Genetic and Biochemical Characterization of the Cell Wall Hydrolase Activity of the Major Secreted Protein of Lactobacillus rhamnosus GG Ingmar J. J. Claes 1 , Geert Schoofs 1 , Krzysztof Regulski 2,3 , Pascal Courtin 2,3 , Marie-Pierre Chapot- Chartier 2,3 , Thomas Rolain 4 , Pascal Hols 4 , Ingemar von Ossowski 5 , Justus Reunanen 5 , Willem M. de Vos 5,6 , Airi Palva 5 , Jos Vanderleyden 1 , Sigrid C. J. De Keersmaecker 1 , Sarah Lebeer 1,7 * 1 Centre of Microbial and Plant Genetics, K. U. Leuven, Leuven, Belgium, 2 INRA, UMR1319 Micalis, F-78350 Jouy-en-Josas, France, 3 AgroParisTech, UMR Micalis, F-78350 Jouy-en-Josas, France, 4 Institut des Sciences de la Vie, UCL, Louvain-la-Neuve, Belgium, 5 Department of Veterinary Biosciences, University of Helsinki, Helsinki, Finland, 6 Laboratory of Microbiology, Wageningen University, Wageningen, The Netherlands, 7 Department of Bioscience Engineering, University of Antwerp, Groenenborgerlaan 171, Antwerp, Belgium Abstract Lactobacillus rhamnosus GG (LGG) produces two major secreted proteins, designated here Msp1 (LGG_00324 or p75) and Msp2 (LGG_00031 or p40), which have been reported to promote the survival and growth of intestinal epithelial cells. Intriguingly, although each of these proteins shares homology with cell wall hydrolases, a physiological function that correlates with such an enzymatic activity remained to be substantiated in LGG. To investigate the bacterial function, we constructed knock-out mutants in the corresponding genes aiming to establish a genotype to phenotype relation. Microscopic examination of the msp1 mutant showed the presence of rather long and overly extended cell chains, which suggests that normal daughter cell separation is hampered. Subsequent observation of the LGG wild-type cells by immunofluorescence microscopy revealed that the Msp1 protein accumulates at the septum of exponential-phase cells. The cell wall hydrolyzing activity of the Msp1 protein was confirmed by zymogram analysis. Subsequent analysis by RP-HPLC and mass spectrometry of the digestion products of LGG peptidoglycan (PG) by Msp1 indicated that the Msp1 protein has D-glutamyl-L-lysyl endopeptidase activity. Immunofluorescence microscopy and the failure to construct a knock-out mutant suggest an indispensable role for Msp2 in priming septum formation in LGG. Citation: Claes IJJ, Schoofs G, Regulski K, Courtin P, Chapot-Chartier M-P, et al. (2012) Genetic and Biochemical Characterization of the Cell Wall Hydrolase Activity of the Major Secreted Protein of Lactobacillus rhamnosus GG. PLoS ONE 7(2): e31588. doi:10.1371/journal.pone.0031588 Editor: Indranil Biswas, University of Kansas Medical Center, United States of America Received October 26, 2011; Accepted January 9, 2012; Published February 16, 2012 Copyright: ß 2012 Claes et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: At the time of experiments, IJJC held a PhD grant of the Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT- Vlaanderen) and SL was a postdoctoral researcher of the Fund for scientific research Vlaanderen, Belgium. Work of PH was supported by the Research Department of the Communaute ´ franc ¸aise de Belgique (Concerted Research Action), and PH is Research Associate of the National fund for scientific research, Walloon, Belgium. TR held a doctoral fellowship from Le Fonds pour la formation a ` la Recherche dans l’Industrie et dans l’Agriculture. Work of MPCC was supported by Institut nationale de la recherche ´ agronomique (INRA) or French National Institut for Agricultural Research. KR was the recipient of a fellowship from INRA and its PhD project received financial support from the Marie Curie FP7 Initial Training Network Cross Talk (grant agreement nu21553-2). The work conducted in the group of AP is supported by the Academy of Finland (118165) and was part of the Center of Excellence in Microbial Food Safety Research at the University of Helsinki and the Academy of 377 Finland funded Research Program on Nutrition, Foods, and Health (ELVIRA). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: sarah.lebeer@biw.kuleuven.be Introduction The ,75-kDa (LGG_00324 or p75) and ,40-kDa (LGG_00031 or p40) Major secreted proteins are the two most abundant proteins found in the spent culture supernatant of probiotic Lactobacillus rhamnosus GG (LGG). These proteins are designated here Msp1 and Msp2 respectively. Previously, these proteins were demonstrated to prevent cytokine-induced apoptosis and promote intestinal epithelial cell homeostasis [1,2]. More re- cently, recombinant Msp2 (p40) was shown to prevent and treat experimental colitis by an epidermal growth factor receptor- dependent mechanism [3]. Interestingly, although the Msp1 and Msp2 proteins both show homology to cell wall hydrolases [1], the physiological function associated with such an enzymatic activity has not yet been elucidated in LGG. The cell wall of Gram-positive bacteria is mainly composed of a thick layer of peptidoglycan (PG), an amino-sugar polymer cross- linked with peptide bridges. This multilayered murein sacculus, in addition to being responsible for maintaining cell shape and morphology, helps to protect bacteria from the external and internal osmotic stresses that can cause cell lysis [4]. The sugar component of PG consists of alternating b-(1,4)-linked N-acetyl- glucosamine and N-acetyl-muramic acid. The peptide side chain of three to five amino acids is attached to N-acetyl-muramic acid. The exact constitution of the peptide side chain and cross-links is species-specific. Cell wall hydrolases catalyze the cleavage of PG sugar or peptide chains. Based on their cleavage specificities, cell wall hydrolases can be divided in N-acetyl muramoyl-L-alanine amidases, carboxypeptidases, endopeptidases, N-acetylglucosami- nidases and N-acetylmuramidases [5]. These cell wall hydrolases PLoS ONE | www.plosone.org 1 February 2012 | Volume 7 | Issue 2 | e31588