Volume 23, Issue 9 p. 5222-5238
Research article

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

Duolong Zhu

Duolong Zhu

Department of Molecular Medicine, Morsani College of Medicine, University of South Florida, Tampa, Florida, USA

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Hiran Malinda Lamabadu Warnakulasuriya Patabendige

Hiran Malinda Lamabadu Warnakulasuriya Patabendige

Department of Molecular Medicine, Morsani College of Medicine, University of South Florida, Tampa, Florida, USA

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Brooke Rene Tomlinson

Brooke Rene Tomlinson

Department of Cell Biology, Microbiology and Molecular Biology, University of South Florida, Tampa, Florida, USA

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Shaohui Wang

Shaohui Wang

Department of Molecular Medicine, Morsani College of Medicine, University of South Florida, Tampa, Florida, USA

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Syed Hussain

Syed Hussain

Department of Molecular Medicine, Morsani College of Medicine, University of South Florida, Tampa, Florida, USA

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Domenica Flores

Domenica Flores

Department of Molecular Medicine, Morsani College of Medicine, University of South Florida, Tampa, Florida, USA

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Yongqun He

Yongqun He

Department of Microbiology and Immunology, and Center for Computational Medicine and Bioinformatics, Unit for Laboratory Animal Medicine, University of Michigan Medical School, Ann Arbor, Michigan, USA

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Lindsey N. Shaw

Lindsey N. Shaw

Department of Cell Biology, Microbiology and Molecular Biology, University of South Florida, Tampa, Florida, USA

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Xingmin Sun

Corresponding Author

Xingmin Sun

Department of Molecular Medicine, Morsani College of Medicine, University of South Florida, Tampa, Florida, USA

For correspondence. E-mail [email protected]; Tel. (+1) 08139744553; Fax: (+1) 8139747357.

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First published: 24 April 2021
Citations: 7

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 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.

Conflict of interest

The authors declare no potential conflict of interest.