IBS - Institut de Biologie Structurale - Grenoble / France

MAS NMR experiments of corynebacterial cell walls (2024)

Simorre Jean-Pierre — 2024-07-31T07:41:14Z

Bacterial cell walls are gigadalton-large cross-linked polymers with a wide range of motional amplitudes, including rather rigid as well as highly flexible parts. Magic-angle spinning NMR is a powerful method to obtain atomic-level information about intact cell walls. Here we investigate sensitivity and information content of different homonuclear 13C13C and heteronuclear 1H15N, 1H13C and 15N13C correlation experiments. We demonstrate that a CPMAS CryoProbe yields ca. 8-fold increased signal-to-noise over a room-temperature probe, or a ca. 3-4-fold larger per-mass sensitivity. The increased sensitivity allowed to obtain high-resolution spectra even on intact bacteria. Moreover, we compare resolution and sensitivity of 1H MAS experiments obtained at 100 kHz vs. 55 kHz. Our study provides useful hints for choosing experiments to extract atomic-level details on cell-wall samples.

Tracking Antibiotics Within Living Bacterial Cells (2024)

Simorre Jean-Pierre — 2024-07-31T07:12:19Z

This study demonstrated how nuclear magnetic resonance (NMR) proves to be a powerful analytical tool for better tracking the fate of antibiotics in the face of resistance phenomena and potentially helping to make them more effective.

The production of enzymes called β-lactamases is one of the main clinical indicators of the emergence of resistance in many bacteria. These β-lactamases have the capacity to degrade β-lactam antibiotics*, rendering them inactive. Produced by the bacterium in its periplasm, a compartment delimited by its inner and outer membranes, these enzymes reinforce the bacterium's defense against antibiotic intrusion. Thanks to the development of a method for in situ nuclear magnetic resonance monitoring of the enzymatic activity taking place in the cell, it has been possible to analyze in real time the degradation of β-lactams by β-lactamases in resistant strains. Using specific β-lactamase enzyme inhibitors, the ability of β-lactams to cross the outer membrane, interact with their target and ultimately block at least partial antibiotic degradation has been assessed. These measurements provide information on the nature and location of these inhibitor-enzyme interactions, by measuring them directly within the cell.

This study, published in J. Am. Chem. Soc., shows that NMR on living cells constitutes a powerful analytical tool for studying new molecules specifically targeting the molecular components of the bacterial periplasm responsible for antibiotic resistance. This approach will make it possible to assess the efficacy of drugs directly in their environment, opening the way to personalized medicine by proposing a specific antibiotic treatment for each patient according to the micro-organism responsible for the resistant infection.

Collaborations: M. Arthur (INSERM, Paris), A. Dessen (IBS, Grenoble)

interaction of MapZ with FtsZ and membranes in Streptococcus pneumoniae (2020)

Simorre Jean-Pierre — 2024-07-30T08:53:34Z

MapZ localizes at midcell and acts as a molecular beacon for the positioning of the cell division machinery in the bacterium Streptococcus pneumoniae. MapZ contains a single transmembrane helix that separates the C-terminal extracellular domain from the N-terminal cytoplasmic domain. Only the structure and function of the extracellular domain is known. Here, we demonstrate that large parts of the cytoplasmic domain is intrinsically disordered and that there are two regions (from residues 45 to 68 and 79 to 95) with a tendency to fold into amphipathic helices. We further reveal that these regions interact with the surface of liposomes that mimic the Streptococcus pneumoniae cell membrane. The highly conserved and unfolded N-terminal region (from residues 17 to 43) specifically interacts with FtsZ independently of FtsZ polymerization state. Moreover, we show that MapZ phosphorylation at positions Thr67 and Thr68 does not impact the interaction with FtsZ or liposomes. Altogether, we propose a model in which the MapZ-mediated recruitment of FtsZ to mid-cell is modulated through competition of MapZ binding to the cell membrane. The molecular interplay between the components of this tripartite complex could represent a key step toward the complete assembly of the divisome.

Collaboration : C. Grangeasse (CNRS, Lyon)

Peptidoglycan Synthase Activator LpoP in Pseudomonas aeruginosa (2020)

Simorre Jean-Pierre — 2024-07-30T08:42:30Z

Peptidoglycan (PG) is an essential component of the bacterial cell wall and is assembled from a lipid II precursor by glycosyltransferase and transpeptidase reactions catalyzed in particular by bifunctional class A penicillin-binding proteins (aPBPs). In the major clinical pathogen Pseudomonas aeruginosa, PBP1B is anchored within the cytoplasmic membrane but regulated by a bespoke outer membrane-localized lipoprotein known as LpoP. Here, we report the structure of LpoP, showing an extended N-terminal, flexible tether followed by a well-ordered C-terminal tandem-tetratricopeptide repeat domain. We show that LpoP stimulates both PBP1B transpeptidase and glycosyltransferase activities in vitro and interacts directly via its C terminus globular domain with the central UB2H domain of PBP1B. Contrary to the situation in E. coli, P. aeruginosa CpoB does not regulate PBP1B/LpoP in vitro. We propose a mechanism that helps to underscore similarities and differences in class A PBP activation across Gram-negative bacteria.

Collaboration : W. Vollmer (NewCastle Univ., UK), Natalie Strynadka (Vancouver, Canada)

Mode of Interaction of Two S. aureus Enzymes with the Bacterial Cell Wall (2023)

Laguri cédric, Simorre Jean-Pierre — 2024-07-30T07:10:27Z

Peptidoglycan (PG) is a giga-dalton polymer constituting scaffold and integrity of bacterial cell wall (CW). PG is metabolised by a large and diverse group of PG hydrolases, guards of bacterial cell growth and division. PG hydrolases have been the focus of many studies over the years, but molecular understanding of their action within PG mesh is missing.
Our recent work fills this gap by providing structural knowledge on the interaction of two evolutionary related peptidases of the M23 family, lysostaphin and LytM, with short PG fragments and the entire sacculus. Through nuclear magnetic resonance, information-driven modelling, mass spectrometry, site-directed mutagenesis and biochemical approaches, we provide residue-resolution details of their interaction with the bacterial cell wall. This allows us to address long-standing question regarding the relationship between selectivity and specificity of lysostaphin and LytM and their physiological function. We propose a new model in which PG cross-linking affects the activity of these two enzymes differently .
With this work, we intend to provide better understanding of the action and regulation of PG hydrolyses in the complex mesh of the bacterial cell wall. In broader perspective, our work will serve to underpin future research concerning the development of new antimicrobial agents.

Collaboration : I. Sabala ( Warsaw, Poland)