both humans and animals. One such toxin is perfringolysin O (PFO, classically referred to as θ toxin), a pore-forming cholesterol-dependent cytolysin (CDC). PFO is secreted as a water-soluble monomer that [...] Read more.
The anaerobic bacterium Clostridium perfringens expresses multiple toxins that promote disease development in both humans and animals. One such toxin is perfringolysin O (PFO, classically referred to as θ toxin), a pore-forming cholesterol-dependent cytolysin (CDC). PFO is secreted as a water-soluble monomer that recognizes and binds membranes via cholesterol. Membrane-bound monomers undergo structural changes that culminate in the formation of an oligomerized prepore complex on the membrane surface. The prepore then undergoes conversion into the bilayer-spanning pore measuring approximately 250–300 Å in diameter. PFO is expressed in nearly all identified C. perfringens strains and harbors interesting traits that suggest a potential undefined role for PFO in disease development. Research has demonstrated a role for PFO in gas gangrene progression and bovine necrohemorrhagic enteritis, but there is limited data available to determine if PFO also functions in additional disease presentations caused by C. perfringens. This review summarizes the known structural and functional characteristics of PFO, while highlighting recent insights into the potential contributions of PFO to disease pathogenesis. Full article
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