Agradecemos al Dr. Craig Roy y el Dr. Dario Zamboni para proporcionar una plantilla para las infecciones por protozoos celulares. Agradecemos al Dr. Jagdeep Obhrai, Dr. Georgiana Purdy, el Dr. Fred Heffron y Todd Wisner para el equipo y los reactivos, el Dr. Lulu Cambronne para la revisión crítica del manuscrito. La citometría de flujo se realizó en las instalaciones de flujo OHSU Recursos Citometría compartido. Este trabajo fue apoyado en parte por una beca de la Fundación de Investigación Médica de Oregon y un NIH subvención R21 AI088275 (EDC).

1. Preparación de Legionella pneumophila Culturas de Infecciones fase de cebado <ol><li> Transformar todo L. pneumophila cepas usadas en el ensayo con los plásmidos pAM239, que codifica una isopropil β-D-1-tiogalactopiranósido (IPTG) inducible proteína verde fluorescente (GFP) 28. Inocular las cepas bacterianas en hierro y cisteína suplementado N-(2-acetamido)-2-aminoetanosulfónico (ACES) tamponada con carbón vegetal agar de extracto de levadura (CYEA) que contiene 6,25 mg / ml de cloranfenicol (CM) (para el mantenimiento del plásmido) y se incuba durante 72 hr a 37 ° C. </li><li> Transferir un inóculo de la cepa bacteriana en ACES suplementadas tamponada caldo de extracto de levadura (AYE) con 6,25 g / ml CM y 1 mM de IPTG y cultivar a la fase estacionaria (O / N) en un agitador orbital a 37 ° C. </li><li> Confirmar la expresión de GFP en los cultivos in vitro utilizando microscopía de fluorescencia. Transferir 10 l de cultivo en un portaobjetos de vidrio bajo una cubiertadeslizamiento y la imagen usando un objetivo de 60X con excitación GFP / cubo de emisión (AMG EVOS fl). </li><li> Diluir una alícuota de 100 l de cada cultivo in vitro a 1:10 con H2O estéril El blanco con 100 l AYE medios de comunicación con IPTG 1 mM y 6,25 g / cm ml y agua. Tome OD600 mediciones de las diluciones utilizando un espectrofotómetro (Bio-Rad inteligente Spec Plus). Calcular el volumen necesario para infectar un pozo a una MOI = 20 para cada cultivo in vitro: V = [(ameba cabeza de serie) x MOI] / [OD 600 x (factor de dilución) x (constante)] = [(1 X 10 6 ) x (20)] / [OD 600 x (10) x (1 x 10 6)] = 2/OD 600. La concentración de bacterias se determinó como OD 600 = 1,0 = 1 x 10 9 UFC / ml. </li></ol> 2. Cebado de la infección por Acanthamoeba escenario mediante castellanii <ol><li> Mantener y cultivar las amebas en medio ATCC 712 PYG en 175 cm 2 frascos a temperatura ambiente. </li><li> Vuelva a colocar los medios de comunicación en la cultura amebasras 24 h antes de iniciar los cultivos líquidos bacterianos. En el mismo día líquidos cultivos bacterianos se han iniciado, recoger y contar las células en un microscopio de luz usando un hemocitómetro. </li><li> Diluir las amebas con medio fresco a una concentración final de 1 x 10 6 células / ml. Semilla 12-y placas de cultivo de células con 1 ml de alícuotas de las amebas utilizando una pipeta de repetición y se incuba a RT O / N. </li><li> Después de la incubación, se lavan los pocillos de las placas de cultivo de 12-pocillos de células 3 veces con 1 ml de PBS estéril usando una pipeta serológica de 10 ml y una ayuda de pipeta manual. </li><li> Después de la aspiración de PBS, añadir 1 ml de la infección por los medios de comunicación (ATCC 712 medios de comunicación PYG menos glucosa, peptona, y extracto de levadura) con IPTG 1 mM y 6,25 g / ml cm a cada pocillo. Incubar las placas a temperatura ambiente durante 1 hr. </li><li> Infect pocillos a una MOI = 20. Centrifugar la placa a 400 xg durante 5 min (Eppendorf 5810R) y flotar en un baño a 37 ° C durante 5 min. Colocar la placa en un 37 ° C, 5% de CO 2 incubadora durante 18 hr. </li><li> Confirme las infecciones utilizando imágenes de células vivas en un microscopio de fluorescencia antes de la sede de la lisis celular y la cosecha de las bacterias. </li></ol> 3. Siembra de células THP-1 para la infección de las células diana Stage <ol><li> (Comience este proceso de 24 horas antes de la creación de cultivos bacterianos líquidos). Cultivar células THP-1 en un 75 matraces de cultivo de 2 cm a alrededor de-confluencia en medio RPMI 1640 con inactivado por calor al 10% de suero fetal bovino (FBS). </li><li> Contar las células en suspensión utilizando un hemocitómetro, diluir las células THP-1 en medio RPMI 1640 con FBS al 10% y 100 ng / ml de forbol-12-miristato-13-acetato (PMA) a una concentración de 1 x 10 6 células / ml y la placa en alícuotas de 1 ml en placas de 12 pocillos de cultivo celular. Incubar las placas durante 48 horas a 37 ° C, 5% de CO 2. </li></ol> 4. Tratamiento para la infección de bacterias Etapa de células diana después de la infección fase de cebado <ol><li> Aspirar el medio de las amebas cebada. </li><li> Lyse la unamoebae usando 500 l de hielo frío, estéril ultrafiltrada (UF) H 2 O y se incuba a temperatura ambiente durante 10 min. </li><li> Piscina Los lisados ​​de acuerdo con la cepa de tipo E y tomar una medición de 512 nm para cada uno de los lisados ​​combinados utilizando un lector de placas de fluorescencia (Molecular Devices Spectramax Gemini EM). </li><li> Calcular un OD 600 de medición para los lisados ​​agrupados: calcOD 600 = 0,0008 (E 512 - Fondo de lisado) + 0.0019. La fórmula se determinó previamente por gráfica una comparación directa de ambas la OD 600 y el E 512 nm mediciones de diluciones de L. pneumophila tipo salvaje GFP expresión de la fase estacionaria del cultivo in vitro (Figura 1 *). Los lisados ​​de ameba no infectadas, sujetas a las mismas condiciones experimentales que la ameba infectada, se usa como blanco y proporcionó un valor de corrección (por ejemplo, lisado de fondo), que fue incorporado en la ecuación. El t volumen necesarioo infectar un pozo a una MOI = 20 se calcula para cada grupo de lisado: V = [(THP-1 sin semillas) x MOI] / [calcOD 600 x (constante)] = [(1 X 10 6) x (20)] / [calcOD 600 x (1 x 10 6)] = 20 / calcOD 600. </li><li> Lavar las células THP-1 3x con PBS y añadir 1 ml de medio fresco RPMI 1640 (10% de FBS) a cada pocillo. Incubar las células THP-1 durante 1 hora a 37 ° C, 5% de CO 2. </li><li> Infectar las células THP-1 en la cantidad, calculada usando los lisados ​​MOI agrupados. Permitir que un conjunto de pocillos para no infectarse, sirviendo como un control negativo para el análisis de citometría de flujo. Procesamiento de la fase de cebado debe ser completado en menos de 30 min. Los lisados ​​se mantienen en hielo para limitar los cambios en la expresión génica antes de la infección bacteriana. </li><li> Centrifugar la placa, flotar para elevar la temperatura, y se incuba como en la fase de cebado. </li><li> Una hora después de la infección, retire el soporte mediante aspiración y lavar los pocillos 3 veces con PBS para eliminar las bacterias extracelulares. </li><li> Añadir 1 ml freSH RPMI 1640 (10% de FBS) a los pocillos de la placa y volver a la incubadora. El tiempo inmediatamente después del reemplazo de los medios de comunicación sirve como tiempo cero (T 0). Se incuban las células THP-1 durante 14-16 horas a 37 ° C, 5% de CO 2. </li></ol> 5. Análisis Experimental 5,1 imágenes de células vivas Imagen de los pozos infectados utilizando un microscopio de fluorescencia (AMG EVOS fl) a 10X o 20X de aumento (Figuras 1A-D). Las imágenes se pueden comparar de manera cualitativa o cuantitativamente para determinar los niveles de infección, tanto en la fase de cebado y las infecciones de células diana etapa. 5,2 de citometría de flujo <ol><li> Los picos de emisión de diferentes infecciones pueden ser comparados por citometría de flujo (BD FACS Calibur). Tripsinizar las infectadas células THP-1 y suavemente de lavado de los pozos mediante la mezcla en PBS con una pipeta. </li><li> Piscina las células por tipo de cepa en tubos de 15 ml Falcon y el sedimento a 1.800 xg durante 2min en la centrífuga de mesa. </li><li> Suspender los gránulos en 1 ml de PBS y transferir a tubos de 1,5 ml de microcentrífuga. </li><li> Centrifugar la suspensión a 1.800 x g (Eppendorf 5424) y volver a suspender los gránulos en 1 ml de PBS. Si las suspensiones resultantes son muy turbia (OD 600 ≥ 1,0) que debe ser diluido en PBS adicional (1:3) para prevenir la obstrucción de las líneas en el citómetro de flujo. </li><li> Use filtrada estéril PBS para el equilibrado del citómetro de flujo y para los pasos de lavado entre inyecciones de muestra. Trazar la dispersión frontal / lateral de las células diana infectadas antes de la inyección de muestras objetivo infección celular. </li><li> Recoge 20.000 eventos totales para cada condición de uso de 488 nm láser de excitación y el canal FL1. </li><li> Puerta de captura de post-poblaciones de células para excluir las células no infectadas y la intensidad de fluorescencia de trama en un histograma (FlowJo) (Figura 1E). </li></ol> 5,3 CFU plating <ol><li> Examinar la eficacia of infección por chapado UFC de las células cosechadas por citometría de flujo. Preparar diluciones seriadas de las muestras en ultrafiltrada O H 2 a 10 -1, 10 -2, y -3 10. </li><li> Placa de 20 l de cada dilución en el 1/3 de una placa de CYEA con 6,25 g / ml CM. </li><li> Incubar las placas a 37 ° C durante 72 hr. </li><li> Contar las colonias utilizando un palillo de dientes y contador de células. </li></ol>

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Los autores declaran que no tienen intereses en conflicto financieros.

La expresión génica bacteriana está estrechamente controlada a través de una combinación de la progresión del ciclo de vida y la respuesta a las señales en el microambiente circundante. Patógenos vacuolar tales como L. pneumophila responder a una multitud de receptores de células derivadas de las señales cuando compartimentada en un fagosoma. Como resultado colectivo de agotamiento de nutrientes en la célula huésped, la bacteria compensa mediante la expresión de factores requeridos para la difusió...

Numerosos patógenos bacterianos han adaptado estrategias generalizadas para explotar células huésped para la supervivencia y replicación en un compartimiento intracelular. En muchos casos, los mecanismos patogénicos son similares entre protozoos y metazoos células. Sin embargo, estos dos microambientes son muy diferentes y pueden resultar en la expresión diferencial de los factores de virulencia 1-4. La enfermedad del legionario bacteria Legionella pneumophila doquier se asocian con ambientes de agua dulce en todo el mundo 5. Es importante destacar que, L. pneumophila cultivada en células de protozoos antes de la infección de los monocitos humanos obtener una ventaja patógena, lo que sugiere que los perfiles de expresión génica global de la bacteria sale de una célula de protozoo son diferentes que la de la vitro en cultivo organismo 6-8. En la naturaleza, las amebas de agua dulce proporcionan nutrientes ricos confines de la rápida amplificación de una bacteria invasora. Adquisición humano de L. pneumophila es más a menudo atribuida a la inhalación de gotitas de agua contaminadas que contienen la bacteria. Es probable que estas gotitas puerto protozoarios asociados a las células bacterias; donde las células de protozoos son más resistentes a las prácticas convencionales de tratamiento de agua 9,10. La infección de macrófagos alveolares pulmonares procede de una manera casi idéntica a la del ciclo de vida intracelular de la bacteria en las células huésped de protozoos 11-13. Con el fin de sobrevivir y replicarse en células eucariotas, L. pneumophila utiliza un tipo especializado sistema de secreción IVb denominado Dot / Icm llegar a casi el 300 &#39;ejecutor&#39; proteínas en el citosol de la célula huésped 14-16. Estas proteínas efectoras colectivamente funcionar para subvertir procesos celulares con el fin de generar un compartimiento de replicación permisiva para la bacteria 17,18. Las deleciones en cualquiera de los 26 genes que constituyen el resultado transportador Dot / Icm en cepas defectuosas para mult intracelulariplication 19-23. Históricamente, la deleción de genes individuales que codifican efectoras rara vez resultó en cepas atenuadas para el crecimiento intracelular. Este fenómeno se ha atribuido a varias hipótesis, incluida la función redundante y paralogous copias de efectores. Algunos factores de virulencia se expresan únicamente en el contexto de la célula huésped asociada a crecimiento intracelular 24. Hemos racionalizado que si un efector particular, se expresa sólo en el contexto de la infección por protozoos, entonces la contribución del efector no podía ser comparada con una cepa de tipo salvaje cuando ambos se cultivaron in vitro. L. pneumophila transiciones desde un replicativa a una fase de transmisión a medida que entra en la fase estacionaria de cultivo de 25. El fenotipo de conmutación de fase representa el agotamiento de los nutrientes encontrados durante el crecimiento intracelular y se ejemplifica a través del conjunto de los flagelos para la movilidad 26. Debido a que L. pneumophila es más invasive y virulenta cuando se cosecha a partir de células de protozoos, hemos tratado de desarrollar un ensayo que más fielmente representa el estado patógeno de la bacteria cuando se encontró con los macrófagos de acogida. Para este fin, hemos desarrollado un ensayo protozoo cebado versátil que puede adaptarse a cualquier huésped adecuado tanto para las infecciones de la primera etapa (cebado de células) y segundo (célula diana). El proceso de infección es tratable mediante el uso de bacterias que expresan establemente la proteína verde fluorescente (GFP). El modelo de la infección por el protozoo Acanthamoeba castellanii sigue una metodología ampliamente utilizado en el campo 27. Para la etapa de cebado, L. pneumophila cepas se cultivan in vitro hasta la fase estacionaria en medio líquido para producir la etiqueta &#39;transmisivo &quot;bacterias (Figura 1A). Las bacterias se vuelva a utilizar para infectar monocapas de A. castellanii durante 18 horas para lograr una etapa tardía del ciclo de vida intracelular. Contienen grandes vacuolasbacterias ING puede ser visualizado en este punto de tiempo usando microscopía de fluorescencia (Figura 1A). Las células se lisan por protozoos y bacterias recuperadas a partir del lisado se miden para emisión a 512 nm utilizando un lector de placas de fluorescencia. La fluorescencia se correlaciona con la densidad óptica para calcular multiplicidad de infección (MOI) en la infección de células diana (Figura 1, Curva * Correlación). Después de la invasión h (T 0) y 18 post-invasión (T 18), las células diana se cuantifican por fluorescencia, en representación de las bacterias intracelulares. La fluorescencia puede ser monitoreada por microscopía y citometría de flujo, y el recuento de viables se puede medir a través de placas de colonias. El ensayo de cebado siempre se acompaña de infecciones por L. de tipo salvaje pneumophila y una cepa defectuosa en el sistema Dot / Icm de secreción de tipo IV (Δ dotA) (Figura 1A). Este importante proporciona controles internos para las comparaciones directas entre los de tipo salvaje unnd cualquier cepas isogénicas mutantes utilizados en el proceso de infección. La inclusión de la cepa no virulenta Δ dotA durante la fase de cebado establece un umbral para la observación de fenotipos de crecimiento atenuadas asociados con cepas mutantes isogénicas que se cultivan in vitro.

<table border="1">
 <tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Reagent</td>
 </tr>
 <tr>
 <td>chloramphenicol</td>
 <td>Fisher Scientific</td>
 <td>BP904-100</td>
 <td>antibiotic</td>
 </tr>
 <tr>
 <td>IPTG</td>
 <td>Fisher Scientific</td>
 <td>BP1755-10</td>
 <td></td>
 </tr>
 <tr>
 <td>ACES</td>
 <td>Sigma</td>
 <td>A9758-1KG</td>
 <td>media component</td>
 </tr>
 <tr>
 <td>ATCC medium: 712 PYG</td>
 <td>ATCC</td>
 <td></td>
 <td>growth media for protozoans</td>
 </tr>
 <tr>
 <td>1X PBS</td>
 <td>Fisher Scientific</td>
 <td>SH30256FS</td>
 <td>phosphate buffered saline</td>
 </tr>
 <tr>
 <td>activated charcoal</td>
 <td>Fisher Scientific</td>
 <td>C272-212</td>
 <td>media component</td>
 </tr>
 <tr>
 <td>yeast extract</td>
 <td>Fisher Scientific</td>
 <td>BP1422-500</td>
 <td>media component</td>
 </tr>
 <tr>
 <td>peptone</td>
 <td>BD Diagnostics</td>
 <td>211677</td>
 <td>media component</td>
 </tr>
 <tr>
 <td>agar</td>
 <td>Fisher Scientific</td>
 <td>BP1423-2</td>
 <td>media component</td>
 </tr>
 <tr>
 <td>L-cysteine, 99%+</td>
 <td>Acros organics</td>
 <td>173601000</td>
 <td>media supplement</td>
 </tr>
 <tr>
 <td>Ferric nitrate nonahydrate</td>
 <td>Fisher Scientific</td>
 <td>I110-100</td>
 <td>media supplement</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Equipment</td>
 </tr>
 <tr>
 <td>EVOS fl</td>
 <td>AMG</td>
 <td>EVOS fl</td>
 <td>fluorescence microscope</td>
 </tr>
 <tr>
 <td>Smart Spec Plus</td>
 <td>Bio-Rad</td>
 <td>170-2525</td>
 <td>spectrophotometer</td>
 </tr>
 <tr>
 <td>5810 R centrifuge</td>
 <td>Eppendorf</td>
 <td>22627023</td>
 <td>bench top centrifuge</td>
 </tr>
 <tr>
 <td>Repeater plus</td>
 <td>Eppendorf</td>
 <td>22230201</td>
 <td>repeating pipette</td>
 </tr>
 <tr>
 <td>SpectraMax Gemini EM</td>
 <td>Molecular Devices</td>
 <td></td>
 <td>microplate reader</td>
 </tr>
 <tr>
 <td>Softmax Pro 5.3</td>
 <td>Molecular Devices</td>
 <td>0200-310</td>
 <td>microplate reader software</td>
 </tr>
 <tr>
 <td>5424 microfuge</td>
 <td>Eppendorf</td>
 <td>22620401</td>
 <td>table top microcentrifuge</td>
 </tr>
 <tr>
 <td>Fast-release pipette pump II</td>
 <td>Scienceware</td>
 <td>379111010</td>
 <td>pipette aid</td>
 </tr>
 <tr>
 <td>FACS Calibur</td>
 <td>BD Bioscience</td>
 <td></td>
 <td>flow cytometer</td>
 </tr>
 <tr>
 <td>FlowJo 7.6.1</td>
 <td>FlowJo</td>
 <td>license</td>
 <td>flow cytometery software</td>
 </tr>
 <tr>
 <td>15 ml tube</td>
 <td>BD Falcon</td>
 <td>352096</td>
 <td>polypropylene conical tube</td>
 </tr>
 <tr>
 <td>1.6 ml microfuge tube</td>
 <td>Neptune</td>
 <td>3745.X</td>
 <td>microcentrifuge tubes</td>
 </tr>
 </tbody>
</table>

tractable mammalian cell infections with protozoan-primed bacteria

Muchos patógenos bacterianos intracelulares utilizar protozoos de agua dulce como un reservorio natural para la proliferación en el medio ambiente. Legionella pneumophila, el agente causante de la neumonía del legionario, gana una ventaja sobre patógena en bacterias cultivadas in vitro cuando primero cosechadas a partir de células de protozoos antes de la infección de los macrófagos de mamífero. Esto sugiere que los factores de virulencia importantes que no pueden ser adecuadamente expresada in vitro. Hemos desarrollado un sistema manejable para el cebado L. pneumophila a través de su huésped natural, protozoo Acanthamoeba castellanii antes de la infección de células de mamíferos. La contribución de cualquier factor de virulencia puede examinarse comparando el crecimiento intracelular de una cepa mutante de bacterias de tipo salvaje después de la imprimación protozoo. Que expresa GFP de tipo salvaje y mutante de L. cepas pneumophila se utilizan para infectar monocapas de protozoarios en una etapa de cebado y se dejó alcanzar las últimas etapas de crecimiento intracelular. Bacterias fluorescentes Después se recogen a partir de estas células infectadas y normalizado por espectrofotometría para generar números comparables de las bacterias para una posterior infección en macrófagos de mamífero. Para la cuantificación, bacterias vivas son monitoreados después de la infección mediante microscopía de fluorescencia, citometría de flujo, y por chapado colonia. Esta técnica pone de relieve y se basa en la contribución de la expresión génica de la célula huésped dependiente imitando el medio ambiente que se encuentra en una ruta de adquisición natural. Este enfoque puede ser modificado para adaptarse a cualquier bacteria que utiliza un huésped intermediario como un medio para obtener una ventaja patógena.

Un resultado típico para el proceso de infección completo se describe en la Figura 1. Vivo micrografías de fluorescencia de células que representan monocapas de A.castellanii infectadas con el tipo salvaje L. pneumophila durante la fase de cebado se muestra en la Figura 1A. Una medida de éxito de la etapa de cebado daría lugar a una población de aproximadamente 90% de las células huésped que contienen grandes vacuolas pobladas con bacterias GFP etiquetados en ...

Watch this Scientific Journal Video about Tractable Mammalian Cell Infections with Protozoan-primed Bacteria at JoVE.com

Tractable Mammalian Cell Infections with Protozoan-primed Bacteria

Esta técnica proporciona un método para cosechar, normalizar y cuantificar el crecimiento intracelular de las bacterias patógenas que son pre-cultivadas en células naturales protozoos anfitrionas antes de las infecciones de las células de mamíferos. Este método puede ser modificado para acomodar una amplia variedad de células huésped para la fase de cebado así como los tipos de células diana.

Las infecciones tratables con células de mamíferos Protozoarios cebados con bacterias

Many intracellular bacterial pathogens use freshwater protozoans as a natural reservoir for proliferation in the environment. Legionella pneumophila, the ...

tractable-mammalian-cell-infections-with-protozoan-primed-bacteria

Research

JoVE Journal

Immunology and Infection

10.4K vistas.  Oregon Health & Science University. Esta técnica proporciona un método para cosechar, normalizar y cuantificar el crecimiento intracelular de las bacterias patógenas que son pre-cultivadas en células naturales protozoos anfitrionas antes de las infecciones de las células de mamíferos. Este método puede ser modificado para acomodar una amplia variedad de células huésped para la fase de cebado así como los tipos de células diana.

Video: Tractable Mammalian Cell Infections with Protozoan-primed Bacteria

Using Luciferase to Image Bacterial Infections in Mice

Imaging is a valuable technique that can be used to monitor biological processes. In particular, the presence of cancer cells, stem cells, specific immune cell types, viral pathogens, parasites and bacteria can be followed in real-time within living animals 1-2. Application of bioluminescence imaging to the study of pathogens has advantages as compared to conventional strategies for analysis of infections in animal models3-4. Infections can be visualized within individual animals over time, without requiring euthanasia to determine the location and quantity of the pathogen. Optical imaging allows comprehensive examination of all tissues and organs, rather than sampling of sites previously known to be infected. In addition, the accuracy of inoculation into specific tissues can be directly determined prior to carrying forward animals that were unsuccessfully inoculated throughout the entire experiment. Variability between animals can be controlled for, since imaging allows each animal to be followed individually. Imaging has the potential to greatly reduce animal numbers needed because of the ability to obtain data from numerous time points without having to sample tissues to determine pathogen load3-4.
This protocol describes methods to visualize infections in live animals using bioluminescence imaging for recombinant strains of bacteria expressing luciferase. The click beetle (CBRLuc) and firefly luciferases (FFluc) utilize luciferin as a substrate5-6. The light produced by both CBRluc and FFluc has a broad wavelength from 500 nm to 700 nm, making these luciferases excellent reporters for the optical imaging in living animal models7-9. This is primarily because wavelengths of light greater than 600 nm are required to avoid absorption by hemoglobin and, thus, travel through mammalian tissue efficiently. Luciferase is genetically introduced into the bacteria to produce light signal10. Mice are pulmonary inoculated with bioluminescent bacteria intratracheally to allow monitoring of infections in real time. After luciferin injection, images are acquired using the IVIS Imaging System. During imaging, mice are anesthetized with isoflurane using an XGI-8 Gas Anethesia System. Images can be analyzed to localize and quantify the signal source, which represents the bacterial infection site(s) and number, respectively. After imaging, CFU determination is carried out on homogenized tissue to confirm the presence of bacteria. Several doses of bacteria are used to correlate bacterial numbers with luminescence. Imaging can be applied to study of pathogenesis and evaluation of the efficacy of antibacterial compounds and vaccines.

Methods for bioluminescence imaging of bacterial infections in living animals are decribed. Pathogens are modified to express luciferase allowing optical whole body imaging of infections in live animals. Animal models can be infected with luciferase expressing pathogens and the resulting course of disease visualized in real-time by bioluminescence imaging.

Luciferasa utilizando a las infecciones bacterianas de la imagen en ratones

Imaging is a valuable technique that can be used to monitor biological processes.  In particular, the presence of cancer cells, stem cells, specific ...

Investigación

Inmunología e Infección

Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully generated and infused T-cells specific for Epstein Barr virus (EBV), cytomegalovirus (CMV) and Adenovirus (Adv) using monocytes and EBV-transformed lymphoblastoid cell (EBV-LCL) gene-modified with an adenovirus vector as antigen presenting cells (APCs). As few as 2x105/kg trivirus-specific cytotoxic T lymphocytes (CTL) proliferated by several logs after infusion and appeared to prevent and treat even severe viral disease resistant to other available therapies. The broader implementation of this encouraging approach is limited by high production costs, complexity of manufacture and the prolonged time (4-6 weeks for EBV-LCL generation, and 4-8 weeks for CTL manufacture &#x2013; total 10-14 weeks) for preparation. To overcome these limitations we have developed a new, GMP-compliant CTL production protocol. First, in place of adenovectors to stimulate T-cells we use dendritic cells (DCs) nucleofected with DNA plasmids encoding LMP2, EBNA1 and BZLF1 (EBV), Hexon and Penton (Adv), and pp65 and IE1 (CMV) as antigen-presenting cells. These APCs reactivate T cells specific for all the stimulating antigens. Second, culture of activated T-cells in the presence of IL-4 (1,000U/ml) and IL-7 (10ng/ml) increases and sustains the repertoire and frequency of specific T cells in our lines. Third, we have used a new, gas permeable culture device (G-Rex) that promotes the expansion and survival of large cell numbers after a single stimulation, thus removing the requirement for EBV-LCLs and reducing technician intervention. By implementing these changes we can now produce multispecific CTL targeting EBV, CMV, and Adv at a cost per 106 cells that is reduced by &gt;90%, and in just 10 days rather than 10 weeks using an approach that may be extended to additional protective viral antigens. Our FDA-approved approach should be of value for prophylactic and treatment applications for high risk allogeneic HSCT recipients.

A rapid, simple and cost-effective protocol for the generation of donor-derived multivirus-specific CTLs (rCTL) for infusion to allogeneic hematopoietic stem cell transplant (HSCT) recipients at risk of developing CMV, Adv or EBV infections. This manufacturing process is GMP-compliant and should ensure the broader implementation of T-cell immunotherapy beyond specialized centers.

Generación de células T Multivirus específicos para prevenir / tratar las infecciones virales después de trasplante alogénico de células madre hematopoyéticas

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully ...

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

This protocol outlines the steps required to longitudinally monitor a bioluminescent bacterial infection using composite 3D diffuse light imaging tomography with integrated &mu;CT (DLIT-&mu;CT) and the subsequent use of this data to generate a four dimensional (4D) movie of the infection cycle. To develop the 4D infection movies and to validate the DLIT-&mu;CT imaging for bacterial infection studies using an IVIS Spectrum CT, we used infection with bioluminescent C. rodentium, which causes self-limiting colitis in mice. In this protocol, we outline the infection of mice with bioluminescent C. rodentium and non-invasive monitoring of colonization by daily DLIT-&mu;CT imaging and bacterial enumeration from feces for 8 days.
The use of the IVIS Spectrum CT facilitates seamless co-registration of optical and &mu;CT scans using a single imaging platform. The low dose &mu;CT modality enables the imaging of mice at multiple time points during infection, providing detailed anatomical localization of bioluminescent bacterial foci in 3D without causing artifacts from the cumulative radiation. Importantly, the 4D movies of infected mice provide a powerful analytical tool to monitor bacterial colonization dynamics in vivo.

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

Multi-modality imaging is a valuable approach for studying bacterial colonization in small animal models. This protocol outlines infection of mice with bioluminescent Citrobacter rodentium and the longitudinal monitoring of bacterial colonization using composite 3D diffuse light imaging tomography with &mu;CT imaging to create a 4D movie of C. rodentium infection.

4D de imágenes multimodalidad<em&gt; Citrobacter rodentium</em&gt; Las infecciones en ratones

This protocol outlines the steps required to  longitudinally monitor a bioluminescent bacterial infection using composite 3D diffuse light imaging ...

Fluorescence Microscopy Methods for Determining the Viability of Bacteria in Association with Mammalian Cells

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols to address these questions include colony count assays, gentamicin protection assays, and electron microscopy. Colony count and gentamicin protection assays only assess the viability of the entire bacterial population and are unable to determine individual bacterial viability. Electron microscopy can be used to determine the viability of individual bacteria and provide information regarding their localization in host cells. However, bacteria often display a range of electron densities, making assessment of viability difficult. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells. These assays were developed originally to assess survival of Neisseria gonorrhoeae in primary human neutrophils, but should be applicable to any bacterium-host cell interaction. These protocols combine membrane-permeable fluorescent dyes (SYTO9 and 4&#39;,6-diamidino-2-phenylindole [DAPI]), which stain all bacteria, with membrane-impermeable fluorescent dyes (propidium iodide and SYTOX Green), which are only accessible to nonviable bacteria. Prior to eukaryotic cell permeabilization, an antibody or fluorescent reagent is added to identify extracellular bacteria. Thus these assays discriminate the viability of bacteria adherent to and inside eukaryotic cells. A protocol is also provided for using the viability dyes in combination with fluorescent antibodies to eukaryotic cell markers, in order to determine the subcellular localization of individual bacteria. The bacterial viability dyes discussed in this article are a sensitive complement and/or alternative to traditional microbiology techniques to evaluate the viability of individual bacteria and provide information regarding where bacteria survive in host cells.

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells.

Fluorescencia métodos de microscopía para determinar la viabilidad de las bacterias en asociación con las células de mamíferos

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols ...

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the respective immune response to each single pathogen and may thereby affect pathogenesis and disease outcome. Coinfected patients may also respond differentially to anti-infective interventions. Coinfection between tuberculosis as caused by mycobacteria and the malaria parasite Plasmodium, both of which are coendemic in many parts of sub-Saharan Africa, has not been studied in detail. In order to approach the challenging but scientifically and clinically highly relevant question how malaria-tuberculosis coinfection modulate host immunity and the course of each disease, we established an experimental mouse model that allows us to dissect the elicited immune responses to both pathogens in the coinfected host. Of note, in order to most precisely mimic naturally acquired human infections, we perform experimental infections of mice with both pathogens by their natural routes of infection, i.e. aerosol and mosquito bite, respectively.

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Tuberculosis and malaria are two of the most prevalent infections in humans and major causes of morbidity and mortality in impoverished populations in the tropics. We established an experimental model system to study outcome of malaria-tuberculosis coinfection in mice after challenge with both pathogens via their natural route of infection.

Un modelo experimental para estudiar la tuberculosis-Malaria Coinfección sobre Transmisión Natural de Mycobacterium tuberculosis Y Plasmodium berghei</i

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the ...

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 ...

Using Zebrafish Models of Human Influenza A Virus Infections to Screen Antiviral Drugs and Characterize Host Immune Cell Responses

Each year, seasonal influenza outbreaks profoundly affect societies worldwide. In spite of global efforts, influenza remains an intractable healthcare burden. The principle strategy to curtail infections is yearly vaccination. In individuals who have contracted influenza, antiviral drugs can mitigate symptoms. There is a clear and unmet need to develop alternative strategies to combat influenza. Several animal models have been created to model host-influenza interactions. Here, protocols for generating zebrafish models for systemic and localized human influenza A virus (IAV) infection are described. Using a systemic IAV infection model, small molecules with potential antiviral activity can be screened. As a proof-of-principle, a protocol that demonstrates the efficacy of the antiviral drug Zanamivir in IAV-infected zebrafish is described. It shows how disease phenotypes can be quantified to score the relative efficacy of potential antivirals in IAV-infected zebrafish. In recent years, there has been increased appreciation for the critical role neutrophils play in the human host response to influenza infection. The zebrafish has proven to be an indispensable model for the study of neutrophil biology, with direct impacts on human medicine. A protocol to generate a localized IAV infection in the Tg(mpx:mCherry) zebrafish line to study neutrophil biology in the context of a localized viral infection is described. Neutrophil recruitment to localized infection sites provides an additional quantifiable phenotype for assessing experimental manipulations that may have therapeutic applications. Both zebrafish protocols described faithfully recapitulate aspects of human IAV infection. The zebrafish model possesses numerous inherent advantages, including high fecundity, optical clarity, amenability to drug screening, and availability of transgenic lines, including those in which immune cells such as neutrophils are labeled with fluorescent proteins. The protocols detailed here exploit these advantages and have the potential to reveal critical insights into host-IAV interactions that may ultimately translate into the clinic.

Systemic and localized zebrafish infection models for human influenza A virus are demonstrated. Using a systemic infection model, zebrafish can be used to screen antiviral drugs. Using a localized infection model, zebrafish can be used to characterize host immune cell responses.

El uso de modelos de pez cebra de Infecciones por Virus de la Influenza Humana A a la pantalla medicamentos antivirales y caracterizar inmune del huésped respuestas de las células

Each year, seasonal influenza outbreaks profoundly affect societies worldwide. In spite of global efforts, influenza remains an intractable healthcare ...

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte extravasation might result in pathophysiological changes in the CNS. Thus, investigation of lymphocyte migration into the CNS is important to understand inflammatory CNS diseases and to develop new therapy approaches. Here we present an in vitro model of the human blood-brain barrier to study lymphocyte extravasation. Human brain microvascular endothelial cells (HBMEC) are confluently grown on a porous polyethylene terephthalate transwell insert to mimic the endothelium of the blood-brain barrier. Barrier function is validated by zonula occludens immunohistochemistry, transendothelial electrical resistance (TEER) measurements as well as analysis of evans blue permeation. This model allows investigation of the diapedesis of rare lymphocyte subsets such as CD56brightCD16dim/- NK cells. Furthermore, the effects of other cells, cytokines and chemokines, disease-related alterations, and distinct treatment regimens on the migratory capacity of lymphocytes can be studied. Finally, the impact of inflammatory stimuli as well as different treatment regimens on the endothelial barrier can be analyzed.

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro.

Análisis de linfocitos extravasación El uso de una<em&gt; In Vitro</em&gt; Modelo de la barrera sangre-cerebro humano

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

The WinCF Model - An Inexpensive and Tractable Microcosm of a Mucus Plugged Bronchiole to Study the Microbiology of Lung Infections

Many chronic airway diseases result in mucus plugging of the airways. Lungs of an individual with cystic fibrosis are an exemplary case where their mucus-plugged bronchioles create a favorable habitat for microbial colonization. Various pathogens thrive in this environment interacting with each other and driving many of the symptoms associated with CF disease. Like any microbial community, the chemical conditions of their habitat have a significant impact on the community structure and dynamics. For example, different microorganisms thrive in differing levels of oxygen or other solute concentrations. This is also true in the CF lung, where oxygen concentrations are believed to drive community physiology and structure. The methods described here are designed to mimic the lung environment and grow pathogens in a manner more similar to that from which they cause disease. Manipulation of the chemical surroundings of these microbes is then used to study how the chemistry of lung infections governs its microbial ecology. The method, called the WinCF system, is based on artificial sputum medium and narrow capillary tubes meant to provide an oxygen gradient similar to that which exists in mucus-plugged bronchioles. Manipulating chemical conditions, such as the media pH of the sputum or antibiotics pressure, allows for visualization of the microbiological differences in those samples using colored indicators, watching for gas or biofilm production, or extracting and sequencing the nucleic acid contents of each sample.

The mucus plugged airways of cystic fibrosis (CF) patients are an ideal environment for microbial pathogens to thrive. The manuscript describes a novel method for studying the CF lung microbiome in an environment that mimics where they cause disease and how alterations of chemical conditions can drive microbial dynamics.

El modelo de WinCF - un microcosmos barato y tractable de un bronquio enchufado del moco para estudiar la microbiología de las infecciones pulmonares

Many chronic airway diseases result in mucus plugging of the airways. Lungs of an individual with cystic fibrosis are an exemplary case where their ...

Confocal Imaging of Double-Stranded RNA and Pattern Recognition Receptors in Negative-Sense RNA Virus Infection

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors (PRRs) such as the retinoic acid (RIG-I) like receptors (RLRs) RIG-I and melanoma differentiation-associated protein 5 (MDA-5) leads to the induction of the innate immune response. The formation and intracellular distribution of dsRNA in positive-sense RNA virus infection has been well characterized by microscopy. Many negative-sense RNA viruses, including some arenaviruses, trigger the innate immune response during infection. However, negative-sense RNA viruses were thought to produce low levels of dsRNA, which hinders the imaging study of PRR recognition of viral dsRNA. Additionally, infection experiments with highly pathogenic arenaviruses must be performed in high containment biosafety level facilities (BSL-4). The interaction between viral RNA and PRRs for highly pathogenic RNA virus is largely unknown due to the additional technical challenges that researchers need to face in the BSL-4 facilities. Recently, a monoclonal antibody (Mab) (clone 9D5) originally used for pan-enterovirus detection has been found to specifically detect dsRNA with a higher sensitivity than the traditional J2 or K1 anti-dsRNA antibodies. Herein, by utilizing the 9D5 antibody, we describe a confocal microscopy protocol that has been used successfully to visualize dsRNA, viral protein and PRR simultaneously in individual cells infected by arenavirus. The protocol is also suitable for imaging studies of dsRNA and PRR distribution in pathogenic arenavirus infected cells in BSL4 facilities.

Double-stranded RNA produced during RNA virus replication can be recognized by pattern recognition receptors to induce an innate immune response. For negative-sense RNA viruses, the interaction between the low-level dsRNA and PRRs remains unclear. We have developed a confocal microscopy method to visualize arenavirus dsRNA and PRR in individual cells.

Proyección de imagen confocal de ARN bicatenario y receptores de reconocimiento de patrón en la infección por Virus de ARN de sentido negativo

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors ...