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Caracterización Del Sistema Toxina-Antitoxina Reibe2 De" Streptococcus Pneumoniae"

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Caracterización Del Sistema Toxina-Antitoxina Reibe2 De UNIVERSIDAD COMPLUTENSE DE MADRID FACULTAD DE CIENCIAS BIOLÓGICAS Departamento de Bioquímica y Biología Molecular I CARACTERIZACIÓN DEL SISTEMA TOXINA- ANTITOXINA REIBE2 DE STREPTOCOCCUS PNEUMONIAE MEMORIA PARA OPTAR AL GRADO DE DOCTOR PRESENTADA POR Inmaculada Moreno Córdoba Bajo la dirección de los doctores Manuel Espinosa Padrón Concha Nieto Mazarrón Madrid, 2014 © Inmaculada Moreno Córdoba, 2013 UNIVERSIDAD COMPLUTENSE DE MADRID FACULTAD DE CIENCIAS BIOLÓGICAS DEPARTAMENTO DE BIOQUÍMICA Y BIOLOGÍA MOLECULAR I TESIS DOCTORAL CARACTERIZACIÓN DEL SISTEMA TOXINA-ANTITOXINA RelBE2 DE Streptococcus pneumoniae MEMORIA PARA OPTAR AL GRADO DE DOCTOR PRESENTADA POR Inmaculada Moreno Córdoba Bajo la dirección de los doctores: Manuel Espinosa Padrón Concha Nieto Mazarrón Madrid, 2013 El trabajo recogido en la presente memoria ha sido realizado por Inmaculada Moreno Córdoba bajo la dirección del Dr. Manuel Espinosa Padrón y de la Dra. Concha Nieto Mazarrón, en el Departamento de Microbiología Molecular y Biología de las Infecciones del Centro de Investigaciones Biológicas del Consejo Superior de Investigaciones Científicas (CSIC) con financiación concedida por el Ministerio de Educación y Ciencia (Beca/Contrato FPI asociada al Proyecto BFU2007-63575), el Ministerio de Ciencia e Innovación / Ministerio de Economía y Competitividad (CSD2008- 00013) y la Comisión Europea (EU-CP223111) Opta al grado de Doctor VºBº Directores de Tesis Inmaculada Moreno Córdoba Manuel Espinosa Padrón Concha Nieto Mazarrón A mis padres La ignorancia afirma o niega rotundamente; la ciencia duda. VOLTAIRE La utopía está en el horizonte. Me acerco dos pasos, ella se aleja dos pasos. Camino diez pasos y el horizonte se corre diez pasos más allá. Entonces, ¿para qué sirve la utopía? Para eso sirve: para caminar. EDUARDO GALEANO Agradecimientos Manolo, gracias por darme la oportunidad de descubrir el mundo de la ciencia. Gracias por permitirme aprender, crecer y trabajar con ilusión. Gracias por tus sabios consejos y por ser un gran maestro. Concha, gracias por transmitirme tu pasión por la ciencia, por hacerme disfrutar de este trabajo, por enseñarme tanto y por ser una gran maestra. Gracias por tu ayuda tanto profesinal como “extracurricular”. Gracias a todos los integrantes del grupo. Gracias a Alicia, Gloria y a los “compañeros” Jose y Fabi por su constante ayuda y consejos. Gracias a Virtu, Sofi, Cris, Ana y Lore B. por estar siempre dispuestas a ayudarme, por los buenos momentos compartidos y por que “siempre nos quedará Berlín”. Gracias a Lore por todo su apoyo y trabajo. Gracias a Marta, Celeste, Tania y Wai Ting por su apoyo. Gracias a Ramón Díaz Orejas y a los miembros de su laboratorio por su apoyo constante, especialmente agradezco a Ramón y a Elizabeth Diago Navarro su colaboración y aportación a este trabajo. Gracias a Miquel Coll y a todos los miembros de su grupo que han hecho posible la resolución de la estructura de RelBE2Spn, Albert, Rosa, Diana y Robert. Gracias a Ernesto García y Miriam Moscoso por su paciencia para conseguir los bioflms. Gracias a Eva Medina por abrirme las puertas de su laboratorio en Braunschweig y permitirme vivir una interesante experiencia. Gracias a mis padres por tenderme la mano en cada etapa de mi vida y ayudarme a levantar, por ser como sois, por saliros del pensamiento único y por enseñarme a ser feliz. Fede, gracias por tu paciencia, comprensión y por darme siempre fuerzas para seguir adelante. Gracias a toda mi familia por su cariño. Gracias a Carmen por ser tía, hermana y amiga a la vez. Gracias a Almu, Diana y María por estar siempre ahí. Gracias a los “biólogos 2002” por hacerme pasar tan buenos momentos en la facultad y continuar haciéndolo. Gracias a Irene por su amistad incondicional. Gacias a todos por los ánimos que siempre me dais y conseguir que no deje de sonreir. ÍNDICE SUMMARY ....................................................................................................... 1 INTRODUCCIÓN .............................................................................................. 7 1. DEFINICIÓN DE LOS SISTEMAS TOXINA-ANTITOXINA ........................ 9 2. TIPOS DE SISTEMAS TA ........................................................................... 9 3. SISTEMAS TA DE TIPO II ........................................................................ 12 3.1. LOCALIZACIÓN Y DISTRIBUCIÓN DE LOS SISTEMAS TA DE TIPO II ............................................................................................................. 12 3.2. FUNCIÓN DE LOS SISTEMAS TA DE TIPO II .............................. 14 3.3. REGULACIÓN DE LA EXPRESION DE LOS SISTEMAS TA DE TIPO II .................................................................................................... 18 3.3.1. Regulación transcripcional ..................................................... 18 3.3.2. Cooperatividad condicional .................................................... 18 3.3.3. Regulación traduccional ......................................................... 20 3.3.4. Regulación post-traduccional ................................................. 20 3.4. FAMILIAS DE SISTEMAS TA DE TIPO II ....................................... 20 3.5. CARACTERÍSTICAS Y HOMOLOGIA ESTRUCTURAL ENTRE SISTEMAS TA DE TIPO II CON ACTIVIDAD mRNA INTERFERASA . 22 3.5.1. Estructura de antitoxinas ........................................................ 22 3.5.2. Estructura de toxinas: unión de la toxina a su sustrato .......... 25 3.6. LA FAMILIA RelBE .......................................................................... 27 3.6.1. RelBE ..................................................................................... 28 3.6.2. YefMYoeB .............................................................................. 32 3.6.3. MqsRA ................................................................................... 34 4. Streptococcus pneumoniae: RELEVANCIA Y SISTEMAS TA .............. 36 4.1. Streptococcus pneumoniae ............................................................. 36 4.2. SISTEMAS TA EN S. pneumoniae .................................................. 38 5. RELEVANCIA DE LOS SISTEMAS TA Y SU UTILIZACIÓN EN BIOTECNOLOGÍA Y MEDICINA ........................................................... 43 OBJETIVOS ................................................................................................... 49 MATERIALES Y MÉTODOS .......................................................................... 53 MATERIALES ................................................................................................ 55 1. ESTIRPES BACTERIANAS ..................................................................... 55 2. LÍNEAS CELULARES .............................................................................. 55 3. MEDIOS DE CULTIVO ............................................................................. 55 3.1. Cultivo de bacterias y selección ...................................................... 55 3.2. Medios de cultivo para líneas celulares .......................................... 57 4. PLÁSMIDOS ............................................................................................ 57 5. OLIGONUCLEÓTIDOS ............................................................................ 58 6. ENZIMAS, PRODUCTOS QUÍMICOS Y REACTIVOS ............................ 60 7. TAMPONES ............................................................................................. 60 8. SOPORTE INFORMÁTICO ...................................................................... 63 MÉTODOS ..................................................................................................... 64 1. CRECIMIENTO Y CONSERVACIÓN DE ESTIRPES BACTERIANAS ... 64 2. PREPARACIÓN Y TRANSFORMACIÓN DE CÉLULAS COMPETENTES ............................................................................................................... 65 3. PREPARACIONES DE DNA .................................................................... 66 3.1. Extracción de DNA ......................................................................... 66 3.2. Obtención de fragmentos de DNA .................................................. 66 3.3. Purificación ..................................................................................... 67 3.4. Ligación de DNA ............................................................................. 68 3.5. Construcción de plásmidos recombinantes .................................... 69 3.6. Construcción de mutantes cromosómicos por reemplazamiento génico .............................................................................................. 69 4. ANÁLISIS DE DNA .................................................................................. 71 4.1. Cuantificación ................................................................................. 71 4.2. Electroforesis .................................................................................. 72 4.3. Secuenciación ................................................................................ 73 4.4. Marcaje radiactivo de DNA ............................................................. 74 5. PURIFICACIÓN DE PROTEÍNAS ............................................................ 74 5.1. Purificación de RelBE2Spn y RelB2Spn ......................................... 74 5.2. Purificación de RelBE2Spn con Selenio-Metionina ........................ 75 6. ANÁLISIS DE PROTEÍNAS ..................................................................... 76 6.1. Cuantificación ................................................................................
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