ADP-Ribosylation of Actin by the Clostridium botulinum C2 Toxin in Mammalian Cells Results in Delayed Caspase-Dependent Apoptotic Cell Death

Abstract

The binary C2 toxin from Clostridium botulinum mono-ADP-ribosylates G-actin in the cytosol of eukaryotic cells. This modification leads to depolymerization of actin filaments accompanied by cell rounding within 3 h of incubation but does not immediately induce cell death. Here we investigated the long-term responses of mammalian cell lines (HeLa and Vero) following C2 toxin treatment. Cells stayed round even though the toxin was removed from the medium after its internalization into the cells. No unmodified actin reappeared in the C2 toxin-treated cells within 48 h. Despite actin being completely ADP-ribosylated after about 7 h, no obvious decrease in the overall amount of actin was observed for at least 48 h. Therefore, ADP-ribosylation was not a signal for an accelerated degradation of actin in the tested cell lines. C2 toxin treatment resulted in delayed apoptotic cell death that became detectable about 15 to 24 h after toxin application in a portion of the cells. Poly(ADP)-ribosyltransferase 1 (PARP-1) was cleaved in C2 toxin-treated cells, an indication of caspase 3 activation and a hallmark of apoptosis. Furthermore, specific caspase inhibitors prevented C2 toxin-induced apoptosis, implying that caspases 8 and 9 were activated in C2 toxin-treated cells. C2I, the ADP-ribosyltransferase component of the C2 toxin, remained active in the cytosol for at least 48 h, and no extensive degradation of C2I was observed. From our data, we conclude that the long-lived nature of C2I in the host cell cytosol was essential for the nonreversible cytotoxic effect of C2 toxin, resulting in delayed apoptosis of the tested mammalian cells.Clostridium botulinum C2 toxin, the prototype of the family of binary actin ADP-ribosylating toxins, mono-ADP-ribosylates G-actin at Arg-177 (1). C2 toxin consists of the enzyme component C2I (431 amino acid residues, 49.3 kDa) and the binding/translocation component C2II (721 amino acid residues, 80.8 kDa). The ADP-ribosyltransferase activity of C2I is located in its C-terminal domain (5), while the enzymatically inactive N-terminal domain (C2IN, amino acid residues 1 to 225) mediates interaction with C2IIa and translocation of C2I into the cytosol (6). Following limited proteolysis, the active C2IIa protein (ϳ60 kDa) forms ring-shaped heptamers that assemble with C2I and mediate binding of the toxin complex to the cellular receptor, a carbohydrate structure (2, 11, 19). During cellular uptake, the heptamers form pores in endosomal membranes, thereby facilitating translocation of C2I into the cytosol (7). Translocation of C2I requires unfolding (14) and subsequent refolding of the protein in the cytosol by the host cell chaperone Hsp90 (13).In the cytosol, C2I catalyzes covalent transfer of the ADPribose moiety from NAD (NAD ϩ ) to Arg-177 of G-actin (1). Mono-ADP-ribosylation of actin induces the depolymerization of F-actin and thereby a complete loss of actin filaments, resulting in rounding of cultured monolayer cells (1). The ADPribosylation at Arg-177 turns G-actin monomers into "cappin...

The Long-Lived Nature of Clostridium perfringens Iota Toxin in Mammalian Cells Induces Delayed Apoptosis

“…Despite some structural and enzymatic differences between the C2 and iota-like toxins, they are quite comparable regarding their nonreversible cytopathic mode of action and time course of toxin-mediated cell death. Iota toxin treatment of Vero cells caused caspase-3 activation within 12 to 15 h after application, which favorably compares to the time course of caspase-3 activation in C2 toxin-treated HeLa cells (10).…”

“…We detected enzyme-active Ia in the cytosol 48 h after application of iota toxin to cells, indicating that the Ia domain harboring ADPribosyltransferase activity remained stable in the cytosol. Prompted by our recent observation that C2 toxin delays apoptosis and persists as an active ADP-ribosyltransferase in the cytosol of intoxicated cells (10), we have now addressed whether the long-lived nature of clostridial actin-ADP-ribosylating toxins in the cytosol is crucial for delayed cell death. To this end, we also investigated whether a fusion toxin containing the catalytic domain of S. enterica SpvB (C/SpvB) mono-ADP ribosylates G-actin as do the C2 and iota toxins (11,27).…”

“…Subsequently, the cell preparation was centrifuged (100,000 ϫ g, 1 h, 4°C) and the supernatant (cytosolic fraction) used to detect Ia or C2I activity. Alternatively, the cytosolic fractions were obtained by digitonin extraction as described earlier (10,12). In brief, Vero cells were grown in 12-well plates and incubated with 0.2 g Ia/ml plus 0.4 g Ib/ml for the indicated times.…”

“…Cytosolic fractions were incubated for 1 h at 37°C with fresh cell lysate as a source of actin and biotinylated NAD ϩ as a cosubstrate (R&D Systems, Wiesbaden, Germany). Alternatively, a bioassay with naive Vero cells was performed as described recently (10). Cytosolic samples from cells incubated with iota or C2 toxin were applied to naive cells in combination with fresh Ib or C2IIa, respectively, for the indicated times at 37°C, and the percentage of rounded cells was determined after various incubation periods.…”

A Type VI Secretion System Effector Protein, VgrG1, from Aeromonas hydrophila That Induces Host Cell Toxicity by ADP Ribosylation of Actin

“…HeLa cells in suspension were lysed by sonication in a buffer containing 20 mM Tris-HCl (pH 7.5), 1 mM EDTA, 1 mM dithiothreitol (DTT), 5 mM MgCl 2 , and protease inhibitors. The whole lysate was centrifuged at 14,000 ϫ g for 10 min at 4°C, and the supernatant was used for the ADP ribosylation assay (19). Briefly, 50 g of normal HeLa cell lysate proteins or 1 g of recombinant nonmuscle actin (Cytoskeleton, Denver, CO) as the target protein for ADP ribosylation was incubated with 1 g of different purified rVgrG1 proteins and 10 M NAD conjugated with biotin (R&D Systems, Minneapolis, MN) for 30 min at 37°C.…”