Los autores desean agradecer a Cui Chao, Piso de Kort, Stoop Esther y Carvalho para las imágenes de Ralph en la Figura 4, Nehrdich Ulrike y Davy de Witt para el cuidado de los peces, y otros miembros del laboratorio útil para los debates. Este trabajo es apoyado por el Programa Smart Mix del Ministerio holandés de Asuntos Económicos y el Ministerio de Educación, Cultura y Ciencia, la Comisión Europea séptimo proyecto marco ZF-SALUD (SALUD-F4-2010-242048), la Unión Europea Marie Curie- Red de Formación Inicial FishForPharma (PITN-GA-2011 a 289209), y por el NHMRC Australia (637.394). El australiano Instituto de Medicina Regenerativa con el apoyo de subvenciones del Gobierno del Estado de Victoria y el Gobierno de Australia.

<ol>
	<li>Meijer, A. H., Spaink, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host-pathogen+interactions+made+transparent+with+the+zebrafish+model.">Host-pathogen interactions made transparent with the zebrafish model.</a> Curr. Drug Targets. 12, 1000-1017 (2011).</li><li>Davis, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+visualization+of+mycobacterium-macrophage+interactions+leading+to+initiation+of+granuloma+formation+in+zebrafish+embryos.">Real-time visualization of mycobacterium-macrophage interactions leading to initiation of granuloma formation in zebrafish embryos.</a> Immunity. 17, 693-702 (2002).</li><li>vander Sar, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+embryos+as+a+model+host+for+the+real+time+analysis+of+Salmonella+typhimurium+infections.">Zebrafish embryos as a model host for the real time analysis of Salmonella typhimurium infections.</a> Cell Microbiol. 5, 601-611 (2003).</li><li>Zakrzewska, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophage-specific+gene+functions+in+Spi1-directed+innate+immunity.">Macrophage-specific gene functions in Spi1-directed innate immunity.</a> Blood. 116, 1-11 (2010).</li><li>Le Guyader, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origins+and+unconventional+behavior+of+neutrophils+in+developing+zebrafish.">Origins and unconventional behavior of neutrophils in developing zebrafish.</a> Blood. 111, 132-141 (2008).</li><li>Herbomel, P., Thisse, B., Thisse, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ontogeny+and+behaviour+of+early+macrophages+in+the+zebrafish+embryo.">Ontogeny and behaviour of early macrophages in the zebrafish embryo.</a> Development. 126, 3735-3745 (1999).</li><li>Alibaud, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Mycobacterium+marinum+TesA+mutant+defective+for+major+cell+wall-associated+lipids+is+highly+attenuated+in+Dictyostelium+discoideum+and+zebrafish+embryos.">A Mycobacterium marinum TesA mutant defective for major cell wall-associated lipids is highly attenuated in Dictyostelium discoideum and zebrafish embryos.</a> Mol. Microbiol. 80, 919-934 (2011).</li><li>Carvalho, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+screen+for+tuberculosis+progression.">A high-throughput screen for tuberculosis progression.</a> PLoS One. 6, 16779-16779 (2011).</li><li>van der Sar, A. M., Spaink, H. P., Zakrzewska, A., Bitter, W., Meijer, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specificity+of+the+zebrafish+host+transcriptome+response+to+acute+and+chronic+mycobacterial+infection+and+the+role+of+innate+and+adaptive+immune+components.">Specificity of the zebrafish host transcriptome response to acute and chronic mycobacterial infection and the role of innate and adaptive immune components.</a> Mol. Immunol. 46, 2317-2332 (2009).</li><li>Rosen, J. N., Sweeney, M. F., Mably, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+of+Zebrafish+Embryos+to+Analyze+Gene+Function.">Microinjection of Zebrafish Embryos to Analyze Gene Function.</a> J. Vis. Exp. (25), e1115-e1115 (2009).</li><li>Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B., Schilling, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stages+of+embryonic+development+of+the+zebrafish.">Stages of embryonic development of the zebrafish.</a> Dev. Dyn. 203, 253-310 (1995).</li><li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Zebrafish+Book.">The Zebrafish Book.</a> A Guide for the Laboratory Use of Zebrafish (Brachydanio rerio). , (1993).</li><li>Gutzman, J. H., Sive, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+Brain+Ventricle+Injection.">Zebrafish Brain Ventricle Injection.</a> J. Vis. Exp. (26), e1218-e1218 (2009).</li><li>Ellett, F., Pase, L., Hayman, J. W., Andrianopoulos, A., Lieschke, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mpeg1+promoter+transgenes+direct+macrophage-lineage+expression+in+zebrafish.">mpeg1 promoter transgenes direct macrophage-lineage expression in zebrafish.</a> Blood. 117, 49-56 (2011).</li><li>Stockhammer, O. W., Zakrzewska, A., Hegedus, Z., Spaink, H. P., Meijer, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptome+profiling+and+functional+analyses+of+the+zebrafish+embryonic+innate+immune+response+to+Salmonella+infection.">Transcriptome profiling and functional analyses of the zebrafish embryonic innate immune response to Salmonella infection.</a> J. Immunol. 182, 5641-5653 (2009).</li><li>Renshaw, S. 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+transgenic+zebrafish+model+of+neutrophilic+inflammation.">A transgenic zebrafish model of neutrophilic inflammation.</a> Blood. 108, 3976-3978 (2006).</li><li>Stoop, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+embryo+screen+for+mycobacterial+genes+involved+in+the+initiation+of+granuloma+formation+reveals+a+newly+identified+ESX-1+component.">Zebrafish embryo screen for mycobacterial genes involved in the initiation of granuloma formation reveals a newly identified ESX-1 component.</a> Dis. Model Mech. 4, 526-536 (2011).</li><li>Levraud, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+the+zebrafish+IFN+receptor:+implications+for+the+origin+of+the+vertebrate+IFN+system.">Identification of the zebrafish IFN receptor: implications for the origin of the vertebrate IFN system.</a> Journal of Immunology. 178, 4385-4394 (2007).</li><li>Levraud, J. P., Colucci-Guyon, E., Redd, M. J., Lutfalla, G., Herbomel, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+analysis+of+zebrafish+innate+immunity.">In vivo analysis of zebrafish innate immunity.</a> Methods Mol. Biol. 415, 337-363 (2008).</li><li>Brothers, K. M., Newman, Z. R., Wheeler, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+disseminated+candidiasis+in+zebrafish+reveals+role+of+phagocyte+oxidase+in+limiting+filamentous+growth.">Live imaging of disseminated candidiasis in zebrafish reveals role of phagocyte oxidase in limiting filamentous growth.</a> Eukaryot. Cell. 10, 932-944 (2011).</li><li>Colucci-Guyon, E., Tinevez, J. Y., Renshaw, S. A., Herbomel, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strategies+of+professional+phagocytes+in+vivo:+unlike+macrophages,+neutrophils+engulf+only+surface-associated+microbes.">Strategies of professional phagocytes in vivo: unlike macrophages, neutrophils engulf only surface-associated microbes.</a> J. Cell Sci. 124, 3053-3059 (2011).</li><li>van Soest, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+static+immersion+and+intravenous+injection+systems+for+exposure+of+zebrafish+embryos+to+the+natural+pathogen+Edwardsiella+tarda.">Comparison of static immersion and intravenous injection systems for exposure of zebrafish embryos to the natural pathogen Edwardsiella tarda.</a> BMC Immunology. 12, 58-58 (2011).</li><li>Adams, K. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug+tolerance+in+replicating+mycobacteria+mediated+by+a+macrophage-induced+efflux+mechanism.">Drug tolerance in replicating mycobacteria mediated by a macrophage-induced efflux mechanism.</a> Cell. 145, 39-53 (2011).</li><li>Ellett, F. L., Lieschke, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Computational+quantification+of+fluorescent+leukocyte+numbers+in+zebrafish+embryos.">Computational quantification of fluorescent leukocyte numbers in zebrafish embryos.</a> Methods in Enzymology. , (2011).</li><li>Yuan, S., Sun, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+of+mRNA+and+Morpholino+Antisense+Oligonucleotides+in+Zebrafish+Embryos.">Microinjection of mRNA and Morpholino Antisense Oligonucleotides in Zebrafish Embryos.</a> J. Vis. Exp. (27), e1113-e1113 (2009).</li><li>Mathias, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resolution+of+inflammation+by+retrograde+chemotaxis+of+neutrophils+in+transgenic+zebrafish.">Resolution of inflammation by retrograde chemotaxis of neutrophils in transgenic zebrafish.</a> J. Leukoc. Biol. 80, 1281-1288 (2006).</li><li>Hall, C., Flores, M. V., Storm, T., Crosier, K., Crosier, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish+lysozyme+C+promoter+drives+myeloid-specific+expression+in+transgenic+fish.">The zebrafish lysozyme C promoter drives myeloid-specific expression in transgenic fish.</a> BMC Dev. Biol. 7, 42-42 (2007).</li><li>Gray, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+intravital+imaging+of+macrophage+and+neutrophil+behaviour+during+inflammation+using+a+novel+transgenic+zebrafish.">Simultaneous intravital imaging of macrophage and neutrophil behaviour during inflammation using a novel transgenic zebrafish.</a> Thromb. Haemost. 105, 811-819 (2011).</li></ol>

Los autores no tienen nada que revelar.

Los métodos de infección se describen en este artículo se utilizan con frecuencia para estudiar la función de los genes receptores de inmunidad innata o de los genes de virulencia bacteriana 1. Los micro-inyección por vía intravenosa métodos (protocolos 5 y 6.1) se utilizan con mayor frecuencia para este tipo de estudios. La vena caudal en la isla sangre posterior es el lugar más conveniente para la inyección intravenosa de 1-día-embriones. Como la vena caudal se vuelve más difícil de penetrar en las etapas posteriores, se prefiere que el conducto de Cuvier como sitio de la inyección intravenosa de embriones en 2-3 fdd. En todos los casos es crítico para inyectar a los embriones con números consistentes de las bacterias, los cuales deben ser controlados por los inóculos de siembra de la inyección para el conteo de ufc. Los siguientes aspectos deben ser considerados para lograr inyecciones reproducibles. En primer lugar, es esencial disponer de alta calidad agujas de vidrio microcapilares inyección. Si la punta de la aguja es demasiado grande, la inyección creará un agujero punción suficientemente grande para que se produzca sangrado ylas bacterias inyectadas fluirá hacia fuera de la circulación sanguínea. En segundo lugar, las bacterias debe ser inyectado directamente en la circulación sanguínea. Si la punta de la aguja no está dentro de la vena o si se propaga de inyección de fluidos en la extensión del saco vitelino, el embrión debe ser desechado en el experimento. En tercer lugar, usando PVP40 como un portador para inyectar M. marinum ayudará en la prevención de las bacterias que se hunda en la aguja de vidrio. PVP40 mejora la homogeneidad de la suspensión, lo que resulta en inóculos más reproducible a lo largo de la duración de las inyecciones. PVP40 también puede ser usada para S. inyecciones typhimurium, pero aquí es menos importante porque el tamaño más grande y brillante de fluorescencia de las bacterias permite un control visual sobre el inóculo de la inyección durante los experimentos. Para la práctica de las inyecciones se recomienda el uso de tinte rojo de fenol (1% v / v) o 1 m esferas fluorescentes disponibles en diferentes colores (Invitrogen). Una vez que la técnica se domina, se tarda aproximadamente 30 minutES para inyectar 50-100 embriones, incluyendo el tiempo requerido para alinear los embriones en placas de agarosa y ajustando el volumen de inyección bacteriana. Mientras que las inyecciones por vía intravenosa en la isla de sangre (protocolo 5) o en el conducto de Cuvier (protocolo 6.1) son los más utilizados, las vías alternativas de infección se describen en este artículo de vídeo han demostrado ser útiles para estudiar los macrófagos y la quimiotaxis de neutrófilos (protocolos 6.2, 6.3 y 6.4), para estudiar el crecimiento de cepas bacterianas atenuadas (protocolo 6.5), y para adaptar el modelo de infección pez cebra para aplicaciones de alto rendimiento (protocolo 6.6) 4-8. Los descritos micro-inyección métodos también se pueden aplicar para las inyecciones de virus 18, 19, esporas de hongos 14, 20 o parásitos protozoarios (Maria Forlenza, comunicación personal). Una adición útil a los procedimientos de inyección descritas en este artículo de vídeo es un procedimiento descrito recientemente para la inyección subcutánea de bacterias en zebrembriones afish 21. Este procedimiento fue utilizado para estudiar la fagocitosis de las bacterias por los neutrófilos, los cuales fueron encontrados a las bacterias eficientemente engullen en las superficies del tejido pero, en contraste con los macrófagos, eran virtualmente incapaz de fagocitar bacterias después de la inyección en la sangre o en las cavidades corporales llenas de líquido. Para subcutáneos embriones inyecciones se colocan similar al de las inyecciones de cola musculares descritos en este artículo de vídeo, pero se inserta la aguja justo debajo de la piel para inyectar las bacterias sobre un somito. Es importante considerar que la microinyección es esencialmente una vía artificial de la infección. Sin embargo, los primeros embriones son altamente resistentes a la exposición externa a los patógenos bacterianos. De hecho, los ensayos de inmersión, donde los embriones se bañan en una suspensión bacteriana, no se pueden reproducir en nuestras manos 22. Mientras que algunos embriones pueden infectarse tras la inmersión, se ha observado una gran variación en las tasas de mortalidad y en el expresoión de los genes marcadores inflamatorios entre embriones individuales en dichos ensayos. Los métodos de inyección de micro-descritos en este artículo conseguir infecciones reproducibles y son particularmente útiles para estudiar las interacciones bacterianas con las células inmunes del huésped innatas. Un enfoque eficaz para cuantificar la infección bacteriana en los embriones individuales es el de analizar las imágenes fluorescentes de embriones infectados con el encargo, el software dedicado cuantificación pixel 17, 23. Este enfoque se ha demostrado que se correlaciona bien con los resultados de los recuentos cfu después de placas de embriones infectados. Del mismo modo, los cambios relativos en el número de leucocitos por embrión han sido cuantificados por análisis digital de imágenes en las líneas transgénicas de leucocitos fluoróforo reportero, este enfoque sería aplicable a la cuantificación de la respuesta del huésped a la infección leukopoietic 24. La función de los genes receptores inmunes innatas puede ser investigado eficazmente in vivo mediante la combinación de los modelos de infección descritoscon la caída morfolino. Morpholinos son oligonucleótidos sintéticos que se dirigen a los ARN antisentido específicos y reducir la expresión de los productos génicos cuando se inyectan en embriones de pez cebra 1-celulares etapa como se muestra en artículos de vídeo otros en esta revista 10, 25. En estudios desmontables morfolino, es de gran importancia que todos los grupos de embrión a comparar se realizaron correctamente antes de las inyecciones bacterianas. Esto es especialmente importante porque los embriones tienen un desarrollo del sistema inmune que hace cada vez más competente en el tiempo. Hay varias líneas de reportero transgénicos actualmente disponibles para distinguir los principales subconjuntos de inmunidad innata, los macrófagos y los neutrófilos, en embriones de pez cebra. El MPX y promotores Lyz se han utilizado para generar líneas de neutrófilos reportero 16, 26, 27, y el CSF1R (FMS) y promotores mpeg1 se utilizaron para producir reporteros macrófagos 14, 28. Mientras CSF1R (FMS) es, además, se expresa en xantóforos, mpeg1 expresión es exclusivamente en los macrófagos. Como se muestra en este artículo de vídeo, las líneas se han desarrollado recientemente mpeg1 reporteros son muy útiles para la fagocitosis por los macrófagos de imagen bacteriana.

<table border="1"><tbody><tr><td> Reactivo </td><td> Proveedor </td><td> Número de catálogo </td></tr><tr><td> Rojo fenol </td><td> Sigma </td><td> P0290 </td></tr><tr><td> Glicerol </td><td> Sigma-Aldrich </td><td> 228210 </td></tr><tr><td> Middlebrook 7H10 agar Difco </td><td> Becton, Dickinson y Compañía </td><td> 262710 </td></tr><tr><td> BBL Middlebrook OADC Enriquecimiento </td><td> Becton, Dickinson y Compañía </td><td> 211886 </td></tr><tr><td> Difco caldo Middlebrook 7H9 </td><td> Becton, Dickinson y Compañía </td><td> 271310 </td></tr><tr><td> BBL Middlebrook ADC Enriquecimiento </td><td> Becton, Dickinson y Compañía </td><td> 211887 </td></tr><tr><td> Tween 80 </td><td> Sigma-Aldrich </td><td> P1754 </td></tr><tr><td> Polivinilpirrolidona (PVP40) </td><td> Calbiochem, Merck KGaA </td><td> 529504 </td></tr><tr><td> N &lt;/ Em&gt;-feniltiourea (PTU) </td><td> Sigma-Aldrich </td><td> P7629 </td></tr><tr><td> Etilo 3-aminobenzoato metanosulfonato sal (tricaína) </td><td> Sigma-Aldrich </td><td> A5040 </td></tr><tr><td> Metil celulosa </td><td> Sigma-Aldrich </td><td> M0387 </td></tr><tr><td> SeaPlaque agarosa (bajo punto de fusión de agarosa) </td><td> Lonza Inc. </td><td> 50100 </td></tr><tr><td> FluoSpheres (1,0 micras, rojo fluorescente (580/605)) </td><td> Invitrogen </td><td> F8821 </td></tr><tr><td> FluoSpheres (1.0 m, de color amarillo-verde fluorescente (505/515)) </td><td> Invitrogen </td><td> F8823 </td></tr></tbody></table>

infection of zebrafish embryos with intracellular bacterial pathogens

Pez cebra (Danio rerio), los embriones se utiliza cada vez más como un modelo para estudiar la función de los vertebrados del sistema inmune innato en las interacciones huésped-patógeno 1. Los principales tipos de células del sistema inmune innato, los macrófagos y neutrófilos, se desarrollan durante los primeros días de la embriogénesis antes de la maduración de los linfocitos que se requieren para las respuestas inmunitarias adaptativas. La facilidad de obtener un gran número de embriones, su accesibilidad debido a la externa para el desarrollo, la transparencia óptica de estadios embrionario y larval, una amplia gama de herramientas genéticas, los amplios recursos y colecciones de mutantes de las líneas transgénicas, el reportero se suman a la versatilidad del pez cebra modelo. Salmonella enterica serovar Typhimurium (S. typhimurium) y Mycobacterium marinum puede residir de manera intracelular en los macrófagos y se utilizan con frecuencia para estudiar las interacciones huésped-patógeno en embriones de pez cebra. Los procesos de infección de estas dospatógenos bacterianos son interesantes para comparar ya que S. la infección es aguda typhimurium y letal en un día, mientras que M. la infección es crónica marinum y se puede exponer a la etapa de larva 2, 3. El sitio de micro-inyección de bacterias en el embrión (Figura 1) determina si la infección rápidamente se convertirá sistémica o inicialmente permanecerá localizada. Una infección sistémica rápida puede ser establecido por micro-inyección de bacterias directamente en la circulación sanguínea a través de la vena caudal en la isla posterior o sangre a través del conducto de Cuvier, un canal de circulación de ancho en el saco vitelino conecta el corazón a la vasculatura tronco. En un DPF, cuando los embriones en esta etapa tienen los macrófagos phagocytically activos, pero los neutrófilos aún no han madurado, la inyección en la isla de sangre se prefiere. Para las inyecciones en el 2-3 fdd, cuando los embriones también se han desarrollado funcionales (mieloperoxidasa que produce) los neutrófilos, el conducto de Cuvier se prefiere como tque el sitio de inyección. Para estudiar la migración dirigida de las células mieloides hacia infecciones locales, las bacterias pueden ser inyectados en el músculo cola, vesícula ótica, o rombencéfalo ventrículo 4-6. Además, la notocorda, una estructura que parece ser normalmente inaccesible a las células mieloides, es altamente susceptible a la infección local 7. Una alternativa útil para aplicaciones de alto rendimiento es la inyección de bacterias en la yema de los embriones en las primeras horas después de la fecundación 8. La combinación de bacterias fluorescentes y las líneas de transgénicos de pez cebra con los macrófagos fluorescentes o neutrófilos crea condiciones ideales para la multi-color de imágenes de las interacciones huésped-patógeno. Este artículo video describe protocolos detallados para la infección por vía intravenosa y locales de los embriones de pez cebra con S. typhimurium o M. marinum bacterias y para la imagen posterior de fluorescencia de la interacción con las células del sistema inmune innato.

Watch this Scientific Journal Video about Infection of Zebrafish Embryos with Intracellular Bacterial Pathogens at JoVE.com

Infection of Zebrafish Embryos with Intracellular Bacterial Pathogens

Embriones de pez cebra transparentes han demostrado ser útiles los ejércitos de modelo para visualizar y las interacciones funcionales de estudio entre las células inmunes y los patógenos bacterianos intracelulares, tales como<em&gt; Salmonella typhimurium</em&gt; Y<em&gt; Mycobacterium marinum</em&gt;. Micro-inyección de bacterias y multi-color de imagen de fluorescencia son técnicas esenciales que intervienen en la aplicación de pez cebra modelos embrión infección.

La infección de embriones de pez cebra con patógenos bacterianos intracelulares

Zebrafish (Danio rerio) embryos are increasingly used as a model for studying the function of the vertebrate innate immune system in host-pathogen ...

infection-of-zebrafish-embryos-with-intracellular-bacterial-pathogens

Research

JoVE Journal

Immunology and Infection

43.5K vistas.  Leiden University. Embriones de pez cebra transparentes han demostrado ser útiles los ejércitos de modelo para visualizar y las interacciones funcionales de estudio entre las células inmunes y los patógenos bacterianos intracelulares, tales como Salmonella typhimurium Y Mycobacterium marinum. Micro-inyección de bacterias y multi-color de imagen de fluorescencia son técnicas esenciales que intervienen en la aplicación de pez cebra modelos embrión infecci...

Video: Infection of Zebrafish Embryos with Intracellular Bacterial Pathogens

'Bioluminescent' Reporter Phage for the Detection of Category A Bacterial Pathogens

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. Although the natural occurrence of both diseases&#39; is now relatively rare, the possibility of terrorist groups using these pathogens as a bioweapon is real. Because of the disease&#39;s inherent communicability, rapid clinical course, and high mortality rate, it is critical that an outbreak be detected quickly. Therefore methodologies that provide rapid detection and diagnosis are essential to ensure immediate implementation of public health measures and activation of crisis management.
Recombinant reporter phage may provide a rapid and specific approach for the detection of Y. pestis and B. anthracis. The Centers for Disease Control and Prevention currently use the classical phage lysis assays for the confirmed identification of these bacterial pathogens 2-4. These assays take advantage of naturally occurring phage which are specific and lytic for their bacterial hosts. After overnight growth of the cultivated bacterium in the presence of the specific phage, the formation of plaques (bacterial lysis) provides a positive identification of the bacterial target. Although these assays are robust, they suffer from three shortcomings: 1) they are laboratory based; 2) they require bacterial isolation and cultivation from the suspected sample, and 3) they take 24-36 h to complete. To address these issues, recombinant &#34;light-tagged&#34; reporter phage were genetically engineered by integrating the Vibrio harveyi luxAB genes into the genome of Y. pestis and B. anthracis specific phage 5-8. The resulting luxAB reporter phage were able to detect their specific target by rapidly (within minutes) and sensitively conferring a bioluminescent phenotype to recipient cells. Importantly, detection was obtained either with cultivated recipient cells or with mock-infected clinical specimens 7.
For demonstration purposes, here we describe the method for the phage-mediated detection of a known Y. pestis isolate using a luxAB reporter phage constructed from the CDC plague diagnostic phage &#934;A1122 6,7 (Figure 1). A similar method, with minor modifications (e.g. change in growth temperature and media), may be used for the detection of B. anthracis isolates using the B. anthracis reporter phage W&#946;::luxAB 8. The method describes the phage-mediated transduction of a biolumescent phenotype to cultivated Y. pestis cells which are subsequently measured using a microplate luminometer. The major advantages of this method over the traditional phage lysis assays is the ease of use, the rapid results, and the ability to test multiple samples simultaneously in a 96-well microtiter plate format.
<img alt="Figure 1" src="/files/ftp_upload/2740/2740fig1.jpg" /> 
Figure 1. Detection schematic. The phage are mixed with the sample, the phage infects the cell, luxAB are expressed, and the cell bioluminesces. Sample processing is not necessary; the phage and cells are mixed and subsequently measured for light.

&#039;Bioluminescent&#039; Reporter Phage for the Detection of Category A Bacterial Pathogens

A simple method for the identification of priority bacterial pathogens is to use genetically engineered reporter phage. These reporter phage, which are specific to their particular host species, are capable of rapidly transducing a bioluminescent signal response to host cells. Herein, we describe the use of reporter phage for the detection of Yersinia pestis.

&quot;Bioluminiscente &#39;reportero fagos para la detección de patógenos bacterianos de la categoría A

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. ...

Investigación

Inmunología e Infección

Single Cell Measurements of Vacuolar Rupture Caused by Intracellular Pathogens

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

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

Sola célula Medidas de Ruptura Vacuolar causadas por patógenos intracelulares

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

Discovery of New Intracellular Pathogens by Amoebal Coculture and Amoebal Enrichment Approaches

Intracellular pathogens such as legionella, mycobacteria and Chlamydia-like organisms are difficult to isolate because they often grow poorly or not at all on selective media that are usually used to cultivate bacteria. For this reason, many of these pathogens were discovered only recently or following important outbreaks. These pathogens are often associated with amoebae, which serve as host-cell and allow the survival and growth of the bacteria. We intend here to provide a demonstration of two techniques that allow isolation and characterization of intracellular pathogens present in clinical or environmental samples: the amoebal coculture and the amoebal enrichment. Amoebal coculture allows recovery of intracellular bacteria by inoculating the investigated sample onto an amoebal lawn that can be infected and lysed by the intracellular bacteria present in the sample. Amoebal enrichment allows recovery of amoebae present in a clinical or environmental sample. This can lead to discovery of new amoebal species but also of new intracellular bacteria growing specifically in these amoebae. Together, these two techniques help to discover new intracellular bacteria able to grow in amoebae. Because of their ability to infect amoebae and resist phagocytosis, these intracellular bacteria might also escape phagocytosis by macrophages and thus, be pathogenic for higher eukaryotes.

Amoebal coculture is a cell culture system using adherent amoebae to selectively grow intracellular pathogens able to resist phagocytic cells such as amoebae and macrophages. It thus represents a key tool to discover new infectious agents. Amoebal enrichment allows discovery of new amoebal species and of their specific intracellular bacteria.

Descubrimiento de nuevos patógenos intracelulares por amebas Coculture y Enfoques amebas Enriquecimiento

Intracellular pathogens such as legionella, mycobacteria and Chlamydia-like organisms are difficult to isolate because they often grow poorly or not at ...

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

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

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

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

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

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

Applying an Inducible Expression System to Study Interference of Bacterial Virulence Factors with Intracellular Signaling

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a signaling pathway. The method is based, on the one hand, on the inducible expression of a specific protein to initiate a signaling event at a defined and predetermined step in the selected signaling cascade. Concomitant expression, on the other hand, of the gene of interest then allows the investigator to evaluate if the activity of the expressed target protein is located upstream or downstream of the initiated signaling event, depending on the readout of the signaling pathway that is obtained. Here, the apoptotic cascade was selected as a defined signaling pathway to demonstrate protocol functionality. Pathogenic bacteria, such as Coxiella burnetii, translocate effector proteins that interfere with host cell death induction in the host cell to ensure bacterial survival in the cell and to promote their dissemination in the organism. The C. burnetii effector protein CaeB effectively inhibits host cell death after induction of apoptosis with UV-light or with staurosporine. To narrow down at which step CaeB interferes with the propagation of the apoptotic signal, selected proteins with well-characterized pro-apoptotic activity were expressed transiently in a doxycycline-inducible manner. If CaeB acts upstream of these proteins, apoptosis will proceed unhindered. If CaeB acts downstream, cell death will be inhibited. The test proteins selected were Bax, which acts at the level of the mitochondria, and caspase 3, which is the major executioner protease. CaeB interferes with cell death induced by Bax expression, but not by caspase 3 expression. CaeB, thus, interacts with the apoptotic cascade between these two proteins.

The method described here is used to induce the apoptotic signaling cascade at defined steps in order to dissect the activity of an anti-apoptotic bacterial effector protein. This method can also be used for inducible expression of pro-apoptotic or toxic proteins, or for dissecting interference with other signaling pathways.

La aplicación de un sistema de expresión inducible para el Estudio de la interferencia de los factores de virulencia bacteriana con Señalización Intracelular

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a ...

Identification of Rare Bacterial Pathogens by 16S rRNA Gene Sequencing and MALDI-TOF MS

There are a number of rare and, therefore, insufficiently described bacterial pathogens which are reported to cause severe infections especially in immunocompromised patients. In most cases only few data, mostly published as case reports, are available which investigate the role of such pathogens as an infectious agent. Therefore, in order to clarify the pathogenic character of such microorganisms, it is necessary to conduct epidemiologic studies which include large numbers of these bacteria. The methods used in such a surveillance study have to meet the following criteria: the identification of the strains has to be accurate according to the valid nomenclature, they should be easy to handle (robustness), economical in routine diagnostics and they have to generate comparable results among different laboratories. Generally, there are three strategies for identifying bacterial strains in a routine setting: 1) phenotypic identification characterizing the biochemical and metabolic properties of the bacteria, 2) molecular techniques such as 16S rRNA gene sequencing and 3) mass spectrometry as a novel proteome based approach. Since mass spectrometry and molecular approaches are the most promising tools for identifying a large variety of bacterial species, these two methods are described. Advances, limitations and potential problems when using these techniques are discussed.

Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) and molecular techniques (16S rRNA gene sequencing) permit the identification of rare bacterial pathogens in routine diagnostics. The goal of this protocol lies in the combination of both techniques which leads to more accurate and reliable data.

La identificación de patógenos bacterianos raras por 16S rRNA gen secuenciación y MALDI-TOF MS

There are a number of rare and, therefore, insufficiently described bacterial pathogens which are reported to cause severe infections especially in ...

Isolation of Single Intracellular Bacterial Communities Generated from a Murine Model of Urinary Tract Infection for Downstream Single-cell Analysis

In this article, we outline a procedure used to isolate individual intracellular bacterial communities from a mouse that has been experimentally infected in the urinary tract. The protocol can be broadly divided into three sections: the infection, bladder epithelial cell harvesting, and mouth micropipetting to isolate individual infected epithelial cells. The isolated epithelial cell contains viable bacterial cells and is nearly free of contaminating extracellular bacteria, making it ideal for downstream single-cell analysis. The time taken from the start of infection to obtaining a single intracellular bacterial community is about 8 h. This protocol is inexpensive to deploy and uses widely available materials, and we anticipate that it can also be utilized in other infection models to isolate single infected cells from cell mixtures even if those infected cells are rare. However, due to a potential risk in mouth micropipetting, this procedure is not recommended for highly infectious agents.

This protocol describes a simple method of isolating single, infected-bladder epithelial cells from a murine model of urinary tract infection.

Aislamiento de las comunidades bacterianas intracelulares solo generados a partir de un modelo murino de infección del tracto urinario para posteriores análisis unicelular

In this article, we outline a procedure used to isolate individual intracellular bacterial communities from a mouse that has been experimentally infected ...

Opsonophagocytic Killing Assay to Assess Immunological Responses Against Bacterial Pathogens

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental procedure in which phagocytic cells are co-cultured with bacterial units. The immune cells will phagocytose and kill the bacterial cultures in a complement-dependent manner. The efficiency of the immune-mediated cell killing is dependent on a number of factors and can be used to determine how different bacterial cultures compare with regard to resistance to cell death. In this way, the efficacy of potential immune-based therapeutics can be assessed against specific bacterial strains and/or serotypes. In this protocol, we describe a simplified OPKA that utilizes basic culture conditions and cell counting to determine bacterial cell viability after co-culture with treatment conditions and HL-60 immune cells. This method has been successfully utilized with a number of different pneumococcal serotypes, capsular and acapsular strains, and other bacterial species. The advantages of this OPKA protocol are its simplicity, versatility (as this assay is not limited to antibody treatments as opsonins), and minimization of time and reagents to assess basic experimental groups.

This opsonophagocytic killing assay is used to compare the ability of phagocytic immune cells to respond to and kill bacteria based on different treatments and/or conditions. Classically, this assay serves as the gold&#160;standard for assessing effector functions of antibodies raised against a bacterium as opsonin.

Opsonofagocítico asesinato ensayo para evaluar las respuestas inmunológicas contra patógenos bacterianos

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental ...

Visualization of Macrophage Lytic Cell Death During Mycobacterial Infection in Zebrafish Embryos via Intravital Microscopy

Zebrafish is an excellent model organism for studying innate immune cell behavior due to its transparent nature and reliance solely on its innate immune system during early development. The Zebrafish Mycobacterium marinum (M. marinum) infection model has been well-established in studying host immune response against mycobacterial infection. It has been suggested that different macrophage cell death types will lead to the diverse outcomes of mycobacterial infection. Here we describe a protocol using intravital microscopy to observe macrophage cell death in zebrafish embryos following M. marinum infection. Zebrafish transgenic lines that specifically label macrophages and neutrophils are infected via intramuscular microinjection of fluorescently labeled M. marinum in either the midbrain or the trunk. Infected zebrafish embryos are subsequently mounted on low melting agarose and observed by confocal microscopy in X-Y-Z-T dimensions. Because long-term live imaging requires using low laser power to avoid photobleaching and phototoxicity, a strongly expressing transgenic is highly recommended. This protocol facilitates the visualization of the dynamic processes in vivo, including immune cell migration, host pathogen interaction, and cell death.

This protocol describes a technique for visualizing macrophage behavior and death in embryonic zebrafish during Mycobacterium marinum infection. Steps for the preparation of bacteria, infection of the embryos, and intravital microscopy are included. This technique may be applied to the observation of cellular behavior and death in similar scenarios involving infection or sterile inflammation.

Visualización de la muerte de células líticas de macrófagos durante la infección micobacteriana en embriones de pez cebra a través de la microscopía intravital

Zebrafish is an excellent model organism for studying innate immune cell behavior due to its transparent nature and reliance solely on its innate immune ...

Determination of Chemical Inhibitor Efficiency against Intracellular Toxoplasma Gondii Growth Using a Luciferase-Based Growth Assay

Toxoplasma gondii is a protozoan pathogen that widely affects the human population. The current antibiotics used for treating clinical toxoplasmosis are limited. In addition, they exhibit adverse side effects in certain groups of people. Therefore, discovery of novel therapeutics for clinical toxoplasmosis is imperative. The first step of novel antibiotic development is to identify chemical compounds showing high efficacy in inhibition of parasite growth using a high throughput screening strategy. As an obligate intracellular pathogen, Toxoplasma can only replicate within host cells, which prohibits the use of optical absorbance measurements as a quick indicator of growth. Presented here is a detailed protocol for a luciferase-based growth assay. As an example, this method is used to calculate the doubling time of wild-type Toxoplasma parasites and measure the efficacy of morpholinurea-leucyl-homophenyl-vinyl sulfone phenyl (LHVS, a cysteine protease-targeting compound) regarding inhibition of parasite intracellular growth. Also described, is a CRISPR-Cas9-based gene deletion protocol in Toxoplasma using 50 bp homologous regions for homology-dependent recombination (HDR). By quantifying the inhibition efficacies of LHVS in wild-type and TgCPL (Toxoplasma cathepsin L-like protease)-deficient parasites, it is shown that LHVS inhibits wild-type parasite growth more efficiently than &#916;cpl growth, suggesting that TgCPL is a target that LHVS binds to in Toxoplasma. The high sensitivity and easy operation of this luciferase-based growth assay make it suitable for monitoring Toxoplasma proliferation and evaluating drug efficacy in a high throughput manner.

Determination of Chemical Inhibitor Efficiency against Intracellular Toxoplasma Gondii Growth Using a Luciferase-Based Growth Assay

Presented here is a protocol to evaluate the inhibition efficacy of chemical compounds against in vitro intracellular growth of Toxoplasma gondii using a luciferase-based growth assay. The technique is used to confirm inhibition specificity by genetic deletion of the corresponding target gene. The inhibition of LHVS against TgCPL protease is evaluated as an example.

Determinación de la eficiencia de los inhibidores químicos contra el crecimiento de toxoplasma intracelular Gondii mediante un ensayo de crecimiento basado en Luciferase

Toxoplasma gondii is a protozoan pathogen that widely affects the human population. The current antibiotics used for treating clinical toxoplasmosis are ...