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Infection and Immunity, December 2009, p. 5593-5601, Vol. 77, No. 12
0019-9567/09/$08.00+0     doi:10.1128/IAI.00710-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

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

Hanna Hilger,1 Sascha Pust,1,{dagger} Guido von Figura,2,{ddagger} Eva Kaiser,1 Bradley G. Stiles,3,4 Michel R. Popoff,5 and Holger Barth1*

Institute of Pharmacology and Toxicology, University of Ulm Medical Center, Albert-Einstein-Allee 11, D-89081 Ulm, Germany,1 Institute of Experimental and Clinical Pharmacology and Toxicology, University of Freiburg, Albertstrasse 25, D-79104 Freiburg, Germany,2 Wilson College, Chambersburg, Pennsylvania,3 Integrated Toxicology Division, Medical Research Institute of Infectious Diseases, Fort Detrick, Maryland,4 CNR Anaerobes and Botulism, Anaerobic Bacteria and Toxins Unit, Institut Pasteur, Paris, France5

Received 24 June 2009/ Returned for modification 26 July 2009/ Accepted 13 September 2009

Mono-ADP ribosylation of actin by bacterial toxins, such as Clostridium perfringens iota or Clostridium botulinum C2 toxins, results in rapid depolymerization of actin filaments and cell rounding. Here we report that treatment of African green monkey kidney (Vero) cells with iota toxin resulted in delayed caspase-dependent death. Unmodified actin did not reappear in toxin-treated cells, and enzyme-active toxin was detectable in the cytosol for at least 24 h. C2 toxin showed comparable, long-lived effects in cells, while a C2 toxin control lacking ADP-ribosyltransferase activity did not induce cell death. To address whether the remarkable stability of the iota and C2 toxins in cytosol was crucial for inducing cell death, we treated cells with C/SpvB, the catalytic domain of Salmonella enterica SpvB. Although C/SpvB also mono-ADP ribosylates actin as do the iota and C2 toxins, cells treated with a cell-permeating C/SpvB fusion toxin became rounded but recovered and remained viable. Moreover, unmodified actin reappeared in these cells, and ADP-ribosyltransferase activity due to C/SpvB was not detectable in the cytosol after 24 h, a result most likely due to degradation of C/SpvB. Repeated application of C/SpvB prevented recovery of cells and reappearance of unmodified actin. In conclusion, a complete but transient ADP ribosylation of actin was not sufficient to trigger apoptosis, implying that long-term stability of actin-ADP-ribosylating toxins, such as iota and C2, in the cytosol is crucial for inducing delayed, caspase-dependent cell death.

* 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 5 October 2009.

Editor: A. Camilli

{dagger} Present address: Center 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.

{ddagger} Present address: Department of Internal Medicine I, University of Ulm Medical Center, Albert-Einstein-Allee 11, D-89081 Ulm, Germany.

Infection and Immunity, December 2009, p. 5593-5601, Vol. 77, No. 12
0019-9567/09/$08.00+0     doi:10.1128/IAI.00710-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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