Revealing roles of S-layer protein (SlpA) in pathogenicity by generating the first gene deletion mutant.

infection (CDI) with high morbidity and high mortality is an urgent threat to public health, and pathogenesis studies are eagerly required for CDI therapy. The major surface layer protein, SlpA, was supposed to play a key role in pathogenesis; however, a lack of isogenic mutants has greatly hampered analysis of SlpA functions. In this study, the whole gene was successfully deleted for the first time via CRISPR-Cas9 system. Deletion of in resulted in smaller, smother-edged colonies, shorter bacterial cell size, and aggregation in suspension. For life cycle, the mutant demonstrated lower growth (changes of optical density at 600 nm, OD600) but higher cell density (colony-forming unit, CFU), decreased toxins production, and inhibited sporulation. Moreover, the mutant was more impaired in motility, more sensitive to vancomycin and Triton X-100-induced autolysis, releasing more lactate dehydrogenase. In addition, SlpA deficiency led to robust biofilm formation but weak adhesion to human host cells.IMPORTANCE infection (CDI) has been the most common hospital-acquired infection, with a high rate of antibiotic resistance and recurrence incidences, become a debilitating public health threat. It is urgently needed to study pathogenesis for developing efficient strategies as CDI therapy. SlpA was indicated to play a key role in pathogenesis. However, analysis of SlpA functions was hampered due to lack of isogenic mutants. Surprisingly, the first deletion strain was generated in this study via CRISPR-Cas9, further negating the previous thought about being essential. Results in this study will provide direct proof for roles of SlpA in pathogenesis, which will facilitate future investigations for new targets as vaccines, new therapeutic agents, and intervention strategies in combating CDI.