Identificación del ORF VPA1373 como candidato a efector del T3SS2 del clon pandémico de Vibrio parahaemolyticus
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Fecha
2022
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Idioma
es
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Universidad Andrés Bello
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Licencia CC
Licencia CC
Resumen
Vibrio parahaemolyticus es una bacteria Gram negativo que es la principal
causa de gastroenteritis causada por el consumo de productos del mar a nivel
mundial. Esta bacteria posee múltiples factores de virulencia, dentro de los que
destacan los Sistemas de Secreción de Proteínas del Tipo III (T3SS). Vibrio
parahaemolyticus posee dos T3SS (T3SS1 y T3SS2), cada uno codificado en un
cromosoma distinto de la bacteria. Los T3SS son unas nanomáquinas proteicas
en forma de aguja, las cuales han evolucionado específicamente para la
interacción con células eucariontes. Estos sistemas median la entrega de
proteínas, llamadas efectores, directamente desde el citosol de la bacteria hacia
el citosol de la célula eucarionte infectada. En este contexto, el T3SS2 es
reconocido como uno de los principales factores de virulencia de esta bacteria
por su capacidad de generar citotoxicidad in vitro y de ser esencial para la
enterotoxicidad causada por V. parahaemolyticus en modelos animales de
infección.
Actualmente se conocen diez proteínas efectoras de Vibrio
parahaemolyticus, no obstante, no se conoce el número total de efectores
existentes. Un análisis computacional realizado en el laboratorio llevó a la
identificación de una posible señal de secreción en la proteína codificada por el
ORF vpa1373, lo que sugiere que VPA1373 sería un nuevo efector del T3SS2.
Es así, como este trabajo tiene como objetivo determinar sí VPA1373
corresponde a una proteína efectora del T3SS2. Para este objetivo, en primer
lugar, se construyó una fusión génica entre el ORF que codifica VPA1373 y el
reportero adenilato ciclasa CyaA, demostrando la producción de la proteína de
fusión mediante ensayos de SDS/PAGE y Western Blot, usando anticuerpos
específicos anti-CyaA. Finalmente, dicho constructo fue transformado en distintos
fondos genéticos de V. parahaemolyticus y se realizó un ensayo de translocación
a células Caco-2.
Vibrio parahaemolyticus is a Gram-negative bacterium that is the leading of gastroenteritis caused by consuming of seafood products worldwide. This bacterium possesses multiple virulence factors, among which the Type III Protein Secretion Systems (T3SS) V. parahaemolyticus encodes two T3SS (T3SS1 and T3SS2), each encoded in a different chromosome of the bacterium. The T3SS is a needle-shaped protein nanomachines that has evolved specifically for interaction with eukaryotic cells. These systems mediate the delivery of proteins, called effectors, directly from the cytosol of the bacterium to the cytosol of the infected eukaryotic cell. In this context, T3SS2 is recognized as one of the major virulence factors of this bacterium because of its ability to generate cytotoxicity in vitro and to be essential for enterotoxicity caused by V. parahaemolyticus in animal models of infection. Ten effector proteins of V. parahaemolyticus are currently known, however, the total number of existing effectors is unknown. A computational analysis performed in the laboratory led to identifying of a possible secretion signal in the protein encoded by ORF vpa1373, suggesting that VPA1373 would be a new effector of T3SS2. Thus, this work aims to determine whether VPA1373 corresponds to a T3SS2 effector protein. For this purpose, first, a gene fusion between the ORF encoding VPA1373 and the adenylate cyclase reporter CyaA was constructed, demonstrating the production of the fusion protein by SDS/PAGE and Western Blot assays, using specific anti-CyaA antibodies. Finally, this construct was transformed into different V. parahaemolyticus genetic backgrounds and a translocation assay to Caco-2 cells was performed.
Vibrio parahaemolyticus is a Gram-negative bacterium that is the leading of gastroenteritis caused by consuming of seafood products worldwide. This bacterium possesses multiple virulence factors, among which the Type III Protein Secretion Systems (T3SS) V. parahaemolyticus encodes two T3SS (T3SS1 and T3SS2), each encoded in a different chromosome of the bacterium. The T3SS is a needle-shaped protein nanomachines that has evolved specifically for interaction with eukaryotic cells. These systems mediate the delivery of proteins, called effectors, directly from the cytosol of the bacterium to the cytosol of the infected eukaryotic cell. In this context, T3SS2 is recognized as one of the major virulence factors of this bacterium because of its ability to generate cytotoxicity in vitro and to be essential for enterotoxicity caused by V. parahaemolyticus in animal models of infection. Ten effector proteins of V. parahaemolyticus are currently known, however, the total number of existing effectors is unknown. A computational analysis performed in the laboratory led to identifying of a possible secretion signal in the protein encoded by ORF vpa1373, suggesting that VPA1373 would be a new effector of T3SS2. Thus, this work aims to determine whether VPA1373 corresponds to a T3SS2 effector protein. For this purpose, first, a gene fusion between the ORF encoding VPA1373 and the adenylate cyclase reporter CyaA was constructed, demonstrating the production of the fusion protein by SDS/PAGE and Western Blot assays, using specific anti-CyaA antibodies. Finally, this construct was transformed into different V. parahaemolyticus genetic backgrounds and a translocation assay to Caco-2 cells was performed.
Notas
Tesis (Licenciada en Biología)
Palabras clave
Vibrio Parahaemolyticus, Citotoxicidad, Proteínas Efectoras, Gastroenteritis