CoordenaÃÃo de AperfeÃoamento de Pessoal de NÃvel Superior / As bactÃrias pertencentes ao gÃnero Vibrio tÃm sido apontadas como um dos grandes problemas no cultivo de camarÃo. AutÃctones de ambiente marinho e estuarino, algumas espÃcies de vÃbrios sÃo capazes de provocar doenÃas, representando perdas econÃmicas para a indÃstria e perigo potencial para saÃde pÃblica quando veiculadas ao produto final destinado ao mercado. O presente estudo teve por objetivo pesquisar as espÃcies de Vibrio presentes no cultivo do Litopenaeus vannamei e do meio onde à cultivado. Foram acompanhados dois ciclos de cultivo do L. vannamei em uma fazenda de camarÃo marinho situada no estuÃrio do rio Coreaà (CE) no perÃodo de maio a novembro de 2005, sendo analisadas 24 amostras de Ãgua de captaÃÃo, 24 de Ãgua do viveiro e 24 de camarÃo nos estÃgios de pÃs-larva, juvenil e adulto. As anÃlises foram concernentes à determinaÃÃo do NÃmero Mais ProvÃvel (NMP) de Vibrio, contagem de colÃnias sacarose positivas e negativas, identificaÃÃo das espÃcies e testes de suscetibilidade das cepas a antibiÃticos. Os resultados mostraram que o Ãndice de vÃbrio foi maior nas amostras de Ãgua do viveiro e de camarÃo nos dois ciclos quando comparadas Ãs amostras de Ãgua de captaÃÃo. Das 76 cepas isoladas de camarÃo nos diferentes estÃgios de desenvolvimento foram obtidas 14 espÃcies, com predominÃncia das espÃcies de V. harveyi e V. cholerae nas pÃs-larvas; V. cholerae, V. anguillarum, V. alginolyticus e V. harveyi nos juvenis; e V. parahaemolyticus, V. harveyi e V. anguillarum nos adultos. A espÃcie que apresentou maior grau de resistÃncia aos antimicrobianos foi V. cholerae, onde 33,33% das 12 cepas testadas mostraram-se resistentes a sulfazotrim, 25% a ampicilina e 33,33% a ceftriaxona. A elevada incidÃncia de vÃbrios nas amostras de Ãgua e camarÃo pode ser indicativa de risco para a atividade, se condiÃÃes ambientais desfavorÃveis forem estabelecidas no ambiente de cultivo. AlÃm disso, o isolamento feito de cepas patogÃnicas da microbiota acompanhante dos camarÃes pode representar risco para a saÃde pÃblica. / Infections with bacteria of the genus Vibrio represent a major challenge to shrimp farmers today. Some species of vibrios, autochthonous of marine and estuarine environments, can cause severe infection in livestock, leading to economic loss and, when communicated to marketed foods, public health hazards. The objective of the present study was to identify and determine the incidence of vibrios present in a marine shrimp (Litopenaeus vannamei) culture in Northeastern Brazil. The study covered two culture cycles of L. vannamei, from May to November 2005, on a shrimp farm located in the estuary of Rio Coreaà (CearÃ). Analyses were based on 24 samples of inflow water, 24 samples of pond water, and 24 samples of postlarval, juvenile and adult shrimp. The outcome measures included most probable number (MPN) of vibrios, number of sucrose-positive and negative strains, species identification and susceptibility to antibiotics. Vibrio concentrations were higher in pond water and shrimp samples than in inflow water samples. Out of 76 strains isolated from samples of shrimp at different development stages 14 species were identified, the most prevalent of which were V. harveyi and V. cholerae (in postlarvae), V. cholerae, V. anguillarum, V. alginolyticus and V. harveyi (in juveniles), and V. parahaemolyticus, V. harveyi and V. anguillarum (in adults). The hardiest species was V. cholerae of which one third out of 12 strains tested were resistant to sulfazotrim, one fourth to ampicillin, and one third to ceftriaxone. Depending on culture conditions, a high incidence of vibrios, as observed in the present study, may lead to substantial losses for the shrimp farming industry. In addition, the possibility of communicating pathogenic strains to marketed foods represents a major concern to public health.

FundaÃÃo Cearense de Apoio ao Desenvolvimento Cientifico e TecnolÃgico / Muitas pesquisas tÃm associado contaminaÃÃo aquÃtica ambiental com infecÃÃes de Vibrio em humanos, sugerindo que a importÃncia do monitoramento sistemÃtico das cepas ambientais se faz necessÃrio para definir seu possÃvel potencial patogÃnico e sua significÃncia clÃnica. O objetivo dessa pesquisa foi estudar a diversidade do gÃnero Vibrio isolado de quatro regiÃes estuarinas no Estado do CearÃ, (Pacoti, ChorÃ, Pirangi e Jaguaribe). As coletas realizadas resultaram num total de 32 amostras de Ãgua e 32 de sedimento, durante os meses de janeiro a abril de 2009. Foram catalogadas 19 espÃcies de bactÃrias pertencentes ao gÃnero Vibrio, das quais Vibrio parahaemolyticus e Vibrio alginolyticus foram as mais abundantes nos quatro estuÃrios: V. parahaemolyticus no Rio Chorà e V. alginolyticus no Rio Pacoti. As cepas identificadas foram submetidas a testes de susceptibilidade a quinze antimicrobianos. Todas as cepas analisadas (197) apresentaram susceptibilidade a sulfazotrim, ciprofloxacin, Ãcido nalidÃxico e cloranfenicol, sendo que cento e sessenta e trÃs (82%) apresentaram resistÃncia a penicilina G, cento e oito (54%) a ampicilina, quinze (7%) a cefalotina, trÃs (1%) a aztreonam, uma (0,5%) a gentamicina, a cefotaxima e a ceftriaxona. Cinquenta e uma cepas (25%) apresentaram comportamento intermediÃrio frente à cefalotina, vinte e oito cepas (14%) a ampicilina, dez (5%) a aztreonam, oito (4%) a tetracilina, duas (1%) a oxitetraciclina e uma (0,5%) a florfenicol, a cefotaxima, a ceftriaxona, a estreptomicina e a gentamicina. Foram escolhidas cinco espÃcies patÃgenas ao homem para verificaÃÃo de seus fatores de patogenicidade. As cepas identificadas como V. parahaemolyticus (64) e V. cholerae (9) foram analisadas atravÃs de tÃcnicas de biologia molecular, usando genes que confirmam as espÃcies e genes que indicam virulÃncia. Das 64 amostras de V. parahaemolyticus analisadas, 63 foram positivas para o gene tl, especÃfico para espÃcie, 57 para o gene tdh e 20 para o trh, genes que indicam patogenicidade. Das nove cepas de V. cholerae, cinco foram positivas para o gene ompW, gene especÃfico para espÃcie, porÃm, nenhuma amostra apresentou os genes de virulÃncia ctxA, zot, tcp e rfbO1. Com isso conclui-se que os estuÃrios dos rios analisados apresentam uma elevada abundÃncia de espÃcies, tendo V. parahaemolyticus e V. alginolyticus como as mais abundantes. O antibiograma das cepas isoladas mostrou uma elevada resistÃncia à penicilina e a ampicilina. Foram encontradas elevada positividade para a presenÃa dos fatores de virulÃncia nas cepas pertencentes Ãs espÃcies de Vibrio patÃgenas a humanos. As cepas de V. parahaemolyticus apresentaram genes de virulÃncia indicando que as cepas podem acarretar danos à saÃde pÃblica. A presenÃa do V. cholerae foi confirmada nas Ãguas e sedimento dos estuÃrios / The frequent association of environmental aquatic contamination with vibriosis in humans suggests the need for systematic monitoring and study of environmental vibrio strains and their pathogenic potential and clinical significance. The objective of this study was to evaluate the diversity of vibrio species in four estuaries (Pacoti, ChorÃ, Pirangi and Jaguaribe) in CearÃ, Northeastern Brazil. Nineteen vibrio species were identified in 32 water samples and 32 sediment samples collected between January and April 2009. Overall, V. parahaemolyticus and V. alginolyticus were the most abundant (the former in ChorÃ, the latter in Pacoti). The isolated strains were submitted to antibiogram testing with 15 antibiotics. All strains (n=197) were susceptible to sulfametoxazol-trimetoprim, ciprofloxacin, nalidixic acid and chloramphenicol. Resistance was observed to penicillin G (n=163; 82%), ampicillin (n=108; 54%), cephalothin (n=15; 7%), aztreonam (n=3; 1%), gentamicin, cefotaxime, ceftriaxone (1 each; 0.5%). Partial resistance was observed to cefalotin (n=52; 25%), ampicillin (n=28; 14%), aztreonam (n=10; 5%), tetracycline (n=8; 4%), oxytetracycline (n=2; 1%), and florfenicol, cefotaxime, ceftriaxone, streptomycin and gentamicin (1 each; 0.5%). Five species known to be pathogenic to humans were chosen for analysis of factors of pathogenicity. Strains belonging to the species V. parahaemolyticus (n=64) and V. cholerae (n=9) were submitted to molecular analysis using genes to confirm the species and indicate virulence. Sixty-three strains of V. parahaemolyticus were positive for species-specific tl, 57 were positive for tdh and 20 for trh. Five strains of V. cholerae were positive for species-specific ompW, but no strains presented the genes ctxA, zot, tcp or rfbO1. In conclusion, the estuaries surveyed presented a great diversity of vibrio species, the most abundant of which were V. parahaemolyticus and V. alginolyticus. Resistance to penicillin and ampicillin was elevated and positivity for virulence factors was considerable among strains of species pathogenic to humans. V. parahaemolyticus strains presented virulence genes indicating risk to public health. V. cholerae was identified in samples of both water and sediment

A poluição dos rios brasileiros é, atualmente, um grande problema para as autoridades governamentais já que a demanda dos recursos hídricos vem crescendo nos últimos anos. Os rios Tietê, Pinheiros e Tamanduateí foram de grande importância para a cidade de São Paulo em seu período de industrialização, contudo o próprio desenvolvimento industrial impactou permanentemente os mesmos. Preocupados com a saúde ambiental, órgãos fiscalizadores estaduais iniciaram forte fiscalização para coibir e responsabilizar os principais culpados. Esta política pública resultou em uma diminuição das concentrações de poluentes orgânicos e inorgânicos, porém muito pouco foi efetivamente feito com relação ao esgoto doméstico, e, como consequência, a toxicidade das águas dos rios permanece elevada. Os estudos de Avaliação e identificação da toxicidade englobam diversas metodologias reunidas em três documentos pela USEPA - Agência de Proteção Ambiental dos Estados Unidos, onde método físico-químico e testes toxicológicos são usados para identificar substâncias tóxicas, ou grupo de substâncias tóxicas que contribuem efetivamente para a toxicidade total de uma amostra. A abordagem rotineiramente utilizada baseia-se no fracionamento da amostra de acordo com suas propriedades físico-químicas: volatilidade, solubilidade e capacidade de formar complexos, ou de ser reduzida, oxidada, ionizada, entre outras. Em conjunto com os ensaios químicos, os ensaios toxicológicos, em especial o Sistema Microtox®, contribuem na investigação da toxicidade para os rios metropolitanos Tietê, Pinheiros e Tamanduateí, que se mostrou eficaz na determinação dos grupos de compostos, sendo os compostos orgânicos apolares, orgânicos voláteis, tensoativos e as partículas, as principais fontes da toxicidade para esses rios. Embora muitas informações sobre a toxicidade tenham sido descobertas, a identificação dos compostos só pode ser realizada por análises químicas mais robustas uma vez que um grande número de substâncias, metabólitos e subprodutos de degradação são facilmente encontrados neste tipo de amostra. Mesmo sofrendo algumas modificações, deve-se incentivar o uso desta metodologia para outros rios a fim de determinar os principais toxicantes responsáveis pela toxicidade. / Pollution of Brazilian rivers is currently a major problem for government authorities as demand for water resources has been increasing in recent years. Tietê, Pinheiros and Tamanduateí rivers were of great importance for the city of São Paulo during its industrialization period, however, the industrial development itself permanently impacted them. Concerned about environmental health, state supervisory bodies have begun strong enforcement to curb and hold the main culprits accountable. This public policy has resulted in a decrease in concentrations of organic and inorganic chemicals, but very little has been done in relation to domestic sewage, and as a consequence, the toxicity of the rivers remains high. The Toxicology Assessment and Identification studies encompass various methodologies gathered in three documents by the USEPA, where physico-chemical method and toxicological tests are used to identify toxic substances, or group of toxic substances that effectively contribute to the total toxicity of a sample. The routinely used approach is based on fractionation of the sample according to its physicochemical properties: volatility, solubility and chemical properties such as the ability to form complexes, or to be reduced, oxidized, ionized, among others. In conjunction with the chemical tests, the toxicological tests in particular the Microtox® System contribute to the investigation of the toxicity to the metropolitan rivers, Tietê, Pinheiros and Tamanduateí, which proved to be effective in the determination of the groups of compounds, the organic compounds being apolar, organic volatiles, surfactants and particulates, the main sources of toxicity for the studied rivers. Although much information on toxicity has been discovered, the identification of compounds can only be performed by more robust chemical analyzes since a large number of substances, metabolites, degradation byproducts are readily found in this type of sample. Even with some modifications, it is necessary to encourage the use of this methodology for other rivers in order to determine the main xenobiotics responsible for toxicity.

Vibrio cholerae acquiert les gènes de la toxine cholérique suite à l’infection par le phage CTXphi et devient par la suite une bactérie pathogène. L'infection se déroule en deux étapes : une interaction entre le pilus TCP et le domaine pIIIN2ctx, puis la formation du complexe TolAIIIV.c/pIIIN1ctx. Cette seconde étape est l’étape limitante de l’infection. L’objectif général de ma thèse a été d’étudier les forces motrices associées à cette étape.1) J’ai étudié les mécanismes moléculaires associés à la spécificité phage/bactérie en ciblant les interactions électrostatiques et le feuillet intermoléculaire par RMN et double hybride bactérien.2) J’ai résolu la structure de TolAIIIV.c libre par RMN. La comparaison des structures de cette protéine à l’état libre et liée ont permis de mettre en évidence un changement conformationnel et de proposer un mécanisme moléculaire d’ajustement induit. De plus, l’étude de la flexibilité de la protéine par RMN à haute pression (HP) a montré l’importance de la cavité interne de la protéine TolAIII pour favoriser l’ajustement induit lors de la formation du complexe TolAIIIV.c/pIIIN1CTX.3) J’ai vérifié si l’ajustement induit observé précédemment était lié à la présence de cette cavité d’une manière générale chez les protéines TolAIII. Une étude de dispersion de relaxation et de RMN à HP de la protéine TolAIIIE.c a permis de vérifier l’importance de cette cavité pour le mécanisme d’ajustement induit essentiel à cette famille de protéine. De plus, nous avons corrélée la flexibilité particulière de la protéine TolAIIIE.c à la présence d’une boucle qui lui confère une certaines flexibilité nécessaires pour interagir avec plusieurs partenaires. / Vibrio cholerae becomes a pathogen after CTXphi phage infection. The phagic infection is a wo step mechanism: first TCP pilus binds to pIIIN2ctx, then TolAIIIV.c binds to pIIIN1ctx. The second step is essential for the acquisition of genes of cholera toxins leading to cholera disease. The main goal of my thesis is to study the driving forces associated to the phage infection.First, I studied the molecular mechanism associated to phage/bacteria specificity targeting electrostatic bonds and hydrophobic interactions within the intermolecular sheet. These experiments use NMR and bacterial two hybrids methods. Our results show that electrostatic bonds are essential for the complex formation.Second, I solved the solution structure of TolAIIIV.c using NMR. The comparison of the structures of free and bound states of TolAIIIV.c, shows an associate conformational change and lead us to propose a model for the molecular mechanism of the induced fit. Then the study of the TolAIII flexibility, using high pressure NMR shows the importance of TolAIII cavity to promote the induced fit during TolAIIIV.c/pIIIN1ctx complex formation.Finally, we wanted to show if the induced fit is correlated to the presence of cavity in TolAIII family. A study using NMR relaxation dispersion and high-pressure NMR experiments on TolAIIIE.c shows the importance of this cavity for the induced fit. The presence of a loop at the top of the N-terminal helix in TolAIIIE.c leads to the protein to have several conformations necessary to interact with many partners.

Le cholera est une maladie diarrhéique aiguë due à la consommation d’eau ou d’aliments contaminés par des souches toxigéniques de Vibrio cholerae. Selon le “paradigme du choléra”, la maladie est provoquée par une exposition à un réservoir environnemental de V. cholerae avec des épidémies directement modulées par des facteurs environnementaux. Cependant, comme divers arguments plaident contre ce dogme, nous avons voulu élucider les mécanismes de la dynamique des épidémies de cholera dans trois foyers situés en Haïti, en République Démocratique du Congo (RDC) et en Afrique de l’Ouest. Nous avons associé une analyse temporo-spatiale des épidémies à une étude génétique des isolats de V. cholerae. En Haïti, nous avons cherché à savoir si les épidémies actuelles étaient dues à des souches toxigéniques de V. cholerae O1 durablement implantées dans l’environnement aquatique. En Afrique de l’Ouest, notre étude a révélé qu’Accra, la capitale du Ghana, était le principal foyer de choléra pour l’ensemble des pays d’Afrique de l’Ouest situés à l’Ouest du Nigeria. Le réseau d’eau d’Accra a probablement joué un rôle dans la propagation rapide de V. cholerae vers la majorité des quartiers de la ville. Les épidémies de choléra ont diffusé vers les autres pays sous la forme de vagues épidémiques et plusieurs épidémies ont été liées à la migration de populations à risque comme certains pêcheurs. En conclusion, notre réflexion globale sur les épidémies de choléra dans ces trois foyers distincts nous donne une vision cohérente des mécanismes d’émergence et de diffusion du choléra. / Cholera is an acute diarrheal disease caused by consumption of water or food contaminated with toxigenic Vibrio cholerae. According to the "cholera paradigm", the disease is contracted by exposure to environmental reservoirs of V. cholerae, with outbreaks driven directly by climatic factors. However, as recent findings argue against this dogma, we aimed to elucidate the dynamics of cholera outbreaks in three global foci: Haiti, Democratic Republic of the Congo (DRC) and West Africa. We combined spatiotemporal analysis of epidemics with genetic assessment of V. cholerae isolates. In Haiti, we assessed whether outbreak re-emergence during the rainy season was due to toxigenic V. cholerae O1 strains that have settled into the aquatic environment. Instead, we found that the re-emergence of outbreaks was likely due to persisting outbreaks during the dry season that were insufficiently controlled, rather than an environmental reservoir of V. cholerae O1. In West Africa, our study revealed that Accra, Ghana was the hotspot of cholera in the entire region of West Africa, west of Nigeria. The Accra water network likely played a role in rapid diffusion of V. cholerae throughout the city. Cholera outbreaks spread from Accra into other countries in a wave-like fashion. Distinct outbreaks were linked via migration of at-risk populations, such as certain fishermen. In conclusion, our global reflection of cholera epidemics in these three distinct foci provides a coherent vision of the mechanisms of cholera emergence and diffusion.

Vibrio harveyi is a bioluminescent bacterium and the organism in which quorum sensing was discovered. It was recently found that a class of molecules, cyclic dipeptides, may be a new kind of quorum sensing signal that may affect other species in the genus. The purpose of this study was to determine if V. harveyi produced one of these molecules, cyclo(Phe-Pro) or cFP, and the effects it has on bioluminescence, growth and gene expression. Electrospray Mass Spectrometry was used to detect cFP, and it was found. While growth and gene expression were not significantly affected by cFP, bioluminescence was slightly induced at low concentrations. It appears that V. harveyi does not produce cFP and it does not significantly affect the luminescence quorum sensing controlled genes, and is most likely not a true signal, in V. harveyi.

Chez les bactéries, les pôles cellulaires servent de domaines subcellulaires impliqués dans plusieurs processus cellulaires. Chez l’agent pathogène du choléra, Vibrio cholerae, en forme de bâtonnet incurvé, le pole contenant l’unique flagelle est impliqué dans la virulence. La protéine d’ancrage polaire HubP interagit avec plusieurs ATPases telles que ParA1 (ségrégation des chromosomes), ParC (localisation polaire du système de chimiotaxie) et FlhG (biosynthèse des flagelles), organisant ainsi l'identité polaire de V. cholerae. Cependant, les mécanismes moléculaires exacts de cet ancrage polaire doivent encore être élucidés. L’objectif de cette thèse est d’établir une vue d'ensemble de l'organisation de pôle cellulaire ce qui implique le mécanisme d’orchestration des différentes fonctions cellulaires par l’identification de l’ensemble des partenaires d'interaction de HubP ainsi que la cartographie fine du pôle cellulaire par microscopie à super résolution (PALM). Afin d’identifier de nouveaux partenaires d'interaction de HubP, j'ai étudié la différence de composition en protéines polaires entre les contextes HubP+ et HubP-. La composition en protéines polaires a été quantifiée de manière relative et absolue en ajoutant des Tag isobares aux protéines extraites de mini-cellules. Ces mini-cellules correspondent des petits compartiments cellulaires issus d’un évènement de division anormal proche du pole et sont enrichies en protéines polaires. Parmi ~800 protéines identifiées, ~ 80 protéines ont été considérées comme enrichies en contexte HubP+ incluant de nombreuses protéines attendues (FlhG, ParC et en aval des protéines de chimiotaxie). J'ai étudié la localisation de 14 protéines par microscopie à fluorescence et pu révéler 4 nouvelles protéines présentant une localisation polaire dépendant de HubP : VbrX, VbrY, et 2 protéines hypothétiques MotV et MotW. La délétion de motV et motW provoque un défaut significatif de propagation dans une gélose molle suggérant une implication dans la chimiotaxie et/ou la motilité. Alors que la microscopie électronique a montré que les deux mutants ont bien un flagelle polaire unique, le suivi-vidéo de leur déplacement a révélé que les deux mutants présentaient des défauts de nage assez distincts: ∆motV est plutôt affecté dans le changement de direction et ∆motW dans la vitesse de déplacement. Des expériences de microscopie fluorescente ont montré que MotV, MotW et HubP présentaient des dynamiques de localisation polaire distinctes au cours du cycle cellulaire. Pour une observation fine du pôle cellulaire par PALM, de nouveaux outils d’analyse d’image à haut débit étaient exigés. La précision des contours des petites cellules bactériennes faiblement contrastées n’est pas suffisante par l’observation en fond clair, j'ai développé une nouvelle technique de marquage avec des protéines fluorescentes photo-activables pour un tracé précis de la membrane interne ou du périplasme. En outre, nous avons créé un logiciel utilisant Matlab appelé Vibio qui intègre le contour de cellule et la liste des molécules obtenues par microscopie à super résolution. La capacité d’analyse à haut débit du logiciel permet d’étudier la distribution des molécules de l’échelle de la cellule unique à une population en orientant les cellules par leur courbure longitudinale. J’ai pu révéler que HubP est principalement localisé du côté convexe du pôle de la cellule, tandis que ses partenaires se situaient principalement au milieu du pôle. Mon travail de thèse a révélé avec succès de nouveaux partenaires d'interaction de HubP et la fonction de certaines protéines dans la motilité cellulaire. J'ai développé une nouvelle technique de microscopie pour une localisation subpolaire précise qui fonctionne bien pour l'analyse d'images PALM dans Vibio. J’ai ainsi pu faire progresser les connaissances de l’orchestration des fonctions polaires chez V. cholerae. / In rod shaped bacteria, cell poles serve as important subcellular domains involved in several cellular processes including motility, chemotaxis, protein secretion, antibiotic resistance, and chromosome segregation. In the cholera pathogen Vibrio cholerae, vibrioid rod shape and single polarized flagellum involve in the virulence. Polar landmark protein HubP was shown to interact with multiple ATPases, such as ParA1 (chromosome segregation), ParC (polar localization of chemotaxis apparatus), and FlhG (flagella biosynthesis), thus organizing the polar identity of V. cholerae by tethering proteins to cell pole. However, the exact molecular mechanisms are yet to be elucidated. In this thesis, I tackled to unveil comprehensive view of the cell pole organization which implies the orchestration of different cellular functions, by identifying further interaction partners of HubP as well as drawing conceivable picture of the cell pole by super-resolution photoactivated localization microscopy. To identify new interaction partners of HubP, I used minicells in which cell poles were enriched as they derived from cell division near the cell pole. Difference in protein composition between HubP+ and HubP- minicells were examined by isobaric tags for relative and absolute quantitation. Among ~800 proteins identified, ~80 proteins were considered to be enriched in HubP+ minicells including many expected proteins (FlhG, ParC and downstream chemotaxis proteins). I chose 14 proteins to investigate their subcellular localization with fluorescent microscopy. In conclusion, I discovered 4 proteins that showed polar localization in a HubP-dependent manner. These proteins are VbrX, VbrY, and 2 hypothetical proteins MotV and MotW. ∆motV and ∆motW showed significant defect in a diameter of travel in soft agar plate that suggesting the possible involvement in chemotaxis and/or motility. Whereas electron microscopy showed that both mutants possess intact monotrichous flagellum, video-tracking revealed that the two mutants showed rather distinct defects during swimming: MotV is rather turning mutant while MotW is a speed mutant. Fluorescent microscopy experiments indicated that MotV, MotW and HubP showed distinct polar dynamics over cell cycle. For fine-scale observation of the cell pole by PALM, it was appreciated that novel tools for high-throughput analysis was demanded. Since brightfield images are not sufficient to have accurate contours of small and low contrast bacterial cells, I developed new labeling technique with photoactivatable fluorescent proteins for precise outlining at either inner membrane or periplasm. Furthermore, we created Matlab-based software called Vibio which integrates cell outline and the list of molecules obtained by super-resolution microscopy. High-throughput capability of the software enabled to analyze distribution of detected molecules from single cell to whole bunch of cells in a manner that cells are oriented by cell curvature. These allowed me to discover that HubP is mostly lopsided at the convex side of the cell pole, while its partners mostly located middle of the pole. Altogether, I successfully unveiled 4 novel interaction partners of HubP. I revealed of the function of hypothetical proteins that are involved in cell motility. I developed new labeling technique for precise polar localization that works well for PALM image analysis in Vibio. Therefore, I observed precise polar localization of HubP and other polar proteins.

La plupart des bactéries ont un unique chromosome circulaire. Lors de la réplication de l’ADN, la circularité lie topologiquement les deux chromatides sœurs résultant de la réplication (caténanes et dimères). Ces liens topologiques doivent être résolus afin de permettre une bonne ségrégation de l’information génétique entre les deux cellules filles au cours de la division cellulaire. Les bactéries possèdent une machinerie très conservée: les recombinases à tyrosines XerC et XerD, capables de résoudre les dimères et une partie des caténanes, en catalysant un crossover au site spécifique dif situé dans la région Ter du chromosome. Lors de ce processus elles réalisent successivement deux échanges de brins. La réaction Xer est spatio-temporellement contrôlée par une protéine du divisome: FtsK. FtsK est une translocase qui pompe l’ADN à travers le septum de division. Lorsqu’elle rencontre une synapse constituée de deux sites dif chargés de XerC et XerD, elle active la catalyse de XerD pour initier le premier échange de brins. Dans un second temps XerC catalyse un second échange de brins indépendamment de FtsK. A ce jour le mécanisme d’activation de XerD n’est pas bien compris. Certains éléments mobiles résolvent leur états multimériques (tels que les plasmides) ou intègrent leur génome dans celui de leur hôte en détournant les recombinases XerCD. On parle d’IMEXs (integrative Mobile Element using Xer). Les éléments mobiles étudiés avant ma thèse utilisaient tous des voies de recombinaison initiées par la catalyse de XerC et ne nécessitant pas l’activation de XerD. Au cours de ma thèse j’ai étudié dans un premier temps le mécanisme d’intégration / excision d’une nouvelle classe d’IMEXs en utilisant comme modèle le phage TLCphi de Vibrio cholerae, la bactérie responsable du choléra. Par des approches de génétique j’ai démontré que TLCphi utilise une voie de recombinaison initiée par la catalyse de XerD et indépendante de FtsK. Mes travaux ont également montré que l’excision du phage participe à l’évolution des souches pandémiques de V.cholerae. Dans une seconde partie, j’ai identifié un facteur phagique qui permet à TLCphi de contourner le contrôle de FtsK sur l’activation de XerD. Ce facteur était une protéine de fonction inconnue présentant un domaine HTH et un domaine DUF3653. Ce dernier est retrouvé dans de nombreux IMEXs. Par des approches de biologie moléculaire j’ai étudié le mécanisme d’action de cette protéine. J’ai reproduit la réaction de recombinaison in vitro et démontré qu’elle active XerD en interagissant directement avec elle. Enfin dans un troisième temps, nous nous sommes intéressés aux disparités observées entre la recombinaison Xer chez E.coli et V.cholerae. En particulier, la recombinaison Xer semble agir seulement sur les dimères chez E.coli alors qu’elle est active également sur les monomères chez V.cholerae. Nous avons démontré que ces divergences de comportement ne viennent pas des Xer elles-mêmes, ni de leurs propriétés d'activations par FtsK. Elles résultent des différentes chorégraphies de ségrégation des chromosomes entre ces deux bactéries et dépendent également des vitesses de croissance. / Most of bacteria have a single circular chromosome. During replication of DNA, this circularity can lead to two sister chromatids topologically linked (catenanes and dimers). These topological links have to be solved in order to allow good segregation of genetic information between the two daughter cells during cell division. Bacteria possess a highly conserved machinery: the tyrosine recombinases XerC XerD that are capable to resolve dimers and some catenanes, by catalyzing a crossover at the specific site dif located in the Ter region of the chromosome. During this process they realize two sequentialstrand exchanges.The Xer reaction is spatiotemporally controlled by a protein of the divisome: FtsK. FtsK is a pump that translocates DNA through the septum of division. When FtsK meets a synapse that consists of two dif loaded by XerC and XerD, it activates XerD catalysis that initiates first strand exchange. Secondly XerC catalyzes a second strand exchange independently of FtsK. To date the activation mechanism of XerD is not well understood. Some mobile elements solve their multimeric states (like plasmids) or integrate their genome into the chromosome of their host by using XerCD recombinases. Such integrative elements are named IMEXs (Integrative Mobile Element using Xer). The mobile elements studied before my thesis all used recombination pathways initiated by catalysis of XerC and not requiring activation of XerD .During my PhD I studied at first the integration mechanism / excision of a new class IMEXs using as a model the TLC phage Vibrio cholerae, the bacterium responsible for cholera. By genetic approaches I demonstrated that TLCphi uses a recombination pathway initiated by XerD catalysis and independently of FtsK. My work has also shown that the phage excision participates in the evolution of pandemic strains of V. cholerae. In the second part, I identified a phage factor that allows TLC to bypass the activation of XerD by FtsK. This factor was a protein of unknown function with a HTH domain and a DUF3653 domain. DUF3653 are found in many IMEXs. Using molecular biology approaches, I studied the mechanism of action of this protein. I reproduced the recombination reaction in vitro and demonstrated that this factor activates XerD by directly interacting with it. Finally, we were interested to study disparities between Xer recombination in E.coli and V.cholerae. In particular, the Xer recombination seems to act only on dimers in E.coli while it is also active on monomers in V.cholerae. We have demonstrated that these differences in behaviors do not come from Xer themselves or their activation by FtsK. They result from different choreographies of chromosome segregation between these two bacteria and are also dependent on growth rates.

Whole cells of Vibrio cholerare serotype Inaba and serotype Ogawa (strains I389 and O395) were injected into rabbits to obtain antiserum. The antiserums were used for immune reaction with antigenic components of 25 strains of V.cholerae isolated from five provinces of Vietnam and the two standard strains I389 and O395 by Western-blot technique. Analysis of immune hybrid results showed that there were 11 antigenic components with molecular weights approximately 79kDa, 62kDa, 52kDa, 45kDa, 42kDa, 38kDa, 35kDa, 31kDa, 26kDa, 23kDa and 20kDa. In which the antigens of 45kDa, 42kDa, 31kDa and 20kDa were similar to OmpT, OmpS, Omp-31kDa and TcpA that have been considered as vaccine-candidate antigens. Among 25 V.cholerae strains, there were 6 antigenic components in common including 79kDa, 62kDa, 45kDa, 35kDa, 31kDa and 20kDa. 23/25 strains contained 42kDa antigen; 5/25 strains contained 38kDa and 23kDa antigens; 11/25 had 26kDa antigen. In addition, 7/25 strains contained antigens identical to V.cholerae I389 serotype Inaba; 6/25 strains contained antigens of I389 and O395; 12/25 strains had changes of antigenic components. These changes were actually the lack of antigens, not appearing new antigens. These results are considered as basis for researches about immune response and prevention of cholera disease. / Toàn bộ tế bào của các chủng Vibrio cholerare typ huyết thanh Inaba và typ huyết thanh Ogawa (chủng I389 và O395) được sử dụng để gây miễn dịch trên thỏ để thu kháng huyết thanh. Các kháng huyết thanh được dùng để thực hiện phản ứng miễn dịch với các thành phần kháng nguyên của 25 chủng V.cholerae phân lập từ 5 tỉnh thành của Việt Nam và hai chủng chuẩn I389 và O395 bằng kỹ thuật Western-blot. Phân tích kết quả lai miễn dịch cho thấy, có tổng số 11 thành phần kháng nguyên có kích thước khoảng 79kDa, 62kDa, 52kDa, 45kDa, 42kDa, 38kDa, 35kDa, 31kDa, 26kDa, 23kDa và 20kDa. Các kháng nguyên này chủ yếu là các protein màng ngoài (Omp) và kháng nguyên lông (TcpA). Trong đó các kháng nguyên 45kDa, 42kDa, 31kDa và 20kDa trùng với các kháng nguyên OmpS, OmpT, Omp-31kDa và TcpA được xem là những kháng nguyên dự tuyển vacxin tả. Có 6 kháng nguyên chung giữa 25 chủng với kích thước 79kDa, 62kDa, 45kDa, 35kDa, 31kDa và 20kDa. 7/25 chủng có các kháng nguyên giống với kháng nguyên của chủng V. cholerae I389 typ huyết thanh Inaba; 6/25 chủng có các kháng nguyên giống với kháng nguyên của cả hai chủng V.cholerae I389 và O395; 12/25 chủng có sự biến đổi thành phần kháng nguyên. Tuy nhiên, sự biến đổi này thực chất là sự thiếu hụt chứ không phải là sự xuất hiện các thành phần kháng nguyên mới. Các kết quả nghiên cứu này có thể được xem là nền tảng ban đầu cho các nghiên cứu về miễn dịch và dự phòng bệnh tả.

Whole cells of Vibrio cholerare serotype Inaba and serotype Ogawa (strains I389 and O395) were injected into rabbits to obtain antiserum. The antiserums were used for immune reaction with antigenic components of 25 strains of V.cholerae isolated from five provinces of Vietnam and the two standard strains I389 and O395 by Western-blot technique. Analysis of immune hybrid results showed that there were 11 antigenic components with molecular weights approximately 79kDa, 62kDa, 52kDa, 45kDa, 42kDa, 38kDa, 35kDa, 31kDa, 26kDa, 23kDa and 20kDa. In which the antigens of 45kDa, 42kDa, 31kDa and 20kDa were similar to OmpT, OmpS, Omp-31kDa and TcpA that have been considered as vaccine-candidate antigens. Among 25 V.cholerae strains, there were 6 antigenic components in common including 79kDa, 62kDa, 45kDa, 35kDa, 31kDa and 20kDa. 23/25 strains contained 42kDa antigen; 5/25 strains contained 38kDa and 23kDa antigens; 11/25 had 26kDa antigen. In addition, 7/25 strains contained antigens identical to V.cholerae I389 serotype Inaba; 6/25 strains contained antigens of I389 and O395; 12/25 strains had changes of antigenic components. These changes were actually the lack of antigens, not appearing new antigens. These results are considered as basis for researches about immune response and prevention of cholera disease. / Toàn bộ tế bào của các chủng Vibrio cholerare typ huyết thanh Inaba và typ huyết thanh Ogawa (chủng I389 và O395) được sử dụng để gây miễn dịch trên thỏ để thu kháng huyết thanh. Các kháng huyết thanh được dùng để thực hiện phản ứng miễn dịch với các thành phần kháng nguyên của 25 chủng V.cholerae phân lập từ 5 tỉnh thành của Việt Nam và hai chủng chuẩn I389 và O395 bằng kỹ thuật Western-blot. Phân tích kết quả lai miễn dịch cho thấy, có tổng số 11 thành phần kháng nguyên có kích thước khoảng 79kDa, 62kDa, 52kDa, 45kDa, 42kDa, 38kDa, 35kDa, 31kDa, 26kDa, 23kDa và 20kDa. Các kháng nguyên này chủ yếu là các protein màng ngoài (Omp) và kháng nguyên lông (TcpA). Trong đó các kháng nguyên 45kDa, 42kDa, 31kDa và 20kDa trùng với các kháng nguyên OmpS, OmpT, Omp-31kDa và TcpA được xem là những kháng nguyên dự tuyển vacxin tả. Có 6 kháng nguyên chung giữa 25 chủng với kích thước 79kDa, 62kDa, 45kDa, 35kDa, 31kDa và 20kDa. 7/25 chủng có các kháng nguyên giống với kháng nguyên của chủng V. cholerae I389 typ huyết thanh Inaba; 6/25 chủng có các kháng nguyên giống với kháng nguyên của cả hai chủng V.cholerae I389 và O395; 12/25 chủng có sự biến đổi thành phần kháng nguyên. Tuy nhiên, sự biến đổi này thực chất là sự thiếu hụt chứ không phải là sự xuất hiện các thành phần kháng nguyên mới. Các kết quả nghiên cứu này có thể được xem là nền tảng ban đầu cho các nghiên cứu về miễn dịch và dự phòng bệnh tả.