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Acd, a peptidoglycan hydrolase of Clostridium difficile with N-acetylglucosaminidase activity

¹ Groupe de Recherche sur les Antimicrobiens et les Micro-organismes (UPRES EA 2656, IFR 23), Université de Rouen, UFR Médecine-Pharmacie, 22 Boulevard Gambetta, F-76183 Rouen Cedex, France
² INSERM U 614 (IFR 23), Université de Rouen, UFR Médecine-Pharmacie, 76183 Rouen Cedex, France
³ Unité de Biochimie et Structure des Protéines, INRA, 78352 Jouy-en-Josas Cedex, France

Correspondence
Jean-Louis Pons
Jean-Louis.Pons{at}univ-rouen.fr

A gene encoding a putative peptidoglycan hydrolase was identified by sequence similarity searching in the Clostridium difficile 630 genome sequence, and the corresponding protein, named Acd (autolysin of C. difficile) was expressed in Escherichia coli. The deduced amino acid sequence of Acd shows a modular structure with two main domains: an N-terminal domain exhibiting repeated sequences and a C-terminal catalytic domain. The C-terminal domain exhibits sequence similarity with the glucosaminidase domains of Staphylococcus aureus Atl and Bacillus subtilis LytD autolysins. Purified recombinant Acd produced in E. coli was confirmed to be a cell-wall hydrolase with lytic activity on the peptidoglycan of several Gram-positive bacteria, including C. difficile. The hydrolytic specificity of Acd was studied by RP-HPLC analysis and MALDI-TOF MS using B. subtilis cell-wall extracts. Muropeptides generated by Acd hydrolysis demonstrated that Acd hydrolyses peptidoglycan bonds between N-acetylglucosamine and N-acetylmuramic acid, confirming that Acd is an N-acetylglucosaminidase. The transcription of the acd gene increased during vegetative cellular growth of C. difficile 630. The sequence of the acd gene appears highly conserved in C. difficile strains. Regarding deduced amino acid sequences, the C-terminal domain with enzymic function appears to be the most conserved of the two main domains. Acd is the first known autolysin involved in peptidoglycan hydrolysis of C. difficile.

The GenBank/EMBL/DDBJ accession number for the acd sequence reported in this paper is AY775569.