<i>Clostridium difficile</i>
            toxin B-induced necrosis is mediated by the host epithelial cell NADPH oxidase complex

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

doi:10.1073/pnas.1313658110

Cited by 110 publications

(138 citation statements)

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“…Apoptosis was assessed by measuring active caspase-3 and -7 levels using the Apo-ONE homogeneous caspase-3/7 assay (catalog number Promega, G7792). ROS production was assayed with carboxy-2=,7=-dichlorodihydrofluorescein diacetate (catalog number C400; Life Technologies) as previously described (37).…”

Section: Methodsmentioning

confidence: 99%

“…The sections were cut by the Vanderbilt University Translational Pathology Shared Resource Core. Staining of tissues for detection of caspase-3 and ROS was done as previously described (34,37). All slides stained with fluorescent markers were analyzed with an LSM 510 confocal microscope.…”

Section: Methodsmentioning

confidence: 99%

“…In tissue culture models, the apoptotic effects of TcdA and TcdB occur at toxin concentrations of picomolar or lower and are evident at 24 to 48 h postintoxication (26,28,29,(34)(35)(36). TcdB also induces a glucosyltransferase-independent necrosis that is mediated by the assembly and activation of the NADPH oxidase (NOX) complex, subsequently producing high levels of reactive oxygen species (ROS) (34,(37)(38)(39)(40). Indicators of necrosis are apparent within 2 to 4 h using nanomolar concentrations of TcdB in both tissue culture and colonic explant models.…”

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confidence: 99%

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Clostridium difficile Toxins TcdA and TcdB Cause Colonic Tissue Damage by Distinct Mechanisms

et al. 2016

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e As the major cause of antibiotic-associated diarrhea, Clostridium difficile is a serious problem in health care facilities worldwide. C. difficile produces two large toxins, TcdA and TcdB, which are the primary virulence factors in disease. The respective functions of these toxins have been difficult to discern, in part because the cytotoxicity profiles for these toxins differ with concentration and cell type. The goal of this study was to develop a cell culture model that would allow a side-by-side mechanistic comparison of the toxins. Conditionally immortalized, young adult mouse colonic (YAMC) epithelial cells demonstrate an exquisite sensitivity to both toxins with phenotypes that agree with observations in tissue explants. TcdA intoxication results in an apoptotic cell death that is dependent on the glucosyltransferase activity of the toxin. In contrast, TcdB has a bimodal mechanism; it induces apoptosis in a glucosyltransferase-dependent manner at lower concentrations and glucosyltransferase-independent necrotic death at higher concentrations. The direct comparison of the responses to TcdA and TcdB in cells and colonic explants provides the opportunity to unify a large body of observations made by many independent investigators. C lostridium difficile is the most common cause of antibioticassociated diarrhea in the United States, and C. difficile infection (CDI) has been steadily increasing in prevalence and severity over the last 15 years (1-3). Symptoms of CDI can range from mild diarrhea to pseudomembranous colitis, and hallmarks of the disease include neutrophil infiltration, fluid release, and necrotic lesions in the colonic epithelium (4, 5). The bacteria produce two main virulence factors, large toxins called TcdA and TcdB (6, 7).The respective function and relative importance of each toxin in pathogenesis have been active topics of investigation. Genetic knockout experiments in C. difficile have shown that both toxins are important for disease pathology, although TcdB alone is sufficient to cause death in both the hamster and mouse models (6-8). For many years, TcdA and TcdB have been thought to act synergistically, with TcdA acting as an enterotoxin and TcdB acting as a cytotoxin (9, 10). The general term enterotoxin refers to the capacity of TcdA to induce inflammation, cytokine release, and fluid secretion in animal intoxication models (11-13). While TcdB does not always induce these same phenotypes in models, such as the ileal loop model, it has been shown to disrupt the integrity of the epithelial structure in human explant and xenograft models (14, 15). TcdB is also notably more potent as a cytotoxin in cell culture models (9, 10, 16).The toxins have an N-terminal glucosyltransferase domain (GTD) that is delivered into the host cytosol by the C-terminal portion of the protein (17, 18). The GTD has been shown to target and inactivate a number of Rho-family GTPases (19,20). This inactivation has been linked to a cell rounding or cytopathic effect (CPE) (21-24) and to an apoptotic cytotoxic ef...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Clostridium difficile Toxins TcdA and TcdB Cause Colonic Tissue Damage by Distinct Mechanisms

et al. 2016

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“…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). We took advantage of the pore-defective mutants identified here to examine the importance of pore formation on this alternative mode of TcdB-mediated cell death.…”

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

confidence: 99%

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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“…The present study hypothesized that excessive ROS production may promote necroptosis in HK-2 human kidney cells. Diphenyleneiodonium (DPI), an NADPH oxidase inhibitor, has been demonstrated to reduce ROS production and exert a protective role in numerous cell types (17)(18)(19) (20). Cell treatment.…”

Section: Introductionmentioning

confidence: 99%

NADPH oxidase inhibitor, diphenyleneiodonium prevents necroptosis in HK-2 cells

Dong

Chen

et al. 2017

Biomedical Reports

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Abstract. The aim of the present study was to investigate the protective effect of the NADPH oxidase inhibitor, diphenyleneiodonium (DPI) against necroptosis in renal tubular epithelial cells. A necroptosis model of HK-2 cells was established using tumor necrosis factor-α, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone and antimycin A (collectively termed TZA), as in our previous research. The necroptosis inhibitor, necrostatin-1 (Nec-1) or the NADPH oxidase inhibitor, DPI were administered to the necroptosis model. Production of reactive oxygen species (ROS) was detected by dichlorodihydrofluorescein diacetate in the different groups, and the manner of cell death was identified by flow cytometry. Western blot analysis was used to determine the levels of phosphorylation of receptor-interacting protein kinase 3 (RIP-3) and mixed lineage kinase domain-like (MLKL), which are essential to necroptosis. The results revealed that TZA increased the percentages of propidium iodide-positive HK-2 cells from 1.22±0.69 to 8.98±0.73% (P<0.001), and augmented the phosphorylation of RIP-3 and MLKL. ROS levels were increased in the TZA group compared with the control group (27.74±1. IntroductionAcute kidney injury (AKI) is a common clinical syndrome that is characterized by the rapid loss of kidney function and abrupt kidney damage. It is associated with high morbidity and mortality worldwide (1). Acute tubular necrosis (ATN) is the most common and severe pathological manifestation of AKI. Although necrosis has long been considered to be unregulated, previous studies have revealed various types of regulated necrosis that are independent of caspase activities (2). Necroptosis is an important type of regulated necrosis and is involved in various pathological conditions (3-7). Receptor-interacting protein kinase (RIP)-1 receives signals from various death stimuli, and subsequently recruits RIP-3 via RIP homotypic interaction motif domain-mediated interactions and promotes RIP-3 phosphorylation (8,9). Phosphorylated RIP-3 mediates the phosphorylation of mixed-lineage kinase domain-like (MLKL), ultimately leading to rupture of the plasma membrane (7). Previous studies have identified necroptosis in ATN induced by various stimuli, including ischemia/reperfusion, contrast medium and cisplatin (10-12). RIP-1 inhibition by necrostatin-1 (Nec-1), or knockout of RIP-3 or MLKL all prevent ATN (10-12).Reactive oxygen species (ROS) promote necroptosis in cardiomyocytes, liver cells, Jurkat cells and lung adenoma cells (13)(14)(15)(16). However, the effect of ROS on the necroptosis of renal tubular epithelium remains unknown. The present study hypothesized that excessive ROS production may promote necroptosis in HK-2 human kidney cells. Diphenyleneiodonium (DPI), an NADPH oxidase inhibitor, has been demonstrated to reduce ROS production and exert a protective role in numerous cell types (17)(18)(19)

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Clostridium difficile toxin B-induced necrosis is mediated by the host epithelial cell NADPH oxidase complex

Cited by 110 publications

References 38 publications

Clostridium difficile Toxins TcdA and TcdB Cause Colonic Tissue Damage by Distinct Mechanisms

Clostridium difficile Toxins TcdA and TcdB Cause Colonic Tissue Damage by Distinct Mechanisms

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

NADPH oxidase inhibitor, diphenyleneiodonium prevents necroptosis in HK-2 cells

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