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 ADP-Ribosylation of Actin by the Clostridium botulinum C2 Toxin in Mammalian Cells Results in Delayed Caspase-Dependent Apoptotic Cell Death

2008 ◽  
Vol 76 (10) ◽  
pp. 4600-4608 ◽  
Author(s):  
Karin Heine ◽  
Sascha Pust ◽  
Stefanie Enzenmüller ◽  
Holger Barth
Keyword(s):  
Cell Death ◽  
Cell Lines ◽  
Mammalian Cells ◽  
Clostridium Botulinum ◽  
Apoptotic Cell ◽  
Apoptotic Cell Death ◽  
Adp Ribosylation ◽  
Mammalian Cell Lines ◽  
Adp Ribosyltransferase ◽  
C2 Toxin

ABSTRACT The binary C2 toxin from Clostridium botulinum mono-ADP-ribosylates G-actin in the cytosol of eukaryotic cells. This modification leads to depolymerization of actin filaments accompanied by cell rounding within 3 h of incubation but does not immediately induce cell death. Here we investigated the long-term responses of mammalian cell lines (HeLa and Vero) following C2 toxin treatment. Cells stayed round even though the toxin was removed from the medium after its internalization into the cells. No unmodified actin reappeared in the C2 toxin-treated cells within 48 h. Despite actin being completely ADP-ribosylated after about 7 h, no obvious decrease in the overall amount of actin was observed for at least 48 h. Therefore, ADP-ribosylation was not a signal for an accelerated degradation of actin in the tested cell lines. C2 toxin treatment resulted in delayed apoptotic cell death that became detectable about 15 to 24 h after toxin application in a portion of the cells. Poly(ADP)-ribosyltransferase 1 (PARP-1) was cleaved in C2 toxin-treated cells, an indication of caspase 3 activation and a hallmark of apoptosis. Furthermore, specific caspase inhibitors prevented C2 toxin-induced apoptosis, implying that caspases 8 and 9 were activated in C2 toxin-treated cells. C2I, the ADP-ribosyltransferase component of the C2 toxin, remained active in the cytosol for at least 48 h, and no extensive degradation of C2I was observed. From our data, we conclude that the long-lived nature of C2I in the host cell cytosol was essential for the nonreversible cytotoxic effect of C2 toxin, resulting in delayed apoptosis of the tested mammalian cells.

scholarly journals EGA Protects Mammalian Cells from Clostridium difficile CDT, Clostridium perfringens Iota Toxin and Clostridium botulinum C2 Toxin

Toxins ◽  
2016 ◽  
Vol 8 (4) ◽  
pp. 101 ◽  
Author(s):  
Leonie Schnell ◽  
Ann-Katrin Mittler ◽  
Mirko Sadi ◽  
Michel Popoff ◽  
Carsten Schwan ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
Clostridium Perfringens ◽  
Mammalian Cells ◽  
Clostridium Botulinum ◽  
Clostridium Botulinum C2 Toxin ◽  
Clostridium Perfringens Iota Toxin ◽  
C2 Toxin

scholarly journals Binding Component of Clostridium perfringens Iota-Toxin Induces Endocytosis in Vero Cells

2002 ◽  
Vol 70 (4) ◽  
pp. 1909-1914 ◽  
Author(s):  
Masahiro Nagahama ◽  
Koichi Nagayasu ◽  
Keiko Kobayashi ◽  
Jun Sakurai
Keyword(s):  
Clostridium Perfringens ◽  
Vero Cells ◽  
Binary Toxin ◽  
Wild Type ◽  
Oligomer Formation ◽  
Clostridium Perfringens Iota Toxin ◽  
Binding Component ◽  
Pronase Treatment

ABSTRACT Clostridium perfringens iota-toxin is a binary toxin consisting of two individual proteins, the binding component (Ib) and the enzyme component (Ia). Wild-type Ib bound to Vero cells at 4 and 37°C and formed oligomers at 37°C but not at 4°C. The Ib-induced K+ release from the cells was dependent on the oligomer formation of Ib in the cells, but the oligomer formation did not induce rounding activity or cytotoxicity. After incubation of the cells with recombinant Ib (rIb) at 37°C, the Ib oligomer in the cell became resistant to pronase treatment with time, but the Ib monomer was sensitive to the treatment. Furthermore, treatment of Vero cells with rIb in the presence of bafilomycin, methylamine, or ethylamine resulted in accumulation of the oligomer in the cells but had no effect on K+ release. Moreover, incubation with Ib plus Ia in the presence of these agents caused no rounding in the cells. These observations suggest that Ib binds to Vero cells, itself oligomerizing to form ion-permeable channels and that the formation of oligomer then induces endocytosis.

scholarly journals Detergent-Resistant Membrane Microdomains Facilitate Ib Oligomer Formation and Biological Activity of Clostridium perfringens Iota-Toxin

2004 ◽  
Vol 72 (4) ◽  
pp. 2186-2193 ◽  
Author(s):  
Martha L. Hale ◽  
Jean-Christophe Marvaud ◽  
Michel R. Popoff ◽  
Bradley G. Stiles
Keyword(s):  
Clostridium Perfringens ◽  
Cell Types ◽  
Cell Receptor ◽  
Vero Cells ◽  
Membrane Microdomains ◽  
Oligomer Formation ◽  
A Cell ◽  
Clostridium Perfringens Iota Toxin ◽  
Detergent Resistant Membrane ◽  
Soluble Fractions

ABSTRACT Clostridium perfringens iota-toxin consists of two separate proteins identified as a cell binding protein, iota b (Ib), which forms high-molecular-weight complexes on cells generating Na+/K+-permeable pores through which iota a (Ia), an ADP-ribosyltransferase, presumably enters the cytosol. Identity of the cell receptor and membrane domains involved in Ib binding, oligomer formation, and internalization is currently unknown. In this study, Vero (toxin-sensitive) and MRC-5 (toxin-resistant) cells were incubated with Ib, after which detergent-resistant membrane microdomains (DRMs) were extracted with cold Triton X-100. Western blotting revealed that Ib oligomers localized in DRMs extracted from Vero, but not MRC-5, cells while monomeric Ib was detected in the detergent-soluble fractions of both cell types. The Ib protoxin, previously shown to bind Vero cells but not form oligomers or induce cytotoxicity, was detected only in the soluble fractions. Vero cells pretreated with phosphatidylinositol-specific phospholipase C before addition of Ib indicated that glycosylphosphatidyl inositol-anchored proteins were minimally involved in Ib binding or oligomer formation. While pretreatment of Vero cells with filipin (which sequesters cholesterol) had no effect, methyl-β-cyclodextrin (which extracts cholesterol) reduced Ib binding and oligomer formation and delayed iota-toxin cytotoxicity. These studies showed that iota-toxin exploits DRMs for oligomer formation to intoxicate cells.

scholarly journals Clostridium perfringensTpeL Glycosylates the Rac and Ras Subfamily Proteins

2010 ◽  
Vol 79 (2) ◽  
pp. 905-910 ◽  
Author(s):  
Masahiro Nagahama ◽  
Akiko Ohkubo ◽  
Masataka Oda ◽  
Keiko Kobayashi ◽  
Katsuhiko Amimoto ◽  
...  
Keyword(s):  
Amino Acid ◽  
Clostridium Difficile ◽  
Recombinant Protein ◽  
Clostridium Perfringens ◽  
Catalytic Domain ◽  
Vero Cells ◽  
Small Gtpases ◽  
Cytotoxic Effects ◽  
Streptolysin O ◽  
First Time

ABSTRACTClostridium perfringensTpeL belongs to a family of large clostridial cytotoxins that encompassesClostridium difficiletoxin A (TcdA) and B (TcdB) andClostridium sordelliilethal toxin (TcsL). We report here the identification of the TpeL-catalyzed modification of small GTPases. A recombinant protein (TpeL1-525) derived from the TpeL N-terminal catalytic domain in the presence of streptolysin O (SLO) induced the rounding of Vero cells and the glycosylation of cellular Rac1. Among several hexoses tested, UDP-N-acetyl-glucosamine (UDP-GlcNAc) and UDP-glucose (UDP-Glc) served as cosubstrates for TpeL1-525-catalyzed modifications. TpeL1-525 catalyzed the incorporation of UDP-Glc into Ha-Ras, Rap1B, and RalA and of UDP-GlcNAc into Rac1, Ha-Ras, Rap1B, and RalA. In Rac1, TpeL and TcdB share the same acceptor amino acid for glycosylation, Thr-35. In Vero cells treated with TpeL1-525 in the presence of SLO, glycosylation leads to a translocation of the majority of Rac1 and Ha-Ras to the membrane. We demonstrate for first time that TpeL uses both UDP-GlcNAc and UDP-Glc as donor cosubstrates and modifies the Rac1 and Ras subfamily by glycosylation to mediate its cytotoxic effects.

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 Roles of Asp179 and Glu270 in ADP-Ribosylation of Actin by Clostridium perfringens Iota Toxin

PLoS ONE ◽  
2015 ◽  
Vol 10 (12) ◽  
pp. e0145708 ◽  
Author(s):  
Alexander Belyy ◽  
Irina Tabakova ◽  
Alexander E. Lang ◽  
Thomas Jank ◽  
Yury Belyi ◽  
...  
Keyword(s):  
Clostridium Perfringens ◽  
Adp Ribosylation ◽  
Clostridium Perfringens Iota Toxin
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