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Institut de Biologie StructuraleGrenoble / France

Bacterial cell wall formation

Structural and functional studies of cell wall machinery complexes

Collaborations: A. Zapun, T. Vernet, J.P. Simorre (IBS); W. Vollmer (Newcastle Univ. UK); E. Breukink (Univ. Utrecht, Holland); S. Gobec (Univ. Ljubljana, Slovenia); I. Boneca (Institut Pasteur)

The bacteria cell wall is composed mostly of peptidoglycan (or murein), a three-dimensional mesh which consists of polymerized chains of repeating disaccharide subunits (N-acetylglucosamine (NAG) and N-acetylmuramic acids (NAM)) cross-linked by stem pentapeptides and whose function is to provide cellular shape and maintain internal osmotic pressure. Peptidoglycan building blocks are synthesized in 3 different compartments in the cell: cytoplasm, membrane, and periplasm. (Fig.1).

Fig.1.- Proteins involved in peptidoglycan biosynthesis reside in the cytoplasm, membrane, and periplasm compartments of the bacterial cell. IM, inner membrane; OM, outer membrane; from Mattei, Neves and Dessen (2010) Curr. Opin. Struct. Biol. 20, 749-755.

Among the cytoplasmic actors of peptidoglycan biosynthesis, the MurC, MurD, MurE and MurF enzymes are adenosine triphosphate (ATP)-dependent ligases that catalyse the sequential addition of amino acids to the UDP-MurNAc precursor in order to generate a short peptide which will be cross-linked within the peptidoglycan layer. In addition, A number of actors involved in peptidoglycan biosynthesis have been shown to interact with each other. Among them, MreB has been widely reported to play a key role in the spatial organization of the overall process in rod-shaped bacteria. Our group showed that MurD, MurE and MurF all recognize and bind to MreB and MurG, but not to each other, while MreB and MurG were shown to interact. In addition, we solved the crystal structures of MurD, MurE and MurF, the latter two in complex with ADP. The structures revealed that the C-terminal domains of the molecules may adopt different orientations, and support the hypothesis that Mur ligases display distinct conformations upon ligand recognition. This fact could play an important role in MreB and MurG recognition and binding. These results provide an initial framework for understanding the structural requirements of a cytosolic complex of murein-synthesizing enzymes involving the cytoskeletal protein MreB. (Favini-Stabile et al. 2013 Environ. Microbiol.)

Fig.2.- The cytoplasmic steps of peptidlgycan biosynthesis are catalyzed by Mur enzymes. From Nikolaidis, Favini and Dessen (2014) Protein Sci.

Development of new inhibitors

Beta-lactam antibiotics have been the stronghold of anti-infectious treatment for the past 80 years, displaying efficacy in the treatment of both Gram-positive and Gram-negative organisms. Nevertheless, pathogenic bacteria have evolved efficient antibiotic resistance mechanisms to circumvent the employment of these drugs, underlining the importance of a continuous effort in the search for new antibacterials whose structure is distinct from the classical beta-lactam scaffold. In our search for new inhibitors, we have targeted both cytoplasmic and periplasmic steps of peptidoglycan biosynthesis, and have used Mur ligases and PBPs as targets.

We solved eleven enzyme-inhibitor complexes of PBP1b from S. pneumoniae (Contreras-Martel, et al., 2011) and twelve of MurD from E. coli (Kotnik et al., 2007 ; Humljan et al., 2008 ; Zidar et al., 2010 ; Zidar et al., 2011 ; Tomašić et al., 2011 and Sosič et al., 2011), (Fig.3). This work has led to the identification of boronate analogs capable of killing multi-drug resistant S. aureus (MRSA) strains (Contreras-Martel et al. 2011).

Fig.3.- A) The active site of PBP1b (S. pneumoniae) binds boronate inhibitors in distinct conformations. B) Binding of inhibitors within the active site of MurD (E. coli).