Elsevier

Toxicology

Volume 316, 28 February 2014, Pages 25-33
Toxicology

Inhibitions of the translocation pore of Clostridium botulinum C2 toxin by tailored azolopyridinium salts protects human cells from intoxication

https://doi.org/10.1016/j.tox.2013.12.006Get rights and content

Abstract

C2 toxin from Clostridium botulinum represents the prototype of clostridial binary actin ADP-ribosylating toxins which destroy the actin-cytoskeleton of mammalian cells and cause severe enteric diseases in humans and animals. After receptor-mediated endocytosis of the C2 toxin complex, the binding/translocation component C2IIa forms a heptameric transmembrane pore in membranes of acidified endosomal vesicles. The separate ADP-ribosyltransferase component C2I translocates through this C2IIa-pore from the endosomal lumen into the cytosol.

Here we demonstrate that positively charged heterocyclic azolopyridinium salts which were developed as pore blockers for the anthrax toxins, efficiently protect cultured mammalian cells from intoxication with C2 toxin. The inhibitors had no effects on enzyme activity of C2I or receptor binding of C2 toxin but inhibited the pH-dependent membrane translocation of C2I in living cells, most likely by blocking the C2IIa-translocation pores. In vitro, the substances blocked C2IIa-pores in black lipid bilayer membranes when applied to the cis-side of the membrane which corresponds to the endosomal lumen of cells. Thus, heterocyclic azolopyridinium salts could represent lead compounds for development of novel therapeutics against binary clostridial toxins.

Introduction

Clostridium (C.) botulinum C2 toxin belongs to the family of binary toxins of the AB-type (for review see Barth et al., 2004, Aktories and Barth, 2011). C2 toxin consists of two distinct proteins: The enzymatically active component C2I and the binding/translocation component C2II (Ohishi et al., 1980) which mediates the transport of C2I into the cytosol of target cells (Barth et al., 2000). Translocation of C2I from acidified endosomes into the cytosol is mediated by proteolytically activated C2II and facilitated by host cell chaperones including Hsp90, cyclophilins and FK506 binding proteins (Barth et al., 2000, Haug et al., 2003a, Kaiser et al., 2009, Kaiser et al., 2012). In the cytosol C2I exerts its catalytic activity and ADP-ribosylates G-actin at position arginine 177 (Aktories et al., 1986, Vandekerckhove et al., 1988). This inhibits actin polymerization (Wegner and Aktories, 1988) resulting in breakdown of the actin cytoskeleton, cell rounding (Wiegers et al., 1991) and cell death (Heine et al., 2008).

Further members of the family of binary actin ADP-ribosylating toxins are iota toxin of Clostridium perfringens (Blöcker et al., 2001, Perelle et al., 1996, Schering et al., 1988, Stiles and Wilkins, 1986a, Stiles and Wilkins, 1986b), CDT of Clostridium difficile (Gülke et al., 2001, Perelle et al., 1997, Popoff et al., 1988a), Clostridium spiroforme toxin (Popoff and Boquet, 1988b), the vegetative insecticidal proteins (VIPs) of Bacillus (B.) cereus and B. thuringiensis (Han et al., 1999, Leuber et al., 2006). The anthrax toxins of Bacillus anthracis are also binary toxins with an overall comparable cellular uptake mechanism but the enzyme components of the anthrax toxins are no ADP-ribosyltransferases (for review see Collier and Young, 2003).

Essential for the transport of C2I (∼50 kDa) into the cell is C2II (∼80 kDa) which needs to be cleaved by trypsin to obtain its biological activity (Ohishi, 1987). This cleavage generates a ∼60 kDa fragment, which forms the ring-shaped C2IIa heptamer (Barth et al., 2000, Schleberger et al., 2006), and a ∼20 kDa fragment, which dissociates from C2II. C2IIa heptamers bind to asparagine-linked carbohydrates on the surface of target cells (Eckhardt et al., 2000) and form a complex with C2I (Barth et al., 2000, Blöcker et al., 2003a, Ohishi and Yanagimoto, 1992). Addition of C2IIa to artificial lipid bilayer membranes results in formation of ion permeable channels that are formed by C2IIa heptamers (Bachmeyer et al., 2001, Barth et al., 2000, Schmid et al., 1994). In cells, C2IIa forms pores in the membranes of acidified endosomes and these pores serve as translocation channels for unfolded C2I through the endosomal membrane (Barth et al., 2000, Blöcker et al., 2003a, Blöcker et al., 2003b, Haug et al., 2003b). Chloroquine and structurally related 4-aminoquinolones block channel formation by C2IIa in vitro and prevent intoxication of cells with C2-toxin in cell-based assays (Bachmeyer et al., 2001). Responsible for channel blockage is the binding of chloroquine and structurally related 4-aminoquinolones to two rings of totally 14 negatively charged amino acids (7 glutamates at position E399 in each PA63-monomer and 7 aspartates at position D426 in each PA63-monomer) and to the Φ-clamp (a ring formed by the 7 phenylalanines at position F428 in each PA63-monomer) which are localized within the vestibule of the C2IIa heptamer on the cis-side of the channel (Neumeyer et al., 2008). Important for the binding of the blockers to the C2IIa-channel is an overall positive charge of the molecules and the presence of bulky heterocycles (Bachmeyer et al., 2003, Orlik et al., 2005).

Protective antigen (PA), the binding/translocation component of the binary anthrax toxins, shows a considerable homology to C2II (Petosa et al., 1997, for review see Barth et al., 2004 or Young and Collier, 2007). Activated PA63 provides also a pathway to carry the enzyme components of anthrax toxin, namely edema factor (EF) and lethal factor (LF) into the cytosol of the target cells, which is also blocked by chloroquine and structurally related 4-aminoquinolones (Orlik et al., 2005, Zhang et al., 2004a, Zhang et al., 2004b). In a previous study we demonstrated that positively charged heterocyclic azolopyridinium salts (see Fig. 1) block the channels formed by PA63 (Blaustein et al., 1989, Blaustein et al., 1990, Krantz et al., 2005) and inhibit intoxication of J774A.1 macrophages by the combination of PA63 and lethal factor LF (Beitzinger et al., 2013). In the present study we investigated whether these heterocyclic azolopyridinium salts block C2IIa channels and intoxication of HeLa cells by C2IIa/C2I. A set of test compounds including thiazolopyridinium N-alkylpyridinium, tetrazolopyridinium, triazolopyridinium and imidazopyridinium salts is shown in Fig. 1. The block of the C2IIa-channels resulted in a dose-dependent decrease of membrane conductance in titration experiments with black lipid bilayers. The results revealed interesting insight in the structural requirement for the azolopyridinium salts to effectively inhibit C2IIa-channels in vitro. Furthermore, very low concentrations of the azolopyridinium salts delayed intoxication of living cells by C2 toxin.

Section snippets

Materials

The recombinant components of C2 toxin, C2I and C2II, were expressed as GST fusion proteins in Escherichia (E.) coli BL21 cells and purified as described (Barth et al., 2000). To obtain biologically active C2IIa, C2II was treated with trypsin as reported earlier (1). The heterocyclic azolopyridinium salts HA 1383, HA 1495 and HA 1568 (see Fig. 1) were synthesized and dissolved in dimethyl sulfoxide as 100 mM stock solutions as previously described (Beitzinger et al., 2013). Cell culture media

The heterocyclic azolopyridinium salts HA 1383, HA 1495 and HA 1568 protect HeLa cells from intoxication with C2 toxin

Since we identified HA 1383, HA 1495 and HA 1568 as the most efficient inhibitors against anthrax toxin among the various HA-substances (Beitzinger et al., 2013), their effects on the mode of action of C2 toxin was investigated. To this end, HeLa cells were treated with the toxin in the absence or presence of either substance and after different incubation periods, the toxin-induced cell rounding was analyzed. As shown in Fig. 2A, the presence of each HA-substance reduced the amount of round

Discussion

In order to identify novel pharmacological inhibitors of anthrax toxins, we found recently that heterocyclic azolopyridinium salts (HA-substances) protect macrophages, the target cells for anthrax lethal toxin, from intoxication with this toxin (Beitzinger et al., 2013). The substances prevent the translocation of the enzyme subunit of lethal toxin from acidified endosomes into the host cell cytosol since they block the translocation channels formed in endosomal membranes by PA63, the transport

Acknowledgements

We thank Detlev Gabel, Jacobs University, for helpful discussions and Ulrike Binder, University of Ulm, for expert technical assistance. This work was financially supported by the Deutsche Forschungsgemeinschaft (DFG, grant BA 2087/2-2).

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