RNA modifications in Staphylococcus aureus during stress responses, antibiotic treatments and virulence: impact on translation and its regulation – SaRNAmod

RNA modifications are involved in numerous biological processes and are present in all classes of RNA. These modifications are constitutive or modulated in response to adaptive processes and can impact RNA base pairing formation, protein recognition, RNA structure and stability. However, their roles in stress, environmental adaptation responses and infections caused by pathogenic bacteria, have just started to be appreciated. We make the assumption that RNA modifications in bacteria may be more ubiquitous and physiologically important than presently suspected. With the development of modern technologies in mass spectrometry and deep sequencing, and the possibility to test the impact of RNA modifications on the infection process, the three partners decided to join their forces and complementary expertise in order to determine the contribution of post-transcriptional modifications in non-coding and stable RNAs in translation and its regulation at a global scale in one of the major human pathogen, Staphylococcus aureus.
The SaRNAmod project is based on three interconnected tasks and on a comparative study that will be performed on two specific strains, the methicillin-susceptible and genetically tractable HG001 strain and the methicillin-resistant USA300 clinical strain (i) We first aim at establishing the global profiles of post-transcriptional modifications in stable RNAs including tRNAs, ribosomal RNAs, and some selected regulatory RNAs acting as translational regulators (sRNA). This mapping will be done using state-of-the art methodologies including mass spectrometry (P1) and deep sequencing P2). Purification of specific RNAs for precise characterization of their modifications is available. (ii) We will analyze the dynamic-regulated modifications in tRNAs and rRNAs in response to specific stresses encountered during infection (i.e. oxidative and nitric oxide sensing) and to anti-toxinic antibiotic treatments. We will make use of sophisticated approaches such as ribosome profiling, to decipher the impact of modification deregulation on the decoding process. (iii) Using specific mutations at modifier enzymes, we will analyze their impact on S. aureus physiology and pathogenesis and relate the expression of specific modification enzymes with specific infections (P3).
The outcomes of the project are expected to generate major breakthroughs: (i) at the technical level with the development of new methods of mass spectrometry and deep sequencing to map the set of modified bases of any RNA (particularly for the detection of pseudouridines); (ii) at the basic research level, with complete mapping of the modified bases of S. aureus non-coding RNAs, their functional impact on the physiology and antibiotic resistance mechanisms, and characterization of modification enzymes as well as proteins regulating the modifications of RNAs; (iii) at the medical level, the identification of new targets for the search for strategies to interfere with virulence and / or bacterial growth.
The network lies in the combination of complementary expertise including RNA biology and deep mechanistic insights in the translation process, development of MS approaches linked to RNA biology (P1: S. Marzi), detection of RNA modifications by deep sequencing and biological functions of their machineries (P2: Y. Motorin), the study of staphylococcal pathogenesis, virulence and resistance to antibiotics (P3: F. Vandenesch). This synergy represents a unique opportunity to unite biochemical, genetics, structural, proteomics and transcriptomics skills, infection models and patient isolated strains, in a common effort to take up this challenge of determining the epitranscriptome and its regulation in this major human pathogen and to open new avenues for therapeutic applications.