title: Localization of the Clostridium difficile cysteine protease Cwp84 and insights into its maturation process.
abstract: Clostridium difficile is a nosocomial pathogen involved in antibiotic-associated diarrhea. C. difficile expresses a cysteine protease, Cwp84, which has been shown to degrade some proteins of the extracellular matrix and play a role in the maturation of the precursor of the S-layer proteins. We sought to analyze the localization and the maturation process of this protease. Two identifiable forms of the protease were found to be associated in the bacteria: a form of ∼80 kDa and a cleaved one of 47 kDa, identified as the mature protease. They were found mainly in the bacterial cell surface fractions and weakly in the extracellular fraction. The 80-kDa protein was noncovalently associated with the S-layer proteins, while the 47-kDa form was found to be tightly associated with the underlying cell wall. Our data supported that the anchoring of the Cwp84 47-kDa form is presumably due to a reassociation of the secreted protein. Moreover, we showed that the complete maturation of the recombinant protein Cwp84(30-803) is a sequential process beginning at the C-terminal end, followed by one or more cleavages at the N-terminal end. The processing sites of recombinant Cwp84 are likely to be residues Ser-92 and Lys-518. No proteolytic activity was detected with the mature recombinant protease Cwp84(92-518) (47 kDa). In contrast, a fragment including the propeptide (Cwp84(30-518)) displayed proteolytic activity on azocasein and fibronectin. These results showed that Cwp84 is processed essentially at the bacterial cell surface and that its different forms may display different proteolytic activities.
authors.affiliation: EA 4043, Université Paris-Sud 11, Faculté de Pharmacie, Département de Microbiologie, 5 Rue Jean Baptiste Clément, 92296 Châtenay-Malabry Cedex, France.
authors.forename: Diana
authors.initials: D
authors.lastname: ChapetónMontes
authors.forename: Thomas
authors.initials: T
authors.lastname: Candela
authors.forename: Anne
authors.initials: A
authors.lastname: Collignon
authors.forename: Claire
authors.initials: C
authors.lastname: Janoir
mesh_terms: D000818:Animals; D016360:Clostridioides difficile / Q000201:enzymology* / Q000235:genetics; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D015151:Immunoblotting; D051379:Mice; D008856:Microscopy, Fluorescence; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D053719:Tandem Mass Spectrometry
chemical_list: D011994:Recombinant Proteins; C524258:Cwp84 protein, Clostridium difficile; D003546:Cysteine Endopeptidases