Bacterial infections are an increasing cause for concern as resistance spreads to the majority of our front line antibiotics. To counter antibiotic resistance, new treatment regimens and drug targets are being investigated, including directly targeting bacterial virulence (pathogen-induced harm to the host), and therapies which target resistance mechanisms. The outcome of successful treatment with these compounds is not always killing or halting growth of bacteria, therefore selection for resistance to these types of therapeutics is complex. This complexity is increased by the secretion of many virulence factors, meaning their effects are shared with neighbouring individuals. In addition virulence factors show high phenotypic plasticity due to regulation by processes like quorum sensing (QS), which further complicates treatments targeting virulence, or the regulatory processes themselves. Using the example of quorum sensing inhibitors this study shows the importance of understanding the function and ecology of targeted virulence factors, to predict the selection for resistance to anti-virulence drugs. Later chapters elaborate on this to show how quorum sensing control affects selection on secreted virulence factors. The use of anti-virulence drugs as adjuvants is discussed, with a study showing that the interaction between QS inhibition and translation inhibitors is dependent on the environment. The selection for resistance to combinations of antibiotics and adjuvants is investigated using co-amoxiclav as an example, showing that treatment with high doses of adjuvant are robust to the evolution of resistance.

L’identification, chez divers organismes, d’enzymes de dégradation des N-acyl homosérineslactones (NAHLs) impliquées dans la signalisation QS pose la question de leurs rôles dans lesinteractions bactéries-eucaryotes. Dans une première partie, une synthèse bibliographique analyse lesconnaissances acquises sur ces enzymes dégradant les NAHLs. Dans une seconde partie, la croissancedes bactéries dégradant les signaux NAHLs a été stimulée par l’application de g-caprolactone (GCL)dans la rhizosphère de plants de pommes de terre à des fins de phytoprotection. L’effet de cetraitement sur la diversité des communautés bactériennes rhizosphériques a été évalué en combinantdifférentes approches d’écologie microbienne moléculaire comme la DGGE, le pyroséquençaged’amplicons rrs, et la métagénomique fonctionnelle. Cette dernière approche appliquée à une banquede 30 000 clones environ a conduit à l’identification d’un gène qsdB codant la dégradation des signauxNAHL. Ce travail révèle ainsi l’existence d’une nouvelle classe d’enzymes de dégradation des NAHLsappartenant à la famille des enzymes possédant une signature amidase (AS) dont des membres sontpar ailleurs impliqués dans la dégradation de composés xénobiotiques. Dans une troisième partie, unsystème expérimental a été développé afin de mesurer le transfert conjugatif du plasmide de virulenceTi (tumor inducing) chez des dérivés du pathogène Agrobacterium tumefaciens, appelés «tricheurs»,incapables de produire des signaux NAHLs mais utilisateurs de ceux produits par les autres bactéries.Ce modèle a permis de montrer l’effet modérateur de lactonases dégradant les NAHLs exprimées chezdes agrobactéries produisant les NAHLs, chez des bactéries réceptrices du plasmide Ti, ou des planteshôtes des agrobactéries sur le transfert conjugatif initié par les tricheurs. L’ensemble de ce travailrévèle à la fois une nouvelle famille d’enzymes impliquées dans la dégradation des NAHLs, ainsiqu’un nouveau rôle de ces enzymes dans la modulation des flux de gènes entre bactériesphytopathogènes en interaction avec une plante hôte. / Identification of bacterial and eukaryotic enzymes that degrade N-acyl homoserine lactones(NAHLs) involved in QS signaling raises the question of their roles in bacteria-eucaryotesinteractions. In a first part of this study, a bibliographic report analyzes the current data on thoseNAHL-degrading enzymes. In a second part, the growth of NAHL-degrading bacteria was stimulatedby g-caprolactone (GCL) amendment in potato rhizosphere to protect this plant against the soft-rotpathogen Pectobacterium. The effect of the GCL treatment on rhizospheric bacterial communities wasevaluated by a combination of different molecular microbial ecology techniques such as DGGE,pyrosequencing and functional metagenomic. This last approach was applied to generate ametagenomic library of ca. 30,000 clones and lead to the identification of the qsdB gene that encodesNAHL degradation, This work revealed the occurrence of a novel class of NAHL-degrading enzymesthat belong to the amidase signature (AS) family, some members of which being involved inxenobiotic compound degradation. In a third part, an experimental system was developed to measurethe conjugative transfer of Ti plasmid in various strains of the pathogen Agrobacterium tumefaciens,including “cheaters”, i.e. bacteria unable to produce NAHL signals but capable to use signals producedby other bacteria. Using this model; variations of the plasmid transfer of cheaters were measured whenNAHL-degrading lactonases were expressed in agrobacteria that produce NAHL signals, in recipientbacteria of Ti plasmid, or in agrobacterial host plant. Taken together, thesis experiments revealed anovel class of enzymes involved in NAHL-degradation and a new role for thoses enzymes in themodulation of gene transfer between pathogenic bacteria interacting with host plants.

Muitos genes bacterianos são regulados pelo mecanismo de comunicação denominado quorum sensing (QS). Neste sistema, moléculas sinalizadoras ativam um comportamento de grupo, conforme a densidade celular, permitindo o controle da expressão gênica. Estudos sugerem o potencial de compostos extraídos de plantas sobre o QS, a exemplo da quercetina, um flavonol presente em concentrações elevadas em algumas frutas e hortaliças. Este composto é o flavonoide majoritário presente em cebola (Allium cepa), mas não existem estudos que mostrem a atividade anti-QS de extratos orgânicos deste vegetal. Este trabalho avaliou o potencial antimicrobiano e anti-QS de extratos orgânicos de cebola branca e cebola roxa, assim como de alguns de seus componentes majoritários identificados, em fenótipos regulados pelo QS como a produção de violaceína em Chrormobacterium violaceum ATCC 12472, a motilidade tipo swarming e a formação de biofilmes em Pseudomonas aeruginosa PAO1 e Serratia marcescens MG1. Extratos de cebola branca e roxa foram obtidos por extração em fase sólida utilizando coluna de poliamida e seus compostos identificados e quantificados pelas técnicas de Cromatografia líquida- ionização por elétron spray-espectrometria de massas e cromatografia líquida de alta eficiência acoplada a detector de arranjo de diodo. A atividade antimicrobiana foi avaliada pelas curvas de multiplicação de cada micro-organismo. O efeito dos compostos quercetina aglicona (inibidor do QS já relatado na literatura e encontrado no extrato de cebola roxa) e quercetina-3-β-D-glicosideo (um dos compostos majoritários encontrados em ambos extratos) sobre os micro-organismos utilizados neste estudo foi também avaliado. Foram obtidos três extratos: cebola branca em metanol (CB-MeOH), cebola branca em metanol amônia (CBMeOH/ NH4) e cebola roxa em metanol (CR-MeOH). Os compostos quercetina 3,4\'- diglicosídeio, quercetina-4-glicosídeo, quercetina-3-β-D-glicosideo e quercetina aglicona foram os predominantes nos extratos das duas variedades de cebola. Cianidina-3-O-glicosideo também foi identificada no extrato de cebola roxa. A concentração inibitória mínima (MIC) dos extratos foi igual ou superior a 125 µg/ml (p/v) de extrato seco. Não foi observada inibição significativa da produção de violaceína em C. violaceum pelos extratos orgânicos de cebola e nem pela quercetina-3-β-D-glicosideo, nas concentrações sub-inibitórias avaliadas. No entanto, a quercetina aglicona inibiu significativamente a produção de violaceína em todas as concentrações. A glicosilação da quercetina pode ter afetado sua atividade sobre a inibição da produção de violaceina, já que estudos mostram menor atividade biológica deste composto quando glicosilado. Para a motilidade tipo swarming em P. aeruginosa PAO1 houve inibição significativa pelo extrato de cebola roxa, em todas as concentrações estudadas. Os demais extratos não apresentaram inibição contra este micro-organismo. Para S. marcescens MG1, foi observada inibição da motilidade swarming somente na concentração de 125 µg/ml de CBMeOH/ NH4. As análises de comparação entre os dois tipos de quercetina revelaram que, embora para as duas bactérias testadas os dois compostos apresentaram atividade inibitória sobre a motilidade tipo swarming, a quercetina-3-β-D-glicosideo foi menos eficiente que a quercetina aglicona na concentração de 125 µg/ml. A formação de biofilmes não foi influenciada pelos extratos e, inesperadamente, não se detectou inibição da formação de biofilmes por ambos tipos de quercetina avaliados. De forma geral, os extratos orgânicos de cebola mostraram pouco efeito sobre os fenótipos controlados pelo quorum sensing e a glicosilação da quercetina provavelmente explica a baixa atividade antimicrobiana e anti-QS dos extratos. / Many bacterial genes are regulated by a communication mechanism called quorum sensing (QS). In this system, signaling molecules activate a group behavior according to cell density, allowing the control of gene expression. Studies suggest the inhibitory potential of compounds extracted from plants on the QS system, like quercetin, a flavonol present in high concentrations in some fruits and vegetables. This compound is the main flavonoid found in onion (Allium cepa); however, there are no studies showing the anti-QS activity of organic extracts of this plant. The objective of this work was to evaluate the antimicrobial and anti-QS potential of organic extracts of white and red onions, and their major components studied in QS-regulated phenotypes such as violacein production in Chromobacterium violaceum, swarming motility and biofilm formation in Pseudomonas aeruginosa PAO1 and Serratia marcescens MG1.White and red onion extracts were obtained by solid phase extraction using a polyamide column and its compounds were identified and quantified by Liquid Chromatography - Electron Spray-Mass Spectrometry and high performance liquid chromatography coupled to diode array detector. O The antimicrobial activity was evaluated by growth curves of each microorganism. The effect of non-glycosylated quercetin (a QS inhibitor already reported in the literature and found in red onion extract) and quercetin-3-β-D-glycoside (one of the major compounds found in both extracts) on the microorganisms used in this study was also evaluated. Three extracts were obtained: white onion in methanol (CB-MeOH), white onion in methanol ammonia (CB-MeOH / NH4) and red onion in methanol (CR-MeOH). Our results showed that quercetin 3,4\'- diglycoside, quercetin-4-glycoside, quercetin-3-β-D-glycoside and non-glycosylated quercetin were predominant in the extracts of the two onion varieties. Cyanidin-3-O-glycoside has also been identified in the purple onion extract. The minimum inhibitory concentration (MIC) of extracts was equal or greater than 125 µg / ml (w / v) of dry extract. There was no significant inhibition of violacein production in C. violaceum by organic onion extracts or by quercetin-3-β- D-glycoside at the sub-inhibitory concentrations evaluated. However, non-glycosylated quercetin showed a significant inhibition of violacein production in all tested concentrations. The glycosylation of Quercetin could have altered its inhibition activity towards violacein production, and in fact, some studies have shown less biological activity of some phenolic compounds when they have been glycosylated. For swarming motility in P. aeruginosa PAO1 there was significant inhibition by red onion extract, in all studied concentrations. The other extracts did not present inhibition against this microorganism. For S. marcescens MG1, inhibition of swarming motility was observed only at the concentration of 125 µg / ml of CB-MeOH / NH4. Comparative analyses between the two types of quercetin showed that, although for the two bacteria tested the two compounds showed inhibitory activity on swarming motility, quercetin-3-β-D-glycoside was less efficient than non-glycosylated quercetin in the concentration of 125 µg / ml. Biofilm formation was not influenced by the extracts and unexpectedly, both types of quercetin evaluated did not show inhibition towards biofilm formation. In general, organic onion extracts showed little effect on quorum sensing controlled phenotypes and glycosylation of quercetin probably explains the low antimicrobial and anti-QS activity of the extracts.

Muitos genes bacterianos são regulados pelo mecanismo de comunicação denominado quorum sensing (QS). Neste sistema, moléculas sinalizadoras ativam um comportamento de grupo, conforme a densidade celular, permitindo o controle da expressão gênica. Estudos sugerem o potencial de compostos extraídos de plantas sobre o QS, a exemplo da quercetina, um flavonol presente em concentrações elevadas em algumas frutas e hortaliças. Este composto é o flavonoide majoritário presente em cebola (Allium cepa), mas não existem estudos que mostrem a atividade anti-QS de extratos orgânicos deste vegetal. Este trabalho avaliou o potencial antimicrobiano e anti-QS de extratos orgânicos de cebola branca e cebola roxa, assim como de alguns de seus componentes majoritários identificados, em fenótipos regulados pelo QS como a produção de violaceína em Chrormobacterium violaceum ATCC 12472, a motilidade tipo swarming e a formação de biofilmes em Pseudomonas aeruginosa PAO1 e Serratia marcescens MG1. Extratos de cebola branca e roxa foram obtidos por extração em fase sólida utilizando coluna de poliamida e seus compostos identificados e quantificados pelas técnicas de Cromatografia líquida- ionização por elétron spray-espectrometria de massas e cromatografia líquida de alta eficiência acoplada a detector de arranjo de diodo. A atividade antimicrobiana foi avaliada pelas curvas de multiplicação de cada micro-organismo. O efeito dos compostos quercetina aglicona (inibidor do QS já relatado na literatura e encontrado no extrato de cebola roxa) e quercetina-3-β-D-glicosideo (um dos compostos majoritários encontrados em ambos extratos) sobre os micro-organismos utilizados neste estudo foi também avaliado. Foram obtidos três extratos: cebola branca em metanol (CB-MeOH), cebola branca em metanol amônia (CBMeOH/ NH4) e cebola roxa em metanol (CR-MeOH). Os compostos quercetina 3,4\'- diglicosídeio, quercetina-4-glicosídeo, quercetina-3-β-D-glicosideo e quercetina aglicona foram os predominantes nos extratos das duas variedades de cebola. Cianidina-3-O-glicosideo também foi identificada no extrato de cebola roxa. A concentração inibitória mínima (MIC) dos extratos foi igual ou superior a 125 µg/ml (p/v) de extrato seco. Não foi observada inibição significativa da produção de violaceína em C. violaceum pelos extratos orgânicos de cebola e nem pela quercetina-3-β-D-glicosideo, nas concentrações sub-inibitórias avaliadas. No entanto, a quercetina aglicona inibiu significativamente a produção de violaceína em todas as concentrações. A glicosilação da quercetina pode ter afetado sua atividade sobre a inibição da produção de violaceina, já que estudos mostram menor atividade biológica deste composto quando glicosilado. Para a motilidade tipo swarming em P. aeruginosa PAO1 houve inibição significativa pelo extrato de cebola roxa, em todas as concentrações estudadas. Os demais extratos não apresentaram inibição contra este micro-organismo. Para S. marcescens MG1, foi observada inibição da motilidade swarming somente na concentração de 125 µg/ml de CBMeOH/ NH4. As análises de comparação entre os dois tipos de quercetina revelaram que, embora para as duas bactérias testadas os dois compostos apresentaram atividade inibitória sobre a motilidade tipo swarming, a quercetina-3-β-D-glicosideo foi menos eficiente que a quercetina aglicona na concentração de 125 µg/ml. A formação de biofilmes não foi influenciada pelos extratos e, inesperadamente, não se detectou inibição da formação de biofilmes por ambos tipos de quercetina avaliados. De forma geral, os extratos orgânicos de cebola mostraram pouco efeito sobre os fenótipos controlados pelo quorum sensing e a glicosilação da quercetina provavelmente explica a baixa atividade antimicrobiana e anti-QS dos extratos. / Many bacterial genes are regulated by a communication mechanism called quorum sensing (QS). In this system, signaling molecules activate a group behavior according to cell density, allowing the control of gene expression. Studies suggest the inhibitory potential of compounds extracted from plants on the QS system, like quercetin, a flavonol present in high concentrations in some fruits and vegetables. This compound is the main flavonoid found in onion (Allium cepa); however, there are no studies showing the anti-QS activity of organic extracts of this plant. The objective of this work was to evaluate the antimicrobial and anti-QS potential of organic extracts of white and red onions, and their major components studied in QS-regulated phenotypes such as violacein production in Chromobacterium violaceum, swarming motility and biofilm formation in Pseudomonas aeruginosa PAO1 and Serratia marcescens MG1.White and red onion extracts were obtained by solid phase extraction using a polyamide column and its compounds were identified and quantified by Liquid Chromatography - Electron Spray-Mass Spectrometry and high performance liquid chromatography coupled to diode array detector. O The antimicrobial activity was evaluated by growth curves of each microorganism. The effect of non-glycosylated quercetin (a QS inhibitor already reported in the literature and found in red onion extract) and quercetin-3-β-D-glycoside (one of the major compounds found in both extracts) on the microorganisms used in this study was also evaluated. Three extracts were obtained: white onion in methanol (CB-MeOH), white onion in methanol ammonia (CB-MeOH / NH4) and red onion in methanol (CR-MeOH). Our results showed that quercetin 3,4\'- diglycoside, quercetin-4-glycoside, quercetin-3-β-D-glycoside and non-glycosylated quercetin were predominant in the extracts of the two onion varieties. Cyanidin-3-O-glycoside has also been identified in the purple onion extract. The minimum inhibitory concentration (MIC) of extracts was equal or greater than 125 µg / ml (w / v) of dry extract. There was no significant inhibition of violacein production in C. violaceum by organic onion extracts or by quercetin-3-β- D-glycoside at the sub-inhibitory concentrations evaluated. However, non-glycosylated quercetin showed a significant inhibition of violacein production in all tested concentrations. The glycosylation of Quercetin could have altered its inhibition activity towards violacein production, and in fact, some studies have shown less biological activity of some phenolic compounds when they have been glycosylated. For swarming motility in P. aeruginosa PAO1 there was significant inhibition by red onion extract, in all studied concentrations. The other extracts did not present inhibition against this microorganism. For S. marcescens MG1, inhibition of swarming motility was observed only at the concentration of 125 µg / ml of CB-MeOH / NH4. Comparative analyses between the two types of quercetin showed that, although for the two bacteria tested the two compounds showed inhibitory activity on swarming motility, quercetin-3-β-D-glycoside was less efficient than non-glycosylated quercetin in the concentration of 125 µg / ml. Biofilm formation was not influenced by the extracts and unexpectedly, both types of quercetin evaluated did not show inhibition towards biofilm formation. In general, organic onion extracts showed little effect on quorum sensing controlled phenotypes and glycosylation of quercetin probably explains the low antimicrobial and anti-QS activity of the extracts.

Quorum sensing (QS) is a type of microbial communication used by bacteria to coordinate their behavior based on population density, regulating complex processes like biofilm formation and virulence, among other behaviors. Quorum quenching (QQ), on the other hand, disrupts this communication, usually by degradation of the QS signaling molecule. QQ offers a potential strategy for controlling bacterial behaviors linked to pathogenicity and biofouling. Despite significant advances in understanding and modeling the spatial-temporal behavior of QS, predictive modeling of QQ remains nascent, with a notable gap in the quantitative assessment of QQ's impact on QS. Here we show quantitative evaluation and characterization of the effect of QQ on QS in agar-based experiments, combined with an experimentally validated computational model. This research utilizes green fluorescence in E. coli MG 1655 as an indicator of QS activation, focusing on the degradation of Acyl-Homoserine Lactone (AHL), a key QS molecule in Gram-negative bacteria linked to pathogenicity, by the AiiA enzyme in engineered AiiA-producing Salmonella Typhimurium 14028. Our findings suggest that QQ more effectively influences QS in spatial configurations of the populations with larger interaction surfaces and shorter diffusion distances. Contrary to our initially held hypothesis, the primary effect of QQ is not a delay in QS onset but rather an attenuation of QS activity, with the area-under-the-curve of fluorescence serving as a quantitative metric. This study also introduces, to the best of our knowledge, one of the first instances of experimentally validated predictive modeling for QQ, applied to agar-based experimental setups. We posit that the quantitative experimental characterization and modeling framework presented in this research will enhance the understanding of bacterial community interactions. Enhanced comprehension of QQ and QS behaviors holds significant promise for advancing practical applications, particularly in mitigating or diminishing undesirable QS-associated activities. This is especially relevant in areas like biofouling, waste treatment, and the reduction of infections and progression of diseases in plants and animals, areas increasingly important as concerns about drug resistance in microbes and food security escalates. / Master of Science / One of the ways bacteria communicate with each other is called quorum sensing (QS), where they use chemical signals to organize and time group behavior, including forming communities encapsulated in protective layers, called biofilms, and engaging in virulent attacks against hosts. Quorum quenching (QQ) in bacteria, however, disrupts this communication system, usually by breaking down the chemical signals that bacteria use to send messages to each other. Even though QS has been studied extensively, determining how to predict and control QQ is still a nascent area of research. Here, we studied and characterized how QQ affects QS by doing experiments with bacteria populations in agar (a jelly-like substance) and applied a computational model to explain and ultimately predict the experimental observations. Engineered QS population (E. coli MG 1655) produced Acyl-Homoserine Lactone (AHL) signaling molecules, and engineered QQ bacteria (S. Tm 14028) used the Autoinducer Inactivation A (AiiA) enzyme to break down the AHL. According to our results, QQ doesn't delay the QS bacteria's group behaviors (in our case, green fluorescent signal production); it weakens the signal instead. Understanding QQ and QS better, especially through measurements and modeling, could lead to expanded methods of deterring harmful bacterial behavior, managing waste better, and stopping diseases in plants, animals, and humans, especially with the concerning rise of drug-resistant microbes and food security. One exciting possibility is using QQ to protect plants from bacterial infections. This could be a way to shield our crops without always relying on antibiotics.

This study provided evidence of cell-density dependent signalling interactions between the autotrophic ammonia-oxidising bacterium Nitrosomonas europaea and a variety of terrestrial heterotrophic bacteria. The autoinducer signal molecule N-(3-hexanoyl)-L-homoserine lactone (HHL) extended the lag phase of N. europaea recovering from starvation while N-(3-oxooctanoyl)-L-homoserine lactone (OOHL) reduced the lag phase. However, the autoinducers N-(3-oxohexanoyl)-L-homoserine lactone N-(3-oxohexanoyl)-L-homoserine lactone (OHHL), N-(3-oxobutanoyl)-L-homoserine lactone(OBHL) and N-(3-oxododecanoyl)-L-homoserine lactone (OdDHL) did not exert an effect. Spent cell-free medium from early stationary phase cultures of Agrobacterium tumefaciens, Pseudomonas fluorescens and Micrococcus luteus reduced the lag phase of N. europaea recovering from starvation. Supplementing solid medium with spent cell-free medium from the heterotrophs A. tumefaciens, P. fluorescens and M. luteus reduced the incubation period required for the development of colonies of the ammonia-oxidiser. For example, the presence of spent cell-free medium from A. tumefaciens reduced the required incubation period from 20 weeks to 2 weeks. Spent cell-free medium from the heterotrophs Comomonas testosteroni, Erwinia carotovora and Rhizobium leguminosarum had no effect on the growth of N. europaea. A. tumefaciens produces the N-acyl autoinducer OOHL which reduces the lag phase of N. europaea. A mutant unable to produce this autoinducer was generated and spent cell-free medium from this organism had no effect on the recovery of N. europaea from starvation or the incubation period required for the development of colonies of N. europaea on solid medium. Enrichment cultures of ammonia-oxidising bacteria were established from soil and the heterotrophs present in the final stages of enrichment were isolated and identified. Spent cell-free medium from these organisms also reduced the lag phase of N. europaea recovering from starvation and reduced the incubation period required for the development of colonies of N. europaea on solid medium.

Helicobacter pylori is a human pathogen that colonizes the stomach and increases the risk of diseases such as ulcers and gastric cancer. The distribution of H. pylori within the stomach is associated with different disease outcomes, with more dispersed colonization correlated with gastric cancer. The focus of this research is to study the chemotactic responses that promote dispersal of H. pylori within the stomach. We have shown previously that H. pylori perceive the quorum signal autoinducer-2 (AI-2) as a chemorepellent. We report that H. pylori chemorepulsion from endogenous AI-2 influences the proportion and spatial organization of cells within biofilms. Strains that fail to produce AI-2 (∆luxS) or are defective for chemotaxis (∆cheA) formed more spatially homogenous biofilms with a greater proportion of adherent versus planktonic cells than wildtype biofilms. Reciprocally, a strain that overproduced AI-2 (luxSOP) formed biofilms with proportionally fewer adherent cells. Along with the known AI-2 chemoreceptor, TlpB, we identified and characterized two novel periplasmic binding proteins, AibA and AibB, that independently both bind AI-2 and are required for the AI-2 chemorepulsion response. Disruptions in any of the proteins required for AI-2 chemotaxis recapitulated the biofilm adherence and spatial organization phenotype of the ∆luxS mutant. Furthermore, exogenously administered AI-2 was sufficient to decrease the proportion of adherent cells in biofilms and promote dispersal of cells from biofilms in a chemotaxis dependent manner. Finally, we found that disruption of AI-2 production or AI-2 chemotaxis resulted in increased clustering of cells in microcolonies on cultured epithelial cells. We conclude that chemotaxis from AI-2 is a determinant of H. pylori biofilm spatial organization and dispersal and may play an important role in H. pylori colonization of the stomach by promoting dispersal away from areas of high cell density, thereby modulating the disease spectrum of the host. This dissertation contains previously unpublished co-authored material.

Quorum sensing (QS) is a type of cell-to-cell communication that allows the bacteria to communicate via small molecules to coordinate activities such as growth, biofilm formation, virulence, and stress response as a population. QS depends on the accumulation of signal molecules as the bacterial population increases. After a critical threshold of the signal molecules are reached, the bacteria induce a cellular response allowing the bacteria to coordinate their activities as a population. In Vibrio anguillarum, three parallel quorum-sensing phosphorelay systems channels information via three hybrid sensor kinases VanN, VanQ, and CqsS that function as receptors for signal molecules produced by the synthases VanM, VanS, and CqsA, respectively. The phosphorelay systems converge onto a single regulatory pathway via the phosphotransferase VanU, which phosphorylates the response regulator VanO. Together with the alternative sigma factor RpoN, VanO activates the expression of a small RNA, Qrr1 (Quorum regulatory RNA), which in conjunction with the small RNA chaperone Hfq, destabilizes vanT mRNA, which encode the major quorum-sensing regulator in V. anguillarum. This thesis furthers the knowledge on the quorum-sensing phosphorelay systems in V. anguillarum. In this study, three additional qrr genes were identified, which were expressed during late logarithmic growth phase. The signal synthase VanM activated the expression of the Qrr1-4, which stands in contrast to Qrr regulation in other vibrios. Moreover, in addition to VanO, we predict the presence of a second response regulator which can be phosphorylated by VanU and repress Qrr1-4 expression. Thus, VanU functions as a branch point that can regulate the quorum-sensing regulon by activating or repressing VanT expression. Furthermore, VanT was shown to directly activate VanM expression and thus forming a negative regulatory loop, in which VanM represses VanT expression indirectly via Qrr1-4. In addition, VanM expression was negatively regulated post-transcriptionally by Hfq. Furthermore, a universal stress protein UspA repressed VanM expression via the repression of VanT expression. We showed that UspA binds Hfq, thus we suggest that UspA plays a role in sequestering Hfq and indirectly affect gene expression. This thesis also investigated the mechanism by which V. anguillarum can attach to and colonize fish skin tissue. We show unequivocally that fish skin epithelial cells can internalize bacteria, thus keeping the skin clear from pathogens. In turn, V. anguillarum utilized the lipopolysaccharide O-antigen to evade internalization by the fish skin epithelial cells. This study provides new insights into the molecular mechanism by which pathogen interacts with marine animals to cause disease.

Autoinducer 2 (AI-2) molecules produced by bacterial cells are thought to be involved in controlling a variety of bacterial cellular processes by coordinated gene and protein expression. Previous work in our laboratory has shown that ground beef contains compounds that can interfere with AI-2-mediated bioluminescence expression in Vibrio. harveyi. The underlying hypothesis of this work was that AI-2 molecules affect the protein expression in Escherichia coli O157:H7 and AI-2 inhibitory molecules negate the influence of AI-2 molecules. The main objectives of this study were to identify, characterize, and isolate the factors responsible for inhibition of AI-2 molecules from ground beef extracts, elucidate the role of LuxS/AI-2 cell signaling system in E. coli O157:H7 protein expression, and determine if inhibitory factors present in ground beef extract can negate the influence of AI-2 molecules on the protein expression. Using a solvent extraction procedure and gas chromatography analysis, AI-2 inhibitory factors present in ground beef extracts were identified as both medium and long chain fatty acids. When identified fatty acids were tested at different concentrations for AI-2 inhibition, AI-2 inhibition ranging from 25% to 90% was observed. Both ground beef extracts and mixture of selected fatty acids also resulted in 2- to 4-fold reduced AI-2 influenced biofilm formation by E. coli K12 cells. Identification of LuxS/AI-2-mediated protein expression in E. coli O157:H7 was conducted using two dimensional gel electrophoresis. Protein expression analysis showed that the LuxS/AI-2 system modulates the expression of proteins involved in different cellular processes such as carbohydrate and amino acid metabolism, stress response, and formation of flagella and motility. When AI-2 inhibitory factors were added along with AI-2 molecules, the expression patterns of three AI-2-influenced proteins (GlmS, SpeE, and NikA) were changed suggesting that AI-2 inhibitors can negate the influence of AI-2 molecules on protein expression of selected proteins. Collectively, these results highlight that proteins associated with different cellular processes in E. coli O157:H7 can be modulated depending on whether cells are in contact with AI-2 molecules in the presence or absence of AI-2 inhibitory factors.