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Clostridium difficile toxin A-induced apoptosis is p53-independent but depends on glucosylation of Rho GTPases

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Abstract

Clostridium difficile toxin A (TcdA) is one of two homologous glucosyltransferases that mono-glucosylate Rho GTPases. HT29 cells were challenged with wild-type and mutant TcdA to investigate the mechanism by which apoptosis is induced. The TcdA-induced re-organization of the actin cytoskeleton led to an increased number of cells within the G2/M phase. Depolymerization of the actin filaments with subsequent G2/M arrest, however, was not causative for apoptosis, as shown in a comparative study using latrunculin B. The activation of caspase-3, -8, and -9 strictly depended on the glucosylation of Rho GTPases. Apoptosis measured by flow cytometry was completely abolished by a pan-caspase inhibitor (z-VAD-fmk). Interestingly, cleavage of procaspase-3 and Bid was not inhibited by z-VAD-fmk, but was inhibited by the calpain/cathepsin inhibitor ALLM. Cleavage of procaspase-8 was susceptible to inhibition by z-VAD-fmk and to the caspase-3 inhibitor Ac-DMQD-CHO, indicating a contribution to the activation of caspase-3 in an amplifying manner. Although TcdA induced mitochondrial damage and cytochrome c release, p53 was not activated or up-regulated. A p53-independent apoptotic effect was also checked by treatment of HCT 116 p53−/− cells. In summary, TcdA-induced apoptosis in HT29 cells depends on glucosylation of Rho GTPases leading to activation of cathepsins and caspase-3.

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Abbreviations

TcdA/TcdB:

C. difficile toxin A/toxin B

CPE:

Cytopathic effect

CTE:

Cytotoxic effect

FACS:

Fluorescence-associated cell sorting

GAPDH:

Glyceraldehyde-3-phosphate dehydrogenase

GT:

Glucosyltransferase

z-VAD-fmk:

Benzyloxycarbonyl-Phe-Val-Ala-Asp-(Ome)-fluoromethyl-ketone

Ac-DMQD-CHO:

Acetyl-Asp-Met-Gln-Asp-CHO

ALLM:

N-Acetyl-Leu-Leu-Met

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Acknowledgments

We are grateful to Helma Tatge for excellent technical assistance, and we also thank Hanping Feng, Tufts University, for critical reading of the manuscript. This study was supported by Deutsche Forschungsgemeinschaft SFB621 (Project B5) and SPP1150 (Project GE 1247/1-2). HCT 116 p53−/− and wild-type cells were kindly provided by Bert Vogelstein, Baltimore, USA.

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Authors and Affiliations

  1. Institute of Toxicology, Hannover Medical School, 30625, Hannover, Germany

    Stefanie Nottrott, Janett Schoentaube, Harald Genth, Ingo Just & Ralf Gerhard

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  1. Stefanie Nottrott
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  2. Janett Schoentaube
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  3. Harald Genth
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  4. Ingo Just
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Correspondence to Ralf Gerhard.

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Nottrott, S., Schoentaube, J., Genth, H. et al. Clostridium difficile toxin A-induced apoptosis is p53-independent but depends on glucosylation of Rho GTPases. Apoptosis 12, 1443–1453 (2007). https://doi.org/10.1007/s10495-007-0074-8

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