This work was funded by the Creative Inquiry Program for Undergraduate Research and the Calhoun Honors College at Clemson University.

1. general Tripanosoma Ganadería <ol><li> Pesar sólidos para la preparación de medios SDM79 (Tabla 1). </li><li> Almacenar a 4 ° C en 50 ml cónico o una bolsa Ziploc. NOTA: Los reactivos son estables durante al menos 6 meses. </li><li> Descongelar el suero fetal bovino (FBS) en un baño de agua a 37 ° C, y mezclar periódicamente por inversión. NOTA: FBS se recibe del proveedor como una solución estéril. Filtro esterilización FBS reduce su capacidad para apoyar el crecimiento del parásito. </li><li> Una vez que la botella entera se descongela, el calor inactiva suero durante 30 min en un baño de agua a 56 ° C, mezclando periódicamente para minimizar la precipitación de los componentes del suero. </li><li> Descongelar penicilina / solución de estreptomicina (pen / strep), hemina (2 mg / ml en NaOH 50 mM), y la solución de vitamina medio basal de Eagle a temperatura ambiente. </li><li> Para unos 1000 ml probeta graduada, añadir 20,2 g de sólidos SDM79 (Tabla 1) a los componentes líquidos (Tabla 2) y mezclar bien en una placa de agitación. </li><li> Ajustar el pH a 7,35, y llevar el volumen a 850 ml con agua. </li><li> Filtrar esterilizar medios por conectar una manguera de vacío a la parte inferior de una botella de filtro. Aplicar vacío hasta que se filtra la solución. </li><li> Retire la parte superior del filtro en la cabina de bioseguridad y añadir 150 ml de FBS inactivado por calor. Quite cubierta de plástico de la tapa y la botella estéril de medios de sellado. </li><li> Preparar medios SDM80 de la misma manera como SDM79 con las siguientes excepciones; no agregue la glucosa o la glucosamina y utilizar MEM sin glutamina. En lugar de 150 ml de FBS inactivado por calor, añadir 135 ml de dializado, FBS inactivado por calor, y 15 ml de FBS inactivado por calor. </li><li> Cultura parásitos PCF en 25 cm 2 matraz con 10 ml de medio apropiado a 27 ° C y 5% de CO 2. Nota: Las celdas deben contarse todos los días usando un hemocitómetro o un citómetro de flujo (ver sección 6) y las culturas deben mantenerse en densidades entre 1 x 10 5 y 5 x 10 6 células / ml. </li></ol> 2. Transfection de PCF parásitos con el constructo indicador fluorescente NOTA: Para seguir glicosoma dinámica, una proteína indicadora que contiene la secuencia de dirección peroxisomal (PTS2) de aldolasa fusionado a la proteína fluorescente amarilla mejorada se expresa en los parásitos. La secuencia que codifica la proteína de fusión se clonó en pXS2bla 12, que contiene el promotor procyclin y las regiones intergénicas de tubulina, que la recombinación homóloga directa en el genoma y el gen de resistencia a blasticidina para la selección. Líneas celulares que albergan procíclico los genes que codifican la polimerasa y la tetraciclina represor T7 (PF29-13) se transforman con la construcción de la orientación, pXS2: PTS2eYFP. <ol><li> Preparar citomix mezclando los componentes enumerados en la Tabla 3. Filtro esterilizar y almacenar a temperatura ambiente. </li><li> Linealizar el ADN plásmido con MluI pipeteando 10 l de ADN (1 mg / l), 5 l de tampón de enzima de restricción, agua 33 l, y 2 μ; L de enzima MluI en un tubo de microcentrífuga. Incubar a 37 ° C durante 1 hr. </li><li> Purifica la digestión de restricción mediante la adición de 1 volumen de tampón de unión a la enzima de digestión de restricción. Mezclar tampón de unión y el ADN digerido mediante agitación con vórtex. Centrifugar brevemente para recoger la muestra en la parte inferior del tubo. </li><li> Inserte un ADN vinculante columna en un tubo de recogida de 2 ml, y la transferencia de ADN digerido / solución tampón a la columna de unión. </li><li> Centrifugar para 10.000 xg durante 1 min. Después de la centrifugación, desechar el tubo de recogida de filtrado y devolver la columna para el tubo de recogida. </li><li> Añadir 700 l de tampón de lavado SPW y se centrifuga a 10.000 xg durante 1 min. </li><li> Descartar filtrado de tubo de recogida, de regreso a la columna del tubo de recogida y repetir SPW etapa de lavado y centrifugación. </li><li> Descartar filtrado, la columna de retorno a tubo de recogida, y se centrifuga la columna de vacío durante 3 min a 10.000 xg para eliminar el etanol residual. </li><li> En un gabinete de bioseguridad, deseche coltubo de recolección y la columna a cabo en un tubo de microcentrífuga estéril. </li><li> Para eluir el ADN, añadir 25 l de agua estéril a la columna y se incuba a temperatura ambiente durante 2 min. Centrifugar a 10.000 xg durante 1 min. </li><li> Añadir otros 25 l de agua estéril en la campana de bioseguridad a la columna y se incuba a temperatura ambiente durante 2 min. </li><li> Se centrifuga a 10.000 xg velocidad durante 1 min. </li><li> En una cabina de bioseguridad transferir la totalidad de volumen de ADN a un tubo nuevo de microcentrífuga estéril. NOTA: Este paso evita la contaminación de la tapa abierta durante la centrifugación. </li><li> Añadir la,, el ADN linealizado estéril purificada (10 g) a 400 l de Cytomix esterilizada por filtración. </li><li> Contar las células como se describe en la sección 6 y cosechar 5 7 -10 8 x10 células por centrifugación a 800 g durante 15 minutos a temperatura ambiente. </li><li> Retirar el sobrenadante y resuspender el sedimento celular en 450 l de ADN + citomix usando un 1 ml pipeta serológica. </li><li> Solución de transferencia que contiene ce<html> LLS, ADN, y Cytomix a una cubeta de 4 mm brecha y lugar cubeta estéril en la cámara de electroporación. </li><li> Seleccione &quot;decaimiento exponencial&quot; e introduzca manualmente los siguientes parámetros: Voltaje: 1,5 kV, la capacitancia: 25 mF, Resistencia: Ω, y la cubeta: 4 mm. Prensa pulso. Una vez que el pulso es completa, Sacar la cubeta de la cámara de electroporación y volver a la cabina de bioseguridad. </li><li> En un nuevo frasco estéril, una pipeta 10 ml de SDM79. Transferencia de las células transformadas al matraz con 10 ml de SDM79. </li><li> Incubar sin selección de drogas durante 24 horas a 27 ° C, 5% de CO 2. Después de 24 horas, añadir G418 (15 mg / ml), higromicina (50 g / ml), y blasticidina (10 g / ml). Pase 1 ml de células transformadas con el fármaco en 9 ml de SDM79 con el fármaco. </li><li> Analizar las células diaria como se describe en las secciones 6 y 7 Cuando las células son fluorescentes, y han alcanzado una densidad celular de 1 x 10 7 / ml, hacer estabilizados para almacenamiento a largo plazo (3.1 a 3.3). </li></ol>ve_title &quot;&gt; 3. Hacer estabilizados <ol><li> Para hacer que la congelación de los medios de comunicación, añadir un volumen igual de glicerol al 100% a Cytomix y el filtro de esterilizar. </li><li> Añadir 200 l de los medios de congelación a 800 l de células (1 x 10 7) en un criovial, mezclar suavemente por inversión y el lugar entre dos bastidores de espuma de poliestireno y congelar a -80 ° C. </li><li> Una vez congelado (~ 24 horas), las células de transferencia a nitrógeno líquido. NOTA: Es importante mover celdas en LN 2 después de 24 horas, como un almacenamiento más prolongado a -80 ° C conduce a una disminución de la viabilidad celular. </li></ol> 4. Existencias descongelar <ol><li> Eliminar vial congelado de -80 ° C y descongelación a temperatura ambiente durante aproximadamente 10 min. </li><li> Pipetear 9 ml de SDM79 con drogas en un nuevo frasco estéril. Añadir las células descongeladas a este frasco. Pasar las células 1:10 añadiendo 1 ml de este cultivo a 9 ml de SDM79 en un nuevo matraz (concentración final de 1 x 10 5 / ml). </li><li> Antes de que las células alcanzan una densidad de 10 7 / ml, comience a establecer citómetroy ensayos de dilución. </li></ol> 5. citómetro de configuración <ol><li> Encienda el citómetro de flujo y abrir el programa CFlow Plus. NOTA: Antes de ejecutar cualquier muestra, los fluidos deben ser toma retroactiva y enjuagarse de acuerdo a los pasos 5.2 a 5.7. </li><li> Vuelva a colocar el tubo de agua nanopura en la que la SIP se almacena con un tubo vacío. </li><li> Seleccione el botón de &quot;autolimpieza&quot;. </li><li> Una vez que toma retroactiva, retire el tubo de microcentrífuga de sorbo y descarte. </li><li> Colocar un nuevo tubo de microcentrífuga que contiene 1 ml de agua nueva nanopura en el SIP. </li><li> Seleccione un nuevo pozo en el programa CFlow. En &quot;Límites Run&quot; establecer el tiempo de 2 min. En &quot;Fluidics&quot; seleccione &quot;rápida&quot; y &quot;Run&quot;. </li><li> Una vez que el límite de tiempo de 2 min se haya completado, haga clic en el botón &quot;Eliminar datos de ejemplo&quot;. </li></ol> 6. Recuento celular utilizando el citómetro de flujo <ol><li> Reemplazar el agua con la muestra a ser contado. Ajuste &quot;Límites Run &quot;a 30 seg,&quot; Fluidics &quot;a&quot; rápida &quot;y seleccione&quot; Ejecutar &quot;. </li><li> Después de que el límite de tiempo de 30 segundos es completa, CFlow además proporcionará los eventos / l en &quot;Última ejecución.&quot; Conteo de repetición para cada cultura. NOTA: Antes de células alcanzan 1 x 10 7 células / ml, proceder con el ensayo de dilución. Las células deben ser descontinuados después de completar un ensayo de dilución. Las células cultivadas durante períodos más largos pierden su capacidad de responder a las condiciones ambientales. </li></ol> 7. midiendo la fluorescencia usando el citómetro de flujo <ol><li> Para evaluar la fluorescencia, establecer &quot;límites Run&quot; a 10.000 eventos, y &quot;Fluidics&quot; a &quot;lentas&quot;. Seleccione &quot;umbral de Ajuste&quot;. En &quot;umbral básico&quot;, seleccione &quot;Permanentemente eliminar eventos en&quot;, &quot;FSC-H&quot; (en caja de abajo) y entrar a menos de 30.000. Haga clic en &quot;Aplicar&quot;, luego en &quot;Cerrar&quot;. </li><li> Seleccione el220; &quot;botón en una de las nuevas ventanas de trazado y seleccione&quot; Histograma FSC-A &quot;Desde la lista desplegable, seleccione&quot; FL1-A &quot;. NOTA: medidas FL1-A fluorescencia en la longitud de onda de 530 nm. </li><li> Seleccione &quot;Ejecutar&quot; y repita para todas las culturas. </li><li> Ejecutar la solución de limpieza a través de la línea de fluidos durante 2 min y el agua durante 2 min. </li></ol> 8. dilución ensayos <ol><li> Pasar las células a una densidad de 1 x 10 5 células / ml en 3 ml de SDM79 en un matraz de 25 cm 2 cultura y medir fluorescencia inmediatamente después del paso y luego a 3 hr y 24 hr. </li></ol> Análisis de datos 9. <ol><li> Seleccione &quot;Analyze&quot; pestaña en CFlow Plus. </li><li> Haga clic aparecerá el botón &quot;Histograma&quot; en &quot;Hacer una nueva parcela&quot;. Seleccione &quot;FSC-A&quot; y una lista desplegable. Seleccione el canal &quot;FL1-A.&quot; </li><li> Resalte un bien de analizar. Seleccione el botón &quot;Gate&quot;, y dibujar manualmente una puertapara la población de interés. </li><li> Flow Plus proporcionará celular &quot;Count&quot; dentro de esta puerta y &quot;% de esta trama&quot;. </li></ol>

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The authors have nothing to disclose.

Glycosomes son, orgánulos específicos del parásito esenciales y dinámicos. Los procesos que regulan la biogénesis, mantenimiento, proliferación y remodelación de estos orgánulos probable incluyen objetivos farmacológicos que podrían ser explotadas con fines terapéuticos. A pesar de la posibilidad de alta abundancia de tales objetivos farmacológicos, el campo de glicosoma biogénesis se ha quedado atrás el estudio de procesos similares en otros organismos, principalmente debido a la falta de un sistema de al...

Trypanosoma brucei causa la enfermedad del sueño africana en los seres humanos y una enfermedad degenerativa, nagana, en el ganado bovino. Los fármacos utilizados en el tratamiento de estas enfermedades son anticuadas y extremadamente tóxico, las vacunas no están disponibles, y el potencial para el desarrollo de resistencia a los medicamentos requiere la búsqueda de nuevas dianas farmacológicas 1. Durante su ciclo de vida, T. brucei, se alterna entre un insecto vector y hospedador mamífero; dos hosts que se presentan muy diferentes entornos en los que el parásito tiene que sobrevivir. Una serie de cambios metabólicos y morfológicos se producen como el parásito está expuesto a diferentes condiciones ambientales. Algunos de los cambios más dramáticos se observan en parásitos microcuerpos específicos de una sola membrana limitada, denominada glycosomes 13. Los niveles de glucosa son relativamente altos (~ 5 mm) en el torrente sanguíneo y parásitos en el torrente sanguíneo (BSF) generan ATP exclusivamente a través de wh glucólisisile metabolismo mitocondrial es reprimida 14. A diferencia de otros eucariotas en el que la glucólisis se produce en el citoplasma, T. brucei compartimenta mayoría de las enzimas glucolíticas en glycosomes 14,15. Los parásitos son absorbidos por la mosca tsetsé durante una harina de sangre y experimentan una caída en la glucosa, que cae a niveles indetectables en los 15 minutos de ser ingerido por la mosca. El metabolismo de los insectos, la forma procíclica (PCF), parásitos es más flexible y glucosa, así como aminoácidos tales como prolina, se pueden utilizar en la síntesis de ATP 16-18. Estudios proteómicos comparativos revelan cambios de ciclo de vida que dependen en glicosomal y proteínas mitocondriales con proteínas glucolíticos aumento en los parásitos del torrente sanguíneo y proteínas mitocondriales implicadas en el ciclo TCA y la cadena respiratoria 13,19. Mientras que muchos estudios se han centrado en las diferencias entre BSF y glycosomes PCF, se sabe poco sobre los cambios en glycosomes PCF que se producen en respuesta a envcambios ironmental. En el intestino grueso de la marcha, los niveles de glucosa son bajos con aumentos transitorios durante una alimentación 20. En la mayoría de los estudios in vitro, los parásitos PCF se cultivan en medios que contienen glucosa. Sin embargo, estudios recientes han demostrado que los cambios del metabolismo PCF significativamente en respuesta a glucosa disponibilidad 17. En ausencia de glucosa, la captación de prolina y prolina aumento de la actividad deshidrogenasa 18. Este cambio en el metabolismo mitocondrial es probable que acompañado por un cambio en la composición y la morfología glicosoma, sin embargo, esto no ha sido evaluado directamente. Electron y microscopía de fluorescencia se utilizan técnicas comunes para estudiar la dinámica glicosoma en T. brucei 2,21-24. Estos protocolos son el tiempo y mano de obra intensiva, caro, y difícil de adaptar a los estudios en tiempo real y protocolos de alto rendimiento. Para superar esta limitación, un sistema indicador fluorescente u orgánulosed para estudiar orgánulos en los sistemas de mamíferos y levaduras se ha modificado para su uso en T. brucei 12. Sistemas de indicador fluorescente orgánulos se han utilizado ampliamente en eucariotas superiores tales como levadura, planta, y células de mamífero 25-27. En tales sistemas, una proteína fluorescente se fusiona a una secuencia de aminoácidos que se dirige a la proteína a orgánulos específicos. La degradación o síntesis de las proteínas específicas se mide a través de la fluorescencia y los cambios en la composición del orgánulo se reflejan en cambios en la fluorescencia celular. Cuando la fase de lectura abierta de la proteína fluorescente amarilla mejorada (eYFP) se fusiona con una secuencia focalización peroxisomal tipo II (PTS2) 12, la proteína PTS2eYFP se importa a, glycosomes maduros-importación competente y de fluorescencia pueden ser monitoreados mediante citometría de flujo. Las variaciones en la composición glicosoma se reflejan en cambios en la fluorescencia celular. Este sistema puede ayudar a resolving los mecanismos que regulan los cambios inducidos por el medio ambiente en glicosoma composición. Este manuscrito describe la generación de un sistema reportero glicosoma en los parásitos PCF en conjunción con la citometría de flujo para controlar la dinámica de glicosoma en tiempo real en parásitos vivos y proporciona un ejemplo de cómo se ha utilizado para seguir los cambios en glicosoma composición en respuesta a diferentes entornos. En resumen, glicosoma composición está influenciada por las concentraciones de glucosa extracelular y paso de cultivos en fase logarítmica en medio fresco provoca cambios en glicosoma composición. Este sistema puede ser modificado para estudiar el comportamiento dinámico de otros orgánulos en tripanosomas y otros parásitos.

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using fluorescent proteins to monitor glycosome dynamics in the african trypanosome

Trypanosoma brucei es un parásito kinetoplastid que causa tripanosomiasis humana africana (THA), o enfermedad del sueño, y una enfermedad degenerativa, nagana, en el ganado bovino 1. Los suplentes de parásitos entre el torrente sanguíneo del huésped mamífero y el vector de la mosca tsetsé. La composición de muchos orgánulos celulares cambia en respuesta a las diferentes condiciones extracelulares 2-5. Glycosomes son los peroxisomas altamente especializados en los que muchas de las enzimas que intervienen en la glucólisis son compartimentados. Cambios en la composición glicosoma de una manera regulada en el desarrollo y el medio ambiente 4-11. Actualmente, las técnicas más comunes que se utilizan para estudiar glicosoma dinámica es la microscopía electrónica y de fluorescencia; técnicas que son caros, tiempo y mano de obra intensiva, y no adaptarse fácilmente a los análisis de alto rendimiento. Para superar estas limitaciones, un sistema indicador fluorescente en glicosomaque mejoró la proteína fluorescente amarilla (eYFP) se fusiona a una secuencia de peroxisomas focalización (PTS2), que dirige la proteína de fusión a glycosomes 12, se ha establecido. Sobre la importación de la proteína de fusión PTS2eYFP, glycosomes vuelven fluorescentes. Orgánulos la degradación y los resultados de reciclaje en la pérdida de la fluorescencia que puede ser medido por citometría de flujo. Un gran número de células (5000 células / seg) se pueden analizar en tiempo real sin extensa preparación de la muestra tales como la fijación y el montaje. Este método ofrece una forma rápida de detectar cambios en la composición orgánulo en respuesta a las fluctuaciones de las condiciones ambientales.

En este sistema, se observó un cambio dependiente de la glucosa en glicosoma composición. Cuando las células se cultivan en medios que contienen glucosa, se observaron dos poblaciones; uno brillante y una tenue (Figura 2A). Células Dim albergan glycosomes inmaduras, que no han importado la PTS2eYFP mientras que las células brillantes albergan una mezcla de glycosomes maduros e inmaduros 12. Cuando la glucosa está presente en los medios de comunicación, mislocalization de glicosoma prot...

Watch this Scientific Journal Video about Using Fluorescent Proteins to Monitor Glycosome Dynamics in the African Trypanosome at JoVE.com

Using Fluorescent Proteins to Monitor Glycosome Dynamics in the African Trypanosome

Glycosome dynamics in African trypanosomes are difficult to study by traditional cell biology techniques such as electron and fluorescence microscopy. As a means of observing dynamic organelle behavior, a fluorescent-organelle reporter system has been used in conjunction with flow cytometry to monitor real-time glycosome dynamics in live parasites.

El uso de proteínas fluorescentes de Seguimiento glicosoma Dinámica en el tripanosoma africano

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in ...

using-fluorescent-proteins-to-monitor-glycosome-dynamics-african

Research

JoVE Journal

Immunology and Infection

8.9K vistas.  Clemson University Eukaryotic Pathogens Innovation Center. Glycosome dynamics in African trypanosomes are difficult to study by traditional cell biology techniques such as electron and fluorescence microscopy. As a means of observing dynamic organelle behavior, a fluorescent-organelle reporter system has been used in conjunction with flow cytometry to monitor real-time glycosome dynamics in live parasites.

Video: Using Fluorescent Proteins to Monitor Glycosome Dynamics in the African Trypanosome

The Use of Carboxyfluorescein Diacetate Succinimidyl Ester (CFSE) to Monitor Lymphocyte Proliferation

Carboxyfluorescein succinimidyl ester (CFSE) is an effective and popular means to monitor lymphocyte division1-3. CFSE covalently labels long-lived intracellular molecules with the fluorescent dye, carboxyfluorescein. Thus, when a CFSE-labeled cell divides, its progeny are endowed with half the number of carboxyfluorescein-tagged molecules and thus each cell division can be assessed by measuring the corresponding decrease in cell fluorescence via Flow cytometry. The capacity of CFSE to label lymphocyte populations with a high fluorescent intensity of exceptionally low variance, coupled with its low cell toxicity, make it an ideal dye to measure cell division. Since it is a fluorescein-based dye it is also compatible with a broad range of other fluorochromes making it applicable to multi-color flow cytometry. This article describes the procedures typically used for labeling mouse lymphocytes for the purpose of monitoring up to 8 cell divisions. These labeled cells can be used both for in vitro and in vivo studies.

The Use of Carboxyfluorescein Diacetate Succinimidyl Ester CFSE to Monitor Lymphocyte Proliferation

CFSE covalently labels long-lived intracellular molecules with the fluorescent dye, carboxyfluorescein. As such, when a CFSE-labeled cell divides, its progeny have half the amount of fluorescence, which can thereby be used to assess cell division. This article describes the procedures typically used for labeling mouse lymphocytes with CFSE.

El uso de diacetato de carboxifluoresceína Ester succinimidil (CFSE) para controlar la proliferación de linfocitos

Carboxyfluorescein succinimidyl ester (CFSE) is an effective and popular means to monitor lymphocyte division1-3.  CFSE covalently labels long-lived ...

Investigación

Inmunología e Infección

A Visual Assay to Monitor T6SS-mediated Bacterial Competition

Type VI secretion systems (T6SSs) are molecular nanomachines allowing Gram-negative bacteria to transport and inject proteins into a wide variety of target cells1,2. The T6SS is composed of 13 core components and displays structural similarities with the tail-tube of bacteriophages3. The phage uses a tube and a puncturing device to penetrate the cell envelope of target bacteria and inject DNA. It is proposed that the T6SS is an inverted bacteriophage device creating a specific path in the bacterial cell envelope to drive effectors and toxins to the surface. The process could be taken further and the T6SS device could perforate other cells with which the bacterium is in contact, thus injecting the effectors into these targets. The tail tube and puncturing device parts of the T6SS are made with Hcp and VgrG proteins, respectively4,5.
The versatility of the T6SS has been demonstrated through studies using various bacterial pathogens. The Vibrio cholerae T6SS can remodel the cytoskeleton of eukaryotic host cells by injecting an "evolved" VgrG carrying a C-terminal actin cross-linking domain6,7. Another striking example was recently documented using Pseudomonas aeruginosa which is able to target and kill bacteria in a T6SS-dependent manner, therefore promoting the establishment of bacteria in specific microbial niches and competitive environment8,9,10.
In the latter case, three T6SS-secreted proteins, namely Tse1, Tse2 and Tse3 have been identified as the toxins injected in the target bacteria (Figure 1). The donor cell is protected from the deleterious effect of these effectors via an anti-toxin mechanism, mediated by the Tsi1, Tsi2 and Tsi3 immunity proteins8,9,10. This antimicrobial activity can be monitored when T6SS-proficient bacteria are co-cultivated on solid surfaces in competition with other bacterial species or with T6SS-inactive bacteria of the same species8,11,12,13.
The data available emphasized a numerical approach to the bacterial competition assay, including time-consuming CFU counting that depends greatly on antibiotic makers. In the case of antibiotic resistant strains like P. aeruginosa, these methods can be inappropriate. Moreover, with the identification of about 200 different T6SS loci in more than 100 bacterial genomes14, a convenient screening tool is highly desirable. We developed an assay that is easy to use and requires standard laboratory material and reagents. The method offers a rapid and qualitative technique to monitor the T6SS-dependent bactericidal/bacteriostasis activity by using a reporter strain as a prey (in this case Escherichia coli DH5&alpha;) allowing a-complementation of the lacZ gene. Overall, this method is graphic and allows rapid identification of T6SS-related phenotypes on agar plates. This experimental protocol may be adapted to other strains or bacterial species taking into account specific conditions such as growth media, temperature or time of contact.

We describe a qualitative assay to monitor bacterial competition mediated by the Pseudomonas aeruginosa type VI secretion system (T6SS). The assay relies on the survival/killing of Escherichia coli target cells carrying a lacZ-reporter. This technique is adjustable to assess the bactericidal/bacteriostasis activity of T6SS-proficient microorganisms.

Un ensayo visual para monitorear T6SS mediada por la competencia bacteriana

Type VI secretion systems (T6SSs) are molecular  nanomachines allowing Gram-negative bacteria to transport and inject proteins  into a wide variety of ...

Use of In vivo Imaging to Monitor the Progression of Experimental Mouse Cytomegalovirus Infection in Neonates

Human Cytomegalovirus (HCMV or HHV-5) is a life-threatening pathogen in immune-compromised individuals. Upon congenital or neonatal infection, the virus can infect and replicate in the developing brain, which may induce severe neurological damage, including deafness and mental retardation. Despite the potential severity of the symptoms, the therapeutic options are limited by the unavailability of a vaccine and the absence of a specific antiviral therapy. Furthermore, a precise description of the molecular events occurring during infection of the central nervous system (CNS) is still lacking since observations mostly derive from the autopsy of infected children. Several animal models, such as rhesus macaque CMV, have been developed and provided important insights into CMV pathogenesis in the CNS. However, despite its evolutionary proximity with humans, this model was limited by the intracranial inoculation procedure used to infect the animals and consistently induce CNS infection. Furthermore, ethical considerations have promoted the development of alternative models, among which neonatal infection of newborn mice with mouse cytomegalovirus (MCMV) has recently led to significant advances. For instance, it was reported that intraperitoneal injection of MCMV to Balb/c neonates leads to infection of neurons and glial cells in specific areas of the brain. These findings suggested that experimental inoculation of mice might recapitulate the deficits induced by HCMV infection in children. Nevertheless, a dynamic analysis of MCMV infection of neonates is difficult to perform because classical methodology requires the sacrifice of a significant number of animals at different time points to analyze the viral burden and/or immune-related parameters. To circumvent this bottleneck and to enable future investigations of rare mutant animals, we applied in vivo imaging technology to perform a time-course analysis of the viral dissemination in the brain upon peripheral injection of a recombinant MCMV expressing luciferase to C57Bl/6 neonates.

Use of In vivo Imaging to Monitor the Progression of Experimental Mouse Cytomegalovirus Infection in Neonates

Human Cytomegalovirus (HCMV) infection of neonates represents an important cause of mental retardation, yet the molecular events leading to virus-induced pathogenesis are still poorly understood. To investigate the dynamics of brain infection, we adapted whole-animal in vivo imaging to perform time-course analysis of neonates infected with a luciferase-recombinant virus.

El uso de<em&gt; En vivo</em&gt; Imágenes para controlar la progresión de la infección por citomegalovirus Experimental del ratón en neonatos

Human Cytomegalovirus (HCMV or HHV-5) is a life-threatening  pathogen in immune-compromised individuals. Upon congenital or neonatal  infection, the virus ...

Using Fluorescent Proteins to Visualize and Quantitate Chlamydia Vacuole Growth Dynamics in Living Cells

The obligate intracellular bacterium Chlamydia elicits a great burden on global public health. C.&#160;trachomatis is the leading bacterial cause of sexually transmitted infection and also the primary cause of preventable blindness in the world. An essential determinant for successful infection of host cells by Chlamydia is the bacterium's ability to manipulate host cell signaling from within a novel, vacuolar compartment called the inclusion. From within the inclusion, Chlamydia acquire nutrients required for their 2-3 day developmental growth, and they additionally secrete a panel of effector proteins onto the cytosolic face of the vacuole membrane and into the host cytosol. Gaps in our understanding of Chlamydia biology, however, present significant challenges for visualizing and analyzing this intracellular compartment. Recently, a reverse-imaging strategy for visualizing the inclusion using GFP expressing host cells was described. This approach rationally exploits the intrinsic impermeability of the inclusion membrane to large molecules such as GFP. In this work, we describe how GFP- or mCherry-expressing host cells are generated for subsequent visualization of chlamydial inclusions. Furthermore, this method is shown to effectively substitute for costly antibody-based enumeration methods, can be used in tandem with other fluorescent labels, such as GFP-expressing Chlamydia, and can be exploited to derive key quantitative data about inclusion membrane growth from a range of Chlamydia species and strains.

Using Fluorescent Proteins to Visualize and Quantitate Chlamydia Vacuole Growth Dynamics in Living Cells

A live cell fluorescent protein based method for illuminating cellular vacuoles (inclusions) containing Chlamydia is described. This strategy enables rapid, automated determination of Chlamydia infectivity in samples and can be used to quantitatively investigate inclusion growth dynamics.

El uso de proteínas fluorescentes para visualizar y cuantificar<em&gt; Chlamydia</em&gt; Vacuola dinámica de crecimiento en las células vivas

The obligate intracellular bacterium Chlamydia elicits a great burden on global public health. C.&#160;trachomatis is the leading bacterial cause of ...

The Mesenteric Lymph Duct Cannulated Rat Model: Application to the Assessment of Intestinal Lymphatic Drug Transport

The intestinal lymphatic system plays key roles in fluid transport, lipid absorption and immune function. Lymph flows directly from the small intestine via a series of lymphatic vessels and nodes that converge at the superior mesenteric lymph duct. Cannulation of the mesenteric lymph duct thus enables the collection of mesenteric lymph flowing from the intestine. Mesenteric lymph consists of a cellular fraction of immune cells (99% lymphocytes), aqueous fraction (fluid, peptides and proteins such as cytokines and gut hormones) and lipoprotein fraction (lipids, lipophilic molecules and apo-proteins). The mesenteric lymph duct cannulation model can therefore be used to measure the concentration and rate of transport of a range of factors from the intestine via the lymphatic system. Changes to these factors in response to different challenges (e.g., diets, antigens, drugs) and in disease (e.g., inflammatory bowel disease, HIV, diabetes) can also be determined. An area of expanding interest is the role of lymphatic transport in the absorption of orally administered lipophilic drugs and prodrugs that associate with intestinal lipid absorption pathways. Here we describe, in detail, a mesenteric lymph duct cannulated rat model which enables evaluation of the rate and extent of lipid and drug transport via the lymphatic system for several hours following intestinal delivery. The method is easily adaptable to the measurement of other parameters in lymph. We provide detailed descriptions of the difficulties that may be encountered when establishing this complex surgical method, as well as representative data from failed and successful experiments to provide instruction on how to confirm experimental success and interpret the data obtained.

Here we describe a technique to cannulate the mesenteric lymph duct in rats which enables quantification of lipid and drug transport via the lymphatic system following intestinal delivery. The technique can be adapted to assess mesenteric lymph concentrations and/or transport of fluid, immune cells, peptides, proteins and lipophilic molecules.

La linfa mesentérica Conducto canulado Rata Modelo: Aplicación de la Evaluación de Intestinal Linfático Transporte de Drogas

The intestinal lymphatic system plays key roles in fluid transport, lipid absorption and immune function. Lymph flows directly from the small intestine ...

Bioluminescence Imaging to Detect Late Stage Infection of African Trypanosomiasis

Human African trypanosomiasis (HAT) is a multi-stage disease that manifests in two stages; an early blood stage and a late stage when the parasite invades the central nervous system (CNS). In vivo study of the late stage has been limited as traditional methodologies require the removal of the brain to determine the presence of the parasites.
Bioluminescence imaging is a non-invasive, highly sensitive form of optical imaging that enables the visualization of a luciferase-transfected pathogen in real-time. By using a transfected trypanosome strain that has the ability to produce late stage disease in mice we are able to study the kinetics of a CNS infection in a single animal throughout the course of infection, as well as observe the movement and dissemination of a systemic infection.
Here we describe a robust protocol to study CNS infections using a bioluminescence model of African trypanosomiasis, providing real time non-invasive observations which can be further analyzed with optional downstream approaches.

This manuscript describes the use of a bioluminescent strain of African trypanosomes to enable the tracking of late stage infection and demonstrates how in vivo live imaging can be used to visualize infections within the central nervous system in real-time.

Imágenes de bioluminiscencia para detectar la infección en fase tardía de la tripanosomiasis africana

Human African trypanosomiasis (HAT) is a multi-stage disease that manifests in two stages; an early blood stage and a late stage when the parasite invades ...

"Phagosome Closure Assay" to Visualize Phagosome Formation in Three Dimensions Using Total Internal Reflection Fluorescent Microscopy (TIRFM)

Phagocytosis is a mechanism used by specialized cells to internalize and eliminate microorganisms or cellular debris. It relies on profound rearrangements of the actin cytoskeleton that is the driving force for plasma membrane extension around the particle. In addition, efficient engulfment of large material relies on focal exocytosis of intracellular compartments. This process is highly dynamic and numerous molecular players have been described to have a role during phagocytic cup formation. The precise regulation in time and space of all of these molecules, however, remains elusive. In addition, the last step of phagosome closure has been very difficult to observe because inhibition by RNA interference or dominant negative mutants often results in stalled phagocytic cup formation. 
We have set up a dedicated experimental approach using total internal reflection fluorescence microscopy (TIRFM) combined with epifluorescence to monitor step by step the extension of pseudopods and their tips in a phagosome growing around a particle loosely bound to a coverslip. This method allows us to observe, with high resolution the very tips of the pseudopods and their fusion during closure of the phagosome in living cells for two different fluorescently tagged proteins at the same time.

&quot;Phagosome Closure Assay&quot; to Visualize Phagosome Formation in Three Dimensions Using Total Internal Reflection Fluorescent Microscopy TIRFM

We describe an experimental setup to visualize with unprecedented high resolution phagosome formation and closure in three dimensions in living macrophages, using total internal reflection fluorescence microscopy. It allows monitoring of the base of the phagocytic cup, the extending pseudopods, as well as the precise site of phagosome scission.

&quot;Fagosoma Cierre de ensayo&quot; para visualizar Fagosoma Formación en tres dimensiones utilizando la reflexión interna total fluorescente Microscopía (TIRFM)

Phagocytosis is a mechanism used by specialized cells to internalize and eliminate microorganisms or cellular debris. It relies on profound rearrangements ...

Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), that carry foreign genes such as a reporter or marker protein in their genomes. These recombinant viruses usually faithfully mimic the life cycle of the original virus in infected cells and exhibit the same host range dependence. The development of a recombinant virus enables the efficient screening of inhibitors and the identification of specific host factors. However, to date the construction of recombinant hepatitis B virus (HBV) has been difficult because of various experimental limitations. The main limitation is the compact genome size of HBV, and a fairly strict genome size that does not exceed 1.3 genome sizes, that must be packaged into virions. Thus, the size of a foreign gene to be inserted should be smaller than 0.4 kb if no deletion of the genome DNA is to be performed. Therefore, to overcome this size limitation, the deletion of some HBV DNA is required. Here, we report the construction of recombinant HBV encoding a reporter gene to monitor the early stage of the HBV replication cycle by replacing part of the HBV core-coding region with the reporter gene by deleting part of the HBV pol coding region. Detection of recombinant HBV infection, monitored by the reporter activity, was highly sensitive and less expensive than detection using the currently available conventional methods to evaluate HBV infection. This system will be useful for a number of applications including high-throughput screening for the identification of anti-HBV inhibitors, host factors and virus-susceptible cells.

Here we describe a newly developed hepatitis B virus (HBV) reporter system to monitor the early stages of the HBV life cycle. This simplified in vitro system will aid in the screening of anti-HBV agents using a high-throughput strategy.

Desarrollo de un sistema reportero virus de la hepatitis B para supervisar las primeras etapas del ciclo de replicación

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple condensation reactions. DHDDS (dehydrodolichyl diphosphate synthase) is a eukaryotic long-chain cis-PT (forming cis double bonds from the condensation reaction) that catalyzes chain elongation of farnesyl diphosphate (FPP, an allylic diphosphate) via multiple condensations with isopentenyl diphosphate (IPP). DHDDS is of biomedical importance, as a non-conservative mutation (K42E) in the enzyme results in retinitis pigmentosa, ultimately leading to blindness. Therefore, the present protocol was developed in order to acquire large quantities of purified DHDDS, suitable for mechanistic studies. Here, the usage of protein fusion, optimized culture conditions and codon-optimization were used to allow the overexpression and purification of functionally active human DHDDS in E. coli. The described protocol is simple, cost-effective and time sparing. The homology of cis-PT among different species suggests that this protocol may be applied for other eukaryotic cis-PT as well, such as those involved in natural rubber synthesis.

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

A simple protocol for overexpression and purification of codon-optimized, human cis-prenyltransferase, under non-denaturing conditions, from Escherichia coli, is described, along with an enzymatic activity assay. This protocol can be generalized for production of other cis- prenyltransferase proteins in quantity and quality suitable for mechanistic studies.

Sobreexpresión y Purificación de Humanos<em&gt; Cis</em&gt; -preniltransferasa en<em&gt; Escherichia coli</em

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...

Visualization of Candida albicans in the Murine Gastrointestinal Tract Using Fluorescent In Situ Hybridization

Candida albicans is a fungal component of the gut microbiota in humans and many other mammals. Although C. albicans does not cause symptoms in most colonized hosts, the commensal reservoir does serve as a repository for infectious disease, and the presence of high fungal titers in the gut is associated with inflammatory bowel disease. Here, we describe a method to visualize C. albicans cell morphology and localization in a mouse model of stable gastrointestinal colonization. Colonization is established using a single dose of C. albicans in animals that have been treated with oral antibiotics. Segments of gut tissue are fixed in a manner that preserves the architecture of luminal contents (microorganisms and mucus) as well as the host mucosa. Finally, fluorescent in situ hybridization is performed using probes against fungal rRNA to stain for C. albicans and hyphae. A key advantage of this protocol is that it allows for simultaneous observation of C. albicans cell morphology and its spatial association with host structures during gastrointestinal colonization.

Visualization of Candida albicans in the Murine Gastrointestinal Tract Using Fluorescent In Situ Hybridization

The purpose of this protocol is to visualize Candida albicans cell shape and localization in the mammalian gastrointestinal tract.

Visualización de Candida albicans en el tracto gastrointestinal murino utilizando hibridación fluorescente in situ

Candida albicans is a fungal component of the gut microbiota in humans and many other mammals. Although C. albicans does not cause symptoms in most ...