Name: rna purification from intracellularly grown listeria monocytogenes in macrophage cells
Uploaded: 2016-06-04T14:00:00.000+00:00
Duration: 10 min 42 s
Description: Watch this Scientific Journal Video about RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells at JoVE.com

The research in the Herskovits lab is supported by 335400 ERC and R01A/109048 NIH grants.

Nota: Durante todo el experimento, las células de macrófagos se incubaron a 37 ° C en un incubador de CO2 de aire forzado 5% y sacados de la incubadora sólo para las manipulaciones experimentales, que se llevan a cabo en una cabina de seguridad biológica de Clase II. Trabajar con L. bacterias monocytogenes es de acuerdo al nivel de seguridad biológica 2 regulaciones. 1. Preparación de células y la infección bacteriana (Día 1 y 2) <ol><li> Día 1 <ol><li> Células de semillas 2,0 x 10 7 de médula ósea derivadas de macrófagos (BMDM) en un plato de 145 mm en 30 ml BMDM + medios de Pen-Strep (Tabla 2). Seed 3 placas para cada cepa bacteriana a ser analizados. Incubar toda la noche a 37 ° C en una incubadora de CO2 de aire forzado 5%. </li><li> Iniciar un cultivo bacteriano durante la noche de tipo salvaje (WT) L. monocytogenes 10403S cepa en 10 ml de medio de cerebro y corazón de infusión (BHI), a 30 ° C en un incubador estándar. Coloque los tubos de cultivo inclinados sin agitación. Nota:Estas condiciones de crecimiento hasta de regular la expresión de los genes flagelos, que promueve la infección eficiente. </li></ol></li><li> Dia 2 <ol><li> Medio Pre-caliente BMDM (sin antibióticos Pen-Strep) (Tabla 2) y solución salina tamponada con fosfato (PBS) a 37 ° C. </li><li> Lavar monocapa de macrófagos dos veces con 25 ml de PBS precalentado para eliminar los antibióticos. Añadir 30 ml de medio fresco BMDM. </li><li> Lavar 1,5 ml de cultivo bacteriano durante la noche con PBS por centrifugación las bacterias a&gt; 14.000 xg durante 1 min, desechar el sobrenadante, y resuspendiendo las bacterias suavemente en 1,5 ml de PBS mediante pipeteo. Repetir dos veces. </li><li> Infectar a los macrófagos cada placa con 0,5 ml de un cultivo de una noche de lava de tipo salvaje de L. monocytogenes. Si el uso de múltiples placas, infectar a cada placa de 15 minutos de diferencia. Este intervalo de tiempo permitirá la cosecha de cada placa individualmente al final de la infección. Nota: La multiplicidad de infección (MOI) se ensaya mediante siembra de diluciones en series de cultivo bacteriano usado para la infección en placas de BHI agar y contando las unidades formadoras de colonias (CFU) después de 24 hr de incubación de las placas a 37 ° C. Usando 0,5 ml de WT L. monocytogenes cepa 10403S resultados en una MOI de ~ 100 (100 UFC de bacterias por célula macrófagos). Al analizar los mutantes bacterianos defectuosos en el crecimiento intracelular, un MOI más alto debe ser considerado. </li><li> Después de 0,5 horas de incubación a 37 ° C, se lavan las células infectadas dos veces con PBS, para eliminar las bacterias no unidas, y añadir 30 ml pre-calentado medio BMDM. bacterias adheridas internalicen durante el próximo 0,5 horas. </li><li> A 1 h después de la infección, añadir 30 l de gentamicina (1: 1.000 para alcanzar una concentración final de 50 mg / ml) para matar las bacterias extracelulares. </li><li> Montar el aparato de filtración mediante la colocación de una cabeza de filtro en un matraz de recogida de líquido con un puerto de salida de vacío. A continuación, coloque un filtro (0,45 micras) en una cabeza de filtro, seguido de un embudo de cilindro y asegurar las diferentes partes con un metal clámpara. Preparar agua libre de RNasa helada. </li><li> A las 6 horas después de la infección, las bacterias de la cosecha de las células infectadas de la siguiente manera. Tratar a cada placa individual. Nota: Por lo general, L. monocytogenes completa 1-log de ​​crecimiento durante 6 h de infección en células de macrófagos. incubaciones más largas podrían dar lugar a la proliferación bacteriana y la muerte celular de los macrófagos, mientras que los períodos de incubación más cortos podrían resultar en cargas bacterianas considerablemente inferiores. El MOI y el tiempo de la infección pueden ser modificados para alcanzar condiciones óptimas. <ol><li> Lavar las células infectadas con PBS una vez. Añadir 20 ml de agua libre de RNasa enfriado con hielo para lisar células macrófagos. El uso de un rascador de células, raspar las células de la placa rápidamente, pero con cuidado. </li><li> Recoger las células lisadas a un tubo cónico de 50 ml. Vortex durante 30 segundos. Centrifugar a 800 xg durante 3 min a 4 ° C. </li><li> Pasar el sobrenadante a través del aparato de filtro usando un sistema de vacío. Usando las pinzas, el rollo de filtro y rápidamente la transferencia a un 15 ml CONItubo de cal. Snap-congelar los tubos con los filtros en nitrógeno líquido. </li><li> Montar el aparato de filtración con un filtro nuevo para la siguiente muestra, como se describe en 1.2.7. </li><li> Almacenar los filtros congelados a -80 ° C para el día siguiente. Como alternativa, proceder directamente a la extracción de ácidos nucleicos. </li></ol></li></ol></li></ol> 2. Ácidos nucleicos extracción (día 3) Nota: Realice todas las manipulaciones con las soluciones de fenol y cloroformo en una campana de humos. <ol><li> Preparar una mezcla 1: 1 de fenol acidificado: cloroformo, 400 l para cada muestra. Mezclar en tubos separados, y luego retirar la capa acuosa resultante por aspiración. Añadir 40 l de 10% SDS. </li><li> Descongelar los tubos que contienen filtros-en hielo para mantenerlos fríos. A cada tubo que contiene filtro de añadir 650 l de acetato-EDTA (AE) (Tabla 2). </li><li> Realice los siguientes pasos lo más rápido posible. <ol><li> Vigorosamente vórtice el tubo que contiene el filtro paraque el filtro bate a la periferia del tubo y el tampón se lava completamente el filtro. Siempre mantenga el tubo frío colocándolo de nuevo en hielo. Nota: puede ser necesario vórtice, además, el tubo mientras invertida para lavar completamente las bacterias fuera del filtro. </li><li> Repita este procedimiento para todos los tubos. Puede ser útil para hacer girar hacia abajo la suspensión en breve (1 min a 120 xg) al final del proceso. </li></ol></li><li> Transferir el tampón de bacterias que contienen a un tubo de microcentrífuga de 1,5 ml que contiene el SDS y la mezcla de fenol / cloroformo (como se preparó en 2,1). Repita este procedimiento para todos los tubos. Puede que sea necesario para hacer girar los tubos de filtro que contiene (1 min a 120 xg) de nuevo para obtener tampón residual del filtro. </li><li> Coloque todos los tubos de 1,5 ml en un dispositivo de vórtice de tubos múltiples, y vórtice a velocidad máxima durante 10 min. </li><li> Incubar los tubos en un bloque de calor C 65 ° durante 10 min. Centrifugar a máxima velocidad (&gt; 14.000 xg) durante 5 min. </li><li> La transferencia de la capa acuosa (aproximadamente 400 l) de cada uno tUbe a un nuevo tubo de 1,5 ml que contiene 40 l de acetato de sodio 3 M (pH 5,2) y 1,0 ml de etanol al 100%. Vortex cada tubo a fondo. Nota: El glucógeno se puede añadir en esta etapa para mejorar la visualización de la pastilla precipitado. </li><li> Incubar las muestras a -80 ° C durante 1 hr. Alternativamente, se incuban las muestras a -20 ° C durante la noche. A continuación, centrifugar las muestras a 4 ° C durante 20 min a la velocidad máxima (&gt; 14.000 xg). </li><li> aspirar cuidadosamente el etanol (capa superior) de cada tubo. Añadir etanol 500 l frío 70% a cada muestra y mezclar bien. Centrifugar a 4 ° C durante 20 min a la velocidad máxima (&gt; 1 4.000 xg). </li><li> aspirar cuidadosamente el etanol a partir de cada tubo. Secar las muestras durante aproximadamente 2 minutos usando un evaporador al vacío sin calor. No sobre-secar las muestras. </li><li> Añadir 25 l de agua libre de RNasa a cada muestra. Incubar a temperatura ambiente durante 20 min. Con cuidado vórtice y spin-down. </li><li> Medir la concentración de ARN en tél muestras usando un microtomo UV-Vis. Esperar a extraer aproximadamente 0,5 - 1 mg de ácidos nucleicos por placa. Nota: Técnico repeticiones se pueden combinar en esta etapa. </li></ol> 3. El tratamiento de DNasa <ol><li> Configurar la reacción de acuerdo con la Tabla 1 en un tubo de microcentrífuga. Incubar a 37 ° C durante 45 min. Añadir 450 l de agua libre de RNasa y 500 l de fenol-cloroformo-IAA mezcla (Tabla 2), fases separadas por centrifugación durante 2 minutos a velocidad máxima (&gt; 14.000 xg). </li><li> Transferir la fase acuosa a un tubo nuevo y añadir 500 l de cloroformo-IAA mezcla (Tabla 2), mezclar y fases separadas por centrifugación de 2 minutos a velocidad máxima (&gt; 14.000 xg). </li><li> Transferir la fase acuosa a un nuevo tubo y añadir 1 ml de etanol y 50 l de acetato de sodio 3 M (pH 5,2). Vórtice. Nota: El glucógeno se puede añadir en esta etapa para mejorar la visualización de la pastilla precipitado. </li><li&gt; incubar durante 1 hora a -80 ° C. Alternativamente, se incuban las muestras a -20 ° C durante la noche. Centrifugar a 4 ° C durante 20 min a velocidad máxima. </li><li> aspirar cuidadosamente el etanol a partir de cada tubo. Añadir etanol 500 l frío 70% a cada muestra y mezclar bien. Centrifugar a 4 ° C durante 20 min a velocidad máxima. aspirar cuidadosamente el etanol a partir de cada tubo. </li><li> Secar las muestras durante aproximadamente 2 minutos usando un evaporador al vacío sin calor. No sobre-secar las muestras. </li><li> Añadir 12 l de agua libre de RNasa, se incuba durante 2 minutos a temperatura ambiente, vórtice, y spin-down. Las muestras contienen ARN purificado, los mantienen en hielo. Como alternativa, disolver ARN en RNasa libre de H 2 O con EDTA 0,1 mM o tampón TE (Tris 10 mM, EDTA 1 mM). </li><li> Medir las concentraciones de ARN usando un microtomo UV-Vis. Esperar ~ 100 ng de RNA por muestra (si se combinan técnica repeticiones). Nota: el ARN extraído de acuerdo con este protocolo es adecuado for análisis de transcripción génica mediante múltiples técnicas. Por lo general, empleamos el análisis RT-qPCR para estudiar L. monocytogenes la transcripción de genes durante la infección de las células de macrófagos 1-4. </li></ol>

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The authors have nothing to disclose.

El protocolo descrito aquí representa un método optimizado para el aislamiento de ARN bacteriana de L. monocytogenes bacterias que crecen intracelularmente en células de macrófagos. Este protocolo se basa en la lisis diferencial de células e incluye dos pasos principales para el enriquecimiento de ARN bacteriano: sedimentación núcleos de macrófagos utilizando centrifugación y una rápida acumulación de bacterias por filtración. Estos pasos son seguidos por un procedimiento de extracción de ARN está...

Patógenos bacterianos intracelulares ─ bacterias causantes de enfermedades infecciosas y capaz de crecer y reproducirse dentro de las células del huésped humano ─ son un importante problema de salud en todo el mundo 5. Para invadir y replicarse dentro de una célula de mamífero, patógenos intracelulares han adquirido sofisticados mecanismos de virulencia y factores. Aunque estos mecanismos son fundamentales para la capacidad de causar una enfermedad, se sabe poco acerca de su regulación y dinámica. Desde los perfiles de expresión de genes de bacterias cultivadas en medio líquido no reflejan el entorno real dentro de células huésped, hay una necesidad creciente de transcriptoma análisis de bacterias cultivadas en sus nichos intracelulares. Estos análisis permitirán al desciframiento de adaptaciones específicas bacterianas provocadas por el anfitrión y le ayudará a identificar nuevas dianas para el diseño terapéutico. El análisis del transcriptoma de bacterias que crecen de forma intracelular es muy difícil, puesto que superan en número a ARN de mamífero ARN bacteriano por lopor lo menos diez veces. En este manuscrito, se describe un método experimental para aislar el ARN bacteriano de Listeria monocytogenes bacterias que crecen dentro de las células de macrófagos murinos. El ARN extraído se puede utilizar para estudiar las adaptaciones intracelulares y mecanismos de virulencia de las bacterias patógenas por diversas técnicas de análisis de la transcripción, tales como RT-PCR, el ARN-Seq, microarrays y otras tecnologías basadas en hibridación. L. monocytogenes es el agente causante de la listeriosis en humanos, una enfermedad con manifestaciones clínicas dirigidas principalmente a los individuos inmunocomprometidos, ancianos y mujeres embarazadas 6. Eso es un patógeno intracelular facultativo Gram-positiva que invade una amplia gama de células de mamíferos que se han utilizado durante décadas como modelo en las interacciones huésped-patógeno estudia 7. Tras la invasión, que reside inicialmente en una vacuola o fagosoma (en el caso de las células fagocíticas), de la que debe escapar en tque acogerá citosol de la célula con el fin de replicar. Varios factores de virulencia han demostrado mediar en el proceso de escape, principalmente la hemolisina de formación de poros, listeriolisina O (LLO) y dos fosfolipasas adicionales 8. En el citosol las bacterias utilizan el anfitrión maquinaria polimerización de la actina para impulsarse en los filamentos de actina dentro de la célula y propagarse de célula a célula (Figura 1). Todos los principales factores de virulencia de L. monocytogenes involucrados en la invasión, la supervivencia intracelular y la replicación, son activados por el regulador de la transcripción de virulencia principal, PrfA. 8-10. En la última década, varios estudios realizados por nosotros y otros han métodos para el análisis del transcriptoma de bacterias que crecen de forma intracelular aplicado con éxito dentro de las células anfitrionas 2,11-15. Dos enfoques principales se utilizan para separar el ARN bacteriano de host-RNA que se basan en: 1) el enriquecimiento selectivo de ARN bacteriana y 2) el aislamiento de ARN por difelisis celular rential. El primer enfoque se basa en la hibridación de sustracción de extractos de ARN total de las moléculas de ARN de mamífero (por ejemplo, utilizando kits disponibles en el comercio) o la captura selectiva de secuencias transcritas bacterianas (escoceses) 11. El segundo enfoque se basa en la lisis diferencial de bacterias y células huésped, en el que se lisan las células huésped mientras que las células bacterianas se mantienen intactos. Las células bacterianas se separan a continuación a partir del lisado de la célula huésped, generalmente por centrifugación, y el ARN se extrae usando técnicas estándar. El principal problema con este enfoque es que, junto con las bacterias intactas, también se aíslan células huésped núcleos, por lo tanto las preparaciones de ARN todavía contienen ARN de mamífero. Una forma de superar este problema es separar bacterias intactas de células huésped núcleos utilizando centrifugación diferencial, aunque este procedimiento por lo general toma tiempo el aumento de la preocupación de los cambios en el perfil de la expresión génica durante la extracción. En este artículo presentamos un ba mejorada y rápidaprotocolo de extracción de ARN cteria, que se basa en el enfoque de lisis diferencial de células. En primer lugar, L. monocytogenes células de macrófagos infectados se lisan con agua fría. A continuación, los núcleos de los macrófagos se eliminan mediante una breve centrifugación y bacterias intactas se recogen rápidamente en los filtros, de la que el ARN se aísla usando caliente extracción con fenol-SDS de los ácidos nucleicos bacterianos.

<table><tbody><tr><td>Listeria monocytogenes 10403S</td><td></td><td></td><td>&lt;sup&gt;20&lt;/sup&gt;</td></tr><tr><td>Bone marrow derived macrophages prepared from C57B/6 female mice</td><td></td><td></td><td>&lt;sup&gt;21&lt;/sup&gt;</td></tr><tr><td>H&lt;sub&gt;2&lt;/sub&gt;O, RNAse free</td><td>Thermo Scientific</td><td>10977-015</td><td>DEPC-treated water can be used</td></tr><tr><td>DMEM</td><td>Gibco</td><td>41965039</td><td></td></tr><tr><td>Glutamine</td><td>Gibco</td><td>25030081</td><td></td></tr><tr><td>Sodium pyruvate</td><td>Gibco</td><td>11360-088</td><td></td></tr><tr><td>&amp;#946;-Mercaptoethanol</td><td>Gibco</td><td>31350010</td><td></td></tr><tr><td>Pen/Strep</td><td>Gibco</td><td>15140-122</td><td></td></tr><tr><td>Gentamicin</td><td>Sigma-Aldrich</td><td>G1397</td><td></td></tr><tr><td>FBS</td><td>Gibco</td><td>10270106</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Phosphate Buffered Saline-PBS</td><td>Sigma-Aldrich</td><td>D8537</td><td></td></tr><tr><td>Brain heart infusion (BHI)</td><td>Merckmillipore</td><td>1104930500</td><td></td></tr><tr><td>Phenol saturated pH 4.3</td><td>Fisher</td><td>BP1751I-400</td><td></td></tr><tr><td>Chloroform</td><td>Fisher</td><td>BP1145-1</td><td></td></tr><tr><td>Iso-amyl alcohol</td><td>Sigma-Aldrich</td><td>W205702</td><td></td></tr><tr><td>Sodium acetate</td><td>Sigma-Aldrich</td><td>W302406</td><td></td></tr><tr><td>EDTA</td><td>Sigma-Aldrich</td><td>EDS</td><td></td></tr><tr><td>DNaseI</td><td>Fermentas</td><td>EN0521</td><td></td></tr><tr><td>SDS 10%</td><td>Sigma-Aldrich</td><td>L4522</td><td></td></tr><tr><td>Ethanol absolute</td><td>Merck Millipore</td><td>1070174000</td><td></td></tr><tr><td>37 &amp;deg;C, 5% CO&lt;sub&gt;2&lt;/sub&gt; forced-air incubator</td><td>Thermo Scientific</td><td>Model 3111</td><td></td></tr><tr><td>Cell scrapers</td><td>Nunc</td><td>179693</td><td></td></tr><tr><td>Kontes glass holder for 45 mm filters</td><td>Fisher</td><td>K953755-0045</td><td></td></tr><tr><td>MF-Millipore filters 45&amp;nbsp;mm, 0.45&amp;nbsp;&amp;micro;m</td><td>Merck Millipore</td><td>HAWP04700</td><td></td></tr><tr><td>SpeedVac system</td><td>Thermo Scientific</td><td>SPD131DDA</td><td></td></tr><tr><td>Vortex-Genie 2</td><td>Scientific Industries</td><td>Model G560E</td><td></td></tr><tr><td>NanoDrop</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>145 mm cell culture dishes</td><td>Greiner</td><td>639 160</td><td></td></tr><tr><td>1.7 ml tubes, RNase-free</td><td>Axygen</td><td>MCT-175-C</td><td></td></tr><tr><td>30 &amp;deg;C incubator</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>65 &amp;deg;C heat block</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>4 &amp;deg;C table centrifuge</td><td>Eppendorf</td><td>5417R</td><td></td></tr><tr><td>Sterile pipettes, 25 ml</td><td>Greiner</td><td></td><td></td></tr><tr><td>Falcon tubes, 50 ml</td><td>Greiner</td><td></td><td></td></tr><tr><td>Liquid nitrogen</td><td></td><td></td><td></td></tr></tbody></table>

rna purification from intracellularly grown listeria monocytogenes in macrophage cells

Analysis of the transcriptome of bacterial pathogens during mammalian infection is a valuable tool for studying genes and factors that mediate infection. However, isolating bacterial RNA from infected cells or tissues is a challenging task, since mammalian RNA mostly dominates the lysates of infected cells. Here we describe an optimized method for RNA isolation of Listeria monocytogenes bacteria growing within bone marrow derived macrophage cells. Upon infection, cells are mildly lysed and rapidly filtered to discard most of the host proteins and RNA, while retaining intact bacteria. Next, bacterial RNA is isolated using hot phenol-SDS extraction followed by DNase treatment. The extracted RNA is suitable for gene transcription analysis by multiple techniques. This method is successfully employed in our studies of Listeria monocytogenes gene regulation during infection of macrophage cells 1-4. The protocol can be easily modified to study other bacterial pathogens and cell types.

El sistema modelo se muestra en la Figura 1 e incluye células de macrófagos infectados con L. monocytogenes bacterias, que se replican en el citoplasma de los macrófagos. La figura 2 representa el esquema experimental. La figura 3 representa los resultados típicos de tales análisis de RT-qPCR de genes de virulencia durante WT L. monocytogenes en los macrófagos en comparación con el crecimiento en medio rico labor...

Watch this Scientific Journal Video about RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells at JoVE.com

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

Here we describe a method for bacterial RNA isolation from Listeria monocytogenes bacteria growing inside murine macrophages. This technique can be used with other intracellular pathogens and mammalian host cells.

La purificación del ARN intracelularmente Grown<em&gt; Listeria monocytogenes</em&gt; En los macrófagos

Analysis of the transcriptome of bacterial pathogens during mammalian infection is a valuable tool for studying genes and factors that mediate infection. ...

rna-purification-from-intracellularly-grown-listeria-monocytogenes

Research

JoVE Journal

Immunology and Infection

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

9.1K vistas.  Tel Aviv University. Here we describe a method for bacterial RNA isolation from Listeria monocytogenes bacteria growing inside murine macrophages. This technique can be used with other intracellular pathogens and mammalian host cells.

Video: RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

A Simple and Efficient Method to Isolate Macrophages from Mixed Primary Cultures of Adult Liver Cells

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although the isolation methods of liver macrophages have been well-described4-6, most of these methods require sophisticated equipment, such as a centrifugal elutriator and technical skills. Here, we provide a novel method to obtain liver macrophages in sufficient number and purity from mixed primary cultures of adult rat liver cells, as schematically illustrated in Figure 1.
After dissociation of the liver cells by two-step perfusion method7,8,a fraction mostly composed of parenchymal hepatocytes is prepared and seeded into T75 tissue culture flasks with culture medium composed of DMEM and 10% FCS.Parenchymal hepatocytes lose the epithelial cell morphology within a few days in culture, degenerate or transform into fibroblast-like cells (Figure 2). As the culture proceeds, around day 6, phase contrast-bright, round macrophage-like cells start to proliferate on the fibroblastic cell sheet (Figure 2). The growth of the macrophage-like cells continue and reach to maximum levels around day 12, covering the cell sheet on the flask surface. By shaking of the culture flasks, macrophages are readily suspended into the culture medium. Subsequent transfer and short incubation in plastic dishes result in selective adhesion of macrophages(Figure 3), where as other contaminating cells remain suspended. After several rinses with PBS, attached macrophages are harvested. More than 106 cells can be harvested repeatedly from the same T75 tissue culture flask at two to three day intervals for more than two weeks(Figure 3).The purities of the isolated macrophages were 95 to 99%, as evaluated by flow cytometry or immunocytochemistry with rat macrophage-specific antibodies (Figure 4).The isolated cells show active phagocytosis of polystylene beads (Figure 5), proliferative response to recombinant GM-CSF, secretion of inflammatory/anti-inflammatory cytokines upon stimulation with LPS, and formation of multinucleated giant cells9.
In conclusion, we provide a simple and efficient method to obtain liver macrophages in sufficient number and purity without complex equipment and skills.This method might be applicable to other mammalian species.

A novel method to obtain macrophages from primary culture of rat liver cells is described. This method utilizes the proliferation of macrophages in the culture, followed by shaking of culture flasks and purification by selective attachment to plastic dishes. This technique efficiently provides liver macrophages without complex equipment and skills.

Un método simple y eficaz para aislar los macrófagos de la Mezcla de cultivos primarios de células de hígado en adultos

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although ...

Investigación

Inmunología e Infección

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of Listeria, which results in systemic infection2. After injection, Listeria quickly disseminates to the spleen and liver due to uptake by CD8&alpha;+ dendritic cells and Kupffer cells3,4. Once phagocytosed, various bacterial proteins enable Listeria to escape the phagosome, survive within the cytosol, and infect neighboring cells5. During the first three days of infection, different innate immune cells (e.g. monocytes, neutrophils, NK cells, dendritic cells) mediate bactericidal mechanisms that minimize Listeria proliferation. CD8+ T cells are subsequently recruited and responsible for the eventual clearance of Listeria from the host, typically within 10 days of infection6.
Successful clearance of Listeria from infected mice depends on the appropriate onset of host immune responses6	. There is a broad range of sensitivities amongst inbred mouse strains7,8. Generally, mice with increased susceptibility to Listeria infection are less able to control bacterial proliferation, demonstrating increased bacterial load and/or delayed clearance compared to resistant mice. Genetic studies, including linkage analyses and knockout mouse strains, have identified various genes for which sequence variation affects host responses to Listeria infection6,8-14. Determination and comparison of infection kinetics between different mouse strains is therefore an important method for identifying host genetic factors that contribute to immune responses against Listeria. Comparison of host responses to different Listeria strains is also an effective way to identify bacterial virulence factors that may serve as potential targets for antibiotic therapy or vaccine design.
We describe here a straightforward method for measuring bacterial load (colony forming units [CFU] per tissue) and preparing single-cell suspensions of the liver and spleen for FACS analysis of immune responses in Listeria-infected mice. This method is particularly useful for initial characterization of Listeria infection in novel mouse strains, as well as comparison of immune responses between different mouse strains infected with Listeria. We use the Listeria monocytogenes EGD strain15 that, when cultured on blood agar, exhibits a characteristic halo zone around each colony due to &beta;-hemolysis1 (Figure 1). Bacterial load and immune responses can be determined at any time-point after infection by culturing tissue homogenate on blood agar plates and preparing tissue cell suspensions for FACS analysis using the protocols described below. We would note that individuals who are immunocompromised or pregnant should not handle Listeria, and the relevant institutional biosafety committee and animal facility management should be consulted before work commences.

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes is a model organism for studying immune responses and genetic susceptibility to intracellular bacteria in mice. This method enables one to measure bacterial load and generate single-cell suspensions of the liver and spleen from mice for FACS analysis to determine changes in immune cells due to Listeria infection.

La medición de la carga bacteriana y la respuesta inmune en ratones infectados con Listeria monocytogenes</em

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of ...

Purification of Pathogen Vacuoles from Legionella-infected Phagocytes

The opportunistic pathogen Legionella pneumophila is an amoeba-resistant bacterium, which also replicates in alveolar macrophages thus causing the severe pneumonia "Legionnaires' disease"1. In protozoan and mammalian phagocytes, L. pneumophila employs a conserved mechanism to form a specific, replication-permissive compartment, the "Legionella-containing vacuole" (LCV). LCV formation requires the bacterial Icm/Dot type IV secretion system (T4SS), which translocates as many as 275 "effector" proteins into host cells. The effectors manipulate host proteins as well as lipids and communicate with secretory, endosomal and mitochondrial organelles2-4.
The formation of LCVs represents a complex, robust and redundant process, which is difficult to grasp in a reductionist manner. An integrative approach is required to comprehensively understand LCV formation, including a global analysis of pathogen-host factor interactions and their temporal and spatial dynamics. As a first step towards this goal, intact LCVs are purified and analyzed by proteomics and lipidomics.
The composition and formation of pathogen-containing vacuoles has been investigated by proteomic analysis using liquid chromatography or 2-D gel electrophoresis coupled to mass-spectrometry. Vacuoles isolated from either the social soil amoeba Dictyostelium discoideum or mammalian phagocytes harboured Leishmania5, Listeria6, Mycobacterium7, Rhodococcus8, Salmonella9 or Legionella spp.10. However, the purification protocols employed in these studies are time-consuming and tedious, as they require e.g. electron microscopy to analyse LCV morphology, integrity and purity. Additionally, these protocols do not exploit specific features of the pathogen vacuole for enrichment.
The method presented here overcomes these limitations by employing D. discoideum producing a fluorescent LCV marker and by targeting the bacterial effector protein SidC, which selectively anchors to the LCV membrane by binding to phosphatidylinositol 4-phosphate (PtdIns(4)P)3,11 . LCVs are enriched in a first step by immuno-magnetic separation using an affinity-purified primary antibody against SidC and a secondary antibody coupled to magnetic beads, followed in a second step by a classical Histodenz density gradient centrifugation12,13 (Fig. 1).
A proteome study of isolated LCVs from D. discoideum revealed more than 560 host cell proteins, including proteins associated with phagocytic vesicles, mitochondria, ER and Golgi, as well as several GTPases, which have not been implicated in LCV formation before13. LCVs enriched and purified with the protocol outlined here can be further analyzed by microscopy (immunofluorescence, electron microscopy), biochemical methods (Western blot) and proteomic or lipidomic approaches.

Purification of Pathogen Vacuoles from Legionella-infected Phagocytes

This article describes a method for the isolation and purification of intact Legionella-containing vacuoles (LCVs) from amoeba and macrophages. The two-step protocol comprises LCV enrichment by immuno-magnetic separation using an antibody against a bacterial LCV marker and further purification by density gradient centrifugation.

La purificación del patógeno a partir de vacuolas<em&gt; Legionella</em&gt; Infectados por fagocitos

The opportunistic pathogen Legionella pneumophila is an amoeba-resistant bacterium, which also replicates in alveolar macrophages thus causing the severe ...

Oral Transmission of Listeria monocytogenes in Mice via Ingestion of Contaminated Food

L. monocytogenes are facultative intracellular bacterial pathogens that cause food borne infections in humans. Very little is known about the gastrointestinal phase of listeriosis due to the lack of a small animal model that closely mimics human disease. This paper describes a novel mouse model for oral transmission of L. monocytogenes. Using this model, mice fed L. monocytogenes-contaminated bread have a discrete phase of gastrointestinal infection, followed by varying degrees of systemic spread in susceptible (BALB/c/By/J) or resistant (C57BL/6) mouse strains. During the later stages of the infection, dissemination to the gall bladder and brain is observed. The food borne model of listeriosis is highly reproducible, does not require specialized skills, and can be used with a wide variety of bacterial isolates and laboratory mouse strains. As such, it is the ideal model to study both virulence strategies used by L. monocytogenes to promote intestinal colonization, as well as the host response to invasive food borne bacterial infection.

Oral Transmission of Listeria monocytogenes in Mice via Ingestion of Contaminated Food

This paper describes a novel method for oral infection of mice using Listeria monocytogenes-contaminated food. The protocol can readily be adapted for use with other food borne bacterial pathogens.

La transmisión oral de<em&gt; Listeria monocytogenes</em&gt; En ratones a través de la ingestión de alimentos contaminados

L. monocytogenes are facultative intracellular bacterial pathogens that cause food borne infections in humans. Very little is known about the ...

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

Bacterial intracellular pathogens can be conceived as molecular tools to dissect cellular signaling cascades due to their capacity to exquisitely manipulate and subvert cell functions which are required for the infection of host target tissues. Among these bacterial pathogens, Listeria monocytogenes is a Gram positive microorganism that has been used as a paradigm for intracellular parasitism in the characterization of cellular immune responses, and which has played instrumental roles in the discovery of molecular pathways controlling cytoskeletal and membrane trafficking dynamics. In this article, we describe a robust microscopical assay for the detection of late cellular infection stages of L. monocytogenes based on the fluorescent labeling of InlC, a secreted bacterial protein which accumulates in the cytoplasm of infected cells; this assay can be coupled to automated high-throughput small interfering RNA screens in order to characterize cellular signaling pathways involved in the up- or down-regulation of infection.

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

Listeria monocytogenes is a Gram positive bacterial pathogen frequently used as a major model for the study of intracellular parasitism. Imaging late L. monocytogenes infection stages within the context of small-interfering RNA screens allows for the global study of cellular pathways required for bacterial infection of target host cells.

Imaging InlC secreción de investigar la infección celular por el patógeno bacteriano<em&gt; Listeria monocytogenes</em

Bacterial intracellular  pathogens can be conceived as molecular tools to dissect cellular signaling  cascades due to their capacity to exquisitely ...

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

Macrophages are key phagocytic innate immune cells. When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the pathogen, produce various cytokines and chemokines to recruit and stimulate other immune cells, and present antigens to stimulate the adaptive immune response. Thus, being able to efficiently manipulate macrophages with techniques such as RNA-interference (RNAi) is critical to our ability to investigate this important innate immune cell. However, macrophages can be technically challenging to transfect and can exhibit inefficient RNAi-induced gene knockdown. In this protocol, we describe methods to efficiently transfect two mouse macrophage cell lines (RAW264.7 and J774A.1) with siRNA using the Amaxa Nucleofector 96-well Shuttle System and describe procedures to maximize the effect of siRNA on gene knockdown. Moreover, the described methods are adapted to work in 96-well format, allowing for medium and high-throughput studies. To demonstrate the utility of this approach, we describe experiments that utilize RNAi to inhibit genes that regulate lipopolysaccharide (LPS)-induced cytokine production.

In this protocol, we describe methods to efficiently transfect murine macrophage cell lines with siRNAs using the Amaxa Nucleofector 96-well Shuttle System, stimulate the macrophages with lipopolysaccharide, and monitor the effects on inflammatory cytokine production.

El uso de ARN de interferencia para Investigar la respuesta inmune innata en macrófagos de ratón

Macrophages are key phagocytic innate immune cells.  When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the ...

Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages

Bacteria are able to secrete a variety of molecules via various secretory systems. Besides the secretion of molecules into the extracellular space or directly into another cell, Gram-negative bacteria can also form outer membrane vesicles (OMVs). These membrane vesicles can deliver their cargo over long distances, and the cargo is protected from degradation by proteases and nucleases. 
Legionella pneumophila (L. pneumophila) is an intracellular, Gram-negative pathogen that causes a severe form of pneumonia. In humans, it infects alveolar macrophages, where it blocks lysosomal degradation and forms a specialized replication vacuole. Moreover, L. pneumophila produces OMVs under various growth conditions. To understand the role of OMVs in the infection process of human macrophages, we set up a protocol to purify bacterial membrane vesicles from liquid culture. The method is based on differential ultracentrifugation. The enriched OMVs were subsequently analyzed with regard to their protein and lipopolysaccharide (LPS) amount and were then used for the treatment of a human monocytic cell line or murine bone marrow-derived macrophages. The pro-inflammatory responses of those cells were analyzed by enzyme-linked immunosorbent assay. Furthermore, alterations in a subsequent infection were analyzed. To this end, the bacterial replication of L. pneumophila in macrophages was studied by colony-forming unit assays. 
Here, we describe a detailed protocol for the purification of L. pneumophila OMVs from liquid culture by ultracentrifugation and for the downstream analysis of their pro-inflammatory potential on macrophages.

Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages

Here, we describe the purification of Legionella pneumophila (L. pneumophila) outer membrane vesicles (OMVs) from liquid cultures. These purified vesicles are then used for the treatment of macrophages to analyze their pro-inflammatory potential.

Legionella pneumophila exterior vesículas de membrana: Aislamiento y análisis de su potencial pro-inflamatoria en los macrófagos

Bacteria are able to secrete a variety of molecules via various secretory systems. Besides the secretion of molecules into the extracellular space or ...

Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' pathogens (Listeria monocytogenes, Shigella flexneri, Burkholderia pseudomallei, Francisella tularensis, and Rickettsia spp.) gain access to the infected cell cytosol by physically and enzymatically degrading the primary vacuolar membrane. Once in the cytosol, these pathogens both proliferate as well as generate sufficient mechanical forces to penetrate the plasma membrane of the host cell in order to infect new cells. Here, we show how this terminal step of the cellular infection cycle of L. monocytogenes (Lm) can be quantified by both colony-forming unit assays and flow cytometry and give examples of how both pathogen- and host-encoded factors impact this process. We also show a close correspondence of Lm infection dynamics of cultured cells infected in vitro and those of hepatic cells derived from mice infected in vivo. These function-based assays are relatively simple and can be readily scaled up for discovery-based high-throughput screens for modulators of eukaryotic cell function.

We describe both in vitro and in vivo infection assays that can be used to analyze the activities of host-encoding factors.

Usando un patógeno bacteriano a punta de prueba para las respuestas celular y organísmica nivel Host

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' ...

Label-Free Quantitative Proteomics Workflow for Discovery-Driven Host-Pathogen Interactions

The technological achievements of mass spectrometry (MS)-based quantitative proteomics opens many undiscovered avenues for analyzing an organism&#8217;s global proteome under varying conditions. This powerful strategy applied to the interactions of microbial pathogens with the desired host comprehensively characterizes both perspectives towards infection. Herein, the workflow describes label-free quantification (LFQ) of the infectome of Cryptococcus neoformans, a fungal facultative intracellular pathogen that is the causative agent of the deadly disease cryptococcosis, in the presence of immortalized macrophage cells. The protocol details the proper protein preparation techniques for both pathogen and mammalian cells within a single experiment, resulting in appropriate peptide submission for liquid-chromatography (LC)-MS/MS analysis. The high throughput generic nature of LFQ allows a wide dynamic range of protein identification and quantification, as well as transferability to any host-pathogen infection setting, maintaining extreme sensitivity. The method is optimized to catalogue extensive, unbiased protein abundance profiles of a pathogen within infection-mimicking conditions. Specifically, the method demonstrated here provides essential information on C. neoformans pathogenesis, such as protein production necessary for virulence and identifies critical host proteins responding to microbial invasion.

Here, we present a protocol to profile the interplay between host and pathogen during infection by mass spectrometry-based proteomics. This protocol uses label-free quantification to measure changes in protein abundance of both host (e.g., macrophages) and pathogen (e.g., Cryptococcus neoformans) in a single experiment.

Flujo de trabajo de proteómica cuantitativa sin etiquetas para interacciones host-patógenos impulsadas por el descubrimiento

The technological achievements of mass spectrometry (MS)-based quantitative proteomics opens many undiscovered avenues for analyzing an organism&#8217;s ...

Flow Cytometry Analysis of Intracellular and Emergent Extracellular Listeria monocytogenes In Vitro

Source: Gayle, P. et al., <a href="https://app.jove.com/v/58775">Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses.</a> J. Vis. Exp. (2019)
In this video, we quantify intracellular and extracellular Listeria monocytogenes, an intracellular pathogenic bacteria, following their infection of cultured macrophages in vitro using flow cytometry.

Análisis por citometría de flujo de Listeria monocytogenes intracelular y extracelular emergente in vitro

Source: Gayle, P. et al., Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses. J. Vis. Exp. (2019)
In this video, we ...

La purificación del ARN intracelularmente Grown

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