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Determinants of Raft Partitioning of the Helicobacter pylori Pore-Forming Toxin VacA.
Raghunathan K, Foegeding NJ, Campbell AM, Cover TL, Ohi MD, Kenworthy AK
(2018) Infect Immun 86:
MeSH Terms: Bacterial Proteins, Bacterial Toxins, Helicobacter pylori, Host-Pathogen Interactions, Membrane Microdomains, Stomach Neoplasms, Vacuoles
Show Abstract · Added July 29, 2018
, a Gram-negative bacterium, is a well-known risk factor for gastric cancer. vacuolating cytotoxin A (VacA) is a secreted pore-forming toxin that induces a wide range of cellular responses. Like many other bacterial toxins, VacA has been hypothesized to utilize lipid rafts to gain entry into host cells. Here, we used giant plasma membrane vesicles (GPMVs) as a model system to understand the preferential partitioning of VacA into lipid rafts. We show that a wild-type (WT) toxin predominantly associates with the raft phase. Acid activation of VacA enhances binding of the toxin to GPMVs but is not required for raft partitioning. VacA mutant proteins with alterations at the amino terminus (resulting in impaired membrane channel formation) and a nonoligomerizing VacA mutant protein retain the ability to preferentially associate with lipid rafts. Consistent with these results, the isolated VacA p55 domain was capable of binding to lipid rafts. We conclude that the affinity of VacA for rafts is independent of its capacity to oligomerize or form membrane channels.
Copyright © 2018 American Society for Microbiology.
0 Communities
1 Members
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7 MeSH Terms
Patients with familial adenomatous polyposis harbor colonic biofilms containing tumorigenic bacteria.
Dejea CM, Fathi P, Craig JM, Boleij A, Taddese R, Geis AL, Wu X, DeStefano Shields CE, Hechenbleikner EM, Huso DL, Anders RA, Giardiello FM, Wick EC, Wang H, Wu S, Pardoll DM, Housseau F, Sears CL
(2018) Science 359: 592-597
MeSH Terms: Adenomatous Polyposis Coli, Animals, Bacterial Toxins, Bacteroides fragilis, Biofilms, Carcinogenesis, Colon, Colonic Neoplasms, DNA Damage, Escherichia coli, Gastrointestinal Microbiome, Humans, Interleukin-17, Intestinal Mucosa, Metalloendopeptidases, Mice, Peptides, Polyketides, Precancerous Conditions
Show Abstract · Added March 20, 2018
Individuals with sporadic colorectal cancer (CRC) frequently harbor abnormalities in the composition of the gut microbiome; however, the microbiota associated with precancerous lesions in hereditary CRC remains largely unknown. We studied colonic mucosa of patients with familial adenomatous polyposis (FAP), who develop benign precursor lesions (polyps) early in life. We identified patchy bacterial biofilms composed predominately of and Genes for colibactin () and toxin (), encoding secreted oncotoxins, were highly enriched in FAP patients' colonic mucosa compared to healthy individuals. Tumor-prone mice cocolonized with (expressing colibactin), and enterotoxigenic showed increased interleukin-17 in the colon and DNA damage in colonic epithelium with faster tumor onset and greater mortality, compared to mice with either bacterial strain alone. These data suggest an unexpected link between early neoplasia of the colon and tumorigenic bacteria.
Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.
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19 MeSH Terms
Bacteroides fragilis Toxin Coordinates a Pro-carcinogenic Inflammatory Cascade via Targeting of Colonic Epithelial Cells.
Chung L, Thiele Orberg E, Geis AL, Chan JL, Fu K, DeStefano Shields CE, Dejea CM, Fathi P, Chen J, Finard BB, Tam AJ, McAllister F, Fan H, Wu X, Ganguly S, Lebid A, Metz P, Van Meerbeke SW, Huso DL, Wick EC, Pardoll DM, Wan F, Wu S, Sears CL, Housseau F
(2018) Cell Host Microbe 23: 203-214.e5
MeSH Terms: Adenomatous Polyposis Coli Protein, Animals, Bacterial Toxins, Bacteroides fragilis, Carcinogenesis, Cell Line, Tumor, Colon, Colorectal Neoplasms, Enzyme Activation, Epithelial Cells, Female, Gene Deletion, HT29 Cells, Humans, Inflammation, Interleukin-17, Male, Metalloendopeptidases, Mice, Mice, Inbred C57BL, Mice, Knockout, Myeloid Cells, Receptors, Interleukin-17, Receptors, Interleukin-8B, STAT3 Transcription Factor, Transcription Factor RelA
Show Abstract · Added March 20, 2018
Pro-carcinogenic bacteria have the potential to initiate and/or promote colon cancer, in part via immune mechanisms that are incompletely understood. Using Apc mice colonized with the human pathobiont enterotoxigenic Bacteroides fragilis (ETBF) as a model of microbe-induced colon tumorigenesis, we show that the Bacteroides fragilis toxin (BFT) triggers a pro-carcinogenic, multi-step inflammatory cascade requiring IL-17R, NF-κB, and Stat3 signaling in colonic epithelial cells (CECs). Although necessary, Stat3 activation in CECs is not sufficient to trigger ETBF colon tumorigenesis. Notably, IL-17-dependent NF-κB activation in CECs induces a proximal to distal mucosal gradient of C-X-C chemokines, including CXCL1, that mediates the recruitment of CXCR2-expressing polymorphonuclear immature myeloid cells with parallel onset of ETBF-mediated distal colon tumorigenesis. Thus, BFT induces a pro-carcinogenic signaling relay from the CEC to a mucosal Th17 response that results in selective NF-κB activation in distal colon CECs, which collectively triggers myeloid-cell-dependent distal colon tumorigenesis.
Copyright © 2018 Elsevier Inc. All rights reserved.
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1 Members
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26 MeSH Terms
A neutralizing antibody that blocks delivery of the enzymatic cargo of toxin TcdB into host cells.
Kroh HK, Chandrasekaran R, Zhang Z, Rosenthal K, Woods R, Jin X, Nyborg AC, Rainey GJ, Warrener P, Melnyk RA, Spiller BW, Lacy DB
(2018) J Biol Chem 293: 941-952
MeSH Terms: Antibodies, Monoclonal, Antibodies, Neutralizing, Bacterial Toxins, Caco-2 Cells, Clostridium difficile, Crystallography, X-Ray, Cytosol, Enterotoxins, Humans, Hydrogen-Ion Concentration, Microscopy, Electron, Rubidium, rac1 GTP-Binding Protein
Show Abstract · Added March 15, 2018
infection is the leading cause of hospital-acquired diarrhea and is mediated by the actions of two toxins, TcdA and TcdB. The toxins perturb host cell function through a multistep process of receptor binding, endocytosis, low pH-induced pore formation, and the translocation and delivery of an N-terminal glucosyltransferase domain that inactivates host GTPases. Infection studies with isogenic strains having defined toxin deletions have established TcdB as an important target for therapeutic development. Monoclonal antibodies that neutralize TcdB function have been shown to protect against infection in animal models and reduce recurrence in humans. Here, we report the mechanism of TcdB neutralization by PA41, a humanized monoclonal antibody capable of neutralizing TcdB from a diverse array of strains. Through a combination of structural, biochemical, and cell functional studies, involving X-ray crystallography and EM, we show that PA41 recognizes a single, highly conserved epitope on the TcdB glucosyltransferase domain and blocks productive translocation and delivery of the enzymatic cargo into the host cell. Our study reveals a unique mechanism of toxin neutralization by a monoclonal antibody, which involves targeting a process that is conserved across the large clostridial glucosylating toxins. The PA41 antibody described here provides a valuable tool for dissecting the mechanism of toxin pore formation and translocation across the endosomal membrane.
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2 Members
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13 MeSH Terms
The role of toxins in Clostridium difficile infection.
Chandrasekaran R, Lacy DB
(2017) FEMS Microbiol Rev 41: 723-750
MeSH Terms: Bacterial Toxins, Clostridium Infections, Clostridium difficile, Humans, Immunity, Intestinal Mucosa
Show Abstract · Added April 3, 2018
Clostridium difficile is a bacterial pathogen that is the leading cause of nosocomial antibiotic-associated diarrhea and pseudomembranous colitis worldwide. The incidence, severity, mortality and healthcare costs associated with C. difficile infection (CDI) are rising, making C. difficile a major threat to public health. Traditional treatments for CDI involve use of antibiotics such as metronidazole and vancomycin, but disease recurrence occurs in about 30% of patients, highlighting the need for new therapies. The pathogenesis of C. difficile is primarily mediated by the actions of two large clostridial glucosylating toxins, toxin A (TcdA) and toxin B (TcdB). Some strains produce a third toxin, the binary toxin C. difficile transferase, which can also contribute to C. difficile virulence and disease. These toxins act on the colonic epithelium and immune cells and induce a complex cascade of cellular events that result in fluid secretion, inflammation and tissue damage, which are the hallmark features of the disease. In this review, we summarize our current understanding of the structure and mechanism of action of the C. difficile toxins and their role in disease.
Published by Oxford University Press on behalf of FEMS 2017.
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MeSH Terms
Functional defects in TcdB toxin uptake identify CSPG4 receptor-binding determinants.
Gupta P, Zhang Z, Sugiman-Marangos SN, Tam J, Raman S, Julien JP, Kroh HK, Lacy DB, Murgolo N, Bekkari K, Therien AG, Hernandez LD, Melnyk RA
(2017) J Biol Chem 292: 17290-17301
MeSH Terms: Animals, Antibodies, Monoclonal, Antibodies, Neutralizing, Bacterial Proteins, Bacterial Toxins, CHO Cells, Caco-2 Cells, Cercopithecus aethiops, Chondroitin Sulfate Proteoglycans, Clostridium difficile, Cricetinae, Cricetulus, Glucosyltransferases, HEK293 Cells, Humans, Membrane Proteins, Protein Binding, Protein Domains
Show Abstract · Added April 3, 2018
is a major nosocomial pathogen that produces two exotoxins, TcdA and TcdB, with TcdB thought to be the primary determinant in human disease. TcdA and TcdB are large, multidomain proteins, each harboring a cytotoxic glucosyltransferase domain that is delivered into the cytosol from endosomes via a translocation domain after receptor-mediated endocytosis of toxins from the cell surface. Although there are currently no known host cell receptors for TcdA, three cell-surface receptors for TcdB have been identified: CSPG4, NECTIN3, and FZD1/2/7. The sites on TcdB that mediate binding to each receptor are not defined. Furthermore, it is not known whether the combined repetitive oligopeptide (CROP) domain is involved in or required for receptor binding. Here, in a screen designed to identify sites in TcdB that are essential for target cell intoxication, we identified a region at the junction of the translocation and the CROP domains that is implicated in CSPG4 binding. Using a series of C-terminal truncations, we show that the CSPG4-binding site on TcdB extends into the CROP domain, requiring three short repeats for binding and for full toxicity on CSPG4-expressing cells. Consistent with the location of the CSPG4-binding site on TcdB, we show that the anti-TcdB antibody bezlotoxumab, which binds partially within the first three short repeats, prevents CSPG4 binding to TcdB. In addition to establishing the binding region for CSPG4, this work ascribes for the first time a role in TcdB CROPs in receptor binding and further clarifies the relative roles of host receptors in TcdB pathogenesis.
© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
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MeSH Terms
Use of a neutralizing antibody helps identify structural features critical for binding of toxin TcdA to the host cell surface.
Kroh HK, Chandrasekaran R, Rosenthal K, Woods R, Jin X, Ohi MD, Nyborg AC, Rainey GJ, Warrener P, Spiller BW, Lacy DB
(2017) J Biol Chem 292: 14401-14412
MeSH Terms: Amino Acid Sequence, Anti-Bacterial Agents, Antibodies, Monoclonal, Humanized, Antibodies, Neutralizing, Bacterial Proteins, Bacterial Toxins, Binding Sites, Antibody, Caco-2 Cells, Clostridium difficile, Conserved Sequence, Crystallography, X-Ray, Enterocytes, Enterotoxins, Epitope Mapping, Glucosyltransferases, Humans, Immunoglobulin Fab Fragments, Models, Molecular, Peptide Fragments, Protein Conformation, Protein Interaction Domains and Motifs, Recombinant Proteins, Repetitive Sequences, Amino Acid
Show Abstract · Added March 15, 2018
is a clinically significant pathogen that causes mild-to-severe (and often recurrent) colon infections. Disease symptoms stem from the activities of two large, multidomain toxins known as TcdA and TcdB. The toxins can bind, enter, and perturb host cell function through a multistep mechanism of receptor binding, endocytosis, pore formation, autoproteolysis, and glucosyltransferase-mediated modification of host substrates. Monoclonal antibodies that neutralize toxin activity provide a survival benefit in preclinical animal models and prevent recurrent infections in human clinical trials. However, the molecular mechanisms involved in these neutralizing activities are unclear. To this end, we performed structural studies on a neutralizing monoclonal antibody, PA50, a humanized mAb with both potent and broad-spectrum neutralizing activity, in complex with TcdA. Electron microscopy imaging and multiangle light-scattering analysis revealed that PA50 binds multiple sites on the TcdA C-terminal combined repetitive oligopeptides (CROPs) domain. A crystal structure of two PA50 Fabs bound to a segment of the TcdA CROPs helped define a conserved epitope that is distinct from previously identified carbohydrate-binding sites. Binding of TcdA to the host cell surface was directly blocked by either PA50 mAb or Fab and suggested that receptor blockade is the mechanism by which PA50 neutralizes TcdA. These findings highlight the importance of the CROPs C terminus in cell-surface binding and a role for neutralizing antibodies in defining structural features critical to a pathogen's mechanism of action. We conclude that PA50 protects host cells by blocking the binding of TcdA to cell surfaces.
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2 Members
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23 MeSH Terms
Clostridium difficile toxin glucosyltransferase domains in complex with a non-hydrolyzable UDP-glucose analogue.
Alvin JW, Lacy DB
(2017) J Struct Biol 198: 203-209
MeSH Terms: Apigenin, Bacterial Proteins, Bacterial Toxins, Binding Sites, Clostridium difficile, Enterotoxins, Glucosyltransferases, Molecular Structure, Protein Binding, Uridine Diphosphate Glucose
Show Abstract · Added April 3, 2018
Clostridium difficile is the leading cause of hospital-acquired diarrhea and pseudomembranous colitis worldwide. The organism produces two homologous toxins, TcdA and TcdB, which enter and disrupt host cell function by glucosylating and thereby inactivating key signalling molecules within the host. As a toxin-mediated disease, there has been a significant interest in identifying small molecule inhibitors of the toxins' glucosyltransferase activities. This study was initiated as part of an effort to identify the mode of inhibition for a small molecule inhibitor of glucosyltransferase activity called apigenin. In the course of trying to get co-crystals with this inhibitor, we determined five different structures of the TcdA and TcdB glucosyltransferase domains and made use of a non-hydrolyzable UDP-glucose substrate. While we were able to visualize apigenin bound in one of our structures, the site was a crystal packing interface and not likely to explain the mode of inhibition. Nevertheless, the structure allowed us to capture an apo-state (one without the sugar nucleotide substrate) of the TcdB glycosyltransferase domain that had not been previously observed. Comparison of this structure with structures obtained in the presence of a non-hydrolyzable UDP-glucose analogue have allowed us to document multiple conformations of a C-terminal loop important for catalysis. We present our analysis of these five new structures with the hope that it will advance inhibitor design efforts for this important class of biological toxins.
Published by Elsevier Inc.
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1 Members
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MeSH Terms
Epitopes and Mechanism of Action of the Clostridium difficile Toxin A-Neutralizing Antibody Actoxumab.
Hernandez LD, Kroh HK, Hsieh E, Yang X, Beaumont M, Sheth PR, DiNunzio E, Rutherford SA, Ohi MD, Ermakov G, Xiao L, Secore S, Karczewski J, Racine F, Mayhood T, Fischer P, Sher X, Gupta P, Lacy DB, Therien AG
(2017) J Mol Biol 429: 1030-1044
MeSH Terms: Antibodies, Monoclonal, Antibodies, Neutralizing, Bacterial Toxins, Binding Sites, Enterotoxins, Epitopes, Protein Aggregates, Protein Binding
Show Abstract · Added April 26, 2017
The exotoxins toxin A (TcdA) and toxin B (TcdB) are produced by the bacterial pathogen Clostridium difficile and are responsible for the pathology associated with C. difficile infection (CDI). The antitoxin antibodies actoxumab and bezlotoxumab bind to and neutralize TcdA and TcdB, respectively. Bezlotoxumab was recently approved by the FDA for reducing the recurrence of CDI. We have previously shown that a single molecule of bezlotoxumab binds to two distinct epitopes within the TcdB combined repetitive oligopeptide (CROP) domain, preventing toxin binding to host cells. In this study, we characterize the binding of actoxumab to TcdA and examine its mechanism of toxin neutralization. Using a combination of approaches including a number of biophysical techniques, we show that there are two distinct actoxumab binding sites within the CROP domain of TcdA centered on identical amino acid sequences at residues 2162-2189 and 2410-2437. Actoxumab binding caused the aggregation of TcdA especially at higher antibody:toxin concentration ratios. Actoxumab prevented the association of TcdA with target cells demonstrating that actoxumab neutralizes toxin activity by inhibiting the first step of the intoxication cascade. This mechanism of neutralization is similar to that observed with bezlotoxumab and TcdB. Comparisons of the putative TcdA epitope sequences across several C. difficile ribotypes and homologous repeat sequences within TcdA suggest a structural basis for observed differences in actoxumab binding and/or neutralization potency. These data provide a mechanistic basis for the protective effects of the antibody in vitro and in vivo, including in various preclinical models of CDI.
Copyright © 2017 Elsevier Ltd. All rights reserved.
0 Communities
2 Members
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8 MeSH Terms
Clostridium difficile Toxin A Undergoes Clathrin-Independent, PACSIN2-Dependent Endocytosis.
Chandrasekaran R, Kenworthy AK, Lacy DB
(2016) PLoS Pathog 12: e1006070
MeSH Terms: Adaptor Proteins, Signal Transducing, Animals, Bacterial Toxins, Blotting, Western, Caco-2 Cells, Clathrin, Clostridium Infections, Clostridium difficile, Endocytosis, Enterotoxins, Fluorescent Antibody Technique, Gene Knockdown Techniques, HEK293 Cells, Humans, Image Processing, Computer-Assisted, Mice, Microscopy, Confocal, Protein Transport, Reverse Transcriptase Polymerase Chain Reaction, Transfection, Virulence Factors
Show Abstract · Added April 26, 2017
Clostridium difficile infection affects a significant number of hospitalized patients in the United States. Two homologous exotoxins, TcdA and TcdB, are the major virulence factors in C. difficile pathogenesis. The toxins are glucosyltransferases that inactivate Rho family-GTPases to disrupt host cellular function and cause fluid secretion, inflammation, and cell death. Toxicity depends on receptor binding and subsequent endocytosis. TcdB has been shown to enter cells by clathrin-dependent endocytosis, but the mechanism of TcdA uptake is still unclear. Here, we utilize a combination of RNAi-based knockdown, pharmacological inhibition, and cell imaging approaches to investigate the endocytic mechanism(s) that contribute to TcdA uptake and subsequent cytopathic and cytotoxic effects. We show that TcdA uptake and cellular intoxication is dynamin-dependent but does not involve clathrin- or caveolae-mediated endocytosis. Confocal microscopy using fluorescently labeled TcdA shows significant colocalization of the toxin with PACSIN2-positive structures in cells during entry. Disruption of PACSIN2 function by RNAi-based knockdown approaches inhibits TcdA uptake and toxin-induced downstream effects in cells indicating that TcdA entry is PACSIN2-dependent. We conclude that TcdA and TcdB utilize distinct endocytic mechanisms to intoxicate host cells.
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21 MeSH Terms