The waterborne bacterial pathogen Vibrio cholerae uses a process of cell-to-cell communication called quorum sensing (QS) to coordinate transcription of four sRNAs (Qrr1-4; quorum regulatory RNAs) in response to changes in extracellular QS signals that accumulate with cell density. The Qrr sRNAs are predicted to negatively control translation of several mRNAs, including hapR, which encodes the master QS transcription factor that controls genes for virulence factors, biofilm formation, protease production, and DNA uptake. The Qrr sRNAs are also predicted to positively control vca0939, which encodes a GGDEF family protein that promote biofilm formation by elevating intracellular levels of the second messenger molecule c-di-GMP. Using complementary in vivo, in vitro, and bioinformatic approaches, I showed that Qrr sRNAs base-pair with and repress translation of the mRNA encoding HapR. A single nucleotide mutation in Qrr RNA abolishes hapR pairing and thus prevents cholera toxin production and biofilm formation that are important in disease, and also alters expression of competence genes required for uptake of DNA in marine settings. I also demonstrated that base-pairing of the Qrr sRNAs with vca0939 disrupts an inhibitory structure in the 5' UTR of the mRNA. Qrr-activated translation of vca0939 was sufficient to promote synthesis of c-di-GMP and early biofilm formation in a HapR-independent manner. Thus, these studies define the non-coding Qrr sRNAs as a critical component allowing V. cholerae to sense and respond to environmental cues to regulate important developmental processes such as biofilm formation.

As technology and engineering allow mankind to survey nature at finer scales, the importance of bacteria has been elucidated in their metabolic diversity, ability to transfer genetic information, involvement in biogeochemical cycling, and sheer abundance. With an individual domain of life unto themselves, this diverse group of microorganisms plays an integral role in facilitating life on land and in the oceans, and is second only to viruses in abundance on Earth. They carve niches in a wide range of environments, including those inhospitable to other life forms, and reside in concert or to the detriment of other microbes and/or hosts they inhabit. Solely culturing microorganisms has proven to severely underestimate microbial numbers, capturing less than 1% of marine microbes. However, the advent of molecular methods has revealed the ubiquity, abundance, and diversity of bacteria. Higher organisms have evolved varying degrees of ecological relationships with bacteria, ranging from mutualism to parasitism. As the microbial players are elucidated, determining the specificity and functional roles of these bacteria is a critical and exciting scientific question. The microbiome of corals is an interesting model of complexity, with the animal host striving to maintain a delicate symbiosis, and using its microbiota to assist in nutrient cycling and protection. A contrasting example to the well-studied cnidarians are ctenophores, gelatinous organisms that are globally distributed in the world's oceans, yet the literature contains few studies on microbiota associated with this unique group of animals. Since ctenophores are one of the earliest diverging, extant multicellular animals, these unique organisms could prove to be a better model system than cnidarians. The first project in this dissertation examined spatial structure of bacteria across wild, healthy colonies of the coral Montastraea annularis. Microscale heterogeneity of the bacterial community was observed in coral mucus samples collected tens of centimeters apart on the same coral colony, which has implications for sampling strategies in microbiological studies, and impacts the application of the bacterial community as a proxy for determining coral condition in coral restoration projects. The second project looked at the linkages between coral bacterial community composition and zooxanthellae clade in Pocillopora damicornis, and results suggested that clade is not a major factor in influencing coral bacterial community composition. Sample location was also considered in the P. damicornis bacterial surveys and determined to be driving community structure. The third project is the first study to describe bacteria associated with ctenophores (Mnemiopsis leidyi and Beroe ovata). The ctenophores contained bacterial communities that were distinct from the surrounding water column, and temporal variability was exhibited by bacteria associated with the ctenophores. Exploring microbial landscapes in cnidarians and ctenophores to understand microbial roles in health and disease is the uniting theme of the three separate projects that will be discussed in this dissertation.

Atlantic cod (Gadus morhua L.) is one of the most promising new fish species introduced to cold water aquaculture due to the large established market in Europe and the USA and the decline in wild stock. So far, the production of farmed cod has been relatively low, with the main hindrance due to diseases. Vibrio anguillarum has been recognised as the biggest disease problem of farmed cod and has slowed the development of a successful cod aquaculture industry. When the first incidences of V. anguillarum occurred in cod aquaculture, vaccines designed for vibriosis in Atlantic salmon (Salmo salar L.) were used in an attempt to combat the disease. However, these vaccines did not provide sufficient protection, possibly because they lacked serotype O2b, which is known to affect cod and to a lesser extent salmonids. Recently, vibriosis vaccines specifically designed to protect Atlantic cod have been formulated, but outbreaks of vibriosis in vaccinated fish are still being reported, suggesting that these formulations are inadequate. The aim of this project was to develop a whole cell inactivated vaccine formulation specifically tailored to protect Atlantic cod against Vibrio anguillarum. The serological classification of V. anguillarum was first investigated by producing a set of monoclonal antibodies (mAbs). Using lipopolysaccharides (LPS) extracted with butan-1-ol, 4 mAbs were selected and shown to react specifically with V. anguillarum serotypes O1, O2a and O2b. A collection of over 150 V. anguillarum isolates were screened using these, which revealed that most of the isolates had been previously correctly classified. A new sub-serotype of V. anguillarum O2 was identified from isolates recovered from outbreaks of vibriosis in Norway as well as Scotland. This new sub-serotype was referred to as O2d since the subserotype O2c has been recently identified in vibriosis cases from Atlantic cod. However, it was shown that the O2c sub-serotype might not belong to the O2 serotype, but in fact belongs to another serotype. To protect Atlantic cod against all the V. anguillarum serotypes (and subserotypes) which they are susceptible to, it is recommended that isolates from serotypes O1, O2a, O2b, O2c and O2d should all be included in a bacterin vaccine for cod. In order to determine which isolates from each of the serotypes to include in the vaccine, a variety of virulence factors of V. anguillarum were investigated in vitro. The interaction of some candidate isolates from O1, O2a and O2b serotypes (O2c and O2d were not identified at the time this part of the study took place) with cod phagocytic cells were studied using flow cytometry. Phagocytosis and respiratory burst of cod macrophages and neutrophils as well as cod serum killing of V. anguillarum were quantified. It was found that isolates within the same serotype displayed varying degrees of resistance to phagocytosis and the subsequent respiratory burst activity as well as that all the V. anguillarum strains tested were resistant to Atlantic cod serum killing. These in vitro assays were found to be very useful in assessing the virulence of V. anguillarum. The isolate within each serotype eliciting the highest percentage of positive phagocytic cells was selected in order to increase the antigen presentation pathway, thus theoretically enhancing the protection elicited by the vaccine. A multivalent formalin-inactivated non-adjuvanted vaccine was prepared which included all the serotypes previously described and was injected intraperitoneally into Atlantic cod. A bath challenge was performed on vaccinated and mock-vaccinated fish, 6 weeks post immunisation, using V. anguillarum isolates from the serotypes O2b, O2c and O2d that were not included in the vaccine. An excellent level of protection was obtained against O2b and O2d (relative percentage survival 100% and 96.4%, respectively), but the challenge with the sub-serotype O2c isolate did not produce any mortality in the control group and needs to be repeated. The vaccine formulation was very efficient at protecting Atlantic cod against vibriosis but further challenges need to be performed with other serotypes included in the vaccine (O1 and O2a), as well as with more isolates from the O2b, O2c and O2d sub-serotype. To conclude, Atlantic cod is a species which will certainly have a major influence in marine aquaculture, but many areas have to be improved. The development of an effective and broad range vaccine to protect cod against Vibrio anguillarum offers another advance which should help Atlantic cod aquaculture to reach its full potential.

<p>Availability of genome sequences for numerous bacterial species comprising of different bacterial strains allows elucidation of species and strain specific adaptations that facilitate their survival in widely fluctuating micro-environments and enhance their pathogenic potential. Different bacterial species use different strategies in their pathogenesis and the pathogenic potential of a bacterial species is dependent on its genomic complement of virulence factors. A bacterial virulence factor, within the context of this study, is defined as any endogenous protein product encoded by a gene that aids in the adhesion, invasion, colonization, persistence and pathogenesis of a bacterium within a host. Anecdotal evidence suggests that bacterial virulence genes are undergoing diversifying evolution to counteract the rapid adaptability of its host&rsquo / s immune defences. Genome sequences of pathogenic bacterial species and strains provide unique opportunities to study the action of diversifying selection operating on different classes of bacterial genes.</p>

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.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv., 2002. / Härtill 4 uppsatser.

Vibrio anguillarum infects many fish species in aquaculture, reducing farm productivity and negatively impacting fish welfare. Deeper understanding of the biology of V. anguillarum, particularly during infections in vivo, will help to improve disease prevention and control. Thus, the aim of this thesis was to provide further insight into the infection biology of V. anguillarum with a view to identifying better ways to reduce the impact of this pathogen in aquaculture. Conventional studies on virulence, particularly those aiming to identify novel virulence factors, often employ transposon mutagenesis where the functions of individual genes in the bacterium are disrupted. These mutant libraries are screened to identify those with attenuated virulence, allowing subsequent identification of the gene responsible. Usually the native fish host would be used but such studies are increasingly difficult to perform due to regulations on vertebrate experiments and ethical concerns. As a result, alternative invertebrate hosts are now an important means to studying microbial infections, but few models have been assessed for bacterial pathogens of fish. In this thesis, larvae of the greater wax moth Galleria mellonella were evaluated as an alternative host to investigate V. anguillarum virulence. Wild-type V. anguillarum isolates killed larvae in a dose-dependent manner, replicated in the haemolymph, and larvae infected with a lethal dose of bacteria could be rescued by antibiotic therapy, thus indicating that V. anguillarum established an infection in G. mellonella. Crucially, virulence of 11 wild-type V. anguillarum isolates correlated significantly between larva and Atlantic salmon infection models, and studies with isogenic mutants knocked out for various virulence determinants revealed conserved roles for some in larva and fish infections, including the pJM1 virulence plasmid and rtxA toxin. Thereafter, 350 strains from a V. anguillarum random transposon insertion library were screened for attenuated virulence in G. mellonella. In total, 12 strains had reduced virulence and in these mutants the transposon had inserted into genes encoding several recognised and putative virulence factors, including a haemolytic toxin (vah1) and proteins involved in iron sequestration (angB/G and angN). Importantly, the transposon in one strain had inserted into an uncharacterised hypothetical protein. Preliminary investigations found this putative novel virulence factor to contain a GlyGly-CTERM sorting domain motif, with sequence similarity to VesB of Vibrio cholerae which is involved in post-translational processing of cholera toxin. Finally, three transposon insertion libraries were mass sequenced on a MiSeq platform to identify V. anguillarum genes lacking transposon insertions. These genes were assumed to be ‘required’ for viability in the conditions under which the mutants were selected, in this case tryptone soya agar. In total, 248 genes lacked a transposon insertion and were the putative ‘required’ genes, and these may be important chemotherapeutic targets for new approaches to combat V. anguillarum infections. This thesis has furthered our understanding of the biology of the important fish pathogen V. anguillarum using an ethically acceptable approach, and the findings may assist with new ways to reduce the burden of this bacterium in aquaculture.

Cholera is a water-borne disease that continues to ravage resource poor communities around the world especially those in developing countries. The disease is caused by Vibrio cholerae microorganisms whose natural habitat is the aquatic ecosystem. It is believed that this microorganism prior to becoming the primary cause of cholera acquired virulence factors expressed by two separate genetic elements. These genetic elements are known as VPIФ and CTXФ were acquired in that order for known physiological reasons. However only V. cholerae in possession of the CTX genetic element are capable of causing cholera disease. At present only two serotypes are known to have the ability to cause cholera and these are V. cholerae serotypes O1 and O139. SODIS (Solar disinfection) is an extremely low cost refined technology that can be used for the disinfection of water especially in areas where there is a considerable amount of sunshine. Although this technology is a composite of various factors the underlying principle is the use of solar ultraviolet radiation (SUVR). The preliminary target of SUVR is the cytoplasmic membrane and this was confirmed by flow cytometric analysis. The consequences of leaky cytoplasmic membrane include cellular death to the microorganism as well as an increase in cholera toxin secretion. The main objective of this study was to investigate the effect of solar ultraviolet radiation on the growth of V. cholerae and on the secretion of cholera toxin and to provide supporting information for the use of SODIS in South Africa while observing the possible role that climate may play in the onset of cholera disease. The initial part of the study evaluated the culturability, biomass increase and cholera toxin secretion in both a nutrient poor and a nutrient rich media by two toxigenic and one non toxigenic strain of V. cholerae. A series of pH and temperature combinations were used to achieve this objective. The result revealed that the microorganisms survived in both media. An increase in biomass was observed for all the bacteria grown in the nutrient rich media whereas in the poor nutrient media the bacteria remained culturable but no increase in biomass was observed. Interestingly lower temperatures seemed to provide more optimal growth conditions while high temperature on most occasions favoured cholera toxin secretion, in both media.The second part of the study required the exposure of the microorganisms to SUVR. A SODIS approach was used with a few modifications. The V. cholerae strains were exposed to solar radiation during all the seasons of the year. Evaluation of the viability, the increase in biomass and the detection of cholera toxin secretion was determined after each exposure to solar radiation. The results seem to suggest that the effect of SUVR depended on the season of the year, the nature of the media, strain, solar conditions and in the duration of solar exposure, in no particular order. The secretion of cholera toxin was mainly dependent on the media used, the season of the year and on the serotype of the strain. This study represents the first report on the evaluation of SUVR for the disinfection of water under South African conditions (Pretoria area) during all seasons of the year with variations in solar radiation levels and temperature. Furthermore what actually happened to V. cholerae during solar exposure in terms of cell morphology, cell viability and secretion of cholera toxin is also reported and this can give an insight of the possible role that SUVR may play in the onset of cholera. The main recommendation emanating from this study is the sensitisation of communities worldwide about the capacity that, SUVR carries to lighten the burden of communicable water borne diseases especially, in resource limited areas through the implementation of SODIS. / Life and Consumer Sciences / M. Sc. (Life Science)

Mytilus edulis est un mollusque bivalve de grand intérêt économique et écotoxicologique. Cette espèce sentinelle est connue pour sa résistance aux contaminants chimiques et biologiques. Néanmoins, depuis quelques années la moule bleue est touchée par des mortalités dans les élevages des Pertuis Charentais ayant pour dénominateur commun la présence de bactéries virulentes de type Vibrio. Le premier axe de cette thèse décrit les interactions des isolats de V. Splendidus avec la moule bleue au niveau cellulaire et physiologique. Les infections expérimentales ont permis la sélection de deux isolats bactériens affiliés à V. splendidus/V. hemicentroti : une souche virulente codée 10/068 1T1 et une souche inoffensive codée 12/056 M24T1. Ces deux bactéries ont été marquées à la GFP et validées en tant que modèles authentiques d’exposition à travers leurs caractéristiques de croissance et de virulence. Par ailleurs, V. hemicentroti 10/068 1T1 est capable d’altérer différentes fonctions hémocytaires incluant la motilité, l’adhésion, l’internalisation, la production de ROS, la maturation du phagosome et la viabilité contrairement à la bactérie non virulente. Les produits extracellulaires bactériens semblent être toxiques et inhibent certaines réponses cellulaires (internalisation et production de ROS). Enfin, nous avons reproduis avec succès l’infection des animaux par le pathogène via un modèle expérimental de cohabitation. Le suivi de l’infection montre que V. hemicentroti 10/068 1T1 a pour cible principale les branchies. Le deuxième axe explore le fonctionnement du système MXR (MultiXenobiotic Resistance) chez la moule bleue. La séquence codante complète d’un nouveau transporteur ABCG2 a été établie et la protéine résultante a été identifiée. La caractérisation moléculaire montre la présence du transcrit dans les hémocytes ainsi que dans les branchies et son homologie avec les autres protéines appartenant à diverses espèces. L’utilisation des sondes fluorescentes bodipy prazosin et pheophorbide A, combinées avec des bloqueurs spécifiques, démontre l’activité d’efflux de ce transporteur et son hétérogénéité dans les tissus et cellules. Par ailleurs, il est également démontré que l’expression des trois transporteurs ABC (abcb, abcc, abcg2) identifiés chez la moule bleue est modulée par les contaminants chimiques. Les animaux exposés au BaP au laboratoire ou prélevés sur un terrain contaminé montrent une surexpression des transcrits abc dans les branchies et une sous expression dans les hémocytes. La saisonnalité, sur le terrain, a également un effet sur les niveaux des transcrits et interfère avec les réponses liées aux contaminants. Seul le transporteur abcb exprimé dans les branchies n’est pas affecté par des variations saisonnières et montre une surexpression dans le site contaminé tout au long de l’année. En conclusion, nos résultats démontrent la vulnérabilité de la moule bleue face un pathogène. L'impact immunotoxique des xénobiotiques et le rôle que peuvent jouer les transporteurs ABC dans le fonctionnement du système immunitaire des moules reste à explorer. / Mytilus edulis is a bivalve mollusc representing an economic and ecotoxicological interest. This sentinel species is known for its resistance to chemical and biological contaminants. However, for few years, abnormal mortality events have been reported for farmed blue mussels in France where different Vibrio strains were isolated. The first section of this thesis describes cellular and physiological interactions of V. Splendidus isolates with the blue mussel. Experimental infections allowed the selection of two isolates affiliated to V. splendidus/V. hemicentroti type strains: a virulent 10/068 1T1 and an innocuous 12/056 M24T1. These two strains were GFP-tagged and validated for their growth characteristics and virulence as genuine models for exposure. V. splendidus 10/068 1T1 is capable to alter different functions of hemocytes including motility, adhesion, internalization, ROS production, phagosome maturation and viability, unlike the avirulent strain. Furthermore, bacterial extracellular products appeared toxic and inhibit cellular responses (internalization and ROS production). Finally, we successfully reproduced experimental infection by water tank cohabitation assays with septic animals. Infection monitoring shows the targeting of gills by bacteria. The second section explores the MXR (MultiXenobiotic Resistance) system functioning in the blue mussel. For the first time, a complete ABCG2 amino acid sequence was established. Molecular characterization shows the presence of the abcg2 transcript in hemocytes and gills and its homology with other proteins from various species. The combination of the fluorescent probes bodipy prazosin and pheophorbide A with specific blockers demonstrate the transporter efflux activity and its heterogeneity in tissues and cells. Moreover, the expression of three ABC transporters (abcb, abcc, abcg2) identified in the blue mussel has been shown to be modulated by chemical contaminants. Mussels exposed to BaP in the laboratory or collected from contaminated mussel beds in the field show upregulated abc transcripts in gills whereas these mRNA undergone a downregulation in hemocytes. Season had also an effect on mRNA levels and interacted with site effects. Only the abcb gene displayed a more abundant mRNA level in gills dissected from animals collected in the more polluted area all over the diachronic study. In conclusion, our results demonstrate the vulnerability of the blue mussel towards a pathogen. The immunotoxic impact of environmental xenobiotics and the precise role of ABC efflux pumps in the immune system of the mussel has yet to be explored.

Philosophiae Doctor - PhD (Biotechnology) / Availability of genome sequences for numerous bacterial species comprising of different bacterial strains allows elucidation of species and strain specific adaptations that facilitate their survival in widely fluctuating micro-environments and enhance their pathogenic potential. Different bacterial species use different strategies in their pathogenesis and the pathogenic potential of a bacterial species is dependent on its genomic complement of virulence factors. A bacterial virulence factor, within the context of this study, is defined as any endogenous protein product encoded by a gene that aids in the adhesion, invasion, colonization, persistence and pathogenesis of a bacterium within a host. Anecdotal evidence suggests that bacterial virulence genes are undergoing diversifying evolution to counteract the rapid adaptability of its host&rsquo;s immune defences. Genome sequences of pathogenic bacterial species and strains provide unique opportunities to study the action of diversifying selection operating on different classes of bacterial genes. / South Africa