Cellular Uptake of Clostridium botulinum C2 Toxin Requires Oligomerization and Acidification

Abstract

The actin-ADP-ribosylating binary Clostridium botulinum C2 toxin consists of two individual proteins, the binding/translocation component C2II and the enzyme component C2I. To elicit its cytotoxic action, C2II binds to a receptor on the cell surface and mediates cell entry of C2I via receptor-mediated endocytosis. Here we report that binding of C2II to the surface of target cells requires cleavage of C2II by trypsin. Trypsin cleavage causes oligomerization of the activated C2II (C2IIa) to give SDS-stable heptameric structures, which exhibit a characteristic annular or horseshoe shape and form channels in lipid bilayer membranes. Cytosolic delivery of the enzyme component C2I is blocked by bafilomycin but not by brefeldin A or nocodazole, indicating uptake from an endosomal compartment and requirement of endosomal acidification for cell entry. In the presence of C2IIa and C2I, short term acidification of the extracellular medium (pH 5.4) allows C2I to enter the cytosol directly. Our data indicate that entry of C2 toxin into cells involves (i) activation of C2II by trypsin-cleavage, (ii) oligomerization of cleaved C2IIa to heptamers, (iii) binding of the C2IIa oligomers to the carbohydrate receptor on the cell surface and assembly with C2I, (iv) receptor-mediated endocytosis of both C2 components into endosomes, and finally (v) translocation and release of C2I into the cytosol after acidification of the endosomal compartment.

A Stepwise Dissection of the Intracellular Fate of Cationic Cell-penetrating Peptides

“…We therefore asked whether an acidic extracellular pH enabled a direct non-endosomal entry of CPPs from the medium into the cytoplasm. Such a pH-dependent direct passage through the plasma membrane was observed for Clostridium botulinum C2 toxin (52). This toxin is normally taken up by endocytosis and requires oligomerization and endosomal acidification for release into the cytosol (52).…”

“…Such a pH-dependent direct passage through the plasma membrane was observed for Clostridium botulinum C2 toxin (52). This toxin is normally taken up by endocytosis and requires oligomerization and endosomal acidification for release into the cytosol (52). Therefore MC57 cells were briefly pulsed with peptide and then incubated with physiological citrate buffer of various acidic pH values (pH 5-7).…”

Actin filaments are involved in the maintenance of Golgi cisternae morphology and intra‐Golgi pH

“…Jasplakinolide (Jpk) was from Molecular Probes (Eugene, OR, USA). C2I and C2IIa components of Clostridium botulinum C2 toxin (C2 toxin) were obtained as described [Barth et al, 2000] cDNAs encoding the N-terminal of human b1,4-GT fused enhanced mutants GFP (EGFP), CFP (EGFP) or YFP (EYFP) mutants were used as described [Llopis et al, 1998]. EMbed-812 embedding media kit and the reagents used in electron microscopy experiments were from Electron Microscopy Sciences (Hatfield, PA,Life Technologies (Paisley, UK) supplemented with fetal bovine serum (FBS) from Gibco, penicillin (100 U/ml), streptomycin (100 lg/ml), L-glutamine (20 mM) and MEM sodium piruvate (10 mM).…”

Cholesterol- and Sphingolipid-rich Microdomains Are Essential for Microtubule-based Membrane Protrusions Induced by Clostridium difficile Transferase (CDT)

“…These toxins are binary in structure and consist of an enzymatic component, which possesses ADP-ribosyltransferase activity, and a separated binding/translocation component. The binding component is proteolytically activated and forms heptamers, which interact with the enzymatic component (8,10). After binding of the toxin to a cell surface receptor, the toxin complex is endocytosed (11,12).…”

“…After binding of the toxin to a cell surface receptor, the toxin complex is endocytosed (11,12). At low pH of endosomal compartments, the binding component inserts into the membrane of endosomes and forms pores, which allow the translocation of the enzymatic component into the cytosol (10,13). Here, the toxin ADP-ribosylates actin at arginine 177 (14).…”