Clostridium difficile Toxin B Causes Epithelial Cell Necrosis through an Autoprocessing-Independent Mechanism

Chumbler, Nicole M.; Farrow, Melissa A.; Lapierre, Lynne A.; Franklin, Jeffrey L.; Haslam, David B.; Goldenring, James R.; Lacy, D. Borden

doi:10.1371/journal.ppat.1003072

Cited by 112 publications

(145 citation statements)

References 44 publications

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“…Several recent studies have shown that under certain conditions, the glucosyltransferase domain (10), the cysteine protease domain (10,32), and even the receptorbinding domain (33) are dispensable for function. Our panel of pore-defective mutants identified here, uniquely allowed us to test the importance of translocation domain function in toxin function.…”

Section: Discussionmentioning

confidence: 99%

“…Cell death assay was performed as previously described (10). Assay plates were read in Spectramax M5 plate reader (Molecular Devices).…”

Section: Methodsmentioning

confidence: 99%

“…The experimental conditions that we used in this study to measure TcdB cytotoxicity depend on functional autoprocessing and GTD functions. Recently, Lacy and coworkers identified a second, alternative mode of cytotoxicity for TcdB that was found to be independent of autoprocessing and glucosyltransferase functions (10). At higher doses of TcdB-which are hypothesized to be possible during infection-cells undergo a rapid necrotic-like cell death via an NADPH oxidase pathway, characterized by a rapid depletion of ATP (25).…”

Section: High-throughput Mapping Of the Functional Determinants In Thementioning

confidence: 99%

“…The high-dose acute cytotoxicity of purified WT TcdB, a glucosyltransferase-defective mutant (D270A), and a pore-formation defective mutant (L1106K). Constructs were tested on human IMR-90 fibroblasts under necrosis-like conditions as described previously (10). Fig.…”

Section: Mapping the Determinants Of Pore Formation Onto A Model Of Thementioning

confidence: 99%

“…In the endosome, cryptic regions from within the large ∼1,000-aa translocation domain emerge and insert into the endosomal membrane, creating a pore that is believed to enable translocation of the N-terminal glucosyltransferase (i.e., the A domain) into the cytosol. Processed and released A chains enzymatically glucosylate and thereby inactivate intracellular Rho and Ras family GTPases (6, 7) leading first to cytopathic effects (i.e., cell rounding) (8), and later, cytotoxic effects (i.e., apoptosis and necrosis) (9,10).…”

mentioning

confidence: 99%

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Translocation domain mutations affecting cellular toxicity identify the Clostridium difficile toxin B pore

Zhang

Park

Tam

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Disease associated with Clostridium difficile infection is caused by the actions of the homologous toxins TcdA and TcdB on colonic epithelial cells. Binding to target cells triggers toxin internalization into acidified vesicles, whereupon cryptic segments from within the 1,050-aa translocation domain unfurl and insert into the bounding membrane, creating a transmembrane passageway to the cytosol. Our current understanding of the mechanisms underlying pore formation and the subsequent translocation of the upstream cytotoxic domain to the cytosol is limited by the lack of information available regarding the identity and architecture of the transmembrane pore. Here, through systematic perturbation of conserved sites within predicted membrane-insertion elements of the translocation domain, we uncovered highly sensitive residues-clustered between amino acids 1,035 and 1,107-that when individually mutated, reduced cellular toxicity by as much as >1,000-fold. We demonstrate that defective variants are defined by impaired pore formation in planar lipid bilayers and biological membranes, resulting in an inability to intoxicate cells through either apoptotic or necrotic pathways. These findings along with the unexpected similarities uncovered between the pore-forming "hotspots" of TcdB and the wellcharacterized α-helical diphtheria toxin translocation domain provide insights into the structure and mechanism of formation of the translocation pore for this important class of pathogenic toxins.T he primary virulence determinants of pathogenic Clostridium difficile are two protein toxins, TcdA and TcdB, which are responsible for the symptoms associated with infection, including diarrhea and pseudomembranous colitis (1). TcdA and TcdB are large (i.e., 308 and 270 kDa, respectively) homologous toxins (sharing 48% sequence identity) that appear to intoxicate target cells using a strategy that is similar in principle to that described for a number of smaller A-B toxins, such as anthrax toxin (2) and diphtheria toxin (DT) (3). In addition to a cytotoxic enzymic A domain and receptor-binding B domain responsible for binding and translocating the A domain into cells, TcdA and TcdB are additionally equipped with an internal autoprocessing domain that proteolytically cleaves and releases the N-terminal glucosyltransferase domain in response to intracellular inositol hexakisphosphate (4).The series of events leading to the delivery of the A domain into cells begins with toxin binding to an as yet unidentified receptor on target cells via the C-terminal receptor-binding domain (i.e., the B domain), which triggers toxin internalization into acidified vesicles via clathrin-mediated endocytosis (5). In the endosome, cryptic regions from within the large ∼1,000-aa translocation domain emerge and insert into the endosomal membrane, creating a pore that is believed to enable translocation of the N-terminal glucosyltransferase (i.e., the A domain) into the cytosol. Processed and released A chains enzymatically glucosylate and thereby inactivat...

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Section: Discussionmentioning

confidence: 99%

“…Cell death assay was performed as previously described (10). Assay plates were read in Spectramax M5 plate reader (Molecular Devices).…”

Section: Methodsmentioning

confidence: 99%

Section: High-throughput Mapping Of the Functional Determinants In Thementioning

confidence: 99%

Section: Mapping the Determinants Of Pore Formation Onto A Model Of Thementioning

confidence: 99%

mentioning

confidence: 99%

See 3 more Smart Citations

Translocation domain mutations affecting cellular toxicity identify the Clostridium difficile toxin B pore

Zhang

Park

Tam

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Glucosyltransferase-dependent and -independent effects of TcdB on the proteome of HEp-2 cells

et al. 2017

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Toxin B (TcdB) of the nosocomial pathogen C. difficile has been reported to exhibit a glucosyltransferase-dependent and -independent effect on treated HEp-2 cells at toxin concentration above 0.3 nM. In order to investigate and further characterize both effects epithelial cells were treated with wild type TcdB and glucosyltransferase-deficient TcdB and their proteomes were analyzed by LC-MS. Triplex SILAC labeling was used for quantification. Identification of 5212 and quantification of 4712 protein groups was achieved. Out of these 257 were affected by TcdB treatment, 92 by TcdB treatment and 49 by both. TcdB mainly led to changes in proteins that are related to "GTPase mediated signaling" and the "cytoskeleton" while "chromatin" and "cell cycle" related proteins were altered by both, TcdB and TcdB . The obtained dataset of HEp-2 cell proteome helps us to better understand glucosyltransferase-dependent and -independent mechanisms of TcdB and TcdB , particularly those involved in pyknotic cell death. All proteomics data have been deposited in the ProteomeXchange with the dataset identifier PXD006658 (https://proteomecentral.proteomexchange.org/dataset/PXD006658).

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Receptors and Binding Structures for Clostridium difficile Toxins A and B

Gerhard

2016

Current Topics in Microbiology and Immunology

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Two characteristics of toxins A and B from C. difficile (TcdA, TcdB) are important for the understanding of the pathogenic effect of these homologous toxins. First, these toxins are huge single-chain but multidomain proteins that display their action intracellularly within the cytosol of host cells. And second, albeit various cell types highly differ in their sensitivity toward these toxins, no toxin-resistant cell type has been described yet. Investigation of receptor-mediated uptake of these toxins is very ambitious. It demands discrimination between cell surface binding, interaction with more than one functional receptor responsible for uptake as well as other functional receptors that recognize bacterial pathogens and are not necessarily related with endocytosis. The current understanding of a complex uptake process is that TcdB interacts with at least two facultative receptors that mediate entry into host cells by redundant endocytotic pathways. Although both homologous toxins do obviously not share the same receptors, this principle of redundant binding domains found for TcdB does also account for TcdA.

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Clostridium difficile Toxin B Causes Epithelial Cell Necrosis through an Autoprocessing-Independent Mechanism

Cited by 112 publications

References 44 publications

Translocation domain mutations affecting cellular toxicity identify the Clostridium difficile toxin B pore

Translocation domain mutations affecting cellular toxicity identify the Clostridium difficile toxin B pore

Glucosyltransferase-dependent and -independent effects of TcdB on the proteome of HEp-2 cells

Receptors and Binding Structures for Clostridium difficile Toxins A and B

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