Staphylococcus aureus est une bactérie pathogène de l'homme impliquée dans de nombreuses infections nosocomiales et communautaires. Comme elle acquiert régulièrement de nouvelles résistances à diverses classes d'antibiotiques, il devient urgent de proposer de nouvelles cibles thérapeutiques. Certains ARN régulateurs (ARNrég) sont importants dans le contrôle de la virulence et de la pathogénie de la bactérie. Au cours de ma thèse, nous avons étudié la fonction d'un ARNrég, appelé SprX (alias RsaOR), exprimé par Staphylococcus aureus. Dans un premier temps, nous avons montré que, dans les souches N315 et HG001, l'expression de SprX varie au cours de la croissance et lors de différentes conditions expérimentales. Dans un second temps, nous avons identifié, par une analyse comparative du protéome, plusieurs protéines dont l'expression est dépendante de SprX et découvert le mécanisme de régulation de l'une de ces protéines par SprX. En effet, SprX interagit avec l'ARNm yabJ-spoVG au niveau des signaux d'initiation de la traduction de SpoVG par un mécanisme antisens qui conduit à la répression de sa traduction. Une boucle accessible de SprX, qui contient un motif riche en C, est impliquée dans la régulation de l'expression de SpoVG et est nécessaire à la modulation de la résistance aux antibiotiques de S. aureus. Nous avons également étudié l'effet des modifications dans la séquence des différentes copies de SprX sur la régulation de l'expression de SpoVG. Ainsi, parmi les deux copies de SprX dans la souche HG001, SprX2 possède une meilleure affinité pour l'ARNm yabJ-spoVG que la copie SprX1. L'ensemble de ces résultats suggèrent que les ARNrég peuvent altérer la résistance des bactéries aux antibiotiques et il est a prévoir que d'autres exemples seront découverts prochainement. / Staphylococcus aureus is a serious human pathogen responsible for both hospital and community-acquired infections. As it becomes alarmingly and increasingly resistant to antibiotics, studies on the mechanisms involved in its virulence is a promising path to develop new treatments. Some, small regulatory RNAs (sRNAs) are important actors in bacterial virulence and pathogenicity. During my thesis, we investigated the functions and the mechanisms of action of a sRNA, named SprX (also known as RsaOR), expressed by the Staphylococcus aureus. First, we demonstrated that, in strains N315 and HG001, SprX expression varies through the growth and among numerous environmental conditions. By a comparative proteomic study, we identified several proteins whose expressions are ‘SprX-dependent’ and elucidated the mechanism of SprX action on one of those proteins. Indeed, SprX interacts specifically with the SpoVG translational initiation site of the yabJ-spoVG mRNA by an antisense mechanism inhibiting its expression. An accessible loop within SprX structure contains a C-rich domain involved in SpoVG regulation and is required and sufficient to modulate bacterial antibiotic resistance. We also studied whether the nucleotides changes between SprX sequence copies could influence SpoVG regulation triggered by SprX. Therefore, among the two copies of SprX in strain HG001, SprX2 has a higher affinity for yabJ-spoVG mRNA than SprX1. Altogether, our results showed that a regulatory RNA can alter bacterial resistance to antibiotics, and additional examples will probably be detected in the near future for more sRNAs and antibiotics.

Borrelia burgdorferi, the causative agent of Lyme disease, exists in a defined enzootic cycle involving Ixodes scapularis ticks and various vertebrates. Humans can serve as an accidental host, if a tick colonized with B. burgdorferi happens to feed on a human. B. burgdorferi are also accidental pathogens: they do not make toxins, or destroy host tissue by other mechanisms. They merely transmit between vector and host to survive. In order to do this, they must effectively sense their current environment, and appropriately alter cellular processes. Understanding the regulatory mechanisms of how B. burgdorferi manages to do this has been a focus of the Stevenson lab for many years. Previous work identified SpoVG as a DNA-binding protein. Although a homologue of this protein had been implicated to serve a regulatory role in other bacteria, the Stevenson lab was the first to demonstrate a function for the protein, both for B. burgdorferi and two other bacteria. Studies contained in this body of work aim to provide insight into regulation of SpoVG by B. burgdorferi as well the impact that it has on gene regulation. By using genetic mutants, we determined that SpoVG is regulated at the levels of transcription and translation in culture by growth rate, temperature, and other regulatory factors. Additionally, we provide evidence that SpoVG regulates its own expression. Numerous genes are under control of SpoVG. Biochemical analyses revealed that SpoVG specifically interacts with DNAs and RNAs associated with genes found to be under its regulatory control. Finally, we provide evidence for SpoVG acting in concert with other known regulatory factors such as other DNA-binding proteins and the cyclic di-nucleotide second messengers cyclic-di-GMP and cyclic-di-AMP. All together, these studies provide insight into how B. burgdorferi broadly regulates cellular processes during different stages of the enzootic cycle. We hypothesize that SpoVG does this through globally manipulating the three-dimensional structure of the bacterial chromosome, and that exactly how SpoVG acts at any given point will be dependent on the other regulatory factors that are also present in the cell.