Translation and its control in Staphylococcus aureus: consequences on virulence and stress responses – RIBOSTAPH

Translational control has become a major attention during the last decade. In bacteria, the determination of the structures of ribosomes led to a better understanding of ribosome functioning and dynamics. New development of methodologies enabling unbiased characterization of the translatome, which refers to the entirety of mRNAs associated with ribosome for protein synthesis, has provided a global picture of translational regulatory events in bacteria. Most of these works were carried out in the model Gram-negative Escherichia coli while less information exists for Gram-positive bacteria, which have evolved different machineries and mechanisms to regulate gene expression. In this consortium, the three partners will join their forces and complementary expertise to determine the contribution of translational regulatory processes to gene expression at a global scale in the major human pathogen, Staphylococcus aureus and to analyze at the molecular level the diversity of regulatory mechanisms. S. aureus is an opportunistic pathogen responsible for severe nosocomial and community-acquired infections, and has evolved numerous strategies to regulate the synthesis of its multiple virulence factors in response to the host and environmental changes. Besides transcriptional regulatory proteins, partners 1 and 3 have demonstrated that RNAs are key effectors in adaptive processes and co-regulate mRNA translation in response to metabolic burden and stresses. Moreover, several mRNAs involved in pathogenesis adopt particular structure features in their 5’ and 3’ untranslated regions (UTR) that modulate their translation rates, and the ribosome should be assisted by trans-acting factors to initiate translation of mRNAs carrying structured 5’UTRs.
The objectives of the project are (i) to define the contribution of the translational control in the regulatory circuits that respond to two major stress responses encountered during infection (oxidative and NO sensing) and to anti-toxinic antibiotic (inhibition of toxins at the translational level) treatments using ribosome profiling in methicillin-sensitive and -resistant clinical strains, (ii) to gain knowledge on the translation machinery and the mechanism of action of trans-acting factors modifying the recognition of structured mRNAs by the ribosome at the initiation step, and (iii) to identify translational regulatory events that affect bacterial growth and pathogenesis. Recently, homogenous preparation of S. aureus ribosomes produces reproducible crystals (partner 2) and to the first cryo-electron microscopy maps at 3.6-3.9 Å of S. aureus 70S ribosome and of a stalled 100S ribosome dimer mediated by the small hibernation-promoting factor (partners 1 and 2). These ribosome structures will be the basis for further investigations of functionally relevant regulatory ribosomal complexes. The outcomes of the project are multiple: (i) it will provide a detailed analysis of the initiation process of structured and regulated mRNAs in S. aureus, a key step for control; (ii) it will characterize the differences existing in the translation and regulation between Gram-negative and Gram-positive bacteria; and (iii) it will shed light into the mechanism of action of anti-toxinic antibiotics, and on regulatory pathways responding to major stresses encountered during the initial phase of the infection.
This network lies in the combination of complementary expertise including RNA biology and deep mechanistic insights of post-transcriptional mechanisms (Partner 1: P. Romby), structural biology of the translation machinery (Partner 2, M. Yusupov; Partner 1, Y. Hashem), and S. aureus microbial pathogenesis (partner 3, F. Vandenesch). This synergy represents a unique opportunity to unite biochemical, genetics, and structural skills, in a common effort to take up this challenge of determining the translatome and its regulation in this major human pathogen and to open new avenues for therapeutic applications.