SatRNAsPG — Analyse structurale du complexe ribonucléoprotéique FemXAB-NPtRNAsGly de Staphylococcus aureus, essentiel à la synthèse du peptidoglycane et contribuant à la résistance à la méthicilline

Staphylococcus aureus relies on three Fem proteins to synthesize the pentaglycine bridges (Gly5) within its peptidoglycan (PG), a fundamental and specific component of the bacterial cell wall. Our major aim is to understand the molecular mechanisms that underlie the specificity of Fem proteins in recognizing native non-proteinogenic Glycyl-tRNAs (NP-tRNAs), which carry unique determinants. How the three Fem proteins successively recognize these unusual tRNAs to form the pentaglycine is still not known. To achieve this, our project will employ an integrative approach, combining in vivo experiments with advanced structural biology techniques, including Cryo-EM and X-ray crystallography, and chemical synthesis of lipid precursors and tRNA-based inhibitors. By exploring Fem protein-tRNA complex formation, we intend to gain detailed structural insights into the recognition process, shedding light on the specific molecular interactions that govern this pivotal step. The SatRNAsPG project comprises three main tasks, each contributing unique insights into the formation of pentaglycine bridges in S. aureus PG. These tasks are independent yet complementary: (i) Understanding NP-tRNA Recognition: Through in vivo and in vitro analyses, we aim to elucidate how native NP-tRNAs are specifically recognized by Fem proteins, exploring complex organization and RNA modification analysis. (ii) Structural Studies: Investigation of native Fem-NP-tRNA-lipid and glycyl-tRNA synthetase (GlyRS) - NP-tRNA complexes will shed light on molecular interactions underlying pentaglycine bridge formation and NP-tRNA aminoacylation, utilizing cryo-EM and X-ray crystallography techniques. (iii) Impact of tRNA Modifications and dynamics of PG: Analyzing how modifications on native tRNAs influence Gly5 formation and PG composition under infection-related conditions and in clinical strains with varying antibiotic sensitivity will provide insights into the dynamics of this crucial bacterial barrier. The network lies in the combination of complementary expertise including RNA biology and deep mechanistic insights in the study of staphylococcal pathogenesis and virulence, development of MS approaches linked to RNA biology (P1: S. Marzi); tRNA and lipid biochemistry and chemical synthesis of tRNA and lipid derived substrates and inhibitors (P2: M. Fonvielle); state of the art structural analysis of RNA-protein complexes (P3, B. Klaholz). This synergy represents a unique opportunity to unite biochemical, genetics, structural and chemistry, in a common effort to take up this challenge of determining how the peptidoglycan if formed and is regulated in this major human pathogen and to open new avenues for therapeutic applications.