Cwp19 Is a Novel Lytic Transglycosylase Involved in Stationary-Phase Autolysis Resulting in Toxin Release in Clostridium difficile.

Sandra Wydau-Dematteis,Marie-Pierre Chapot-Chartier,Imane El Meouche,Marie-José Butel,Bruno Saubaméa,Johann Peltier,François Fenaille,Audrey Hamiot,Bruno Dupuy,Pascal Courtin,René Lai-Kuen,Jean-Louis Pons

doi:10.1128/mbio.00648-18

Abstract

ABSTRACTClostridium difficile is the major etiologic agent of antibiotic-associated intestinal disease. Pathogenesis of C. difficile is mainly attributed to the production and secretion of toxins A and B. Unlike most clostridial toxins, toxins A and B have no signal peptide, and they are therefore secreted by unusual mechanisms involving the holin-like TcdE protein and/or autolysis. In this study, we characterized the cell surface protein Cwp19, a newly identified peptidoglycan-degrading enzyme containing a novel catalytic domain. We purified a recombinant His6-tagged Cwp19 protein and showed that it has lytic transglycosylase activity. Moreover, we observed that Cwp19 is involved in cell autolysis and that a C. difficile cwp19 mutant exhibited delayed autolysis in stationary phase compared to the wild type when bacteria were grown in brain heart infusion (BHI) medium. Wild-type cell autolysis is correlated to strong alterations of cell wall thickness and integrity and to release of cytoplasmic material. Furthermore, we demonstrated that toxins were released into the extracellular medium as a result of Cwp19-induced autolysis when cells were grown in BHI medium. In contrast, Cwp19 did not induce autolysis or toxin release when cells were grown in tryptone-yeast extract (TY) medium. These data provide evidence for the first time that TcdE and bacteriolysis are coexisting mechanisms for toxin release, with their relative contributions in vitro depending on growth conditions. Thus, Cwp19 is an important surface protein involved in autolysis of vegetative cells of C. difficile that mediates the release of the toxins from the cell cytosol in response to specific environment conditions.

Highlights

Clostridium difficile is the major etiologic agent of antibiotic-associated intestinal disease
To identify the 70-kDa PG-degrading enzyme, surface proteins of C. difficile 630Δerm were separated by SDS-PAGE and proteins with a size around 70 kDa were excised from the gel and digested with trypsin and tryptic peptides were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/ MS)
An increase in the intracellular levels of toxins was observed between 54 and 80 h for the wild-type strain (Fig. 4D). This secondary toxin production was associated with a slight increase in the optical density at 600 nm (OD600) (Fig. 4A) that might have been due to the growth of surviving cells using lysed cells, as cytoplasmic content can act as a source of nutrients in a nutrient-depleted environment. These results show that Cwp19 plays a critical role in toxin release of C. difficile 630Δerm cultivated in brain heart infusion (BHI) medium

Summary

Introduction

Clostridium difficile is the major etiologic agent of antibiotic-associated intestinal disease. Cwp did not induce autolysis or toxin release when cells were grown in tryptone-yeast extract (TY) medium These data provide evidence for the first time that TcdE and bacteriolysis are coexisting mechanisms for toxin release, with their relative contributions in vitro depending on growth conditions. Cwp is an important surface protein involved in autolysis of vegetative cells of C. difficile that mediates the release of the toxins from the cell cytosol in response to specific environment conditions. The major virulence factors mediating these symptoms are toxins A and B (TcdA and TcdB), which alter the actin cytoskeleton of intestinal epithelial cells through glucosylation of Rho family proteins, leading to a severe inflammatory response and inducing the symptoms associated with CDI [6]. A link between toxin release and peptidoglycan (PG) hydrolase (PGH)-induced bacteriolysis has been suggested [14]

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Conclusion