scholarly journals RIP1, RIP3, and MLKL Contribute to Cell Death Caused by Clostridium perfringens Enterotoxin

mBio ◽  
2019 ◽  
Vol 10 (6) ◽  
Author(s):  
Archana Shrestha ◽  
Iman Mehdizadeh Gohari ◽  
Bruce A. McClane
Keyword(s):  
Cell Death ◽  
Clostridium Perfringens ◽  
Vero Cells ◽  
Kinase Domain ◽  
Content Type ◽  
Mixed Lineage Kinase ◽  
Human Enterocyte ◽  
Induced Apoptosis ◽  
Apoptosis And Necrosis ◽  
Calpain Inhibitors

ABSTRACT Clostridium perfringens type F strains cause gastrointestinal disease when they produce a pore-forming toxin named C. perfringens enterotoxin (CPE). In human enterocyte-like Caco-2 cells, low CPE concentrations cause caspase-3-dependent apoptosis, while high CPE concentrations cause necrosis. Since necrosis or apoptosis sometimes involves receptor-interacting serine/threonine-protein kinase-1 or 3 (RIP1 or RIP3), this study examined whether those kinases are important for CPE-induced apoptosis or necrosis. Highly specific RIP1 or RIP3 inhibitors reduced both CPE-induced apoptosis and necrosis in Caco-2 cells. Those findings suggested that the form of necrosis induced by treating Caco-2 cells with high CPE concentrations involves necroptosis, which was confirmed when high, but not low, CPE concentrations were shown to induce oligomerization of mixed-lineage kinase domain-like pseudokinase (MLKL), a key late step in necroptosis. Furthermore, an MLKL oligomerization inhibitor reduced cell death caused by high, but not low, CPE concentrations. Supporting RIP1 and RIP3 involvement in CPE-induced necroptosis, inhibitors of those kinases also reduced MLKL oligomerization during treatment with high CPE concentrations. Calpain inhibitors similarly blocked MLKL oligomerization induced by high CPE concentrations, implicating calpain activation as a key intermediate in initiating CPE-induced necroptosis. In two other CPE-sensitive cell lines, i.e., Vero cells and human enterocyte-like T84 cells, low CPE concentrations also caused primarily apoptosis/late apoptosis, while high CPE concentrations mainly induced necroptosis. Collectively, these results establish that high, but not low, CPE concentrations cause necroptosis and suggest that RIP1, RIP3, MLKL, or calpain inhibitors can be explored as potential therapeutics against CPE effects in vivo. IMPORTANCE C. perfringens type F strains are a common cause of food poisoning and antibiotic-associated diarrhea. Type F strain virulence requires production of C. perfringens enterotoxin (CPE). In Caco-2 cells, high CPE concentrations cause necrosis while low enterotoxin concentrations induce apoptosis. The current study determined that receptor-interacting serine/threonine-protein kinases 1 and 3 are involved in both CPE-induced apoptosis and necrosis in Caco-2 cells, while mixed-lineage kinase domain-like pseudokinase (MLKL) oligomerization is involved in CPE-induced necrosis, thereby indicating that this form of CPE-induced cell death involves necroptosis. High CPE concentrations also caused necroptosis in T84 and Vero cells. Calpain activation was identified as a key intermediate for CPE-induced necroptosis. These results suggest inhibitors of RIP1, RIP3, MLKL oligomerization, or calpain are useful therapeutics against CPE-mediated diseases.

scholarly journals Using More Than 1 (Path)Way to Kill a Host Cell: Lessons From Clostridium perfringens Enterotoxin

2020 ◽  
Vol 13 ◽  
pp. 117863612093151
Author(s):  
Bruce McClane ◽  
Archana Shrestha
Keyword(s):  
Recent Work ◽  
Clostridium Perfringens ◽  
Caspase 3 ◽  
Kinase Domain ◽  
Intermediate Step ◽  
Clostridium Perfringens Enterotoxin ◽  
Mixed Lineage Kinase ◽  
Intestinal Infections ◽  
Apoptosis And Necrosis

Clostridium perfringens enterotoxin (CPE) is responsible for the symptoms of common intestinal infections due to C. perfringens type F isolates. CPE is a pore-forming toxin that uses certain claudins as a receptor. Previous studies showed that, in enterocyte-like Caco-2 cells, low CPE concentrations cause caspase 3-mediated apoptosis but high CPE concentrations cause necrosis. The recent work published in mBio by Shrestha, Mehdizadeh Gohari, and McClane determined that RIP1 and RIP3 are involved in both CPE-mediated apoptosis and necrosis in Caco-2 cells. Furthermore, mixed lineage kinase-domain (MLKL) oligomerization was shown to be important for necrosis caused by CPE, identifying this necrosis as programmed necroptosis. In addition, calpain activation due to Ca2+ influx through the CPE pore was identified as a critical intermediate step for MLKL oligomerization and, thus, CPE-induced necroptosis. These findings may have applicability to understand the action of some other pore-forming toxins that induce necroptosis and may also be important for understanding CPE action in vivo.

scholarly journals The Potential Therapeutic Agent Mepacrine Protects Caco-2 Cells against Clostridium perfringens Enterotoxin Action

mSphere ◽  
2017 ◽  
Vol 2 (4) ◽  
Author(s):  
John C. Freedman ◽  
Matthew R. Hendricks ◽  
Bruce A. McClane
Keyword(s):  
Clostridium Perfringens ◽  
Pore Formation ◽  
Therapeutic Potential ◽  
Food Poisoning ◽  
Host Cells ◽  
Artificial Membranes ◽  
Content Type ◽  
Gi Symptoms ◽  
Human Enterocyte ◽  
Bacterial Food

ABSTRACT Clostridium perfringens enterotoxin (CPE) causes the gastrointestinal (GI) symptoms of a common bacterial food poisoning and several nonfoodborne human GI diseases. A previous study showed that, via an undetermined mechanism, the presence of mepacrine blocks CPE-induced electrophysiologic activity in artificial membranes. The current study now demonstrates that mepacrine also inhibits CPE-induced cytotoxicity in human enterocyte-like Caco-2 cells and that mepacrine does not directly inactivate CPE. Instead, this drug reduces both CPE pore formation and CPE pore activity in Caco-2 cells. These results suggest mepacrine as a therapeutic candidate for treating CPE-mediated GI diseases. Clostridium perfringens enterotoxin (CPE) causes the diarrhea associated with a common bacterial food poisoning and many antibiotic-associated diarrhea cases. The severity of some CPE-mediated disease cases warrants the development of potential therapeutics. A previous study showed that the presence of mepacrine inhibited CPE-induced electrophysiology effects in artificial lipid bilayers lacking CPE receptors. However, that study did not assess whether mepacrine inactivates CPE or, instead, inhibits a step in CPE action. Furthermore, CPE action in host cells is complex, involving the toxin binding to receptors, receptor-bound CPE oligomerizing into a prepore on the membrane surface, and β-hairpins in the CPE prepore inserting into the membrane to form a pore that induces cell death. Therefore, the current study evaluated the ability of mepacrine to protect cells from CPE. This drug was found to reduce CPE-induced cytotoxicity in Caco-2 cells. This protection did not involve mepacrine inactivation of CPE, indicating that mepacrine affects one or more steps in CPE action. Western blotting then demonstrated that mepacrine decreases CPE pore levels in Caco-2 cells. This mepacrine-induced reduction in CPE pore levels did not involve CPE binding inhibition but rather an increase in CPE monomer dissociation due to mepacrine interactions with Caco-2 membranes. In addition, mepacrine was also shown to inhibit CPE pores when already present in Caco-2 cells. These in vitro studies, which identified two mepacrine-sensitive steps in CPE-induced cytotoxicity, add support to further testing of the therapeutic potential of mepacrine against CPE-mediated disease. IMPORTANCE Clostridium perfringens enterotoxin (CPE) causes the gastrointestinal (GI) symptoms of a common bacterial food poisoning and several nonfoodborne human GI diseases. A previous study showed that, via an undetermined mechanism, the presence of mepacrine blocks CPE-induced electrophysiologic activity in artificial membranes. The current study now demonstrates that mepacrine also inhibits CPE-induced cytotoxicity in human enterocyte-like Caco-2 cells and that mepacrine does not directly inactivate CPE. Instead, this drug reduces both CPE pore formation and CPE pore activity in Caco-2 cells. These results suggest mepacrine as a therapeutic candidate for treating CPE-mediated GI diseases.

scholarly journals The Long-Lived Nature of Clostridium perfringens Iota Toxin in Mammalian Cells Induces Delayed Apoptosis

2009 ◽  
Vol 77 (12) ◽  
pp. 5593-5601 ◽  
Author(s):  
Hanna Hilger ◽  
Sascha Pust ◽  
Guido von Figura ◽  
Eva Kaiser ◽  
Bradley G. Stiles ◽  
...  
Keyword(s):  
Cell Death ◽  
Clostridium Perfringens ◽  
Mammalian Cells ◽  
Catalytic Domain ◽  
Vero Cells ◽  
African Green Monkey Kidney ◽  
Adp Ribosylation ◽  
Clostridium Perfringens Iota Toxin ◽  
Adp Ribosyltransferase ◽  
C2 Toxin

ABSTRACT Mono-ADP ribosylation of actin by bacterial toxins, such as Clostridium perfringens iota or Clostridium botulinum C2 toxins, results in rapid depolymerization of actin filaments and cell rounding. Here we report that treatment of African green monkey kidney (Vero) cells with iota toxin resulted in delayed caspase-dependent death. Unmodified actin did not reappear in toxin-treated cells, and enzyme-active toxin was detectable in the cytosol for at least 24 h. C2 toxin showed comparable, long-lived effects in cells, while a C2 toxin control lacking ADP-ribosyltransferase activity did not induce cell death. To address whether the remarkable stability of the iota and C2 toxins in cytosol was crucial for inducing cell death, we treated cells with C/SpvB, the catalytic domain of Salmonella enterica SpvB. Although C/SpvB also mono-ADP ribosylates actin as do the iota and C2 toxins, cells treated with a cell-permeating C/SpvB fusion toxin became rounded but recovered and remained viable. Moreover, unmodified actin reappeared in these cells, and ADP-ribosyltransferase activity due to C/SpvB was not detectable in the cytosol after 24 h, a result most likely due to degradation of C/SpvB. Repeated application of C/SpvB prevented recovery of cells and reappearance of unmodified actin. In conclusion, a complete but transient ADP ribosylation of actin was not sufficient to trigger apoptosis, implying that long-term stability of actin-ADP-ribosylating toxins, such as iota and C2, in the cytosol is crucial for inducing delayed, caspase-dependent cell death.

scholarly journals Cytotoxic and Apoptotic Effects of Recombinant Subtilase Cytotoxin Variants of Shiga Toxin-Producing Escherichia coli

2015 ◽  
Vol 83 (6) ◽  
pp. 2338-2349 ◽  
Author(s):  
J. Funk ◽  
N. Biber ◽  
M. Schneider ◽  
E. Hauser ◽  
S. Enzenmüller ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Vero Cells ◽  
Toxic Activity ◽  
Content Type ◽  
B Subunits ◽  
Induced Apoptosis ◽  
The Individual ◽  
Apoptotic Effects ◽  
Subtilase Cytotoxin

In this study, the cytotoxicity of the recently described subtilase variant SubAB2-2of Shiga toxin-producingEscherichia coliwas determined and compared to the plasmid-encoded SubAB1and the chromosome-encoded SubAB2-1variant. The genes for the respective enzymatic active (A) subunits and binding (B) subunits of the subtilase toxins were amplified and cloned. The recombinant toxin subunits were expressed and purified. Their cytotoxicity on Vero cells was measured for the single A and B subunits, as well as for mixtures of both, to analyze whether hybrids with toxic activity can be identified. The results demonstrated that all three SubAB variants are toxic for Vero cells. However, the values for the 50% cytotoxic dose (CD50) differ for the individual variants. Highest cytotoxicity was shown for SubAB1. Moreover, hybrids of subunits from different subtilase toxins can be obtained which cause substantial cytotoxicity to Vero cells after mixing the A and B subunits prior to application to the cells, which is characteristic for binary toxins. Furthermore, higher concentrations of the enzymatic subunit SubA1exhibited cytotoxic effects in the absence of the respective B1subunit. A more detailed investigation in the human HeLa cell line revealed that SubA1alone induced apoptosis, while the B1subunit alone did not induce cell death.

scholarly journals Analysis of the N-terminal region of human MLKL, as well as two distinct MLKL isoforms, reveals new insights into necroptotic cell death

2016 ◽  
Vol 36 (1) ◽  
Author(s):  
Katja Hrovat Arnež ◽  
Michaela Kindlova ◽  
Nilesh J. Bokil ◽  
James M. Murphy ◽  
Matthew J. Sweet ◽  
...  
Keyword(s):  
Cell Death ◽  
Human Monocyte ◽  
Kinase Domain ◽  
Terminal Region ◽  
Activation Loop ◽  
Phosphorylation Sites ◽  
Mixed Lineage Kinase

We show that mixed lineage kinase domain-like (MLKL) isoform 2, which lacks the pseudokinase domain and activation loop phosphorylation sites, is a more potent activator of cell death compared with MLKL isoform 1. Both MLKL isoforms are expressed in human monocyte-derived macrophages.

scholarly journals Translocation of mixed lineage kinase domain-like protein to plasma membrane leads to necrotic cell death

Cell Research ◽  
2013 ◽  
Vol 24 (1) ◽  
pp. 105-121 ◽  
Author(s):  
Xin Chen ◽  
Wenjuan Li ◽  
Junming Ren ◽  
Deli Huang ◽  
Wan-ting He ◽  
...  
Keyword(s):  
Cell Death ◽  
Plasma Membrane ◽  
Kinase Domain ◽  
Necrotic Cell Death ◽  
Necrotic Cell ◽  
Mixed Lineage Kinase

scholarly journals Elevated Serum Levels of Mixed Lineage Kinase Domain-Like Protein Predict Survival of Patients during Intensive Care Unit Treatment

2018 ◽  
Vol 2018 ◽  
pp. 1-8 ◽  
Author(s):  
Mihael Vucur ◽  
Christoph Roderburg ◽  
Lukas Kaiser ◽  
Anne Theres Schneider ◽  
Sanchari Roy ◽  
...  
Keyword(s):  
Intensive Care Unit ◽  
Cell Death ◽  
Intensive Care ◽  
Critical Illness ◽  
Critically Ill Patients ◽  
Serum Levels ◽  
Kinase Domain ◽  
Healthy Controls ◽  
Mixed Lineage Kinase ◽  
Icu Treatment

Mixed lineage kinase domain-like (MLKL), a crucial regulator of necroptotic cell death, was shown to play a role in inflammatory diseases. However, its role as a biomarker in critical illness and sepsis is currently unknown. We analyzed serum levels of MLKL in 136 critically ill patients at admission to the intensive care unit (ICU) and after three days of ICU treatment. Results were compared with 36 healthy controls and correlated with clinical and laboratory patients’ data. MLKL serum levels of critically ill patients at admission to the ICU were similar compared to healthy controls. At ICU admission, MLKL serum concentrations were independent of disease severity, presence of sepsis, and etiology of critical illness. In contrast, median serum levels of MLKL after three days of ICU treatment were significantly lower compared to those at admission to the ICU. While serum levels of MLKL at admission were not predictive for short-term survival during ICU treatment, elevated MLKL concentrations at day three were an independent negative predictor of patients’ ICU survival. Thus, elevated MLKL levels after three days of ICU treatment were predictive for patients’ mortality, indicating that sustained deregulated cell death is associated with an adverse prognosis in critical illness.

scholarly journals Plant mixed lineage kinase domain-like proteins limit biotrophic pathogen growth

2019 ◽  
Author(s):  
Lisa Mahdi ◽  
Menghang Huang ◽  
Xiaoxiao Zhang ◽  
Ryohei Thomas Nakano ◽  
Leïla Brulé Kopp ◽  
...  
Keyword(s):  
Cell Death ◽  
Host Cell ◽  
Kinase Domain ◽  
Protein Family ◽  
Mixed Lineage Kinase ◽  
Helical Bundles ◽  
Cryo Electron Microscopy ◽  
Host Cell Death ◽  
A Cell ◽  
Immunity Mechanism

AbstractMixed lineage kinase domain-like (MLKL) protein mediates necroptotic cell death in vertebrates. We report here the discovery of a conserved protein family across seed plants that is structurally homologous to vertebrate MLKL. TheArabidopsis thalianagenome encodes three MLKLs with overlapping functions in limiting growth of obligate biotrophic fungal and oomycete pathogens. Although displaying a cell death activity mediated by N-terminal helical bundles, termed HeLo domain,AtMLKL-dependent immunity can be separated from host cell death. Cryo-electron microscopy structures ofAtMLKLs reveal a tetrameric configuration, in which the pseudokinase domain and brace region bury the HeLo-domains, indicative of an auto-repressed complex. We also show the association of twoAtMLKLs with microtubules. These findings, coupled with resistance-enhancing activity and altered microtubule association of a phosphomimetic mutation in the pseudokinase domain ofAtMLKL1, point to a cell death-independent immunity mechanism.One Sentence SummaryPlants have a protein family that is structurally homologous to vertebrate mixed lineage kinase domain-like protein, which induces necroptotic cell death, but these plant proteins can confer immunity without host cell death.

scholarly journals Site-specific ubiquitination of MLKL targets it to endosomes and targets Listeria and Yersinia to the lysosomes

2022 ◽  
Author(s):  
Seongmin Yoon ◽  
Konstantin Bogdanov ◽  
David Wallach
Keyword(s):  
Cell Death ◽  
Defense Mechanisms ◽  
Kinase Domain ◽  
Intracellular Bacteria ◽  
Endosomal Trafficking ◽  
Polyubiquitin Chains ◽  
Mixed Lineage Kinase ◽  
Lysine Residues ◽  
Covalent Modifications ◽  
Bacterial Yield

AbstractPhosphorylation of the pseudokinase mixed lineage kinase domain-like protein (MLKL) by the protein kinase RIPK3 targets MLKL to the cell membrane, where it triggers necroptotic cell death. We report that conjugation of K63-linked polyubiquitin chains to distinct lysine residues in the N-terminal HeLo domain of phosphorylated MLKL (facilitated by the ubiquitin ligase ITCH that binds MLKL via a WW domain) targets MLKL instead to endosomes. This results in the release of phosphorylated MLKL within extracellular vesicles. It also prompts enhanced endosomal trafficking of intracellular bacteria such as Listeria monocytogenes and Yersinia enterocolitica to the lysosomes, resulting in decreased bacterial yield. Thus, MLKL can be directed by specific covalent modifications to differing subcellular sites, whence it signals either for cell death or for non-deadly defense mechanisms.

scholarly journals Oligomerization-driven MLKL ubiquitylation antagonises necroptosis

2021 ◽  
Author(s):  
Zikou Liu ◽  
Laura Francesca Dagley ◽  
Kristy Lynn Shield-Artin ◽  
Samuel Nicholas Young ◽  
Aleksandra Bankovacki ◽  
...  
Keyword(s):  
Cell Death ◽  
Plasma Membrane ◽  
Protein Kinase ◽  
Kinase Domain ◽  
Membrane Disruption ◽  
Fusion Strategy ◽  
Mixed Lineage Kinase ◽  
Independent Form ◽  
Lysine Residues ◽  
Kinetic Regulation

Mixed lineage kinase domain-like (MLKL) is the executioner in the caspase-independent form of programmed cell death called necroptosis. Receptor Interacting serine/threonine Protein Kinase 3 (RIPK3) phosphorylates MLKL, triggering MLKL oligomerization, membrane translocation and membrane disruption. MLKL also undergoes ubiquitylation during necroptosis, yet neither the mechanism nor significance of this event have been demonstrated. Here we show that necroptosis-specific, multi-mono-ubiquitylation of MLKL occurs following its activation and oligomerization. Ubiquitylated MLKL accumulates in a digitonin insoluble cell fraction comprising plasma/organellar membranes and protein aggregates. This ubiquitylated form is diminished by a plasma membrane located deubiquitylating enzyme. MLKL is ubiquitylated on at least 4 separate lysine residues once oligomerized, and this correlates with proteasome- and lysosome- dependent turnover. Using a MLKL-DUB fusion strategy, we show that constitutive removal of ubiquitin from MLKL licenses MLKL auto-activity independent of necroptosis signalling in mouse and human cells. Therefore, besides its role in the kinetic regulation of MLKL-induced death following an exogenous necroptotic stimulus, ubiquitylation also contributes to the restraint of basal levels of activated MLKL to avoid errant cell death.

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