Characterization of Acp, a Peptidoglycan Hydrolase of Clostridium perfringens with N-Acetylglucosaminidase Activity That Is Implicated in Cell Separation and Stress-Induced Autolysis

Emilie Camiade (1, 2) , Johann J. Peltier (1) , Ingrid Bourgeois (1) , Evelyne Couture-Tosi (2) , Pascal Courtin (3) , Ana Antunes (2) , Marie-Pierre Chapot-Chartier (3) , Bruno Dupuy (2) , Jean-Louis Pons (1)

1 GRAM 1.0 - Groupe de Recherche sur les Antimicrobiens et les Micro-Organismes
2 Toxines et Pathogénie Bactérienne
3 MICALIS - MICrobiologie de l'ALImentation au Service de la Santé

This work reports the characterization of the first known peptidoglycan hydrolase (Acp) produced mainly during vegetative growth of Clostridium perfringens. Acp has a modular structure with three domains: a signal peptide domain, an N-terminal domain with repeated sequences, and a C-terminal catalytic domain. The purified recombinant catalytic domain of Acp displayed lytic activity on the cell walls of several Gram-positive bacterial species. Its hydrolytic specificity was established by analyzing the Bacillus subtilis peptidoglycan digestion products by coupling reverse phase-high-pressure liquid chromatography (RP-HPLC) and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis, which displayed an N-acetylglucosaminidase activity. The study of acp expression showed a constant expression during growth, which suggested an important role of Acp in growth of C. perfringens. Furthermore, cell fractionation and indirect immunofluorescence staining using anti-Acp antibodies revealed that Acp is located at the septal peptidoglycan of vegetative cells during exponential growth phase, indicating a role in cell separation or division of C. perfringens. A knockout acp mutant strain was obtained by using the insertion of mobile group II intron strategy (ClosTron). The microscopic examination indicated a lack of vegetative cell separation in the acp mutant strain, as well as the wild-type strain incubated with anti-Acp antibodies, demonstrating the critical role of Acp in cell separation. The comparative responses of wild-type and acp mutant strains to stresses induced by Triton X-100, bile salts, and vancomycin revealed an implication of Acp in autolysis induced by these stresses. Overall, Acp appears as a major cell wall N-acetylglucosaminidase implicated in both vegetative growth and stress-induced autolysis.

Domaines: Sciences du Vivant [q-bio]; Biochimie, Biologie Moléculaire

Emilie Camiade, Johann J. Peltier, Ingrid Bourgeois, Evelyne Couture-Tosi, Pascal Courtin, et al.. Characterization of Acp, a Peptidoglycan Hydrolase of Clostridium perfringens with N-Acetylglucosaminidase Activity That Is Implicated in Cell Separation and Stress-Induced Autolysis. Journal of Bacteriology, 2010, 192 (9), pp.2373-2384. ⟨10.1128/JB.01546-09⟩. ⟨pasteur-02444015⟩