Cwp84, a surface-associated protein of Clostridium difficile, is a cysteine protease with degrading activity on extracellular matrix proteins.
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.
Janoir
Claire
C
Université de Paris-Sud, Faculté de Pharmacie, Département de Microbiologie, 5 rue JB Clément, 92296, Châtenay-Malabry Cedex, France.
Péchiné
Séverine
S
Grosdidier
Charlotte
C
Collignon
Anne
A
D000907:Antibodies, Bacterial / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002364:Caseins / Q000378:metabolism; D016360:Clostridioides difficile / Q000201:enzymology*; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D016326:Extracellular Matrix Proteins / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D008562:Membrane Glycoproteins / Q000378:metabolism; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000276:immunology / Q000302:isolation & purification
D000907:Antibodies, Bacterial; D001426:Bacterial Proteins; D002364:Caseins; D015853:Cysteine Proteinase Inhibitors; D016326:Extracellular Matrix Proteins; D008562:Membrane Glycoproteins; D011994:Recombinant Proteins; C055764:S-layer proteins; C524258:Cwp84 protein, Clostridium difficile; D003546:Cysteine Endopeptidases
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