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Institut de Biologie StructuraleGrenoble / France

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Cell wall metabolism, morphogenesis and beta-lactam resistance

MurT/GatD

The membrane-bound precursor of peptidoglycan is synthesized in the cytoplasm by a succession of enzymatic reactions. In many gram-positive species, prior to translocation at the cell surface, the second residue of the peptide is amidated from a D-glutamate into a D-iso-glutamine by the essential enzymatic complex MurT/GatD.
We are investigating the structure/function relationship of MurT/GatD from S. pneumoniae as a potential target for the development of new antibiotics.
Collaborations: C. Gravier-Pelletier, Univ. Paris Descartes; L. Håvarstein, Univ. Ås, Norway; T. Santos-Silva, Univ. Nova de Lisboa, Portugal; W. Vollmer, Univ. Newcastle, UK.

Crystal structure of MurT/GatD; model of substrates in the active site; morphological aberration upon depletion of MurT/GatD

Morlot et al. 2018 Nature Comm. 9, 3180.

Peptidoglycan assembly in vitro

The peptidoglycan is assembled at the bacterial cell surface by enzymes called PBPs and SEDS that catalyze the polymerization of the glycan chains (glycosyl transferase activity) and their cross-linking (transpeptidase activity). The transpeptidase activity is inhibited by beta-lactam antibiotics (penicillin…). We study in vitro the mechanisms of peptidoglycan assembly, in particular the transpeptidase activity of the PBPs (penicillin-binding proteins).
Collaboration: E. Breukink, Univ. Utrecht, The Netherlands.

Teichoic acids synthesis in vivo

We are interested in how the biosynthesis of the two polymers of the bacterial cell wall are coordinated and the roles of teichoic acids in pneumococcal biology. To that end, we have developed a new method for fluorescent labeling of teichoic acids that can be combined with established techniques for peptidoglycan labeling.
Collaboration: Y.-S. Wong, Univ. Grenoble Alpes

Co-labeling of teichoic acids and peptidoglycan

Bonnet et al. 2018 ACS Chem. Biol. 13, 2010.

Localization of FtsZ and other morphogenetic proteins

The shape of bacteria is determined by that of their cell wall. The localization and activity of the enzymes that assemble the cell wall is governed by a number of morphogenetic proteins. We are investigating the localization of these proteins with unprecedented accuracy using super-resolution photo-activated localization microscopy (PALM). These experiments are conducted in various laboratory mutant strains that show morphological defects.
Collaborations: D. Bourgeois, Team Pixel (IBS); C. Grangeasse, MMSB Lyon.

Diffraction-limited and PALM super-resolved images of FtsZ division rings

Jacq et al. 2015 mBio 6, e01108.

Peptidoglycan assembly in vivo

We are developing new labeling methods to study peptidoglycan assembly with very high spatio-temporal resolution using direct stochastic optical reconstruction microscopy (dSTORM). Mutant strains with aberrant shapes are studied to uncover regulatory mechanisms and revise models of bacterial morphogenesis.
Collaborations: Y.-S. Wong, Univ. Grenoble Alpes; D. Bourgeois, Team Pixel (IBS); C. Grangeasse, MMSB Lyon.

Diffraction-limited and dSTORM super-resolved images of regions of peptidoglycan assembly

Membranes

The enzymes that polymerize the cell wall and most morphogenetic proteins are membrane proteins and the peptidoglycan precursor is membrane-bound. We are interested in the role of the nature of the membrane lipids in morphogenesis. Using a combination of fluorescent lipid probes, we have uncovered the existence of different physical lipid phases localized according the cellular geometry. Lipid phases may in turn localize morphogenetic proteins.
Collaboration: J. Jouhet, BIG CEA Grenoble.

Segregation of DOPE-labeled (red) lipid Lalpha phase on the hemispheres; FtsZ in green

Beta-lactam resistance

Resistance to penicillin in the pneumococcus results from the expression of altered endogenous PBPs (penicillin-binding proteins), which are the transpeptidase enzymes that cross-link the peptidoglycan. We are investigating the relationship between amino acid substitutions in PBPs, their reactivity with beta-lactams and their transpeptidase activity.

Distribution of amino acid substitutions in PBP2b from a penicillin-resistant strain of pneumococcus

Calvez et al. 2017 J. Biol. Chem. 292, 2854.