Brucella abortus is a bacterial species that infects cattle, elk, and bison herds worldwide and is a causative agent of brucellosis. B. abortus is a common form of zoonosis, as incidental spillover into the human population results in millions of infections annually. Current treatment options are limited to culling infected animals and treating humans with a rigorous antibiotic regimen, which still results in up to a 30% relapse rate. Detection of the pathogen is difficult due to the replicative niche residing within the host's immune cells, specifically macrophages and dendritic cells. Numerous small regulatory RNAs (sRNAs) were found to be expressed by B. abortus, and it was hypothesized that they may be important for virulence. One sRNA, when deleted, was shown to be linked to outer membrane stress resistance and was named MssR (membrane sensitivity sRNA). When the ΔmssR strain was tested in both macrophage and mouse models of infection, there were no virulence defects. Additionally, proteomic and transcriptomic studies of the ΔmssR strain showed very few dysregulated targets. Expression of mssR was tested under numerous biologically relevant conditions, and it was shown to be expressed significantly more during exponential phase of growth, compared to stationary phase. Initial microscopical analysis of mutant cells after treatment with sodium dodecyl sulfate (SDS_ did not reveal any morphological differences. It is unknown what contributes to the observed phenotypes and additional experiments are required to determine what is causing the perturbations in the outer membrane of the ΔmssR strain. / Master of Science / Brucella abortus is a bacterial species that causes the disease brucellosis in cattle and humans worldwide. To understand how B. abortus establishes infection, we are studying how the bacteria control the expression of genes during the process of infection. One method of bacterial gene regulation is the use of small regulatory RNAs (sRNAs). These small transcripts are similar to mRNAs but are shorter in length and typically do not encode for a protein. One such sRNA in B. abortus was shown to be linked to sensitivity to outer membrane stress and was named Membrane Sensitivity sRNA (MssR). After engineering a strain of B. abortus that does not produce MssR, there were no differences in the ability of the bacteria to infect macrophages or mice. Additionally, there were no noticable differences in the structure of the bacterial cells. When sRNAs regulate gene expression, differences can be seen at the mRNA and protein levels when the sRNAs are deleted. Very few targets were found be dysregulated at the transcript and protein level within the ΔmssR mutant. It is unknown what is causing the mutant to be more sensitive to outer membrane perturbations and additional tests are necessary to determine how MssR is linked to this phenotype.

Brucella abortus is a facultative, intracellular, zoonotic pathogen that resides inside macrophages during infection. This is a specialized niche where B. abortus encounters various stresses, such as acidic conditions and reactive oxygen species, as it navigates through the macrophage. In order to survive this harsh environment, B. abortus utilizes post-transcriptional regulation through the use of small regulatory RNAs (sRNAs). sRNAs bind to messenger RNA (mRNA) targets via complementary base pairing. sRNAs are a class of regulatory molecules in bacteria that elicit rapid post-transcriptional regulation. sRNA-mRNA binding can positively or negatively influence gene expression. Positive regulation can occur through sRNA binding to protect the mRNA from RNases. sRNA binding can also alleviate the secondary structure and reveal the ribosomal binding site. Alternatively, sRNA-mRNA interactions can have negative consequences on gene expression through degradation via RNases or sRNA binding can occlude the ribosomal binding site. Although some sRNAs have been discovered in B. abortus, few have been characterized in regards to virulence. In this study, B. abortus was stressed in conditions relevant to the macrophage, including, including low pH, oxidative stress, and nutrient limitation. Transcriptomic analysis revealed high levels of transcripts in intergenic regions, a hallmark of sRNAs, which led to the discovery of VcrS for virulence and cell wall regulating sRNA. A ΔvcrS was engineered and this mutant was used to infect both naïve murine macrophages, as well as BALB/c mice. Both virulence studies demonstrated significantly decreased bacterial recovery of ΔvcrS compared to the wildtype strain. Quantitative proteomics revealed that one protein, BAB1_1454, is 30-fold over-produced in ΔvcrS compared to wildtype. This essential protein encodes MurF, which catalyzes the final cytoplasmic step of generating the mura-pentapeptide precursor for peptidoglycan synthesis. VcrS is hypothesized to interact with murF mRNA and interfere with translation initiation. Sequence data indicates a putative 6 nucleotide motif in VcrS that has complementarity to the ribosomal binding site of murF. Identification of the binding site and further characterization of VcrS will showcase the importance of sRNA regulation in the virulence of B. abortus. / Master of Science / Brucella abortus is a bacterial pathogen that primarily infects cattle but is also transmitted to humans. Human disease most commonly results from the consumption of unpasteurized milk and milk products. Human brucellosis has very limited treatment options, with a high incidence of disease relapse. B. abortus survives and replicates within immune cells, which create a harsh environment. However, the bacteria are able to sense and adapt to survive and replicate within these immune cells, maintaining a chronic infection. A better understanding of the adaptation process B. abortus utilizes to survive within the human host can lead to improvement of treatment options. The present work characterizes a novel regulatory small RNA- VcrS, which was found required for survival and replication inside immune cells

Vibrio splendidus est une bacterie Gram négative du genre Vibrio. Les Vibrios sont des bactéries motiles, en forme de virgule, vivant essentiellement dans les ecosystèmes marins. Depuis une quinzaine d'années, Vibrio splendidus a été associé a des épisodes estivaux de mortalité de naissains d'huitres en France, jusqu'à compromettre l'économie ostréicole. Cependant on sait encore peu de choses sur l’évolution de cette espèce, sa capacité d'adaptation, et ses mécanismes de virulence. De nombreux travaux au cours de ces dernières années ont souligné l'importance des petits ARN non codants (sRNA) dans la régulation des gènes bactériens, en particulier en réponse à l'environnement ainsi qu'au cours de la pathogenèse. Nous avons réalisé une étude de transcriptomique par séquençage à haut débit afin d'établir un répertoire des sRNA chez V. splendidus. Cette analyse transcriptomique nous a permis d'identifier des centaines de sRNAs putatifs, dont la majorité sont spécifiques de cette espèce, une minorité résultant de transferts horizontaux avec d'autres bactéries marines, comme les Shewanellaceae. Les données transcriptomiques ont montré la présence chez V. splendidus de 4 copies du petit ARN CsrB, contrairement aux autres Vibrios qui en ont trois ou deux. Nous avons pu montrer que ces CsrBs jouent un role important dans la physiologie cellulaire en contrôlant la production et/ou la sécrétion de deux proteases jouant un role dans la virulence. De plus, nous avons montré que le réseau de régulation impliquant ces CsrBs est significativement différent des autres Vibrios, indiquant que la présence d'une copie supplémentaire a sans doute entraine un remodelage de ce réseau de régulation. / Vibrio splendidus is a Gram negative bacterium of the genus Vibrio. The Vibrios are motile, comma-shaped bacteria, living mainly in marine ecosystems. Over the past fifteenyears, Vibrio splendidus has been associated with oyster summer mortality episodes inFrance, generating heavy economic losses. However, still little is known about the evolutionof this species, its adaptive capacity, and the mechanisms of virulence. Many works in recentyears have stressed the importance of small noncoding RNAs (sRNAs) in bacterial generegulation, particularly in response to the environment as well as during pathogenesis. Wedid a transcriptomic study by high-throughput sequencing in order to explore the sRNArepertoire in V. splendidus. This transcriptomic analysis allowed us to identify hundreds ofputative sRNAs, the majority of which being specific of this species, a minority resulting fromhorizontal transfers with other marine bacteria, such as the Shewanellaceae. Transcriptomicdata showed the presence of 4 copies of the small RNA CsrB in V. splendidus, unlike otherVibrios which have two or three. We have shown that these CsrBs play an important role incell physiology by controlling the production and/or secretion of two proteases playing a rolein virulence. In addition, we have shown that the regulatory network involving these CsrBs issignificantly different from other Vibrios, indicating that the presence of an extra copy hasindeed resulted in a reshaping of the regulatory network.

Les ARN double brin (ARNdb) sont les molécules clés initiatrices du RNA silencing, à partir desquelles les différentes classes de petits ARN (sRNAs), conférant la séquence spécificité de ce mécanisme, vont être produit. Chez la plante modèle Arabidopsis thaliana, le clivage des divers ARNdb en sRNAs est opéré par quatre enzymes de type RNase III, nommées DCL1 à DCL4, dont l’activité peut être assistée par des protéines fixant l’ARNdb (DRBs). Au cours de cette thèse, j’ai pu caractériser la fonction d’une nouvelle DRB, DRB7.2. Les résultats obtenus m’ont permis de démontrer que cette protéine régule la production d’une classe particulière de sRNAs endogènes, les endoIR-siRNAs, en séquestrant spécifiquement leurs précurseurs ARNdb, inhibant ainsi leur clivage par les différents DCLs. En parallèle, j’ai également développé des outils moléculaires afin d’étudier le mode d’action du DCL le plus polyvalent chez les plantes, DCL4. La caractérisation détaillée de ces outils a permis de révéler le rôle clé de déterminant structuraux distinct (protéiques ou nucléiques) impliqués dans la spécificité de reconnaissance et de clivage des divers substrats ARNdb par cette enzyme. / RNA silencing is initiated by double-stranded RNA (dsRNA) molecules that will be processed into various classes of small RNAs (sRNAs), which confer the sequence-specificity of this mechanism. In the model plant Arabidopsis thaliana, dsRNA processing is mediated by four distinct RNaseIII-like enzymes, named DCL1 to DCL4, which can be assisted by dsRNA-binding proteins (DRBs). During my PhD, I was able to characterize in details the function of a new DRB protein, DRB7.2. Our results revealed that this protein regulates the accumulation of a specific class of endogenous sRNAs, the endoIR-siRNAs, by selectively sequestering their dsRNA precursors and inhibiting their cleavage by the DCLs. In parallel, I also developed molecular tools to study the mode of action of the most versatile DCL in plants, DCL4. Detailed characterization of these tools revealed key roles of distinct structural determinants (at the protein or RNA level), implicated in the specificity and cleavage efficiency of the various dsRNA susbtrates by DCL4.

Knowledge of the processes of RNA interference, the regulation of gene expression by small RNAs (sRNAs), has grown at an unprecedented rate over the last 30 years. Some of the findings were literally revolutionary, as they revealed events that overturned many long-held notions. Many phenomena have been shown to be highly conserved and common to organisms of different species, but others are specific to certain lineages or have not yet been fully explored. There is also a lack of knowledge about the interconnection of numerous pathways - for example between silencing at the transcriptional (TGS, leading to the promoter methylation) and post-transcriptional levels (PTGS, affecting mRNA stability or translation). The present work summarizes the findings of two published and two unpublished works and attempts to describe some of the less known sites of RNA interference using various plant model organisms. Research on Solanum tuberosum transgenic lines has revealed the ability of 5-azacytidine to restore the expression of transcriptionally silenced transgenes at the whole plant level. De novo regeneration from leaves of such plants can lead to re-silencing of reactivated transgenes and thus serves as a selection method to exclude lines prone to spontaneous silencing. The nature of changes in the...

The bacterial RNA binding protein Hfq is a key component for bacterial sRNA mediated riboregulation of mRNA expression. A kinetic and thermodynamic analysis of Hfq binding to its sRNA targets DsrA, RprA, and OxyS, and to its mRNA target rpoS was carried out. The ability of Hfq to significantly enhance the stability of the DsrA-rpoS and RprA-rpoS complex was demonstrated, and the entire untranslated leader region of rpoS was shown to be important for Hfq binding and in Hfq facilitated sRNA-mRNA duplex formation. Hfq was not shown to enhance OxyS binding to rpoS. DsrA and OxyS were shown to bind mostly to the proximal surface region of Hfq, while RprA bound to both proximal and distal surface regions. The rpoS leader region was shown to possess at least two distinct Hfq binding sites, with one site binding the proximal region and the other to the distal region of Hfq. These sites were shown to be important for Hfq to stimulate DsrA-rpoS binding. The outer-circumference region and the C-terminal tail of Hfq does not play a major role in binding DsrA, RprA, OxyS and rpoS, and in stimulating DsrA-rpoS binding. Evidence was obtained implicating Hfq to bind DsrA, RprA, OxyS, and oligo rA18 in a 1:1 protein to RNA stoichiometry. Binding properties of Hfq to E. coli genomic DNA were examined. Double stranded DNA was shown to bind mostly on the distal surface region and the C-terminal tail of Hfq with an affinity 10 fold less than Hfq targeted RNA. Single stranded DNA binds Hfq more tightly than double stranded DNA and binding seems to be sequence specific. Evidence indicates Hfq binds certain sequences of the E. coli genome.

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L'identification des interactions survenant au niveau moléculaire joue un rôle crucial pour la compréhension du vivant. L'objectif de ce travail a consisté à développer des méthodes permettant de modéliser et de prédire ces interactions pour le métabolisme et la régulation de la transcription. Nous nous sommes basés pour cela sur la modélisation de ces systèmes sous la forme de graphes et d'automates. Nous avons dans un premier temps développé une méthode permettant de tester et de prédire la distribution du flux au sein d'un réseau métabolique en permettant la formulation d'une à plusieurs contraintes. Nous montrons que la prise en compte des données biologiques par cette méthode permet de mieux reproduire certains phénotypes observés in vivo pour notre modèle d'étude du métabolisme énergétique du parasite Trypanosoma brucei. Les résultats obtenus ont ainsi permis de fournir des éléments d'explication pour comprendre la flexibilité du flux de ce métabolisme, qui étaient cohérentes avec les données expérimentales. Dans un second temps, nous nous sommes intéressés à une catégorie particulière d'ARN non codants appelés sRNAs, qui sont impliqués dans la régulation de la réponse cellulaire aux variations environnementales. Nous avons développé une approche permettant de mieux prédire les interactions qu'ils effectuent avec d'autres ARN en nous basant sur une prédiction des interactions, une analyse par enrichissement du contexte biologique de ces cibles, et en développant un système de visualisation spécialement adapté à la manipulation de ces données. Nous avons appliqué notre méthode pour l'étude des sRNAs de la bactérie Escherichia coli. Les prédictions réalisées sont apparues être en accord avec les données expérimentales disponibles, et ont permis de proposer plusieurs nouvelles cibles candidates. / The identification of the interactions occurring at the molecular level is crucial to understand the life process. The aim of this work was to develop methods to model and to predict these interactions for the metabolism and the regulation of transcription. We modeled these systems by graphs and automata.Firstly, we developed a method to test and to predict the flux distribution in a metabolic network, which consider the formulation of several constraints. We showed that this method can better mimic the in vivo phenotype of the energy metabolism of the parasite Trypanosoma brucei. The results enabled to provide a good explanation of the metabolic flux flexibility, which were consistent with the experimental data. Secondly, we have considered a particular class of non-coding RNAs called sRNAs, which are involved in the regulation of the cellular response to environmental changes. We developed an approach to better predict their interactions with other RNAs based on the interaction prediction, an enrichment analysis, and by developing a visualization system adapted to the manipulation of these data. We applied our method to the study of the sRNAs interactions within the bacteria Escherichia coli. The predictions were in agreement with the available experimental data, and helped to propose several new target candidates.

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