Salmonella enterica serovar Typhimurium (S. typhimurium) is a gram-negative bacteria capable of infecting a variety of warm-blooded vertebrate hosts. In humans, consumption of S. typhimurium through contaminated food or water typically leads to an acute but self-limiting gastroenteritis and oftentimes an enlargement of the spleen (splenomegaly). Splenomegaly has been attributed to the expansion of phagocytes, B and T lymphocytes, and immature CD71+Ter119+ red blood cells (RBCs). The spleen is an important organ with distinct roles in RBC recycling, the capture of blood-borne pathogens, and as a site of initiation of the adaptive immune response. The spleen has a characteristic tissue architecture composed of three compartments. The white pulp (WP), largely populated by B and T lymphocytes is surrounded by the red pulp (RP), which primarily contains F4/80+ macrophages. The border between the WP and RP, the marginal zone (MZ), is populated by MOMA+ and MARCO+ macrophages which are important for the capture of blood-borne pathogens. This precise organization of the spleen allows for efficient antigen capture and activation of adaptive immunity, due to the close proximity of antigen presenting cells and lymphocytes. It is known that Salmonella spp. infections delay the adaptive immune response in comparison to other bacteria, such as Listeria monocytogenes. Therefore, we investigated the effect of an attenuated S. typhimurium strain (÷9088) on in situ splenic organization. We utilized four-color immunofluorescence microscopy (IFM) in combination with flow cytometry to characterize the in situ changes of spleen architecture and cell population profiles during S. typhimurium infection in mice. Within the first week of infection, splenomegaly is evident and after three weeks of infection, the spleen comprises over 5% of the mouse's total body weight. During this time S. typhimurium has not been cleared from the spleen and mice are anemic with decreased pack cell volume. We confirmed previous studies that reported extramedullary erythropoiesis was a major cause of splenomegaly. However, we also show that RP F4/80+ macrophages significantly expand and take over the WP regions of the spleen, increasingly co-localizing with immature (CD71+Ter119+) and mature (CD71-Ter119+) RBC subsets. As a result of these dramatic changes in cell proportions and their in situ distribution, the splenic architecture becomes unrecognizable. The boundary between WP and RP is lost as proportions of MOMA+ macrophages of the MZ are reduced following infection. Likewise, B and T cell zones of the WP are also drastically reduced, most likely due to their increased co-localization with F4/80+ macrophages. As a result of infection, the increased cellularity of splenomegaly results in changing proportions of cell populations, potentially disrupting the link between infection and immune response. Together, these data provide further insight into the disease process of S. typhimurium infection in mice. Understanding how the changes in splenic architecture affect the adaptive immune response has implications for the design of more effective Salmonella-based vaccines and therapies.

Orientador: Marcelo Brocchi / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-20T04:43:16Z (GMT). No. of bitstreams: 1 Damiani_IgorAlexandreCampos_M.pdf: 2676549 bytes, checksum: 95c7715f3b322149e80749501a5500a2 (MD5) Previous issue date: 2011 / Resumo: Salmonella enterica Typhimurium é uma bactéria anaeróbica facultativa que apresenta tropismo por áreas tumorais. Esta interessante propriedade abre novas perspectivas em relação à pesquisa contra o câncer, pois há muito tempo buscam-se veículos seletivos para a eliminação de neoplasias. A inibição do crescimento tumoral e até mesmo seu total retrocesso foram observados em modelos murinos de câncer tratados com linhagens atenuadas de S. enterica. Além disso, seu potencial como veículo de moléculas antitumorais exógenas (vacina de DNA, RNAi, citocinas e enzimas, por exemplo) também foi descrito. No entanto, as linhagens testadas em humanos até o presente não induziram os mesmos efeitos observados nos modelos animais. Isto indica que estudos adicionais são necessários para otimização desta terapia, incluindo o teste de novas linhagens mutantes atenuadas de S. enterica....Observação: O resumo, na íntegra, poderá ser visualizado no texto completo da tese digital / Abstract: Salmonella enterica Typhimurium, a facultative anaerobic bacterium, presents tropism for tumor areas. This interesting property creates new perspectives in cancer research, in which great efforts have been done to seek a drug carrier that could selectively target and destroy malignant cells. Inhibition of tumor growth and even its total elimination were observed in murine cancer models infected by attenuated strains of S. enterica. Besides, its potential as a carrier of exogenous antitumor molecules (DNA vaccine, iRNA, cytokines and enzymes, for example) is also described. Nevertheless, when these strains were tested in humans, they did not induce the same effects observed in murine models. Thus, additional studies are needed to optimize this therapy, including the test of novel S. enterica attenuated strains...Note: The complete abstract is available with the full electronic digital thesis or dissertations / Mestrado / Microbiologia / Mestre em Genética e Biologia Molecular

Magíster en Bioquímica área de Especialización en Bioquímica de Proteínas Recombinantes / Memoria de título de bioquímico / Los sistemas de secreción tipo VI (T6SS) corresponden a un mecanismo de interacción célula-célula ampliamente distribuido entre bacterias Gram negativo. Si bien inicialmente al T6SS se le atribuyó un papel en la virulencia de los microorganismos, estudios posteriores dieron cuenta de su versatilidad, indicando que el sistema también toma parte en relaciones mutualistas o comensales entre bacterias y eucariontes, además de relaciones de competencia interbacteriana. Salmonella Typhimurium codifica un T6SS en la isla de patogenicidad SPI-6 (T6SSSPI-6), sin embargo el rol que cumple en la patogénesis de Salmonella aún no ha sido aclarado. Resultados obtenidos en nuestro laboratorio indican que mutantes de este sistema presentan una menor colonización de órganos internos, tanto en ratones BALB/c como en pollos White Leghorn infectados oralmente. Considerando que los componentes celulares del sistema inmune son la principal puerta de entrada de Salmonella para el desarrollo de la infección sistémica, se planteó como hipótesis de este trabajo que “el Sistema de Secreción Tipo VI codificado en la isla genómica SPI-6 de Salmonella enterica serovar Typhimurium se expresa en el interior de macrófagos de origen murino y aviar, favoreciendo la supervivencia bacteriana en estas células”. Para probar esta hipótesis el objetivo fue evidenciar la expresión, funcionalidad y contribución del T6SS durante la interacción de S. Typhimurium con macrófagos murinos y aviares. Para determinar la expresión del T6SS durante la infección de macrófagos, se construyó el vector pLZ01 que permitio la generación de fusiones transcripcionales y traduccionales a la proteína fluorescente verde (GFP) en Salmonella, mediante recombinación homóloga de productos de PCR. De esta manera, se fusionaron componentes estructurales del T6SSSPI-6 (VgrG, Hcp-1, Hcp-2) a GFP y se evaluó su transcripción y traducción en ensayos de infección in vitro mediante microscopía de epifluorescencia. Por otra parte, para determinar si el sistema es translocado al citoplasma de macrófagos durante la infección de S. Typhimurium, se estudió la translocación de una fusión traduccional de VgrG a la β-lactamasa TEM1, construida en el plasmidio pFlagTEM1. La translocación de las fusiones fue determinada mediante un ensayo de pérdida de FRET de la cefalosporina CCF2, observado mediante microscopía de epifluorescencia y cuantificado mediante fluorometría. Finalmente, para determinar la contribución del T6SS en los procesos de internalización y supervivencia en macrófagos se realizaron ensayos de protección con gentamicina. En ellos se comparó la capacidad de la cepa silvestre para invadir y sobrevivir en el interior de macrófagos, versus mutantes que carecen de todo el T6SSSPI-6 o poseen un T6SSSPI-6 no funcional debido a la mutación de clpV, ATPasa esencial para este sistema. Todos los experimentos se realizaron en líneas de macrófagos murinos (RAW264.7) y aviares (HD11), utilizando cepas derivadas de S. Typhimurium 14028s. Los resultados mostraron que ninguno de los componentes estructurales estudiados (VgrG, Hcp-1, Hcp-2) del T6SSSPI-6 de S. Typhimurium se transcribe y traduce en el medio de cultivo celular, sin embargo su transcripción y traducción es gatillada al infectar tanto macrófagos murinos como aviares. A pesar de observar la transcripción y traducción de VgrG, no se detectó su translocación al citoplasma de las células infectadas. Contrariamente a lo esperado, se observó que la presencia del T6SSSPI-6 no contribuye a la supervivencia en el interior de macrófagos murinos o aviares, pero sí tendría una implicancia en la etapa de internalización de Salmonella, puesto que al utilizar mutantes con un T6SSSPI-6 no funcional se observó un fenotipo de mayor internalización en ambos modelos celulares. Estos resultados permiten aceptar una parte de la hipótesis planteada, ya que el Sistema de Secreción Tipo VI codificado en la isla genómica SPI-6 de Salmonella enterica serovar Typhimurium se expresa en el interior de macrófagos de origen murino y aviar, y rechazar una segunda parte de la hipótesis, pues este sistema no tendría un rol en la supervivencia bacteriana en estas células. No obstante, el aumento en la capacidad de internalización de mutantes del T6SS indica que el sistema tendría un rol durante la infección de los macrófagos. / Type VI Secretion Systems (T6SS) correspond to a widely distributed cell-cell interaction mechanism in Gram-negative bacteria. Although initially the T6SS was attributed a role in the virulence of microorganisms, subsequent studies realized its versatility, indicating that this system also takes part in comensal or mutualistic relationships between bacteria and eukaryotes, as well as interbacterial competition. Salmonella Typhimurium encodes a T6SS in the pathogenicity island SPI-6 (T6SSSPI-6), however the role of this island in the pathogenesis of Salmonella has not been clarified. Results obtained in our laboratory indicate that mutants of this system generate a phenotype of reduced colonization of internal organs, both in orally infected BALB/c mice and White Leghorn chicken. Because the initial contact of Salmonella with cellular components of the immune system is the main gateway for the development of systemic infection of Salmonella, the objective of this work was to determine the expression, functionality and contribution of the T6SS during S. Typhimurium interaction with murine and avian macrophages. The vector pLZ01was built to determine the expression of the T6SS during infection of macrophages. This plasmid enables the generation of transcriptional and translational fusions to the green fluorescent protein (GFP) reporter in Salmonella by homologous recombination of PCR products. In this way, structural components of the T6SSSPI-6 (VgrG, Hcp-1, Hcp-2) were merged to GFP and their transcription and translation were assessed by in vitro infection assays and epifluorescence microscopy. On the other hand, to determine whether the system is translocated to the cytoplasm of macrophages during infection of S. Typhimurium, translocation of VgrG was studied using a translational fusion of VgrG to the β-lactamase TEM1, built in the pFlagTEM1 plasmid. The translocation of the β-lactamase fusion was determined by processing of the CCF2/AM fluorescence substrate, detected by epifluorescence microscopy and quantified using fluorometry. Finally, gentamicin protection assays were performed to determine the contribution of the T6SS in the processes of internalization and survival in macrophages. In these experiments, invasion and survive inside macrophages at the wild type strain was compared to a deletion mutant of the T6SS gene cluster and a mutant on the clpV gene, which encodes the ATPase essential for the functioning of the system, All experiments were carried out in murine (RAW264.7) and avian (HD11) macrophage cell-lines, using strains derived from the sequenced wild-type S. Typhimurium 14028s strain. The results showed that none of the studied structural components (VgrG, Hcp-1, Hcp-2) of T6SSSPI-6 of S. Typhimurium are produced in cell culture media, but their transcription and translation are triggered when murine or avian macrophages are infected. Despite observing transcription and translation of VgrG, translocation of this protein into the cytoplasm of infected cells could not be detected. Contrary to expectations, it was observed that the presence of the T6SSSPI-6 did not contribute to Salmonella survival within murine or avian macrophages. However, internalization experiments showed that non-functional T6SSSPI-6 mutants showed a greater uptake into both cellular models. These results indicate that the T6SSSPI-6 of S. Typhimurium is expressed during infection of murine and avian macrophages (the first part of the hypothesis is true), however it did not have an impact on the ability of S. Typhimurium to survive inside murine or avian macrophages (the second part of the hypothesis is false). However, the increase in the internalization of the T6SS mutants suggests a novel role for the T6SS during infection of macrophages. / Fondecyt

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

No description available.

No description available.

Salmonella do grupo paratifóide é responsável por toxi-infecção alimentar no homem, veiculada por alimentos contaminados. Este estudo determinou o perfil genotípico de nove amostras de S. Typhimurium, a sua patogenicidade, assim como sua capacidade de colonização e invasão em aves SPF. Verificou-se pela análise dos genes: agfA, avrA, cdtB, fimA, fliC, invA, iroN, lpfC, mgtC, pefA, sefC, sifA, sipB, sipC, sitC, slyA, sopB, sopE1, sptP ou spvC em amostras de Salmonella Typhimurium que todas foram negativas para os genes sopE1 ou spvC. O gene cdtB estava presente em apenas uma amostra (11,11%) e o gene pefA em duas amostras (22,22%). O gene sefC foi encontrado em três amostras (33,33%). Em oito amostras (88,88%) os genes agfA, fimA, lpfC ou sptP estiveram presentes. Os genes avrA, fliC, invA, iroN, mgtC, sifA, sipB, sipC, sitC ou slyA ou sopB estiveram presentes em 100% das amostras analisadas. Determinou-se quatro perfis genotípicos. No ensaio de patogenicidade observou-se que as amostras inoculadas por via oral, não causaram mortalidade de pintinhos SPF de um dia de idade, com a exceção da amostra SA 633 e SA 006 que apresentaram 30 e 10%, respectivamente. No entanto, observou-se que a infecção por via subcutânea provocou uma maior mortalidade de pintinhos, sendo que as amostras SA 003, SA 004, SA 005 e a amostra vacinal mostraram somente 10% de mortalidade, a amostra SA 002 30%, as amostras SA 632 e SA 634 70% e a amostra SA 633 100%. A amostra SA 006 não provocou nenhuma mortalidade. A amostra mais patogênica pela via subcutânea foi a SA 633. O ensaio de colonização foi realizado em pintinhos SPF, com as amostras SA 002, SA 003, SA 004, SA 005, SA 006, SA 632, SA 633, SA 634 e amostra vacinal. Verificou-se ausência de invasão no fígado e baço 24 horas pós- infecção, exceto para as amostras SA 632 (30%) e amostra vacinal (20%). Após sete dias da infecção houve invasão em dois pintinhos com a amostra SA 002 e um pintinho com as amostras SA 004 e SA 005. Apenas na amostra SA 632 constatou-se colonização em ceco após 24 horas em 10% das amostras e após 7 dias em 70% pós-infecção. Concluiu-se que entre as amostras de Salmonella Typhimurium analisadas existiam diferentes perfis genotípicos baseando-se na presença ou ausência de genes de virulência, e que a amostra vacinal assemelha-se a amostras de S. Typhimurium estudadas quanto a presença dos genes. Os resultados do teste de patogenicidade das amostras de ST indicaram que a via de inoculação modifica a patogenicidade de uma mesma amostra e que a mortalidade após a inoculação pela via subcutânea é sempre superior que pela via oral. / Parathyphoid Salmonella are major food-borne pathogens spread by contaminated food products. This study determined the genotypic profile of nine samples of S. Typhimurium, pathogenicity, colonization and invasion in SPF chicks. It was found by analysis of genes: agfA, avrA, cdtB, fimA, fliC, invA, iroN, lpfC, mgtC, pefA, sefC, sifA, sipB, sipC, sitC, slyA, sopB, sopE1, sptP or spvC samples of S. Typhimurium all were negative to sopE1 or spvC. The cdtB gene was identified in one sample and pefA gene in two samples (22,22%). Sef C gene was detected in three samples (33,33%). In eight samples (88,88%) agfA, fimA, lpf or sptP were detected. AvrA, fliC, invA, iroN, mgtC, sifA, sipB, sipC, sitC, slyA and sopB were detected in all samples evaluated. Four genotypic profiles were established. Pathogenicity tests showed that samplesinoculated by oral gavage did not present mortality in one day old SPF chicks, except samples SA 633 and SA006 with 30 and 10%, respectively. However, it was observed that subcutaneous inoculation showed high mortality in SPF chicks than oral inoculation, and samples SA 003, SA004, SA005 and vaccinal strain showed 10% of mortality, 30% for sample SA002, 70% in the samples SA632 and SA 634 and 100% when the sample SA 633 was inoculated. No mortality was observed in sample SA006. Colonization test was performed in SPF chicks using the samples: SA002, SA 003, SA 004, SA005, SA006, SA 632, SA 633, SA 634 and vaccinal strain. The more pathogenic strain subcutaneously was the SA 633. There was not invasion in liver and spleen 24 hours p.i., except for sample SA 632 (30%) and vaccinal strain (20%). Seven days p.i. invasion was detected in two chicks inoculated with samples identified as SA 004 and SA 005. Sample SA 632 showed 10% of cecal colonization 24 hours p.i. and 70% after one week p.i. It was concluded that Salmonella Typhimurium strains including the vaccinal strain, showed different genotypical profiles, based on the presence or absence of genes of virulence genes. The results of the pathogenicity test indicated that inoculation route modify the pathogenicity of the same strain, and the mortality post-inoculation was always higher in chicks inoculated by subcutaneous route when compared to the oral route.

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 105-118).

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 88-102).

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 112-125).