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Early host-pathogen interactions during mycobacterial infection of zebrafish embryos /Clay, Hilary, January 2007 (has links)
Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 113-128).
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Réservoirs de Mycobacterium ulcerans : développement de nouvelles techniques de laboratoire / Mycobacterium ulcerans reservoirs : development of new laboratory techniquesZingue, Dezemon 24 November 2017 (has links)
L'ulcère de Buruli est une maladie infectieuse tropicale présente dans des foyers endémiques. Cette infection essentiellement cutanée est causée par Mycobacterium ulcerans. M. ulcerans est un pathogène opportuniste dont le réservoir est environnemental. Notre revue de la littérature a répertorié les sources environnementales potentielles de cette mycobactérie. Seulement cinq souches de M. ulcerans ont été isolées à partir de prélèvements de l’environnement. Il existe une corrélation inverse entre réchauffement climatique et incidence de l’ulcère de Buruli, peut-être liée à la sensibilité intrinsèque de M. ulcerans aux variations de température, ou bien à des modifications de son écosystème. Dans la perspective d’améliorer les protocoles d’isolement de M. ulcerans à partir de l’environnement, nous avons entrepris une analyse phénotypique à haut débit des substrats carbonés métabolisés par M. ulcerans et le profil obtenu nous a orientés après une recherche bibliographique des principales sources environnementales de ces substrats, vers des interactions plus spécifiques de M. ulcerans avec les bactéries, algues, mollusques et champignons. Les résultats de ce premier travail ont servi de base pour la mise au point de milieux de culture innovants qui nous ont permis d’isoler pour la première fois, une microcolonie de M. ulcerans à partir de fèces d’agouti. Nous avons mis au point une méthode de lecture automatisée des échantillons colorés par la méthode de Ziehl-Neelsen. Notre travail de thèse a produit des protocoles qui ont pour objectif d’être mis en œuvre dans les pays d’endémie, pour préciser les sources et modes de transmission de M. ulcerans aux populations. / Buruli ulcer is a tropical infectious disease present in endemic foci/This mainly cutaneous infection is caused by Mycobacterium ulcerans. M. ulcerans is an opportunistic pathogen from the environment. Our literature review has listed the potential environmental sources of this mycobacterium.. However, only five strains of M. ulcerans have been isolated from environmental samples. There is an inverse correlation between global warming and incidence of Buruli ulcer, possibly related to the intrinsic sensitivity of M. ulcerans to temperature, or to changes in its ecosystem. In order to improve the isolation protocols of M. ulcerans from the environment, we conducted a high-throughput phenotypic analysis of the carbon substrates metabolized by M. ulcerans and the profile obtained oriented us afterwards a bibliographic search of the main environmental sources of these substrates, towards more specific interactions of M. ulcerans with other bacteria, algae, molluscs and fungi. The results of this first work served as a basis for the development of innovative culture media which, allowed us to isolate for the first time a microcolony of M. ulcerans from feces of agouti. We also developed a method for automated reading of samples stained by Ziehl-Neelsen staining. Our thesis work has produced protocols that are intended to be implemented in African endemic countries, in order to clarify the sources and modes of transmission of M. ulcerans to populations.
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Caractérisation de la microalgue rouge Porphyridium marinum sous différentes conditions de culture et valorisation de ces métabolites / Characterization of the red microalga Porphyridium marinum under different growing conditions and valorisation of its metabolitesGargouch, Nesrine 14 December 2018 (has links)
La présente étude s’attache à étudier l’effet d’une source de carbone organique sélectionnée sur la croissance et la production de métabolites de la microalgue rouge Porphyridium marinum. Cette dernière s’est montrée incapable de se multiplier en hétérotrophie, en absence totale de lumière. Cependant, en condition mixotrophique la production de biomasse, lipides et de phycobiliprotéines par P. marinum a été améliorée en comparaison avec la condition autotrophique. Les teneurs en exopolysaccharides ont été presque similaires dans les deux conditions. Dans le but de valoriser ces métabolites, l’effet antioxydant, antibactérien, antibiofilm et anticancéreux ont été testés. L’exopolysaccharide de P. marinum ainsi que ses dérivés de faible poids moléculaire (EPS-2P et EPS-5P) ont tous présenté des activités antibactériennes et antibiofilm à différentes concentrations. Cependant l’EPS-2P et l’EPS-5P ont été jugés plus efficaces pour l’activité anticancéreuse contre les cellules de cancer de sein. D’autre part, la production du pigment majoritaire des microalgues rouges, la B-phycoerythrine (B-PE), a été optimisée moyennant des plans d’expériences adaptés. Une teneur de 40 mg/g MS a été obtenue en faisant varier la concentration en NaNO3, K2HPO4 et solution métallique ainsi que l’intensité lumineuse. Après purification, la molécule optimisée a montré une activité antioxydante en termes de piégeage des radicaux libres DPPH, chélation et réduction des ions de fer et inhibition de la décoloration du β-carotène. Nos données suggèrent alors que les métabolites produits par la microalgue rouge P. marinum peuvent être potentiellement utilisé dans plusieurs applications à savoir cosmétiques et pharmaceutiques. / The present study investigates the effect of a selected organic carbon source on the growth and production of metabolites of the red microalga Porphyridium marinum. The latter has been unable to multiply in heterotrophy, in total absence of light. However, in mixotrophic condition the productions of biomass, lipids and phycobiliproteins by P. marinum have been improved in comparison with the autotrophic condition. The contents of exopolysaccharide were almost similar under both conditions. In order to valorize these metabolites, the antioxidant, antibacterial, antibiofilm and anticancer effect were tested. The exopolysaccharide of P. marinum as well as its low molecular weight derivatives (EPS-2P and EPS-5P) have all exhibited antibacterial and antibiofilm activities at different concentrations. However, EPS-2P and EPS-5P were found to be more effective for anti-cancer activity against breast cancer cells. On the other hand, the production of the majority pigment of red microalgae, B-phycoerythrin (B-PE), has been optimized by means of adapted experimental plans. A content of 40 mg/g DW was obtained by varying the concentration of NaNO3, K2HPO4 and metal solution as well as the light intensity. After purification, the optimized molecule showed antioxidant activity in terms of free radical scavenging DPPH, chelation and reduction of iron ions and β-carotene bleachinginhibition. Our data suggest that the metabolites produced by the red microalga P. marinum may be potentially used in several applications namely cosmetic and pharmaceutical.
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Immunopathogenesis of chronic Mycobacterium marinum infection in adult zebrafish (Danio rerio)Jaeckel, Gilta January 2014 (has links)
Tuberculosis (TB) is still a global epidemic disease despite its discovery over 100 years ago. It is caused by Mycobacterium tuberculosis, which invades and replicates within macrophages, key cells of the innate immune system. The hallmark of tuberculosis is the granuloma which is an accumulation of Mycobacterium-infected cells surrounded by immune cells, and the containment of the bacteria is assured as long as the host immune response remains intact. Despite a well-developed immune response in the infected host, reactivation of latent tuberculosis infection (LTBI) may occur through the introduction of other bacterial pathogens, re-infection with M. tuberculosis or due to other immunosuppression, e.g. AIDS or cancer. The zebrafish–M. marinum model provides an ideal system for examining the pathogenesis of tuberculosis and the associated immune response of the host due to its vertebrate-like immune system, and the close phylogenetic relationship of M. marinum to M. tuberculosis. Granuloma formation and immune response to M. marinum have been investigated mainly in zebrafish embryos or larvae, which lack an adaptive immune response, and little work has been performed in adult fish. This complicates the transfer of findings in these models to chronic, latent or re-activated disease stages in humans, where adaptive immunity plays an important part. The aim of the research presented here was to investigate the immune response of the adult zebrafish to M. marinum infection, with the focus on the kidney as one of the major immune organs in fish. The results obtained support further use of the adult zebrafish-M. marinum model for human tuberculosis infections in the future. In the present study, adult zebrafish were infected with low doses of M. marinum (NCIMB 1297 or NCIMB 1298) and the kidney was investigated for histopathological changes in the form of granulomas over a period of two months(Chapter 3). No granulomas were detected in the fish infected with M. marinum NCIMB 1298 while in zebrafish infected with NCIMB 1297, macrophage aggregation and granuloma formation were detected as early as day 11 post-infection. Occurrence and severity of granulomas and the presence of replicating bacteria increased over time, resulting in a high density of non-caseating and caseating granulomas in the head and posterior kidney after two months of infection. Interleukin 1 beta (IL-1β), Interleukin-12 (IL-12), Tumor necrosis factor alpha (TNFα) and Interferon gamma (IFNγ) have been shown to be important cytokines functioning in defence against tuberculosis, especially IFNγ which is considered to play an important part in acute, chronic and latent tuberculosis. Changes in gene expression of these immune genes in adult zebrafish were investigated over the first two weeks of infection with M. marinum NCIMB 1298 and NCIMB 1297. The results obtained in the first week after infection were inconclusive for both strains investigated. In agreement with the results presented in Chapter 3, no specific immune response was detectable in fish infected with M. marinum NCIMB 1298. However, after 14 days, a high-fold change in IL-12 and TNFα expression were detected in fish infected with M. marinum NCIMB 1297, while IL-1β showed no changes compared to the control fish. Furthermore, no IFNγ expression was detectable over the first two weeks of infection. The delay in the expression of IL-12 and the lack of IFNγ expression can be explained by the ability of M. marinum to manipulate the host immune response, as described for M. tuberculosis and other intracellular bacteria. Besides in vivo investigations of the host-pathogen interactions, in vitro primary macrophage cultures from individual zebrafish kidneys were developed to investigate macrophage-specific gene expression to M. marinum infection (Chapter 4). Although the results looked promising, further optimization is required before the results of the in vitro assays can be fully compared to the in vivo results. Our understanding of reactivation in latent tuberculosis infection (LTBI) both in healthy and immune compromised individuals is insufficient and is delaying the development of treatments for the disease. Therefore, the transcriptome profile of long-term infections (26 weeks) with M. marinum NCIMB 1297 in adult zebrafish was investigated to determine whether the gene expression in this model is comparable to LTBI in humans or other vertebrate model organisms (Chapter 5). In addition, transcriptome profiling was investigated in a group of long-term infected zebrafish exposed to stress to induce re-activation of the disease. Expression profiles in the long-term infected fish and the infected plus stressed fish differed from each other and displayed similar gene profiles to those found in the latent or re-activated disease states, respectively, in human and other vertebrate models. Infected fish displayed a profile highlighted by IFNγ, TNFα, NOS2b and IL-8 expression alongside activating and regulatory T cell responses, including involvement of cytotoxic T cells (CTLs). The transcriptome profile of the group of fish that had been infected and then stressed was distinguished by the lack of IFNγ expression and reduction in TNFα and NOS2b expression, as well as a lack of T cell response compared to the infected fish. In conclusion, the results obtained from Chapters 3 and 4 showed that M. marinum NCIMB 1298 is non-pathogenic to zebrafish. Infection with M. marinum NCIMB 1297, on the other hand, resulted in a similar immune response to that described for human and other mammalian vertebrate models (Chapters 3-5). These results support the use of the adult zebrafish-M. marinum model to investigate LTBI and disease reactivation, and will aid our understanding host-pathogen interactions for tuberculosis in the future.
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Detection and transmission of Mycobacterium marinum and Mycobacterium chelonae in zebrafish (Danio rerio)Peterson, Tracy Shawn 02 April 2015 (has links)
Mycobacteriosis is a common disease of laboratory zebrafish (Danio rerio). Different infection patterns occur in zebrafish depending on mycobacterial species. Mycobacterium marinum and M. haemophilum produce virulent infections associated with high mortality, whereas M. chelonae is more wide spread and not associated with high mortality. Identification of mycobacterial infections to the species level provides important information for making management decisions. Observation of acid-fast bacilli in histological sections or tissue imprints is the most common diagnostic method for mycobacteriosis in fish, but only allows for diagnosis to the genus level. Mycobacterial culture, followed by molecular or biochemical identification is the traditional approach for species identification, but recently it has been shown that DNA of diagnostic value can be retrieved from paraffin blocks. Type of fixative, time in fixative before processing, species of mycobacteria, and severity of infection were investigated as parameters to determine if the hsp gene PCR assay (primer set HS5F/hsp667R) could detect and amplify mycobacterial DNA from paraffin-embedded zebrafish. Whole zebrafish were experimentally infected with either M. chelonae or M. marinum, and then preserved in 10% neutral buffered formalin or Dietrich's fixative for
3, 7, 21 and 45 days. Subsequently, fish were evaluated by H&E and Fite's acid-fast stains to detect mycobacteria within granulomatous lesions. The PCR assay was quite effective, and obtained PCR product from 75% and 88% of the M. chelonae and M. marinum infected fish, respectively. Fixative type, time in fixative, and mycobacterial species showed no statistical relationship with the efficacy of the PCR test.
Regarding natural transmission, zebrafish are capable of contracting mycobacterial infections by feeding on infected fish tissue, but other natural routes have not been clearly elucidated. Free living amoebae have been shown to be vectors for mycobacteria and their virulence is enhanced when residing in these protozoans. Paramecium caudatum are commonly used as a first food for zebrafish, and I investigated this ciliate's potential to serve as a vector of Mycobacterium marinum and M. chelonae. The ability of live P. caudatum to transmit these mycobacteria to larval, juvenile and adult zebrafish was evaluated. Infections were defined by histologic observation of granulomas containing acid-fast bacteria in extraintestinal locations. In both experiments, fish fed paramecia containing mycobacteria became infected at a higher incidence than controls. Larvae (exposed at 4 days post hatch) fed paramecia with M. marinum exhibited an incidence of 30% (24/80) and juveniles (exposed at 21 days post hatch) showed 31% incidence (14/45). Adult fish fed gelatin diets containing bacteria within paramecia or mycobacteria alone for 2 wk resulted in infections when examined 8 wk after exposure: M. marinum OSU 214; in paramecia 47% (21/45; 3.5 x 10⁵ dose/fish/day), M. marinum CH in paramecia 47% (9/19; 3.6 x 10⁵ dose/fish/day), M. chelonae in paramecia 38% (5/13; 3.5 x 10⁵ dose/fish/day).
I investigated the ability of mycobacteria to persist within paramecia, as this has previously been demonstrated in amoebae. Gram negative bacteria ingested by paramecia were processed within an hour. In contrast, I determined using GFP-labeled Mycobacterium marinum that mycobacteria can persist within paramecia digestive vacuoles. The concentration of M. marinum at 1 hour was similar to that at the time of ingestion. Twenty-four hours post-ingestion and later there was significant decline in M. marinum concentrations compared to time of ingestion, but M. marinum continued to persist inside digestive vacuoles for up to one week. My results demonstrate for the first time that Paramecium caudatum can act as a vector for mycobacteria. This provides a useful animal model for evaluation of natural mycobacterial infections and demonstrates the possibility of mycobacterial transmission in zebrafish facilities via contaminated paramecia cultures. / Graduation date: 2013 / Access restricted to the OSU Community at author's request from April 2, 2013 - April 2, 2015
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Characterization of bacterial ultrastructure involved in storage granule formation and DNA segregationFakih, Doaa 08 1900 (has links)
Projet I :
Les endospores représentent un état de dormance des bactéries leur permettant de résister à des conditions extrêmes et de persister pendant des années. La formation d'endospores a façonné l'évolution puisqu’elle se produit exclusivement chez les Firmicutes. Plusieurs études ont rapporté la formation d'endospores chez des espèces en dehors des Firmicutes, en particulier chez deux espèces de Protéobactéries, Rhodobacter johrii et Serratia marscescens, et une espèce d'Actinobacteries, Mycobacterium marinum. Le fait d’identifier les endospores en dehors des Firmicutes pourrait affecter la forme de l'arbre de vie et aiderait dans notre lutte contre les agents pathogènes humains. Par conséquent, nous avons visé d’étudier l'endosporulation chez ces trois espèces en utilisant des approches avancées d'imagerie et d'analyse, y compris la microscopie corrélative alliant la microscopie optique et électronique (CLEM), la tomographie de cryo- électron (cryo-ET) et la lipidomique. Nous avons utilisé la bactérie sporulante bien caractérisée Bacillus subtilis comme contrôle positif de la sporulation. L'examen de R. johrii, S. marcescens et M. marinum en utilisant CLEM et cryo-ET a montré que les objets à phase brillante ne ressemblaient à aucun stade de l'endosporulation. Les cryo-tomogrammes ont montré que les objets à phase brillante chez S. marcescens étaient des débris cellulaires agrégés de cellules mortes, alors qu'ils présentaient des structures granulaires typiques des cellules bactériennes chez les R. johrii et M. marinum. L'analyse lipidomique chez R. johrii a identifié les structures granulaires comme des granules de stockage potentiels enrichis en triacylglycérides (TAG). Nous pensons que les TAG peuvent fournir une source d'énergie pour résister à l'épuisement des nutriments. Des approches biochimiques et bioinformatiques supplémentaires ont soutenu nos conclusions selon lesquelles R. johrii, S. marcescens et M. marinum sont des bactéries non sporulantes.
Projet II :
Les plasmides jouent un rôle vital dans la propagation des gènes de résistance au sein et entre les espèces bactériennes. Par conséquent, il est essentiel de comprendre les systèmes bactériens impliqués dans le transfert et la maintenance des plasmides pour mieux aider dans notre lutte contre la propagation de la résistance aux antibiotiques. Dans cette thèse de doctorat, nous avons cherché à caractériser l'opéron alp7ARC, en utilisant l'homologue de l'actine bactérienne Alp7A pour séparer le plasmide pLS20 codant pour la résistance à la tétracycline dans B. subtilis. La stabilité du plasmide s'est avérée dépendante de l'opéron alp7ARC, indiquant un rôle essentiel dans la ségrégation plasmidique. Nos résultats préliminaires sur Alp7A ont montré qu'il s'assemble dans une nouvelle nanostructure tubulaire plutôt que des filaments, suggérant un nouveau mécanisme de ségrégation de l'ADN par Alp7A. Nous avons également étudié la structure d'Alp7A in vivo en utilisant une combinaison d'approches, notamment la biologie moléculaire, la Cryo-ET et la fLM. Nous avons également utilisé la CLEM pour localiser Alp7A dans des cellules entières à une résolution macromoléculaire. En outre, nous avons étudié la structure et la fonction d'Alp7A in vitro en transfectant B. subtilis et E. coli avec diverses constructions plasmidiques incorporant des mutations dans le gène d’Alp7A. Nous avons déployé différentes méthodes pour la purification de la protéine Alp7A, y compris la séparation par chromatographie, et le fractionnement au sulfate d'ammonium. J'ai discuté des divers défis que nous avons rencontrés dans ces expériences, tels que la contamination, l'instabilité de la protéine Alp7A et l'épaisseur bactérienne. Enfin, j'ai proposé des approches expérimentales alternatives qui aideraient à étudier le mécanisme de ségrégation des plasmides par Alp7ARC. / Project I:
Endospores represent a dormant state of bacteria that allows them to withstand extreme conditions and persist for years. Endospore formation has shaped evolution, whereby it exclusively occurs in Firmicutes. Several studies have reported endospore formation in species outside of Firmicutes, particularly in two species of Proteobacteria, Rhodobacter johrii and Serratia marcescens, and one species of Actinobacteria, Mycobacterium marinum. Identifying endospores outside of Firmicutes would affect the shape of the tree of life and aid in our fight against human pathogens. Therefore, we aimed to investigate endosporulation in these three species using advanced imaging and analytical approaches, including correlative light and electron microscopy (CLEM), cryo-electron tomography (cryo-ET), and lipidomics. We used the well-characterized sporulating bacterium Bacillus subtilis as a positive control of sporulation. Examination of R. johrii, S. marcescens, and M. marinum using CLEM and cryo-ET showed that phase-bright objects did not resemble any stages of endosporulation. Cryo-tomograms revealed that the phase-bright objects in S. marcescens were aggregated cellular debris of dead cells, whereas they displayed granular structures typical of bacterial cells in R. johrii and M. marinum. Lipidomic analysis in R. johrii identified the granular structures as potential storage granules enriched with triacyl-glycerides (TAGs). We speculate that TAGs may provide an energy source to withstand the nutrient depletion. Additional biochemical and bioinformatics approaches supported our conclusions that R. johrii, S. marcescens, and M. marinum are non-sporulating bacteria.
Project II:
Plasmids play a vital role in the spread of resistance genes within and across bacterial species. Therefore, it is essential to understand the bacterial systems involved in the transfer and maintenance of plasmids to better aid in our fight against the spread of antibiotic resistance. In this doctorate, we aimed to characterize the alp7ARC operon, employing the bacterial actin homolog Alp7A to segregate the tetracycline resistance-encoding plasmid pLS20 in B. subtilis. The stability of the plasmid was shown to be dependent on the alp7ARC operon, indicating an essential role in plasmid segregation. Preliminary results on Alp7A showed that it assembles into a novel tubular nanostructure rather than filaments, suggesting a novel mechanism for DNA segregation by Alp7A. We further studied the structure of Alp7A in vivo using combination of approaches, including molecular biology, cryo-ET, and fLM. We also used CLEM to localize Alp7A in whole cells to a macromolecular resolution. Besides, we investigated the structure and function of Alp7A in vitro by transfecting E. coli with various plasmid constructs and purification by several methods, including affinity chromatography and ammonium sulfate precipitation. I discussed the diverse challenges we encountered in these experiments, such as bacterial thickness, contamination, and Alp7A protein instability. Finally, I proposed alternative experimental approaches for investigating the mechanism of plasmid segregation by Alp7ARC.
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