Participación del factor transcripcional ArcA en la virulencia de Salmonella enterica serovar Typhimurium
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Fecha
2015
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Idioma
es
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Universidad Andrés Bello
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Licencia CC
Resumen
Varios factores involucrados en los procesos infectivos han sido identificados a través
de estudios de interacción patógeno-hospedero. Salmonella enterica serovar Typhimurium,
agente causante de gastroenteritis en humanos e infección sistémica en ratones, posee
sistemas de defensa que le permiten sobrellevar tanto la respuesta inmune como la
producción de especies reactivas de oxígeno (ROS) por parte del hospedero. Uno de los
mecanismos por los cuales Salmonella modula la respuesta a ROS está mediado por el factor
transcripcional ArcA. En nuestro laboratorio, la proteína ArcA fue caracterizada bioquímica
y funcionalmente en condiciones de crecimiento aeróbico, observando que la respuesta a
estrés oxidativo generado por peróxido de hidrógeno (H2O2) está modulada por la expresión
de genes claves identificados en esta condición. Sin embargo, estos genes no han sido
caracterizados en términos de infección de macrófagos murinos, y no ha sido caracterizado
como un' factor de virulencia. En este contexto, nos propusimos demostrar que la expresión
de estos genes es modulada por el regulador global ArcA durante la infección de macrófagos
murinos y que ArcA es un factor de virulencia requerido para un ciclo infectivo en
macrofagos. Para responder esta hipótesis, realizamos ensayos de supervivencia con cepas
mutantes AarcA en macrófagos murinos RAW 264.7, medimos por qRT-PCR la expresión
de genes previamente descritos que son regulados por AreA en respuesta a ROS usando
bacterias recuperadas desde macrófagos infectados, y se realizaron ensayos de infección en
el modelo murino (BALB/c).
Nuestros resultados muestran una disminución del 50% de la invasión al macrófago en la
cepa carente del factor transcripcional AreA, comparado con la cepa silvestre de S.
Typhimurium, y además la falta de este factor promueve una notable disminución en su
viabilidad en el modelo murino. Además varios genes regulados por ArcA en la infección de
macrófagos fueron identificados, por ejemplo el gen ompW codificante de una proteína de
membrana externa, el cual está negativamente regulado por ArcA a lo largo de todos los
estadíos de infección. Estos resultados se correlacionan con trabajos que demostraron que
dicho gen es requerido como mecanismo para la defensa de ROS y también concuerdan con
el patrón de expresión que presentó en respuesta a H2O2. Por lo tanto, durante este trabajo de
tesis se logró demostrar que el factor transcripcional ArcA participa en la regulación de la
expresión génica durante la infección de macrófagos murinos, siendo las condiciones de
estrés oxidativo presentes al interior del macrófago las que gatillarían la activación del factor
transcripcional AreA para su consecuente acción. Además fuimos capaces de demostrar en
estudios de infección de macrófagos murinos que el factor transcripcional ArcA por sí solo
es requerido para el proceso de invasión celular. Y finalmente demostramos, a partir de
infección del modelo murino, que la proteína ArcA es un factor de virulencia.
Several factors involved in the infective process have being identified through hostpathogen interaction studies. Salmonella enterica serovar Typhimurium, the causative agent of gastroenteritis in humans and systemic fever in mice, has different systems that allow its defense and overcome both the immune response and the production of reactive oxygen species (ROS) by the host. One of the mechanisms by which Salmonella modulates ROS response is mediated by the transcriptional factor ArcA. In our laboratory, the protein ArcA was characterized biochemically and functionally in aerobic growth conditions, observing that response to oxidative stress generated by hydrogen peroxide (H2O2) is modulated by the expression of key genes described for this condition, however, these genes have not been characterized in terms of infection of murine macrophages, nor characterized as a virulence factor. In this context, we propose to demonstrate that in Salmonella enterica serovar Typhimurium the expression of these genes is modulated by the global regulator ArcA during infection of murine macrophages which is also a virulence factor required for the success of the infection cycle. To answer this hypothesis, we pérformed survival assays with mutant strains AarcA in murine macrophages RAW 264.7, measuring by qRT-PCR the expression of genes previously described to be regulated by ArcA on responce to ROS using bacteria recovered from infected macrophages and infection assays in the mice model (BALB/c). Our results show a 50% decrease in macrophage invasion in the strain lacking the transcriptional factor ArcA, compared with the wild strain of S. Typhimurium, moreover the lack of this factor promotes a striking decrease in their viability to survive in mice. Furthermore, several genes regulate by ArcA in the macrophage infection were identified, for example the ompW gene encoding an outer membrane protein, which expression is negatively regulated by ArcA through all stages of infection. These results correlate with previous work showing that this gene is required as a mechanism of defense against ROS and also consistent with the expression pattern presented in response to H2O2. Therefore, during this Thesis it was possible to demonstrate that the two-components system ArcAB are involved in the regulation of gene expression during infection of murine macrophages, where the oxidative stress conditions present in the macrophage would trigger activation of ArcA and then a successful action. Notewortily, we were able to demonstrate using infection studies of murine macrophages, that only the ArcA factor transcriptional is required for the process of cellular invasion. Finally we show, using the murine model of infection that ArcA protein is a virulence factor.
Several factors involved in the infective process have being identified through hostpathogen interaction studies. Salmonella enterica serovar Typhimurium, the causative agent of gastroenteritis in humans and systemic fever in mice, has different systems that allow its defense and overcome both the immune response and the production of reactive oxygen species (ROS) by the host. One of the mechanisms by which Salmonella modulates ROS response is mediated by the transcriptional factor ArcA. In our laboratory, the protein ArcA was characterized biochemically and functionally in aerobic growth conditions, observing that response to oxidative stress generated by hydrogen peroxide (H2O2) is modulated by the expression of key genes described for this condition, however, these genes have not been characterized in terms of infection of murine macrophages, nor characterized as a virulence factor. In this context, we propose to demonstrate that in Salmonella enterica serovar Typhimurium the expression of these genes is modulated by the global regulator ArcA during infection of murine macrophages which is also a virulence factor required for the success of the infection cycle. To answer this hypothesis, we pérformed survival assays with mutant strains AarcA in murine macrophages RAW 264.7, measuring by qRT-PCR the expression of genes previously described to be regulated by ArcA on responce to ROS using bacteria recovered from infected macrophages and infection assays in the mice model (BALB/c). Our results show a 50% decrease in macrophage invasion in the strain lacking the transcriptional factor ArcA, compared with the wild strain of S. Typhimurium, moreover the lack of this factor promotes a striking decrease in their viability to survive in mice. Furthermore, several genes regulate by ArcA in the macrophage infection were identified, for example the ompW gene encoding an outer membrane protein, which expression is negatively regulated by ArcA through all stages of infection. These results correlate with previous work showing that this gene is required as a mechanism of defense against ROS and also consistent with the expression pattern presented in response to H2O2. Therefore, during this Thesis it was possible to demonstrate that the two-components system ArcAB are involved in the regulation of gene expression during infection of murine macrophages, where the oxidative stress conditions present in the macrophage would trigger activation of ArcA and then a successful action. Notewortily, we were able to demonstrate using infection studies of murine macrophages, that only the ArcA factor transcriptional is required for the process of cellular invasion. Finally we show, using the murine model of infection that ArcA protein is a virulence factor.
Notas
Tesis (Bioquímica, Magíster en Bioquímica)
Esta tesis se realizó en el Laboratorio de Microbiología Molecular, de la Facultad de Ciencias Biológicas de la Universidad Andrés Bello y fue financiada por el proyecto FONDECYT N°1120384, titulado "ArcA and SIyA-mediated response to radical oxygen species in Salmonella", dirigido por la Dra. Claudia Saavedra Sanchez, y por el Proyecto Interno UNAB DI-786-1518.
Esta tesis se realizó en el Laboratorio de Microbiología Molecular, de la Facultad de Ciencias Biológicas de la Universidad Andrés Bello y fue financiada por el proyecto FONDECYT N°1120384, titulado "ArcA and SIyA-mediated response to radical oxygen species in Salmonella", dirigido por la Dra. Claudia Saavedra Sanchez, y por el Proyecto Interno UNAB DI-786-1518.
Palabras clave
Salmonella Enteritidis, Factor Transcripcional ArcA