Inhibitions of the translocation pore of Clostridium botulinum C2 toxin by tailored azolopyridinium salts protects human cells from intoxication
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).
References (52)
- et al.
Mechanism of C2-toxin inhibition by fluphenazine and related compounds: investigation of their binding kinetics to the C2II-channel using the current noise analysis
J. Mol. Biol.
(2003) - et al.
Cellular uptake of Clostridium botulinum C2 toxin requires oligomerization and acidification
J. Biol. Chem.
(2000) - et al.
Formation of large, ion-permeable membrane channels by the matrix protein (porin) of Escherichia coli
Biochim. Biophys. Acta
(1978) - et al.
Clostridium botulinum C2 toxin: low pH-induced pore formation is required for translocation of the enzyme component C2I into the cytosol of host cells
J. Biol. Chem.
(2003) - et al.
Binding of Clostridium botulinum C2 toxin to asparagine-linked complex and hybrid carbohydrates
J. Biol. Chem.
(2000) - et al.
The host cell chaperone Hsp90 is essential for translocation of the binary Clostridium botulinum C2 toxin into the cytosol
J. Biol. Chem.
(2003) - et al.
Synthesis and nitrogen elimination of 3-aryltetrazolo[1,5-a]pyridinium salts and its angular benzologues
Tetrahedron
(1986) - et al.
Clostridium botulinum C2 toxin. Identification of the binding site for chloroquine and related compounds and influence of the binding site on properties of the C2II channel
J. Biol. Chem.
(2008) - et al.
Anthrax toxin protective antigen: inhibition of channel function by chloroquine and related compounds and study of binding kinetics using the current noise analysis
Biophys. J.
(2005) - et al.
Evidence that Arg-295, Glu-378, and Glu-380 are active-site residues of the ADP-ribosyltransferase activity of iota toxin
FEBS Lett.
(1996)
Clostridium spiroforme toxin is a binary toxin which ADP-ribosylates cellular actin
Biochem. Biophys. Res. Commun.
Structure and action of the binary C2 toxin from Clostridium botulinum
J. Mol. Biol.
Interaction of Clostridium botulinum C2 toxin with lipid bilayer membranes. Formation of cation-selective channels and inhibition of channel function by chloroquine
J. Biol. Chem.
Clostridium perfringens iota toxin: synergism between two proteins
Toxicon
Botulinum C2 toxin ADP-ribosylates cytoplasmic beta/gamma-actin in arginine 177
J. Biol. Chem.
ADP-ribosylated actin caps the barbed ends of actin filaments
J. Biol. Chem.
Protein translocation through anthrax toxin channels formed in planar lipid bilayers
Biophys. J.
Botulinum C2 toxin ADP-ribosylates actin
Nature
New insights into the mode of action of the actin ADP-ribosylating virulence factors Salmonella enterica SpvB and Clostridium botulinum C2 toxin
Eur. J. Cell Biol.
Interaction of Clostridium botulinum C2 toxin with lipid bilayer membranes and Vero cells: inhibition of channel function by chloroquine and related compounds in vitro and intoxification in vivo
FASEB J.
Quarternary salts of halogenated pyridines and quinolines
J. Am. Chem. Soc.
Binary bacterial toxins: biochemistry, biology, and applications of common Clostridium and Bacillus proteins
Microbiol. Mol. Biol. Rev.
Designed azolopyridinium salts block protective antigen pores in vitro and protect cells from anthrax toxin
PLOS ONE
Pore formation by LamB of Escherichia coli in lipid bilayer membranes
J. Bacteriol.
Anthrax toxin: channel-forming activity of protective antigen in planar phospholipid bilayers
Proc. Natl. Acad. Sci. U. S. A.
Voltage-dependent block of anthrax toxin channels in planar phospholipid bilayer membranes by symmetric tetraalkylammonium ions: single-channel analysis
J. Gen. Physiol.
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These authors contributed equally.