Efecto del sistema de dos componentes PhoP/PhoQ en la regulación de la expresión del factor sigma alternativo RpoS bajo distintas condiciones de estrés en salmonella entérica Serovar Typhi
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2011
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es
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Universidad Andrés Bello
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Licencia CC
Resumen
El género Salmonella comprende dos especies: Salmonella bongori y Salmonella
enterica. Esta última abarca seis subespecies, dentro de las cuales se encuentra la
subespecie enterica (I), la cual agrupa a las Salmonelas causantes de enfermedades en
animales homeotermos, como es el caso de S. Typhi, que es el agente causal de la fiebre
tifoidea en el humano. La fiebre tifoidea es una enfermedad del sistema retículo-endotelial,
caracterizada por una fiebre alta, que puede causar la muerte del paciente cuando no es
tratada oportunamente.
La infección con S. Typhi comienza con la ingestión de comidas o aguas
contaminadas. Una vez que la bacteria ha llegado al intestino, invade las células del
epitelio, donde puede entrar al torrente sanguíneo del hospedero para diseminarse en
órganos profundos como el hígado, el bazo y/o la médula ósea.
Durante el proceso de infección, S Typhi debe enfrentar diferentes tipos de estrés.
Para poder enfrentar las diferentes condiciones, las bacterias se deben enfocar en la
expresión de genes que le permitan. Para modular la expresión de genes, las bacterias se
valen de factores sigma alternativos y de reguladores transcripcionales, incluyendo los
implicados en los sistemas de dos componentes.
Los factores sigma de la RNA polimerasa tienen la función de reconocer los
promotores de diferentes genes blancos. Dependiendo de la condición ambiental se utilizará
un factor sigma determinado, el cual reconocerá y activará la transcripción de un grupo
especial de genes, logrando la supervivencia de la bacteria en diferentes tipos de estrés.
Dentro de los factores sigma se encuentra el factor sigma alternativo RpoS, el cual se activa
en fase logarítmica tardía alcanzando un máximo en fase estacionaria, además de activarse en pH ácido, shock osmótico, limitación de nutrientes y shock térmico, entre otras
condiciones.
Los sistemas de dos componentes son una organización de proteínas,
correspondiente a un sensor de membrana, encargado de sensar las condiciones ambientales
y acoplarlas a un regulador de respuesta que va a generar la regulación de sus genes blanco.
Dentro de estos se encuentra el sistema de dos componentes PhoP/PhoQ, que se activa en
un medio con baja concentración de magnesio, donde PhoQ es el sensor de membrana y
PhoP el regulador de respuesta.
En este trabajo, se hipotetizó que el sistema de dos componentes PhoP / PhoQ está
implicado en la regulación de la expresión de RpoS en S. Typhi.
En esta tesis se examinó la regulación del sistema de dos componentes PhoP/PhoQ
sobre RpoS, en diferentes condiciones de crecimiento, donde está descrito que existe una
activación de éste factor sigma y/o de PhoP / PhoQ. Los análisis de expresión proteica
demuestran que la ausencia de phoP, aumenta los niveles de RpoS. Además el aumento del
número de copias de phoP disminuye la cantidad de RpoS. Los niveles proteicos anteriores
se asociaron a la supervivencia a estrés oxidativo causado por H2O2, demostrando que la
ausencia de phoP produce mayor supervivencia de las bacterias y el aumento en el número
de copias de phoP tiene un efecto negativo en la supervivencia. Los resultados anteriores
también fueron corroborados por el análisis de la activación del promotor de un gen
dependiente de RpoS e independiente de phoP.
Los resultados sugieren que el sistema de dos componentes PhoP/PhoQ produce una
regulación negativa sobre el factor sigma alternativo RpoS en S. Typhi.
The genus Salmonella includes two species, S bongori and S enterica. The last one includes six subspecies, among them subspecies enterica (I), which involves bacteria causing diseases in warm-blooded animals, such as S Typhi, causative agent of typhoid fever in human beings. This is a reticuloendothelial system disease characterized by high fever, causing the patient's death if not treated early. The S. Typhi infection begins by the ingestion of food or water contaminated. Once S. Typhi invades intestinal epithelial cells come into the bloodstream of the host, this is capable to spreading towards deep organs such as liver or spleen. During S. Typhi infection process, bacteria must face different kinds of stress. In order to survive at different environmental conditions, bacteria must be focused on the transcription of genes that allow survival in order to achieve a successful infection cycle. These processes are performed by using alternative sigma factors and two-component systems. The sigma factors of RNA polymerase are capable to recognize the promoters of different target genes. Depending on sigma factor, this can recognize and activate the transcription of a pool of genes that allow the survival of the bacteria in different environment. The RpoS belong to the alternative sigma factor, which is activated in late log phase and peaking in stationary phase. Furthermore, this factor is activated by acid pH, osmotic shock, nutrient limitation, heat shock, among other conditions. The two-component system consists in inner membrane sensors capable to register environmental conditions. These systems are connected to a response regulator, which regulates the expression of their target genes. Among these two-component systems, the system PhoP / PhoQ, is activated in a medium with low concentration of magnesium. It consists of the inner membrane sensor PhoQ and the cytoplasmic regulator PhoP. In this work, we hypothesized that the two-component system PhoP / PhoQ is involved in regulating the expression of 6 s in S. Typhi. This thesis examined the regulation of the two components on RpoS, under infection conditions where has been reported the activation of this sigma factor and PhoP/PhoQ. Protein expression analysis demonstrated that absence of phoP, increases levels of 6 s as well as the increasing of number of copies of phoP result in reduces amount of 6 s. Previous protein levels were associated with survival to oxidative stress by H2O2, demonstrating that the absence of phoP produces greater survival of the bacteria and increasing the number of copies of phoP has a negative effect on survival. The above results were confirmed by promoter activation analyses of an RpoS-dependent and PhoPindependent gene. The results suggest that two-component system PhoP/PhoQ produces a negative regulation on the alternative sigma factor RpoS in S. Typhi.
The genus Salmonella includes two species, S bongori and S enterica. The last one includes six subspecies, among them subspecies enterica (I), which involves bacteria causing diseases in warm-blooded animals, such as S Typhi, causative agent of typhoid fever in human beings. This is a reticuloendothelial system disease characterized by high fever, causing the patient's death if not treated early. The S. Typhi infection begins by the ingestion of food or water contaminated. Once S. Typhi invades intestinal epithelial cells come into the bloodstream of the host, this is capable to spreading towards deep organs such as liver or spleen. During S. Typhi infection process, bacteria must face different kinds of stress. In order to survive at different environmental conditions, bacteria must be focused on the transcription of genes that allow survival in order to achieve a successful infection cycle. These processes are performed by using alternative sigma factors and two-component systems. The sigma factors of RNA polymerase are capable to recognize the promoters of different target genes. Depending on sigma factor, this can recognize and activate the transcription of a pool of genes that allow the survival of the bacteria in different environment. The RpoS belong to the alternative sigma factor, which is activated in late log phase and peaking in stationary phase. Furthermore, this factor is activated by acid pH, osmotic shock, nutrient limitation, heat shock, among other conditions. The two-component system consists in inner membrane sensors capable to register environmental conditions. These systems are connected to a response regulator, which regulates the expression of their target genes. Among these two-component systems, the system PhoP / PhoQ, is activated in a medium with low concentration of magnesium. It consists of the inner membrane sensor PhoQ and the cytoplasmic regulator PhoP. In this work, we hypothesized that the two-component system PhoP / PhoQ is involved in regulating the expression of 6 s in S. Typhi. This thesis examined the regulation of the two components on RpoS, under infection conditions where has been reported the activation of this sigma factor and PhoP/PhoQ. Protein expression analysis demonstrated that absence of phoP, increases levels of 6 s as well as the increasing of number of copies of phoP result in reduces amount of 6 s. Previous protein levels were associated with survival to oxidative stress by H2O2, demonstrating that the absence of phoP produces greater survival of the bacteria and increasing the number of copies of phoP has a negative effect on survival. The above results were confirmed by promoter activation analyses of an RpoS-dependent and PhoPindependent gene. The results suggest that two-component system PhoP/PhoQ produces a negative regulation on the alternative sigma factor RpoS in S. Typhi.
Notas
Tesis (Bioquímico, Magíster en Bioquímica)
Esta tesis fue fmanciada por el proyecto FONDECYT N° 1060999: "Salmonella enterica serovar Typhi, PATOGENO ESPECÍFICO Y CRÓNICO DEL SER HUMANO: CARACTERIZACIÓN DE MECANISMOS MOLECULARES DE SU PATOGENICIDAD"
Esta tesis fue fmanciada por el proyecto FONDECYT N° 1060999: "Salmonella enterica serovar Typhi, PATOGENO ESPECÍFICO Y CRÓNICO DEL SER HUMANO: CARACTERIZACIÓN DE MECANISMOS MOLECULARES DE SU PATOGENICIDAD"