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Estudio de la expresión diferencial de proteínas de Fusobacterium nucleatum en biofilm vs. en estado planctónico

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2017
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Introducción: Las células bacterianas pueden crecer formando biofilms o en estado de vida libre planctónico. En el modo de crecimiento de biofilm, los microorganismos muestran una mayor capacidad virulenta, un aumento en la resistencia a compuestos antimicrobianos, presentan una mejor respuesta al estrés ambiental y desarrollan numerosos mecanismos de respuesta inmunológica frente al huésped. Objetivo: El propósito de este estudio ha sido el análisis de la expresión diferencial de proteínas de Fusobacterium nucleatum DSM20482 cuando está formando biofilm respecto a cuando está en estado planctónico. De esta forma, se pueden encontrar posibles blancos para futuras terapias que permitan bloquear o mermar la incorporación de bacterianas patógenas a la placa bacteriana, e identificar proteínas que se expresan exclusivamente en la fase de biofilm claramente implicadas en producción de una respuesta inmunogénica en el huésped y que podrían utilizarse en una primera aproximación como biomarcadores en las enfermedades periodontales. Material y Métodos: Para el estudio de las proteínas diferenciales de F. nucleatum se desarrolló el biofilm de esta especie sobre portaobjetos (slides) cubiertos de hidroxiapatita con una concentración bacteriana de 107 unidades formadoras de colonias (ufc)/mL y 24 horas de incubación. El cultivo planctónico se realizó en las mismas condiciones. Después de recuperar las bacterias que están en el biofilm y en el cultivo planctónico, se procedió a la ruptura de la célula con un tampón adecuado y a la extracción de las proteínas bacterianas en ambos estados. Posteriormente, se limpiaron las muestras para eliminar impurezas que interfiriesen en la electroforesis bidimensional, se solubilizaron las proteínas y se cuantificaron por el método Bradford. Para comprobar la cantidad y calidad de las muestras proteicas se llevó a cabo la separación de las proteínas por electroforesis bidimensional en geles de poliacrilamida. Cuando se obtuvo la suficiente cantidad de proteína, haciendo grupos de muestras, se procedió al marcaje de éstas utilizando la tecnología 2Dr la cantidad y calidad de las muestras proteicas se llevó a cabo la separación de las proteínas por electroforesis bidimensional en geles de poliacrilamida. Cuando se obtuvo la suficiente cantidad de proteína, haciendo grupos de muestras, se procedió al marcaje de éstas utilizando la tecnología 2DDIGE ™ (2D Fluorescence Difference Gel Electrophoresis) y a la separación de las proteínas mediante electroforesis bidimensional. Los geles se escanearon con el escáner Typhoon™ 9400 y las imágenes se analizaron con el software DeCyder™ v6.5. Las proteínas diferenciales se identificaron por espectrometría de masas. Resultados y Conclusiones: Se observaron 68 spots que se expresaban diferencialmente. De estos 68, se han identificado 26 proteínas que se sobreexpresan cuando está en estado biofilm y 18 que se reprimen. Es importante destacar que las proteínas que se sobreexpresan son proteínas implicadas en metabolismo y en biosíntesis de macromoléculas, transportadores de tipo ABC y proteínas de función desconocida. El estudio de estas proteínas puede ayudar a entender la formación del biofilm en F. nucleatum y su implicación en la virulencia de la bacteria.
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