Pseudomonas aeruginosa est un bacille opportuniste responsable d'infections graves. Sa pathogénicité repose sur de nombreux facteurs de virulence dont le système de sécrétion de type III (SSTT). Ce système est activé par le contact de la bactérie avec une cellule ou une déplétion calcique et permet l'injection de toxines directement dans le cytosol de la cellule. Différents phénotypes sont observés lors d'une infection pulmonaire dans le cas de la mucoviscidose : un phénotype inductible par le contact cellulaire ou la déplétion calcique et un autre non inductible.<br />En l'absence de mutations, cette dualité de phénotype peut être envisagée sous un aspect épigénétique.<br />A l'aide d'un outil informatique, nous avons déterminé les dynamiques possibles d'un modèle du SSTT supportant l'hypothèse de bistabilité et mis en évidence l'existence possible d'épigénèse. Grâce à cette méthode nous avons également définit les expériences permettant de tester cette hypothèse. Nous avons démontré qu'une modification épigénétique pouvait être à l'origine d'une acquisition stable de l'inductibilité du SSTT in vitro. Ce changement héréditaire de phénotype a été confirmé, in vivo, à l'aide d'un modèle d'infection pulmonaire aiguë.<br />Dans un second temps, nous avons mis en évidence une répression du SSTT à densité cellulaire élevée. Celle-ci est induite par un signal produit et sécrété par la bactérie. L'utilisation de mutants a permis de montrer que les signaux connus du quorum sensing ne sont pas impliqués dans cette répression. Ainsi, l'expression du SSTT dépend de la densité bactérienne et la répression à densité cellulaire élevée est induite par un mécanisme de type quorum sensing non connu.

<p>The Escherichia coli small RNA (sRNA) MicA was identified recently in a genomewide search for sRNAs. It is encoded between the genes <i>gshA</i> and <i>luxS</i> in E. coli and its close relatives. The function of sRNAs in bacteria is generally believed to be in maintenance of homeostasis via stress-induced modulation of gene expression. Our studies on MicA have been aimed at attributing function(s) to this molecule.</p><p>We carried out high throughput assays aimed at identifying genes that are differentially regulated upon knocking out or overexpressing MicA. Among the protein candidates identified was the outer membrane protein, OmpA. Subsequent analysis allowed us to show this regulation to be antisense in nature with MicA binding within the translation initiation region of <i>ompA</i> mRNA. Furthermore, blocking the ribosome from loading caused a translational decoupling that instigates degradation of the mRNA. The regulation was apparent in early stationary phase and seen to be dependent on the RNA chaperone Hfq. </p><p>We went on to characterize the regulation of MicA, looking at its own transcription. Testing various stress conditions, we were able to identify putative promoter elements that we confirmed using transcriptional fusions. The results showed MicA to be dependent on the extracytoplasmic function ECF sigma E (σ^E) and could not detect MicA in mutants deleted for this factor.</p><p>Lastly, we identified an additional target for MicA being the adjacently encoded <i>luxS</i> mRNA. The LuxS protein is essential for the synthesis of the quorum sensing AI-2 molecule. Transcription of the <i>luxS </i>mRNA is commences within the <i>gshA</i> gene, on the other side of MicA coding region. We were able to show that MicA interacts with <i>luxS </i>mRNA and is recognized by RNase III which processes this complex leading to a shorter <i>luxS</i> mRNA isoform. The significance of this processing event is as yet undetermined. Our data elucidated a new promoter driving transcription of <i>luxS,</i> and we demonstrated this promoter to be stationary phase responsive.</p><p>In summary, the work presented here characterizes the sRNA MicA as a dual regulatory sRNA molecule, moonlighting between its cis-encoded target and its trans-encoded target. .</p>

RESUME : Le Quorum Sensing est un système de communication bactérien permettant à une population de coordonner l’expression de gènes cibles en fonction de sa densité ou des propriétés du milieu (diffusion, flux....). Chez les bactéries à Gram négatif, le Quorum Sensing est basé sur la synthèse et la détection de petites molécules signal appelées N-acyl-homosérine lactones (AHLs). Les régulateurs transcriptionnels de type LuxR sont les médiateurs de ce système de régulation. Lorsque la concentration en AHLs augmente, ces molécules se fixent au domaine N-terminal d’un régulateur LuxR et provoquent des changement conformationnels entraînant une modification de l’activité du régulateur. Un tel système de régulation a été mis en évidence chez la bactérie Gram négative Brucella melitensis. Cette bactérie pathogène intracellulaire synthétise une dodécanoyl-homosérine lactone (C12-HSL) et possède deux régulateurs de type LuxR : VjbR et BabR. VjbR est impliqué dans la virulence de B. melitensis et est indispensable à l’expression de deux facteurs de virulence: le système flagellaire et le système de sécrétion de type IV VirB. Les C12-HSL ont quant à elles un effet répresseur sur ces deux structures membranaires. Durant ce travail, la mutation du domaine Nterminal du régulateur VjbR a permis de démontrer la capacité de VjbR à médier l’effet des C12-HSL sur l’opéron virB. Les souches mutées dans le gène vjbR forment des agrégats en cultures liquides. Nous avons montré que ce phénotype est lié à la production d’un exopolysaccharide, suggérant pour la première fois que Brucella pourrait former des structures de type biofilm. Cette étude a également permis de mettre en évidence un rôle majeur de VjbR dans la régulation de structures de surface puisque ce régulateur est impliqué dans le contrôle de l’expression de nombreuses protéines de membrane externe (Omp). L’utilisation de la technique d’immunoprécipitation chromatinienne (ChIP) a permis de montrer que VjbR régule directement deux de ces Omps ainsi que l’opéron virB. La virulence de Brucella est en partie basée sur sa capacité à dévier le trafic intracellulaire de ses cellules hôtes (phagocytes professionnels et nonprofessionnels) et à s’y multiplier. Lors de son cycle infectieux, Brucella est confrontée à de nombreux stress et environnements différents, suggérant la nécessité d’une régulation génétique fine en réponse à des stimuli environnementaux. Le Quorum Sensing, de par son implication dans la virulence de ce pathogène pourrait être impliqué dans de telles régulations. Afin d’aborder de façon globale le rôle de VjbR et de BabR chez B. melitensis, des études transcriptomique et protéomique des mutants ΔvjbR et ΔbabR ont été réalisées. Ces études ont permis de mettre en évidence que le Quorum Sensing chez B. melitensis est un système de régulation global, puisqu’il permet de réguler 10% du génome dans les conditions testées (dont 9% sous le contrôle de VjbR). De nombreuses cibles de ces régulateurs sont impliquées dans la virulence et l’adaptation aux conditions de stress (oxydatif, métabolique...), suggérant un rôle important du Quorum Sensing dans l’accomplissement du cycle infectieux de B. melitensis. SUMMARY : Quorum Sensing is a bacterial communication system wich allows the coordinated gene expression within a population regarding its density and environmental properties (diffusion, flow...). In Gram negative bacteria, Quorum Sensing is based on the synthesis and the detection of small diffusible molecules called N-acyl-homoserine lactones (AHLs). LuxR transcriptional regulators are the mediators of these regulation systems. When AHL concentration increases, these molecules bind to the N-terminal domain of a LuxR-type regulator and leads to conformational changes driving the modification of the regulator activity. A similar regulation system has been discovered in the Gram negative bacteria Brucella melitensis. This intracellular pathogen synthesizes a dodecanoylhomoserine lactone (C12-HSL) and possesses two LuxR-type regulators: VjbR and BabR. VjbR is involved in the virulence of this pathogen and is crucial for the expression of two virulence factors : the flagellar system and the type four secretion system VirB. C12-HSL have a repressor effect on these two membrane structures. During this work, mutation of the N-terminal domain of VjbR allowed us to demonstrate the ability of VjbR to mediate C12-HSL effect on the virB operon. vjbR mutated strains aggregate in liquid cultures. We have demonstrated that this phenotype is linked to the production of an exopolysaccharide, suggesting for the first time that Brucella could form biofilm-type structures. This study also demonstrates that VjbR has a major role in the regulation of surface structures because this regulator controls the expression of many outer membrane proteins (Omp). Using the chromatin immunoprecipitation technique (ChIP), we have shown that two of these Omps, as well as the virB operon, are directly regulated by VjbR. The virulence of Brucella is partly based on its ability to deviate the intracellular traffic of its host cells (professional and nonprofessional phagocytes) and to proliferate within these cells. During its infectious cycle, Brucella faces numerous stresses and environments, suggesting the necessity of a finely tuned genetic regulation depending on environmental stimuli. Quorum Sensing, through its involvement in the virulence of this intracellular pathogen, could be involved in such regulations. In order to investigate the role of VjbR and BabR in B. melitensis, global transcriptomic and proteomic studies of ΔvjbR and ΔbabR mutants were performed. These studies demonstrate that Quorum Sensing is a global regulation system in B. melitensis because it controls the expression of 10% of the genome in the condition tested (9% through VjbR). Numerous targets of these two regulators are involved in virulence and adaptation to environmental stresses (oxydative, metabolic...), suggesting an important role of Quorum sensing in the achievement of the infectious cycle of B. melitensis.

Des travaux de recherche en fermentation liquide ont montré que P. canescens est une souche hyperproductrice de xylanases non contaminées par des activités cellulolytiques et amylolytiques. Selon les scientifiques, lintérêt de lutilisation industrielle de ces hémicellulases dans différents secteurs (particulièrement dans la formulation daliments pour le bétail, en industries des jus de fruits et brassicoles, en amidonnerie, en industrie du papier, en pharmacie, dans les textiles et dans la production du bioéthanol) va croître significativement. Mais le développement de ces enzymes est fréquemment limité par le coût de production. Ce travail sest intéressé à loptimisation de la production des xylanases de P. canescens à partir de matières premières peu coûteuses telles les résidus agro-industriels par fermentation solide, une technique traditionnellement utilisée dans la fermentation des aliments en Asie. Létude a démontré que le tourteau de soja est un bon inducteur de la production des xylanases. La teneur initiale en eau, le pH initial, la température de la culture et laération active influencent la synthèse de l'enzyme. Compte tenu des résultats obtenus à léchelle du laboratoire, la transposition à léchelle industrielle serait facilitée naturellement par de fines épaisseurs de cultures statiques, ce qui réduit de moitié le coût de production comparativement à la fermentation liquide. Les expérimentations ont confirmé que la production de xylanases par P. canescens répondait à des phénomènes dinduction et de répression dépendant du substrat et des conditions physico-chimiques de croissance, et non pas à des phénomènes de régulation de type quorum sensing. Lenzyme sous forme liquide concentrée présente une bonne stabilité pendant six mois sans protection préalable (stérilisation, stabilisation ou inhibition de protéases).

The Escherichia coli small RNA (sRNA) MicA was identified recently in a genomewide search for sRNAs. It is encoded between the genes gshA and luxS in E. coli and its close relatives. The function of sRNAs in bacteria is generally believed to be in maintenance of homeostasis via stress-induced modulation of gene expression. Our studies on MicA have been aimed at attributing function(s) to this molecule. We carried out high throughput assays aimed at identifying genes that are differentially regulated upon knocking out or overexpressing MicA. Among the protein candidates identified was the outer membrane protein, OmpA. Subsequent analysis allowed us to show this regulation to be antisense in nature with MicA binding within the translation initiation region of ompA mRNA. Furthermore, blocking the ribosome from loading caused a translational decoupling that instigates degradation of the mRNA. The regulation was apparent in early stationary phase and seen to be dependent on the RNA chaperone Hfq. We went on to characterize the regulation of MicA, looking at its own transcription. Testing various stress conditions, we were able to identify putative promoter elements that we confirmed using transcriptional fusions. The results showed MicA to be dependent on the extracytoplasmic function ECF sigma E (σE) and could not detect MicA in mutants deleted for this factor. Lastly, we identified an additional target for MicA being the adjacently encoded luxS mRNA. The LuxS protein is essential for the synthesis of the quorum sensing AI-2 molecule. Transcription of the luxS mRNA is commences within the gshA gene, on the other side of MicA coding region. We were able to show that MicA interacts with luxS mRNA and is recognized by RNase III which processes this complex leading to a shorter luxS mRNA isoform. The significance of this processing event is as yet undetermined. Our data elucidated a new promoter driving transcription of luxS, and we demonstrated this promoter to be stationary phase responsive. In summary, the work presented here characterizes the sRNA MicA as a dual regulatory sRNA molecule, moonlighting between its cis-encoded target and its trans-encoded target. .

The secondary metabolites of citrus such as limonoids and flavonoids constitute an important part of human diet. The present work was undertaken to elucidate the effect of citrus limonoids and flavonoids on the bacterial cell-cell signaling in Vibrio harveyi, Escherichia coli O157:H7 and Salmonella Typhimurium LT2. The first experiment was focused on purification of limonoids from grapefruit and sour orange seeds. The limonoids were extracted using organic solvents and purified by chromatographic techniques. A total of ten limonoids (7 aglycones and 3 glucosides) were purified. Currently, simultaneous measurement of aglycones and glucosides of limonoids is not available. To address this limitation, an analytical method using high performance liquid chromatography was developed with the capability of measuring both aglycones and glucosides in a single run. Furthermore, its applicability in the fruit and juice samples was demonstrated. The third study investigated the V. harveyi cell-cell signaling inhibitory potential of purified limonoids. Isolimonic acid, ichangin, obacunone and nomilin were showed potent inhibitory activity. Furthermore, isolimonic acid and ichangin inhibit the signal transduction pathway by up-regulating the response regulator luxO. Isolimonic acid was also found to be a potent inhibitor of Escherichia coli O157:H7 cell-cell signaling in the fourth study. The results demonstrated that isolimonic acid inhibits the autoinducer/epinephrine mediated cell-cell signaling, biofilm and virulence in QseBC and QseA dependent fashion. Further investigations using limonin analogues, in the fifth study, demonstrated that the analogue limonin-7-methoxime inhibited the E. coli biofilm in type 1 pili and antigen 43 dependent-fashion, by preventing the binding of the adhesins to plastic surfaces. Another limonoid, obacunone was demonstrated to attenuate the Salmonella virulence by repressing Salmonella Pathogenicity Island 1 (SPI-1) in EnvZ/OmpR dependent mecahnism. The seventh study showed that naringenin, among the flavonoids, was the most potent inhibitor of V. harveyi and E. coli O157:H7 cell-cell signaling. Furthermore, naringenin was found to repress the (SPI-1) in PstS-HilD dependent fashion in the eighth study. In conclusion, the current project identified several limonoids and flavonoids with cell-cell signaling inhibitory property in three bacterial species.

Many bacteria produce signal molecules that serve in a cell-to-cell communication system termed quorum sensing. This signalling system allows a bacterial population to co-ordinately regulate functions according to their cell number in a defined environment. As bacterial growth progresses towards the stationary phase, signalling molecules accumulate in the growth medium and, above a certain threshold level, regulate the expression of genes involved in diverse functions. Most of the functions monitored by quorum sensing are most beneficial when they are performed as a population than by single cells, such as virulence factor production, biofilm formation, conjugation and bioluminescence. Vibrio anguillarum is a bacterial pathogen that causes terminal hemorrhagic septicaemia in marine fish. V. anguillarum possesses multiple quorum sensing circuits similar to the LuxI/LuxR and the V. harveyi-type systems. In this study, a characterisation of the quorum sensing-regulated transcriptional activator VanT was made. VanT belongs to the V. harveyi LuxR family of transcriptional regulators, which play a central role in quorum sensing signalling in Vibrio species. VanT was shown to regulate serine, metalloprotease, pigment, exopolysaccharide (EPS) and biofilm production. VanT repressed an EPS locus that plays a critical role in bacterial colonization of the fish integument and virulence. The V. harveyi-like quorum sensing systems were shown to limit rather than induce vanT expression throughout growth in V. anguillarum. In contrast to homologous proteins in other Vibrio spp., the quorum sensing phosphorelay protein VanU and the response regulator VanO had antagonistic roles in the regulation of vanT expression. Unlike other members of the luxR family, vanT was expressed at low cell density and no significant induction due to quorum sensing regulation was seen. Interestingly, VanT expression was induced by the alternative sigma factor RpoS as the cells entered stationary phase. RpoS was shown to regulate VanT expression post-transcriptionally by promoting vanT mRNA stability. VanT and RpoS were important for bacterial survival under stress conditions, indicating that VanT is likely an essential factor of V. anguillarum stress response.

The objectives of this work are: 1) to determine whether plant essential oil components influence the ability of Escherichia coli and several Pseudomonas species to form biofilms, and inhibit bacterial quorum sensing; 2) to understand the role of autoinducer-2 (AI-2) in biofilm formation by E. coli W3110. The biofilm formation assays determined that cinnamon, cassia and citronella oils differentially affected growth-normalized biofilm formation by E. coli. Cinnamaldehyde (CA) also inhibited the swimming motility of E. coli. Subinhibitory concentrations of CA were effective at inhibiting two types of acyl homoserine lactone (HSL) mediated quorum sensing (QS), and also AI-2 mediated QS. Because CA is widely used in the food and flavor industries, its potential to affect bacterial QS regulated processes should be recognized. The role of AI-2 mediated QS expression in physiology of E. coli W3110 was pleiotropic, including carbon utilization, fimbriae production, and the biofilm development. Overall, the research presented in this dissertation supported the concept that QS, biofilm formation, and cell adhesion may be broadly correlated. The anti-biofilm and anti-QS capability of CA implies that plant essential oil components might be promising for preventing the formation of detrimental biofilms.

Nosocomial infections associated with implanted medical- devices are on the rise due to a growing immunocompromised patient population. The organisms of interest in this study are Pseudomonas aeruginosa and Candida albicans. These organisms are opportunistic pathogens and are frequently implicated as the cause of infection and colonization of medical devices. P. aeruginosa is a motile gram-negative bacterium that is able to suppress the growth of C. albicans. Quourm sensing mimicry and biofilm formation on the hyphal surface of C. albicans by P. aeruginosa aids in suppression. C. albicans is a dimorphic fungus capable of quorum sensing with E,E-farnesol and is a central focus in this work. The goal of this project is to determine changes in protein expression when P. aeruginosa is exposed to E,E,-farnesol using 2D DIGE®. Changes in the cytosolic proteome of P. aeruginosa expose metabolic shifts that result in suppression of C. albicans. This work summarizes the effect of growth phase and concentration of E,E-farnesol on P. aeruginosa PAO1 and GSU3. Preliminary results reveal a general response of P. aeruginosa to C. albicans as changes in relevant metabolic nodes that affect pyocyanin production and the induction of virulence factors that lead to the killing of C. albicans. The overall goal of this study was to generate a profile of protein expression where a variety of conditions to further characterize the response could be easily assayed.

It is well known boronic acids have its unique chemistry and related applications in organic synthesis. The boronic acid functionally group also plays very important roles in medicinal chemistry and chemical biology. For example, boronic acids have been developed as potential therapeutic agents, chemical biology tools. All these applications are directly related to the unique electronic and chemical properties of the boronic acid group. Herein, several application of boronic acids have been studied: 1) several groups of compounds were found as bacterial quorum sensing inhibitors; 2) a boronate compound was developed as a probe for detecting reactive oxygen species (ROS); and 3) boronic acid-modified aptamers can be used for glycoprotein recognition.