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

Abstract

Summary 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.

The Transcriptional Regulator Lrp Contributes to Toxin Expression, Sporulation, and Swimming Motility in Clostridium difficile

“…The severity of infection was clearly indicated in the mice infected with the R20291 lrp mutant strain, attributed to smaller cecum size and less well-formed feces, as well as more extensive necrosis and inflammation, as revealed by histological examination. It has been suggested in the past that clindamycin administration prior to challenge with C. difficile select for Clostron-based mutants bearing the ermB cassette, although other studies in which Clostron-based mutants with reduced virulence in vivo have also been reported (Ünal et al, 2018; Zhu et al, 2019). However, for further clarification, we are in the process of obtaining a markerless lrp mutant using the recently developed RiboCas system (Cañadas et al, 2019).…”

Mapping Epitopes of a Novel Peptidoglycan Cross-Linking Enzyme Cwp22 Recognized by Human Sera Obtained from Patients with Clostridioides difficile Infection and Cord Blood

“…When we started looking for new anti-CD vaccine antigens, the Cwp22 protein was identified bioinformatically as one of the cell wall proteins [35]. Next, it was found in the CD proteome [36], characterized [29] and recently, predicted to be a good vaccine antigen by Zhou et al [13]. Now, we are the first to identify Cwp22 protein as an immunoreactive one using CDI patient sera.…”

“…This can be explained using different extraction method. Wright, et al used TS buffer (10 mM Tris/HCl (pH 6.9), 10 mM MgCl2, 0.5 M sucrose) with the addition of mutanolysin, lysozyme, lysostaphin, RNase and 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride which mostly extracted proteins of pI between 4 and 6, whereas Cwp22 protein pI is 8.89 [13].…”

“…In this study we identified a new immunoreactive protein that turned to be one of the cell wall proteins (Cwp22). Most recently, the paper about Cwp22 protein has been published characterizing its functionality [13]. Cwp22 protein is a l , d -transpeptidase, peptidoglycan cross-linking enzyme, which mutation leads to decreased toxin production at early stage of bacteria growth along with its delayed sporulation and lower motility.…”

Vegetative Cell and Spore Proteomes of Clostridioides difficile Show Finite Differences and Reveal Potential Protein Markers

“…36 In fact, recently, Cwp22 has also been shown to be involved in toxin production, sporulation, motility, and cell viability in C. difficile strain R20291. 37 CwpV promotes C. difficile aggregation and its strain-dependent structural variations may assist in evading the host antibody response 38 or to launch an antiphage strategy. 39 Disseminating spores, on the contrary, make use of the hydrophobic exosporium to adhere to and colonize surfaces.…”