This work was supported by NIGMS, National Institutes of Health (GM094623). Significant portions of this work were performed in the Environmental Molecular Sciences Laboratory, a DOE/BER national scientific user facility located at the Pacific Northwest National Laboratory (PNNL). PNNL is operated for the DOE by Battelle under Contract DE-AC05-76RLO1830.

1. Prepárese por adelantado <ol><li> Cálido tampón fosfato salino (PBS) a 37 ° C. </li><li> Modificación caliente de Dulbecco de medio de Eagle (DMEM) y Medio Esencial Mínimo (MEM) suplementado con 10% de suero bovino fetal (FBS), 100 IU / ml de penicilina, y 100 mg / ml de estreptomicina a 37 ° C. Nota: Estos representan Crecimiento Normal Media (NGM) para células RAW y HeLa, respectivamente. </li></ol> 2. Preparación de las células <ol><li> Se cultivan las células RAW 264.7 a 70-90% de confluencia en NGM. <ol><li> Mantener las células a 37 ° C en un 95% de aire / 5% de CO 2 atmósfera húmeda. </li></ol></li><li> Se cultivan las células HeLa a 70-90% de confluencia en NGM. <ol><li> Mantener las células a 37 ° C en un 95% de aire / 5% de CO 2 atmósfera húmeda. </li></ol></li><li> Antes de la recolección, lave monocapa de células una vez con PBS estéril. </li><li> Recoger las células pre-confluentes en un tubo cónico estéril. <ol><li> Raspar suavemente las células RAWcon un policía de caucho en PBS, con pipeteo repetido para dispersar agregaciones celulares. </li><li> Separar las células HeLa con solución de tripsina al 0,25% hasta que el examen visual muestra la disociación de la superficie de cultivo. Por ejemplo, utilizar 2 a 3 ml de un matraz T-75. Ajuste el volumen en consecuencia para garantizar solución de tripsina cubre toda la superficie de crecimiento. <ol><li> Se inactiva la reacción de disociación con NGM que contiene 10% de FBS. </li><li> Utilice al menos dos veces el volumen de NGM a la tripsina, con pipeteo repetido para dispersar agregaciones celulares. </li></ol></li></ol></li><li> Ligeramente sedimentar las células por centrifugación a 900 xg durante 4 min. </li><li> Resuspender en mismo volumen que la solución de tripsina / enfriamiento utilizando PBS estéril. </li><li> Contar las células utilizando un hemocitómetro o un contador de partículas. </li><li> Ligeramente sedimentar las células por centrifugación a 900 xg durante 4 min. </li><li> Resuspender en volumen adecuado de PBS durante 5,5 x 10 6 a 6,0 x 10 6 células / ml. Nota: Por ejemplo, un matraz T-75 producirá aproximadam ente 7,5 x 10 6 células 29. </li><li> Mantenga suspensión de células en hielo hasta la electroporación. </li></ol> 3. Preparación para la electroporación <ol><li> Cubetas Pre protección contra el frío de electroporación (brecha de 0,4 cm) en el hielo. </li><li> Encienda el aparato de electroporación y ajustar la tensión de 0,3 kV. NOTA: La capacitancia y la resistencia no eran configuraciones ajustables en nuestro electroporador (fijado en 10 mF y 600 Ω por el fabricante). </li><li> Llenar placas de recuperación con NGM y equilibrar en humidificado 95% de aire / 5% de CO 2 atmósfera a 37 ° C. </li></ol> 4. La electroporación <ol><li> Coloque 400 l de suspensión celular en la cubeta previamente enfriada y añadir 20 g de proteína seleccionada para la cubeta (50 mg / ml). </li><li> Cubeta Flick suavemente ~ 10 veces para mezclar sin dañar las células. Nota: La cubeta también puede invertirse varias veces para mezclar bien, pero no pipetear hacia arriba y hacia abajo o vortex para evitar daños en las células. </li><li&gt; fuera seco de cubeta con una toalla de papel u otro limpiador para evitar la formación de arco eléctrico en el electroporador. </li><li> Muestra electroporar a 0,3 kV para 1.5 a 1.7 ms. Nota: Esto era típico para este estudio. </li><li> Inmediatamente después de la electroporación película cubeta suavemente ~ 10 veces para mezclar a fondo. </li></ol> 5. Chapado de células <ol><li> Cubeta tienda con células electroporadas en hielo hasta que esté listo para colocar en placas de recuperación. </li><li> Para la mayoría de los análisis posteriores, se lavan las células 1x con pre-calentado NGM para eliminar la proteína efectora extraña. <ol><li> Eliminar las células para su análisis y suspender en 3-5 ml de NGM. </li><li> Sedimentar las células por centrifugación a 900 xg durante 4 min. </li><li> Volver a suspender en un volumen adecuado de NGM por tamaño de la placa deseada (por ejemplo, 2 ml de 35 mm plato). </li></ol></li><li> Retire cantidad adecuada de células para el análisis de aguas abajo. <ol><li> Ejemplo 1: placa en placas de fondo de cristal para el análisis de microscopía. </li><li> Ejemplo 2: Placa de into de plástico de cultivo celular para el análisis de proteínas tales como purificaciones de afinidad. </li></ol></li><li> Permitir que las células se recuperen en placas equilibrada en humidificado 95% de aire / 5% de CO 2 atmósfera a 37 ° C durante al menos 4 h. </li></ol> 6. Análisis de Microscopía 6.1) Fijación / inmunofluorescencia tinción <ol><li> Lavar las células con PBS 1x estéril después de un período de recuperación de 4 horas. </li><li> Fijar las células en 100% de metanol durante 2 min a temperatura ambiente. Use suficiente metanol para cubrir completamente las células (por ejemplo, 2 ml por placa de 35 mm). </li><li> Lavar 3 veces con PBS estéril. </li><li> Permeabilizar las células con 0,4% Triton X-100 en PBS durante 15 min. Por ejemplo, utilizar 1 ml de una placa de 35 mm de diámetro. <ol><li> Ajuste la longitud de permeabilización y la fuerza de Triton X-100 de acuerdo con epítopo y la ubicación de proteína diana. Nota: se necesitan mejores resultados debe determinarse empíricamente para cada objetivo, sin embargo las condiciones anteriores deben ser adecuados para la mayoría cobjetivos ytosolic. </li></ol></li><li> Bloque con 5% albúmina de suero bovino (BSA) en PBS durante 1 hora a RT. Por ejemplo, utilizar 1 ml de una placa de 35 mm de diámetro. </li><li> Lavar 3 veces con PBS. </li><li> Incubar anticuerpo primario en solución de unión del anticuerpo (0,1% Triton X-100 y 1% de BSA en PBS) durante la noche a 4 &#160; ° C con agitación suave. Por ejemplo, use 0.5 ml para una placa de 35 mm de diámetro. <ol><li> Siga las recomendaciones del fabricante para la dilución de anticuerpos. Nota: Por ejemplo, se utilizó el anticuerpo etiqueta de péptido de unión a estreptavidina-(SBP-tag) a 1: 1000 dilución, mientras que el anticuerpo PKN1 se utilizó a 1: 200 dilución. </li></ol></li><li> Lave 4 veces con PBS. </li><li> Incubar con el anticuerpo secundario conjugado fluorescente apropiada en solución de unión del anticuerpo durante 1 hora a temperatura ambiente, protegido de la luz. <ol><li> Por ejemplo, utilice Alexa 488 o Alexa 647 durante 1 hora a temperatura ambiente. <ol><li> Siga las recomendaciones del fabricante para la hormigadilución ibody. (Por ejemplo aproximadamente 1: 1000 para este estudio). </li></ol></li><li> Añadir otras manchas según sea necesario, por ejemplo, 5 mM de germen de trigo agglutinnin (WGA) conjugado con Alexa 647 o DAPI según la recomendación del fabricante, durante 1 hora a temperatura ambiente. </li></ol></li><li> Lave 5x con PBS y se almacena a 4 ° C, protegido de la luz hasta el momento de la imagen. </li></ol> 6.2) Microscopía Confocal y Análisis de Imágenes <ol><li> Muestras de imagen en un microscopio confocal invertido utilizando un objetivo de inmersión en aceite de 63x. <ol><li> Imagen canal verde utilizando la línea de 488 nm de un láser de argón, con emisión de ancho de banda entre 492 a 542 nm. </li><li> Imagen canal rojo usando un láser de diodo de 633 nm, con emisión de ancho de banda entre 640 a 718 nm. </li><li> Imagen azul canal utilizando un láser de diodo de 405 nm, con emisión de ancho de banda entre 407 a 453 nm. </li><li> Asegúrese de que el canal múltiple z-pilas cubren la totalidad del volumen celular. </li></ol></li><li> Procesar imágenes con software de procesamiento de imagen correspondiente. </li></ol> Purificación 7. Affinity <ol><li> Lavar las células electroporadas dos veces con PBS 4 ° C después de un período de recuperación de 4 hr. </li><li> Lisan con ~ 1,0 ml de tampón de lisis (1% Triton X-100 con cóctel inhibidor de la proteasa e inhibidor de la fosfatasa en PBS) en hielo. Utilice inhibidores de acuerdo con las instrucciones del fabricante. Nota: Por ejemplo, tanto los inhibidores de la fosfatasa y de la proteasa utilizados en este estudio fueron suministrados a 100X, pero otras formulaciones deben funcionar igualmente bien. </li><li> Inmediatamente raspar las células con goma de policía y recoger en tubos cónicos. </li><li> Lisan mediante agitación vigorosa y sonicación. Nota: Otros métodos de lisis pueden funcionar igual de bien, pero necesitarán la eficiencia por determinar empíricamente acerca de la adecuación de los requisitos de ensayo. <ol><li> Sonicar 3 x 30 segundos con agitación con vórtex intermitente. </li></ol></li><li> Centrifugar a 10000 xg durante 10 min a 4 ° C para recogerrestos celulares y agregados insolubles; guardar el sobrenadante. </li><li> Combinar volúmenes iguales (~ 1,0 ml) de lisado de células a electroporación con 50 l de estreptavidina suspensión de resina de agarosa a 4 ° C durante la noche con rotación de extremo a extremo. Nota: Esta resina captura la proteína de electroporación (y complejos asociados) a través de su péptido etiqueta de afinidad de unión a estreptavidina. </li><li> Centrifugar a 2500 xg durante 2 min y desechar el sobrenadante. </li><li> Lavar dos veces con 40 volúmenes de lecho (~ 1 ml) PBS. </li><li> Añadir 30 l de 4x LDS tampón de carga (141 mM de base Tris, 2% LDS, 10% de glicerol, EDTA 0,51 mM, 0,22 mM SERVA azul G, fenol 0,175 mM rojo, pH 8,5), 20 l DIH 2 O y 1 l de 0,5 M de tris (2-carboxietil) fosfina (TCEP), lo que permite algo de volumen permanezca en perlas. </li><li> Se calienta a 95 ° C durante 10 min y enfriar en hielo. </li><li> Girar&gt; 10.000 xg a 4 ° C durante 5 min. </li><li> Recoger el sobrenadante. </li><li> Realizar una transferencia Western utilizando anticuerpos apropiados Nota: Élre, se utiliza anti-PKN1 anticuerpo primario. </li></ol>

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I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Salmonella+type+III+secretion+effector+interacts+with+the+mammalian+serine/threonine+protein+kinase+PKN1.">A Salmonella type III secretion effector interacts with the mammalian serine/threonine protein kinase PKN1.</a> Cellular Microbiology. 8, 837-846 (2006).</li><li>Zinchuk, V., Grossenbacher-Zinchuk, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+colocalization+analysis+of+confocal+fluorescence+microscopy+images.">Quantitative colocalization analysis of confocal fluorescence microscopy images.</a> Current Protocols in Cell Biology / Editorial Board, Juan S. Bonifacino ... [et al]. 4 (Unit 4.19), (2011).</li><li>Kimple, M. E., Sondek, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overview+of+affinity+tags+for+protein+purification.">Overview of affinity tags for protein purification.</a> Current Protocols in Protein Science / Editorial Board, John E. Coligan ... [et al]. 9 (Unit 9.9), (2004).</li></ol>

Los autores no tienen nada que revelar.

Efectores secreta a partir de bacterias patógenas han evolucionado para funcionar en el entorno de la célula huésped y por lo tanto es útil para estudiar in situ dentro del huésped. Introducción de efectores específicos de interés en las células huésped permite las interacciones patógeno-hospedador relevantes para ser estudiados en forma aislada, sin interferencia de otras proteínas bacterianas. El objetivo era explorar la electroporación como un medio para introducir proteínas efectoras bacterian...

Muchas bacterias Gram-negativas emplean sistemas de secreción especializados para inyectar proteínas relacionados con la virulencia (referidos como efectores) directamente en células huésped 1-5. Estos efectores tienen una amplia gama de funciones biológicas que incluyen: la supresión de la inmunidad del huésped, cambios en el citoesqueleto, la modificación de tráfico y la señalización intracelular, los cambios transcripcionales, y las alteraciones del proteasoma anfitrión 6-9. Las funciones de algunos efectores son conocidos, sin embargo, los objetivos de acogida y acción bioquímica (s) de muchos otros queda por determinar. Si bien la comparación de tipo salvaje y las infecciones bacterianas recombinantes es un enfoque válido para estudiar los mecanismos de virulencia efector intracelular, a menudo es ventajoso introducir un efector individuo en la célula huésped. Así, los métodos simples para la introducción y la caracterización de proteínas efectoras bacterianas en el contexto de las células huésped es altamente deseable. Simplificando el análisis wi experimentalº un solo efector es crítica como en otros efectores pueden tener funciones opuestas o redundantes. Para llevar a cabo esta simplificación, los investigadores han introducido previamente macromoléculas en las células mediante muchos métodos diferentes, incluyendo la transducción viral 10, la microinyección 11, carga por raspado 12,13, fusión celular con microinyección químicamente inducido 14, proteína patentada reactivos &quot;transfección&quot; 15, precipitaciones de fosfato de calcio 16, y la electroporación 17-20. Las moléculas introducidas se extienden a partir de ácidos nucleicos que incluyen las especies de ADN, ARN, y RNAi a las proteínas, tintes impermeables a las células, y los anticuerpos para dianas intracelulares 21,22. Algunos métodos tienen limitaciones incluyendo el tipo de macromolécula que se puede introducir, y en particular análisis aguas abajo pueden ser limitadas debido a la alta toxicidad celular, los mecanismos dañinos de la acción, baja eficacia o eficiencia de introducción. La transfección, un oftemétodo n-utilizado para expresar genes bacterianos en células de mamíferos, también sufre la limitación de que algunos tipos de células de acogida pertinentes, como los macrófagos y las células primarias, son particularmente resistentes a la transfección. Más allá de esto, es difícil de controlar los niveles de proteína bacteriana producida tras la introducción de ADN extraño. Mucho trabajo se ha establecido la electroporación de ácidos nucleicos en ambas células bacterianas y de mamíferos como una técnica de laboratorio común; Sin embargo, hay investigaciones en curso en los mejores métodos para la entrega de proteínas en las células en condiciones fisiológicas. Informes sobre la transfección de proteínas son prometedores, pero requieren reactivos y optimización caros. El deseo de introducir efectores bacterianos potencialmente tóxicos en una amplia variedad de objetivos celulares con un coste mínimo nos llevó a considerar la electroporación como un método para el estudio de estas proteínas in vivo. Proteína electroporación 23-25 ​​es un conocióhod para introducir las proteínas en las células vivas a través de electropermeabilización, también conocido como electro-transfección o electro-inyección 26. Esta técnica utiliza pulsos eléctricos de alta intensidad para crear poros en las membranas celulares. Estos poros reversibles permiten macromoléculas que normalmente están excluidos desde el espacio intracelular para entrar en la célula. Tras la eliminación del campo eléctrico externo, la membrana puede volver a cerrar, permitiendo que la célula para retener moléculas que pasan a través de los poros 27,28. A electroporador estándar se utilizó en este estudio para introducir constantemente efectores bacterianos en ambos ratón células similares a macrófagos y células epiteliales humanas. El método es rápido, eficiente y de bajo costo, sin disminución apreciable en la viabilidad celular. Las proteínas introducidas se pueden visualizar a través de microscopía de inmunofluorescencia o se utilizan para ensayos funcionales. Esto ha sido demostrado utilizando la proteína fluorescente verde (GFP) como un estándar no tóxico, así comodos proteínas efectoras Salmonella, SspH1 y GtgE. Proponemos electroporación proteína como una herramienta adicional en el repertorio para el estudio de las proteínas de virulencia bacteriana y sus funciones en células huésped eucariotas.

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			<td height="21" style="height:21px;width:532px;">Material or Equipment</td>
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			<td style="width:108px;">Catalog Number</td>
			<td style="width:315px;">Comments</td>
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			<td height="20" style="height:20px;">0.25% Trypsin-EDTA Solution</td>
			<td>Cellgro</td>
			<td>25-050-Cl</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">0.4cm Gap-disposable electroporation cuvettes</td>
			<td>Bio-Rad</td>
			<td>165-2088</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">100% Methanol</td>
			<td>Any</td>
			<td>N/A</td>
			<td>Flammable, Toxic</td>
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			<td height="20" style="height:20px;">Bovine Serum Albumin (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>A4919</td>
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			<td>Cellgro</td>
			<td>10-013</td>
			<td>Warm to 37 &#176;C&#160;</td>
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			<td>Bio-Rad</td>
			<td>E. coli Pulser</td>
			<td></td>
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			<td height="20" style="height:20px;">Fetal Bovine Serum (FBS)</td>
			<td>Cellgro</td>
			<td>35-016-CV</td>
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		<tr height="20">
			<td height="20" style="height:20px;">Fluorescent confocal microscope&#160;</td>
			<td>Ziess</td>
			<td>Model&#160; LSM 710&#160;</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">Glass Bottom Dishes for Microscopy</td>
			<td>Wilco Wells</td>
			<td>HBSt-3522</td>
			<td></td>
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			<td height="20" style="height:20px;">HALT Protease Inhibitor Cocktail</td>
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			<td>Corrosive, Toxic</td>
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			<td>10-010</td>
			<td>Warm to 37 &#176;C&#160;</td>
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		<tr height="20">
			<td height="20" style="height:20px;">Other fluorescent stains (WGA, DAPI) in conjunction with anti-fade reagent</td>
			<td>Any</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin/Streptomycin</td>
			<td>Cellgro</td>
			<td>30-002-Cl</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">RAW 264.7 Cell line</td>
			<td>ATCC</td>
			<td>TIB-71</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Primary Antibody Against Target of Interest</td>
			<td>Any</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Secondary Antibody Conjugated to Fluorophore</td>
			<td>Any</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phosphate Buffered Saline</td>
			<td>Any</td>
			<td>N/A</td>
			<td>Chill to 4 &#176;C&#160;</td>
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		<tr height="20">
			<td height="20" style="height:20px;">Sterile Phosphate Buffered Saline</td>
			<td>Any</td>
			<td>N/A</td>
			<td>Warm to 37 &#176;C&#160;</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Streptavidin Agarose Resin Suspension&#160;</td>
			<td>Pierce</td>
			<td align="right">20353</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">Table Top Centrifuge Capable of Accepting Conical Tubes (swinging bucket preferred)</td>
			<td>Any</td>
			<td>N/A</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">TCEP</td>
			<td>Sigma-Aldrich</td>
			<td align="right">646547</td>
			<td>Corrosive, Toxic</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T8585</td>
			<td>Irritant, Toxic</td>
		</tr>
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</table>

electroporation of functional bacterial effectors into mammalian cells

El estudio de las interacciones proteína en el contexto de las células vivas puede generar información crítica acerca de la localización, la dinámica y socios que interactúan. Esta información es particularmente valioso en el contexto de la interacción huésped-patógeno. Muchas proteínas de patógenos funcionan dentro de células huésped en una variedad de manera tal, que permite la evasión del sistema inmune del huésped y la supervivencia en el entorno intracelular. Para estudiar estas interacciones proteína-patógeno de la célula huésped, varios enfoques se utilizan comúnmente, incluyendo: la infección in vivo con una cepa que expresa una proteína etiquetada o mutante, o la introducción de genes de patógenos a través de la transfección o transducción. Cada uno de estos enfoques tiene ventajas y desventajas. Se buscó un medio para introducir directamente las proteínas exógenas en células. La electroporación se utiliza comúnmente para introducir ácidos nucleicos en células, pero ha sido más raramente aplicado a las proteínas, aunque la base biofísica es exactamente el mismo.A electroporador estándar fue utilizado para introducir efectores bacterianos-etiquetados por afinidad en células de mamífero. Macrófagos epiteliales y ratón Humanos fueron cultivadas por métodos tradicionales, extraído, y se colocaron en 0,4 cm cubetas de electroporación brecha con una proteína exógena bacteriano patógeno de interés (por ejemplo, Salmonella Typhimurium GtgE). Después de la electroporación (0,3 kV) y un corto (4 h) período de recuperación, la proteína intracelular se verificó mediante el etiquetado con fluorescencia la proteína a través de su etiqueta de afinidad y el examen de la distribución espacial y temporal por microscopía confocal. La proteína electroporación también ha demostrado ser funcional dentro de la célula y capaz de tráfico subcelular correcta y la interacción proteína-proteína. Mientras que las proteínas exógenas tendían a acumularse en la superficie de las células, las muestras electroporadas tenían grandes aumentos en la concentración intracelular de efector con respecto a la incubación solo. El protocolo es simple y lo suficientemente rápido para ser hecho en apmoda an paralelo, lo que permite la caracterización de alto rendimiento de las proteínas de patógenos en células huésped incluyendo focalización y la función de las proteínas de virulencia subcelular.

Como prueba inicial de concepto, proteína fluorescente verde purificado se introdujo con éxito en células de mamífero utilizando electroporación. GFP, un aproximado de 27 kD una proteína de peso molecular se introduce comúnmente en células de mamífero (normalmente expresadas a partir de ADN del plásmido) como una herramienta de biología molecular sin toxicidad celular significativa. Las células HeLa se incubaron (Figura 1A) o electroporación (Figura 1B) con 25 g GFP / ml, s...

Watch this Scientific Journal Video about Electroporation of Functional Bacterial Effectors into Mammalian Cells at JoVE.com

Electroporation of Functional Bacterial Effectors into Mammalian Cells

Electroporation was used to insert purified bacterial virulence effector proteins directly into living eukaryotic cells. Protein localization was monitored by confocal immunofluorescence microscopy. This method allows for studies on trafficking, function, and protein-protein interactions using active exogenous proteins, avoiding the need for heterologous expression in eukaryotic cells.

La electroporación de efectores bacterianos funcionales en células de mamífero

The study of protein interactions in the context of living cells can generate critical information about localization, dynamics, and interacting partners. ...

electroporation-of-functional-bacterial-effectors-into-mammalian-cells

Research

JoVE Journal

Immunology and Infection

10.0K vistas.  Pacific Northwest National Laboratory. Electroporation was used to insert purified bacterial virulence effector proteins directly into living eukaryotic cells. Protein localization was monitored by confocal immunofluorescence microscopy. This method allows for studies on trafficking, function, and protein-protein interactions using active exogenous proteins, avoiding the need for heterologous expression in eukaryotic cells. 

Video: Electroporation of Functional Bacterial Effectors into Mammalian Cells

Invasion of Human Cells by a Bacterial Pathogen

Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus . The general protocol can be applied to the study of cell invasion by virtually any culturable bacterium. The stages at which specific aspects of invasion can be studied, such as the role of actin rearrangement or caveolae, will be highlighted. Host cells are grown in flasks and when ready for use are seeded into 24-well plates containing Thermanox coverslips. Using coverslips allows subsequent removal of the cells from the wells to reduce interference from serum proteins deposited onto the sides of the wells (to which S. aureus would attach). Bacteria are grown to the required density and washed to remove any secreted proteins (e.g. toxins). Coverslips with confluent layers of endothelial cells are transferred to new 24-well plates containing fresh culture medium before the addition of bacteria. Bacteria and cells are then incubated together for the required amount of time in 5% CO2 at 37&deg;C. For S. aureus this is typically between 15-90 minutes. Thermanox coverslips are removed from each well and dip-washed in PBS to remove unattached bacteria. If total associated bacteria (adherent and internalised) are to be quantified, coverslips are then placed in a fresh well containing 0.5% Triton X-100 in PBS. Gentle pipetting leads to complete cell lysis and bacteria are enumerated by serial dilution and plating onto agar. If the number of bacteria that have invaded the cells is needed, coverslips are added to wells containing 500 &mu;l tissue culture medium supplemented with gentamicin and incubation continued for 1 h, which will kill all external bacteria. Coverslips can then be washed, cells lysed and bacteria enumerated by plating onto agar as described above. If the experiment requires direct visualisation, coverslips can be fixed and stained for light, fluorescence or confocal microscopy or prepared for electron microscopy.

A general protocol for the study of invasion of host cells by a bacterial pathogen, focusing on Staphylococcus aureus and human endothelial cells.

La invasión de las células humanas por un patógeno bacteriano

Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus . The general protocol can be ...

Investigación

Inmunología e Infección

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host cells1. These adhesins rely on interactions with host cell surface receptors or soluble proteins acting as a bridge between bacteria and host. Adhesion is a critical first step prior to invasion and/or secretion of toxins, thus it is a key event to be studied in bacterial pathogenesis. Furthermore, adhered bacteria often induce exquisitely fine-tuned cellular responses, the studies of which have given birth to the field of 'cellular microbiology'2. Robust assays for bacterial adhesion on host cells and their invasion therefore play key roles in bacterial pathogenesis studies and have long been used in many pioneer laboratories3,4. These assays are now practiced by most laboratories working on bacterial pathogenesis.
Here, we describe a standard adherence assay illustrating the contribution of a specific adhesin. We use the Escherichia coli strain 27875, a human pathogenic strain expressing the autotransporter Adhesin Involved in Diffuse Adherence (AIDA). As a control, we use a mutant strain lacking the aidA gene, 2787&Delta;aidA (F. Berthiaume and M. Mourez, unpublished), and a commercial laboratory strain of E. coli, C600 (New England Biolabs). The bacteria are left to adhere to the cells from the commonly used HEp-2 human epithelial cell line. This assay has been less extensively described before6.

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

This protocol is a simple bacterial adhesion assay consisting in counting the numbers of bacterial colony forming units that are adhered onto cultured cells. The assay is robust, independent of the adhesin studied, and numerous variations are used in most laboratories working on bacterial pathogenesis.

Ensayo in vitro de la adhesión bacteriana a las células epiteliales de mamíferos

To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host ...

Isolation of Functional Cardiac Immune Cells

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5 These immune cells, which include mast cells, T-cells and macrophages; store and release a variety of biologically active mediators including cytokines and proteases such as tryptase.6-8 These mediators have been shown to be key players in extracellular matrix metabolism by activating matrix metalloproteinases or causing collagen accumulation by modulating the cardiac fibroblasts' function.9-11 However, available techniques for isolating cardiac immune cells have been problematic because they use bacterial collagenase to digest the myocardial tissue. This technique causes activation of the immune cells and thus a loss of function. For example, cardiac mast cells become significantly less responsive to compounds that cause degranulation.12 Therefore, we developed a technique that allows for the isolation of functional cardiac immune cells which would lead to a better understanding of the role of these cells in cardiac disease.13, 14
This method requires a familiarity with the anatomical location of the rat's xiphoid process, axilla and falciform ligament, and pericardium of the heart. These landmarks are important to increase success of the procedure and to ensure a higher yield of cardiac immune cells. These isolated cardiac immune cells can then be used for characterization of functionality, phenotype, maturity, and co-culture experiments with other cardiac cells to gain a better understanding of their interactions.

This method for isolating functional immune cells from the heart provides an alternative to the conventional methods of collagenase digestion, which causes unwanted immune cell activation, resulting in a decreased responsiveness of these cells. Our method of isolation yields functional cardiac immune cells by avoiding problems associated with enzymatic digestion.

Aislamiento de células inmunes funcionales cardíacos

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5  These immune cells, which ...

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques enable the observation of live cellular behavior inside the organ but can be challenging in some types of cancer due to organ and tumor accessibility such as intestine. Direct observation of intestinal tumors has not been previously reported. A surgical procedure described here allows direct observation of myeloid cell dynamics within the intestinal tumors in live mice by using transgenic fluorescent reporter mice and injectable tracers or antibodies. For this purpose, a four-color, multi-region, micro-lensed spinning disk confocal microscope that allows long-term continuous imaging with rapid image acquisition has been used. ApcMin/+ mice that develop multiple adenomas in the small intestine are crossed with c-fms-EGFP mice to visualize myeloid cells and with ACTB-ECFP mice to visualize intestinal epithelial cells of the crypts. Procedures for labeling different tumor components, such as blood vessels and neutrophils, and the procedure for positioning the tumor for imaging through the serosal surface are also described. Time-lapse movies compiled from several hours of imaging allow the analysis of myeloid cell behavior in situ in the intestinal microenvironment.

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

By using transgenic reporter mice and injectable fluorescent labels, long-term intravital spinning disk confocal microscopy enables direct visualization of myeloid cell behavior into intestinal adenoma in the ApcMin/+ colorectal cancer model.

Imágenes en tiempo real de las células mieloides en Dinámica<em&gt; Apc<sup&gt; Min / +</sup</em&gt; Intestinal Tumores de Spinning disco microscopía confocal

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques ...

Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. Polysaccharides such as glycogen, glycoproteins, and glycolipids stain bright magenta making it easy to enumerate positive and negative cells within the tissue. In muscle cells PAS staining is used to determine the glycogen content in different types of muscle cells, while in blood cell samples PAS staining has been explored as a diagnostic tool for a variety of conditions. Blood contains a proportion of white blood cells that belong to the immune system. The notion that cells of the immune system possess glycogen and use it as an energy source has not been widely explored. Here, we describe an adapted version of the PAS staining protocol that can be applied on peripheral blood mononuclear immune cells from human venous blood. Small cells with PAS-positive granules and larger cells with diffuse PAS staining were observed. Treatment of samples with amylase abrogates these patterns confirming the specificity of the stain. An alternate technique based on enzymatic digestion confirmed the presence and amount of glycogen in the samples. This protocol is useful for hematologists or immunologists studying polysaccharide content in blood-derived lymphocytes.

Periodic acid Schiff staining is a technique that visualizes the polysaccharide content of tissues. This article demonstrates periodic acid Schiff staining protocol adapted for use on peripheral blood mononuclear cells purified from human venous blood. Such samples are enriched for lymphocytes and other white blood cells of the immune system.

La detección de glucógeno en las células mononucleares de sangre periférica con ácido periódico de Schiff tinción

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

Generación y multi-fenotípicos cribado de alto contenido de<em&gt; Coxiella burnetii</em&gt; transposón mutantes

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

Bacterial Artificial Chromosomes: A Functional Genomics Tool for the Study of Positive-strand RNA Viruses

Reverse genetics, an approach to rescue infectious virus entirely from a cloned cDNA, has revolutionized the field of positive-strand RNA viruses, whose genomes have the same polarity as cellular mRNA. The cDNA-based reverse genetics system is a seminal method that enables direct manipulation of the viral genomic RNA, thereby generating recombinant viruses for molecular and genetic studies of both viral RNA elements and gene products in viral replication and pathogenesis. It also provides a valuable platform that allows the development of genetically defined vaccines and viral vectors for the delivery of foreign genes. For many positive-strand RNA viruses such as Japanese encephalitis virus (JEV), however, the cloned cDNAs are unstable, posing a major obstacle to the construction and propagation of the functional cDNA. Here, the present report describes the strategic considerations in creating and amplifying a genetically stable full-length infectious JEV cDNA as a bacterial artificial chromosome (BAC) using the following general experimental procedures: viral RNA isolation, cDNA synthesis, cDNA subcloning and modification, assembly of a full-length cDNA, cDNA linearization, in vitro RNA synthesis, and virus recovery. This protocol provides a general methodology applicable to cloning full-length cDNA for a range of positive-strand RNA viruses, particularly those with a genome of &gt;10 kb in length, into a BAC vector, from which infectious RNAs can be transcribed in vitro with a bacteriophage RNA polymerase.

Here, a protocol is presented for creating an infectious bacterial artificial chromosome containing a full-length cDNA of the positive-strand genomic RNA of Japanese encephalitis virus. This protocol can be used to construct a functional cDNA of other positive-strand RNA viruses, making it a powerful genomic tool for studying virus biology.

Cromosomas artificiales bacterianos: una herramienta de Genómica Funcional para el Estudio de cadena positiva virus de ARN

Reverse genetics, an approach to rescue infectious virus entirely from a cloned cDNA, has revolutionized the field of positive-strand RNA viruses, whose ...

Implementation of a Permeable Membrane Insert-based Infection System to Study the Effects of Secreted Bacterial Toxins on Mammalian Host Cells

Many bacterial pathogens secrete potent toxins to aid in the destruction of host tissue, to initiate signaling changes in host cells or to manipulate immune system responses during the course of infection. Though methods have been developed to successfully purify and produce many of these important virulence factors, there are still many bacterial toxins whose unique structure or extensive post-translational modifications make them difficult to purify and study in in vitro systems. Furthermore, even when pure toxin can be obtained, there are many challenges associated with studying the specific effects of a toxin under relevant physiological conditions. Most in vitro cell culture models designed to assess the effects of secreted bacterial toxins on host cells involve incubating host cells with a one-time dose of toxin. Such methods poorly approximate what host cells actually experience during an infection, where toxin is continually produced by bacterial cells and allowed to accumulate gradually during the course of infection. This protocol describes the design of a permeable membrane insert-based bacterial infection system to study the effects of Streptolysin S, a potent toxin produced by Group A Streptococcus, on human epithelial keratinocytes. This system more closely mimics the natural physiological environment during an infection than methods where pure toxin or bacterial supernatants are directly applied to host cells. Importantly, this method also eliminates the bias of host responses that are due to direct contact between the bacteria and host cells. This system has been utilized to effectively assess the effects of Streptolysin S (SLS) on host membrane integrity, cellular viability, and cellular signaling responses. This technique can be readily applied to the study of other secreted virulence factors on a variety of mammalian host cell types to investigate the specific role of a secreted bacterial factor during the course of infection.

Here, a method using a permeable membrane insert-based infection system to study the effects of Streptolysin S, a secreted toxin produced by Group A Streptococcus, on keratinocytes is described. This system can be readily applied to the study of other secreted bacterial proteins on various host cell types during infection.

Implementación de un Sistema de infección basada en la inserción de membrana permeable para estudiar los efectos de toxinas secretadas bacterianas en células huésped de mamífero

Many bacterial pathogens secrete potent toxins to aid in the destruction of host tissue, to initiate signaling changes in host cells or to manipulate ...

Unravelling the Function of a Bacterial Effector from a Non-cultivable Plant Pathogen Using a Yeast Two-hybrid Screen

Unravelling the molecular mechanisms of disease manifestations is important to understand pathologies and symptom development in plant science. Bacteria have evolved different strategies to manipulate their host metabolism for their own benefit. This bacterial manipulation is often coupled with severe symptom development or the death of the affected plants. Determining the specific bacterial molecules responsible for the host manipulation has become an important field in microbiological research. After the identification of these bacterial molecules, called &#34;effectors,&#34; it is important to elucidate their function. A straightforward approach to determine the function of an effector is to identify its proteinaceous binding partner in its natural host via a yeast two-hybrid (Y2H) screen. Normally the host harbors numerous potential binding partners that cannot be predicted sufficiently by any in silico algorithm. It is thus the best choice to perform a screen with the hypothetical effector against a whole library of expressed host proteins. It is especially challenging if the causative agent is uncultivable like phytoplasma. This protocol provides step-by-step instructions for DNA purification from a phytoplasma-infected woody host plant, the amplification of the potential effector, and the subsequent identification of the plant&#39;s molecular interaction partner with a Y2H screen. Even though Y2H screens are commonly used, there is a trend to outsource this technique to biotech companies that offer the Y2H service at a cost. This protocol provides instructions on how to perform a Y2H in any decently equipped molecular biology laboratory using standard lab techniques.

Bacterial effector proteins are important for establishing successful infections. This protocol describes the experimental identification of proteinaceous binding partners of a bacterial effector protein in its natural plant host. Identifying these effector interactions via yeast two-hybrid screens has become an important tool in unravelling molecular pathogenicity strategies.

Desenredar la función de un efector bacteriana de una planta de patógenos no cultivables Uso de una levadura de dos híbridos pantalla

Unravelling the molecular mechanisms of disease manifestations is important to understand pathologies and symptom development in plant science. Bacteria ...

Immunofluorescence Analysis of Stress Granule Formation After Bacterial Challenge of Mammalian Cells

Fluorescent imaging of cellular components is an effective tool to investigate host-pathogen interactions. Pathogens can affect many different features of infected cells, including organelle ultrastructure, cytoskeletal network organization, as well as cellular processes such as Stress Granule (SG) formation. The characterization of how pathogens subvert host processes is an important and integral part of the field of pathogenesis. While variable phenotypes may be readily visible, the precise analysis of the qualitative and quantitative differences in the cellular structures induced by pathogen challenge is essential for defining statistically significant differences between experimental and control samples. SG formation is an evolutionarily conserved stress response that leads to antiviral responses and has long been investigated using viral infections1. SG formation also affects signaling cascades and may have other still unknown consequences2. The characterization of this stress response to pathogens other than viruses, such as bacterial pathogens, is currently an emerging area of research3. For now, quantitative and qualitative analysis of SG formation is not yet routinely used, even in the viral systems. Here we describe a simple method for inducing and characterizing SG formation in uninfected cells and in cells infected with a cytosolic bacterial pathogen, which affects the formation of SGs in response to various exogenous stresses. Analysis of SG formation and composition is achieved by using a number of different SG markers and the spot detector plug-in of ICY, an open source image analysis tool.

We describe a method for the qualitative and quantitative analysis of stress granule formation in mammalian cells after the cells are challenged with bacteria and a number of different stresses. This protocol can be applied to investigate the cellular stress granule response in a wide range of host-bacterial interactions.

Análisis de inmunofluorescencia de la formación de gránulos de estrés tras el desafío bacteriano de las células de mamíferos

Fluorescent imaging of cellular components is an effective tool to investigate host-pathogen interactions. Pathogens can affect many different features of ...