Caracterización proteómica de los compartimentos intracelulares tempranos de macrófagos RAW 264.7 en los que transitan dos cepas de Brucella abortus utilizando la plataforma del Instituto Clodomiro Picado y comparación con los resultados obtenidos en una plataforma distinta en trabajos anteriores
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2023
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Brucella sp. es una α2-Proteobacteria intracelular causante de infecciones en humanos y animales. Mundialmente al año se presentan 500 000 nuevos casos de brucelosis en humanos, específicamente en Costa Rica del 2003 al 2015 se reportaron 124 pacientes afectados por este agente. Además, las infecciones en ganado bovino repercuten en la industria ganadera al limitar la producción de leche. Durante el proceso de infección Brucella spp. produce cambios en el tráfico intracelular para evitar su eliminación en el lisosoma, finalizando en el retículo endoplasmático; a través de toda esta vía la bacteria modula el entorno a través de diferentes factores de virulencia. Se propone que B. abortus modifica proteómicamente los compartimentos que ocupa en el interior de los macrófagos RAW 264.7 por medio del sistema de secreción VirB. Para comprobar esto se aislaron las vacuolas de macrófagos infectados por B. abortus silvestre y una cepa mutante de VirB a través de un método de ultracentrifugación empleando un gradiente de sacarosa. Se realizó una extracción proteica de dichas vacuolas para finalmente concretar una comparación de la composición proteómica entre ambas. Se utilizó la cepa virB10- como control y el enfoque permitió identificar las proteínas que difieren entre los compartimentos y que tienen un alto poder discriminatorio en el análisis y una relevancia biológica. Para determinar qué proteínas caracterizan exclusivamente los compartimentos contenedores de las cepas 2308W o virB10-, se compararon las listas de proteínas obtenidas y se evaluó su importancia biológica. En este estudio, se implementó un protocolo de enriquecimiento de las vacuolas durante las primeras etapas de infección en macrófagos murinos RAW 264.7. También se compararon las proteínas resultantes de análisis HPLC- MS/MS en cepas diferentes para destacar las diferencias a nivel cualitativo y cuantitativo de los compartimentos intracelulares a las 4...
Inglés: Brucella sp. belongs to the α2-Proteobacteriaceae class and is an intracellular pathogen of humans and animals. Around the world, the annual incidence of human brucellosis is 500 000 new cases. Specifically, in Costa Rica from 2003-2015 there were 124 reported cases of patients infected with these bacteria. Also, infections in cattle with this bacterium have negative consequences in milk and beef production. During the infectious process Brucella sp. produces changes in the intracellular trafficking with the goal of avoiding its elimination in the lysosome, finalizing instead in the endoplasmic reticulum. All around this pathway the bacteria modulates its surroundings through different virulence factors. We propose that B. abortus modifies proteomically the compartments that it occupies in the interior of RAW 264.7 macrophages via the secretion system VirB. To verify this effect, wildtype and VirB mutant B. abortus-infected macrophage vacuoles were purified using a sucrose gradient ultracentrifugation method. A protein extraction of these vacuoles was performed to ultimately carry out a comparison of the proteomic composition between both strains. The virB10- strain was used as a control, and the approach allowed the identification of proteins that differ between the compartments and have high discriminatory power in the analysis and biological relevance. To determine which proteins exclusively characterize the compartments containing the 2308W or virB10- strains, the obtained protein lists were compared, and their biological significance was evaluated. In this study, a vacuole enrichment protocol was implemented during the initial stages of infection in RAW 264.7 murine macrophages. The resulting proteins from HPLC-MS/MS analysis were also compared in different strains to highlight the qualitative and quantitative differences in the intracellular compartments at 4 hours post- infection, allowing for a discussion of the differences in the number of...
Inglés: Brucella sp. belongs to the α2-Proteobacteriaceae class and is an intracellular pathogen of humans and animals. Around the world, the annual incidence of human brucellosis is 500 000 new cases. Specifically, in Costa Rica from 2003-2015 there were 124 reported cases of patients infected with these bacteria. Also, infections in cattle with this bacterium have negative consequences in milk and beef production. During the infectious process Brucella sp. produces changes in the intracellular trafficking with the goal of avoiding its elimination in the lysosome, finalizing instead in the endoplasmic reticulum. All around this pathway the bacteria modulates its surroundings through different virulence factors. We propose that B. abortus modifies proteomically the compartments that it occupies in the interior of RAW 264.7 macrophages via the secretion system VirB. To verify this effect, wildtype and VirB mutant B. abortus-infected macrophage vacuoles were purified using a sucrose gradient ultracentrifugation method. A protein extraction of these vacuoles was performed to ultimately carry out a comparison of the proteomic composition between both strains. The virB10- strain was used as a control, and the approach allowed the identification of proteins that differ between the compartments and have high discriminatory power in the analysis and biological relevance. To determine which proteins exclusively characterize the compartments containing the 2308W or virB10- strains, the obtained protein lists were compared, and their biological significance was evaluated. In this study, a vacuole enrichment protocol was implemented during the initial stages of infection in RAW 264.7 murine macrophages. The resulting proteins from HPLC-MS/MS analysis were also compared in different strains to highlight the qualitative and quantitative differences in the intracellular compartments at 4 hours post- infection, allowing for a discussion of the differences in the number of...
Descripción
Tesis (licenciatura en microbiología y química clínica)--Universidad de Costa Rica. Facultad de Microbiología, 2023
Palabras clave
BRUCELLA ABORTUS, CELULAS, ESPECTROMETRIA DE MASAS, MACROFAGOS, PROTEINAS - COMPOSICION