University, Diss., 2003--Kassel. / Erscheinungsjahr an der Haupttitelstelle: 2002.

Universiẗat, Diss., 2006--Kassel.

No description available.

No description available.

Tesis de Magíster en Bioquímica Área de Especialización, Bioquímica de Proteínas y Biotecnología; y Memoria para Optar al Título de Bioquímico / Los mecanismos moleculares que permiten a Salmonella Typhimurium causar enfermedades en mamíferos son numerosos y generan manifestaciones clínicas que abarcan desde una colitis hasta septicemia y la posterior muerte del hospedero. Estos mecanismos se relacionan con su capacidad de infectar en primera instancia células epiteliales del tracto digestivo y su posterior supervivencia y replicación en células fagocíticas. Esto es posible gracias a la existencia de factores de virulencia que se encuentran codificados en genes localizados mayoritariamente en islas de patogenicidad o en el plasmidio de virulencia de Salmonella. La gran mayoría de estos mecanismos han sido caracterizados en la infección del modelo murino o aviar. En este trabajo buscamos identificar los genes de S. Typhimirium que están implicados en la supervivencia de esta bacteria dentro de organismos eucariontes simples tales como amebas. Considerando que Salmonella podría interactuar con estos organismos en el medio ambiente, en este trabajo se usó como modelo de estudio la ameba social Dictyostelium discoideum, que de acuerdo a nuestras observaciones sería incapaz de degradar a Salmonella. Esto es interesante si se toma en cuenta que las amebas presentan una estrecha relación con los macrófagos, ya que ambas son células eucariontes que comparten muchos de los mecanismos implicados en el proceso de fagocitosis. Para estudiar la supervivencia de Salmonella al interior de D. discoideum implementamos un ensayo de competencia infectando esta ameba con S. Typhimurium 14028s y mutantes definidas. Nuestros resultados indican que las mutantes ΔinvA y ΔssaD (relacionadas directamente con la patogenicidad de Salmonella en otros modelos de infección) y ΔaroA (mutante metabólica), presentan problemas de supervivencia en D. discoideum en comparación con la cepa silvestre parental. Posteriormente, para identificar el conjunto de genes involucrados en la supervivencia intracelular de S. Typhimurium en este organismo modelo realizamos infecciones de D. discoideum utilizando una genoteca de ~3690 mutantes de S. Typhimurium 14028s por deleción de genes individuales, que contienen un cassette de resistencia a kanamicina. Desarrollamos un protocolo que permite amplificar y secuenciar las regiones adyacentes a la inserción de este cassette e identificar a nivel genómico cada una de las mutantes presentes en la población recuperada desde D. discoideum. Mediante este análisis a gran escala fue posible identificar un total de 81 mutantes bajo selección negativa. Entre las mutantes identificadas podemos mencionar a ΔorgB, gen que codifica un componente esencial para el funcionamiento del sistema de secreción tipo III (T3SS) codificado en la isla de patogenicidad SPI-1 y 3 mutantes (ΔssrA, ΔssaR y ΔpipB2) cuya función se asocia al T3SS codificado en la isla de patogenicidad SPI-2. El análisis a escala genómica también nos permitió tener una idea general del ambiente en el que se encuentra S. Typhimurium al interior de D. discoideum. Esta idea surge de la selección negativa que presentan mutantes en genes como rpoE y creB, que codifican proteínas que participan de la regulación transcripcional de genes asociados con la respuesta al estrés extracitoplasmático y al crecimiento en medio mínimo, respectivamente. En conjunto, los resultados de esta tesis constituyen un primer paso para comprender la interacción entre Salmonella y la ameba D. discoideum a nivel molecular / The molecular mechanisms that allow Salmonella Typhimurium to cause disease in mammals are numerous, and are responsible for clinical traits ranging from a self-limited colitis to septicemia and eventually the death of the host. These mechanisms are related to the ability of the pathogen to infect epithelial cells in the small intestine and its intracellular survival and growth in phagocytic cells of the host. This ability is the result of genes coding for virulence factors generally located in pathogenicity islands or in the Salmonella virulence plasmid. The vast majority of these mechanisms have been characterized using murine and avian infection models. In this work, we aimed to identify S. Typhimurium genes involved in the survival of this pathogen in simple eukaryotic organisms like amoebas. Considering that Salmonella can interact with these organisms in the environment, in this work we used the social amoeba Discoideum discoideum as a model. According to our data, this organism is unable to degrade Salmonella after phagocytosis. This is remarkable considering that amoebas show a close relationship with macrophages, both being eukaryotic cells that share many phagocytosis mechanisms. To study the intracellular survival of Salmonella in D. discoideum we developed a competition assay where the amoeba was infected with S. Typhimurium 14028s and selected derivative mutants. Our data show that mutants ΔinvA and ΔssaD (directly related to pathogenicity in other infection models) and ΔaroA (metabolic mutant) have an impaired intracellular survival in D. discoideum as compared to the wild-type strain. Later, in order to identify the complement of genes involved in the intracellular survival of S. Typhimurium in this organism, we infected D. discoideum using a single-gene deletion mutant library of S. Typhimurium 14028s (~3690 mutants), containing a kanamycin resistance cassette. We developed a protocol to amplify and sequence the genomic region adjacent to the resistance cassette. This protocol allowed us to identify at the genomic level the mutants present in the population of bacteria recovered from D. discoideum. Using this genomic analysis, we identified 81 mutants under negative selection. Relevant mutants in this group include ΔorgB, a gene that encodes an essential component required for the function of the T3SS encoded in SPI-1, and 3 mutants (ΔssrA, ΔssaR and ΔpipB2) in genes associated to the T3SS encoded in SPI-2. Furthermore, our genomic analysis allowed us to have a general idea of the environment faced by Salmonella within D. discoideum. This notion comes from the negative selection observed for mutants in genes such as rpoE and creB, coding proteins involved in the transcriptional regulation of genes associated to extracitoplasmatic stress and growth in minimal media, respectively. Taken together, the results in this work are the first step in the understanding of the molecular mechanisms involved in the interaction between Salmonella and D. discoideum / Conicyt; Fondecyt

Les amibes se nourrissent principalement de bactéries. Certaines bactéries pouvant résister à la digestion dans la voie phagocytique amibienne s’y retrouvent enrobées et sécrétées dans l’environnement par la suite. Cet enrobage augmente la résistance bactérienne à différents stress. Les bactéries enrobées pourraient favoriser la propagation de maladies infectieuses, mais aucune donnée n’est disponible à ce sujet. Les bactéries pathogènes Escherichia coli O157:H7, Salmonella typhimurium et Pseudomonas aeruginosa sont capables de résister à la digestion amibienne, mais leur capacité à être enrobée est inconnue. L’étude de la résistance de ces bactéries face à l’amibe Dictyostelium discoideum et l’analyse de la viabilité de cette dernière en présence des bactéries ont été réalisées pour mieux cibler les souches bactériennes potentiellement enrobables. Des essais d’enrobage ont été tentés, mais aucune bactérie enrobée n’a été observée en microscopie électronique. D’autres analyses seront requises pour comprendre la propagation des maladies infectieuses via les bactéries enrobées. / Amoebae mainly feed bacteria. Many bacteria are resistant to the digestion in the phagocytic pathway of amoebae. There they can be packaged and then secreted into the environment. Packaging increases the resistance of bacteria to different stresses. Packaged bacteria could improve the spread of infectious diseases, but no data is available regarding that so far. The pathogenic bacteria Escherichia coli O157:H7, Salmonella typhimurium and Pseudomonas aeruginosa are able to resist to digestion by amoeba digestion, but their ability to be packaged is unknown. The study of the bacteria resistance to the amoeba Dictyostelium discoideum and the analysis of the viaibility of the latter in the presence of bacteria were done to better target the bacterial isolates that can be packaged. Assays of packaging of bacteria were done, but no packaged bacteria were observed by electron microscopy. Other analyzes are required to understand the spread of infectious diseases by packaged bacteria.

L'amibe Dictyostelium discoideum sécrète des corps multilamellaires (CMLs) à partir de sa voie endocytique lorsqu'elle est cultivée en présence de bactéries. L'objectif de la présente étude consistait à comprendre le rôle des CMLs dans la physiologie de D. discoideum en caractérisant l'antigène de l'anticorps H36 présent sur les CMLs et celui de l'anticorps H72 qui se retrouve dans la voie endocytique. L'identification des antigènes a été réalisée par une immunoprécipitation suivie par une analyse en spectrométrie de masse. L'identité de l'antigène H36 a été déterminée et a été confirmée par des analyses en Western blot pour être la protease cprG aussi nommée CP7. Dans le cas de l'anticorps H72, l'identification n'a pas été fructueuse. Cette étude démontre que les CMLs sécrétés contiennent donc la protease cprG. Il s'agit de la deuxième description de l'association d'une protease avec des structures membranaires sécrétées par l'amibe D. discoideum.

One purpose of this study was to determine whether the ras protein from Dictyostelium discoideum (p23) binds guanine nucleotides like the ras proteins from mammals (p21) and yeast. The other purpose of this investigation was to purify or enrich for p23ras from D. discoideum by immunoaffinity chromatography. A number of different approaches were used to determine guanine nucleotide binding by p23RAS . A simple filter binding assay, binding to Western blots, and photoaffinity labeling all failed to demonstrate specific binding with lysates of D. discoideum cells. In contrast p21RAS from transformed NIH-3T3 cell lysate was successfully photoaffinity labeled in the presence of ³²P-α-guanosine 5¹-triphosphate (GTP) suggesting that the technique had been performed correctly. It was concluded that either p23RAS has a very low affinity for guanine nucleotides such that GTP binding was not detectable in these experiments or that the ras protein from D. discoideum simply does not bind guanine nucleotides. The purification of p23RAS from D. discoideum cells was attempted in order to provide a purified protein preparation for guanine nucleotide binding and for reconstitution studies. An anti-ras monoclonal antibody (Y13-259) was used as the ligand for the immunoaffinity chromatography. This approach was not successful in that the ras protein could not be enriched relative to other proteins because the immunoaffinity columns did not bind p23RAS. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

The role of cyclic AMP and a differentiation-indueing factor (DIF) in the differentiation of stalk cells was investigated in the cellular slime mold Dictyostelium discoideum. In this organism, starvation triggers the aggregation of amoebae into multicellular masses within which a simple, well-regulated pattern of partially differentiated cells is formed and which ultimately form fruiting bodies comprised of spore and stalk cells. In a monolayer system at low cell densities, stalk cell formation is dependent on the presence of both cyclic AMP and DIF. Both factors act within a short time of each other, induction by cyclic AMP preceding induction by DIF, beginning between 8 to 10 hours of incubation in monolayers, and progressively committing an increasing proportion of the cells in monolayer to form stalk cells. The relative effectiveness of analogues of cyclic AMP to induce stalk cell formation in monolayers indicates that the well-characterized cell surface cyclic AMP receptor most probably mediates the action of cyclic AMP. Although this receptor appears early during aggregation, it does not become activated until later during development in vivo, probably because the cyclic AMP concentrations within developing cell masses must build up to levels higher than those in aggregation streams. The finding that caffeine inhibits stalk cell formation in low density monolayers and that the permeable analogue 8-Bromo-cyclic AMP can partially reverse this inhibition suggests that activation of this receptor leads to an increase in internal cyclic AMP levels as one of the steps in stalk cell differentiation. The finding that the expression in low density monolayers of AP IV, a cell-type non-specific isozyme of acid phosphatase, was cyclic AMP-dependent is consistent with the view that cyclic AMP induces non-specific postaggregative gene expression during development in vivo. The findings that the expression of pre-stalk arid stalk cell specific antigens and of the pre-stalk cell specific isozyme AP II was DIF-dependent provide good evidence for the idea that both pre-stalk and stalk cell formation are induced by DIF. The fact that isolated pre-stalk cells require DIF for stalk cell formation in low density monolayers further supports this idea. Whereas cells independent of DIF for stalk cell formation in monolayers appear immediately after cyclic AMP-independent cells during differentiation in low density monolayers, DIF-independent cells appear considerably later during development in vivo. This evidence and the fact that developing cell masses contain elevated levels of DIF lead to the postulate that the factor(s) which triggers the formation of fruiting bodies also controls the pre-stalk to stalk cell conversion. / Science, Faculty of / Botany, Department of / Zoology, Department of / Graduate

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Chemistry, 1982 / Vita. / Includes bibliographical references. / by Charles Joseph Reynold Verret. / Ph. D. / Ph. D. Massachusetts Institute of Technology, Department of Chemistry