1. Cwp19 Is a Novel Lytic Transglycosylase Involved in Stationary-Phase Autolysis Resulting in Toxin Release in Clostridium difficile
- Author
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Jean-Louis Pons, Bruno Dupuy, Marie-Pierre Chapot-Chartier, Marie-José Butel, Imane El Meouche, Sandra Wydau-Dematteis, René Lai-Kuen, Johann Peltier, Audrey Hamiot, François Fenaille, Bruno Saubaméa, Pascal Courtin, Ecosystème intestinal, probiotiques, antibiotiques (EA 4065), Université Paris Descartes - Paris 5 (UPD5), Groupe de Recherche sur l'Adaptation Microbienne (GRAM 2.0), Université de Caen Normandie (UNICAEN), Normandie Université (NU)-Normandie Université (NU)-Université de Rouen Normandie (UNIROUEN), Normandie Université (NU), MICrobiologie de l'ALImentation au Service de la Santé (MICALIS), Institut National de la Recherche Agronomique (INRA)-AgroParisTech, Pathogénèse des Bactéries Anaérobies / Pathogenesis of Bacterial Anaerobes (PBA (U-Pasteur_6)), Institut Pasteur [Paris]-Université Paris Diderot - Paris 7 (UPD7), Université Paris Diderot - Paris 7 (UPD7), Plateau technique Imagerie Cellulaire et Moléculaire, Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Laboratoire d'Etude du Métabolisme des Médicaments (LEMM), Service de Pharmacologie et Immunoanalyse (SPI), Médicaments et Technologies pour la Santé (MTS), Université Paris-Saclay-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Médicaments et Technologies pour la Santé (MTS), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), This work was supported by the University of Rouen and the University of Paris-Descartes. J. Peltier is funded by a Pasteur-Roux fellowship. The work of P. Courtin andM.-P. Chapot-Chartier was supported by INRA., Institut Pasteur [Paris] (IP)-Université Paris Diderot - Paris 7 (UPD7), Faculté de Pharmacie de Paris - Université Paris Descartes (UPD5 Pharmacie), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Paris-Saclay-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Médicaments et Technologies pour la Santé (MTS), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Université de Rouen Normandie (UNIROUEN), Normandie Université (NU)-Normandie Université (NU)-Université de Caen Normandie (UNICAEN), Université Paris Descartes - Faculté de Pharmacie de Paris (UPD5 Pharmacie), Peltier, Johann, and Martin, Marie
- Subjects
autolysins ,lytic transglycosylase ,peptidoglycan ,surface proteins ,toxins ,clostridium difficile ,0301 basic medicine ,autolysin ,Autolysis (biology) ,toxins secretion ,peptidoglycan hydrolase ,Applied Microbiology ,MESH: Catalytic Domain ,medicine.disease_cause ,Cwp19 ,chemistry.chemical_compound ,Cell Wall ,Catalytic Domain ,glucose ,MESH: Bacterial Proteins ,MESH: Gene Expression Regulation, Bacterial ,Chemistry ,Cell autolysis ,Clostridium difficile ,QR1-502 ,3. Good health ,[SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology ,Lytic cycle ,MESH: Peptidoglycan Glycosyltransferase ,Research Article ,MESH: Bacteriolysis ,MESH: Clostridium Infections ,030106 microbiology ,Microbiology ,03 medical and health sciences ,Bacteriolysis ,MESH: Cell Wall ,Bacterial Proteins ,Virology ,Genetics ,medicine ,Humans ,Secretion ,Molecular Biology ,[SDV.MP] Life Sciences [q-bio]/Microbiology and Parasitology ,MESH: Clostridium difficile ,MESH: Humans ,Clostridioides difficile ,Toxin ,Lytic transglycosylase activity ,Gene Expression Regulation, Bacterial ,Clostridium Infections ,Peptidoglycan Glycosyltransferase ,Peptidoglycan - Abstract
Clostridium 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., IMPORTANCE Clostridium difficile-associated disease is mainly known as a health care-associated infection. It represents the most problematic hospital-acquired infection in North America and Europe and exerts significant economic pressure on health care systems. Virulent strains of C. difficile generally produce two toxins that have been identified as the major virulence factors. The mechanism for release of these toxins from bacterial cells is not yet fully understood but is thought to be partly mediated by bacteriolysis. Here we identify a novel peptidoglycan hydrolase in C. difficile, Cwp19, exhibiting lytic transglycosylase activity. We show that Cwp19 contributes to C. difficile cell autolysis in the stationary phase and, consequently, to toxin release, most probably as a response to environmental conditions such as nutritional signals. These data highlight that Cwp19 constitutes a promising target for the development of new preventive and curative strategies.
- Published
- 2018
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