Staphylococcus aureus, pathogène opportuniste de l’Homme, est un réel problème de santé publique du fait de l’émergence de souches virulentes résistantes à différentes classes d’antibiotiques. La compréhension des mécanismes moléculaires impliqués dans sa virulence est indispensable pour la mise au point de nouveaux agents anti-infectieux. Il est apparu récemment que certains ARN régulateurs (ARNrég) de S. aureus sont impliqués dans l’expression de facteurs de virulence. Mon projet de thèse a porté sur l’étude d’un ARNrég de fonction inconnue, SprB. Des expériences préliminaires à ce travail ont montré une variation du niveau d’expression de la protéine SasG en absence de l’ARN SprB. Dans une première partie de ce travail, j’ai cherché à confirmer expérimentalement le lien entre l’ARN SprB et la protéine SasG. Les niveaux d’expression de la protéine SasG ont été observés dans différents contextes génétiques mutants pour SprB et/ou différents facteurs de régulation de S. aureus. L’ensemble des résultats obtenus suggère le rôle indirect de SprB sur l’expression de SasG. SprB semble impliqué dans un réseau global de régulations qui gouverne l’expression de la protéine SasG. La deuxième partie de cette étude qui porte sur l’identification des cibles directes de SprB a été appréhendée à l’aide d’outils prédictifs. Parmi les cibles potentielles révélées par ce criblage in silico, notre intérêt s’est porté sur la sous-unité Opp4F du transporteur ABC Opp4. J’ai montré que l’ARN SprB empêche, in vitro, la fixation du ribosome sur l’ARN Opp4F et diminuerait potentiellement la synthèse de la sous unité Opp4F. Cela pourrait avoir un impact dans la régulation du métabolisme énergétique de S. aureus. L’ensemble de ces résultats suggère une implication de l’ARN SprB dans plusieurs processus physiologiques de S. aureus aussi cruciaux que la régulation de l’expression de gènes de virulence et le métabolisme énergétique de la bactérie. / Staphylococcus aureus is a major human opportunist pathogen causing a wide spectrum of nosocomial and community-associated infections. The emergence of strains resistant to most currently available antibiotics presents a serious public health threat. Therefore, there is an urgent need for new treatment. The development of such arsenal requires the elucidation of the molecular mechanisms involved in bacterial virulence. sRNA, represent a new and promising field of investigation, because of its implication in main cellular processes such as virulence factor regulation. My PhD thesis focused on the study of SprB : a small regulatory RNA (sRNA) of unknown function. Preliminary results have shown that the absence of SprB leads to differences in the of SasG protein level. First, I investigate the link between SprB RNA and SasG protein. Therefore, SasG protein levels have been studied in several genetic backgrounds, parental or mutant for SprB and/or global regulation factors. Taken together, our findings suggest an indirect effect of SprB on SasG regulation. In addition, SprB seems to be implicated in global bacterial regulation network which governs SasG protein expression. In the second part of my thesis, we tried to identify direct targets of SprB through in silico screening. Among a list of the potential targets revealed, we focused on Opp4F, sub-unit of the ABC transporter Opp4. We demonstrated, in vitro, that SprB RNA is able to inhibit the binding of the ribosome on Opp4F mRNA, potentially decreasing the synthesis of Opp4F sub-unit. Therefore, Opp4F is not or less produced which could have serious impact on the regulation of energetic metabolism. Altogether, the results obtained highlighted the potential role of SprB in several crucial physiological processes such as regulation of energetic metabolism and regulation of bacterial virulence.

Staphylococcus aureus encodes for a secreted hyaluronidase, hysA. Hyaluronidases are bacterial enzymes that cleave hyaluronic acid (HA) at the β-1,4 glycosidic bond, yielding unsaturated disaccharides. Initially, little was known about the regulation of this enzyme as well as its roles in S. aureus physiology and pathogenesis. The goal of this dissertation was to determine the regulation of hysA, and to determine the biological and physiological roles of this enzyme. Studies presented in Chapter II focus on determining the regulation of hysA and role of hysA in S. aureus pathogenesis. By screening the Nebraska Transposon Mutant Library (NTML) we identified 8 mutations that significantly altered HysA activity. Further analysis revealed that CodY directly represses hysA by binding to the CodY consensus binding sequence upstream of the hysA translational start site. Additionally, we found that a hysA mutant was attenuated in a neutropenic murine pneumonia model of infection and that there was reduced degradation of HA in the lungs of mice infected with the hysA mutant compared to wildtype by immunofluorescence. Studies presented in Chapter III focus on examining if HA can be incorporated into the S. aureus biofilm matrix and if HysA is involved in biofilm dispersal. We show that HA is incorporated into the biofilm matrix both in vitro and in vivo by confocal microscopy and HA ELISA. Additionally, we found that HA can enhance biofilm formation of the hysA mutant as well as other staphylococcal species in vitro. We show that induction of hysA can prevent biofilm formation in the presence of HA and that exogenous addition of purified HysA can disperse established HA containing biofilms. Finally, we found that a hysA mutant has reduced dissemination in an implant-associated infection model. Together these studies support our hypothesis that HA is incorporated into staphylococcal biofilms and that HysA is involved in dispersing S. aureus from the biofilm.

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Staphylococcus aureus (S. aureus) is a frequent cause of infections and sepsis in animals and humans worldwide. Staphylococcal enterotoxins and toxic shock syndrome toxin-1 are bacterial superantigens (SAgs) produced by S. aureus that simultaneously bind to T cell receptor (TCR) and the major histocompatibility complex (MHC) class II, leading to extensive T cell stimulation, release of cytokines, consequently resulting in toxic shock and immunosuppression. In this study, we generated mutant SAgs by introducing alanine substitution at residues involved in interaction with MHC class II and TCR binding and demonstrated attenuation of toxicity in vitro and in vivo. An immunization with mutant SAgs elicits production of neutralizing antibodies against wild type SAgs and protected animals from S. aureus peritonitis at a lethal dose. These results suggest that mutant SAgs will be useful to develop a novel vaccine against S. aureus infections.

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