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Journal of Bacteriology, October 2007, p. 7174-7180, Vol. 189, No. 20
0021-9193/07/$08.00+0     doi:10.1128/JB.00578-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Cwp84, a Surface-Associated Protein of Clostridium difficile, Is a Cysteine Protease with Degrading Activity on Extracellular Matrix Proteins{triangledown}

Claire Janoir,1 Séverine Péchiné,1 Charlotte Grosdidier,1 and Anne Collignon1,2*

Université de Paris-Sud XI, Faculté de Pharmacie, Département de microbiologie, USC INRA, EA 40-43, 92296 Châtenay-Malabry Cedex, France,1 Hôpital Jean Verdier, AP-HP, Bondy, France2

Received 15 April 2007/ Accepted 19 July 2007

Clostridium difficile pathogenicity is mediated mainly by its A and B toxins, but the colonization process is thought to be a necessary preliminary step in the course of infection. The aim of this study was to characterize the Cwp84 protease of C. difficile, which is highly immunogenic in patients with C. difficile-associated disease and is potentially involved in the pathogenic process. Cwp84 was purified as a recombinant His-tagged protein, and specific antibodies were generated in rabbits. Treatment of multiple-band-containing eluted fractions with a reducing agent or with trypsin led to accumulation of a unique protein species with an estimated molecular mass of 61 kDa, corresponding most likely to mature autoprocessed Cwp84 (mCwp84). mCwp84 showed concentration-dependent caseinolytic activity, with maximum activity at pH 7.5. The Cwp84 activity was inhibited by various cysteine protease inhibitors, such as the specific inhibitor E64, and the anti-Cwp84-specific antibodies. Using fractionation experiments followed by immunoblot detection, the protease was found to be associated with the S-layer proteins, mostly as a nonmature species. Proteolytic assays were performed with extracellular matrix proteins to assess the putative role of Cwp84 in the pathogenicity of C. difficile. No degrading activity was detected with type IV collagen. In contrast, Cwp84 exhibited degrading activity with fibronectin, laminin, and vitronectin, which was neutralized by the E64 inhibitor and specific antibodies. In vivo, this proteolytic activity could contribute to the degradation of the host tissue integrity and to the dissemination of the infection.

* Corresponding author. Mailing address: Université de Paris-Sud, Faculté de Pharmacie, Département de Microbiologie, 5 rue JB Clément, 92296 Châtenay-Malabry Cedex, France. Phone: (33) 1 46 83 56 34. Fax: (33) 1 46 83 55 37. E-mail: anne.collignon{at}u-psud.fr

{triangledown} Published ahead of print on 10 August 2007.

Journal of Bacteriology, October 2007, p. 7174-7180, Vol. 189, No. 20
0021-9193/07/$08.00+0     doi:10.1128/JB.00578-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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