Bochum, Universiẗat, Diss., 2002.

Made available in DSpace on 2014-06-12T23:01:40Z (GMT). No. of bitstreams: 2 arquivo8566_1.pdf: 606886 bytes, checksum: e93461286809c9a8403ab536bdc0c982 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2006 / O advento da creditação e a busca para determinar a qualidade dos dados produzidos vêm aumentando a demanda para validação de métodos de controle de qualidade laboratorial. O limite mínimo de detecção (LOD) é a menor concentração de um analito que pode ser mensurada, estabelecendo desta forma o ponto onde a análise é possível. Baseado nisto objetivou-se desenvolver metodologia para estabelecimento do limite mínimo de detecção de métodos de análise de Salmonella spp. aplicados a alimentos. Inicialmente foi estabelecida a cinética de uma cepa de Salmonella typhimurium ATCC 14028 por um período de 24 horas através da contagem de bactérias (UFC/mL) e leitura da densidade óptica (absorbância) para observação do desenvolvimento do microrganismo em estudo. Os dados experimentais foram ajustados matematicamente pelos modelos de Baranyi e quadrático. Com base na curva padrão foi possível determinar o tempo de incubação necessário para o inóculo atingir a fase estacionária. A partir deste, foram realizadas diluições seriadas até 10-11 das quais as quatro ultimas diluições e a amostra branco foram analisadas concomitantemente por métodos AOAC 967.26 (tradicional) e AOAC 996.08 (rápido). O LOD para os métodos testados foi encontrado na faixa de 1 a 8 UFC/25mL de amostra, não apresentando diferença significativa entre eles (p< 0,05). Na validação do método proposto foi obtida sensibilidade de 100% para ambos os métodos e precisão de 95% para o tradicional e 96,25% para o rápido. Os resultado demonstraram a aplicabilidade do modelo quadrático utilizado no ajuste da cinética microbiana e a metodologia proposta para limite mínimo de detecção aplicada aos métodos de detecção de Salmonella spp. foi satisfatória podendo ser aplicada na rotina laboratorial assegurando maior confiabilidade nos resultados dos ensaios microbiológicos, atendendo as exigências dos protocolos para creditação laboratorial

No description available.

Memoria para optar al Título Profesional de Bioquímico / Autorizada por el autor, pero con restricción para ser publicada a texto completo en el Portal de Tesis Electrónicas, hasta diciembre de 2014 / Salmonella enterica es un patógeno intracelular Gram negativo capaz de infectar un amplio rango de hospederos. En particular, S. enterica serovar Gallinarum infecta aves de corral causando una enfermedad sistémica que puede provocar la muerte. Una vez que entra al organismo, Salmonella utiliza una serie de factores de virulencia que le permiten sobrevivir al pH ácido del estómago, resistir a sales biliares y péptidos antimicrobiales, invadir y traspasar la barrera epitelial intestinal, sobrevivir y replicarse dentro de macrófagos y diseminarse dentro de los órganos del hospedero, principalmente a nivel del bazo e hígado. Hasta el momento, se desconoce gran parte de los factores de virulencia que utiliza S. Gallinarum para infectar un hospedero. Es por eso que en este trabajo se propuso identificar los genes de S. Gallinarum involucrados en la colonización sistémica en un modelo murino a través de un análisis global de mutantes bajo selección negativa in vivo. Para ello, se utilizó una genoteca de ~48.000 mutantes por inserción del transposón EZ-Tn5<T7/KAN-2>, la que se inyectó en ratones BALB/c por vía intraperitoneal. La comparación entre la población de bacterias inyectadas (input) y la población de bacterias recuperadas a partir del bazo de los animales (output) mediante hibridaciones competitivas en un microarray genómico nos permitió obtener una base de datos en la que identificamos 280 mutantes bajo selección negativa in vivo. La lista de mutantes bajo selección negativa incluye genes descritos previamente como necesarios para la virulencia de S. enterica, como los sistemas de secreción tipo III codificados en la SPI-1 y SPI-2, genes que codifican efectores de estos sistemas (sseB, sseE), genes relacionados a la síntesis y modificación del LPS (rfaL, rfaJ, rfbK, rfbM), genes que codifican reguladores globales de la virulencia (phoP, phoQ, ompR, envZ), genes que codifican proteínas de respuesta a estrés (oxyR, rpoE, htrA), entre otros. La lista también incluye genes no reportados previamente como necesarios para la virulencia de S. Gallinarum, pero si para la virulencia de otros serovares de S. enterica, como tatB y tatC que codifican proteínas del sistema Twin-Arginine Transport; y genes con funciones desconocidas como la región génica STM3118 a STM3121 perteneciente a la SPI-13. El sistema Twin-Arginine Transport es un sistema que transporta proteínas plegadas hacia el periplasma de bacterias Gram negativo. Por otra parte, aún se desconoce la función exacta de SPI-13, pero se ha visto que es necesaria para la replicación de S. Typhimurium dentro de macrófagos murinos. A través de ensayos de competencia y complementación in vivo entre la cepa silvestre y las mutantes ΔtatABC o ΔSPI-13, se confirmó la participación de estas regiones génicas en la colonización sistémica de ratones BALB/c. Cabe destacar que la lista de mutantes bajo selección negativa in vivo no incluye ciertos genes previamente descritos como necesarios para la virulencia de S. enterica, como el gen aroA que codifica una proteína involucrada en la síntesis de compuestos aromáticos. Se realizó un ensayo de competencia con una mutante ΔaroA y se determinó que presenta una colonización sistémica deficiente en ratones BALB/c, confirmando la participación de aroA en la virulencia de esta bacteria. Finalmente, mediante este análisis global de mutantes bajo selección negativa in vivo logramos identificar genes de S. Gallinarum requeridos para la colonización sistémica eficiente de un hospedero murino, comprobando de forma independiente la participación del operón tatABC, la isla de patogenicidad SPI-13 y el gen aroA en este proceso. El análisis individual de los genes identificados en esta base de datos permitirá ampliar el conocimiento sobre los mecanismos de patogenicidad de S. Gallinarum / Salmonella is a Gram negative intracelular pathogen able to infect a broad range of hosts. Specifically, S. enterica serovar Gallinarum infects poultry leading to a systemic illness that may cause death. Once Salmonella enters the organism, it uses a variety of virulence factors which allows it to survive in the gastric acid, resist bile salts and antimicrobial peptides, invade and cross the intestinal epithelium, survive and grow within macrophages, and colonize internal organs of the host, mainly spleen and liver. The main virulence factors that S. Gallinarum uses to infect a host remained unknown until know. In this work we proposed to identify genes involved in the systemic colonization of S. Gallinarum in the murine model through a genome-wide screening of mutants under negative selection in vivo. To accomplish this, we used a pool of ~48.000 mutants generated by random insertion of the EZ-Tn5<T7/KAN-2> transposon to inoculate BALB/c mice intraperitoneally. The comparison between the pool of inoculated bacteria (input) and the pool of bacteria recovered from the spleen of the animals (output) through high-throughput microarray-based screening of mutants allowed us to obtain a database of 280 mutants under negative selection in vivo. Within this database we found mutants in several genes known to be required for Salmonella enterica virulence, like those related to the type III secretion system encoded in SPI-1 and SPI-2, genes encoding efectors secreted by these systems (sseB, sseE), genes related to LPS synthesis and modification (rfaL, rfaJ, rfbK, rfbM), genes encoding global virulence regulators (phoP, phoQ, ompR, envZ), and genes encoding proteins involved in response to stress (oxyR, rpoE, htrA), among others. We also found genes not previously reported as required for S. Gallinaum virulence, like tatB and tatC, encoding components of the Twin-Arginine Transport system; and genes with unknown function like the genetic region comprised by STM3118 to STM3121, belonging to SPI-13. The Twin-Arginine Transport system transports a number of folded proteins to the periplasma of Gram-negative bacteria. Besides, the exact function of SPI-13 is still unknown, but has been seen that is necessary for the growth of S. Typhimurium inside murine macrophages. Through in vivo competition and complementation assays between wild type and ΔtatABC or ΔSPI-13 mutants, we were able to confirm the important role of these genes in the systemic colonization of BALB/c mice by S. Gallinarum. Noteworthly, there were genes that we didn’t observe on our database that have been reported as required S. enterica virulence like aroA, a gene involved in the synthesis of aromatic coumpounds. Using an in vivo competition assay we observed a systemic colonization defect for the ΔaroA mutant in BALB/c mice, indicating that this gene is indeed required for S. Gallinarum virulence in this host. Overall, our genome-wide screening of mutants under negative selection in vivo allowed us to identify 280 genes of S. Gallinarum required for the systemic colonization of a murine host. Also, we confirmed the role played by the tatABC operon, SPI-13 and aroA in the systemic colonization by this serovar. The in-depth analysis of the genes identified in our screening will expand our current knowledge on the mechanisms of S. Gallinarum pathogenesis / FONDECYT

Jin Yongjie. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 138-171). / Abstracts in English and Chinese. / Abstract (in English) --- p.i / Abstract (in Chinese) --- p.iii / Acknowledgments --- p.iv / Table of Contents --- p.v / List of Tables --- p.x / List of Figures --- p.xi / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1. --- Salmonella --- p.1 / Chapter 1.1 --- Morphology --- p.1 / Chapter 1.2 --- Antigenic structure --- p.1 / Chapter 1.3 --- Identification --- p.2 / Chapter 1.4 --- Nomenclature and classification --- p.2 / Chapter 1.5 --- Pathogenesis and virulence --- p.3 / Chapter 1.6 --- Infections --- p.4 / Chapter 1.6.1 --- Enteric fever --- p.4 / Chapter 1.6.2 --- Gastroenteritis --- p.4 / Chapter 1.7 --- Treatment --- p.5 / Chapter 1.7.1 --- Enteric fever --- p.5 / Chapter 1.7.2 --- Gastroenteritis --- p.5 / Chapter 1.8 --- Epidemiology and control --- p.6 / Chapter 1.8.1 --- Enteric fever --- p.6 / Chapter 1.8.2 --- Salmonella food poisoning --- p.6 / Chapter 2. --- Fluoroquinolones --- p.6 / Chapter 2.1 --- History of fluoroquinolones --- p.8 / Chapter 2.2 --- Mechanisms of action --- p.9 / Chapter 3. --- Antimicrobial resistance --- p.10 / Chapter 3.1 --- Microorganism-mediated resistance --- p.10 / Chapter 3.1.1 --- Intrinsic resistance --- p.11 / Chapter 3.1.2 --- Acquired resistance --- p.11 / Chapter 3.2 --- Resistance due to environmental factors --- p.12 / Chapter 3.3 --- Biological and clinical resistance --- p.12 / Chapter 3.4 --- Breakpoints --- p.13 / Chapter 4. --- Fluoroquinolone-resistance --- p.14 / Chapter 4.1 --- Increasing fluoroquinolone-resistance in bacteria --- p.14 / Chapter 4.2 --- Mechanisms of resistance to fluoroquinolones --- p.16 / Chapter 4.2.1 --- Mutations of target genes --- p.17 / Chapter 4.2.2 --- Active efflux systems and decreased membrane permeability --- p.20 / Chapter 5. --- Fluoroquinolone-resistance in Salmonella --- p.21 / Chapter 5.1 --- Prevalence of fluoroquinolone-resistant salmonellae in man and Animals --- p.21 / Chapter 5.1.1 --- Prevalence in the world --- p.21 / Chapter 5.1.2 --- Increasing resistance trend in Hong Kong --- p.24 / Chapter 5.2 --- Clinical outcome --- p.25 / Chapter 5.3 --- Mechanisms of fluoroquinolone-resistance in Salmonella --- p.25 / Chapter 5.3.1 --- Target gene mutations --- p.25 / Chapter 5.3.2 --- Other resistance mechanisms --- p.28 / Chapter 5.3.3 --- In vitro development of Salmonella resistant mutants --- p.29 / Chapter 6. --- Restricting the development of resistant mutants --- p.31 / Chapter 6.1 --- Mutant prevention concentration (MPC) --- p.31 / Chapter 6.1.1 --- Definition --- p.31 / Chapter 6.1.2 --- Development of MPC concept --- p.31 / Chapter 6.1.3 --- Significance --- p.34 / Chapter 6.2 --- Mutant selection window (MSW) --- p.35 / Chapter 6.2.1 --- The concept of mutant selection window (MSW) --- p.35 / Chapter 6.2.2 --- Significance --- p.36 / Chapter 7. --- Detection of gene mutations --- p.37 / Chapter 8. --- Objectives --- p.37 / Chapter Chapter 2 --- Materails and Methods --- p.39 / Chapter 1. --- Materials --- p.39 / Chapter 1.1 --- Bacterial strains --- p.39 / Chapter 1.1.1 --- Bacterial strains used for this study --- p.39 / Chapter 1.1.2 --- Storage of bacterial strains --- p.39 / Chapter 1.2 --- Materials --- p.40 / Chapter 2. --- Methods --- p.40 / Chapter 2.1 --- Identification --- p.40 / Chapter 2.2 --- Microbiological methods --- p.40 / Chapter 2.2.1 --- Determination of minimal inhibitory concentration (MIC) --- p.40 / Chapter 2.2.1.1 --- Preparation of antibiotic plates --- p.40 / Chapter 2.2.1.2 --- Preparation of inoculum --- p.43 / Chapter 2.2.1.3 --- Inoculation of antibiotic plates --- p.43 / Chapter 2.2.2 --- Determination of mutant prevention concentration (MPC) --- p.43 / Chapter 2.2.2.1 --- Preparation of bacterial suspension --- p.43 / Chapter 2.2.2.2 --- Inoculation of bacterial suspension --- p.43 / Chapter 2.2.2.3 --- Determination of the size of the inoculum --- p.43 / Chapter 2.2.2.4 --- Reading of results --- p.45 / Chapter 2.3 --- Molecular methods --- p.45 / Chapter 2.3.1 --- Polymerase chain reaction (PCR) --- p.45 / Chapter 2.3.2 --- Agarose gel electrophoresis --- p.47 / Chapter 2.3.3 --- Multiplex PCR amplimer conformation (MPAC) analysis --- p.47 / Chapter 2.3.3.1 --- Preparation of samples --- p.47 / Chapter 2.3.3.2 --- Electrophoresis --- p.49 / Chapter 2.3.3.3 --- Silver staining --- p.49 / Chapter 2.3.4 --- DNA Sequencing --- p.50 / Chapter 2.3.4.1 --- Purification of PCR products --- p.50 / Chapter 2.3.4.2 --- Sequencing reactions --- p.50 / Chapter 2.3.4.3 --- Electrophoresis --- p.50 / Chapter 2.3.4.4 --- Silver staining --- p.52 / Chapter 2.4 --- Stepwise selection and characterization of mutants --- p.53 / Chapter 2.4.1 --- In vitro selection of first-step strains --- p.53 / Chapter 2.4.2 --- Characterization of selected strains --- p.53 / Chapter 2.4.3 --- Subsequent selection of stepwise mutants --- p.54 / Chapter 3. --- Research plan --- p.54 / Chapter Chapter 3 --- Results --- p.55 / Chapter 1. --- Identification and fluoroquinolone MICs of Salmonella strains --- p.55 / Chapter 1.1 --- Identification of Salmonella strains --- p.55 / Chapter 1.2 --- Fluoroquinolone MICs for Salmonella strains --- p.55 / Chapter 1.2.1 --- Salmonella Typhimurium --- p.55 / Chapter 1.2.2 --- Salmonella Hadar --- p.57 / Chapter 2. --- Mutant prevention concentration (MPC) --- p.57 / Chapter 2.1 --- Salmonella Typhimurium --- p.57 / Chapter 2.1.1 --- MPC value --- p.57 / Chapter 2.1.2 --- MPC/MIC ratio --- p.57 / Chapter 2.1.3 --- Cmax/MPC ratio --- p.60 / Chapter 2.1.4 --- Frequencies of development of resistant strains --- p.60 / Chapter 2.2 --- Salmonella Hadar --- p.63 / Chapter 2.2.1 --- MPC value --- p.63 / Chapter 2.2.2 --- MPC/MIC ratio --- p.63 / Chapter 2.2.3 --- Cmax/MPC ratio --- p.63 / Chapter 2.2.4 --- Frequencies of development of resistant strains --- p.65 / Chapter 3. --- Stepwise selection of resistant mutants --- p.65 / Chapter 3.1 --- Salmonella Typhimurium --- p.70 / Chapter 3.1.1 --- Characterization of 1 St-step strains --- p.70 / Chapter 3.1.1.1 --- Antimicrobial susceptibilities --- p.70 / Chapter 3.1.1.2 --- Characterization of gene mutations --- p.75 / Chapter 3.1.1.3 --- Mutations and fluoroquinolone susceptibilities --- p.80 / Chapter 3.1.1.4 --- Mutations and nalidixic acid susceptibilities --- p.81 / Chapter 3.1.2 --- Characterization of 2nd-step mutants --- p.81 / Chapter 3.1.2.1 --- Antimicrobial susceptibilities --- p.82 / Chapter 3.1.2.2 --- Characterization of gene mutations --- p.85 / Chapter 3.1.2.3 --- Mutations and fluoroquinolone susceptibilities --- p.87 / Chapter 3.1.2.4 --- Nalidixic acid susceptibilities --- p.87 / Chapter 3.1.3 --- Characterization of 3rd-step mutants --- p.88 / Chapter 3.1.3.1 --- Antimicrobial susceptibilities --- p.88 / Chapter 3.1.3.2 --- Characterization of gene mutations --- p.92 / Chapter 3.1.3.3 --- Mutations and fluoroquinolone susceptibilities --- p.94 / Chapter 3.1.3.4 --- Nalidixic acid susceptibilities --- p.95 / Chapter 3.1.4 --- Characterization of 4th-step mutants --- p.95 / Chapter 3.1.4.1 --- Antimicrobial susceptibilities --- p.95 / Chapter 3.1.4.2 --- Characterization of gene mutations --- p.98 / Chapter 3.1.4.3 --- Nalidixic acid susceptibilities --- p.98 / Chapter 3.2 --- Salmonella Hadar --- p.98 / Chapter 3.2.1 --- Characterization of 1 St-step strains --- p.98 / Chapter 3.2.1.1 --- Antimicrobial susceptibilities --- p.99 / Chapter 3.2.1.2 --- Characterization of gene mutations --- p.102 / Chapter 3.2.1.3 --- Mutations and fluoroquinolone susceptibilities --- p.107 / Chapter 3.2.1.4 --- Mutations and nalidixic acid susceptibilities --- p.108 / Chapter 3.2.2 --- Characterization of 2nd-step mutants --- p.109 / Chapter 3.2.2.1 --- Antimicrobial susceptibilities --- p.109 / Chapter 3.2.2.2 --- Characterization of gene mutations --- p.112 / Chapter 3.2.2.3 --- Mutations and fluoroquinolone susceptibilities --- p.112 / Chapter 3.2.2.4 --- Nalidixic acid susceptibilities --- p.113 / Chapter Chapter 4 --- Discussion --- p.114 / Chapter 1. --- Susceptibility of salmonellae to fluoroquinolones --- p.114 / Chapter 2. --- The potential of fluoroquinolones to restrict the development of Salmonella resistant strains --- p.114 / Chapter 2.1 --- MPC and MPC/MIC of fluoroquinolones --- p.115 / Chapter 2.2 --- Cmax/MPC of fluoroquinolones --- p.117 / Chapter 2.3 --- Selection frequency of fluoroquinolones --- p.118 / Chapter 2.4 --- Effects of fluoroquinolones on the development of resistancein Salmonella Typhimurium and Salmonella Hadar --- p.119 / Chapter 3. --- Characterization of in vitro fluoroquinolone-resistant Salmonella mutants --- p.120 / Chapter 3.1 --- Development of resistance phenotype --- p.120 / Chapter 3.1.1 --- Microbiology --- p.120 / Chapter 3.1.2 --- Antimicrobial susceptibilities --- p.120 / Chapter 3.1.2.1 --- First-step strains --- p.120 / Chapter 3.1.2.2 --- Second-step mutants --- p.121 / Chapter 3.1.2.3 --- Third-step mutants --- p.121 / Chapter 3.1.2.4 --- Fourth-step mutants --- p.122 / Chapter 3.2 --- Contribution of target gene mutations to resistance development --- p.122 / Chapter 3.2.1 --- First-step mutants --- p.122 / Chapter 3.2.2 --- Second-step mutants --- p.125 / Chapter 3.2.3 --- Third-step mutants --- p.126 / Chapter 3.2.4 --- Fourth-step mutants --- p.128 / Chapter 3.3 --- The sequential development of gene mutations --- p.129 / Chapter 3.4 --- Other fluoroquinolone-resistance mechanisms --- p.130 / Chapter 4. --- Mutations and susceptibilities to fluoroquinolones and nalidixic acid --- p.132 / Chapter 4.1 --- Nalidixic acid - a marker for resistance to fluoroquinolones --- p.132 / Chapter 4.2 --- Breakpoint and clinical efficiency --- p.133 / Chapter 5. --- Strategies to reduce development of fluoroquinolone-resistance --- p.134 / Chapter 6. --- Conclusion --- p.136 / Chapter 7. --- Areas for future research --- p.136 / References --- p.138

Thesis (Ph.D.) - University of Glasgow, 2008. / Ph.D. thesis submitted to the Department of Infection and Immunity, Faculty of Biomedical and Life Sciences, University of Glasgow, 2008. Includes bibliographical references. Print version also available.

Thesis (Ph.D.)--Hong Kong University of Science and Technology, 2005. / Includes bibliographical references (leaves 126-145). Also available in electronic version.

A high frequency chromosomal gene replacement method of general utility was developed for Salmonella enteritidis. This method uses a segregation-deficient temperature-sensitive replicon, pHSG415, as a carrier of the recombinant gene of interest. It allows for site-specific replacement of chromosomal genes without the need for antibiotic resistance markers in the recombinant genes or the use of specific bacterial strains. This gene replacement strategy was used to investigate the foreign antigen-carrying potential of SefA and AgfA, the major fimbrin subunit proteins of Salmonella SEF14 and SEF17 fimbriae. On the basis of epitope mapping and structural predictions, ten different sites within each fimbrin protein were selected for replacement with PT3, an immunoprotective T-cell epitope from the gp63 protein of Leishmania major. PCR-generated sefA and agfA fimbrin genes containing the 48 bp DNA fragment encoding PT3 were used to replace the native fimbrin genes in the chromosome. PCR and DNA sequence analysis confirmed that 10–20% of potential clones contained the corresponding chimeric fimbrin gene. Fimbrial expression and assembly in the chimeric S. enteritidis strains was analyzed by Congo red binding, Western blotting and immunoelectron microscopy using immune serum raised to whole SEF14, whole SEF17 or PT3 peptide. Remarkably, all ten AgfA chimeric fimbrin proteins were expressed under normal conditions and eight were effectively assembled into external SEF17 fimbrial fibers. In contrast, none of the chimeric SefA proteins were expressed and no assembled SEF14 fimbriae were detected. This represents the first fimbrial epitope replacement system in the Salmonellae and the first chimeric fimbrin genes to be reconstituted into a wild-type genetic background. Results are presented from a preliminary vaccine trial in which BALB/c mice were immunized with a PT3-expressing S. enteritidis strain and challenged with virulent L. major friedlin. This model represents a promising “organelle” expression system for epitope display with broad applications as subunit or attenuated vaccines. / Graduate

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

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 105-123).