Rol del sistema de dos componentes BaeSR de Salmonella Typhimurium en la regulación de la expresión de las catalasas frente a ciprofloxacino
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2013
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Universidad Andrés Bello
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Para llevar a cabo una infección exitosa, Salmonella Typhimurium (S. Typhimurium) debe adaptarse a diversos cambios medioambientales en el hospedero, por lo que debe ser capaz de detectar y responder ante distintos estímulos, siendo los sistemas de dos componentes (SDC) uno de los mecanismos usado con este fin. Están compuestos por una proteína anclada a la membrana interna (sensor) y una proteína citosólica (regulador de respuesta). Los SDC han sido ampliamente relacionados con la resistencia bacteriana a antibióticos. Uno de estos SDC es BaeSR, donde BaeS es el sensor y BaeR es el regulador de respuesta, y en conjunto participan en la respuesta a los antibióticos bactericidas novobiocina y ciprofloxacino (CIP). A través de un mecanismo de acción secundario, estos antibióticos estarían provocando la muerte de la bacteria, mediante un aumento de las especies reactivas de oxígeno, entre las que se encuentra el peróxido de hidrógeno. Las enzimas encargadas de la degradación de este radical son las catalasas, donde en S. Typhimurium se han descrito 3 tipos: KatE, KatG y KatN, que se expresan diferencialmente según la fase de crecimiento. Por medio de un análisis in silico se encontraron posibles sitios de unión de BaeR, en los promotores de las tres catalasas.
El objetivo de esta Tesis fue demostrar que BaeSR participa en la respuesta frente a CIP en S. Typhimurium, por medio de la inducción de la expresión de las catalasas. Los resultados demostraron que los niveles de ROS totales y actividad catalasa, específicamente de KatE, son regulados positivamente por BaeSR frente a CIP, en fase exponencial. Además, se determinó que esta regulación es indirecta, ya que BaeR no se une al promotor de katE.
To carry out a successful infection, Salmonella Typhimurium (S. Typhimurium), must adapt to several environmental changes in the host, so you must be able to detect and respond to different stimuli. Two-component systems (TCS) is one of the mechanisms used for this purpose. They are composed of a protein anchored to the inner membrane (sensor) and a cytosolic protein (response regulator). The TCS has been widely associated with bacterial resistance to antibiotics. One of these is BaeSR, where BaeS is the sensor and BaeR is the response regulator, and together participates in the response to bactericidal antibiotics novobiocin and ciprofloxacin (CIP). Through secondary action mechanism, these antibiotics would killing the bacteria, by increased reactive oxygen species, including hydrogen peroxide. The enzymes responsible for the degradation of this radical are catalases. In S. Typhimurium have been described 3 types: KatE, KatG, and KatN, which are differentially expressed depending on the growth phase. Through an in silico analysis it was found potential binding sites BaeR, in the promoters of the three catalases. The goal of this Thesis was to demonstrate that BaeSR participates in the response to CIP in S. Typhimurium through catalases induction. The results showed that the levels of total ROS and catalase activity, specifically KatE, are positively regulated by BaeSR in response to CIP, in exponential phase. In addition, we determined that this regulation is indirect, since BaeR doesn't bind to katE promotor.
To carry out a successful infection, Salmonella Typhimurium (S. Typhimurium), must adapt to several environmental changes in the host, so you must be able to detect and respond to different stimuli. Two-component systems (TCS) is one of the mechanisms used for this purpose. They are composed of a protein anchored to the inner membrane (sensor) and a cytosolic protein (response regulator). The TCS has been widely associated with bacterial resistance to antibiotics. One of these is BaeSR, where BaeS is the sensor and BaeR is the response regulator, and together participates in the response to bactericidal antibiotics novobiocin and ciprofloxacin (CIP). Through secondary action mechanism, these antibiotics would killing the bacteria, by increased reactive oxygen species, including hydrogen peroxide. The enzymes responsible for the degradation of this radical are catalases. In S. Typhimurium have been described 3 types: KatE, KatG, and KatN, which are differentially expressed depending on the growth phase. Through an in silico analysis it was found potential binding sites BaeR, in the promoters of the three catalases. The goal of this Thesis was to demonstrate that BaeSR participates in the response to CIP in S. Typhimurium through catalases induction. The results showed that the levels of total ROS and catalase activity, specifically KatE, are positively regulated by BaeSR in response to CIP, in exponential phase. In addition, we determined that this regulation is indirect, since BaeR doesn't bind to katE promotor.
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Trabajo de títulación (Magíster en Bioquímica y Bioquímico)
Palabras clave
Salmonella Typhimurium, Genética, Resistencia a los Antibióticos, Catalasa, Expresión Génica.