El gen arcA que codifica para el factor transcripcional ArcA de Salmonella Typhimurium es requerido frente a las condiciones de estrés oxidativo encontradas en la infección de neutrófilos murinos
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Fecha
2018
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es
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Universidad Andrés Bello
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Licencia CC
Resumen
Los neutrófilos eliminan a patógenos sometiéndolos a distintos tipos de estrés, principalmente por la producción de especies reactivas de oxígeno (ROS). Salmonella Typhimurium sobrevive a estas condiciones gracias a su capacidad de regulación génica, mediado por factores transcripcionales entre los cuales está ArcA, el cual cumple un rol importante en la resistencia bacteriana contra ROS, participa en la regulación del metabolismo celular, biosíntesis flagelar y motilidad.
Resultados preliminares muestran que ArcA es requerido en la invasión de neutrófilos murinos y durante la infección sistémica en ratones. Sin embargo, se desconoce cuáles son los genes blancos modulados por éste, que le permitirían a Salmonella adaptarse y responder al estrés en el fagosoma del neutrófilo. Por lo que la hipótesis de este trabajo es "el gen arcA, que codifica para el factor transcripcional ArcA de Salmonella Typhimurium es requerido para la regulación de genes relacionados con la respuesta bacteriana a las condiciones de estrés oxidativo encontradas durante la infección de neutrófilos murinos".
Se purificaron e infectaron neutrófilos, extraídos de médula ósea de ratón C57BL16 con las cepas Salmonella Typhimurium 14028s, AarcA y AarcA/pBR::arcA para determinar si el estrés oxidativo generado en el fagosoma del neutrófilo es afectado por la ausencia del gen arcA. Entonces, se evaluó la producción de ROS total, OZ , H2O2 y HOCI y encontramos que en AarcA la producción de ROS total disminuye significativamente. Sin embargo, no hubo cambios en la producción de las especies determinadas individualmente, lo cual sugiere que ArcA influiría en la activación del neutrófilo. Adicionalmente, se midió la concentración de NAD+ en Salmonella durante la infección de neutrófilos, solo se genera mayor concentración de este metabolito en la cepa DarcA en comparación a la cepa silvestre y complementada. Este resultado sugiere que las rutas de consumo de este cofactor se encuentran disminuidas en la mutante lo que generaría una acumulación de NAD+.
También se realizó un análisis a nivel transcripcional para poder identificar los posibles blancos regulados por ArcA de genes involucrados en la respuesta a HOCl, virulencia y metabolismo durante la infección de neutrófilos murinos infectados con las cepas Salmonella Typhimurium 14028s AarcA y DarcA/pBR::arcA. Se evaluaron genes que codifican para:
- Factores de virulencia, donde son inducidos aquellos relacionados con la formación y mantención de la VCS.
- Enzimas destoxificantes y de reparación, de los cuales solo estaba inducido el gen katE y los genes msrA y trxB pertenecientes a sistemas de reparación de proteínas.
- Porinas, donde la mayoría se encontraban inducidas y solo la expresión de dos genes estaban reprimidos: ompX y phoE.
- Metabolismo central y de aminoácidos, en donde ArcA solamente interviene en la regulación de genes relacionados con e metabolismo de glutamato, lisina, alanina y cisteína.
Por lo tanto, los resultados sugieren que la presencia del gen arcA es requerida para la regulación de genes, que en su conjunto permiten a la bacteria adaptarse y responder a las condiciones de estrés generadas en el fagosoma del neutrófilo.
Neutrophils eliminate pathogens by undergoing several types of stress, mainly by the production of reactive oxygen species (ROS). Salmonella Typhimurium survives to this conditions thanks to the genetic regulation capacity, mediated by transcriptional factors among which is ArcA, that has an important role in the bacterial resistance against ROS, also participating in the regulation of cellular metabolism, flagelar biosynthesis and motility. Preliminary results show that ArcA is required for murine neutrophil invasion and during systemic infection in mice. However, it is not known which target genes are modulated by it and would allow Salmonella to adapt and respond to the stress in the neutrophil phagosome. The hypothesis of this work is "The arcA gene, which codifies to the transcriptional factor ArcA of Salmonella Typhimurium, is required for gene regulation related to bacterial response to stress conditions of oxidative stress found during the infection of murine neutrophils". Neutrophils were purified from bone marrow of C57BL/6 mice and infected with the strains Salmonella Typhimurium 14028s, DarcA and AarcAlpBR::arcA to determine if the oxidative stress generated in the neutrophil phagosome is affected by the absence of the arcA gene. In turn, it was evaluated total ROS, Oz , H2O2 and HOCI production and it was found that in DarcA strain the total ROS production is diminished significantly. However, there were not changes in the production of reactive species determined individually; this suggests that ArcA would influence the neutrophil activation. Additionally, it was measured the concentration of NAD+ in Salmonella during neutrophil infection, it was found a greater concentration of this metabolite in AarcA strain in comparison to the wild type and complemented strain. This result suggests that the consumption routes of the cofactor are diminished in the mutant strain that would provoke an accumulation of NAD+. A transcriptional level analysis was also carried out to be able to identify possible targets regulated by ArcA in genes involved to the HOCI response, virulence and metabolism during neutrophil infection with strains Salmonella Typhimurium 14028s AarcA and DarcA/pBR::arcA. There were evaluated genes that codify to: - Virulence factors, where we found genes induced related to the formation and maintenance of the VCS. -Detoxifying and repair enzymes, in which only one gene was induced, katE, and the genes msrA and trxB belonging to the protein repair system. -Porins, where most of the genes were induced and only the expression of two genes was repressed (ompX and phoE). -Central and amino acid metabolism, where ArcA only mediates the gene regulation related to glutamate, lysine, alanine and cysteine biosynthesis. In sum, these results suggest that the presence of the arcA gene is required for gene regulation, that in turn allow the bacteria to adapt and respond to stress conditions generated in the neutrophil phagosome.
Neutrophils eliminate pathogens by undergoing several types of stress, mainly by the production of reactive oxygen species (ROS). Salmonella Typhimurium survives to this conditions thanks to the genetic regulation capacity, mediated by transcriptional factors among which is ArcA, that has an important role in the bacterial resistance against ROS, also participating in the regulation of cellular metabolism, flagelar biosynthesis and motility. Preliminary results show that ArcA is required for murine neutrophil invasion and during systemic infection in mice. However, it is not known which target genes are modulated by it and would allow Salmonella to adapt and respond to the stress in the neutrophil phagosome. The hypothesis of this work is "The arcA gene, which codifies to the transcriptional factor ArcA of Salmonella Typhimurium, is required for gene regulation related to bacterial response to stress conditions of oxidative stress found during the infection of murine neutrophils". Neutrophils were purified from bone marrow of C57BL/6 mice and infected with the strains Salmonella Typhimurium 14028s, DarcA and AarcAlpBR::arcA to determine if the oxidative stress generated in the neutrophil phagosome is affected by the absence of the arcA gene. In turn, it was evaluated total ROS, Oz , H2O2 and HOCI production and it was found that in DarcA strain the total ROS production is diminished significantly. However, there were not changes in the production of reactive species determined individually; this suggests that ArcA would influence the neutrophil activation. Additionally, it was measured the concentration of NAD+ in Salmonella during neutrophil infection, it was found a greater concentration of this metabolite in AarcA strain in comparison to the wild type and complemented strain. This result suggests that the consumption routes of the cofactor are diminished in the mutant strain that would provoke an accumulation of NAD+. A transcriptional level analysis was also carried out to be able to identify possible targets regulated by ArcA in genes involved to the HOCI response, virulence and metabolism during neutrophil infection with strains Salmonella Typhimurium 14028s AarcA and DarcA/pBR::arcA. There were evaluated genes that codify to: - Virulence factors, where we found genes induced related to the formation and maintenance of the VCS. -Detoxifying and repair enzymes, in which only one gene was induced, katE, and the genes msrA and trxB belonging to the protein repair system. -Porins, where most of the genes were induced and only the expression of two genes was repressed (ompX and phoE). -Central and amino acid metabolism, where ArcA only mediates the gene regulation related to glutamate, lysine, alanine and cysteine biosynthesis. In sum, these results suggest that the presence of the arcA gene is required for gene regulation, that in turn allow the bacteria to adapt and respond to stress conditions generated in the neutrophil phagosome.
Notas
Tesis (Bioquímico, 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°1160315, titulado "Identification and characterization of non-cognate partners of the S. Typhimurium ArcAB two-component system in response to hydrogen peroxide and hypochlorous acid" Dirigido por la Dra. Claudia Saavedra Sánchez.
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°1160315, titulado "Identification and characterization of non-cognate partners of the S. Typhimurium ArcAB two-component system in response to hydrogen peroxide and hypochlorous acid" Dirigido por la Dra. Claudia Saavedra Sánchez.
Palabras clave
Salmonella Typhimurium, Factor Transcripcional ArcA, Neutrófilos, Inmunología, Estrés Oxidativo