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Functional Properties of Helicobacter pylori VacA Toxin m1 and m2 Variants.
Caston RR, Sierra JC, Foegeding NJ, Truelock MD, Campbell AM, Frick-Cheng AE, Bimczok D, Wilson KT, McClain MS, Cover TL
(2020) Infect Immun 88:
MeSH Terms: Amino Acid Sequence, Bacterial Proteins, Bacterial Toxins, Gene Expression Regulation, Bacterial, Genetic Variation, Helicobacter Infections, Helicobacter pylori, Humans, Protein Domains, Protein Multimerization, Protein Transport, Vacuoles
Show Abstract · Added April 15, 2020
colonizes the gastric mucosa and secretes a pore-forming toxin (VacA). Two main types of VacA, m1 and m2, can be distinguished by phylogenetic analysis. Type m1 forms of VacA have been extensively studied, but there has been relatively little study of m2 forms. In this study, we generated strains producing chimeric proteins in which VacA m1 segments of a parental strain were replaced by corresponding m2 sequences. In comparison to the parental m1 VacA protein, a chimeric protein (designated m2/m1) containing m2 sequences in the N-terminal portion of the m region was less potent in causing vacuolation of HeLa cells, AGS gastric cells, and AZ-521 duodenal cells and had reduced capacity to cause membrane depolarization or death of AZ-521 cells. Consistent with the observed differences in activity, the chimeric m2/m1 VacA protein bound to cells at reduced levels compared to the binding levels of the parental m1 protein. The presence of two strain-specific insertions or deletions within or adjacent to the m region did not influence toxin activity. Experiments with human gastric organoids grown as monolayers indicated that m1 and m2/m1 forms of VacA had similar cell-vacuolating activities. Interestingly, both forms of VacA bound preferentially to the basolateral surface of organoid monolayers and caused increased cell vacuolation when interacting with the basolateral surface compared to the apical surface. These data provide insights into functional correlates of sequence variation in the VacA midregion (m region).
Copyright © 2020 American Society for Microbiology.
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12 MeSH Terms
infection damages colonic stem cells via TcdB, impairing epithelial repair and recovery from disease.
Mileto SJ, Jardé T, Childress KO, Jensen JL, Rogers AP, Kerr G, Hutton ML, Sheedlo MJ, Bloch SC, Shupe JA, Horvay K, Flores T, Engel R, Wilkins S, McMurrick PJ, Lacy DB, Abud HE, Lyras D
(2020) Proc Natl Acad Sci U S A 117: 8064-8073
MeSH Terms: Animals, Bacterial Proteins, Bacterial Toxins, Cells, Cultured, Clostridioides difficile, Clostridium Infections, Colon, Disease Models, Animal, Female, Frizzled Receptors, Humans, Intestinal Mucosa, Mice, Organoids, Primary Cell Culture, Recombinant Proteins, Stem Cells
Show Abstract · Added March 24, 2020
Gastrointestinal infections often induce epithelial damage that must be repaired for optimal gut function. While intestinal stem cells are critical for this regeneration process [R. C. van der Wath, B. S. Gardiner, A. W. Burgess, D. W. Smith, 8, e73204 (2013); S. Kozar , 13, 626-633 (2013)], how they are impacted by enteric infections remains poorly defined. Here, we investigate infection-mediated damage to the colonic stem cell compartment and how this affects epithelial repair and recovery from infection. Using the pathogen we show that infection disrupts murine intestinal cellular organization and integrity deep into the epithelium, to expose the otherwise protected stem cell compartment, in a TcdB-mediated process. Exposure and susceptibility of colonic stem cells to intoxication compromises their function during infection, which diminishes their ability to repair the injured epithelium, shown by altered stem cell signaling and a reduction in the growth of colonic organoids from stem cells isolated from infected mice. We also show, using both mouse and human colonic organoids, that TcdB from epidemic ribotype 027 strains does not require Frizzled 1/2/7 binding to elicit this dysfunctional stem cell state. This stem cell dysfunction induces a significant delay in recovery and repair of the intestinal epithelium of up to 2 wk post the infection peak. Our results uncover a mechanism by which an enteric pathogen subverts repair processes by targeting stem cells during infection and preventing epithelial regeneration, which prolongs epithelial barrier impairment and creates an environment in which disease recurrence is likely.
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17 MeSH Terms
Intestinal bile acids directly modulate the structure and function of TcdB toxin.
Tam J, Icho S, Utama E, Orrell KE, Gómez-Biagi RF, Theriot CM, Kroh HK, Rutherford SA, Lacy DB, Melnyk RA
(2020) Proc Natl Acad Sci U S A 117: 6792-6800
MeSH Terms: Bacterial Toxins, Bile Acids and Salts, Caco-2 Cells, Clostridioides difficile, Clostridium Infections, HCT116 Cells, Humans, Intestines, Receptors, Cell Surface
Show Abstract · Added March 24, 2020
Intestinal bile acids are known to modulate the germination and growth of Here we describe a role for intestinal bile acids in directly binding and neutralizing TcdB toxin, the primary determinant of disease. We show that individual primary and secondary bile acids reversibly bind and inhibit TcdB to varying degrees through a mechanism that requires the combined oligopeptide repeats region to which no function has previously been ascribed. We find that bile acids induce TcdB into a compact "balled up" conformation that is no longer able to bind cell surface receptors. Lastly, through a high-throughput screen designed to identify bile acid mimetics we uncovered nonsteroidal small molecule scaffolds that bind and inhibit TcdB through a bile acid-like mechanism. In addition to suggesting a role for bile acids in pathogenesis, these findings provide a framework for development of a mechanistic class of antitoxins.
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9 MeSH Terms
Structural elucidation of the transferase toxin reveals a single-site binding mode for the enzyme.
Sheedlo MJ, Anderson DM, Thomas AK, Lacy DB
(2020) Proc Natl Acad Sci U S A 117: 6139-6144
MeSH Terms: Bacterial Toxins, Clostridioides difficile, Cryoelectron Microscopy, Enterotoxins, Protein Conformation, beta-Strand, Protein Multimerization, Transferases
Show Abstract · Added March 24, 2020
is a Gram-positive, pathogenic bacterium and a prominent cause of hospital-acquired diarrhea in the United States. The symptoms of infection are caused by the activity of three large toxins known as toxin A (TcdA), toxin B (TcdB), and the transferase toxin (CDT). Reported here is a 3.8-Å cryo-electron microscopy (cryo-EM) structure of CDT, a bipartite toxin comprised of the proteins CDTa and CDTb. We observe a single molecule of CDTa bound to a CDTb heptamer. The formation of the CDT complex relies on the interaction of an N-terminal adaptor and pseudoenzyme domain of CDTa with six subunits of the CDTb heptamer. CDTb is observed in a preinsertion state, a conformation observed in the transition of prepore to β-barrel pore, although we also observe a single bound CDTa in the prepore and β-barrel conformations of CDTb. The binding interaction appears to prime CDTa for translocation as the adaptor subdomain enters the lumen of the preinsertion state channel. These structural observations advance the understanding of how a single protein, CDTb, can mediate the delivery of a large enzyme, CDTa, into the cytosol of mammalian cells.
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7 MeSH Terms
Small Molecule Inhibitor Screen Reveals Calcium Channel Signaling as a Mechanistic Mediator of TcdB-Induced Necrosis.
Farrow MA, Chumber NM, Bloch SC, King M, Moton-Melancon K, Shupe J, Washington MK, Spiller BW, Lacy DB
(2020) ACS Chem Biol 15: 1212-1221
MeSH Terms: Actin Cytoskeleton, Animals, Anti-Infective Agents, Bacterial Toxins, Calcium Channel Blockers, Calcium Channels, Calcium Signaling, Clostridioides difficile, Cytokines, Dihydropyridines, Dose-Response Relationship, Drug, Drug Evaluation, Preclinical, Glucosyltransferases, Humans, Kinetics, Mice, NADPH Oxidases, Necrosis, Reactive Oxygen Species, Virulence Factors
Show Abstract · Added March 24, 2020
is the leading cause of nosocomial diarrhea in the United States. The primary virulence factors are two homologous glucosyltransferase toxins, TcdA and TcdB, that inactivate host Rho-family GTPases. The glucosyltransferase activity has been linked to a "cytopathic" disruption of the actin cytoskeleton and contributes to the disruption of tight junctions and the production of pro-inflammatory cytokines. TcdB is also a potent cytotoxin that causes epithelium necrotic damage through an NADPH oxidase (NOX)-dependent mechanism. We conducted a small molecule screen to identify compounds that confer protection against TcdB-induced necrosis. We identified an enrichment of "hit compounds" with a dihydropyridine (DHP) core which led to the discovery of a key early stage calcium signal that serves as a mechanistic link between TcdB-induced NOX activation and reactive oxygen species (ROS) production. Disruption of TcdB-induced calcium signaling (with both DHP and non-DHP molecules) is sufficient to ablate ROS production and prevent subsequent necrosis in cells and in a mouse model of intoxication.
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20 MeSH Terms
Structural insights into the transition of Clostridioides difficile binary toxin from prepore to pore.
Anderson DM, Sheedlo MJ, Jensen JL, Lacy DB
(2020) Nat Microbiol 5: 102-107
MeSH Terms: ADP Ribose Transferases, Bacterial Proteins, Bacterial Toxins, Caco-2 Cells, Cryoelectron Microscopy, Humans, Models, Molecular, Polysaccharides, Pore Forming Cytotoxic Proteins, Protein Binding, Protein Domains, Protein Multimerization, Receptors, LDL
Show Abstract · Added March 24, 2020
Clostridioides (formerly Clostridium) difficile is a Gram-positive, spore-forming anaerobe and a leading cause of hospital-acquired infection and gastroenteritis-associated death in US hospitals. The disease state is usually preceded by disruption of the host microbiome in response to antibiotic treatment and is characterized by mild to severe diarrhoea. C. difficile infection is dependent on the secretion of one or more AB-type toxins: toxin A (TcdA), toxin B (TcdB) and the C. difficile transferase toxin (CDT). Whereas TcdA and TcdB are considered the primary virulence factors, recent studies suggest that CDT increases the severity of C. difficile infection in some of the most problematic clinical strains. To better understand how CDT functions, we used cryo-electron microscopy to define the structure of CDTb, the cell-binding component of CDT. We obtained structures of several oligomeric forms that highlight the conformational changes that enable conversion from a prepore to a β-barrel pore. The structural analysis also reveals a glycan-binding domain and residues involved in binding the host-cell receptor, lipolysis-stimulated lipoprotein receptor. Together, these results provide a framework to understand how CDT functions at the host cell interface.
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13 MeSH Terms
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.
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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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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, Clostridioides 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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13 MeSH Terms