Pseudomonas aeruginosa (PA) est une bactérie Gram négatif, pathogène opportuniste, impliquée dans un grand nombre d’infections nosocomiales. Cette bactérie est aussi le principal micro-organisme responsable des surinfections broncho-pulmonaires chez les patients atteints de mucoviscidose. Cette prééminence est due en partie à la capacité de PA à former des biofilms, ce qui lui confèrent une résistance exceptionnelle aux antimicrobiens. Au sein de notre laboratoire, une analyse protéomique différentielle a permis de démontrer, en 2004, l’existence d’un protéome spécifique lorsque la bactérie se développe en mode biofilm. Parmi les protéines spécifiquement exprimées en mode biofilm, la protéine hypothétique PA3731 a été plus particulièrement étudiée. Cette protéine est impliquée dans la formation de biofilm, la production de rhamnolipides, la résistance à la tobramycine et la mobilité de type « swarming ». Des recherches bioinformatiques ont montré que le gène pA3731 appartient à un cluster de 4 gènes allant de pA3729 à pA3732 (système BAC), qui pourraient être impliqués dans l’élaboration et/ou la régulation d’un même système protéique. Cette hypothèse a constitué le point de départ de ce travail de thèse. La présente étude a permis de confirmer l’implication du système BAC dans la formation du biofilm, la résistance aux antibiotiques et la production de rhamnolipides chez PA. Les études protéomiques ont mis en évidence l’implication de ce système dans l’expression de la pompe MexEF-OprN, de la porine OprD, et dans la régulation du Quorum Sensing. Des études intéractomiques, menées en parallèle, ont montré une forte interaction entre la protéine PA3731 et PA3732. Ces études ont également permis de valider une forte interaction entre ces protéines et les rhamnolipides. L’ensemble de ces résultats nous permettent d’avancer une hypothèse quant à l’implication du système BAC dans le transport des rhamnolipides vers le milieu extracellulaire. / Pseudomonas aeruginosa (PA) is a Gram-negative bacterium, opportunistic pathogen, involved in a large number of nosocomial infections. This microorganism is also the main infectious agent involved in bronchopulmonary infections in cystic fibrosis patients. This pre-eminence is partly due to the ability of PA to form biofilms, which confers to the bacterial cells an increased resistance to antibiotics. In our laboratory, a differential proteomic analysis allowed to demonstrate in 2004, the existence of a specific proteome when the bacterium grows in the biofilm mode. This study allowed identifying about 40 proteins, specifically accumulated when bacteria adhere to a surface. Among these proteins, the hypothetical protein PA3731 has been particularly investigated. This protein is involved in the biofilm formation, the rhamnolipids production, the resistance to tobramycin and the swarming mobility. Bioinformatic research showed that the pA3731 gene belongs to a cluster of 4 genes ranging from pA3729 to pA3732 (BAC system), which could be involved in the development and / or regulation of the same protein system. This hypothesis was the starting point of this thesis work. The present study confirmed the involvement of the BAC system in the biofilm formation, the antibiotic resistance and the rhamnolipid production in PA. Proteomic studies highlighted the implication of this system in the expression of the MexEF-OprN pump and that of the OprD porin, and in the regulation of Quorum Sensing. Interactomic investigations, conducted in parallel, showed a strong interaction between PA3731 and PA3732 proteins. These studies have also pointed out a strong interaction between these proteins and rhamnolipids. All these results suggest that the BAC system could play a major role in the transfer of rhamnolipids to the extracellular environment.

Proteins and their intricate network of interactions are fundamental to many molecular processes that govern life. Mass spectrometry-based structural proteomics represents a powerful set of techniques for characterizing protein structures and interactions. The last decade has witnessed a large-scale adoption in the application of these techniques toward solving a variety of biological questions. Addressing these questions has often been coincident with the further development of these techniques. Insight into the structures of individual proteins and their interactions with other proteins in a proteome-wide context has been made possible by recent developments in the relatively new field of chemical crosslinking combined with mass spectrometry. In these experiments crosslinking reagents are used to capture protein-protein interactions by forming covalent linkages between proximal amino acid residues. The crosslinked proteins are then enzymatically digested into peptides, and the covalently-coupled crosslinked peptides are identified by mass spectrometry. These identified crosslinked peptides thus provide evidence of interacting regions within or between proteins. In this dissertation the development of tools and methods that facilitate this powerful technique are described. The primary arc of this work follows the development and application of mass spectrometry-based approaches for the identification of protein crosslinks ranging from those which exist endogenously to those which are introduced synthetically. Firstly, the development of a novel strategy for comprehensive determination of naturally occurring protein crosslinks in the form of disulfide bonds is described. Secondly, the application of crosslinking reagents to create synthetic crosslinks in proteins coupled with molecular dynamics simulations is explored in order to structurally characterize the intrinsically disordered tau protein. Thirdly, improvements to a crosslinking-mass spectrometry method for defining a protein-protein interactome in a complex sample is developed. Altogether, these described approaches represent a toolset to allow researchers to access information about protein structure and interactions. / Graduate