M.Tech. / With the recent cholera outbreak in Zimbabwe and the outbreak taking a sub-regional dimension with cholera cases being reported from neighbouring countries like Botswana and South Africa, there was a need to monitor drinking water from environmental water sources as well as household water-storage containers at the point-of-use in rural communities. Although conventional culture-based microbiological methods for the identification of Vibrio species from environmental water samples are reliable, they require several days to complete (Khan and Cerniglia, 1994). Culture dependent and culture independent methods for the detection of Vibrio cholerae and Vibrio parahaemolyticus from water samples were optimised during the current study. With these methods, the occurrence and distribution of V. cholerae and V. parahaemolyticus in source waters as well as in household container stored-waters at the point-of-use in the Nwanedi Catchment, was determined. The culture based approach analyses involved the enrichment of water samples in alkaline peptone water (APW) for 18 hours at 37°C followed by culture on selective thiosulfate-citrate-bile salts-sucrose (TCBS) agar. Typical colonies on TCBS agar were confirmed using the API 20NE as well as the two multiplex polymerase chain reactions (m-PCR). The culture independent PCR approach was done by filtering 100 ml of the water sample onto polycarbonate membranes followed by DNA extraction from the bacteria captured on the membranes using an adaptation of the in-house DNA extraction method used in the laboratory. This DNA was used as template for the m-PCR’s. For the culture based PCR detection, 100 ml water was filtered onto nitrocellulose membranes followed by 18 hours enrichment in APW. DNA was then extracted from the enrichment broth and subsequently used as template for the m-PCR’s. All water samples were analysed with all three methods to compare the results and determine the most effective method for the detection of the two-selected Vibrio species present in water samples. PCR analyses were performed using two m-PCR assays targeting the SodB (V. cholerae species), FlaE (V. parahaemolyticus species) and 16S rRNA (Vibrio and Enterobacteriacea species) genes (Multiplex 1) and the V. cholerae O1 and V. cholerae O139 rfb genes, ctxA (cholera toxin) gene and 16S rRNA gene (Multiplex 2). The 16S rRNA primers were included in the Multiplex PCR’s as an internal control. The m-PCR assays were 100% specific for total and toxigenic V. cholerae and total V. parahaemolyticus when using target bacteria and various other non-target bacteria. The m-PCR assays when coupled with an 18 hours enrichment step could detect as few as 4-10 V. cholerae and V. parahaemolyticus cells in pure cultures as well as in spiked environmental water samples. Fifty water-storage containers and 56 environmental water samples (river, spring and borehole) from rural households in the Vhembe district of the Limpopo Province of South Africa were tested for the presence of selected Vibrio’s, using (1) the standard culture based approach, (2) PCR detection without enrichment and (3) PCR with a brief pre-enrichment. Container water samples were collected before [referred to as free volume (FV) of water] and after dislodging of the biofilm [referred to as dislodged biofilm (BD)] from the inner sidewalls of containers. Of the samples analysed with the standard cultured based technique combined with colony confirmation using m-PCR 1, 34 (12.8%) tested positive for the presence of V. cholerae (SodB gene), 2 (1.3%) for the presence of V. parahaemolyticus (FlaE gene) and all the samples tested positive for the 16S rRNA gene. In contrast, only 1 (0.6%) tested positive for the presence of V. cholerae and 0 (0%) for the presence of V. parahaemolyticus when the isolates were confirmed with API 20NE. With the culture dependant PCR method, 65 (41.7%) of the samples tested positive for the presence of V. cholerae, 3 (1.9%) for the presence of V. parahaemolyticus and all the samples tested positive for the 16S rRNA gene. Seventeen (10.9%) of the samples tested positive for the presence of V. cholerae (SodB) and 16S rRNA genes, 0 (0%) for the presence of V. parahaemolyticus (FlaE gene) with the culture independent direct PCR detection protocol. All the samples that tested positive for V. cholerae with any of the three methods were tested for the presence of toxigenic V. cholerae species with the second multiplex PCR. Six of the source water samples tested positive for V. cholerae O1 as well as the cholera toxin genes. Of the 56 source water samples, 14 (25%) were positive for V. cholerae and 0 (0%) were positive for V. parahaemolyticus with one or all of the methods. Six (10.7%) of the V. cholerae positive samples tested positive for V. cholerae O1 rfb gene, and ctxA gene (cholera toxin). Thirty (60%) of the 50 FV and 28 (56%) of the DB water samples tested positive for V. cholerae, and 3 (6%) of the FV and 0 (0%) of the DB samples tested positive for V. parahaemolyticus with one or all of the methods. None of the positive V. cholerae samples tested positive for the presence of toxigenic V. cholerae. The results presented suggest that the use of culture-based techniques alone is inadequate for detection of selected Vibrio’s in the environmental water samples and that such techniques are not enough to guarantee satisfactory protection of human health. The combination of filtration, enrichment, DNA extraction and m-PCR method provide a sensitive and specific method for the detection of V. cholerae and V. parahaemolyticus in environmental water samples. This method proved to be the most effective for detection and identification of selected Vibrio’s when compared to the culture based method and PCR without enrichment method. The inclusion of an enrichment period allows for the detection of culturable bacteria which is crucial as PCR detection does not give indications on the viability of the detected material. The enrichment period will also dilute any inhibitors for the m-PCR’s that may be present. Detection of V. cholerae and V. parahaemolyticus in the source water used by the population and in the water-storage containers indicates possible seeding of containers with Vibrio species from the source water. Furthermore, the detection of these organisms in DB samples indicates that these organisms attach to containers’ inner sidewalls, forming biofilms, further sustaining their occurrence and proliferation. The detection of V. cholerae and V. parahaemolyticus in household water-storage containers certainly places the consumers at risk of infection of diseases caused by these organisms.

A rapid assay kit for Vibrio cholerae (Vc) was developed to detect and quantify Vc cells in oyster samples within 24 h. The kit, formulated within a two -phase (liquid and solid) 96-well plate, can detect biomarker expression of Vc when the enrichment broth and incubation temperature are optimized. The kit showed 91 % selectivity and 92 % specificity when tested with 23 inclusive Vc and 106 exclusive non-Vc strains. The kit was further optimized using 47 samples of oysters, clams, and soil. There was no significant difference in most probable number between the kit, conventional PCR and BAX PCR regardless of agar heating method (autoclaved vs. boiled). The kit’s limit of detection was below 5 cfu/g. The kit is a reliable method for the detection of V. cholerae in bivalve samples.

The World Health Organization (WHO) states that 30,000 children under the age of five die each day worldwide. Around a quarter of these die from diarrheal disease caused by microbial infection. In addition to this high mortality rate, there are data emerging on the morbidity effects of diarrheal disease, for example a few episodes of diarrhea in the first two years of life can remove 10 IQ points and lead to growth deficiency. Vibrio cholerae, the causative agent of the diarrheal disease cholera, is a serious problem in third world countries, where sanitary and hygiene infrastructure is very poor, and claims several thousand lives every year. In order to better understand the pathogenicity regulation in V. cholerae, structural and functional investigations of a hypothetical protein family present in pathogenicity islands and a transcriptional regulator protein for DNA-binding were investigated. Two adjacent genes, vc1804 and vc1805, encode hypothetical proteins within the Vibrio pathogenicity island-2 (VPI-2) of Vibrio cholerae, and are part of a cluster of genes only present in pathogenic strains of the bacterium. Paralogous adjacent genes, vc0508 and vc0509, are also present within a second pathogenicity island, the Vibrio seventh pandemic island-2 (VSP-2), of V. cholerae O1 El Tor and O139 serogroup isolates. Sequence similarity suggests that the VC0508, VC0509, VC1804 and VC1805 proteins will share a similar fold. The crystal structures of VC0508, VC0509 and VC1805 have been determined to a resolution of 1.9, 2.4 and 2.1 Å, respectively. Several recombinant constructs of vc1804 were made, but no soluble proteins were expressed. This hypothetical protein family reveals structural homology to human mitochondrial protein p32. Human p32 is a promiscuous protein known to bind to a variety of partners including the globular head component of C1q. We have shown that VC1805 binds to C1q. One possibility is that VC1805 is involved in adherence of the bacterium to membrane-bound C1q in the gut. To explore the roles of VC0508, VC0509, VC1804 and VC1805 in vivo, gene knockout and animal model studies of those proteins are underway. The ferric uptake regulator (Fur), a metal-dependent DNA-binding protein, acts as both a repressor and activator of numerous genes involved in maintaining iron homeostasis in bacteria. It has also been demonstrated in Vibrio cholerae that Fur plays an additional role in pathogenesis, and this opens up the potential of Fur as a drug target for cholera. The first crystal structure of a Fur protein, from Pseudomonas aeruginosa, revealed a dimeric molecule with each monomer containing a dimerization domain, a helical DNA-binding domain and two metal binding sites: Zn1 is proposed to be a regulatory Fe-binding site, and Zn2 is proposed to be a structural Zn-binding site. Here we present the crystal structure of V. cholerae Fur (VcFur) that reveals a very different orientation of the DNA-binding domains. Accompanying these structural changes are alterations in the amino acids coordinating the zinc at the Zn2 site, and this lends support to this being the site regulated by iron. There is no evidence of metal binding to the cysteines that are conserved in many Fur homologues, including the much-studied E. coli Fur. An analysis of the metal binding properties shows that like other Fur proteins, VcFur can be activated by a range of divalent metals. EPR spectroscopy measurements of the movements of the DNA-binding domain, in the presence of DNA and different metals, are underway.

L’objectif de cette thèse est de définir la chorégraphie de ségrégation des deux chromosomes circulaires de Vibrio cholerae, c’est à dire le positionnement de l’information génétique au cours de la croissance de la cellule, ainsi que les mécanismes dirigeant ces ségrégations. Il a longtemps été supposé que les bactéries étaient trop petites pour avoir une organisation intra-cellulaire, et le manque de techniques appropriées ne permettait pas d’infirmer cette hypothèse. Or la taille des chromosomes comparée à celle de la bactérie impose une compaction et aujourd’hui, de nouvelles techniques de microscopie et d’analyse génétique permettent d’affirmer que les chromosomes bactériens étudiés jusqu’à maintenant ont tous une organisation et une chorégraphie de ségrégation précises et différentes selon les espèces. Toutes les espèces étudiées à ce jour ont un chromosome circulaire unique : la réplication du chromosome commence à une origine unique bidirectionnelle, les deux fourches de réplication se déplacent le long des deux bras de réplication (ou réplichores) et finissent la réplication au terminus, diamétralement à l’opposée de l’origine de réplication sur la carte du chromosome. Peu d’espèces ont été étudiées, et Vibrio cholerae émerge progressivement comme un nouveau modèle : son génome est réparti sur deux chromosomes, et la chorégraphie de plusieurs chromosomes dans une cellule n’a jamais été décrite. De plus, cette espèce semble être au croisement évolutif entre Caulobacter crescentus et Escherichia coli : Vibrio cholerae a d’une part une morphologie en croissant, des systèmes de partition aux origines et un positionnement de l’origine du chromosome I, semblables à C. crescentus, et d’autre part un système de compaction du terminus et un set de gènes impliqués dans la maintenance du chromosome ayant co-évolué, qu’on ne retrouve que dans peu d’espèces proches d’E. coli. Une autre caractéristique intéressante de V. cholerae est que le chromosome II semble avoir été acquis récemment et n’est donc peut être pas gouverné par les mêmes mécanismes que le chromosome I, comme en témoignent le positionnement de son origine et son terminus, inédits pour des chromosomes bactériens. Parmi les Vibrios (environ 60 espèces principalement retrouvées dans les environnements aquatiques), certaines espèces sont des pathogènes dévastateurs pour les poissons, le corail, les crustacés ou les fruits de mer. Mais la plus documentée est Vibrio cholerae, car elle provoque chez l’Humain une maladie provoquée par l’ingestion d’eau contaminée qui peut être mortelle si le patient n’est pas réhydraté à temps. Bien que facilement traitable, le choléra fait encore de nombreuses victimes dans les pays en développement où les structures de santé et les règles d’hygiène font parfois défaut. Ainsi l’étude de Vibrio cholerae présente un intérêt médical, mais également par extension aux autres Vibrios, un intérêt environnemental non négligeable. / The aim of this thesis is to define the segregation choreography of the two circular chromosomes of Vibrio cholerae, which is the positionning of the genetic information during cell growth, as well as the mecanisms directing those segregations. It was supposed for a long time that bacteria were too small to have a intra-cellular organization and the lack of appropriate tools could not prove this hypothesis wrong. The size of the chromosomes compared to the size of the cell means there has to be a compaction and today, new tools for microscopy and genetic analysis allow us to affirm that all bacterial chromosomes studied so far have an organization and a segregation choreography which are precise and different between specie. Most bacterial specie studied to this day have a unique circular chromosome : the replication of the chromosome starts at a unique and bidirectionnal origin, both replication forks move along the two replication arms (or replichores) and end the replication at the terminus which is diametrically to the opposite of the origin on the chromosome map. A few specie have been studied, and Vibrio cholerae progressively emerges as a new model : its genome is divided between two chromosomes, and the choreography of several chromosomes in a cell has never been described. Moreover, this species seems to be at the crossover between Caulobacter crescentus and Escherichia coli : Vibrio cholerae as on one hand, a crescent shape, partition systems positionned at both origins and a positionning of the chromosome I origin similar to C. crescentus, and on the other hand a compaction system of the terminus and a set of genes involved on the maintenance of chromosomes that one only finds in very few specie closely related to E. coli. An other interesting characteristic of V. cholerae is that the chromosome II seems to have been acquired recently and thus might not be governed by the same mecanisms as the chromosome I, as shown by the positionning of its origin and terminus which are completely new to bacterial chromosomes. Among Vibrios (about 60 species mostly found in aquatic environments), some species are devastating pathogens for fish, coral, crustacean and shellfish. But the most documented one is Vibrio cholerae, because it induces a disease in humans caused by the ingestion of contaminated water, which can be deadly if the patient is not rehydrated on time. Although easily treatable, cholera still makes a lot of victims in developing countries where health structures and basic hygiene sometimes lack dramatically. The study of Vibrio cholerae has a medical interest, but also by extention to other Vibrios, a non-negligible environmental interest.

"May, 2004" / Includes corrigenda. / includes bibliographical references (leaves 118-156) / [13], 155 leaves : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, School of Molecular and Biomedical Science, Discipline of Microbiology and Immunology, 2005

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

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.

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.