Thèse Identification du Mécanisme de Compétition pour la Biosynthèse du Peptidoglycan Bactérien au Cours du Cycle Cellulaire H/F - Université Grenoble Alpes

Établissement : Université Grenoble Alpes
École doctorale : CSV- Chimie et Sciences du Vivant
Laboratoire de recherche : Institut de Biologie Structurale
Direction de la thèse : Pauline MACHEBOEUF ORCID 0000000345239923
Début de la thèse : 2026-10-01
Date limite de candidature : 2026-04-09T23:59:59

Le perpétuel développement de nouveaux antibiotiques est d'importance cruciale dans le contexte de la résistance microbienne à l'arsenal classique de molécules inhibitrices. Nous focalisons notre recherche sur les étapes de biosynthèse du peptidoglycane (PG) qui est une cible reconnue pour le développement de nouveaux antibiotiques. Cependant, au lieu de cibler notre recherche sur les protéines individuelles de cette synthèse, nous nous intéressons à l'étude du complexe cytoplasmique composés de Mur ligases, responsables de la production de précurseurs du PG, et son recrutement à différentes étapes du cycle cellulaire bactérien, i.e., la division et l'élongation de la paroi. Pour ce projet, nous tirons avantage de la diversité génétique bactérienne et de la présence de chimères naturelles de Mur afin de comprendre comment ce complexe peut être dirigé soit vers le complexe de l'élongasome soit vers le complexe du divisome qui entrent en compétition pour les précurseurs de PG à différents moments de la croissance bactérienne. En particulier, nous nous focaliserons sur la chimère composée de MurD-FtsW chez Betaproteobacteria bacterium afin de comprendre l'interaction entre le complexe cytoplasmique et le divisome. Puisque les Mur et les protéines du divisome sont très conservées parmi les génomes bactériens, nous pourrons extrapoler nos résultats structuraux au protéines orthologues du pathogène humain Pseudomonas aeruginosa et muter les interfaces moléculaires, afin de déterminer comment la déstabilisation des interactions Mur/divisome affecte la survie, la croissance et la division bactérienne. Pour cela, nous proposons de combiner des techniques de biologie structurales, de génétique bactérienne et d'imagerie in vivo en espérant qu'une meilleure compréhension de cette machinerie naturelle fascinante nous permettra de tracer le chemin vers la découverte de nouvelles molécules antibactériennes.

Widespread resistance to antibiotics by pathogenic bacteria is a worldwide concern that needs to be addressed by the constant development of new therapeutic molecules. One of the most successful antibiotic target is the bacterial cell wall biosynthesis process and in particular, the biosynthesis of its key component, the peptidoglycan (PG). The PG is a three-dimensional cross-linked mesh, surrounding the bacteria, essential for bacterial shape and tolerance to modifications in osmotic conditions. It is composed of polymerized chains of repeating disaccharide subunits cross-linked by short peptides. Its biosynthesis is orchestrated by several enzymes, working sequentially, and located in three different cellular compartments (cytoplasm, membrane, and periplasm). The first stage of PG biosynthesis takes place in the cytoplasm, where a series of 7 Mur ligases (MurA to MurG) and MraY synthesize the PG precursor (the Lipid II), a membrane-bound disaccharide pentapeptide. Lipid II is then translocated to the periplasmic side of the cell by flippases. Finally, PG synthesis machineries assemble the PG mesh by polymerizing the Lipid II into glycan strands and cross-linking neighbouring peptide stems. These machineries include scaffolding proteins (such as FtsZ), Penicillin-Binding Proteins (PBPs) and SEDS (Shape, Elongation, Division, and Sporulation, such as FtsW) proteins that catalyze the PG polymerization and cross-linking, or proteins that regulate the activity or stability of these enzymes (such as FtsQ).
In order to generate daughter cells with similar shape and dimension, PG biosynthesis must be controlled temporally and spatially. The coordination of interactions between proteins involved in PG biosynthesis located within the different cellular compartments is thus essential and is regulated by two dynamic complexes, namely the divisome, involved in cell division and the elongasome or Rod complex, that plays a key role in cell wall elongation. Importantly, the elongasome and the divisome do not co-localize, and in rod-shaped bacteria, they respectively assemble PG in the lateral or in the midcell region. Hence, during cell growth, the PG precursors synthesized in the cytoplasm by the Mur ligases must be shared between these two complexes. Notably, lipid II that is present at only 1000-2000molecules per cell is kept constant throughout the Escherichia coli cell cycle. Therefore, the molecular signals that initiate the division or the elongation process do not depend on the oscillation in the concentrations of the murein precursors. In this context, how can the Mur ligases supply PG precursors to both machineries, and what's more, in quantities appropriate for balanced cell elongation and division? Atwo-competing sites model has been proposed which states that the elongasome and divisome processes are in fact two competing reactions (or pathways), and that the balance between them regulates cell shape in rod-shaped bacteria. It has been suggested that molecular interactions between Mur ligases and elongasome or divisome components would timely recruit a Mur complex at elongation or division sites. Evidence for such interactions has been reported for several bacteria. In particular, FtsZ, FtsW and FtsQ which are crucial members of the divisome, interact with MurC, D, E, and F in Mycobacterium tuberculosis and MurG in Caulobacter crescentus. Many of these proteins are encoded by conserved genes located within the dcw (division and cell wall) cluster. The presence of a genetic cluster containing mur and fts genes has been proposed to favor the channeling of PG precursors towards the synthesis of septal PG during cell division, an essential process for bacterial survival, that might be substrate-limited if the precursors were synthesized throughout the whole cell and consumed by the elongation process. Interestingly, our team has recently discovered that numerous proteobacteria possess fused mur genes encoding natural Mur chimeras. Recently, we solved the crystal structure of the MurE-MurF fusion from Bordetella pertussis, thereby unraveling the molecular interface between the MurE and MurF moieties, and their interaction with other Mur ligases. Our genomic analysis also revealed several MurC-MurB, MurF-MraY, MurD-FtsW, FtsW-MurG and MurC-FtsQ fusions. Altogether, these observations support the idea of a Mur ligases complex that constitutively or transiently associates with the divisome to ensure that part of the PG precursors are delivered at the division site. The other advantages of complex formation include substrate tunneling in order to restrict the diffusion of PG precursor intermediates, protein stabilization and maintenance of protein stoechiométrie.
Despite this evidence, the nature of the molecular interactions established between the Mur and divisome proteins, and how they influence the PG synthesis process, are questions that remain to be resolved. In this project, we will study the structure of a specific natural chimera composed of MurD and FstW from Betaproteobacteria bacterium to understand how the divisome complex associates with Mur ligases, and exploit this structural information through bacterial genetics and cell imaging to determine the role of such interactions in cell viability, growth and division processes associated with PG synthesis.

We propose to elucidate the structural determinants of the interaction between a Mur ligase and a divisome protein, both contained in a natural chimeric protein and test the effect of this interaction on bacterial cell growth and division.

Task 1: Characterization of the complex between MurD ligase and divisome FtsW through the elucidation of the chimeric protein from Betaproteobacteria bacterium.
To obtain structural information on Mur ligases in complex with divisome components, we will take advantage of the natural chimeras discovered in our analyses of the dcw cluster. We will focus on the MurD-FtsW chimeric protein found in Betaproteobacteria bacterium, for which we already cloned the synthetic gene and obtained preliminary expression data in cell-free expression. We will purify the chimeric protein using standard chromatography protocols and will analyze its homogeneity, stability and stoichiometry using biochemical and biophysical methods, including analytical ultracentrifugation and multi-angle light scattering. The protein will be subjected to high-throughput crystallization at the EMBL Grenoble platform. We will collect X-ray diffraction data at the European Synchrotron Radiation Facility (ESRF) and process them using our structure solution and refinement pipelines. Phases will be solved by molecular replacement, using AlphaFold3 models of the individual FtsW and MurG regions, as this strategy proved efficient for the structural study of the MurEMurF fusion.
Depending on the oligomerization state of the chimera, we will envisage small angle X-ray scattering (SAXS) or electron microscopy techniques to characterize its structure in case we fail to obtain diffracting crystals. In particular, we already obtained encouraging electron microscopy data on another chimeric complex MurF-MraY that form organized fibers.

Task 2: Physiological implications in PG synthesis, growth and division
The hypothesis driving this project is that the Mur complex shares its activity between cell elongation and cell division through molecular interactions with their respective PG synthesis machineries, the elongasome and the divisome. This hypothesis predicts that the molecular interfaces identified in task 1 between Mur ligases and divisome components should dissociate the Mur complex from the divisome machinery, leading to impaired PG synthesis, cell morphology defects and/or cell death.
To test these hypotheses, we will use Pseudomonas aeruginosa, an elongated rod-shaped Gram-negative bacterial model studied by the group of Ina Attrée (IBS), with whom we extensively collaborate. Based on the composition of the molecular interfaces characterized in the chimera MurD-FtsW (task 1), we will introduce charge reversal mutations in individual mur or division genes from P. aeruginosa, by modifying its genome using a CRISPR-Cas system. If some of these mutations may be lethal, we will create merodiploid strains, in which the native gene is cloned at an ectopic position under the control of an inducible promoter (to allow protein depletion) and the mutated gene is constitutively expressed from the endogenous locus. In collaboration with Cécile Morlot in our group at IBS, the phenotype of the mutant strains will be characterized in terms of cell viability (growth curves, video phase-contrast microscopy), cell shape (phase contrast and cellular electron microscopy) and PG synthesis. To monitor PG synthesis, we will use PG labeling techniques with fluorescent probes and conventional fluorescence microscopy that are well mastered by the Morlot group. Variations in the localization and intensity of the fluorescent signal will inform us on the elongasome and divisome PG synthesis activity.
All together, these analyses will allow determining how molecular interactions between the Mur and divisome complexes influence the balance between PG synthesis dedicated to cell elongation and cell division, whether these interactions are essential for cell survival, and if so, how bacterial cells die when these interactions are disrupted.

De bonnes connaissances et biologie moléculaire, biochimie, purification de protéines recombinantes sont indispensables. Un intérêt pour la biologie structural est demandé.