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Infection and Immunity, October 2008, p. 4600-4608, Vol. 76, No. 10
0019-9567/08/$08.00+0     doi:10.1128/IAI.00651-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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

Karin Heine, Sascha Pust,{dagger} Stefanie Enzenmüller, and Holger Barth*

Institute of Pharmacology and Toxicology, University of Ulm Medical Center, Albert-Einstein-Allee 11, D-89081 Ulm, Germany

Received 27 May 2008/ Returned for modification 28 July 2008/ Accepted 5 August 2008

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.

* Corresponding author. Mailing address: Institute of Pharmacology and Toxicology, University of Ulm Medical Center, Albert-Einstein-Allee 11, D-89081 Ulm, Germany. Phone: 49-731-50065503. Fax: 49-731-50065502. E-mail: holger.barth{at}uni-ulm.de

{triangledown} Published ahead of print on 18 August 2008.

Editor: S. R. Blanke

{dagger} Present address: Centre for Cancer Biomedicine, Faculty Division Norwegian Radium Hospital, University of Oslo, 0316 Oslo, Norway, and Department of Biochemistry, Institute for Cancer Research, Norwegian Radium Hospital, Montebello, 0310 Oslo, Norway.

Infection and Immunity, October 2008, p. 4600-4608, Vol. 76, No. 10
0019-9567/08/$08.00+0     doi:10.1128/IAI.00651-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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