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Comparativa Genómica Y Expresión Diferencial De Genes En Respuesta a Temperatura Y Su Relación Con El Proceso Infeccioso De Yersinia Ruckeri

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Comparativa Genómica Y Expresión Diferencial De Genes En Respuesta a Temperatura Y Su Relación Con El Proceso Infeccioso De Yersinia Ruckeri Programa de Doctorado Biología Funcional y Molecular Comparativa genómica y expresión diferencial de genes en respuesta a temperatura y su relación con el proceso infeccioso de Yersinia ruckeri Desirée Cascales Freire Tesis doctoral Oviedo 2017 2 4 6 7 8 Agradecimientos 9 10 11 12 Índice 13 14 Índice Índice ............................................................................................................................................................... 13 Abreviaturas y siglas .................................................................................................................................. 21 1. Introducción ........................................................................................................................................ 25 1.1. Situación de la acuicultura y sus principales patologías bacterianas asociadas ........ 27 1.2. Yersinia ruckeri..................................................................................................................................... 27 1.3. La enfermedad entérica de la boca roja (enteric redmouth disease) ............................. 29 1.4. Factores de virulencia de Y. ruckeri ............................................................................................. 33 1.5. Influencia de la temperatura en la expresión de genes de virulencia en bacterias patógenas de animales ectotermos .................................................................................................... 35 1.6. Efecto de la exposición a antibióticos en la virulencia ......................................................... 36 1.7. Bombas de expulsión de productos tóxicos ............................................................................ 37 1.8. Estrés osmótico. Proteína OsmY. .................................................................................................. 38 1.9. La luminiscencia como herramienta para el estudio de la regulación génica y el análisis de los procesos infecciosos..................................................................................................... 39 1.10. Secuenciación y análisis de genomas ...................................................................................... 40 2. Objetivos ............................................................................................................................................... 41 3. Material y métodos ........................................................................................................................... 45 3.1. Cepas, plásmidos y condiciones de cultivo .............................................................................. 47 3.2. Técnicas generales de manipulación del ADN ........................................................................ 49 3.3. Secuenciación y caracterización genómica de la cepa Y. ruckeri 150 ............................ 49 3.3.1. Secuenciación del ADN ............................................................................................................ 49 3.3.2. Anotación génica del cromosoma e identificación de secuencias repetidas, profagos y elementos CRISPR ........................................................................................................... 50 3.4. Análisis comparativo del genoma de Y. ruckeri 150 con las cepas Y. ruckeri ATCC24973, Y. ruckeri CSF007-82, Y. ruckeri Big Creek 74 y Y. ruckeri SC09. .................... 50 3.5. Obtención y análisis de una genoteca de mutantes de Y. ruckeri 150 mediante transposición ................................................................................................................................................ 52 3.5.1. Construcción del transposón mini-Tn5 luxlac Km2 ....................................................... 52 3.5.2. Transferencia e inserción del transposón Tn5luxlac en el genoma de Y. ruckeri 150 ............................................................................................................................................................... 53 3.5.3. Análisis de la inserción del transposón Tn5luxlac .......................................................... 54 3.5.4. Estabilidad del transposón e influencia de éste sobre el crecimiento de los clones seleccionados ............................................................................................................................ 55 3.6. Selección, identificación y análisis in vivo de mutantes de Y. ruckeri con mayor actividad β-galactosidasa a 18 ºC que a 28 ºC ............................................................................... 55 15 Índice 3.6.1. Selección de clones con mayor expresión a 18 ºC que a 28ºC................................. 55 3.6.2. Identificación del sitio de inserción del transposón Tn5 luxlac en los clones seleccionados. ......................................................................................................................................... 56 3.6.3. Determinación de la virulencia de los mutantes obtenidos ....................................... 57 3.7. Caracterización in vitro e in vivo de clones de Y. ruckeri 150 obtenidos con cefotaxima como sistema de selección. ............................................................................................. 57 3.7.1. Obtención de la cepa Y. ruckeri 150CTX en medio con cefotaxima ....................... 57 3.7.2. Agregación en medio líquido ................................................................................................ 58 3.7.3. Tinción de las colonias con negro sudán .......................................................................... 58 3.7.4. Observación de células al microscopio .............................................................................. 58 3.7.5. Perfil de resistencia a antibióticos ........................................................................................ 58 3.8. Identificación, secuenciación y análisis de genes que codifican distintas porinas de Y. ruckeri ......................................................................................................................................................... 59 3.9. Análisis del perfil de proteínas mediante SDS-PAGE ............................................................ 59 3.10. Análisis de los mutantes acrR- y osmY- .................................................................................... 60 3.10.1. Cuantificación de la actividad β-galactosidasa de los clones acrR- y osmY- mediante ensayo ONPG ...................................................................................................................... 60 3.10.2. Análisis in sílico de los genes acrR y osmY ..................................................................... 60 3.10.3. Obtención de las cepas complementadas Y. ruckeri acrR+, Y. ruckeri osmY+ y Y. ruckeri osmY++ ................................................................................................................................... 60 3.10.4. Caracterización fenotípica de las cepas Y. ruckeri acrR-, Y. ruckeri acrR+, Y. ruckeri osmY-, Y. ruckeri osmY+ y Y. ruckeri osmY++ ............................................................. 61 2.10.4.1. Ensayo de susceptibilidad a distintos compuestos ............................................. 61 3.10.4.2. Ensayo de sensibilidad a compuestos orgánicos y sales biliares .................. 61 3.10.4.3. Efecto de colorantes y azúcares en la morfología de la colonia .................... 62 3.10.5. Determinación de la virulencia ........................................................................................... 63 3.10.6. Análisis de la expresión de los genes acrR y osmY vía detección de la luminiscencia ........................................................................................................................................... 64 3.10.6.1. Influencia de los compuestos acrilflavina y NaCl ................................................. 64 3.10.6.2. Expresión en relación a los distintos órganos de trucha arcoíris ................. 65 4. Resultados ............................................................................................................................................ 67 4.1. Secuenciación y caracterización genómica de la cepa Y. ruckeri 150 ............................ 69 4.2. Análisis comparativo del genoma de Y. ruckeri 150 con los de las cepas Y. ruckeri ATCC24973, Y. ruckeri CSF007-82, Y. ruckeri Big Creek 74 y Y. ruckeri SC09 ..................... 70 4.2.1. Alineamiento de genomas por pares ................................................................................. 70 16 Índice 4.2.2. Genes diferenciales en el conjunto de los genomas analizados .............................. 71 4.2.2.1. Identificación de genes compartidos de forma exclusiva entre las cepas Y. ruckeri 150, Y. ruckeri ATCC 29473 y Y. ruckeri CSF007-82 ............................................... 73 4.2.2.2. Identificación de genes compartidos de forma exclusiva por las cepas Y. ruckeri Big Creek 74 y Y. ruckeri SC09 ..................................................................................... 75 4.2.2.3. Identificación de los genes únicos de la cepa Y. ruckeri Big Creek 74.......... 77 4.2.2.4. Identificación de genes únicos de la cepa Y. ruckeri SC09 .............................. 78 4.2.2.5. Identificación de genes ausentes en el genoma de la cepa Y. ruckeri ATCC 29473 y presentes en las otras cuatro cepas estudiadas ................................................... 79 4.3. Generación de una
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