Institut de Biologie StructuraleGrenoble / France

Bacterial cell wall formation

Structural and functional studies of cell wall machinery complexes

Collaborations: W. Vollmer (Newcastle Univ. UK); E. Breukink (Univ. Utrecht, Holland); S. Gobec (Univ. Ljubljana, Slovenia); I. Boneca (Inst. Pasteur, France); I. Attree, G. Schoehn, I. Gutsche, C. Breyton, C. Ebel (BIG & IBS, Grenoble)

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. PG plays a key role in shape maintenance, resistance to osmotic pressure, and cell division, and has been a preferential target for antibiotic development for decades. 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; PG, peptidoglycan; from Laddomada, Miyachiro & Dessen (2016).

Proteins that are involved in PG biosynthesis associate into discrete multi-membered complexes that regulate cell division and elongation, and their inhibition or deregulation can lead to defects in cell shape and often lysis and death.

Penicillin-binding proteins (PBPs) catalyze the two last reactions in PG biosynthesis, and have been reported to interact with several members of the cell division and elongation complexes during the bacterial cell cycle. One of these partners is MreC, a membrane-associated protein that has been shown to form fibers in certain bacteria and is thought to serve as a scaffold for macromolecules involved in cell wall elongation. However, protein interactions within these complexes have been reported to only form at defined points in the cell cycle, thus being fleeting in nature, fragile, and difficult to isolate.

We overcame these difficulties and structurally & functionally characterized the PBP2:MreC complex from the human pathogen Helicobacter pylori, as well as PBP2 in its unbound form (Contreras-Martel et al. (2017) Nature Comm; Fig. 2).

Fig.2.- The structures of PBP2 in unbound form (left) as well as of PBP2:MreC (right) from H. pylori. The N-terminal region of PBP2, comprising the head and anchor regions, must undergo a conformational change in order to bind its partner molecule MreC.

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Clefs CEA

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).