Cwp84, a surface-associated protein of Clostridium difficile is a cysteine protease with degrading activity on extracellular matrix proteins

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

^* To whom correspondence should be addressed. Email: anne.collignon{at}u-psud.fr.

	Abstract

Clostridium difficile pathogenicity is mainly mediated 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 Clostridium difficile-associated disease and is potentially involved in the pathogenic process. Cwp84 was purified as a recombinant His-tag protein and specific antibodies were generated in rabbits. Treatment of multiple bands-containing eluted fractions with a reducing agent or with trypsin leads to accumulation of a unique protein species with an estimated molecular weight of 61 kDa, corresponding most likely to mature autoprocessed mCwp84. mCwp84 shows concentration-dependant caseinolytic activity, with a maximum 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 an immunoblot detection, the protease was found to be associated with the S-layer proteins, mostly as a non-mature species. Proteolytic assays were performed on extracellular matrix proteins to assess a putative role for Cwp84 in the pathogenicity of C. difficile. No degrading activity was detected on type IV collagen. On the contrary, Cwp84 has a degrading activity on fibronectin, laminin and vitronectin, which is 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.