La investigación en nuestro laboratorio fue apoyada por el Instituto Max von Pettenkofer, Ludwig-Maximilians de Munich, la Universidad, la Fundación Alemana de Investigación (DFG, HI 1511/3-1) y el Bundesministerium für Bildung und Forschung (BMBF) &quot;Genómica Médica de infección&quot; iniciativa ( 0315834C).

1. Los preparativos para el Aislamiento de LCV <ol><li> Streak a Legionella pneumophila produce DsRed-Express 13,14 de las existencias de glicerol en placas de agar CYE con 5 mg / ml de cloranfenicol (Cam) cuatro días antes del aislamiento LCV. Incubar las bacterias a 37 ° C. </li><li> Semillas de 1 x 10 7 D. discoideum la producción de GFP-calnexina o RAW264.7 macrófagos murinos en cultivo 75 cm 2 frascos de un tejido de la infección antes de los días. Utilizar 10 ml HL5 medio con 20 mg / ml de G418 para D. discoideum e incubar las amebas a 23 ° C. Para RAW264.7 macrófagos utilizar 10 ml de medio RPMI 1640 suplementado con 10% de FCS (inactivado por calor) y 1% de glutamina, y cultivar las células a 37 ° C y 5% de CO 2. Use al menos tres 75 cm 2 frascos por la infección y de la muestra (un mínimo de 6 x 10 7 células). </li><li> Inocular un cultivo de una noche con L. pneumophila de la placa de CYE. Tomar un tubo de ensayo de 15 ml con 3 ml de medio de la EJAy 5 ug / ml de leva. Se inoculan con 100 l de una suspensión bacteriana para producir una 600nm OD de 0,1. Incubar el cultivo durante la noche en una rotación de la rueda de arriba a 37 ° C durante 21-22 horas. </li></ol> 2. LCV aislamiento <ol><li> Cambiar el medio de la D. células discoideum para eliminar el antibiótico, que podría interferir con la infección siguiente. </li><li> Mida el diámetro exterior de la cultura durante la noche. Las bacterias se han alcanzado su infectividad pico a 600 nm un diámetro exterior de ≥ 3, que corresponde a 2 x 10 9 bacterias por ml. </li><li> Infectar las células mediante la adición de aproximadamente 500 l de la L. pneumophila noche a la mañana la cultura a las células que crecen en 10 ml HL5 medio (D. discoideum) o 10 ml de RPMI 1640 (macrófagos). Esto corresponde a una multiplicidad de infección (MOI) de 50. Posteriormente, la infección está sincronizado por centrifugación de las bacterias sobre las células a 500 xg durante 10 min. La centrifugación se siguiópor una incubación de la D. células discoideum a 25 ° C y los macrófagos RAW264.7 a 37 ° C y 5% de CO 2, respectivamente. El tiempo de infección de 1 hora incluye la etapa de centrifugación. </li><li> Después de la infección se elimina el medio y lavar las células una vez para eliminar las bacterias extracelulares. Use helado de amortiguación sorc para D. discoideum y helado de PBS para RAW264.7 macrófagos. Añadir 3 ml HS tampón, suplementado con inhibidores de la proteasa (Roche) a cada matraz y recoger las células mediante el uso de un raspador de células. Piscina de las muestras correspondientes en un tubo de ensayo de 15 ml. </li><li> Para la homogeneización de las muestras de uso de plástico de 3 ml jeringas Luer-Lock y la bola de acero inoxidable homogeneizador. Asegúrese de que usted trabaja en el hielo. Antes de comenzar, lave el homogenizador de la pelota con agua destilada, para evitar cualquier contaminación de detergente y enjuague con tampón enfriado con hielo SA para deshacerse de las burbujas de aire. Utilice la bola 8 micras de liquidación. </li><li> Rellena los primeros 3 ml en la jeringa und montarlo en ªe homogeneizador. Pulse la muestra de ida y vuelta nueve veces a través del homogeneizador. Intercambiar las jeringas después para evitar la contaminación con material no-homogéneo. Recoger y la piscina de la muestra homogeneizada en un tubo de ensayo de 15 mL y tomar una muestra de 150 l para el análisis microscópico. Antes de proceder con una muestra diferente desmontar y lavar el homogeneizador de pelota. </li><li> Bloque el homogeneizado con 2% de CS o FCS durante 30 minutos en un agitador en hielo o en una rueda de sobrecarga de hilatura (10-20 rpm) a 4 ° C. </li><li> Tras el bloqueo utiliza un anticuerpo purificado por afinidad primaria contra SIDC (dilución 1:3000) o contra cualquier otro marcador bacteriana exclusivamente la unión a la membrana de vehículos comerciales ligeros. Vortex el anticuerpo solución antes de añadirlo al homogeneizado. Incubar la muestra durante 1 hora en un agitador en hielo o en una rueda de sobrecarga de hilatura (10-20 rpm) a 4 ° C. </li><li> Mientras tanto, preparar el gradiente Histodenz. Utilizar un tubo de ensayo 15 ml por gradiente y tres 75 cm 2 frascos de una muestra. Abetoc, añadir 5,75 ml de 35% Histodenz al tubo, y segundo, añadir cuidadosamente 5,75 ml de solución al 10% Histodenz en la parte superior. Luego coloque cuidadosamente los tubos en posición horizontal durante 1 hora. </li><li> Después de la incubación del homogeneizado con el reactivo de bloqueo y el anticuerpo primario, se centrifuga a 600 xg durante 15 min a 4 ° C para sedimentar la muestra. Eliminar el sobrenadante y resuspender el precipitado en 1,5 ml de tampón HS. Transferir las muestras a un nuevo máximo de 15 ml tubo de ensayo y tomar una muestra de 40 l para el análisis microscópico más adelante. </li><li> Incubar la bolita con el anticuerpo secundario - MACS de cabra anti-conejo IgG microesferas (dilución 1:25) - durante 30 minutos en un agitador a 4 ° C. Mientras tanto, poner las columnas en el soporte magnético y MACS equilibre con 0,5 ml de tampón HS. </li><li> Posteriormente, se aplican las muestras a la columna. Recoger 150 l de el flujo a través de análisis microscópico posterior. Lavar la columna tres veces con 0,5 ml de tampón HS. </li><li> Eliminar las columnas de tél imán y eluido los vehículos comerciales con 0,5 ml de tampón HS inmediata de chorros. Tomar una muestra de 20 l para el análisis microscópico. La etapa de purificación por inmuno-separación magnética se espera que el rendimiento por 1 x 10 7 infectado D. discoideum células de aproximadamente 1 × 10 6 VCL intactos, que se enriquecen unas siete veces en el eluido en comparación con el flujo a través de 13. </li><li> Colocar los tubos de gradiente Histodenz de nuevo a una posición vertical y con mucho cuidado la carga que el efluente en la parte superior. Centrifugar los tubos a 3350 × g durante 1 hora a 4 ° C. </li><li> Después de la centrifugación tomar 1,5 ml fracciones a partir de la parte inferior del tubo de ensayo mediante el uso de una pipeta Pasteur de vidrio larga. En conjunto recoger ocho fracciones, cada una desde la parte inferior del tubo. No hay capas visibles se observan en la continua pendiente Histodenz. El mayor rendimiento de LCV típicamente se espera en la fracción 4 en una proporción de 2 x 10 ~ 5 LCV intactos por 1 x 10 7 eninfectado amebas 13. Pequeñas cantidades de LCV también están presentes en las fracciones 3 y 5, respectivamente. Así, el rendimiento global de LCV purificadas es de ~ 1 x 10 6 LCV intactas por 6 x 10 7 infectado D. células discoideum 13. La recuperación de vehículos comerciales se puede realizar a temperatura ambiente (TA), y los vehículos comerciales parecen estables durante varias horas. </li></ol> 3. El análisis microscópico de las muestras recogidas <ol><li> Preparar un 24-así de fondo plano placa de cultivo de tejido con poli-L-lisina cubreobjetos revestidos. </li><li> Pipetear todas las muestras tomadas durante la purificación LCV más 150 l de cada fracción de gradiente de densidad en la placa de 24 pocillos. Añadir 0,5 ml de tampón HS a cada pocillo para diluir la alta concentración Histodenz. Después, centrifugar la placa a 600 xg durante 10 min a 4 ° C. Retire con cuidado el sobrenadante y fijar las muestras con 4% paraformaldehído (PFA) durante 20 min a temperatura ambiente. Después de la fijación lavar las muestras dos veces. Nosotrose sorc para D. discoideum y PBS para RAW264.7 macrófagos. </li><li><ol class="lalpha"><li> Montar las muestras de las buenas prácticas agrarias que expresan calnexina D. discoideum, tomadas en diferentes etapas durante el aislamiento vehículos comerciales ligeros, directamente en las diapositivas de cristal con Vectashield por ejemplo. </li><li> Se incuban las muestras de RAW264.7 macrófagos, tomadas en diferentes etapas durante el aislamiento LCV, con solución de bloqueo (1% de BSA en PBS) durante 10 min a temperatura ambiente. Utilice un anticuerpo primario anti-SIDC para teñir los vehículos comerciales e incubar durante 1 hora en cámara húmeda a temperatura ambiente (purificado por afinidad de conejo anti-SIDC; 1:1000 en tampón de bloqueo). Posteriormente, lavar las muestras tres veces con PBS. Se incuba con un anticuerpo secundario (por ejemplo, anti-conejo Cy5; 1:200 en solución de bloqueo) durante 30 minutos en una cámara húmeda y oscuridad a temperatura ambiente. Por último, lavar las muestras tres veces con PBS y montarlos en las diapositivas de cristal con Vectashield por ejemplo. </li></ol></li><li> Morfología, la integridad y la cantidad de los vehículos comerciales aisladas a partir de las buenas prácticas agrarias-que expresa calnexina D. descoideum o de RAW264.7 macrófagos son analizados por un microscopio de fluorescencia equipado con los filtros adecuados. </li></ol> 4. Los resultados representativos La calidad y el rendimiento de la purificación LCV por inmuno-afinidad separación y centrifugación en gradiente de densidad puede ser seguido por microscopía de fluorescencia (Fig. 1). Una visión general de las muestras recogidas durante el aislamiento LCV de D. discoideu m o macrófagos se muestra (fig. 2). El homogeneizado de L. pneumophila infectados por los fagocitos muestra VCL intactas, sino también una gran cantidad de restos celulares y bacterias extracelulares. Después de la granulación de la muestra, la imagen es similar al homogeneizado, a veces un poco más denso. Desde LCV intactas debe pegarse a la columna, el flujo a través contiene principalmente bacterias extracelulares y residuos celulares. Después de eluir la muestra de la columna, el eluido contiene LCV intactos en grandes cantidades. En nuestras manos, el Purificati LCV<html> en que parece ser más eficaz para D. discoideum que para los macrófagos. En D. discoideum las vacuolas patógenos aparecen más redondo y el rendimiento de LCV aisladas es más de 10 veces mayor en comparación con los macrófagos (Fig. 3). En los macrófagos intactos bajo consumo teñidas con diferentes anticuerpos también parecen tener más de forma irregular. <img alt="Figura 1" src="/files/ftp_upload/4118/4118fig1.jpg" /> Figura 1. Esquema general de aislamiento de vehículos comerciales ligeros. A) Aislamiento de LCV por inmuno-separación magnética usando un anticuerpo purificado por afinidad contra la proteína efectora SIDC bacteriana, localizando exclusivamente a la membrana LCV. Un MACS secundaria micro talón acoplado anticuerpo y un imán se utilizan para aislar LCV de los restos celulares. B) Después de separación LCV inmuno-magnéticas se purifica adicionalmente por centrifugación Histodenz gradiente de densidad. VCL se enriquecen en la fracción 4. upload/4118/4118fig2.jpg &quot;/&gt; Las muestras Figura 2. Recogidos durante la purificación LCV. Imágenes de homogeneizado, pellet, el filtrado y eluido de D. discoideum o macrófagos se representan. Las muestras presentan L. pneumophila produce DsRed-Express (rojo) en el D. discoideum la producción de GFP-calnexina señal (panel verde, parte superior) o en los macrófagos teñidos con un anticuerpo anti-SIDC (cian, panel inferior). Bares, 10 m. <img alt="Figura 3" src="/files/ftp_upload/4118/4118fig3.jpg" /> Figura 3. Purificada vehículos comerciales de D. discoideum o macrófagos. Las muestras presentan la fracción 4 después de la centrifugación Histodenz gradiente de densidad de L. pneumophila produce DsRed-Express (rojo) en (A) D. discoideum la producción de GFP-calnexina señal (verde) o (B) en los macrófagos teñidos con un anticuerpo anti-SIDC (cian). Bares, 10 m.

<ol>
	<li>Hilbi, H., Hoffmann, C., Harrison, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Legionella+spp.+outdoors:+colonization,+communication+and+persistence.">Legionella spp. outdoors: colonization, communication and persistence.</a> Environ. Microbiol. Rep. 3, 286-296 (2011).</li><li>Zhu, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+identification+of+protein+substrates+of+the+Dot/Icm+type+IV+transporter+of+Legionella+pneumophila.">Comprehensive identification of protein substrates of the Dot/Icm type IV transporter of Legionella pneumophila.</a> PLoS ONE. 6, e17638 (2011).</li><li>Hilbi, H., Weber, S., Finsel, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anchors+for+effectors:+subversion+of+phospho-inositide+lipids+by+Legionella.">Anchors for effectors: subversion of phospho-inositide lipids by Legionella.</a> Front Microbiol. 2, 91 (2011).</li><li>Hubber, A., Roy, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+host+cell+function+by+Legionella+pneumophila+type+IV+effectors.">Modulation of host cell function by Legionella pneumophila type IV effectors.</a> Annu. Rev. Cell Dev. Biol. 26, 261-283 (2010).</li><li>Kima, P. E., Dunn, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploiting+calnexin+expression+on+phagosomes+to+isolate+Leishmania+parasitophorous+vacuoles.">Exploiting calnexin expression on phagosomes to isolate Leishmania parasitophorous vacuoles.</a> Microb. Pathog. 38, 139-145 (2005).</li><li>L&#252;hrmann, A., Haas, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+to+purify+bacteria-containing+phagosomes+from+infected+macrophages.">A method to purify bacteria-containing phagosomes from infected macrophages.</a> Methods Cell Sci. 22, 329-341 (2000).</li><li>Sturgill-Koszycki, S., Haddix, P. L., Russell, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+interaction+between+Mycobacterium+and+the+macrophage+analyzed+by+two-dimensional+polyacrylamide+gel+electrophoresis.">The interaction between Mycobacterium and the macrophage analyzed by two-dimensional polyacrylamide gel electrophoresis.</a> Electrophoresis. 18, 2558-2565 (1997).</li><li>Fernandez-Mora, E., Polidori, M., L&#252;hrmann, A., Schaible, U. E., Haas, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maturation+of+Rhodococcus+equi-containing+vacuoles+is+arrested+after+completion+of+the+early+endosome+stage.">Maturation of Rhodococcus equi-containing vacuoles is arrested after completion of the early endosome stage.</a> Traffic. 6, 635-653 (2005).</li><li>Mills, S. D., Finlay, B. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+Salmonella+typhimurium+and+Yersinia+pseudotuberculosis-containing+phagosomes+from+infected+mouse+macrophages:+Y.+pseudotuberculosis+traffics+to+terminal+lysosomes+where+they+are+degraded.">Isolation and characterization of Salmonella typhimurium and Yersinia pseudotuberculosis-containing phagosomes from infected mouse macrophages: Y. pseudotuberculosis traffics to terminal lysosomes where they are degraded.</a> Eur. J. Cell Biol. 77, 35-47 (1998).</li><li>Shevchuk, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+analysis+of+Legionella-containing+phagosomes+isolated+from+Dictyostelium.">Proteomic analysis of Legionella-containing phagosomes isolated from Dictyostelium.</a> Int. J. Med. Microbiol. 299, 489-508 (2009).</li><li>Brombacher, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rab1+guanine+nucleotide+exchange+factor+SidM+is+a+major+phosphatidylinositol+4-phosphate-binding+effector+protein+of+Legionella+pneumophila.">Rab1 guanine nucleotide exchange factor SidM is a major phosphatidylinositol 4-phosphate-binding effector protein of Legionella pneumophila.</a> J. Biol. Chem. 284, 4846-4856 (2009).</li><li>Urwyler, S., Finsel, I., Ragaz, C., Hilbi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+Legionella-containing+vacuoles+by+immuno-magnetic+separation.">Isolation of Legionella-containing vacuoles by immuno-magnetic separation.</a> Curr. Protoc. Cell Biol. Chapter 3, Unit 3 (2010).</li><li>Urwyler, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteome+analysis+of+Legionella+vacuoles+purified+by+magnetic+immunoseparation+reveals+secretory+and+endosomal+GTPases.">Proteome analysis of Legionella vacuoles purified by magnetic immunoseparation reveals secretory and endosomal GTPases.</a> Traffic. 10, 76-87 (2009).</li><li>Mampel, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Planktonic+replication+is+essential+for+biofilm+formation+by+Legionella+pneumophila+in+a+complex+medium+under+static+and+dynamic+flow+conditions.">Planktonic replication is essential for biofilm formation by Legionella pneumophila in a complex medium under static and dynamic flow conditions.</a> Appl. Environ. Microbiol. 72, 2885-2895 (2006).</li></ol>

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En contraste con los métodos publicados anteriormente, este protocolo se basa en dos pasos, primero la separación de LCV por un enfoque inmuno-magnética, y en segundo lugar, la purificación de LCV por centrifugación en gradiente de densidad. El aislamiento LCV se pueden seguir fácilmente por microscopía de fluorescencia, cuando sea bacterias marcadas fluorescentemente y las células productoras de GFP o tinción de anticuerpos se utiliza. El protocolo se describe aquí es un sencillo, recto hacia adelante método...

purification of pathogen vacuoles from legionella-infected phagocytes

El patógeno oportunista Legionella pneumophila es una bacteria resistente a la ameba, que también se replica en los macrófagos alveolares por lo tanto causantes de la neumonía grave &quot;Enfermedad de los Legionarios&quot; 1. En los fagocitos protozoos y mamíferos, L. pneumophila emplea un mecanismo conservado para formar un procedimiento específico, la replicación permisiva del compartimiento, la &quot;Legionella contiene vacuola&quot; (LCV). La formación de vehículos comerciales requiere la bacteria icm / Dot sistema de secreción tipo IV (T4SS), que se transloca hasta 275 &quot;efector&quot; proteínas en las células huésped. Los efectores manipulan proteínas del huésped, así como los lípidos y comunicarse con secreción, organelos endosomal mitocondrial y 2-4. La formación de los LCV representa un proceso complejo, robusto y redundante, lo que es difícil de entender de una manera reduccionista. Un enfoque integral es necesaria para comprender globalmente la formación de vehículos comerciales ligeros, incluyendo un análisis global de la trayectoriaacogida ogen las interacciones de los factores y su dinámica temporal y espacial. Como un primer paso hacia esta meta, vehículos comerciales intactas son purificados y analizados por la proteómica y la lipidómica. La composición y la formación de agentes patógenos que contienen vacuolas ha sido investigado por el análisis proteómico mediante cromatografía líquida o 2-D electroforesis en gel acoplado a espectrometría de masas. Vacuolas aisladas, ya sea del suelo de la sociedad ameba Dictyostelium discoideum, o los fagocitos de mamíferos albergaba Leishmania 5, Listeria 6, 7 Mycobacterium, Rhodococcus 8, 9 o Salmonella spp Legionella. 10. Sin embargo, los protocolos de purificación empleadas en estos estudios son mucho tiempo y tedioso, ya que requieren microscopía electrónica por ejemplo, para analizar la morfología LCV, la integridad y la pureza. Además, estos protocolos no explotar las características específicas de la vacuola es patógeno pararichment. El método que aquí se presenta supera estas limitaciones mediante el empleo de D. discoideum produciendo un marcador fluorescente de vehículos comerciales ligeros y por la orientación de la SIDC bacteriana proteína efectora, que selectivamente ancla a la membrana de vehículos comerciales ligeros de la unión a fosfatidilinositol 4-fosfato (PtdIns (4) P) 3,11. LCV están enriquecidos en un primer paso por inmuno-separación magnética usando un anticuerpo purificado por afinidad primaria contra SIDC y un anticuerpo secundario acoplado a perlas magnéticas, seguido en un segundo paso por una centrifugación en gradiente de densidad clásica Histodenz 12,13 (fig. 1) . Un estudio del proteoma de vehículos comerciales aislados de D. discoideum reveló más de 560 proteínas de la célula huésped, incluidas las proteínas asociadas a vesículas fagocíticas, ER mitocondrias y aparato de Golgi, así como las GTPasas varios que no han sido implicados en la formación antes del 13 de LCV. LCV enriquecido y purificado con elprotocolo descrito aquí pueden ser analizadas por microscopía (inmunofluorescencia, microscopía electrónica), los métodos bioquímicos (Western blot) y los enfoques proteómicos o lipidomic.

Watch this Scientific Journal Video about Purification of Pathogen Vacuoles from Legionella-infected Phagocytes at JoVE.com

Purification of Pathogen Vacuoles from Legionella-infected Phagocytes

Este artículo describe un método para el aislamiento y purificación de intacta<em&gt; Legionella</em&gt; Que contienen vacuolas (LCV) de amebas y los macrófagos. El protocolo de dos pasos comprende enriquecimiento LCV por inmuno-separación magnética usando un anticuerpo contra un marcador bacteriano LCV y purificación adicional por centrifugación en gradiente de densidad.

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 ...

purification-of-pathogen-vacuoles-from-legionella-infected-phagocytes

Research

JoVE Journal

Immunology and Infection

Purification of Pathogen Vacuoles from Legionella-infected Phagocytes

12.4K vistas.  Ludwig-Maximilians-Universität. Este artículo describe un método para el aislamiento y purificación de intacta Legionella Que contienen vacuolas (LCV) de amebas y los macrófagos. El protocolo de dos pasos comprende enriquecimiento LCV por inmuno-separación magnética usando un anticuerpo contra un marcador bacteriano LCV y purificación adicional por centrifugación en gradiente de densidad.

Video: Purification of Pathogen Vacuoles from Legionella-infected Phagocytes

Invasion of Human Cells by a Bacterial Pathogen

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

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

La invasión de las células humanas por un patógeno bacteriano

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

Investigación

Inmunología e Infección

One-day Workflow Scheme for Bacterial Pathogen Detection and Antimicrobial Resistance Testing from Blood Cultures

Bloodstream infections are associated with high mortality rates because of the probable manifestation of sepsis, severe sepsis and septic shock1. Therefore, rapid administration of adequate antibiotic therapy is of foremost importance in the treatment of bloodstream infections. The critical element in this process is timing, heavily dependent on the results of bacterial identification and antibiotic susceptibility testing. Both of these parameters are routinely obtained by culture-based testing, which is time-consuming and takes on average 24-48 hours2, 4. The aim of the study was to develop DNA-based assays for rapid identification of bloodstream infections, as well as rapid antimicrobial susceptibility testing. The first assay is a eubacterial 16S rDNA-based real-time PCR assay complemented with species- or genus-specific probes5. Using these probes, Gram-negative bacteria including Pseudomonas spp., Pseudomonas aeruginosa and Escherichia coli as well as Gram-positive bacteria including Staphylococcus spp., Staphylococcus aureus, Enterococcus spp., Streptococcus spp., and Streptococcus pneumoniae could be distinguished. Using this multiprobe assay, a first identification of the causative micro-organism was given after 2 h.
Secondly, we developed a semi-molecular assay for antibiotic susceptibility testing of S. aureus, Enterococcus spp. and (facultative) aerobe Gram-negative rods6. This assay was based on a study in which PCR was used to measure the growth of bacteria7. Bacteria harvested directly from blood cultures are incubated for 6 h with a selection of antibiotics, and following a Sybr Green-based real-time PCR assay determines inhibition of growth. The combination of these two methods could direct the choice of a suitable antibiotic therapy on the same day (Figure 1). In conclusion, molecular analysis of both identification and antibiotic susceptibility offers a faster alternative for pathogen detection and could improve the diagnosis of bloodstream infections.

The design of a straightforward one-day workflow scheme for bacterial pathogen diagnostics enables the rapid recognition of bloodstream infections. The inclusion of eight clinically relevant bacterial targets and their antibiotic resistance profiles offers the clinician an initial insight on the same day, which can lead to more adequate therapy.

Un día de flujo de trabajo Método de detección de patógenos bacterianos y pruebas de resistencia a los antimicrobianos de cultivos de sangre

Bloodstream infections are associated with high mortality rates because of the probable manifestation of sepsis, severe sepsis and septic shock1. ...

Purification and Visualization of Lipopolysaccharide from Gram-negative Bacteria by Hot Aqueous-phenol Extraction

Lipopolysaccharide (LPS) is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of lipid A, which is embedded in the outer membrane, a core oligosaccharide and repeating O-antigen units that extend outward from the surface of the cell1, 2. LPS is an immunodominant molecule that is important for the virulence and pathogenesis of many bacterial species, including Pseudomonas aeruginosa, Salmonella species, and Escherichia coli3-5, and differences in LPS O-antigen composition form the basis for serotyping of strains. LPS is involved in attachment to host cells at the initiation of infection and provides protection from complement-mediated killing; strains that lack LPS can be attenuated for virulence6-8. For these reasons, it is important to visualize LPS, particularly from clinical isolates. Visualizing LPS banding patterns and recognition by specific antibodies can be useful tools to identify strain lineages and to characterize various mutants. 
In this report, we describe a hot aqueous-phenol method for the isolation and purification of LPS from Gram-negative bacterial cells. This protocol allows for the extraction of LPS away from nucleic acids and proteins that can interfere with visualization of LPS that occurs with shorter, less intensive extraction methods9. LPS prepared this way can be separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and directly stained using carbohydrate/glycoprotein stains or standard silver staining methods. Many anti-sera to LPS contain antibodies that cross-react with outer membrane proteins or other antigenic targets that can hinder reactivity observed following Western immunoblot of SDS-PAGE-separated crude cell lysates. Protease treatment of crude cell lysates alone is not always an effective way of removing this background using this or other visualization methods. Further, extensive protease treatment in an attempt to remove this background can lead to poor quality LPS that is not well resolved by any of the aforementioned methods. For these reasons, we believe that the following protocol, adapted from Westpahl and Jann10, is ideal for LPS extraction.

We describe a modified hot aqueous-phenol extraction method for purifying lipopolysaccharide (LPS) from Gram-negative bacteria. Once extracted, the LPS can be subsequently analyzed by SDS-PAGE and visualized by direct staining or Western immunoblot.

La purificación y la visualización de lipopolisacárido de bacterias Gram-negativas por Hot acuosa de fenol-Extracción

Lipopolysaccharide (LPS)  is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of  lipid A, which is ...

Affinity Purification of Influenza Virus Ribonucleoprotein Complexes from the Chromatin of Infected Cells

Like all negative-strand RNA viruses, the genome of influenza viruses is packaged in the form of viral ribonucleoprotein complexes (vRNP), in which the single-stranded genome is encapsidated by the nucleoprotein (NP), and associated with the trimeric polymerase complex consisting of the PA, PB1, and PB2 subunits. However, in contrast to most RNA viruses, influenza viruses perform viral RNA synthesis in the nuclei of infected cells. Interestingly, viral mRNA synthesis uses cellular pre-mRNAs as primers, and it has been proposed that this process takes place on chromatin1. Interactions between the viral polymerase and the host RNA polymerase II, as well as between NP and host nucleosomes have also been characterized1,2.
Recently, the generation of recombinant influenza viruses encoding a One-Strep-Tag genetically fused to the C-terminus of the PB2 subunit of the viral polymerase (rWSN-PB2-Strep3) has been described. These recombinant viruses allow the purification of PB2-containing complexes, including vRNPs, from infected cells. To obtain purified vRNPs, cell cultures are infected, and vRNPs are affinity purified from lysates derived from these cells. However, the lysis procedures used to date have been based on one-step detergent lysis, which, despite the presence of a general nuclease, often extract chromatin-bound material only inefficiently. 
Our preliminary work suggested that a large portion of nuclear vRNPs were not extracted during traditional cell lysis, and therefore could not be affinity purified. To increase this extraction efficiency, and to separate chromatin-bound from non-chromatin-bound nuclear vRNPs, we adapted a step-wise subcellular extraction protocol to influenza virus-infected cells. Briefly, this procedure first separates the nuclei from the cell and then extracts soluble nuclear proteins (here termed the "nucleoplasmic" fraction). The remaining insoluble nuclear material is then digested with Benzonase, an unspecific DNA/RNA nuclease, followed by two salt extraction steps: first using 150 mM NaCl (termed "ch150"), then 500 mM NaCl ("ch500") (Fig. 1). These salt extraction steps were chosen based on our observation that 500 mM NaCl was sufficient to solubilize over 85% of nuclear vRNPs yet still allow binding of tagged vRNPs to the affinity matrix.
After subcellular fractionation of infected cells, it is possible to affinity purify PB2-tagged vRNPs from each individual fraction and analyze their protein and RNA components using Western Blot and primer extension, respectively. Recently, we utilized this method to discover that vRNP export complexes form during late points after infection on the chromatin fraction extracted with 500 mM NaCl (ch500)3.

Influenza viruses replicate their RNA genome in association with host-cell chromatin. Here, we present a method to purify intact viral ribonucleoprotein complexes from the chromatin of infected cells. Purified viral complexes can be analyzed by both Western blot and primer extension of protein and RNA content, respectively.

Purificación por afinidad de los complejos de ribonucleoproteína Influenza Virus de la cromatina de las células infectadas

Like all negative-strand RNA viruses, the genome of influenza viruses is packaged in the form of viral ribonucleoprotein complexes (vRNP), in which the ...

Live-cell Video Microscopy of Fungal Pathogen Phagocytosis

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of phagocytes to the site where pathogens are located; (ii) recognition of pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRRs); (iii) engulfment of microorganisms bound to the phagocyte cell membrane, and (iv) processing of engulfed cells within maturing phagosomes and digestion of the ingested particle. Studies that assess phagocytosis in its entirety are informative1, 2, 3, 4, 5 but are limited in that they do not normally break the process down into migration, engulfment and phagosome maturation, which may be affected differentially. Furthermore, such studies assess uptake as a single event, rather than as a continuous dynamic process. We have recently developed advanced live-cell imaging technologies, and have combined these with genetic functional analysis of both pathogen and host cells to create a cross-disciplinary platform for the analysis of innate immune cell function and fungal pathogenesis. These studies have revealed novel aspects of phagocytosis that could only be observed using systematic temporal analysis of the molecular and cellular interactions between human phagocytes and fungal pathogens and infectious microorganisms more generally. For example, we have begun to define the following: (a) the components of the cell surface required for each stage of the process of recognition, engulfment and killing of fungal cells1, 6, 7, 8; (b) how surface geometry influences the efficiency of macrophage uptake and killing of yeast and hyphal cells7; and (c) how engulfment leads to alteration of the cell cycle and behavior of macrophages 9, 10.
In contrast to single time point snapshots, live-cell video microscopy enables a wide variety of host cells and pathogens to be studied as continuous sequences over lengthy time periods, providing spatial and temporal information on a broad range of dynamic processes, including cell migration, replication and vesicular trafficking. Here we describe in detail how to prepare host and fungal cells, and to conduct the video microscopy experiments. These methods can provide a user-guide for future studies with other phagocytes and microorganisms.

We describe methods for live-cell video microscopy of Candida albicans phagocytosis by macrophages. These methods enable stage-specific analysis of macrophage migration, recognition, engulfment and phagosome maturation and reveal novel aspects of phagocytosis.

Vídeo en vivo de células Microscopía de fagocitosis hongo patógeno

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of ...

Loop-mediated Isothermal Amplification (LAMP) Assays for the Species-specific Detection of Eimeria that Infect Chickens

Eimeria species parasites, protozoa which cause the enteric disease coccidiosis, pose a serious threat to the production and welfare of chickens. In the absence of effective control clinical coccidiosis can be devastating. Resistance to the chemoprophylactics frequently used to control Eimeria is common and sub-clinical infection is widespread, influencing feed conversion ratios and susceptibility to other pathogens such as Clostridium perfringens. Despite the availability of polymerase chain reaction (PCR)-based tools, diagnosis of Eimeria infection still relies almost entirely on traditional approaches such as lesion scoring and oocyst morphology, but neither is straightforward. Limitations of the existing molecular tools include the requirement for specialist equipment and difficulties accessing DNA as template. In response a simple field DNA preparation protocol and a panel of species-specific loop-mediated isothermal amplification (LAMP) assays have been developed for the seven Eimeria recognised to infect the chicken. We now provide a detailed protocol describing the preparation of genomic DNA from intestinal tissue collected post-mortem, followed by setup and readout of the LAMP assays. Eimeria species-specific LAMP can be used to monitor parasite occurrence, assessing the efficacy of a farm&#8217;s anticoccidial strategy, and to diagnose sub-clinical infection or clinical disease with particular value when expert surveillance is unavailable.

Loop-mediated Isothermal Amplification LAMP Assays for the Species-specific Detection of Eimeria that Infect Chickens

Diagnosis of Eimeria infection in chickens remains demanding. Parasite morphology- and host pathology-led approaches are commonly inconclusive, while molecular approaches based on PCR have proven demanding in cost and expertise. The aim of this protocol is to establish loop-mediated isothermal amplification (LAMP) as a straightforward molecular diagnostic for eimerian infection.

Isotérmico de amplificación (LAMP) Ensayos Loop mediada para la detección específica de las especies de<em&gt; Eimeria</em&gt; Que infectan pollos

Eimeria species parasites, protozoa which cause the enteric disease coccidiosis, pose a serious threat to the production and welfare of chickens. In the ...

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.

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. ...

Development and Identification of a Novel Subpopulation of Human Neutrophil-derived Giant Phagocytes In Vitro

Neutrophils (PMN) are best known for their phagocytic functions against invading pathogens and microorganisms. They have the shortest half-life amongst leukocytes and in their non-activated state are constitutively committed to apoptosis. When recruited to inflammatory sites to resolve inflammation, they produce an array of cytotoxic molecules with potent antimicrobial killing. Yet, when these powerful cytotoxic molecules are released in an uncontrolled manner they can damage surrounding tissues. In recent years however, neutrophil versatility is increasingly evidenced, by demonstrating plasticity and immunoregulatory functions. We have recently identified a new neutrophil-derived subpopulation, which develops spontaneously in standard culture conditions without the addition of cytokines/growth factors such as granulocyte colony-stimulating factor (GM-CSF)/interleukin (IL)-4. Their phagocytic abilities of neutrophil remnants largely contribute to increase their size immensely; therefore they were termed giant phagocytes (G&#981;). Unlike neutrophils, G&#981; are long lived in culture. They express the cluster of differentiation (CD) neutrophil markers CD66b/CD63/CD15/CD11b/myeloperoxidase (MPO)/neutrophil elastase (NE), and are devoid of the monocytic lineage markers CD14/CD16/CD163 and the dendritic CD1c/CD141 markers. They also take-up latex and zymosan, and respond by oxidative burst to stimulation with opsonized-zymosan and PMA. G&#981; also express the scavenger receptors CD68/CD36, and unlike neutrophils, internalize oxidized-low density lipoprotein (oxLDL). Moreover, unlike fresh neutrophils, or cultured monocytes, they respond to oxLDL uptake by increased reactive oxygen species (ROS) production. Additionally, these phagocytes contain microtubule-associated protein-1 light chain 3B (LC3B) coated vacuoles, indicating the activation of autophagy. Using specific inhibitors it is evident that both phagocytosis and autophagy are prerequisites for their development and likely NADPH oxidase dependent ROS. We describe here a method for the preparation of this new subpopulation of long-lived, neutrophil-derived phagocytic cells in culture, their identification and their currently known characteristics. This protocol is essential for obtaining and characterizing G&#981; in order to further investigate their significance and functions.

Development and Identification of a Novel Subpopulation of Human Neutrophil-derived Giant Phagocytes In Vitro

We describe here a method for obtaining and identifying a newly characterized subpopulation of neutrophil-derived giant phagocytes. These cells develop in culture from fresh human blood neutrophils, and are characterized by phagocytosis, autophagy, immensely large size, and extended lifespan. This method is essential to further investigate this unique neutrophil-derived subpopulation.

El desarrollo y la identificación de una subpoblación de Novela derivada de neutrófilos humanos fagocitos gigante<em&gt; In Vitro</em

Neutrophils (PMN) are best known for their phagocytic functions against invading pathogens and microorganisms. They have the shortest half-life amongst ...

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

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

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

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

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

Isolation, Characterization, and Purification of Macrophages from Tissues Affected by Obesity-related Inflammation

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. Diet-induced obesity (DIO) is a valuable model in studying the role of macrophage heterogeneity; however, adequate macrophage isolations are difficult to acquire from inflamed tissues. In this protocol, we outline the isolation steps and necessary troubleshooting guidelines derived from our studies for obtaining a suitable population of tissue-resident macrophages from mice following 18 weeks of high-fat (HFD) or high-fat/high-cholesterol (HFHCD) diet intervention. This protocol focuses on three hallmark tissues studied in obesity and atherosclerosis including the liver, white adipose tissues (WAT), and the aorta. We highlight how dualistic usage of flow cytometry can achieve a new dimension of isolation and characterization of tissue-resident macrophages. A fundamental section of this protocol addresses the intricacies underlying tissue-specific enzymatic digestions and macrophage isolation, and subsequent cell-surface antibody staining for flow cytometric analysis. This protocol addresses existing complexities underlying fluorescent-activated cell sorting (FACS) and presents clarifications to these complexities so as to obtain broad range characterization from adequately sorted cell populations. Alternate enrichment methods are included for sorting cells, such as the dense liver, allowing for flexibility and time management when working with FACS. In brief, this protocol aids the researcher to evaluate macrophage heterogeneity from a multitude of inflamed tissues in a given study and provides insightful troubleshooting tips that have been successful for favorable cellular isolation and characterization of immune cells in DIO-mediated inflammation.

This protocol allows a researcher to isolate and characterize tissue-resident macrophages in various hallmark inflamed tissues extracted from diet-induced models of metabolic disorders.