The individual behaviour of prokaryotic organisms such as bacteria often gives rise to complexity that is commonly associated with multicellular behaviour. The transition from unicellular to multicellular behaviour occurs in response to chemical signals, called autoinducers, which bacteria generate and receive internally within a given population. These autoinducers control the gene expression necessary for the emergence of group-behaviour-phenotype. This phenomenon is called quorum sensing (QS). An example of the quorum sensing control of gene regulation has been the luminescence (lux) operon in Vibrio fischeri. The luxI and ainS quorum signalling systems work in conjunction to regulate luminescence in V. fischeri. LuxI and AinS are acyl-synthases that catalyse the production of the autoinducers C6-HSL and C8-HSL respectively. These autoinducers bind to LuxR, a transcriptional activator of the lux operon, which activates expression of the lux genes causing an increase in luminescence. It was shown that quorum signalling also affects motility and biofilm formation in bacteria. However, the evidence with respect to these phenotypes is conflicting and inconclusive, the reason being the state of quorum is ambiguously defined. It is not properly known whether the observed collective behaviour is purely a result of physical crowding of bacteria, or that both chemical signalling and crowding contribute to this phenomenon. This work attempts to address these issues by studying luminescence, motility, and biofilm, a diverse set of behaviours, yet closely linked to each other in V. fischeri-squid symbiosis. We studied the luminescence response of V. fischeri to both endogenous and externally added signals at per-cell and population level. Experiments with ES114, a wild-type strain of V. fischeri, and ainS mutant showed that (i) luminescence per cell does not mutually correlate with the cell-density, indicating that bacteria do not show greater response to the signal at higher densities; (ii) the activity of the lux signalling circuit shows a strong dependence on the growth stage, (iii) the cells do not show enhanced growth, i.e., they do not derive fitness benefits at higher densities in response to the signal. We anticipated that the culture with a higher cell-density should exhibit greater per-cell-luminescence. However, we found that the luminescence curve of the culture with lower density crosses that of the cultures with higher densities during the exponential phase. Kinetic modelling of the luxI mRNA expression showed that the expression profile qualitatively agrees with the luminescence trend observed in the cultures, supporting the observation that growth-phase plays a major role in regulating the luminescence gene expression. We also studied the effect of autoinducers on motility of V. fischeri. V. fischeri uses flagella to move into the inner crypts of the light organ of the squid. The bacterium secretes autoinducers, encounters secretions of the light organ, and slows down during the final stage of colonization process. Studies have shown that flagellar elaboration is repressed as a consequence of ainS signalling. However, those studies were soft-agar migration assays and carried out with the mutant strain of ainS. We measured real-time planktonic motility of ES114 and the signalling mutant strains of V. fischeri in response to autoinducers added exogenously at different concentrations. We found that the autoinducers do not affect the motility of the strains. We also showed that reduction in motility is purely a consequence of physical crowding of bacteria, and chemical signalling may not be involved in the process. It was shown that reduction in motility leads to biofilm formation. Motile bacteria must lose flagella in order to form biofilm, and signalling controls biofilm formation in many species. Our study on motility showed that reduction in motility occurs because of physical crowding in V. fischeri. Hence, we explored the possibility that physical crowding might lead to formation of biofilm rather than signalling in this species. We quantified exopolysaccharide production by crystal violet assay, which revealed that planktonic cells produce exopolysaccharides, in addition to biofilm cells. The study revealed that V. fischeri cells always produce exopolysaccharides irrespective of their physiological state. We examined the effect of signalling on biofilm in ES114 and the mutant strains using gene-expression analysis. We quantified the expression of various genes involved in biofilm formation and found that both ES114 and the mutants expressed rscS and sypP indicating that exopolysaccharide production is not under the control of autoinducers. Therefore, we hypothesized that biofilm formation in V. fischeri may be a result of physical agglomeration of cells. Our observations indicate that the state of quorum is inadequately defined and there is no direct measure of the underlying process. Multicellular behaviour in V. fischeri is regulated by a complex interplay of cell-density, signalling, and other factors such as the growth phase of the culture, indicating that the state of quorum employs different mechanisms to regulate various phenotypes. Our study reveals that QS is an intricate process, and the accepted mechanisms for QS are incomplete at best.

La palourde japonaise, Venerupis philippinarum, a été introduite en France au début des années 70 à des fins aquacoles. Depuis 1987, d’importants épisodes de mortalité, causés par la bactérie pathogène Vibrio tapetis, touchent cette espèce le long des côtes françaises et européennes. Cette vibriose, appelée Maladie de l’Anneau Brun (MAB), est considérée comme une maladie d’eau froide. L’interaction tripartite entre V. philippinarum, V. tapetis et l’environnement a été explorée à travers le rôle du système enzymatique des phénoloxydases (POs) dans le but de mieux comprendre la réponse immunitaire de la palourde japonaise, la virulence de l’agent pathogène mais aussi l’impact de l’environnement et plus particulièrement de la température. Les POs sont des oxydoréductases impliquées dans la synthèse de la mélanine et de ses dérivés mais aussi dans les processus de reconnaissance du non-soi et d’encapsulation chez les invertébrés. Dans un premier temps, l’activité PO du sérum d’hémolymphe a été caractérisée d’un point de vue biochimique comme étant majoritairement de type laccase ; une activité minoritaire de type tyrosinase a également été identifiée. Des infections de palourdes par trois souches de V. tapetis, à différentes températures, ont mis en évidence une modulation de la réponse du système PO en fonction du temps et du compartiment étudiés. De façon générale, l’infection bactérienne s’est traduite par une augmentation de l’activité PO. Toutefois, le niveau basal d’activité PO est variable d’une population à une autre et cette variabilité semble traduire une susceptibilité différente à la MAB. L’augmentation de la température de 15 à 22°C a entraîné une augmentation des capacités immunitaires de la palourde japonaise. La températurea également eu un impact sur la pathogénicité de V. tapetis et ce, de façon différentielle selon les souches. L’inhibition de l’activité PO observée in vitro en présence de produits extracellulaires bactériens souligne la complexité de l’interaction entre V. philippinarum et V. tapetis. Ainsi, le suivi de l’activité PO constitue un biomarqueur pertinent des capacités immunitaires des invertébrés marins dans l’interaction tripartite hôte-pathogène-environnement. / The manila clam, Venerupis philippinarum, was introduced in France in the early 70’s for aquaculture purposes. Since 1987, mass mortality events, caused by the pathogenic bacterium Vibrio tapetis, have occurred in this species along French and European coasts. This vibriose, called Brown Ring Disease (BRD), is considered as a cool water disease. The tripartite interaction between V. philippinarum, V. tapetis and the environment was explored through the role of the enzymatic system of phenoloxidases (POs) in order to better understand the manila clam immune response, the pathogen virulence and also the environment impact and particularly the temperature’s one. POs are oxidoreductase enzymes involved in the synthesis of the melanin and its derivatives and also in the processes of non-self recognition and encapsulation in invertebrates. Firstly, PO activity was biochemically characterized in the hemolymph serum as a laccase-like activity; a minority tyrosinaselike activity was also identified. Clam inoculation with three V. tapetis strains, at various temperatures, highlighted a modulation of the answer of the PO system according to time post-infection and studied compartments. Overall, bacterial infection resulted in an increase in PO activity. However, the PO activity basal level varies between populations and this variability seems to reflect different susceptibility to the BRD. An increase in temperature from 15 to 22°C led to an increase in manila clam immune abilities. The temperature also had an impact on the V. tapetis pathogenicity and this impact was variable according to the strains. PO activity inhibition observed, in vitro, in the presence of bacterial extracellular products underlines the complexity of the interaction between V. philippinarum and V. tapetis. Thus, the monitoring of the PO activity constitutes a relevant biomarker of marine invertebrate immune abilities in the tripartite interaction host-pathogen-environment.

Dans les écosystèmes marins côtiers, l‘activité anthropique et les paramètres naturels induisent chez lesorganismes aquatiques des situations de «multistress». Parmi ces paramètres, deux d‘entre eux ont été étudiés:La contamination métallique et l’infection bactérienne. Dans ce contexte la compréhension des réponsesmoléculaires et physiologiques de l’ormeau rouge Haliotis rufescens a été étudiée, en particulier, face l’impactmétallique (cuivre), et au pathogène (Vibrio parahaemolyticus) en interaction avec des bactéries probiotiquespendant le stade juvénile de ce mollusque en conditions contrôlées. Dans un première temps, une évaluation surl’effet d’un consortium probiotique formé de trois souches bactériennes: Vibrio sp C21, Agarivorans albus F1et Vibrio sp F15 a été testée et les paramètres de survie et de croissance des juvéniles de l’ormeau ont été suivis.Les résultats montrent que les probiotiques augmentent les performances de ces deux paramètres de trait de viecomparés aux individus témoins sans probiotiques. Dans un second temps, une approche génomique parbanques soustractives a été réalisée afin d’identifier les transcrits régulés par l’impact métallique. Cetteméthode a permis d’identifier 108 gènes potentiellement impliqués dans la réponse cellulaire et physiologiquedes organismes face au stress. La cinétique de l’expression génique a été suivie à l’aide de 14 transcrits et leurvariation temporelle pendant la phase critique révèle des régulations parfois complexes et différentielles chezles organismes. Ensuite, les réponses immunitaires des hémocytes d’ormeau face l'exposition au cuivre ont étérecherchées. Les résultats montrent que l’exposition au métal provoque des disfonctionnement importants desactivités hémocytaires. Enfin, l’interaction entre les bactéries pathogènes et probiotiques ont étés étudiées etleurs effets sur le mollusque ont été également recherchés. La présence des bactéries pathogènes régulel’expression génique des transcrits impliqués dans des fonctions physiologiques clés des organismes (lesdéfenses immunitaires et l’état énergétique). De plus, les résultats de cette interaction nous renseignent sur lerôle capital accompli par les bactéries probiotiques sur les performances en termes d’amélioration de la survieet de la croissance des jeunes ormeaux en condition de stress. / In coastal ecosystems, anthropogenic activity and natural factors induce “multi-stress” situations. Inthis study, we focused on two such stress-inducing factors: metal contamination and bacterial infection.Specifically, under controlled conditions, we examined the molecular and physiological responses of redabalone Haliotis rufescens toward the impact of a metal (copper) and a pathogen (Vibrio parahaemolyticus)with respect to this mollusk’s interaction with probiotic bacteria during the juvenile stage. First, we assessed theeffect of a consortium of three probiotic bacterial strains—Vibrio sp. C21, Agarivorans albus F1, and Vibrio sp.F15—on two abalone lifecycle-related parameters, namely, survival and growth. The results showed that,compared to the control treatment (no probiotics), the probiotics improved the performance of these twolifecycle parameters. In a second step, the molecular mechanisms underlying the juvenile abalone response tocopper stress were studied under experimental conditions, using a suppression subtractive hybridizationmethod. This method identified 108 partial sequences of genes involved in the cellular and physiologicalresponses to stress. The kinetics of gene expression were followed using 14 of these transcripts, and theirtemporal variations during the critical phase showed complex and differential regulation exerted by copper inthese organisms. We then investigated the immune response of abalone hemocytes to copper exposure. Theresults showed that exposure to the metal causes a significant dysfunction of hemocyte activities. Finally, theinteraction between probiotics and pathogenic bacteria (transcriptomic and cellular aspects) was studied and theeffects of these bacteria on mollusk performance were also investigated. The presence of pathogenic bacteriawas found to regulate gene expression of transcripts involved in key physiological functions, including thosethat regulate immune responses and energy metabolism. Examination of these interactions thus indicates therole of probiotic bacteria in performance in terms of the improved survival and growth of juvenile abaloneunder stress conditions.

Neste estudo, objetivou-se caracterizar ao nível molecular os vibrios isolados de amostras de água do mar, plâncton e bivalves do Canal de São Sebastião (n=78), Baixada Santista (n=37) e Ubatuba (n=17), analisar a susceptibilidade aos antibióticos e os principais genes associados à virulência. Observou-se sensibilidade à ciprofloxaxina, meropenem e ácido nalidíxico, resistência à ampicilina e à cefalotina, e alta porcentagem de múltipla resistência (Ubatuba: 64,7%; Baixada Santista:48,6%; Canal de São Sebastião: 43%) aos antimicrobianos. Quatro isolados foram positivos para o gene de virulência stn/sto. Por MLSA, foi possível identificar V.alginolyticus, V.fluvialis, V.campbellii e V.harveyi em Ubatuba; V.fluvialis, V.alginolyticus, V.campbellii, V.rotiferianus, V.harveyi, V.diabolicus, V.atypicus, V.coralliilyticus, V.maritimus, V.parahaemolyticus e V.tubiashii no Canal de São Sebastião; e, V.alginolyticus, V.parahaemolyticus, V.rotiferianus, V.campbellii, V.harveyi, V.communis, V.maritimus, V.fluvialis, V.fortis, V.natriegens e V.navarrensis na Baixada Santista. / The aim of this study was to characterize at the molecular level Vibrio species isolated from seawater, plankton, bivalves samples from Canal de São Sebastião (n=78), Baixada Santista (n=37) and Ubatuba (n=17), to analyze antimicrobial susceptibility and the major virulence-associated genes. The results showed ciprofloxacin, meropenem, nalidixic acid sensitivity, ampicillin, and cephalothin resistance, and a significant percentage of multidrug resistance (Ubatuba: 64.7%; Baixada Santista: 48.6%; Canal de São Sebastião: 43%). Four seawater isolates were found positive for the stn/sto virulence gene. MLSA allowed the identification of V.alginolyticus, V.fluvialis, V.campbellii, V.harveyi in Ubatuba; V.fluvialis, V.alginolyticus, V.campbellii, V.rotiferianus, V.harveyi, V.diabolicus, V.atypicus, V.coralliilyticus, V.maritimus, V.parahaemolyticus and V.tubiashii in Canal de São Sebastião, and V.alginolyticus, V.parahaemolyticus, V.rotiferianus, V.campbellii, V.harveyi, V.communis, V.maritimus, V.fluvialis, V.fortis, V.natriegens, and V.navarrensis in Baixada Santista.

D. Tech. (Department of Chemistry, Faculty of Applied and Computer Sciences), Vaal University of Technology. / Cholera is a waterborne disease caused by toxigenic strains of Vibrio cholerae. The spread of cholera in developing countries has largely been imputed to the unavailability of proper water treatment and sanitary infrastructure as well as poor hygiene. In order to prevent the contraction and spread of cholera the use of solar disinfection (SODIS) to treat water in waterborne endemic communities has been recommended by the World Health Organization (WHO). SODIS is a water sterilizing method that relies on natural sunlight to improve the microbiological quality of water. During SODIS the culturability of the water contaminating microorganisms is inactivated by the ultraviolet component of solar radiation. The success of SODIS treatment of water in alleviating the risks associated with the contraction of waterborne diseases such as cholera has been attributed to the effectiveness, with which the water is treated, simple application as well as low cost of materials required. Currently SODIS research has been dominated by studies geared towards understanding how the microbial inactivation occurs, enhancement of the disinfection process and health impact assessments. However, little to no research has been directed towards exploring the role played by the immune system following the consumption of the solar irradiated water pathogens such as V. cholerae. SODIS of microorganisms in water results in immunologically important microbial states and components that could induce an immune reaction or response. In view of the role of dendritic cells in shaping an immune response, the effect that solar irradiated V. cholerae in water has on the immunochemistry of the dendritic cells in vitro was investigated. Prior to the stimulation of the dendritic cells with the solar irradiated cultures of V. cholerae, the first objective required an evaluation on the impact that solar irradiation has on the production and secretion of the cholera toxin by V. cholerae in water. The results from this evaluation showed that solar ultraviolet radiation was incapable of inducing the secretion of the cholera toxin. Furthermore, there was extensive DNA degradation in the solar irradiated cultures of V. cholerae. The second objective was to investigate the ability for solar irradiated cultures of V. cholerae in water to induce the phenotypic maturation of immature dendritic cells in vitro. In order to achieve this objective, solar and non-solar irradiated, chemically/ heat inactivated and phosphate buffered saline (PBS) prepared cultures of V. cholerae as well as lipopolysaccharide (LPS) and cholera toxin-β (CTB) subunit were each used to stimulate immature dendritic cells. After 48 hours of stimulation the dendritic cells were assessed for the expression of CD54, CD80, CD83, CD86, MHC-I and MHC-II on their cell membrane. The results showed an increase in the expression of all the maturation phenotypic markers with CD54, CD86 and MHC-I being the most prominent ones on the surface of the dendritic cells stimulated with solar irradiated cultures of V. cholerae. The third objective was to assess the profile of the cytokines and chemokines secreted by the dendritic cells following their stimulation with solar and non-solar irradiated, chemically/heat inactivated and PBS prepared cultures of V. cholerae as well as LPS and CTB subunit. After 48 hours of dendritic cell stimulation the tissue culture media from each treatment was quantitatively and qualitatively analysed for the presence of interleukin (IL)-1α, IL-1β, IL-6, IL-7, IL-10, IL-12p40, IL-12p70, IL-15, IL-23, IL-27, macrophage inflammatory protein (MIP)-1α, MIP-1β, MIP-2, Regulated on Activation, Normal T cell Expressed and Secreted (RANTES) and tumor necrosis factor (TNF)-α. The analysis revealed that solar irradiated cultures of V. cholerae induced dendritic cells to secrete significant levels of pro-inflammatory cytokines in comparison to the unstimulated dendritic cells. Furthermore the profile of the cytokines and chemokines secreted by the dendritic cells in response to the solar irradiated cultures of V. cholerae in water was similar to that required to induce a T- helper (Th) Th2 immune response. The fourth objective was to assess the expression of the toll like receptor (tlr) genes by the dendritic cells following their stimulation with solar and non-solar irradiated, chemically/heat inactivated and PBS prepared cultures of V. cholerae as well as LPS and CTB subunit. After 48 hours of stimulation total RNA was extracted from the dendritic cells and subjected to real time quantitative polymerase chain reaction (RT qPCR) assay for tlr 1, 2, 3, 4, 5, 6, 9, 11, 12 and 13. The results showed no significant increase or decrease in the expression of most tlr genes in comparison to the unstimulated dendritic cells. This observation is synonyms with dendritic cell maturation. Taken together these findings show that solar irradiated cultures of V. cholerae were able to induce the maturation of immature dendritic cells in vitro. Furthermore dendritic cells stimulated with solar irradiated cultures of V. cholerae produced pro-inflammatory cytokines and chemokines. The results from this study suggests that the consumption of SODIS treated could provide immunological benefits.

Vibrio cholerae is the cholera causing agent, divided into two biotypes, including the classical biotype and ElTor biotype. Both of these biotypes caused cholera epidemics in the world. The classical biotype caused 6th cholera pandemic (from 1921 to 1961), and ElTor biotype caused 7th cholera pandemic (from 1961 to the 70s). Haemolysin A, a hemolytic protein of V. cholerae ElTor biotype, is encoded by the hlyA gene. This gene is often used for analyzing genetic relationship between strains in the same species or between species in the same Vibrio genus. Results of analyzing nucleotide and amino acid sequences of hlyA gene of V. cholerae strain causing cholera in Vietnam (named hlyA.VN) showed that: the hlyA.VN gene sequence was similar to the hlyA gene sequences of V. cholerae strains of the 6thand 7thcholera epidemics. The hlyA gene of the 6th cholera epidemic strain was deficient in 11 nuleotides (this deficiency leading to the loss of 4 amino acids in the haemolysin A protein) comparing to hlyA.VN gene and hlyA gene of the 7th cholera epidemic strain. The results of genetic distance analysis as well as phylogenetic tree construction also confirmed V. cholerae causing cholera in Vietnam was closely relationship to the strains causing cholera pandemics in the world. It is great significance for the surveillance of molecular epidemiology to prevent cholera effectively. / Vibrio cholerae là tác nhân gây bệnh tả, được chia thành hai typ sinh học, đó là typ sinh học cổ điển và typ sinh học ElTor. Cả hai typ này đã từng gây ra các đại dịch tả trên thế giới. Typ sinh học cổ điển đã từng gây ra đại dịch tả lần thứ 6 (từ năm 1921 đến 1961), còn typ sinh học ElTor đã từng gây ra đại dịch tả lần thứ 7 (từ 1961 đến những năm 70). Haemolysin A, một protein có chức năng làm tan máu của V. cholerae typ sinh học ElTor, được mã hóa bởi gen hlyA. Gene này thường được sử dụng cho các phân tích quan hệ di truyền giữa các chủng trong cùng một loài V. cholerae hay giữa các loài trong cùng một chi Vibrio. Kết quả phân tích trình tự nucleotide và axit amin gen hlyA của chủng V. cholerae gâybệnh ở Việt Nam (hlyA.VN) cho thấy: trình tự gen hlyA.VN có sự tương đồng lớn với trình tự gen hlyA của chủng gây đại dịch tả 6 và 7. Gen hlyA của chủng gây đại dịch tả 6 bị thiếu hụt 11 nuleotide (sự thiếu hụt này dẫn tới sự mất đi 4 axit amin trong phân tử haemolysin A) so với gen hlyA.VN và gene hlyA của chủng gây đại dịch tả 7. Kết quả phân tích khoảng cách di truyền cũng như xây dựng cây phát sinh chủng loại cũng đã khẳng định: chủng gây bệnh ở Việt Nam có quan hệ rất gần với các chủng gây đại dịch tả trên thế giới. Nhận định này có ý nghĩa rất lớn đối với công tác giám sát dịch tễ học phân tử để ngăn chặn bệnh tả hiệu quả.

Les éléments intégratifs et conjugatifs (ICE) de la famille SXT/R391 sont reconnus pour leur rôle prépondérant dans la propagation de la résistance aux antibiotiques parmi des populations de Gammaproteobactéries, en particulier chez Vibrio cholerae, l’agent pathogène causant le choléra. Ces éléments génétiques autonomes possèdent tous les gènes nécessaires à leur dissémination au sein d’une population bactérienne et s’intègrent normalement dans un site précis du chromosome bactérien. L’activateur SetCD et la machinerie de conjugaison encodée par les ICE permettent non seulement leur transfert conjugatif, mais également la mobilisation d’îlots génomiques, les MGI (mobilizable genomic islands). Lorsque leur transfert est enclenché sans excision au préalable, les MGI et les ICE peuvent mobiliser plusieurs centaines de kb d’ADN chromosomique adjacent à leurs sites d’insertions. Cet ADN mobilisé peut alors recombiner avec le génome de la cellule réceptrice, aboutissant à des remplacements d’allèles. En plus du squelette de gènes conservés de cette famille d’ICE, ces éléments portent une cargaison d’ADN variable qui peut coder pour des fonctions adaptatives potentiellement avantageuses pour l’hôte bactérien. Les ICE SXT/R391 portent également les gènes codant pour un système de recombinaison qui promeut la diversité de la famille en générant des ICE hybrides. Ces éléments mobiles sont extrêmement stables dans les populations bactériennes. Cette stabilité est attribuable à leur intégration au chromosome et à plusieurs composantes qu’ils contiennent, par exemple les systèmes toxine-antitoxine de la cargaison d’ADN variable ou encore le système conservé de partition des éléments excisés. La majorité des gènes portés par les ICE SXT/R391 est contrôlée par leur système de régulation qui se situe au cœur de ce projet doctoral. Ce système de régulation comprend SetR, le répresseur responsable du maintien de l’état quiescent dans lequel l’ICE est intégré au chromosome et est propagé verticalement dans la population bactérienne, c’est-à-dire au rythme de la réplication chromosomique et de la division cellulaire. Lorsque l’ADN bactérien est endommagé, il y a activation de la réponse SOS de réparation de l’ADN par RecA, un facteur de l’hôte, qui induit parallèlement l’autoprotéolyse de SetR, levant ainsi la répression exercée sur les gènes setC et setD. Ces derniers codent pour SetCD, le complexe activateur des ICE SXT/R391 qui active l’expression de la machinerie de conjugaison ainsi que d’autres fonctions codées par ces ICE. Ce projet doctoral a permis l’identification de nouvelles composantes importantes pour la régulation des ICE SXT/R391. Premièrement, nous avons généré par génie génétique plusieurs mutants qui ont permis de caractériser CroS par des essais de transfert conjugatif, de PCR quantitatif en temps réel (qRT-PCR) et d’expression avec le gène rapporteur lacZ. Nous avons déterminé que CroS est un régulateur transcriptionnel qui, avec SetR, constitue un interrupteur génétique permettant l’induction du transfert conjugatif dépendante de RecA. Nous avons également validé par gel à retardement la liaison par SetR et CroS d’un site opérateur additionnel. Des essais β galactosidase ont montré que ce site contribue à la répression des gènes croS, setC et setD. De plus, les résultats de ce projet doctoral ont clarifié certains points concernant la régulation par SetCD. Des essais d’immunoprécipitation de la chromatine couplée à la digestion avec une exonucléase (ChIP-exo) combinés avec le séquençage de l’ARN (RNA-seq) et la détermination des sites +1 d’initiation de la transcription (5’-RACE et extension d’amorces) ont permis d’établir le régulon de SetCD chez les ICE SXT/R391 et chez les MGI qu’ils mobilisent. La nécessité de SetCD dans la cellule réceptrice pour qu’il y ait intégration de l’ICE de manière site-spécifique dans l’extrémité 5’ du gène prfC a été mise en évidence à l’aide de la construction de mutants, d’essais de transfert conjugatif, de buvardage de type Southern, d’électrophorèse en champs pulsés et de PCR en temps réel. Nous avons également observé, grâce à des essais de PCR quantitatif et d’activité β galactosidase, une boucle de rétroaction positive médiée par l’activation de l’excision et de la réplication de l’ICE par SetCD. En somme, ce projet doctoral a mené à une meilleure compréhension des composantes et des mécanismes en scène pour la gouvernance de cette famille d’ICE qui sont, entres autres, d’importants vecteurs de la dissémination des résistances aux antibiotiques.

The spread of cholera is always associated with contaminated food or water and this is the reason this disease has been endemic in developing countries for centuries due to their lack of proper sanitation facilities and poor or no infrastructure for sewage systems. Cholera can spread quickly and sporadically after any natural disaster that destroys the sewage system or safe drinking water supply of both developed and undeveloped countries. In Southeast Asia in December 2004 and in Pakistan and Haiti 2010, cholera outbreaks followed the natural disasters; with most of the cholera victims being children. Although it is known that the best way to prevent cholera outbreak is the development of the infrastructure, provision of a safe drinking water supply and proper sanitation, this is a very long-term process, and most of the developing countries cannot afford such improvements. These situations can be made worse by natural disasters. Therefore there is a pressing need for the development of a cholera vaccine and there have been numerous research projects working towards this end for several decades. A few of them have been successful to date but because of the severe side effects and narrow range of protection, more effective and wider range vaccine development is still ongoing. In this study, crystallographic and enzymatic studies have been carried out on several novel proteins involved in the control of the production of the factors required for quorum sensing. Quorum sensing is a process in which bacterial cells communicate among themselves by the synthesis, release and detection of small chemical compounds called autoinducers. In this work, structural analysis was carried out on proteins involved in the synthesis and detection of the major autoinducer of Vibrio cholerae, named CAI-1. The crystal structure of CqsA involved in CAI-1 synthesis has been successfully solved and its enzymatic properties have been characterized. The structure of one domain of the cytoplasmic region of the CAI-1 receptor CqsS was also elucidated, and other domains were expressed. The crystal structure of another enzyme (VCA0859, an aldo-keto reductase) thought to have been involved in the synthesis of CAI-1 was also determined. Another protein named VCA0939 was also studied, due to its importance in biofilm development, and its ability to control quorum-sensing in an alternative pathway in the mutated version of pathogenic strains of V. cholerae that were responsible for the seventh cholera pandemic. The aim of this project was to understand the three dimensional structure of some proteins that are involved in quorum sensing and control of the expression of virulence genes for the pathogenesis of V. cholerae. Understanding the three dimensional structure of the proteins and the mode of autoinducer binding to its specific receptor could be highly valuable in the development of a chemical compound that could lead to the discovery of a novel drug with the ability to target cross species specification.

Tilapia culture in Indonesia was started with the Mozambique Tilapia (Oreochromis mossambicus) in the 1930’s, and the Nile Tilapia (Oreochromis niloticus) the 1960’s. The genetic improvement program of the Nile Tilapia, has led Indonesia to be one of the main tilapia producers in the world. On the other hand, shrimp aquaculture in the country was not started until the 1960’s, it became more popular after the eye ablation technology for broodstock maturation was developed in the early 1980’s. The first experimental study was conducted to investigate the feasibility of low salinity shrimp farming in a polyculture system with tilapia. Polyculture increased the survival for shrimp (77% compared to 62%), but at the same time decreased the survival of tilapia (87% compared to 97%). Together, the data on survival, specific growth rates, and feed conversion ratios showed that the shrimp performed well at low salinity. The second experimental study investigated the feasibility of brackishwater shrimp farming in a polyculture system with tilapia. Polyculture increased the survival for shrimp (82% compared to 65%), and had higher survival for the tilapia (60% compared to 43%). The Red hybrid Tilapia strain used in the study experienced mortalities after one month, suggesting the need for a salt tolerant strain. The presence of tilapia stimulated the growth of microalgae (Chlorella dominance), promoted higher numbers of heterotrophic bacteria in the water, and had lower presumptive vibrios on TCBS agar. A challenge study was conducted by mixing pathogenic luminescent Vibrio harveyi UAZ-651 into shrimp and tilapia feed. The survival of shrimp in monoculture were significantly lower (20%) compared to in polyculture systems (75 - 95%). Mortality was not found in tilapia. Based on 16S rRNA gene sequence, shrimp monoculture water was dominated by marine Vibrio spp., while the polyculture system had Bacillus spp. and Vibrio spp. with high homology to V. cholerae. The presence of Bacillus spp. which produce a lactonase enzyme AiiA, seems to inhibit vibrio growth. While providing advantages, shrimp-tilapia polyculture might also contribute to streptococcosis transmission. Injecting shrimp with Streptococcus iniae and S. agalactiae resulted in mortalities. S. iniae caused higher mortality in the shrimp cultured in 20 ppt (40%) compared to 10 ppt (20%), and no mortality in 5 ppt. S. agalactiae caused higher mortality in 5 ppt (40%) compared to 10 ppt (20%) and 20 ppt (20%). Quorum sensing (QS) is a density dependent cell to cell communication process in bacteria. Based on challenge studies in shrimp, the luminescent Vibrio harveyi BB120 wild-type strain caused 75-90 % mortality through injection of 106 CFU/shrimp. The mortality patterns in the QS mutants suggest that QS defined, when specific virulence genes were expressed or repressed. As QS in V. harveyi consists of three different circuits, further experiments deployed six mutants lacking either a synthase or a receptor for each circuit. The highest survival in the CqsS (a receptor for CAI-1 circuit) mutant group indicates that the CAI-1 circuit is the most crucial for virulence, followed by the AI-2 and HAI-1 cascades. Chitin acquisition and oxygen scavenging may be two reasons for luminescence in V. harveyi evolution and why they infect shrimp.

Vibrio vulnificus are marine bacteria causing fatal septicemia through wound infections or consumption of contaminated seafood. V. vulnificus is an excellent model for the study of surface polysaccharides, as it is capable of synthesizing capsular polysaccharide (CPS), lipopolysaccharide (LPS) and exopolysaccharide (EPS). V. vulnificus strains exhibit a multitude of carbotypes that evolve through unknown mechanisms. CPS is a confirmed virulence factor, but the genetics of its biosynthesis are unknown. The main objective of these experiments was to gain insight into the biosynthesis, regulation and evolution of ATCC 27562 outer surface polysaccharides. A miniTn10 transposon (Tn) system was used for mutagenesis and single insertions were confirmed through Southern analysis. A novel 25 kb CPS biosynthesis locus was identified through sequencing of regions surrounding Tn insertions; a region encoding putative LPS core biosynthetic functions was identified adjacent to the CPS cluster. The CPS locus contained features of O-antigen biosynthetic loci and was unusual in carrying characteristics of both group I and IV capsular biosynthetic loci. Mutations in this region resulted in elimination of CPS and LPS, and both were shown to be dependent on the activity of the polymerase Wzy. Evidence is presented here supporting horizontal transfer (HT) as a contributor to V. vulnificus CPS evolution. CPS regions of V. vulnificus 27562, YJ016 and CMCP6 contain strain specific genes surrounded by conserved regions, suggestive of HT. Moreover, a CPS locus virtually identical to that of 27562 was discovered in Shewanella putrefaciens strain 200. 27562 CPS is distinctive as it contains N-acetylmuramic acid. Genes encoding murA and murB activities were identified within the cluster and shown to be functionally redundant, supporting HT acquisition of this region. A screen of V. vulnificus gDNA library using CPS biosynthesis and transport mutants identified a cyclic diguanylate cyclase, dcpA. dcpA-mediated increase in cyclic diguanylate lead to EPS production, rugosity phenotypes and enhanced biofilm formation. Interestingly, virulence and motility were not affected suggesting complexity of cyclic diguanylate regulation in V. vulnificus, supported by the large number of cyclic diguanylate related proteins in Vulnificus strains.