Cross-Reactivity of Anthrax and C2 Toxin: Protective Antigen Promotes the Uptake of Botulinum C2I Toxin into Human Endothelial Cells

Angelika Kronhardt,Michel R Popoff,Monica Rolando,Roland Benz,Michael Leuber,Gilles Flatau,Christoph Beitzinger,Emmanuel Lemichez,Caroline Stefani

doi:10.1371/journal.pone.0023133

Angelika Kronhardt, Michel R Popoff + Show 7 more

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https://doi.org/10.1371/journal.pone.0023133

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Abstract

Binary toxins are among the most potent bacterial protein toxins performing a cooperative mode of translocation and exhibit fatal enzymatic activities in eukaryotic cells. Anthrax and C2 toxin are the most prominent examples for the AB7/8 type of toxins. The B subunits bind both host cell receptors and the enzymatic A polypeptides to trigger their internalization and translocation into the host cell cytosol. C2 toxin is composed of an actin ADP-ribosyltransferase (C2I) and C2II binding subunits. Anthrax toxin is composed of adenylate cyclase (EF) and MAPKK protease (LF) enzymatic components associated to protective antigen (PA) binding subunit. The binding and translocation components anthrax protective antigen (PA63) and C2II of C2 toxin share a sequence homology of about 35%, suggesting that they might substitute for each other. Here we show by conducting in vitro measurements that PA63 binds C2I and that C2II can bind both EF and LF. Anthrax edema factor (EF) and lethal factor (LF) have higher affinities to bind to channels formed by C2II than C2 toxin's C2I binds to anthrax protective antigen (PA63). Furthermore, we could demonstrate that PA in high concentration has the ability to transport the enzymatic moiety C2I into target cells, causing actin modification and cell rounding. In contrast, C2II does not show significant capacity to promote cell intoxication by EF and LF. Together, our data unveiled the remarkable flexibility of PA in promoting C2I heterologous polypeptide translocation into cells.

Highlights

Binary toxins of the AB7/8 type are highly potent and specialized bacterial protein toxins and are organized in two different polypeptide chains that are separately secreted in the external media of Gram-positive bacteria [1]
Small amounts of a concentrated enzyme solution were added to the cis-side of the membrane and the PA63-induced membrane conductance decreased in a dosedependent manner
In previous studies we already demonstrated that the enzymatic components edema factor (EF) and lethal factor (LF) of anthrax toxin bind to their B component protective antigen (PA63) and C2I of C2 toxin binds to its B component C2II in vitro [18,20,34]

Summary

Introduction

Binary toxins of the AB7/8 type are highly potent and specialized bacterial protein toxins and are organized in two different polypeptide chains that are separately secreted in the external media of Gram-positive bacteria [1]. Component A is responsible for the intracellular enzymatic activity of the toxin, whereas heptamers or octamers, of the component B are necessary for receptor-binding and translocation of component A into target cells. One of the most prominent toxins of this type of toxin is anthrax toxin from Bacillus anthracis [2] This toxin possesses a binding and translocation component, protective antigen (PA) and two enzymatic subunits, edema factor (EF) and lethal factor (LF). The binding component PA is essential for delivery of both enzymes into the target cells [3,6,7] It is secreted as an 83 kilo Dalton water-soluble precursor form (PA83) and needs to undergo proteolytic activation by cell-bound furin. After the activation of PA83, the remaining 63 kilo Dalton PA63 forms an oligomeric channel responsible for the binding and translocation of EF and/or LF into the cytosol of target cells [8,9,10,11,12]

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