Institut de Biologie StructuraleGrenoble / France

Contact person(s) related to this article / DI GUILMI Anne-Marie / DURMORT Claire / MORLOT Cécile / ZAPUN André

Cell wall metabolism and morphogenesis

Our past work on the mechanisms of beta-lactam resistance led us to investigate more generally cell wall metabolism in the pneumococcus. The assembly of the peptidoglycan, the major constituent of the cell wall, is catalyzed by the PBPs (Penicillin-Binding Proteins) that are the targets of the beta-lactams. The reaction of the PBPs with beta-lactams is well known, the mechanisms of resistance to these drugs is also well known (Zapun et al. 2008, collab. Bacterial Pathogenesis Group).

Paradoxically, the physiological reactions of peptidoglycan synthesis catalyzed by the PBPs are poorly characterized. Understanding how PBPs, which are altered under antibiotic selection pressure, remain functional for cell wall synthesis should help us to combat resistance. The precise physiological functions of the various PBPs and the consequences of their inactivation by beta-lactams also remain largely ignored. The roles and precise substrates of the diverse peptidoglycan hydrolases are also poorly known. To address these questions, we combine biochemical approaches in vitro with genetic experiments in vivo and protein localization studies using advanced microscopies.

We have successfully reproduced pneumococcal peptidoglycan synthesis in vitro (Zapun et al. 2013, collab. E. Breuking (NL) and D. Roper (UK)). Our assay uses a fluorescent peptidoglycan precursor and products are monitored by gel electrophoresis. We are currently investigating in vitro how PBP variants from clinical resistant strains carry out peptidoglycan synthesis, although they fail to react with beta-lactams, which are substrate mimics.
Most importantly, the precursor of peptidoglycan assembly, lipid II, must be converted to a form that can be used by pneumococcal PBPs by the amidation of the second residue of the stem peptide (Zapun et al. 2013). This conversion is carried out by the MurT/GatD amidotransferase, which we are characterizing in details.

[bleu marine]Assembly of pneumococcal peptidoglycan by PBPs (green and yellow) at the cell surface.[/bleu marine]

The integrity of the cell wall depends on a delicate balance between synthetic and hydrolytic activities. Hydrolases are required for cell separation, but also to allow insertion of nascent peptidoglycan. We are currently investigating the function of LytA and Pmp23, through mutational, biochemical and localization studies.
LytA is an amidase required for the autolysis of the pneumococcus in stationary phase. Of particular interest is its recruitment mechanism at the cell surface as LytA lacks a secretion signal.
Pmp23 is a putative lytic transglycosylase that plays a role in morphogenesis and the organization of cell wall assembly.

[bleu marine]Localization of the LytA peptidoglycan hydrolase on the pneumococcal cell surface.[/bleu marine]

The cell wall defines the cell shape. Regulation of cell wall assembly and hydrolysis is therefore key to understand bacterial morphogenesis.
The precise function of the different PBPs in building a cell wall of the correct shape remains partly mysterious. An investigation of the effect of inhibiting PBP2x or PBP2b with a specific beta-lactam led to revised working model of cell wall building (Philippe et al. 2015).
Among the PBPs of pneumococcus, PBP2x shares structural motifs with the eukaryotic-like serine/threonine kinase StkP, which is important in shape determination. We have shown that the two proteins interact and we are pursuing the study of their respective functions (Morlot et al. 2013, collab. C. Grangeasse, BMSSI, Lyon).

[bleu marine]Localization of PBP2x (fused to GFP) in wild type (left) and StkP mutant cell lines (center and right).[/bleu marine]

The tubulin-like cytoskeletal protein FtsZ is considered as the main organizer of the bacterial cell division. FtsZ forms a ring on the cytoplasmic side of the membrane at the division site and recruits other proteins required for the division, including those involved in cell wall metabolism. To investigate in greater details the interplay between cell wall assembly and division, we have developed and implemented super-resolution fluorescence microscopy in pneumococcus (together with the PIXEL team, D. Bourgeois, at IBS). Photo-activated light microscopy (PALM) was used to localize FtsZ fused to a fluorescent protein during the cell cycle in various mutants.

[bleu marine]Super-resolution PALM imaging of the FtsZ-ring in pneumococcus.[/bleu marine]