Summary
Clostridioides difficile is a Gram-positive, spore-forming, toxin-producing anaerobe that can cause nosocomial antibiotic-associated intestinal disease. Although the production of toxin A (TcdA) and toxin B (TcdB) contribute to the main pathogenesis of C. difficile, the mechanism of TcdA and TcdB release from intracell remains unclear. In this study, we identified and characterized a new cell wall hydrolase Cwl0971 (endopeptidase, CDR20291_0971) from C. difficile R20291, which is involved in bacterial autolysis. The gene 0971 deletion mutant (R20291Δ0971) generated with CRISPR-AsCpfI exhibited significantly delayed cell autolysis and increased cell viability compared to R20291, and the purified Cwl0971 exhibited hydrolase activity for Bacillus subtilis cell wall. Meanwhile, 0971 gene deletion impaired TcdA and TcdB release due to the decreased cell autolysis in the stationary / late phase of cell growth. Moreover, biofilm formation, germination, and sporulation of the mutant strain decreased significantly compared to the wild type strain. In vivo, the depletion of Cwl097 decreased fitness over the parent strain in a mouse infection model. Collectively, Cwl0971 is involved in cell wall lysis and cell viability, which can affect several phenotypes of R20291. Our data indicate that the endopeptidase Cwl0971 is an attractive target for C. difficile infection therapeutics and prophylactics.
Competing Interest Statement
The authors have declared no competing interest.