Clostridium difficile is responsible for 15-20% of antibiotic-associated diarrheas, and nearly all cases of pseudomembranous colitis. Among the cell wall proteins involved in the colonization process, Cwp84 is a protease that cleaves the S-layer protein SlpA into two subunits. A cwp84 mutant was previously shown to be affected for in vitro growth but not in its virulence in a hamster model. In this study, the cwp84 mutant elaborated biofilms with increased biomass compared with the parental strain, allowing the mutant to grow more robustly in the biofilm state. Proteomic analyses of the 630Δerm bacteria growing within the biofilm revealed the distribution of abundant proteins either in cell surface, matrix or supernatant fractions. Of note, the toxin TcdA was found in the biofilm matrix. Although the overall proteome differences between the cwp84 mutant and the parental strains were modest, there was still a significant impact on bacterial surface properties such as altered hydrophobicity. In vitro and in vivo competition assays revealed that the mutant was significantly impaired for growth only in the planktonic state, but not in biofilms or in vivo. Taken together, our results suggest that the phenotypes in the cwp84 mutant come from either the accumulation of uncleaved SlpA, or the ability of Cwp84 to cleave as yet undetermined proteins.

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