Vibrio cholerae é uma bactéria autóctone do ambiente aquático e pode causar sérios riscos à saúde quando cepas patogênicas são acidentalmente consumidas. V. cholerae se encontra associada aos copépodes em concentrações que podem alcançar mais de 1000 vezes a densidade das bactérias livres na água. Se ingerido, um único copépode pode conter a dose mínima de bactérias necessária para a manifestação da doença. Verificar a presença e a distribuição dos sorogrupos O1 e O139 no complexo estuarino de Santos-Bertioga e plataforma continental adjacente em associação com o zooplâncton e seus distintos grupos taxonômicos foi o objetivo desse trabalho. O zooplâncton (>330 µm) foi coletado e a detecção dos sorogrupos O1 e O139 realizada nas amostras totais e nos táxons mais abundantes através das técnicas DVC-DFA e DFA (Contagem direta de bactérias viáveis e ensaio de imunofluorescência direta). Amostras fixadas em formol foram maceradas e preservadas numa solução tampão estéril, previamente aos experimentos. Para o DVC-DFA, animais vivos foram selecionados, lavados, macerados e uma alíquota transferida para meio de cultura. A presença da bactéria no zooplâncton foi correlacionada a parâmetros abióticos e bióticos. O sorogrupo O1 foi detectado em 88% e O139 em 77% das amostras de plâncton no complexo estuarino de Santos-Bertioga, valores mais altos do que os publicados na literatura mundial para outros estuários. Para a plataforma, a presença dos sorogrupos foi menor devido à salinidade mais elevada. Foram testados isoladamente 43 táxons, pertencentes a 9 filos. Dados inéditos da associação entre Vibrio cholerae e quetognatos, estágios larvas de equinodermos, urocordados e ovos de peixes foram registrados. Este trabalho sugere a existência de um gradiente costa-oceano para V. cholerae aderido ao zooplâncton de águas costeiras e ampla capacidade de V. cholerae O1 e O139 em aderir a diversos táxons do zooplâncton marinho. / Vibrio cholerae is an autochthonous bacterium in the sea and may cause serious health problems when pathogenic strains are accidentally ingested. V. cholerae are found associated with copepods in concentrations up to a thousand times higher than the free bacteria in the water. If ingested, a single copepod may have enough bacteria necessary for human infection. The objective of this study was to verify the presence and distribution of Vibrio cholerae O1 and O139 serogroups over Santos-Bertioga estuarine complex and adjacent continental shelf in association with zooplankton and over its distinct taxa. Zooplankton (>330 µm) sampling was carried out and detection of V. cholerae O1 and O139 assessed in whole samples and on most abundant taxa by the DFA and DVC-DFA (Direct Viable Count and Direct Fluorescence Assay) methods. Briefly, formalin-fixed samples were grinded and preserved in a sterilized buffer solution previously to the experiments. Live animals were selected, washed and grinded and an aliquot transferred to culture media for the DVC-DFA assay. Presence of these bacteria on zooplankton was correlated with physical and biological parameters of the seawater. Serogroup O1 was found on 88% while O139 on 77% of the samples from Santos-Bertioga estuarine complex, values higher than the ones found in other estuaries in global literature. For the adjacent shelf, detection was smaller due to higher salinity. 43 taxa, belonging from 9 phyla were individually tested. Inedited data from the association of V. cholerae and chaetognaths, Urochordata, larval stages of Polychaeta, Echinodermata, and fish eggs were documented. This study suggests the existence of an inshore-offshore gradient in V. cholerae attached to zooplankton from coastal waters and the high ability of V. cholerae O1 and O139 to adhere on diverse marine zooplanktonic taxa.

Vibrio cholerae é uma bactéria autóctone do ambiente aquático e pode causar sérios riscos à saúde quando cepas patogênicas são acidentalmente consumidas. V. cholerae se encontra associada aos copépodes em concentrações que podem alcançar mais de 1000 vezes a densidade das bactérias livres na água. Se ingerido, um único copépode pode conter a dose mínima de bactérias necessária para a manifestação da doença. Verificar a presença e a distribuição dos sorogrupos O1 e O139 no complexo estuarino de Santos-Bertioga e plataforma continental adjacente em associação com o zooplâncton e seus distintos grupos taxonômicos foi o objetivo desse trabalho. O zooplâncton (>330 µm) foi coletado e a detecção dos sorogrupos O1 e O139 realizada nas amostras totais e nos táxons mais abundantes através das técnicas DVC-DFA e DFA (Contagem direta de bactérias viáveis e ensaio de imunofluorescência direta). Amostras fixadas em formol foram maceradas e preservadas numa solução tampão estéril, previamente aos experimentos. Para o DVC-DFA, animais vivos foram selecionados, lavados, macerados e uma alíquota transferida para meio de cultura. A presença da bactéria no zooplâncton foi correlacionada a parâmetros abióticos e bióticos. O sorogrupo O1 foi detectado em 88% e O139 em 77% das amostras de plâncton no complexo estuarino de Santos-Bertioga, valores mais altos do que os publicados na literatura mundial para outros estuários. Para a plataforma, a presença dos sorogrupos foi menor devido à salinidade mais elevada. Foram testados isoladamente 43 táxons, pertencentes a 9 filos. Dados inéditos da associação entre Vibrio cholerae e quetognatos, estágios larvas de equinodermos, urocordados e ovos de peixes foram registrados. Este trabalho sugere a existência de um gradiente costa-oceano para V. cholerae aderido ao zooplâncton de águas costeiras e ampla capacidade de V. cholerae O1 e O139 em aderir a diversos táxons do zooplâncton marinho. / Vibrio cholerae is an autochthonous bacterium in the sea and may cause serious health problems when pathogenic strains are accidentally ingested. V. cholerae are found associated with copepods in concentrations up to a thousand times higher than the free bacteria in the water. If ingested, a single copepod may have enough bacteria necessary for human infection. The objective of this study was to verify the presence and distribution of Vibrio cholerae O1 and O139 serogroups over Santos-Bertioga estuarine complex and adjacent continental shelf in association with zooplankton and over its distinct taxa. Zooplankton (>330 µm) sampling was carried out and detection of V. cholerae O1 and O139 assessed in whole samples and on most abundant taxa by the DFA and DVC-DFA (Direct Viable Count and Direct Fluorescence Assay) methods. Briefly, formalin-fixed samples were grinded and preserved in a sterilized buffer solution previously to the experiments. Live animals were selected, washed and grinded and an aliquot transferred to culture media for the DVC-DFA assay. Presence of these bacteria on zooplankton was correlated with physical and biological parameters of the seawater. Serogroup O1 was found on 88% while O139 on 77% of the samples from Santos-Bertioga estuarine complex, values higher than the ones found in other estuaries in global literature. For the adjacent shelf, detection was smaller due to higher salinity. 43 taxa, belonging from 9 phyla were individually tested. Inedited data from the association of V. cholerae and chaetognaths, Urochordata, larval stages of Polychaeta, Echinodermata, and fish eggs were documented. This study suggests the existence of an inshore-offshore gradient in V. cholerae attached to zooplankton from coastal waters and the high ability of V. cholerae O1 and O139 to adhere on diverse marine zooplanktonic taxa.

A poluição, alteração física do habitat e a introdução de espécies invasoras via água de lastro, representam os maiores impactos antropogênicos para os ambientes costeiros. Foram pesquisadas em amostras da região costeira de S. Paulo, regiões portuárias brasileiras e de tanques de lastro de navios, bactérias das espécies Vibrio cholerae (Vc), V. parahaemolyticus (Vp) e V. vulnificus (Vv) que são as que têm maior implicação na saúde pública. As amostras foram avaliadas levando-se em conta parâmetros físico-químicos e microbiológicos e suas relações com a presença de Vc, Vp e Vv. As relações clonais foram verificadas através das técnicas de ERIC, BOX e REP-PCR. Foram identificadas 90 cepas de Vp e 11 de Vc. Foram observadas correlações entre alguns parâmetros microbiológicos e a presença de vibrios. A análise clonal permitiu verificar a alta diversidade das cepas. Concluiu-se que Vc e Vp são autóctones do ambiente costeiro brasileiro e podem ser tornar reservatórios para determinados fatores associados à virulência, gerando cepas com potencial epidêmico. / Pollution, physical alteration of habitat and the introduction of alien species through ballast water constitute the biggest anthropogenic impacts on coastal environments. We examined samples taken from the coastal region of S. Paulo state, Brazilian ports and ship ballast tanks, for bacteria of the species Vibrio cholerae (Vc), V. parahaemolyticus (Vp) and V. vulnificus (Vv) which have the most significant implication for public health. The samples were evaluated for microbiological and physical-chemical parameters as well as the presence of Vc, Vp and Vv. Clonal relationships of bacterial isolates were determined through ERIC, BOX and REP-PCR. A total of 90 strains of Vp and 11 of Vc were identified. Correlations between some microbiological parameters and the presence of vibrios were observed. The clonal analysis revealed extensive strain diversity. We concluded that Vc and Vp are autochthonous bacteria of the Brazilian coastal environment that can become reservoirs for factors associated with virulence, and are capable of generating strains with epidemic potential.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / A desnutriÃÃo, desidrataÃÃo e agentes antineoplÃsicos, tais como o metotrexato (MTX), sÃo imediatos causadores das doenÃas diarrÃicas. Objetivando investigar a mucosite intestinal (MI) induzida por MTX, a eficÃcia das soluÃÃes de reidrataÃÃo oral (SRO) acrescidas com glutamina (Gln), alanilglutamina (Ala-Gln), Hyprol 4107 (HYP) e hyfoama (HYFO) utilizou-se a perfusÃo intestinal. E para a suplementaÃÃo com vitaminas A (VITA) e E (VITE), Gln e peptÃdeos, utilizou-se as avaliaÃÃes morfomÃtricas, metabÃlicas e a de permeabilidade intestinal [(teste lactulose/manitol (L/M)], em camundongos (n=344) e em coelhos (n=72). A mucosite intestinal por MTX (2,75 mg/kg/24h s.c., durante 3 dias) foi validada em camundongos, atravÃs da constataÃÃo do quadro diarrÃico e do prejuÃzo no estado nutricional (-1.89Â0,52 g) estabelecido apÃs o 3o dia de tratamento. A anÃlise morfomÃtrica demonstrou achatamento de vilos (364,8Â19.9 mm) e hiperplasia de criptas (251Â19.2 mm) intestinais com aumento do nÃmero de apoptoses (7.48Â1, 23/cripta), no duodeno e no jejuno. A taxa L/M no grupo MTX foi maior c que no controle (0,35). A perfusÃo intestinal com toxina da cÃlera aumentou a secreÃÃo de Na+ (-12,8Â1.4 mEq/g/min), Cl- (-12,9Â4,6 mEq/g/min) e de Ãgua (-0,02 Â0,04 ml/g/min). As SRO acrescidas de Gln e peptÃdeos, restauram este efeito secretÃrio. A suplementaÃÃo de Gln e peptÃdeos, VITA e VITE, melhoram o ganho ponderal, a ingestÃo de raÃÃo, as alteraÃÃes morfolÃgicas e a apoptose, no modelo de mucosite intestinal. Nenhuma alteraÃÃo foi observada no padrÃo de mitoses nas criptas intestinais, apÃs o 3o dia de tratamento com MTX. A suplementaÃÃo nutricional reduziu na mucosite, a elevaÃÃo das taxas de L/M (0,35 para 0,73) nas vias paracelular e transcelular. Nossos resultados sugerem que, o restabelecimento da barreira morfofuncional, induzido pela suplementaÃÃo, foi obtido atravÃs de um aumento do transporte de cÃtions (Na+ e K+) na mucosa, do fornecimento de substrato energÃtico para o enterÃcito, inibiÃÃo da apoptose (0,25Â0,05), de cÃlulas indiferenciadas incremento no potencial redox e reduÃÃo da peroxidaÃÃo lipÃdica das cÃlulas intestinais diferenciadas. EntÃo sugerimos que o uso de SRO acrescidas de Gln e peptÃdeos podem prevenir a desnutriÃÃo e reduzir as doenÃas diarrÃicas, e a suplementaÃÃo com VITE e VITA melhorar a morfo fisiologia da barreira intestinal / A desnutriÃÃo, desidrataÃÃo e agentes antineoplÃsicos, tais como o metotrexato (MTX), sÃo imediatos causadores das doenÃas diarrÃicas. Objetivando investigar a mucosite intestinal (MI) induzida por MTX, a eficÃcia das soluÃÃes de reidrataÃÃo oral (SRO) acrescidas com glutamina (Gln), alanilglutamina (Ala-Gln), Hyprol 4107 (HYP) e hyfoama (HYFO) utilizou-se a perfusÃo intestinal. E para a suplementaÃÃo com vitaminas A (VITA) e E (VITE), Gln e peptÃdeos, utilizou-se as avaliaÃÃes morfomÃtricas, metabÃlicas e a de permeabilidade intestinal [(teste lactulose/manitol (L/M)], em camundongos (n=344) e em coelhos (n=72). A mucosite intestinal por MTX (2,75 mg/kg/24h s.c., durante 3 dias) foi validada em camundongos, atravÃs da constataÃÃo do quadro diarrÃico e do prejuÃzo no estado nutricional (-1.89Â 0,52 g) estabelecido apÃs o 3o dia de tratamento. A anÃlise morfomÃtrica demonstrou achatamento de vilos (364,8Â 19.9 Âm) e hiperplasia de criptas (251Â 19.2 Âm) intestinais com aumento do nÃmero de apoptoses (7.48Â 1, 23/cripta), no duodeno e no jejuno. A taxa L/M no grupo MTX foi maior c que no controle (0,35). A perfusÃo intestinal com toxina da cÃlera aumentou a secreÃÃo de Na+ (-12,8Â 1.4 ÂEq/g/min), Cl- (-12,9Â 4,6 ÂEq/g/min) e de Ãgua (-0,02 Â 0,04 ml/g/min). As SRO acrescidas de Gln e peptÃdeos, restauram este efeito secretÃrio. A suplementaÃÃo de Gln e peptÃdeos, VITA e VITE, melhoram o ganho ponderal, a ingestÃo de raÃÃo, as alteraÃÃes morfolÃgicas e a apoptose, no modelo de mucosite intestinal. Nenhuma alteraÃÃo foi observada no padrÃo de mitoses nas criptas intestinais, apÃs o 3o dia de tratamento com MTX. A suplementaÃÃo nutricional reduziu na mucosite, a elevaÃÃo das taxas de L/M (0,35 para 0,73) nas vias paracelular e transcelular. Nossos resultados sugerem que, o restabelecimento da barreira morfofuncional, induzido pela suplementaÃÃo, foi obtido atravÃs de um aumento do transporte de cÃtions (Na+ e K+) na mucosa, do fornecimento de substrato energÃtico para o enterÃcito, inibiÃÃo da apoptose (0,25Â 0,05), de cÃlulas indiferenciadas incremento no potencial redox e reduÃÃo da peroxidaÃÃo lipÃdica das cÃlulas intestinais diferenciadas. EntÃo sugerimos que o uso de SRO acrescidas de Gln e peptÃdeos podem prevenir a desnutriÃÃo e reduzir as doenÃas diarrÃicas, e a suplementaÃÃo com VITE e VITA melhorar a morfo fisiologia da barreira intestinal

The production of membranous vesicles is observed to occur among organisms from all domains of the tree of life spanning prokaryotes (bacteria, archaea) and eukaryotes (plants, animals and fungi). Bacterial release of membrane-derived vesicles (MVs) has been studied most extensively in cases of Gram-negative species and implicating their outer membrane in formation of extracellular MVs. However, recent studies focusing on Gram-positive bacteria have established that they also undergo MV formation. Membrane vesicles are released during normal bacterial growth, they are derived from the bacterial membrane(s) and may function as transporters of different proteins, DNA and RNA to the neighbouring bacteria or to the cells of a mammalian host. The transport of virulence factors in a condensed manner via MVs to the host cells presumably protects these proteins from degradation and, thereby, targets the host cells in a specific manner. The aim of my thesis is to investigate secretion of MV-associated virulence factors and to study interactions of MVs produced by two selected Gram-negative and Gram-positive bacteria, i.e. Vibrio cholerae and Listeria monocytogenes, with eukaryotic host cells. Depending on whether the bacterium acts as an extracellular or intracellular pathogen, MVs may be considered to have specific functions, which may lead to the different outcomes of MV-host interactions. V. cholerae transport systems for virulence factors include the Type VI secretion system and MVs (also referred to as the “Type 0” secretion system). We have identified that the biologically active form of PrtV protease in different V. cholerae serogroups is transported via MVs. PrtV protease is essential for V. cholerae environmental survival and protection from natural predator grazing. We demonstrated that PrtV is primarily translocated via the inner membrane to the periplasmic space, where it undergoes autoproteolysis, and the truncated version of PrtV protein is packaged inside the MVs and released from the surface of bacteria. MV-associated PrtV protease showed a contribution to bacterial resistance towards the antimicrobial peptide LL-37, thereby, enhancing bacterial survival by avoiding this innate immune defense of the host. We also studied another virulence factor of V. cholerae, the pore-forming toxin VCC, which was found to be transported by MVs. MV-associated VCC is biologically active and triggers an autophagic response in the target cells. We suggested that autophagy serves as a cellular defense mechanism against the MV-associated bacterial virulence factor of V. cholerae. Listeria monocytogenes is a Gram-positive intracellular and facultative anaerobic food-borne pathogen causing listeriosis. It causes only sporadic outbreaks in healthy individuals, however, it is dangerous for a fetus or newborn child, and for pregnant and immunocompromised people, leading to a deadly infection in one third of the cases. We have analyzed MVs produced by L. monocytogenes and their interaction with eukaryotic cells. Confocal microscopy analysis showed that MVs are internalized into HeLa and HEK293 cells and are accumulated in lysosomes. Moreover, L. monocytogenes produces MVs inside the host cells and even inside the phagosomes. We found that the major virulence factor of L. monocytogenes, the cholesterol-dependent pore-forming protein listeriolysin O (LLO), is entrapped inside the MVs and resides there in an oxidized inactive state. LLO is known to induce autophagy by making pores in the phagosomal membrane of targeted eukaryotic cells. In our studies, we have shown that MVs effectively abrogated autophagy induced by Torin1, by purified LLO or by another pore-forming toxin from V. cholerae. We also found that MVs promote bacterial intracellular survival inside mouse embryonic fibroblasts. In addition, MVs have been shown to have a strong protective activity against host cell necrosis initiated by pore-forming toxin. Taken together, these findings suggested that in vivo MVs production from L. monocytogenes might be a relevant strategy of bacteria to manipulate host responses and to promote bacterial survival inside the host cells.

L’organisation mondiale de la santé estime que le choléra entraîne 100, 000 décès par an pour environ 4 millions de cas recensés, plaçant ainsi cette maladie comme un enjeu de santé publique majeur. Cette infection est causée par Vibrio cholerae, une bactérie à Gram négatif vivant en milieu aquatique. Face à cette agression, l’épithélium intestinal et les bactéries du microbiote agissent comme une barrière, en exprimant notamment des peptides antimicrobiens (PAM). V. cholerae, comme de nombreux pathogènes, montre une résistance accrue aux PAM. Malgré une avancée constante sur la compréhension des mécanismes de résistances bactériens aux PAM, de nombreuses inconnues demeurent, principalement en raison des techniques de mutagénèse aléatoire utilisées pour leur identification. L’objectif de cette étude est d’identifier de nouveaux mécanismes de résistance impliqués dans la résistance de V. cholerae aux PAM. Grâce à des expériences de séquençage par spectrométrie masse suivi d’études bio-informatiques, nous avons identifié les protéines OmpV et Lap comme des candidates intéressantes pour être impliquées dans la résistance de V. cholerae. Alors que la souche V. cholerae A1552 délétée du gène ompV (DompV) ne semble pas présenter de défaut de croissance ou de perméabilité, nos résultats ont montré une diminution des concentrations minimales inhibitrices en différents PAM tels que LL-37. Les tests fonctionnels semblent suggérer l’implication des vésicules de sécrétion dans le mécanisme de d’OmpV face à LL-37 chez V. cholerae. / According to the World Health Organization, cholera remains a significant health problem, causing 100,000 death per 4 millions infections per year. V. cholerae, the etiologic agent of cholera and a Gram-negative bacterium, is generally transmitted via contaminated food or water. During infection, the microbiota and intestinal epithelium acts as a barrier by producing antimicrobial peptides (AMP). Resistance to AMP has emerged as a virulence factor in pathogens and specifically in V. cholerae. As AMP are considered as novel molecular therapeutic agents, it is truly important to better understand strategies of resistance of V. cholerae. The aim of this study is to identify unknown mechanisms involved in V. cholerae resistance to AMP. For this purpose, after protein sequencing by mass spectrometry (MS-MS) and bioinformatics, we identified the OmpV porin, member of the outer membrane protein family and the Lap protease as new protein candidates for strategies of resistance. We characterized the V. cholerae A1552 strain deleted for ompV (DompV). We confirmed OmpV implication in AMP resistance by minimum inhibitory concentration and did not observed any differences in growth or permeability in the presence of AMP between the wild-type (wt) and the (DompV) strains. The work presented here suggest an implication of outer membrane vesicules in V. cholerae resistance mechanisms to some AMP as LL-37.

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.

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.

La investigación tuvo como objetivo incorporar una metodología de identificación rápida conocida como ARDRA (Amplified Ribosomal DNA Restriction Analysis) para identificar especies del género Vibrio. Se estandarizó la técnica con cepas referenciales. Luego, se aislaron cepas bacterianas asociadas con el cultivo de Litopenaeus vannamei. Posteriormente, se realizaron pruebas bioquímicas para encontrar cepas candidatas de pertenecer al género Vibrio. Al finalizar esta primera etapa, la técnica ARDRA estandarizada, fue aplicada en las cepas candidatas, confirmando de esta manera la factibilidad de la metodología bajo las condiciones estudiadas. En una segunda etapa, se secuenció la región 16S rDNA para confirmar e identificar las cepas candidatas por análisis filogenético. Se reportaron tres especies diferentes con alta similitud pertenecientes al Vibrio core group (Vibrio communis, Vibrio harveyi y Vibrio parahaemolyticus). Con estos resultados, fue posible diseñar una identificación rápida por ARDRA para identificar el Vibrio core group y la especie Vibrio communis. La metodología de diseño del ARDRA fue soportado por una valoración diagnóstica bioinformática, obteniendo de esta evaluación, una sensibilidad y una especificidad de 97,1 y 76,9%, respectivamente para la identificación del Vibrio core group, mientras que para identificar la especie Vibrio communis, se obtuvo una sensibilidad y una especificidad de 100 y 97,4%, respectivamente. Finalmente, se ha demostrado que es posible identificar ciertas especies del genero Vibrio asociados con la acuicultura de Litopenaeus vannamei, por ARDRA y esta metodología de identificación, tiene la ventaja de ser mucho más rápido y económico en comparación con la identificación por análisis filogenético, teniendo a su vez la desventaja de ser dependiente del uso del secuenciamiento en un primer momento para el diseño del ARDRA. Palabras clave: Litopenaeus vannamei, ARDRA, Vibrio communis, Vibrio harveyi, Vibrio parahaemolyticus, Vibrio core group, evaluación diagnóstica bioinformática. / --- The research aimed to incorporate a quick identification methodology known as ARDRA (Amplified Ribosomal DNA Restriction Analysis) to identify Vibrio species. Technique was standardized with reference strains. Then, bacterial strains were isolated associated with the cultivation of Litopenaeus vannamei. Subsequently, biochemical tests were performed to find candidate strains belonging to the genus Vibrio. Upon completion of this first stage, the standard technique (ARDRA) was applied for candidate strains, thus confirming the feasibility of the method under the conditions studied. In a second step, the 16S rDNA region sequenced to confirm and identify candidate strains for phylogenetic analysis. Three different species were reported with high similarity belonging to the Vibrio core group (Vibrio communis, Vibrio harveyi and Vibrio parahaemolyticus). With these results, it was possible to design a quick identification by ARDRA to identify the Vibrio core group and Vibrio communis. The design methodology ARDRA was supported by a bioinformatics diagnostic assessment, obtaining this evaluation, a sensitivity and specificity of 97,1 and 76.9% respectively for the identification of Vibrio core group, while identifying the species Vibrio communis, yielded a sensitivity and specificity of 100 and 97,4%, respectively. Finally, it has proved possible to identify certain Vibrio species associated with aquaculture Litopenaeus vannamei, by ARDRA identification and this methodology has the advantage of being much faster and cheaper compared with the identification by phylogenetic analysis, having in turn, the disadvantage of being dependent on the use of the sequencing at first for ARDRA design. Keywords: Litopenaeus vannamei, ARDRA, Vibrio communis, Vibrio harveyi, Vibrio parahaemolyticus, Vibrio core group, bioinformatic diagnostic evaluation. / Tesis