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Cwp22, a novel peptidoglycan cross-linking enzyme, plays pleiotropic roles in Clostridioides difficile.

ABSTRACT: Clostridioides difficile is a Gram-positive, spore-forming, toxin-producing anaerobe pathogen, and can induce nosocomial antibiotic-associated intestinal disease. While production of toxin A (TcdA) and toxin B (TcdB) contribute to the main pathogenesis of C. difficile, adhesion and colonization of C. difficile in the host gut are prerequisites for disease onset. Previous cell wall proteins (CWPs) were identified that were implicated in C. difficile adhesion and colonization. In this study, we predicted and characterized Cwp22 (CDR20291_2601) from C. difficile R20291 to be involved in bacterial adhesion based on the Vaxign reverse vaccinology tool. The ClosTron-generated cwp22 mutant showed decreased TcdA and TcdB production during early growth, and increased cell permeability and autolysis. Importantly, the cwp22 mutation impaired cellular adherence in vitro and decreased cytotoxicity and fitness over the parent strain in a mouse infection model. Furthermore, lactate dehydrogenase cytotoxicity assay, live-dead cell staining and transmission electron microscopy confirmed the decreased cell viability of the cwp22 mutant. Thus, Cwp22 is involved in cell wall integrity and cell viability, which could affect most phenotypes of R20291. Our data suggest that Cwp22 is an attractive target for C. difficile infection therapeutics and prophylactics.

SUBMITTER: Zhu D

PROVIDER: S-EPMC7219524 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature

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Cwp22, a novel peptidoglycan cross-linking enzyme, plays pleiotropic roles in Clostridioides difficile.

Zhu Duolong D Zhu Duolong D Bullock Jessica J He Yongqun Y Sun Xingmin X

Environmental microbiology 20190628 8

Clostridioides difficile is a Gram-positive, spore-forming, toxin-producing anaerobe pathogen, and can induce nosocomial antibiotic-associated intestinal disease. While production of toxin A (TcdA) and toxin B (TcdB) contribute to the main pathogenesis of C. difficile, adhesion and colonization of C. difficile in the host gut are prerequisites for disease onset. Previous cell wall proteins (CWPs) were identified that were implicated in C. difficile adhesion and colonization. In this study, we pr ...[more]

PMID: 31173438

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Project description:Clostridioides (Clostridium) difficile is a major cause of hospital-acquired infections leading to antibiotic-associated diarrhea. C. difficile exhibits a very high level of resistance to lysozyme. Bacteria commonly resist lysozyme through modification of the cell wall. In C. difficile, σV is required for lysozyme resistance, and σV is activated in response to lysozyme. Once activated, σV, encoded by csfV, directs transcription of genes necessary for lysozyme resistance. Here, we analyze the contribution of individual genes in the σV regulon to lysozyme resistance. Using CRISPR-Cas9-mediated mutagenesis we constructed in-frame deletions of single genes in the csfV operon. We find that pdaV, which encodes a peptidoglycan deacetylase, is partially responsible for lysozyme resistance. We then performed CRISPR inhibition (CRISPRi) to identify a second peptidoglycan deacetylase, encoded by pgdA, that is important for lysozyme resistance. Deletion of either pgdA or pdaV resulted in modest decreases in lysozyme resistance. However, deletion of both pgdA and pdaV resulted in a 1,000-fold decrease in lysozyme resistance. Further, muropeptide analysis revealed that loss of either PgdA or PdaV had modest effects on peptidoglycan deacetylation but that loss of both PgdA and PdaV resulted in almost complete loss of peptidoglycan deacetylation. This suggests that PgdA and PdaV are redundant peptidoglycan deacetylases. We also used CRISPRi to compare other lysozyme resistance mechanisms and conclude that peptidoglycan deacetylation is the major mechanism of lysozyme resistance in C. difficile IMPORTANCE Clostridioides difficile is the leading cause of hospital-acquired diarrhea. C. difficile is highly resistant to lysozyme. We previously showed that the csfV operon is required for lysozyme resistance. Here, we used CRISPR-Cas9 mediated mutagenesis and CRISPRi knockdown to show that peptidoglycan deacetylation is necessary for lysozyme resistance and is the major lysozyme resistance mechanism in C. difficile We show that two peptidoglycan deacetylases in C. difficile are partially redundant and are required for lysozyme resistance. PgdA provides an intrinsic level of deacetylation, and PdaV, encoded by a part of the csfV operon, provides lysozyme-induced peptidoglycan deacetylation.

Dataset Information

Cwp22, a novel peptidoglycan cross-linking enzyme, plays pleiotropic roles in Clostridioides difficile.

Publications

Cwp22, a novel peptidoglycan cross-linking enzyme, plays pleiotropic roles in Clostridioides difficile.

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