Department of Molecular Medicine, Morsani College of Medicine, University of South Florida, Tampa, Florida, USA.
Department of Cell Biology, Microbiology and Molecular Biology, University of South Florida, Tampa, Florida, USA.
Environ Microbiol. 2021 Sep;23(9):5222-5238. doi: 10.1111/1462-2920.15529. Epub 2021 Apr 24.
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.
艰难梭菌是一种革兰氏阳性、产芽孢、产毒的厌氧菌，可引起医院获得性抗生素相关性肠道疾病。虽然毒素 A（TcdA）和毒素 B（TcdB）的产生有助于艰难梭菌的主要发病机制，但 TcdA 和 TcdB 从细胞中释放的机制仍不清楚。在本研究中，我们从艰难梭菌 R20291 中鉴定并表征了一种新的细胞壁水解酶 Cwl0971（CDR20291_0971），它参与细菌自溶。与 CRISPR-AsCpfI 一起生成的基因 0971 缺失突变体（R20291Δ0971）表现出明显延迟的细胞自溶和增加的细胞活力，与 R20291 相比，并且纯化的 Cwl0971 对枯草芽孢杆菌细胞壁表现出水解酶活性。同时，由于细胞生长的静止/后期细胞自溶减少，0971 基因缺失会损害 TcdA 和 TcdB 的释放。此外，突变株的孢子形成能力与野生型菌株相比显著降低。在体内，与亲本菌株相比，Cwl0971 的缺陷降低了在小鼠感染模型中的适应性。总之，Cwl0971 参与细胞壁裂解和细胞活力，影响毒素释放、孢子形成、发芽和 R20291 的致病性，表明 Cwl0971 可能是艰难梭菌感染治疗和预防的有吸引力的靶标。
