Tailored ß-Cyclodextrin Blocks the Translocation Pores of Binary Exotoxins from C. Botulinum and C. Perfringens and Protects Cells from Intoxication

Ekaterina M Nestorovich,Sergey M Bezrukov,Michel R Popoff,Vladimir A Karginov,Holger Barth,Michael Hensel

doi:10.1371/journal.pone.0023927

Abstract

Background Clostridium botulinum C2 toxin and Clostridium perfringens iota toxin are binary exotoxins, which ADP-ribosylate actin in the cytosol of mammalian cells and thereby destroy the cytoskeleton. C2 and iota toxin consists of two individual proteins, an enzymatic active (A-) component and a separate receptor binding and translocation (B-) component. The latter forms a complex with the A-component on the surface of target cells and after receptor-mediated endocytosis, it mediates the translocation of the A-component from acidified endosomal vesicles into the cytosol. To this end, the B-components form heptameric pores in endosomal membranes, which serve as translocation channels for the A-components.Methodology/Principal FindingsHere we demonstrate that a 7-fold symmetrical positively charged ß-cyclodextrin derivative, per-6-S-(3-aminomethyl)benzylthio-ß-cyclodextrin, protects cultured cells from intoxication with C2 and iota toxins in a concentration-dependent manner starting at low micromolar concentrations. We discovered that the compound inhibited the pH-dependent membrane translocation of the A-components of both toxins in intact cells. Consistently, the compound strongly blocked transmembrane channels formed by the B-components of C2 and iota toxin in planar lipid bilayers in vitro. With C2 toxin, we consecutively ruled out all other possible inhibitory mechanisms showing that the compound did not interfere with the binding of the toxin to the cells or with the enzyme activity of the A-component.Conclusions/SignificanceThe described ß-cyclodextrin derivative was previously identified as one of the most potent inhibitors of the binary lethal toxin of Bacillus anthracis both in vitro and in vivo, implying that it might represent a broad-spectrum inhibitor of binary pore-forming exotoxins from pathogenic bacteria.

Highlights

Various pathogenic bacteria produce binary exotoxins, which are composed of two non-linked proteins, an enzyme (A-) component and a separate binding and translocation (B-) component
Cells were further incubated at 37uC and the toxin effect was analyzed in terms of cell rounding, a specific and well-established endpoint to monitor the mode of action of the actin-ADP-ribosylating toxin in the cytosol of mammalian cells
We have performed a series of in vitro and cell-based experiments to demonstrate that the low molecular weight compound AMBnTßCD, a positively charged tailor-made ß-cyclodextrin derivative, is an efficient pharmacological inhibitor for the clostridial C2 and iota toxins

Summary

Introduction

Various pathogenic bacteria produce binary exotoxins, which are composed of two non-linked proteins, an enzyme (A-) component and a separate binding and translocation (B-) component. The B-component binds to a receptor on the surface of mammalian target cells, forms a complex with the A-component, and mediates the transport of the A-component into the cytosol, where the enzymatic active A-component modifies its substrate. C2 and iota toxin consists of two individual proteins, an enzymatic active (A-) component and a separate receptor binding and translocation (B-) component. The latter forms a complex with the A-component on the surface of target cells and after receptor-mediated endocytosis, it mediates the translocation of the A-component from acidified endosomal vesicles into the cytosol. The Bcomponents form heptameric pores in endosomal membranes, which serve as translocation channels for the Acomponents

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