Cwl0971, a novel peptidoglycan hydrolase, plays pleiotropic roles in Clostridioides difficile R20291

Abstract

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 cell remains unclear. In this study, we identified and characterized a new cell wall hydrolase Cwl0971 (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, sporulation of the mutant strain decreased significantly compared to the wild type strain. In vivo, the defect of Cwl0971 decreased fitness over the parent strain in a mouse infection model. Collectively, Cwl0971 is involved in cell wall lysis and cell viability, which affects toxin release, sporulation, germination, and pathogenicity of R20291, indicating that Cwl0971 could be an attractive target for C. difficile infection therapeutics and prophylactics.

FliW and CsrA Govern Flagellin (FliC) Synthesis and Play Pleiotropic Roles in Virulence and Physiology of Clostridioides difficile R20291

“…To analyze the role of fliW and csrA in R20291 (NC_013316.1), CRISPR-AsCpfI-based plasmid pDL1 (pMTL82151-Ptet-AscpfI) 3 https://img.jgi.doe.gov/ was constructed for gene deletion in C. difficile (Zhu et al, 2021). pDL1-fliW and pDL1-csrA gene deletion plasmids were constructed, and the fliW gene (288 bp deletion; R20291ΔW) was deleted successfully.…”

Flagellin modulates regulation ofClostridiodes difficilepathogenesis in the absence of motility

“…We constructed deletions of fliC, fliW, csrA, fliC-fliW, fliC-csrA, fliW-csrA , or fliC-fliW-csrA in CD1015 using a CRISPR-Cas12a system (Fig. 3B) [31, 32]. Growth profiles of the different mutants were measured in BHIS and TY media and no significant differences in growth nor motility were found Fig.…”

“…Gene fliC , fliW , and csrA were assembled into EcoR I- BamH I digested plasmid pMTL84153, resulting in overexpression plasmid pMTL84153- fliC (referred to as 153- fliC ), pMTL84153- fliW (153- fliW ), and pMTL84153- csrA (153- csrA ) with primers 20-22, respectively. Gene edit plasmid pDL-1 containing Cas12a (AsCpfI) under control of tetracycline inducing promoter was used for C. difficile gene deletion according to the previous reports [31, 32]. The target sgRNA was designed and analyzed on the Cas-OFFinder (http://www.rgenome.net/cas-offinder/) and CINDEL (http://big.hanyang.ac.kr/cindel/) websites.…”

Non-Toxin-Based Clostridioides difficile Vaccination Approaches

“…Zhu et al, used Vaxign ( , accessed on 3 January 2023) to predict in silico suitable vaccine antigens based on their localization and adhesion properties. Based on the CD R20291 genome sequence, they found 31 candidates with the outstanding putative cell wall hydrolase (P_003217470.1, Cwl0971) [ 44 ]. Cwl0971 deletion mutant showed decreased bacteriolysis, toxin release, sporulation, and decreased fitness over the wild strain in the mouse infection model [ 44 ].…”