Identificación y caracterización del regulador de respuesta no canónico asociado al sensor quinasa ArcB de Salmonella enterica serovar Typhimurium en respuesta a peróxido de hidrógeno y en macrófagos murinos
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Archivos
Fecha
2020
Autores
Profesor/a Guía
Facultad/escuela
Idioma
en
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Universidad Andrés Bello
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Licencia CC
Licencia CC
Resumen
Los organismos deben detectar los cambios en sus entornos de manera rápida y
coordinar una respuesta para permitir la adaptación y la supervivencia. Uno de estos
organismos que enfrenta múltiples cambios en su estilo de vida es Salmonella enterica serovar
Typhimurium, un patógeno Gram negativo que causa gastroenteritis severa en humanos y
fiebre tifoidea en ratones. El ciclo de vida de Salmonella comienza en aguas y/o alimentos
contaminados, donde los huéspedes se alimentan y permiten que Salmonella ingrese al
sistema digestivo del huésped. En estos nuevos ambientes, Salmonella debe enfrentar las
duras condiciones del estómago y luego del intestino delgado. A pesar de estas condiciones,
Salmonella sobrevive e infecta la lámina basal del intestino delgado, donde enfrenta un nuevo
desafío, el sistema inmune innato comandado principalmente por macrófagos. Estas células
huésped fagocitan a Salmonella y tratan de eliminarla mediante el uso de especies reactivas
de oxígeno (ROS), principalmente mediante la producción de peróxido de hidrógeno (H202).
Sin embargo, Salmonella tiene diferentes mecanismos que detectan y eliminan el H202 . Uno
de estos mecanismos es mediante la detección de señales a través de los sistemas de dos
componentes (TCS). Estos sistemas están compuestos por un sensor unido a membrana con
función histidina quinasa (HQ) y un regulador de respuesta (RR) citoplasmático, el cual
promueve cambios genéticos para una rápida adaptación. Uno de los TCS estudiados por
nuestro grupo es el TCS ArcB/ArcA, que se vinculó para la adaptación de los niveles de
oxigenación y más tarde para el estado redox bacteriano. La acción bioquímica de ArcA bajo
estrés de H202 está bien estudiada, sin embargo, el mecanismo molecular de la función de
ArcB está pobremente descrita. Además, algunos trabajos han sugerido que la transducción
de señales en el TCS ArcB/ArcA no siempre funciona de forma canónica, lo que plantea la idea
de una conversación cruzada con otros componentes de TCS. En este trabajo, identificamos
el regulón ArcB en estrés por H202 en Salmonella, este HQ detecta y promueve una respuesta
versátil entre las condiciones de aerobiosis y H202. Además, análisis transcriptómicos sugieren
que este TCS se conversa de manera cruzada con otros componentes de TCS,
específicamente, un(os) RR(s). Nuestros análisis identificaron tres RR que podrían modular
las respuestas a la exposición a H202, que son los reguladores BaeR, BasR y KdpE. Además,
ensayos moleculares y bioquímicos mediante qRT-PCR y Phos-tag™,- SDS - PAGE nos
permitieron determinar que ArcB podría comunicarse con los reguladores BasR y KdpE tras la
exposición al H202. Además, nuestra observación podrían correlacionarse con la infección de
macrófagos. Los resultados obtenidos de este trabajo amplían el conocimiento de cómo
funciona ArcB en condiciones de estrés oxidativo y aclaran algunos aspectos de la hipotética
conversación cruzada, donde identificamos tres reguladores (BaeR, BasR y KdpE), que
podrían conversar de manera cruzada con ArcB en Salmonella.
The organisms must detect the changes in their environments in a rapid and coordinate way to allow adaptation and survival. One organism that faces multiple changes in their lifestyle is Sa/monella enterica serovar Typhimurium, a Gram-negative pathogen that causes severe gastroenteritis in humans and typhoid fever in mice. Salmonel/a lifecycle start in contaminated water and/or foods, where the hosts feed itself and allow Sa/monella to enter to the host digestive system. In these new environments, the bacteria must face the harsh conditions on the stomach and later small intestine. Despite these conditions, Salmonel/a survives and infect the basal lamina of the small intestine where face a new challenge, the innate immune system commanded primarily by macrophages and neutrophils. These host cells phagocytize Sa/monella and try to eliminate it by the use of reactive oxygen species (ROS), mainly by the production of hydrogen peroxide (H202). However, Salmonella has different mechanisms that sense and scavenge H202. One of these mechanisms is by the use of signal detection via two-component systems (TCS). These systems are composed by a membrane-bound sensor with histidine kinase (HK) function and a cytoplasmic response regulator (RR) that prometes genetic changes for a faster adaptation. One of the TCS studied for our group is the TCS ArcB/ArcA, which was linked in the adaptation of different oxygenation levels and later in the bacteria! redox state. The biochemical action of ArcA under H202 stress is well studied, however, the molecular mechanism of how ArcB function is poorly described. Additionally, sorne works have suggested that the signal transduction in the TCS ArcB/ArcA does not always work in a canonical form, rising the idea of sorne crosstalk with other components of TCS. In this work, we identified the ArcB-regulon upon H202 stress in Salmonel!a, this HK sense and prometes a versatile response between aerobiosis and H202 stress. Further, our transcriptomic analysis suggests that this TCS crosstalk with other components of TCS, specifically, a RR. Our analysis identified three RR that could modulate the responses after the H202 exposure, which are the BaeR, BasR and KdpE regulators. Further, molecular and biochemical assays using qRT-PCR and Phos-tag™ compound allow us to determínate that ArcB could crosstalk with BasR and KdpE regulators upon H202 exposure. As well, our observation correlate with the macrophage's infection. The results obtained from this work expand the knowledge of how the HK ArcB works in oxidative stress condition and clarify sorne aspect of the hypothetical crosstalk found in this TCS ArcB/ArcA, where we identified three regulators (BaeR, BasR and KdpE) that could work with ArcB in Salmonel/a upon H202 exposure.
The organisms must detect the changes in their environments in a rapid and coordinate way to allow adaptation and survival. One organism that faces multiple changes in their lifestyle is Sa/monella enterica serovar Typhimurium, a Gram-negative pathogen that causes severe gastroenteritis in humans and typhoid fever in mice. Salmonel/a lifecycle start in contaminated water and/or foods, where the hosts feed itself and allow Sa/monella to enter to the host digestive system. In these new environments, the bacteria must face the harsh conditions on the stomach and later small intestine. Despite these conditions, Salmonel/a survives and infect the basal lamina of the small intestine where face a new challenge, the innate immune system commanded primarily by macrophages and neutrophils. These host cells phagocytize Sa/monella and try to eliminate it by the use of reactive oxygen species (ROS), mainly by the production of hydrogen peroxide (H202). However, Salmonella has different mechanisms that sense and scavenge H202. One of these mechanisms is by the use of signal detection via two-component systems (TCS). These systems are composed by a membrane-bound sensor with histidine kinase (HK) function and a cytoplasmic response regulator (RR) that prometes genetic changes for a faster adaptation. One of the TCS studied for our group is the TCS ArcB/ArcA, which was linked in the adaptation of different oxygenation levels and later in the bacteria! redox state. The biochemical action of ArcA under H202 stress is well studied, however, the molecular mechanism of how ArcB function is poorly described. Additionally, sorne works have suggested that the signal transduction in the TCS ArcB/ArcA does not always work in a canonical form, rising the idea of sorne crosstalk with other components of TCS. In this work, we identified the ArcB-regulon upon H202 stress in Salmonel!a, this HK sense and prometes a versatile response between aerobiosis and H202 stress. Further, our transcriptomic analysis suggests that this TCS crosstalk with other components of TCS, specifically, a RR. Our analysis identified three RR that could modulate the responses after the H202 exposure, which are the BaeR, BasR and KdpE regulators. Further, molecular and biochemical assays using qRT-PCR and Phos-tag™ compound allow us to determínate that ArcB could crosstalk with BasR and KdpE regulators upon H202 exposure. As well, our observation correlate with the macrophage's infection. The results obtained from this work expand the knowledge of how the HK ArcB works in oxidative stress condition and clarify sorne aspect of the hypothetical crosstalk found in this TCS ArcB/ArcA, where we identified three regulators (BaeR, BasR and KdpE) that could work with ArcB in Salmonel/a upon H202 exposure.
Notas
Tesis (Doctor en Biotecnología)
This PhD thesis was funded by FONDECYT #1160315 project entitle "Identification and Characterization of non-cognate partners of the Salmonella Typhimurium ArcAB twocomponent system in response to hydrogen peroxide and hypochlorous acid.". Also, the CONICYT BECAS DOCTORADO NACIONAL #21160858 for the financing of the PhD program.
This PhD thesis was funded by FONDECYT #1160315 project entitle "Identification and Characterization of non-cognate partners of the Salmonella Typhimurium ArcAB twocomponent system in response to hydrogen peroxide and hypochlorous acid.". Also, the CONICYT BECAS DOCTORADO NACIONAL #21160858 for the financing of the PhD program.
Palabras clave
Salmonella, Inmunología, Salmonella Entérica, Investigaciones, Macrófagos