scholarly journals A novel Hsp70 inhibitor prevents cell intoxication with the actin ADP-ribosylating Clostridium perfringens iota toxin

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Katharina Ernst ◽  
Markus Liebscher ◽  
Sebastian Mathea ◽  
Anton Granzhan ◽  
Johannes Schmid ◽  
...  
Keyword(s):  
Clostridium Perfringens ◽  
Clostridium Perfringens Iota 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 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 Acid Sphingomyelinase Promotes Cellular Internalization of Clostridium perfringens Iota-Toxin

Toxins ◽  
2018 ◽  
Vol 10 (5) ◽  
pp. 209 ◽  
Author(s):  
Masahiro Nagahama ◽  
Masaya Takehara ◽  
Kazuaki Miyamoto ◽  
Kazumi Ishidoh ◽  
Keiko Kobayashi
Keyword(s):  
Clostridium Perfringens ◽  
Acid Sphingomyelinase ◽  
Cellular Internalization ◽  
Clostridium Perfringens Iota Toxin

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.

Crystal Structure and Site-directed Mutagenesis of Enzymatic Components from Clostridium perfringens Iota-toxin

2003 ◽  
Vol 325 (3) ◽  
pp. 471-483 ◽  
Author(s):  
Hideaki Tsuge ◽  
Masahiro Nagahama ◽  
Hiroyuki Nishimura ◽  
Junzo Hisatsune ◽  
Yoshihiko Sakaguchi ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Clostridium Perfringens ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Clostridium Perfringens Iota Toxin

scholarly journals Interaction of Clostridium perfringens Iota Toxin and Lipolysis-Stimulated Lipoprotein Receptor (LSR)

Toxins ◽  
2018 ◽  
Vol 10 (10) ◽  
pp. 405 ◽  
Author(s):  
Masahiro Nagahama ◽  
Masaya Takehara ◽  
Keiko Kobayashi
Keyword(s):  
Lipid Rafts ◽  
Clostridium Perfringens ◽  
Green Fluorescence Protein ◽  
Lipoprotein Receptor ◽  
Green Fluorescence ◽  
Extracellular Region ◽  
Cytoplasmic Vesicles ◽  
Clostridium Perfringens Iota Toxin ◽  
Fluorescence Protein ◽  
Binding Component

Iota toxin produced by Clostridium perfringens is a binary, actin ADP-ribosylating toxin that is organized into the enzymatically active component Ia and the binding component Ib. Lipolysis-stimulated lipoprotein receptor (LSR) has been identified as a cellular receptor of Ib. Here, we investigated the functional interaction between Ib and LSR, where siRNA for LSR blocked the toxin-mediated cytotoxicity and the binding of Ib. The addition of Ib to LSR-green fluorescence protein (GFP)-transfected cells at 4 °C resulted in colocalization with LSR and Ib on the cell surface. Upon transfer of the cells from 4 °C to 37 °C, LSR and Ib were internalized and observed in cytoplasmic vesicles. When the cells were incubated with Ib at 37 °C and fractionated using the Triton-insoluble membrane, Ib oligomer was localized in insoluble factions that fulfilled the criteria of lipid rafts, and LSR was clustered in lipid rafts. To examine the interaction between N-terminal extracellular region of LSR and Ib, we constructed a series of LSR N-terminal deletions. Ten amino acids residues can be deleted from this end without any reduction of Ib binding. However, deletion of 15 N-terminal residues drastically reduces its ability to bind Ib. These results demonstrate that Ib binds to the LSR N-terminal 10 to 15 residues and endocytoses into trafficking endosomes together with LSR.

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