Staphylococcus aureus utilise trois protéines Fem pour synthétiser les ponts pentaglycine (Gly5) du peptidoglycane (PG), un composant fondamental et spécifique de la paroi cellulaire bactérienne. Notre objectif principal est de comprendre les mécanismes moléculaires qui sous-tendent la spécificité des protéines Fem dans la reconnaissance des Glycyl-ARNt natifs non protéinogènes (ARNtNP), qui portent des déterminants uniques. Le mécanisme par lequel les trois protéines Fem reconnaissent successivement ces ARNt inhabituels pour former la pentaglycine, n'est pas encore connu. Le projet utilisera une approche intégrative combinant des expériences in vivo et des techniques avancées de biologie structurale, incluant la cryo-EM et la cristallographie des rayons X, ainsi que la synthèse chimique de précurseurs lipidiques et d'inhibiteurs à base d'ARNt. En explorant la formation de complexes protéine-Fem-ARNt, nous avons l'intention d'obtenir des informations structurales détaillées sur le processus de reconnaissance, éclairant les interactions moléculaires spécifiques qui régissent cette étape cruciale. Le projet SatRNAsPG comprend trois tâches principales, contribuant chacune à des perspectives uniques sur la formation des ponts de pentaglycine dans le PG de S. aureus. Ces tâches sont indépendantes mais complémentaires : (i) Compréhension de la reconnaissance des ARNtNP : À travers des analyses in vivo et in vitro, nous visons à élucider comment les ARNtNP natifs sont spécifiquement reconnus par les protéines Fem, en explorant l'organisation complexe et l'analyse des modifications de l'ARN. (ii) Études structurales : L'investigation des complexes natifs Fem-ARNtNP-lipide et glycyl-ARNt synthétase (GlyRS) - ARNtNP éclairera les interactions moléculaires sous-jacentes à la formation du pont de pentaglycine et à l'aminoacylation de l'ARNtNP, en utilisant des techniques de cryo-EM et de cristallographie aux rayons X. (iii) Impact des modifications de l'ARNt et de la dynamique du PG : L'analyse de la manière dont les modifications sur les ARNt natifs influencent la formation de Gly5 et la composition du PG dans des conditions liées à l'infection et dans des souches cliniques présentant une sensibilité aux antibiotiques variable fournira des informations sur la dynamique de cette barrière bactérienne cruciale. Le réseau repose sur la combinaison d'expertises complémentaires, comprenant la biologie de l'ARN et des connaissances mécanistiques approfondies dans l'étude de la pathogenèse et de la virulence staphylococciques, le développement d'approches MS liées à la biologie de l'ARN (P1: S. Marzi); la biochimie des ARNt et des lipides et la synthèse chimique de substrats et d'inhibiteurs dérivés de l'ARNt et des lipides (P2: M. Fonvielle); l'analyse structurale de pointe des complexes ARN-protéine (P3, B. Klaholz). Cette synergie représente une opportunité unique d'unir la biochimie, la génétique, la structure et la chimie, dans un effort commun pour relever ce défi de déterminer comment le peptidoglycane est formé et régulé chez ce pathogène humain majeur et d'ouvrir de nouvelles voies pour des applications thérapeutiques.

The increase of antibiotic resistance is becoming a serious health threat worldwide, but the number of newly released antibiotics remains low. One way to address this problem is to target resistance proteins to restore the efficacy of antibiotics. ß-lactams are the most successful class of antibiotic drugs but they are vulnerable to inactivation by a growing class of ß-lactamases. For a long time, medically relevant ß-lactamases encompassed only serine ß-lactamases (SBLs) for which several inhibitors have now been developed. However, more recently metallo-ß-lactamases (MBLs) emerged as a global threat for which no inhibitor has been developed yet. However, their increasing importance in drug failure is a strong incentive to target them. Here, we propose to develop RNase resistant inhibitory aptamers targeting both types of ß-lactamases using an innovative pipeline combining the use of in vitro selection (SELEX) in tandem with microfluidic-assisted ultrahigh-throughput screening and next generation sequencing (NGS). Indeed, whereas SELEX will allow to enrich aptamers from very large libraries in sequence displaying the potential to bind the target protein, microfluidic screening will allow refining this pre-selection by searching for those aptamers really able to inhibit enzyme activity. Finally, using NGS and bioinformatics will allow analyzing the whole process at once to rapidly identify most promising sequences. Most key elements of this pipeline have already been validated during preliminary experiments, allowing the majority of the project to be focused on the actual development of new aptamers able to inhibit three medically relevant extracellular enzymes: the metallo-ß-lactamase NDM-1 from Klebsiella pneumoniae and the serine-ß-lactamases BlaZ and the protease Aureolysin, both from Staphylococcus aureus. By the end of this project we will not only have validated this new technology through the actual development of efficient aptamers, but we will also have developed new prototypes of drugs that could later be optimized and enter into the arsenal required to fight bacterial infection that are foreseen to be a major thread surpassing every other disease in the up-coming decades. Moreover, the potential application spectrum of the DIRA pipeline refined and used here will be much wider than antibiotic discovery as discussed in the present proposal.

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.

Ribonucleoprotein particles (RNPs) are made of RNA associated with proteins and play a central role in biological systems (maintenance of cellular homeostasis, establishment of infectious or pathological processes). The identification of the components of these RNPs has exploded over the past decade thanks to the use of high-throughput analytical approaches. The fine characterization of the interaction of the components of these RNPs then requires the analysis of a large number of mutants. While several high-throughput methodologies have been developed for the analysis of RNA mutant libraries, progress has been scarcer on the protein side. To fill this gap, we propose "SURF", a highly multidisciplinary project aiming at developing a new chemistry for the efficient capture of target RNAs on the surface of water-in-oil droplets produced, manipulated, and analyzed at rates of several million per hour in microfluidic devices. Gene libraries encoding mutants of the studied protein fused to a fluorescent domain will be expressed in vitro at a rate of one mutant (produced in large numbers of copies) per droplet. Thus, a mutant able to interact with the target RNA sequence will lead to a relocation of the fluorescent protein on the surface of the droplet, making it easily discriminable from a drop containing a mutant unable to recognize its target (fluorescence remaining diffuse in the droplet). Applied and validated with various biological models, this technology will not only allow to finely characterize the formation of RNPs, but also to reprogram their specificity, and even to identify molecules able to modulate this interaction. Finally, this project will also be an opportunity to explore surfactants made of alternative chemistries having a lower impact on the environment.

Neutrophils are the most abundant leukocytes and represent the first line of defence against pathogenic bacteria. Their antimicrobial activity relies on their ability to trigger a myeloperoxidase (MPO)-dependent oxidative burst. Concomitant to the well-documented production of hypochlorous acid (HOCl), other highly oxidizing MPO products have been proposed to form upon bacterial infection. Among the potential candidates, evidence for the implication of N-chlorotaurine (N-ChT) and urate hydroperoxide (UA-OOH) has been only obtained in vitro. N-ChT was shown to be generated by the reaction between taurine (an abundant amino acid derivative) and HOCl, while UA-OOH was proposed to be produced by the MPO-catalysed oxidation of urate (derived from purines catabolism) in an oxidative stress situation. NeutrOX is a multidisciplinary project gathering analytical and organic chemists, microbiologists and immunologists. In this proposal, we propose to decipher for the first time the in vivo antimicrobial activity of these candidates MPO-controlled pro-oxidants using Salmonella as a model. By following the Salmonella-neutrophil interactions, NeutrOX aims at investigating two fundamental questions related to the neutrophil-mediated oxidative burst: 1) Are N-ChT and UA-OOH effective MPO-derived oxidants forming during Salmonella infection? 2) How does Salmonella respond to these identified oxidants? First, following chemical synthesis and purification of N-ChT and UA-OOH, robust analytical and spectrometric methods will be set up to detect these species in varying biological media. This broad array of optimized techniques will be applied afterwards to monitor N-ChT and UA-OOH formation in human neutrophils infected by Salmonella. Second, we will characterize the in vitro antimicrobial activities of N-ChT and UA-OOH against Salmonella and we will identify bacterial MPO-mediated-stress response, an important step to better understand the pathogen adaptation and survival pathways. We will benefit from this molecular study to develop biological detection probes called bacterial “biosensors” (promoters fused to a gene encoded for fluorescent protein) whose expressions are modulated by the presence of each MPO-derived oxidant. These biosensors will represent new and efficient tools for the in vivo detection of such oxidants at a single-bacterial level and will be compared to the analytical and spectrometric methods for N-ChT and UA-OOH detection. The ultimate aim of NeutrOX will be to investigate the in vivo characterization and relevance of newly identified MPO-products in a mouse model of salmonellosis and to determine their respective contributions to Salmonella infection host response. Information and concepts obtained during this project will provide novel insights into the importance of the oxidative burst during the host innate immune response. Moreover, understanding how Salmonella cope with the MPO-derived oxidants could provide information for potential drug targets identification and open the way to new antibacterial compounds design.