The <i>Clostridium difficile</i> Protease Cwp84 Modulates both Biofilm Formation and Cell-Surface Properties

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.艰难梭菌（Clostridium difficile）可引发15%~20%的抗生素相关性腹泻，且几乎所有伪膜性肠炎病例均由其导致。在参与定植过程的细胞壁蛋白中，Cwp84是一种可将S层蛋白SlpA切割为两个亚基的蛋白酶。既往研究显示，cwp84基因突变株的体外生长会受到影响，但在仓鼠模型中其毒力未发生改变。本研究中，与亲本菌株相比，该cwp84基因突变株形成的生物被膜生物量更高，使其在生物被膜状态下生长更为旺盛。对生物被膜内生长的630Δerm菌株进行蛋白质组学分析发现，丰度较高的蛋白分别分布于细胞表面、生物被膜基质及上清组分中。值得注意的是，毒素TcdA被检出存在于生物被膜基质中。尽管cwp84基因突变株与亲本菌株间的整体蛋白质组差异较小，但该突变仍对细菌表面特性产生了显著影响，例如疏水性发生改变。体外及体内竞争实验结果显示，该突变株仅在浮游状态下的生长能力显著受损，而在生物被膜状态或体内环境中则无此现象。综合来看，本研究结果表明，cwp84基因突变株的表型可能源于未切割的SlpA积累，或是Cwp84无法切割目前尚未确定的靶蛋白。