Nos gustaría reconocer Mathias S. Dick, Roland F. Dreier y Sebastian Rühl para la discusión. Este trabajo fue apoyado por una FNS Cátedra PP00P3_139120 / 1 y de la Universidad de Basilea subvención del proyecto ID2153162 a PB

1. Ensayo Digitonina con mCherry expresan Salmonella (FACS basada en Análisis) <ol><li> Preparación de las bacterias Nota: Salmonella de tipo salvaje SL1344 (resistente a la estreptomicina) que expresa mCherry partir de un plásmido (que codifica pFPV mCherry bajo el promotor RPSM) se utiliza en este ensayo. La fagocitosis bacteriana y la proliferación donde no alterada por la expresión constitutiva de mCherry (datos no mostrados) 16. <ol><li> Inocular la cepa 1 día antes de la infección en 3 ml de medio LB suplementado con estreptomicina (90 mg / ml) y ampicilina (100 g / ml, mCherry vector que expresa) en un agitador a 37 ° CO / N. </li></ol></li><li> Plating células para la infección. <ol><li> BMDMs Semilla de tipo salvaje en una placa de 24 pocillos a una densidad de 1,25 x 10 5 células / pocillo en 1 ml de medio de macrófagos primaria (DMEM suplementado con 10% de suero de ternera fetal (FCS, inactivado por calor, 56 ° C, 30 min ), 1% HEPES, 1% de aminoácidos no esenciales (NEAA), 1% de L-glutamina y 10% L929 sobrenadante (medio de macrófagos factor estimulante de colonias (M-CSF acondicionado) producir células L929). Pozos de siembra de acuerdo con la Tabla 1 para dar cuenta de las condiciones individuales. Añadir cubreobjetos a los pocillos usados ​​como controles (Tabla 1). </li><li> Se incuban las células durante la noche en una incubadora a 37 ° C y 5% de CO 2. </li></ol></li></ol><table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td></td><td></td><td></td><td colspan="2"> Permeabilización </td><td colspan="3"> La tinción </td></tr><tr><td></td><td> Cubreobjetos de cristal </td><td> Salmonela </td><td> Digitonina </td><td> La saponina </td><td> anti-Salmonella </td><td> anti-calnexina </td><td> anti-PDI </td></tr><tr><td> A1 (muestra) </td><td> - / + * </td><td> + </td><td> + </td><td></td><td> + </td><td></td><td></td></tr><tr><td> A2 (muestra) </td><td> - / + * </td><td> + </td&gt; <td> + </td><td></td><td> + </td><td></td><td></td></tr><tr><td> A3 (muestra) </td><td> - / + * </td><td> + </td><td> + </td><td></td><td> + </td><td></td><td></td></tr><tr><td> A4 (sin control de permeabilización) </td><td> - / + * </td><td> + </td><td></td><td></td><td> + </td><td></td><td></td></tr><tr><td> A5 (control completo permeabilización) </td><td> - / + * </td><td> + </td><td></td><td> + </td><td> + </td><td></td><td></td></tr><tr><td> B1 (manchado de calnexina) </td><td> + </td><td></td><td></td><td></td><td></td><td> + </td><td></td></tr><tr><td> B2 (manchado de calnexina) </td><td> + </td><td></td><td> + </td><td></td><td></td><td> + </td><td></td></tr><tr><td> B3 (manchado de calnexina) </td><td> + </td><td></td><td></td><td> + </td><td></td><td> + </td><td></td></tr><tr><td> C1 (manchadopara PDI) </td><td> + </td><td></td><td></td><td></td><td></td><td></td><td> + </td></tr><tr><td> C2 (manchado para PDI) </td><td> + </td><td></td><td> + </td><td></td><td></td><td></td><td> + </td></tr><tr><td> C3 (manchado para PDI) </td><td> + </td><td></td><td></td><td> + </td><td></td><td></td><td> + </td></tr></tbody></table> Tabla 1: esquema de la galjanoplastia de macrófagos derivados de médula ósea (BMDMs) en un formato de 24 pocillos para la posterior infección con Salmonella, la permeabilización y tinción * Dependiendo de si el protocolo 1 o protocolo 2 se hace.. <ol start="3"><li> Preparación de las bacterias de la infección. <ol><li> El día siguiente, determinar la concentración de Salmonella en el cultivo durante la noche mediante la dilución de la cultura 1:10 en PBS y midiendo la OD 600 contra un único control PBS. Esto asegura que el OD 600 se mide en el lgama inear del espectrofotómetro. </li><li> Calcular la concentración de Salmonella por ml. Una DO600 de 1 corresponde a 1 x 10 9 bacterias. </li><li> Diluir las bacterias en un medio de macrófagos para alcanzar una concentración de Salmonella 1,25 x 10 6 células / ml. </li></ol></li><li> La infección de células con Salmonella. <ol><li> Retire medio de BMDMs. Añadir 1 ml de medio de macrófagos a los pocillos de control no infectados (B1-B3, C1-C3). Añadir 1 ml de suspensión de Salmonella a los pocillos A1 - A5 para llegar a una multiplicidad de infección (moi) de 10 bacterias por célula. </li><li> Centrifugar la placa durante 15 min a 300 xg a 37 ° C para sincronizar la infección. La transferencia de la placa en una incubadora a 37 ° C y 5% de CO 2. </li></ol></li><li> Matar Salmonella extracelular con gentamicina. <ol><li> En 1 hora después de la infección, retire la placa de la incubadora y añadir 0,1 ml de medio de macrófagos que contienen 0,1 mg / ml de gentamicinapara matar las bacterias extracelulares. Añadir Gentamicina a todos los pocillos, incluso con los controles no infectados, para asegurar que todas las células se tratan de la misma manera. La transferencia de la placa en una incubadora a 37 ° C y 5% de CO 2. </li></ol></li><li> Lavar las células. <ol><li> A las 2 horas después de la infección, retire la placa de la incubadora y lavar todos los pozos 2x con 1 ml de DMEM sin formato (precalentado a 37 ° C) y sustituirlo por medio de macrófagos (precalentado a 37 ° C) que contiene 10 mg / ml &#160; Gentamicina para prevenir el crecimiento de cualquier bacteria extracelular restantes. La transferencia de la placa en una incubadora a 37 ° C y 5% de CO 2. </li></ol></li><li> Preparación de tampones frescos con detergentes y anticuerpos. <ol><li> Justo antes del punto de tiempo deseado de análisis, preparar una solución madre fresca de digitonina a 1 mg / ml en tampón de KHM (acetato de potasio 110 mM, HEPES 20 mM, 2 mM de MgCl 2, pH 7,3). Filtra las acciones y diluir en KHM tampón a una concentración de trabajode 50 g / ml de digitonina. Además, preparar una solución de 0,1% de saponina en KHM Buffer, anticuerpo anti- Salmonella acoplado a FITC (CSA-1-FITC) a 1/500, anti-calnexina en centésimas o anti-PDI en centésimas en KHM Buffer suplementado con 3% de BSA. </li></ol></li><li> Permeabilización de la célula. <ol><li> Retire la placa de la incubadora y lavar los pocillos 3 veces con 0,5 ml de KHM Buffer (precalentado a 37 ° C). Retire KHM Buffer y añadir 0,25 ml llanura KHM Buffer de pozos A4 y B1 / C1, KHM Buffer con digitonina para pozos A1 - A3 y B2 / C2 o KHM Buffer con saponina a pozos A5 y B3 / C3 (de acuerdo con la Tabla 1). Incubar exactamente 1 min a TA y de inmediato se lavan todos los pocillos 3 veces con 0,5 ml de KHM Buffer (pre-calentado a 37 ° C). </li></ol></li><li> Tinción de anticuerpos primaria. <ol><li> Retire la KHM Buffer y añadir las soluciones de anticuerpos primarios (de cabra anti Salmonella FITC, 250 l / pocillo, 0,1 g / ml) a los pocillos apropiados (Figura 1 </strong&gt;, Tabla 1). Incubar la placa durante 15 minutos en una incubadora a 37 ° C y 5% de CO 2. </li><li> Lavar las células 1 vez con 1 ml de PBS. </li></ol></li><li> Las células infectadas (pozos A1 - A5): análisis FACS <ol><li> Lavar las células 3 veces con PBS y se añaden 0,5 ml de helado de PBS que contiene 0,1% Tritón. Transferir a un tubo de 5 ml con tapón de FACS filtro celular para romper los agregados de células. Mantener las muestras en hielo. </li><li> Analizar las muestras de un FACS. Uso de los controles totalmente permeabilizadas, establezca la puerta para que las bacterias totales basados ​​en adelante y dispersión lateral (FSC / SSC) primero, y la señal mCherry (610 nm ± 20 nm filtro). </li><li> A continuación, analizar la señal FITC en la población bacteriana total usando los controles permeabilizadas totalmente permeabilizadas y no para establecer las puertas para las bacterias citosólicas (FITC +, mCherry +) y bacterias vacuolares (FITC, mCherry +) (Figura 3). </li><li> Con las puertas establecen, analizar las muestras y determinar el porcentaje de citosólica vs.bacterias vacuolares utilizando software FlowJo según el protocolo del fabricante. </li></ol></li><li> Controles de permeabilización no infectadas (pozos B1 - B3, C1 - C3): microscopía <ol><li> Fijación <ol><li> Retire PBS y añadir 250 l de PFA 4% en PBS. Incubar durante 10 minutos a 37 ° C. </li><li> Lavar los pocillos 2 veces con 1 ml de PBS y se añaden 250 l de glicina 0,1 M en PBS durante 10 min para inactivar la fijador. Lavar los pocillos 2 veces con 1 ml de PBS. </li></ol></li><li> Tinción de anticuerpos secundaria. <ol><li> Controles de permeabilización mancha durante 1 hr a RT con anticuerpos secundarios apropiados acoplados a fluoróforos en PBS suplementado con 0,1% de saponina y 3% de BSA a las células totalmente permeabilizar. </li><li> Lavar 3x cubreobjetos con 1 ml de PBS y montar los cubreobjetos para el análisis en medio de montaje con DAPI (1,5 g / ml). </li></ol></li><li> Análisis de microscopía. <ol><li> Analizar los cubreobjetos con los controles a una magnificación 63X 40X o utilizando un fluo confocalmicroscopio cencia. Si la permeabilización ha trabajado, no debería haber ninguna tinción para células no permeabilizadas, la tinción calnexina para ambas células digitonin- y permeabilizadas-saponina y PDI sólo para la tinción de células de saponina-permeabilizadas (Figura 4). </li></ol></li></ol></li></ol> 2. Ensayo Digitonina con sin etiqueta Salmonella (análisis basado Microscopía-) <ol><li> Preparación de las bacterias de las culturas O / N. <ol><li> Inocular no marcado Salmonella de tipo salvaje SL1344 (resistente a la estreptomicina) 1 día antes de la infección en 3 ml de medio LB suplementado con estreptomicina (90 mg / ml) en un agitador a 37 ° CO / N. </li><li> Plating células para la infección. <ol><li> BMDMs semilla en una placa de 24 pocillos que contienen cubreobjetos de vidrio estériles a una densidad de 1,25 x 10 5 células / pocillo en 1 ml de medio de macrófagos (DMEM suplementado con 10% de suero de ternera fetal (FCS, de-complementado, 56 ° C,30 min), 1% HEPES, 1% los L-aminoácidos no esenciales (NEAA), 1% de L-glutamina y 10% de sobrenadante L929). Pozos de siembra de acuerdo con la Tabla 1 para dar cuenta de las condiciones individuales. </li><li> Se incuban las células O / N en una incubadora a 37 ° C y 5% de CO 2. </li></ol></li><li> Preparación de las bacterias de la infección. <ol><li> El día siguiente, determinar la concentración de Salmonella en el cultivo O / N mediante la dilución de la cultura 1:10 en PBS y midiendo la OD 600 contra un único control PBS. Esto asegura que el OD 600 se mide en el intervalo lineal del espectrofotómetro. </li><li> Determinar la concentración de Salmonella por mililitro. Un OD 600 de 1 corresponde a 1 x 10 9 bacterias. </li><li> Diluir las bacterias en un medio de macrófagos para alcanzar una concentración de Salmonella 1,25 x 10 6 células / ml. </li></ol></li><li> La infección de células con Salmonella. <ol&gt; <li> Retire medio de todos los pocillos. Añadir 1 ml de medio de macrófagos claro a los pocillos de control no infectadas (B1 - B3, C1 - C3). Añadir 1 ml de suspensión de Salmonella a los pocillos A1 - A5 para llegar a una multiplicidad de infección (moi) de 10 bacterias por célula. </li><li> Centrifugar la placa durante 15 min a 300 xg a 37 ° C para sincronizar la infección. La transferencia de la placa en una incubadora a 37 ° C y 5% de CO 2. </li></ol></li><li> Matar Salmonella extracelular con gentamicina. <ol><li> En 1 hora después de la infección, retire la placa de la incubadora y añadir 0,1 ml de medio de macrófagos que contienen 1 mg / ml de gentamicina para matar las bacterias extracelulares a todos los pocillos. La transferencia de la placa en una incubadora a 37 ° C y 5% de CO 2. </li></ol></li><li> Lavar las células. <ol><li> A las 2 horas después de la infección, retire la placa de la incubadora y lavar cada pocillo 3 veces con 1 ml de medio fresco que contiene macrófagos 10 g / ml Gentamicina para evitarel crecimiento de las bacterias extracelulares que quedan. La transferencia de la placa en una incubadora a 37 ° C y 5% de CO 2. </li></ol></li><li> Preparación de tampones frescos con detergentes y anticuerpos. <ol><li> Justo antes del punto de tiempo deseado de análisis, preparar una solución madre fresca de digitonina a 1 mg / ml en tampón de KHM (acetato de potasio 110 mM, HEPES 20 mM, 2 mM de MgCl 2, pH 7,3). Filtra las acciones y diluir en KHM Buffer a una concentración de trabajo de 50 mg / ml de digitonina. </li><li> Además, preparar una solución de 0,1% de saponina en KHM Buffer, anti- Salmonella FITC (CSA-1, 0,1 g / ml), anti-calnexina a 1/100 o anti-PDI en 1/100 en KHM Buffer suplementado con 3% de BSA (ver tabla materiales para la concentración de anticuerpos). </li></ol></li><li> Permeabilización de la célula. <ol><li> Retire la placa de la incubadora y lavar los pocillos 3 veces con 0,5 ml de KHM Buffer (precalentado a 37 ° C). Retire KHM Buffer y añadir 0,25 ml llanura KHM Buffera los pozos A4 y B1 / C1, KHM Buffer con digitonina para pozos A1 - A3 y B2 / C2 o KHM Buffer con saponina a pozos A5 y B3 / C3 (de acuerdo con la Tabla 1). Incubar exactamente 1 min a TA y de inmediato se lavan todos los pocillos 3 veces con 0,5 ml de KHM Buffer (pre-calentado a 37 ° C). </li></ol></li><li> Tinción de anticuerpos primaria. <ol><li> Retire la KHM Buffer y añadir las soluciones de anticuerpos primarios (de cabra anti Salmonella CSA1-FITC, 250 l / pocillo, 0,1 g / ml) a los pocillos apropiados (Figura 2, Tabla 1). Incubar la placa durante 15 minutos en una incubadora a 37 ° C y 5% de CO 2. Lavar 3 veces con 1 ml de PBS. </li></ol></li><li> Fijación. <ol><li> Retire PBS y añadir 250 l de PFA 4% en PBS. Incubar durante 10 minutos a 37 ° C. </li><li> Lavar los pocillos 2 veces con 1 ml de PBS y se añaden 250 l de 0,1 M de glicina en PBS durante 10 min para inactivar la fijador. Lavar los pocillos 2 veces con 1 ml de PBS. </li></ol></li><li> TotalSalmonella tinción de muestras infectadas. <ol><li> Wash cubreobjetos 3x con PBS con 0,1% de saponina / 3% BSA e incubar las muestras con un anticuerpo anti-CSA1 (cabra anti-CSA1, 0,1 g / ml, ratón anti-LPS funciona tan bien, 0,1 mg / ml) durante 1 hr a RT en PBS con 0,1% de saponina / 3% de BSA. </li><li> Lavar 3x en PBS con 0,1% de saponina / 3% de BSA. Incubar las muestras con un anticuerpo anti-cabra secundario acoplado a fluoróforo de elección en PBS con 0,1% de saponina / 3% de BSA durante 30 min a TA en la oscuridad. </li><li> Lavar 3x cubreobjetos con 1 ml de PBS y montar los cubreobjetos para el análisis en medio de montaje con DAPI (1,5 g / ml). </li></ol></li><li> Análisis de microscopía. <ol><li> Adquirir datos con un microscopio confocal contando Salmonella citosólica (anti- Salmonella FITC positivo) y Salmonella total (doble positivo) (Figura 5). Controles permeabilización deben mostrar: ninguna tinción de células no permeabilizadas, calnexina spara las células que contiene digitonina-permeabilizadas y tinción PDI para las células de saponina-permeabilizadas (Figura 4). Wells A4 y A5 servirán como controles internos para la tinción de Salmonella. </li></ol></li></ol></li></ol>

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R., Schlegel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+orientation+of+the+human+papillomavirus+type+16+E5+oncoprotein.">Membrane orientation of the human papillomavirus type 16 E5 oncoprotein.</a> J Virol. 84, 1696-1703 (2010).</li><li>Keller, C., 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=Single+cell+measurements+of+vacuolar+rupture+caused+by+intracellular+pathogens.">Single cell measurements of vacuolar rupture caused by intracellular pathogens.</a> J Vis Exp. , e50116 (2013).</li></ol>

We have nothing to disclose.

Permeabilización diferencial es un método fácil y robusto para analizar y cuantificar la distribución subcelular de patógenos bacterianos entre compartimentos vacuolar y el citosol. El mismo ensayo ha sido utilizado con éxito con bacterias tales como Francisella novicida 2,4 y Shigella flexneri 12. Sin embargo, ya que muchos patógeno intracelular alterar o modificar las estructuras endomembrane anfitrionas, la robustez de la permeabilización digitonin tendrá que ser deter...

Patógenos bacterianos intracelulares se replican ya sea directamente en el citosol de la célula huésped, o en compartimentos vacuolar especializados 1. Durante la etapa inicial de la infección mayoría de los patógenos internalizados obtener ya sea por fagocitosis por células especializadas (como los macrófagos) o promoviendo activamente su propia absorción en células no fagocíticas. En las células fagocíticas, el fagosoma normalmente se fusiona con los lisosomas para formar un compartimiento degradativa, donde se digieren las partículas fagocitadas. Bacterias citosólicas especializados, tales como Francisella novicida o Shigella flexneri, escapan degradación phagosomal mediante la inducción de la ruptura de la fagosoma y posteriormente escapan al 2,3 citosol. Esto requiere mecanismo de virulencia asociada como la Isla Francisella Patogenicidad (FPI) o la Shigella flexneri SST3, que inyecta proteínas efectoras que promueven vacuolar ruptura 2-4. Las características de citosol de la célula huéspedun número de receptores de reconocimiento de patrones conservados que normalmente reconocen la presencia de patógenos e inducen la señalización inmune innato 5. Además, xenophagy, una forma anti-microbiana de la autofagia puede degradar las bacterias que entran en el citosol. Bacterias citosólicas normalmente se adaptan bien a mitigar o evitar estas respuestas utilizando diferentes estrategias. Por ejemplo, Francisella modifica sus LPS para evitar el reconocimiento de acogida y Shigella impide el reclutamiento autofagia utilizando proteínas efectoras secretadas 6,7. Otra estrategia para escapar de la degradación lisosomal es empleado por el patógeno modelo vacuolar Salmonella enterica serovar Typhimurium, denominado a partir de entonces como Salmonella typhimurium. Salmonella utiliza un SST3 para inyectar efectores que remodelan el fagosoma inicial en su nicho intracelular, la Salmonella -Con vacuola (SCV) 8,9. La manipulación continua de las vías de acogida esnecesaria para mantener esta vacuola mediante la contratación de lípidos y otros nutrientes para la vacuola. De hecho, un mutante de Salmonella SIFA falla para mantener la estabilidad vacuolar, y entra en el citosol de las células huésped dentro de horas después de la infección, lo que resulta en la activación de vías inmunes innatas y reclutamiento autofagia 8. Escapar vacuolar de Salmonella varía dependiendo del tipo de células que son los estudios, y varios informes han demostrado que en las células epiteliales incluso WT Salmonella puede escapar del SCV y hyperreplicate en el citosol 10. Informes recientes muestran que la detección inmune innato de patógenos vacuolar también depende de la capacidad del huésped para reconocer y desestabilizar vacuolas que contienen patógenos (PCV) 11-14. Dado que tanto el anfitrión o bacterias genotipo pueden afectar a la distribución de bacterias intracelulares entre el vacuolar y el compartimento citosólico, es necesario cuantificar el número de vacuolar vs.bacterias citosólica. Dado que, en configuraciones más experimentales células huésped se infectan con más de una bacteria, y posteriormente puede albergar varias bacterias en diferentes compartimentos subcelulares, se necesita una técnica que permite la cuantificación del nivel de bacterias individuales. Permeabilización diferencial (también conocido como ensayo de protección de phagosomal) proporciona esta resolución 2,4,10,12. El ensayo se basa en la permeabilización selectiva de la membrana plasmática de la célula huésped con el detergente digitonina, lo que deja las membranas vacuolares intacta y por lo tanto permite la tinción selectiva de bacterias citosólicas con anticuerpos. Aquí proporcionamos dos protocolos para la cuantificación de citosólicas y vacuolar Salmonella typhimurium usando diferencial permeabilización. El principio de este método se describe en la Figura 1: macrófagos derivados de médula ósea (BMDMs) están infectadas con fase estacionaria mCherry-expresión de Salmonella. Fase estacionaria Salmonella necesita to ser utilizado debido a que regulan negativamente la expresión de la SPI-1 T3SS, cuya actividad de otro modo ser reconocido por el inflamasoma NLRC4 e inducir la muerte celular rápida (pyroptosis) de la BMDMs 15. Después de la infección las células se lavan y se tratan con 50 g / ml de digitonina durante 1 minuto. Las células se lavaron de nuevo inmediatamente y se incubaron con anticuerpos anti-Salmonella acoplados a FITC para marcar bacterias con acceso al citosol. Después de un lavado adicional células se lisan paso y el porcentaje de bacterias mCherry + (vacuolar) y FITC + / + mCherry (citosólica) se determina por FACS. Se presenta también una adaptación de este método que puede ser utilizado si no hay cepas que expresan proteínas fluorescentes están disponibles (Figura 2). Los pasos adicionales se introducen después del etiquetado FITC en el que las células se fijan y completamente permeabilizaron. A partir de entonces todas las bacterias se tiñen con anticuerpos anti-Salmonella y los correspondientes anticuerpos secundarios. Detexión se hace entonces por microscopía en lugar de análisis FACS.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Digitonin</td>
			<td>Sigma</td>
			<td>D5628</td>
			<td>50ug/mL</td>
		</tr>
		<tr>
			<td>PFA</td>
			<td>mpbio</td>
			<td>219998380</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Life Technologies</td>
			<td>15630</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Acetate</td>
			<td>Sigma</td>
			<td>791733</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma</td>
			<td>M8266</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma</td>
			<td>A2153</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-Salmonella CSA-1-FITC</td>
			<td>KPL</td>
			<td>01-91-99-MG</td>
			<td>1/500</td>
		</tr>
		<tr>
			<td>anti-Salmonella CSA-1</td>
			<td>KPL</td>
			<td>02-91-99-MG</td>
			<td>1/500</td>
		</tr>
		<tr>
			<td>anti-Calnexin</td>
			<td>Enzo Lifesciences</td>
			<td>SPA-860D</td>
			<td>1/100</td>
		</tr>
		<tr>
			<td>anti-PDI</td>
			<td>Enzo Lifesciences</td>
			<td>SPA-890</td>
			<td>1/100</td>
		</tr>
		<tr>
			<td>Saponin</td>
			<td>Sigma</td>
			<td>47036</td>
			<td></td>
		</tr>
		<tr>
			<td>Vectashield mounting medium</td>
			<td>Vectorlabs</td>
			<td>H-1200</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti Rabbit antibody-488</td>
			<td>Molecular Probes</td>
			<td>A-11070</td>
			<td>1/500</td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Sigma</td>
			<td>G8898</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Life Technologies</td>
			<td>15710-49</td>
			<td>100ug/mL and 10ug/mL</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Promega</td>
			<td>H5141</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Gibco</td>
			<td>20012-019</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Sigma</td>
			<td>D6429</td>
			<td></td>
		</tr>
		<tr>
			<td>NEAA</td>
			<td>Amimed</td>
			<td>5-13K00-H</td>
			<td></td>
		</tr>
		<tr>
			<td>LSM700 Confocal microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td>imaging done at 63x</td>
		</tr>
		<tr>
			<td>FACS Fortessa</td>
			<td>BD technologies</td>
			<td></td>
			<td>detection mCherry 610 nm</td>
		</tr>
		<tr>
			<td>FACS Fortessa</td>
			<td>BD technologies</td>
			<td></td>
			<td>detection FITC 530 nm</td>
		</tr>
	</tbody>
</table>

quantification of cytosolic vs. vacuolar salmonella in primary macrophages by differential permeabilization

Patógenos bacterianos intracelulares pueden replicar en el citosol o en las vacuolas que contienen patógenos especializados (PCV). Para alcanzar el citosol, bacterias como Shigella flexneri y Francisella novicida necesidad de inducir la ruptura del fagosoma. En contraste, Salmonella typhimurium se replica en un compartimiento vacuolar, conocida como Salmonella vacuola -Con (SCV). Sin embargo, ciertos mutantes de Salmonella fallan para mantener la integridad SCV y por lo tanto son liberados en el citosol. El porcentaje de citosólica vs. bacterias vacuolares en el nivel de bacterias individuales se puede medir por permeabilización diferencial, también conocido como ensayo de protección fagosoma. El enfoque hace uso de la propiedad de digitonina detergente para unirse selectivamente el colesterol. Puesto que la membrana de plasma contiene más colesterol que otras membranas celulares, digitonina se puede utilizar para permeabilizar selectivamente la membrana del plasma mientras que deja las membranas intracelularesintacta. En breve, después de la infección con el patógeno que expresa una proteína marcador fluorescente (por ejemplo mCherry entre otros), la membrana plasmática de las células huésped se permeabilizaron con una breve incubación en digitonina que contiene tampón. Después las células se lavaron y se incubaron con un anticuerpo primario (acoplado a un fluoróforo de elección) dirigido contra la bacteria de elección (por ejemplo, Salmonella anti- FITC) y se lavaron de nuevo. Si se utilizan bacterias no marcados, un paso adicional se puede hacer, en la que todas las membranas se permeabilizaron y todas las bacterias teñidas con un anticuerpo correspondiente. Después de la tinción, el porcentaje de bacterias vacuolares y citosólicas se puede cuantificar mediante FACS o microscopía por conteo de eventos individuales o de doble positivo. Aquí proporcionamos detalles experimentales para el uso de esta técnica con la bacteria Salmonella typhimurium. La ventaja de este ensayo es que, en contraste con otro ensayo, proporciona una cuantificación del nivel de solo bacteria, y si analizada por microscopía proporciona el número exacto de citosólicas y bacterias vacuolares en una célula dada.

Figura 1 y Figura 2 muestran los esquemas del ensayo digitonin descritos en el protocolo 1 y el protocolo 2 ilustra los pasos críticos en el protocolo y los resultados obtenidos. La Figura 3 muestra los resultados típicos FACS. Los controles positivos y negativos se utilizan para ajustar las puertas para mCherry + / bacterias FITC (vacuolar Salmonella) y mCherry + / FITC + citosólico bacterias (Salmonella). Sobre la base de estas puertas el porcentaje de bacterias citos...

Watch this Scientific Journal Video about Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization at JoVE.com

Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization

We describe the quantification of cytosolic and vacuolar Salmonella typhimurium in bone-marrow derived macrophages using differential digitonin permeabilization.

Cuantificación de Citosólica vs. Vacuolar<em&gt; Salmonella</em&gt; En macrófagos primarios por permeabilización diferencial

Intracellular bacterial pathogens can replicate in the cytosol or in specialized pathogen-containing vacuoles (PCVs). To reach the cytosol, bacteria like ...

quantification-cytosolic-vs-vacuolar-salmonella-primary-macrophages

Research

JoVE Journal

Immunology and Infection

Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization

10.8K vistas.  University of Basel. We describe the quantification of cytosolic and vacuolar Salmonella typhimurium in bone-marrow derived macrophages using differential digitonin permeabilization.

Video: Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization

Measurement of Cytosolic Ca2+ in Isolated Contractile Lymphatics

Lymphatic vessels comprise a multifunctional transport system that maintains fluid homeostasis, delivers lipids to the central circulation, and acts as a surveillance system for potentially harmful antigens, optimizing mucosal immunity and adaptive immune responses1. Lymph is formed from interstitial fluid that enters blind-ended initial lymphatics, and then is transported against a pressure gradient in larger collecting lymphatics. Each collecting lymphatic is made up of a series of segments called lymphangions, separated by bicuspid valves that prevent backflow. Each lymphangion possesses a contractile cycle that propels lymph against a pressure gradient toward the central circulation2. This phasic contractile pattern is analogous to the cardiac cycle, with systolic and diastolic phases, and with a lower contraction frequency4. In addition, lymphatic smooth muscle generates tone and displays myogenic constriction and dilation in response to increases and decreases in luminal pressure, respectively5. A hybrid of molecular mechanisms that support both the phasic and tonic contractility of lymphatics are thus proposed.
Contraction of smooth muscle is generally regulated by the cytosolic Ca2+ concentration ([Ca2+]i) plus sensitivity to Ca2+, of the contractile elements in response to changes in the environment surrounding the cell6. [Ca2+]i is determined by the combination of the movement of Ca2+ through plasma membrane ligand or voltage gated Ca2+ channels and the release and uptake of Ca2+ from internal stores. Cytosolic Ca2+ binds to calmodulin and activates enzymes such as myosin light chain (MLC) kinase (MLCK), which in turn phosphorylates MLC leading to actin-myosin-mediated contraction8. However, the sensitivity of this pathway to Ca2+ can be regulated by the MLC phosphatase (MLCP)9. MLCP activity is regulated by Rho kinase (ROCK) and the myosin phosphatase inhibitor protein CPI-17.
Here, we present a method to evaluate changes in [Ca2+]i over time in isolated, perfused lymphatics in order to study Ca2+-dependent and Ca2+-sensitizing mechanisms of lymphatic smooth muscle contraction. Using isolated rat mesenteric collecting lymphatics we studied stretch-induced changes in [Ca2+]i and contractile activity. The isolated lymphatic model offers the advantage that pressure, flow, and the chemical composition of the bath solution can be tightly controlled. [Ca2+]i was determined by loading lymphatics with the ratiometric, Ca2+-binding dye Fura-2. These studies will provide a new approach to the broader problem of studying the different molecular mechanisms that regulate phasic contractions versus tonic constriction in lymphatic smooth muscle.

Measurement of Cytosolic Ca2+ in Isolated Contractile Lymphatics

We introduce an approach to evaluate the cytosolic Ca2+ concentration in isolated lymphatics to study Ca2+-dependent and Ca2+-sensitizing mechanisms of lymphatic smooth muscle contraction.

Medición de la citosólica de Ca 2 + En aislados linfáticos contráctil

Lymphatic vessels comprise a multifunctional transport system that maintains fluid homeostasis, delivers lipids to the central circulation, and acts as a ...

Investigación

Inmunología e Infección

Single Cell Measurements of Vacuolar Rupture Caused by Intracellular Pathogens

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed compartment allows bacteria to move within the cytosol, proliferate and further invade neighboring cells. Mycobacterium tuberculosis is phagocytosed by immune cells, and has recently been shown to rupture phagosomal membrane in macrophages. We developed a robust assay for tracking phagosomal membrane disruption after host cell entry of Shigella flexneri or Mycobacterium tuberculosis. The approach makes use of CCF4, a FRET reporter sensitive to &beta;-lactamase that equilibrates in the cytosol of host cells. Upon invasion of host cells by bacterial pathogens, the probe remains intact as long as the bacteria reside in membrane-enclosed compartments. After disruption of the vacuole, &beta;-lactamase activity on the surface of the intracellular pathogen cleaves CCF4 instantly leading to a loss of FRET signal and switching its emission spectrum. This robust ratiometric assay yields accurate information about the timing of vacuolar rupture induced by the invading bacteria, and it can be coupled to automated microscopy and image processing by specialized algorithms for the detection of the emission signals of the FRET donor and acceptor. Further, it allows investigating the dynamics of vacuolar disruption elicited by intracellular bacteria in real time in single cells. Finally, it is perfectly suited for high-throughput analysis with a spatio-temporal resolution exceeding previous methods. Here, we provide the experimental details of exemplary protocols for the CCF4 vacuolar rupture assay on HeLa cells and THP-1 macrophages for time-lapse experiments or end points experiments using Shigella flexneri as well as multiple mycobacterial strains such as Mycobacterium marinum, Mycobacterium bovis, and Mycobacterium tuberculosis.

We describe a method for tracking the endomembrane rupture elicited by the intracellular bacteria Shigella flexneri and Mycobacterium tuberculosis upon host cell invasion. Our assay makes use of CCF4, a host cytoplasmic FRET probe in live or fixed cells. This reporter is degraded by an enzyme activity present on the bacterial surface.

Sola célula Medidas de Ruptura Vacuolar causadas por patógenos intracelulares

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed ...

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying Salmonella-host interactions are&#160;important to elucidate the molecular pathogenesis of Salmonella infection. The Gentamicin protection assay to phenotype Salmonella association, invasion and replication in phagocytic cells was adapted to allow high-throughput screening to define the roles of deletion mutants of Salmonella enterica serotype Typhimurium in host interactions using RAW 264.7 murine macrophages. Under this protocol, the variance in measurements is significantly reduced compared to the standard protocol, because wild-type and multiple mutant strains can be tested in the same culture dish and at the same time. The use of multichannel pipettes increases the throughput and enhances precision. Furthermore, concerns related to using less host cells per well in 96-well culture dish were addressed. Here, the protocol of the modified in vitro Salmonella invasion assay using phagocytic cells was successfully employed to phenotype 38 individual Salmonella deletion mutants for association, invasion and intracellular replication. The in vitro phenotypes are presented, some of which were subsequently confirmed to have in vivo phenotypes in an animal model. Thus, the modified, standardized assay to phenotype Salmonella association, invasion and replication in macrophages with high-throughput capacity could be utilized more broadly to study bacterial-host interactions.

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

A high-throughput assay to in vitro phenotype Salmonella or other bacterial association, invasion, and replication in phagocytic cells with high-throughput capacity was developed. The method was employed to evaluate Salmonella gene knockout mutant strains for their involvements in host-pathogen interactions.

Ensayo de alto rendimiento para Fenotipo<em&gt; Salmonella enterica</em&gt; Asociación Typhimurium, Invasión y replicación en macrófagos

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying ...

Highly Efficient Transfection of Human THP-1 Macrophages by Nucleofection

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. Transfection with siRNA and plasmid DNA is an efficient tool for studying their function, but transfection of macrophages is not a trivial matter. Although many different approaches for transfection of eukaryotic cells are available, only few allow reliable and efficient transfection of macrophages, but reduced cell vitality and severely altered cell behavior like diminished capability for differentiation or polarization are frequently observed. Therefore a transfection protocol is required that is capable of transferring siRNA and plasmid DNA into macrophages without causing serious side-effects thus allowing the investigation of the effect of the siRNA or plasmid in the context of normal cell behavior. The protocol presented here provides a method for reliably and efficiently transfecting human THP-1 macrophages and monocytes with high cell vitality, high transfection efficiency, and minimal effects on cell behavior. This approach is based on Nucleofection and the protocol has been optimized to maintain maximum capability for cell activation after transfection. The protocol is adequate for adherent cells after detachment as well as cells in suspension, and can be used for small to medium sample numbers. Thus, the method presented is useful for investigating gene regulatory effects during macrophage differentiation and polarization. Apart from presenting results characterizing macrophages transfected according to this protocol in comparison to an alternative chemical method, the impact of cell culture medium selection after transfection on cell behavior is also discussed. The presented data indicate the importance of validating the selection for different experimental settings.

This protocol presents an efficient and reliable method to transfect human THP-1 macrophages with siRNA or plasmid DNA by electroporation with high transfection efficiency while maintaining high cell vitality and full macrophage capacity for differentiation and polarization.

La transfección altamente eficiente de humanos THP-1 macrófagos por Nucleofection

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. ...

Proteomic Profiling of Macrophages by 2D Electrophoresis

The goal of the two-dimensional (2D) electrophoresis protocol described here is to show how to analyse the phenotype of human cultured macrophages. The key role of macrophages has been shown in various pathological disorders such as inflammatory, immunological, and infectious diseases. In this protocol, we use primary cultures of human monocyte-derived macrophages that can be differentiated into the M1 (pro-inflammatory) or the M2 (anti-inflammatory) phenotype. This in vitro model is reliable for studying the biological activities of M1 and M2 macrophages and also for a proteomic approach. Proteomic techniques are useful for comparing the phenotype and behaviour of M1 and M2 macrophages during host pathogenicity. 2D gel electrophoresis is a powerful proteomic technique for mapping large numbers of proteins or polypeptides simultaneously. We describe the protocol of 2D electrophoresis using fluorescent dyes, named 2D Differential Gel Electrophoresis (DIGE). The M1 and M2 macrophages proteins are labelled with cyanine dyes before separation by isoelectric focusing, according to their isoelectric point in the first dimension, and their molecular mass, in the second dimension. Separated protein or polypeptidic spots are then used to detect differences in protein or polypeptide expression levels. The proteomic approaches described here allows the investigation of the macrophage protein changes associated with various disorders like host pathogenicity or microbial toxins.

Macrophages are the key cells involved in host pathogenicity. Macrophages display phenotypic and functional diversity that can be analysed and detected by proteomic analysis. This article describes how to perform 2D electrophoresis of primary cultures of human macrophages differentiated into M1 or M2 phenotype.

Proteómico de perfiles de los macrófagos por electroforesis 2D

The goal of the two-dimensional (2D) electrophoresis protocol described here is to show how to analyse the phenotype of human cultured macrophages. The ...

Phagosome Migration and Velocity Measured in Live Primary Human Macrophages Infected with HIV-1

Macrophages are phagocytic cells that play a major role at the crossroads between innate and specific immunity. They can be infected by the human immunodeficiency virus (HIV)-1 and because of their resistance to its cytopathic effects they can be considered to be persistent viral reservoirs. In addition, HIV-infected macrophages exhibit defective functions that contribute to the development of opportunistic diseases.
The exact mechanism by which HIV-1 impairs the phagocytic response of macrophages was unknown. We had previously shown that the uptake of various particulate material by macrophages was inhibited when they were infected with HIV-1. This inhibition was only partial and phagosomes did form within HIV-infected macrophages. Therefore, we focused on analyzing the fate of these phagosomes. Phagosome maturation is accompanied by migration of these compartments towards the cell center, where they fuse with lysosomes, generating phagolysosomes, responsible for degradation of the ingested material. We used IgG-opsonized Sheep Red Blood Cells as a target for phagocytosis. To measure the speed of centripetal movement of phagosomes in individual HIV-infected macrophages, we used a combination of bright field and fluorescence confocal microscopy. We established a method to calculate the distance of phagosomes towards the nucleus, and then to calculate the velocity of the phagosomes. HIV-infected cells were identified thanks to a GFP-expressing virus, but the method is applicable to non-infected cells or any type of infection or treatment.

We describe a method to measure the velocity of phagosomes moving towards the cell center in living cells infected with or without the human immunodeficiency virus (HIV) type 1, using spinning disk confocal fluorescence microscopy to identify fluorescent infected cells and bright field microscopy to detect phagosomes.

Fagosoma Migración y velocidad medida en vivo primaria macrófagos humanos infectados con VIH-1

Macrophages are phagocytic cells that play a major role at the crossroads between innate and specific immunity. They can be infected by the human ...

Phenotypic Characterization of Macrophages from Rat Kidney by Flow Cytometry

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal disease. Macrophages are heterogeneous cells that have been implicated in kidney injury. Macrophages may be classified into two different phenotypes: classically activated macrophages (M1 macrophages), that release pro-inflammatory cytokines and promote fibrosis; and alternatively activated macrophages (M2 macrophages) that are associated with immunoregulatory and tissue-remodeling functions. These macrophage phenotypes need to be discriminated and analyzed to determine their contribution to renal injury. However, there are scarce studies reporting consistent phenotypic and functional information about macrophage subtypes in inflammatory renal disease models, especially in rats. This fact may be related to the limited macrophage markers used in rats, contrary to mice. Therefore, novel strategies are necessary to quantify and characterize the renal content of these infiltrating cells in a reliable way. This manuscript details a protocol for kidney digestion and further phenotypic and quantitative analysis of macrophages from rat kidneys by flow cytometry. Briefly, kidneys were incubated with collagenase and total macrophages were identified according to the dual presence of CD45 (leukocytes common antigen) and CD68 (PAN macrophage marker) in live cells.This was followed by surface staining of CD86 (M1 marker) and CD163 (M2 marker). Rat peritoneal macrophages were used as positive control for macrophage marker detection by flow cytometry. Our protocol resulted in low cellular mortality and allowed characterization of different intracellular and surface protein markers, thus limiting the loss of cellular integrity observed in other protocols. Moreover, this procedure allows the use of macrophages for further techniques, including cell sorting and mRNA or protein expression studies, among others.

This manuscript describes a detailed protocol for phenotypic and quantitative analysis of resident macrophages from rat kidneys by flow cytometry. The resulting stained cells can be also used for other applications, including cell sorting, gene expression analysis or functional studies, thus increasing the information obtained in the experimental model.

Caracterización fenotípica de los macrófagos a partir de riñón de rata por Citometría de Flujo

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

Aislamiento de Salmonella typhimurium-que contienen fagosomas de los macrófagos

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

Time-lapse 3D Imaging of Phagocytosis by Mouse Macrophages

Phagocytosis plays a key role in host defense, as well as in tissue development and maintenance, and involves rapid, receptor-mediated rearrangements of the actin cytoskeleton to capture, envelop and engulf large particles. Although phagocytic receptors, downstream signaling pathways, and effectors, such as Rho GTPases, have been identified, the dynamic cytoskeletal remodeling of specific receptor-mediated phagocytic events remain unclear. Four decades ago, two distinct mechanisms of phagocytosis, exemplified by Fc&#947; receptor (Fc&#947;R)- and complement receptor (CR)-mediated phagocytosis, were identified using scanning electron microscopy. Binding of immunoglobulin G (IgG)-opsonized particles to Fc&#947;Rs triggers the protrusion of thin membrane extensions, which initially form a so-called phagocytic cup around the particle before it becomes completely enclosed and retracted into the cell. In contrast, complement opsonized particles appear to sink into the phagocyte following binding to complement receptors. These two modes of phagocytosis, phagocytic cup formation and sinking in, have become well established in the literature. However, the distinctions between the two modes have become blurred by reports that complement receptor-mediated phagocytosis may induce various membrane protrusions. With the availability of high resolution imaging techniques, phagocytosis assays are required that allow real-time 3D (three dimensional) visualization of how specific phagocytic receptors mediate the uptake of individual particles. More commonly used approaches for the study of phagocytosis, such as end-point assays, miss the opportunity to understand what is happening at the interface of particles and phagocytes. Here we describe phagocytic assays, using time-lapse spinning disk confocal microscopy, that allow 3D imaging of single phagocytic events. In addition, we describe assays to unambiguously image Fc&#947; receptor- or complement receptor-mediated phagocytosis.

Here we describe methods using spinning disk confocal microscopy to image single phagocytic events by mouse resident peritoneal macrophages. The protocols can be extended to other phagocytic cells.

Proyección de imagen 3D Time-lapse de la fagocitosis por macrófagos de ratón

Phagocytosis plays a key role in host defense, as well as in tissue development and maintenance, and involves rapid, receptor-mediated rearrangements of ...

Quantification of Antibody-dependent Enhancement of the Zika Virus in Primary Human Cells

The recent emergence of the flavivirus Zika and neurological complications, such as Guillain-Barr&#233; syndrome and microcephaly in infants, has brought serious public safety concerns. Among the risk factors, antibody-dependent enhancement (ADE) poses the most significant threat, as the recent re-emergence of the Zika virus (ZIKV) is primarily in areas where the population has been exposed and is in a state of pre-immunity to other closely related flaviviruses, especially dengue virus (DENV). Here, we describe a protocol for quantifying the effect of human serum antibodies against DENV on ZIKV infection in primary human cells or cell lines.

We describe a method to evaluate the effect of pre-existing immunity against dengue virus on the Zika virus infection by using human serum, primary human cells, and infection quantification by quantitative real-time polymerase chain reaction.

Cuantificación de estimulación dependiente de los anticuerpos del Virus Zika en células humanas primarias

The recent emergence of the flavivirus Zika and neurological complications, such as Guillain-Barr&#233; syndrome and microcephaly in infants, has brought ...