RÉSUMÉCsrA est un régulateur post-transcriptionnel contrôlant l’expression et/ou la stabilité des ARNm auxquels il se lie. Il appartient à la famille des régulateurs globaux et contrôle une grande variété de fonctions apparemment non liées. Dans le cas de CsrA, il s’agit principalement de fonctions métaboliques et de fonctions liées aux comportements sociaux des bactéries. Cependant, les limites de la régulation exercée par CsrA sur la physiologie cellulaire sont floues car ses cibles directes semblent abondantes mais difficiles à identifier et, parmi celles-ci, d’autres régulateurs important étendent indirectement l’influence de CsrA.Au cours de cette thèse, nous avons étudié les effets de la délétion du gène csrA à l’échelle de la population bactérienne (1), de la cellule bactérienne (2) et au niveau génétique (3) dans une souche d’Escherichia coli uropathogène. Les infections urinaires font parties des infections bactériennes les plus courantes, présentent un mécanisme de chronicité faisant intervenir la formation de biofilms et E. coli est le principal agent responsable de ces infections. (1) Nous avons montré que l’architecture des biofilms formés par la souche uropathogène d’E. coli était différente de celle décrite pour la souche de laboratoire et que la délétion de csrA affectait fortement cette architecture. (2) Nous avons également montré que le gène csrA n’était pas essentiel mais que sa délétion entraînait un défaut de croissance ainsi qu’une perte d’homéostasie de l’enveloppe. (3) Finalement, nous avons étudié des mutants compensatoires obtenus au travers d’une expérience d’évolution expérimentale partant du mutant ΔcsrA et montré que les différents phénotypes testés étaient restaurés dans ces mutants compensatoires sans qu’aucun changement génétique ne soit identifié.SUMMARYCsrA is a global post-transcriptional regulator controlling the expression/stability of its mRNA targets. It regulates a wide variety of apparently unrelated functions mainly related to metabolism and social behaviors. However, the limits of the regulation mediated by CsrA are not clear as its regulon is large and contains many other regulators extending indirectly its influence.In this thesis, we study the consequences of the deletion of csrA at the population level (1), cellular level (2) and genetic level (3) in an uropathogenic strain of Escherichia coli. Urinary tract infections are among the most frequent bacterial infections, present chronicity mechanism involving biofilms formation and are most of the time caused by E. coli. (1) We showed that the architecture of biofilms formed by the uropathogenic strain is different from that of the lab-strain of E. coli and that CsrA is necessary to generate this particular architecture. (2) We also showed that csrA gene is not essential and that its deletion provokes a growth defect and a loss of the envelope homeostasis. (3) Finally, we studied compensatory mutants selected through experimental evolution and showed that tested phenotypes are restored in these mutants without any genetic change being identified. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Together with the current antibiotic resistance crisis, bacterial persistence appears to play an increasingly important role in the frequent failure of antibiotic treatments. Persister cells are rare bacteria that transiently become drug tolerant, allowing them to survive lethal concentrations of bactericidal antibiotics. Upon antibiotic removal, persister cells are able to resume growth and give rise to a new bacterial population as sensitive to the antibiotic as the original population. Interest in persister cells seriously increased in the past few years as these phenotypic variants were shown to be involved in the recalcitrance of chronic infections, such as tuberculosis and pneumonia and in the well-known biofilm tolerance to antibiotics. Persistence has therefore been extensively studied throughout the last decade, which led to the discovery of large variety of different molecular mechanisms involved in persisters formation. However, the specific physiology of bacterial persisters remains elusive up to now, mainly because of the transient nature and the low frequencies of persister cells in growing bacterial cultures. This work aims to gain a better understanding of the physiology of Escherichia coli persisters by combining population analyses with single-cell observations.In the first part of this thesis, we developed an experimental method allowing for measuring persistence with increased reproducibility. The method was further refined, which allowed us to observe four distinct phases in the ofloxacin time-kill curve, suggesting the existence of a tolerance continuum at the population level at treatment time. Characterization of these four phases notably revealed that the growth rate and the intrinsic antibiotic susceptibility of the strain define the number of surviving cells at the onset of the persistence phase, while persister cells survival mainly relies on active stress responses (SOS and stringent responses in particular).We next investigated the molecular mechanisms underlying the well-known correlation between persistence and the growth rate. Interestingly, we showed that the growth rate determines the number of survival cells at the onset of the persistent phase, whereas it does not affect the death rate of persister cells during antibiotic treatment. Furthermore, slow growth was shown to influence survival to ofloxacin independently of the replication rate, thereby suggesting that target inactivation solely cannot explain this correlation. However, our preliminary data indicate that ppGpp induction upon ofloxacin exposure substantially increases in slow growing bacterial populations, supporting a model in which slow growth would allow bacteria to respond faster to the antibiotic treatment, thereby generating more persisters than fast growing bacterial populations.Finally, both population and single-cell analyses were performed to assess the influence of the SOS response on persistence to ofloxacin. Firstly, population analyses revealed that the SOS response is required for survival of both sensitive and persister cells, but only during recovery, after ofloxacin removal, presumably allowing cells to induce SOS-dependent DNA repair pathways, required to deal with the accumulated ofloxacin-induced DNA lesions. The SOS response therefore appears as a good target for anti-persisters strategies, as shown by the 100-fold decrease in persistence upon co-treatment of a bacterial population with an SOS-inhibitor and ofloxacin. Secondly, single-cell analyses revealed that persister cells sustain similar DNA damages than sensitive cells upon ofloxacin treatment and induce SulA- and SOS-independent filamentation upon antibiotic removal, probably reflecting the presence of remaining cleaved complexes, formed during ofloxacin exposure. Importantly, we showed filamentation to occur in persister cells upon ampicillin treatment as well, thereby suggesting these filaments to be part of a more general survival pathway, which molecular basis remains unknown. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished