Interaction of Clostridium perfringensIota-Toxin with Lipid Bilayer Membranes

Knapp, Oliver; Benz, Roland; Gibert, Maryse; Marvaud, Jean-Christophe; Popoff, Michel R.

doi:10.1074/jbc.m103939200

Cited by 75 publications

(89 citation statements)

References 48 publications

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“…Following the separation of Ia and Ibp from early-log-phase (Ͻ10-h) cultures of C. perfringens type E, as done by DEAE ion-exchange chromatography, trypsin proteolysis of fractions containing Ibp or Ia markedly (i) increases enzymelinked immunosorbent assay (ELISA) readings for Ibp, but not Ia, versus the same untreated fractions, thus suggesting a conformational shift in Ibp that unveils cryptic epitopes recognized by Ib-specific antibodies; and (ii) increases the guinea pig dermonecrotic activity of the Ibp fraction when combined with untreated Ia. It was subsequently discovered, after cloning and sequencing of the -toxin gene, that proteolytic activation of Ibp into Ib occurs at A211 (323), which then facilitates Ia docking (419), formation of voltage-dependent ion-permeable channels in membranes (213), and formation of SDS-stable heptamers on cell membranes (288,420) and in solution (49,288). However, Ib oligomers formed in solution are seemingly less stable and do not promote cytotoxicity compared to solution-generated oligomers of PA63 or C2IIa.…”

Section: Perfringens Ibp and Iamentioning

confidence: 99%

Binary Bacterial Toxins: Biochemistry, Biology, and Applications of CommonClostridiumandBacillusProteins

Barth

Aktories

Popoff

et al. 2004

Microbiol Mol Biol Rev

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375

388

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Certain pathogenic species of Bacillus and Clostridium have developed unique methods for intoxicating cells that employ the classic enzymatic “A-B” paradigm for protein toxins. The binary toxins produced by B. anthracis, B. cereus, C. botulinum, C. difficile, C. perfringens, and C. spiroforme consist of components not physically associated in solution that are linked to various diseases in humans, animals, or insects. The “B” components are synthesized as precursors that are subsequently activated by serine-type proteases on the targeted cell surface and/or in solution. Following release of a 20-kDa N-terminal peptide, the activated “B” components form homoheptameric rings that subsequently dock with an “A” component(s) on the cell surface. By following an acidified endosomal route and translocation into the cytosol, “A” molecules disable a cell (and host organism) via disruption of the actin cytoskeleton, increasing intracellular levels of cyclic AMP, or inactivation of signaling pathways linked to mitogen-activated protein kinase kinases. Recently, B. anthracis has gleaned much notoriety as a biowarfare/bioterrorism agent, and of primary interest has been the edema and lethal toxins, their role in anthrax, as well as the development of efficacious vaccines and therapeutics targeting these virulence factors and ultimately B. anthracis. This review comprehensively surveys the literature and discusses the similarities, as well as distinct differences, between each Clostridium and Bacillus binary toxin in terms of their biochemistry, biology, genetics, structure, and applications in science and medicine. The information may foster future studies that aid novel vaccine and drug development, as well as a better understanding of a conserved intoxication process utilized by various gram-positive, spore-forming bacteria

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Section: Perfringens Ibp and Iamentioning

confidence: 99%

Binary Bacterial Toxins: Biochemistry, Biology, and Applications of CommonClostridiumandBacillusProteins

Barth

Aktories

Popoff

et al. 2004

Microbiol Mol Biol Rev

Self Cite

375

388

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“…It was shown for anthrax toxin (29) as well as for the family of actin-ADP-ribosylating toxins (10,30,31) that the binding components form pores in artificial lipid membranes. These pores were proposed to interact with the enzyme components (31,32) and to be involved in their translocation. Moreover, it was reported that anthrax lethal factor (LF) must be unfolded for translocation (19), and we have obtained similar results for C2I.…”

Section: Fig 7 Influence Of Bafilomycin A1 or Hsp90 Inhibitorsmentioning

confidence: 99%

The Host Cell Chaperone Hsp90 Is Essential for Translocation of the Binary Clostridium botulinum C2 Toxin into the Cytosol

Haug

Leemhuis

Tiemann

et al. 2003

Journal of Biological Chemistry

132

150

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Clostridium botulinum C2 toxin is the prototype of the binary actin-ADP-ribosylating toxins and consists of the binding component C2II and the enzyme component C2I. The activated binding component C2IIa forms heptamers, which bind to carbohydrates on the cell surface and interact with the enzyme component C2I. This toxin complex is taken up by receptor-mediated endocytosis. In acidic endosomes, heptameric C2IIa forms pores and mediates the translocation of C2I into the cytosol. We report that the heat shock protein (Hsp) 90-specific inhibitors, geldanamycin or radicicol, block intoxication of Vero cells, rat astrocytes, and HeLa cells by C2 toxin. ADP-ribosylation of actin in the cytosol of toxin-treated cells revealed that less active C2I was translocated into the cytosol after treatment with Hsp90 inhibitors. Under control conditions, C2I was localized in the cytosol of toxin-treated rat astrocytes, whereas geldanamycin blocked the cytosolic distribution of C2I. At low extracellular pH (pH 4.5), which allows the direct translocation of C2I via C2IIa heptamers across the cell membrane into the cytosol, Hsp90 inhibitors retarded intoxication by C2I. Geldanamycin did not affect toxin binding, endocytosis, and pore formation by C2IIa. The ADP-ribosyltransferase activity of C2I was not affected by Hsp90 inhibitors in vitro. The cytotoxic actions of the actin-ADP-ribosylating Clostridium perfringens iota toxin and the Rho-ADP-ribosylating C2-C3 fusion toxin was similarly blocked by Hsp90 inhibitors. In contrast, radicicol and geldanamycin had no effect on anthrax lethal toxin-induced cytotoxicity of J774-A1 macrophage-like cells or on cytotoxic effects of the glucosylating Clostridium difficile toxin B in Vero cells. The data indicate that Hsp90 is essential for the membrane translocation of ADP-ribosylating toxins delivered by C2II.

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“…However, we have no direct evidence that C2I translocates via the lumen of the pore across the membrane. Recently, Knapp et al showed that Ib, the binding component of C. perfringens iota toxin and another member of the family of binary actin-ADP-ribosylating toxins, forms pores in black lipid bilayer membranes and that these pores are partially "closed" by the enzyme component Ia (29).…”

Section: Fig 6 Ph-dependent C2i Translocation Into the Cytosol Of Vmentioning

confidence: 99%

Clostridium botulinum C2 Toxin

Blöcker

Pohlmann

Haug

et al. 2003

Journal of Biological Chemistry

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The binary Clostridium botulinum C2 toxin consists of two individual proteins, the transport component C2II (80 kDa) and the enzyme component C2I, which ADPribosylates G-actin in the cytosol of cells. Trypsin-activated C2II (C2IIa) forms heptamers that bind to the cell receptor and mediate translocation of C2I from acidic endosomes into the cytosol of target cells. Here, we report that translocation of C2I across cell membranes is accompanied by pore formation of C2IIa. We used a radioactive rubidium release assay to detect C2IIa pores in the membranes of Chinese hamster ovary cells. Pore formation by C2IIa was dependent on the cellular C2 toxin receptor and an acidic pulse. Pores were formed when C2IIa was bound to cells at neutral pH and when cells were subsequently shifted to acidic medium (pH < 5.5), but no pores were detected when C2IIa was added to cells directly in acidic medium. Most likely, acidification induces a change from "pre-pore" to "pore" conformation of C2IIa, and formation of the pore conformation before membrane binding precludes insertion into membranes. When C2I was present during binding of C2IIa to cells prior to the acidification step, C2IIa-mediated rubidium release was decreased, suggesting that C2I interacted with the lumen of the C2IIa pore. A decrease of rubidium efflux was also detected when C2I was added to C2IIa-treated cells after the acidification step, suggesting that C2I interacted with C2IIa in its pore conformation. Moreover, C2I also interacted with C2IIa channels in artificial lipid membranes and blocked them partially. C2I was only translocated across the cell membrane when C2IIa plus C2I were bound to cells at neutral pH and subsequently shifted to acidic pH. When cell-bound C2IIa was exposed to acidic pH prior to C2I addition, only residual intoxication of cells was observed at high toxin concentrations, and binding of C2I to C2IIa was slightly decreased. Overall, C2IIa pores were essential but not sufficient for translocation of C2I. Intoxication of target cells with C2 toxin requires a strictly coordinated pH-dependent sequence of binding, pore formation by C2IIa, and translocation of C2I.

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Interaction of Clostridium perfringensIota-Toxin with Lipid Bilayer Membranes

Cited by 75 publications

References 48 publications

Binary Bacterial Toxins: Biochemistry, Biology, and Applications of CommonClostridiumandBacillusProteins

Binary Bacterial Toxins: Biochemistry, Biology, and Applications of CommonClostridiumandBacillusProteins

The Host Cell Chaperone Hsp90 Is Essential for Translocation of the Binary Clostridium botulinum C2 Toxin into the Cytosol

Clostridium botulinum C2 Toxin

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