Publication: Estudio de la expresión diferencial de proteinas de Porphyrormonas Gingivalis en biofilms versus en estado planctónico
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2019
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Abstract
Objetivo: El objetivo de esto trabajo fue estudiar la expresión diferencial de proteínas de Porphyromonas gingivalis cuando está formando biofilm respecto a cuando está en estado planctónico.
Materiales y Métodos: A partir de un cultivo en medio BHI modificado de P. gingivalis ATCC33277 en fase exponencial se desarrolló el biofilm monoespecie sobre discos estériles de hidroxiapatita en una placa multipocillo de 24 pocillos. El cultivo planctónico se desarrolló en viales de plástico de 50 mL tipo Falcón. En ambos casos se ajustó la concentración bacteriana a 107 unidades formadoras de colonias (ufc)/mL y se incubó en condiciones de anaerobiosis a 37C durante 96 horas (3 réplicas biológicas de cada una de las condiciones). Para recuperar las bacterias del biofilm se extrajo el disco, se añadió 1 mL de PBS (Phosphate Buffer Saline), se vorteó durante 3 minutos recogiendo la suspensión, añadiéndola a un eppendorf y centrifugando a 13000 rpm a 4ºC durante 5 minutos. Para recuperar las bacterias en estado planctónico, se centrifugó la suspensión bacteriana a 9000 rpm a 4ºC durante 10 minutos. Posteriormente, se procedió a la extracción de proteínas en ambos casos. Para ello se siguieron los siguientes pasos: 1) los precipitados se resuspendieron en PBS junto con inhibidores de proteasas para lisar las células bacterianas con un sonicador de punta, 2) las muestras se limpiaron para eliminar posibles restos de sales, detergentes, lípidos e impurezas, 3) las proteínas se solubilizaron en urea 8M mediante vortex y utilizando un sonicador de baño, y 4) se cuantificaron empleando el método de Bradford y el fluorímetro Qubit 3.0. Para comprobar la cuantificación proteica se realizó un gel de control de carga (SDS-PAGE 10%). El estudio de expresión diferencial se realizó sin marcaje proteico ni peptídico acoplado a un espectrómetro de masas (Label-Free (LCMS/MS)).
Resultados: Se pudieron cuantificar un total de 614 proteínas, de las cuales 73 se expresaban diferencialmente en las condiciones de crecimiento. De estas proteínas diferenciales, 28 se sobre-expresaban cuando la bacteria estaba formando biofilm y 45 proteínas se reprimían en este modo de crecimiento. Conclusión: P. gingivalis presentó expresión diferencial de diversas proteínas en el estado de biofilm en relación al estado planctónico. Estas proteínas que se expresan diferencialmente en el biofilm pueden ser buenas candidatas para su empleo en el diagnóstico y tratamiento de la periodontitis.
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