Los autores desean agradecer a Richard Rest por el generoso regalo de anthrolysin O, Michael Caparon por el don generoso de los francos SLO plásmido y Jonathon de asistencia técnica. Este trabajo fue financiado por el NIH subvenciones T32CA82084 (PAK), y R01AI072083 (RDS).

1. Purificación de la estreptolisina O (SLO) <ol><li> Inocular 20 ml de cultivo durante la noche de células BL21 oro que contenía el plásmido pBADgIII SLOhis-9 en caldo LB 0,5 L y añadir 500 l 50 mg / ml de ampicilina. Agitar cultivo a 225 rpm a 37 ° C hasta OD 600 = 0,6, normalmente ~ 1,5 h. Centrifugar 1 ml de bacterias (considerados T 0) a 10.000 xg 5 min, se disuelve el pellet en 140 l 1x tampón de muestra de SDS / OD 1 y sonicado para ADN de cizallamiento para el análisis de proteínas pureza. </li><li> Inducir bacterias con la adición de 5 ml de 20% de arabinosa a la cultura, agitar a 225 rpm a temperatura ambiente durante 3 h. Recoger 1 ml de las bacterias, se centrifuga y se disuelve en tampón de muestra de SDS 1x como anteriormente para el punto de tiempo T 3 para el análisis de pureza. </li><li> Recoger las bacterias restantes en 500 ml botella de centrífuga, girar 12.000 xg durante 12 min a 4 ° C (8.500 rpm Sorvall rotor GS-3). Decantar el sobrenadante, tienda precipitado a -80 ° C durante la noche o más tiempo si es necesario. </li><li> Resuspender el frozen sedimento en hielo en 10 ml Lyse / Tampón de lavado (50 mM NaH 2 PO 4, 300 mM NaCl, 10 mM imidazol, pH 8,0) suplementado con 400 l 25% de Triton X-100, 100 l 100 mg / ml de lisozima, 50 l 200 mM de fluoruro de fenilmetilsulfonilo (PMSF). Transferir el precipitado resuspendido a 50 ml tubo Ridge, Oak. </li><li> Sonicar lisado 5 veces durante 30 segundos en hielo con intervalos de 30 seg. Centrifugación lisado sonicado en tubo Oak Ridge a 39.000 xg durante 20 min a 4 º C (18.000 rpm Sorvall rotor SS-34). </li><li> Mientras que el lisado está girando, lavar 1 ml de Ni-NTA agarosa con 10 ml Lyse / tampón de lavado y centrifugado a 1.200 rpm durante 5 min a 4 ° C. (Todos los lavados adicionales / eluciones utilizar centrifugación en estas condiciones). Aspirar lavar. </li><li> Añadir sobrenadante bacteriano procedente de la Etapa 1,5 a Ni-NTA agarosa, agitar suavemente durante 2,5 horas a 4 ° C. Girar para recoger el líquido sobrenadante. Combine 30 l de sobrenadante con 10 μ 4x SDS muestra de amortiguación y ahorrar para el análisis de pureza. Lavar las perlas 4 veces en Lyse / tampón de lavado y ence en Tris / sal tampón (50 mM Tris, 300 mM NaCl, pH 8,0). </li><li> Eluir 3 veces mediante la adición de 1 ml de tampón de elución (250 mM de imidazol en tampón de Tris / tampón de sal) y 5 l 1 M de ditiotreitol (DTT) a perlas, incubando en hielo durante 10 min, centrifugado y recogiendo el sobrenadante. SLO es una proteína muy sensible a redox, y se debe mantener en hielo en todo momento. Una vez reducida, la proteína perderá rápidamente su actividad si se calienta a 37 ° C, incluso durante un corto período de tiempo. </li><li> Lavar 500 l polimixina-conjugado de agarosa en tampón de elución, centrifugado a 10.000 xg durante 1 min a 4 ° C y se resuspende en 500 l. Para agotar endotoxina, añadir 200 perlas mu l a cada elución y agitar 4 ° C durante 30 min. Centrifugado a 10.000 xg durante 1 min a 4 ° C, y guardar el sobrenadante. Combinar 30 l de cada elución con 10 l de 4x tampón de muestra de SDS para análisis SDS-PAGE. </li><li> Probar la pureza de eluciones por SDS-PAGE. Hervir muestras se guarda para análisis de gel a 95 º C durante 5 min y 10 l de carga en un gel de 10%. Mancha de gel con Coomassie para visualizar las proteínas (Figura 2A). SLO es 69 kDa. Realizar un ensayo de Bradford para determinar la concentración de proteína (rendimiento típico es de 4 mg / ml para dos eluciones primero, pero puede variar según la cepa bacteriana y del plásmido utilizado). </li><li> Prueba de la actividad hemolítica de cada elución (véase más adelante). Eluciones piscina donde la actividad de pureza, concentración y hemolítica son satisfactorios. SLO alícuota en un solo uso alícuotas (l típicamente 5-10), congela en hielo seco y se almacena a -80 ° C. </li></ol> 2. Ensayo hemolítico <ol><li> Lavar glóbulos rojos de oveja (RBC) en RBC tampón de ensayo (0,3% albúmina de suero bovino (BSA), 2 mM CaCl 2, 10 mM Hepes, pH 7,4), centrifugado a 1.200 rpm durante 5 min y se resuspenden en 2,5% en los glóbulos rojos 10 ml de tampón de ensayo de RBC. </li><li> Añadir 10 l tampón de ensayo de RBC / pocillo en 96-y V-de la placa inferior en hielo. Diluciones seriadas cada elución por duplicado. Los rangos típicos de dilución son 1:1,000 a 1:512,000. Incluye 3 pozos con ninguna toxina y pozos 3 con 10l 2% de Triton X-100 como pozos mínimo y máximo, respectivamente. </li><li> Cubrir la placa e incubar a 37 ° C 30 min. Centrifugar a 1.200 rpm durante 5 min. Transferir 70 l de sobrenadante a una placa de fondo plano de 96 también. Lea A 405. Una unidad es la dilución de la toxina requerida para un 50% de lisis de los hematíes. Actividad de la toxina es de 1 unidad * dilución factor/0.01 ml. </li></ol> 3. Lítica celular Ensayo <ol><li> Cosecha, recuento de células, spin. Resuspender en 2x10 6 / ml en tampón de R / B (RPMI con 2 mM CaCl 2, 0,5% de BSA) y 20 ug / ml de yoduro de propidio. Añadir 100 células mu l a un 96-así fondo en V de la placa. </li><li> Diluir en serie SLO a 2x concentración final en tampón de R / B, añadir 100 l toxina o 100 l tampón de R / B a las células. Incubar 5 min 37 ° C. El rango típico concentraciones finales de 2.000 U / ml a 31,25 U / ml. </li><li> Ejecutar las células en citómetro de flujo y reunir datos con filtros para la ficoeritrina (PE). A cambio de un registro representa cel transitoriamente permeabilizadasls mientras que un cambio de log 3 indica las células muertas 4. Cálculo de la lisis específica de las células restando el porcentaje de células muertas en el control (% PI alta ctl) de la experimental (% exp PI alta), como sigue: lisis específica = (% exp PI alta -% PI alta ctl) / (100 -% PI alta ctl) * 100 </li></ol> 4. Micropipetas de entrega de la toxina a las células en placas de cultivo <ol><li> Un día antes del experimento, placa de 2x10 5 macrófagos en un colágeno recubierto con fondo de cristal plato de 35 mm. </li><li> Encienda el microscopio y microinyector. Permitir tiempo de la etapa calentada a calentar a 37 ° C. La elección del microscopio por lo general depende de lo que está disponible localmente. El microscopio tiene una fase invertida, una etapa capaz de calentar los platos a 37 ° C, de excitación / emisión cubos de filtros apropiados para el colorante elegido, y el espacio para conectar físicamente el microinyector. Una lente de BertrandEs útil para la microinyección, pero no es obligatorio. El ordenador de conducción del microscopio necesita suficiente memoria para recoger y almacenar datos. </li><li> Marcar células durante 30 minutos con colorante a 37 º C. Dependiendo del ensayo, el etiquetado puede hacerse con 5 Fura2 AM mu l en 1 ml de PBS o 2 l de calceína AM en 1 ml de medio completo. Para Fura2, excitación / emisión es recogida para 340/510 nm y 380/510, y la relación de 340/380 señales determina el flujo de calcio. Para calceína, excitación / emisión es 495/515 nm, mientras que homodímero de etidio es de 525/620 nm y APC se encuentra a 650/660 nm. Otras etiquetas se pueden elegir también. </li><li> Lavar las células con PBS y el lugar en 1 ml de RPMI suplementado con 2 mM CaCl 2. Montar el microscopio. </li><li> Diluir la toxina y dextrano en agua, y se centrifuga a 20.000 xg durante 10 min 4 º C. Típicamente, 1 l SLO y 4 l 10 mg / ml de dextrano 555-se diluye en 6 l de agua. Dextrano u otra molécula fluorescente en fase líquida se utiliza para verificar que la femto-punta no se ha obstruido, y Injects como se desee. </li><li> Carga de femto-punta por la parte posterior con 0,07 l de la toxina diluida con un microloader. </li><li> Carga de femto-punta en microinyector. Ajustar el ángulo de inyector de manera que la punta se sentará sobre el centro de las células con espacio para moverse en todas las direcciones. Borrar z-límite. Ajustes de inyección deben estar inyectando durante 0,5 segundos a 120 psi con una presión de 20 psi atrás. Baja punta hasta que entra en el medio. </li><li> Uso de la lente de Bertrand, el centro de la punta y el seguimiento de la punta a medida que desciende más cerca de las células. Una vez que se pierde el foco, vuelva a la óptica normal. La sombra aguja debería ser evidente en el campo. Enfoque por encima de las células y bajar la aguja hasta que esté en el foco. Centrarse de nuevo en las células y cuidadosamente llevar la aguja adyacente a una célula. Z Ajuste el límite de la inyección. Comenzar la imagen, mueva la punta a la posición deseada, inyectar a liberar toxinas en el punto de tiempo deseado. Levante la aguja, mueva a una nueva región de las células y se inyecta. Mueva la aguja a la posición inicial para evitar cualquier indeseadotoxina fuga de la aguja. </li></ol>

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No hay conflictos de interés declarado.

Las técnicas aquí descritas permiten el examen de las respuestas de las células inmunes a las toxinas bacterianas. El paso más crítico es el manejo y la dosificación de la toxina. Actividad de la toxina puede ser muy variables, incluso entre diferentes alícuotas de la misma preparación, debido a su fragilidad. Esto requiere ya sea probando cada parte alícuota de toxina contra una línea celular de referencia o glóbulos rojos o el uso de gradientes de toxina. Gradientes de toxina, como entregado por micropipeta...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios (opcional) </td></tr><tr><td> Ni-NTA agarosa </td><td> Qiagen </td><td> 30210 </td><td></td></tr><tr><td> polimixina-agarosa </td><td> Sigma </td><td> P1411-5ML </td><td></td></tr><tr><td> Zeba desalar spin columna </td><td> Pescador </td><td> PI-89891 </td><td></td></tr><tr><td> Los glóbulos rojos de oveja </td><td> Pescador </td><td> 50-415-688 </td><td></td></tr><tr><td> pBADgIII-SLO </td><td> N / A </td><td> N / A </td><td> ver nota 9 </td></tr><tr><td> Cy5 tinte monorreactivo </td><td> GE Healthcare </td><td> PA25001 </td><td></td></tr><tr><td> Fura2-AM </td><td> Life Technologies </td><td> F1221 </td><td></td></tr><tr><td> Calceína AM / EThidium homodímero </td><td> Life Technologies </td><td> L3224 </td><td></td></tr><tr><td> Anti-CD11c-APC </td><td> BD Biosciences </td><td> 550261 </td><td></td></tr><tr><td> colágeno recubierto con fondo de cristal plato </td><td> Mattek </td><td> P35GCol-1.5-10-C </td><td></td></tr><tr><td> femto-tip II </td><td> Pescador </td><td> E5242957000 </td><td></td></tr><tr><td> Microloader </td><td> Pescador </td><td> E5242956003 </td><td></td></tr><tr><td> dextrano Alexa 555 </td><td> Life Technologies </td><td> D34679 </td><td></td></tr><tr><td> InjectMan NI 2 </td><td> Eppendorf </td><td> 920000029 </td><td></td></tr><tr><td> FemtoJet </td><td> Eppendorf </td><td> 5247 000.013 </td><td></td></tr><tr><td></td><td></td><td></td><td> Tabla 1. Lista y fuente de los reactivos específicos y Equipment necesario. Equipos y reactivos utilizados en este protocolo, junto con la empresa y el número de catálogo se enumeran. </td></tr><tr><td></td><td></td><td></td><td><table border="1"><tbody><tr><td> Buffer </td><td> Composición </td><td> Paso Usado </td></tr><tr><td> Lyse / Wash </td><td> 50 mM NaH 2 PO 4 300 mM NaCl 10 mM de imidazol, pH 8,0 </td><td> 1,4 </td></tr><tr><td> Tris / sal </td><td> 50 mM Tris, pH 8,0 300 mM NaCl </td><td> 1,7 </td></tr><tr><td> Tampón de elución </td><td> 50 mM Tris, pH 8,0 300 mM NaCl 250 mM de imidazol </td><td> 1,8 </td></tr><tr><td> RBC tampón de ensayo </td><td> 0,3% de BSA 2 mM CaCl 2 10 mM HEPES, pH 7,4 </td><td> 2,1 </td></tr><tr><td> tampón de R / B </td><td> Células RPMI medio de cultivo 2 mM CaCl 2 0,5% de BSA </td><td&gt; 3,1 </td></tr></tbody></table></td></tr><tr><td></td><td></td><td></td><td> Tabla 2. Lista de los tampones utilizados en este protocolo. Los tampones usados, su composición y el primer paso en el que se utilizan en el protocolo se enumeran. </td></tr></tbody></table>

visualization of bacterial toxin induced responses using live cell fluorescence microscopy

Las toxinas bacterianas se unen al colesterol en las membranas, la formación de poros que permiten la fuga del contenido celular y el flujo de los materiales desde el entorno externo. La célula o bien puede recuperarse de este insulto, que requiere procesos activos de reparación de la membrana, o morir en función de la cantidad de exposición a la toxina y tipo de célula 1. Además, estas toxinas inducir fuertes respuestas inflamatorias en los huéspedes infectados a través de la activación de células inmunes, incluyendo los macrófagos, que producen una variedad de citoquinas pro-inflamatorias 2. Muchas bacterias Gram positivas producir colesterol toxinas vinculantes que se ha demostrado que contribuyen a su virulencia a través de mecanismos en gran medida no caracterizadas. Cambios morfológicos en la membrana plasmática de las células expuestas a estas toxinas incluyen su secuestro en salientes superficiales enriquecidos en colesterol, que puede liberarse en el espacio extracelular, lo que sugiere una defensa celular intrínsecamecanismo de 3,4. Este proceso se produce en todas las células en ausencia de actividad metabólica, y puede ser visualizado usando EM después de 4 fijación química. En las células inmunes, tales como macrófagos, que median la inflamación en respuesta a la exposición a la toxina, inducidos vesículas de membrana se sugieren para contener las citoquinas de la familia IL-1 y puede ser responsable tanto por derramar la toxina y la difusión de estas citoquinas pro-inflamatorias 5,6,7. Un enlace entre la IL-1β de liberación y un tipo específico de la muerte celular, denominado pyroptosis ha sugerido, ya que ambos son procesos que dependen de la caspasa-1 8. Para resolver la complejidad de esta respuesta de los macrófagos, que incluye la unión toxina, el desprendimiento de las vesículas de membrana, la liberación de citoquinas, y la muerte potencialmente celular, se han desarrollado técnicas de etiquetado y los métodos de microscopía de fluorescencia que permiten la visualización en tiempo real de las interacciones célula-toxina, incluyendo mediciones de la disfunción y la muerte (Figura 1). Utilizarde imágenes de células vivas es necesario debido a las limitaciones en otras técnicas. Enfoques bioquímicos no puede resolver los efectos que ocurren en células individuales, mientras que la citometría de flujo no ofrece alta resolución, en tiempo real la visualización de células individuales. Los métodos descritos aquí se pueden aplicar a análisis de la cinética de las respuestas inducidas por otros estímulos que implican complejos cambios fenotípicos en las células.

Típicamente 10 7 -10 8 U / ml SLO puede obtenerse con una concentración de proteína de 4 mg / ml. La cantidad de toxina requerida para la lisis celular varía según el tipo de célula, pero generalmente es 125-500 U / ml SLO (Figura 2B). Tipos de células como los macrófagos pueden ser más resistentes (4000 U / ml), aunque otros (especialmente líneas celulares T) son más sensibles. Estas sensibilidades corresponden con SLO disponible comercialmente. Actividad de la toxina d...

Watch this Scientific Journal Video about Visualization of Bacterial Toxin Induced Responses Using Live Cell Fluorescence Microscopy at JoVE.com

Visualization of Bacterial Toxin Induced Responses Using Live Cell Fluorescence Microscopy

Los métodos para purificar el colesterol unión toxina estreptolisina O de recombinante<em&gt; E. coli</em&gt; Y la visualización de la unión de la toxina a vivir las células eucariotas se describen. Entrega localizada de la toxina induce cambios rápidos y complejos en células específicas que revelan nuevos aspectos de la biología de la toxina.

La visualización de las respuestas inducidas por toxinas bacterianas mediante microscopía de fluorescencia de células en vivo

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external ...

visualization-bacterial-toxin-induced-responses-using-live-cell

Research

JoVE Journal

Immunology and Infection

11.7K vistas.  University of Pittsburgh School of Medicine. Los métodos para purificar el colesterol unión toxina estreptolisina O de recombinante E. coli Y la visualización de la unión de la toxina a vivir las células eucariotas se describen. Entrega localizada de la toxina induce cambios rápidos y complejos en células específicas que revelan nuevos aspectos de la biología de la toxina.

Video: Visualization of Bacterial Toxin Induced Responses Using Live Cell Fluorescence Microscopy

Visualizing Cell-to-cell Transfer of HIV using Fluorescent Clones of HIV and Live Confocal Microscopy

By fusing the green fluorescent protein to their favorite proteins, biologists now have the ability to study living complex cellular processes using fluorescence video microscopy. To track the movements of the human immunodeficiency virus core protein during cell-to-cell transmission of human immunodeficiency virus, we have GFP-tagged the Gag protein in the context of an infectious molecular clone of HIV, called HIV Gag-iGFP. We study this viral clone using video confocal microscopy. In the following visualized experiment, we transfect a human T cell line with HIV Gag-iGFP, and we use fluorescently labeled uninfected CD4+ T cells to serve as target cells for the virus. Using the different fluorescent labels we can readily follow viral production and transport across intercellular structures called virological synapses. Simple gas permeable imaging chambers allow us to observe synapses with live confocal microscopy from minutes to days. These approaches can be used to track viral proteins as they move in from one cell to the next.

This visualized experiment is a guide for utilizing a fluorescent molecular clone of HIV for live confocal imaging experiments.

Visualización de la célula a célula de Transmisión del VIH utilizando clones fluorescente del VIH y la microscopía confocal en vivo

By fusing the green fluorescent protein to their favorite proteins, biologists now have the ability to study living complex cellular processes using ...

Investigación

Inmunología e Infección

Assessing Stomatal Response to Live Bacterial Cells using Whole Leaf Imaging

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of guard cells, which are able to close the stomatal pore in response to a number of external factors including light intensity, carbon dioxide concentration, and relative humidity (RH). The stomatal pore is also the main route for pathogen entry into leaves, a crucial step for disease development. Recent studies have unveiled that closure of the pore is effective in minimizing bacterial disease development in Arabidopsis plants; an integral part of plant innate immunity. Previously, we have used epidermal peels to assess stomatal response to live bacteria (Melotto et al. 2006); however maintaining favorable environmental conditions for both plant epidermal peels and bacterial cells has been challenging. Leaf epidermis can be kept alive and healthy with MES buffer (10 mM KCl, 25 mM MES-KOH, pH 6.15) for electrophysiological experiments of guard cells. However, this buffer is not appropriate for obtaining bacterial suspension. On the other hand, bacterial cells can be kept alive in water which is not proper to maintain epidermal peels for long period of times. When an epidermal peel floats on water, the cells in the peel that are exposed to air dry within 4 hours limiting the timing to conduct the experiment. An ideal method for assessing the effect of a particular stimulus on guard cells should present minimal interference to stomatal physiology and to the natural environment of the plant as much as possible. We, therefore, developed a new method to assess stomatal response to live bacteria in which leaf wounding and manipulation is greatly minimized aiming to provide an easily reproducible and reliable stomatal assay. The protocol is based on staining of intact leaf with propidium iodide (PI), incubation of staining leaf with bacterial suspension, and observation of leaves under laser scanning confocal microscope. Finally, this method allows for the observation of the same live leaf sample over extended periods of time using conditions that closely mimic the natural conditions under which plants are attacked by pathogens.

We have developed a simple and reproducible protocol to access stomatal response to live bacteria. This method minimizes wounding and manipulation of the leaf as compared to the use of epidermal peels reported previously.

Evaluación de la respuesta estomática de las células bacterianas en vivo utilizando imágenes de Hoja Entera

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of ...

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not representative of the behavior, status or phenotype of single cells. Due to this new insight the number of single cell studies rises continuously (for recent reviews see 1,2,3). However, many of the single cell techniques applied do not allow monitoring the development and behavior of one specific single cell in time (e.g. flow cytometry or standard microscopy).
Here, we provide a detailed description of a microscopy method used in several recent studies 4, 5, 6, 7, which allows following and recording (fluorescence of) individual bacterial cells of Bacillus subtilis and Streptococcus pneumoniae through growth and division for many generations. The resulting movies can be used to construct phylogenetic lineage trees by tracing back the history of a single cell within a population that originated from one common ancestor. This time-lapse fluorescence microscopy method cannot only be used to investigate growth, division and differentiation of individual cells, but also to analyze the effect of cell history and ancestry on specific cellular behavior. Furthermore, time-lapse microscopy is ideally suited to examine gene expression dynamics and protein localization during the bacterial cell cycle. The method explains how to prepare the bacterial cells and construct the microscope slide to enable the outgrowth of single cells into a microcolony. In short, single cells are spotted on a semi-solid surface consisting of growth medium supplemented with agarose on which they grow and divide under a fluorescence microscope within a temperature controlled environmental chamber. Images are captured at specific intervals and are later analyzed using the open source software ImageJ.

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

This protocol provides a step-by-step procedure to monitor single cell behavior of different bacteria in time using automated fluorescence time-lapse microscopy. Furthermore, we provide guidelines how to analyze the microscopy images.

Imágenes de células vivas de Bacillus subtilis Y Streptococcus pneumoniae Automatizada utilizando Time-lapse microscopía

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not ...

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Biofilm formation is a general attribute to almost all bacteria 1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly constituted by proteins and exopolysaccharides, among other factors 7-10. The microbial community encased within the biofilm often shows the differentiation of distinct subpopulation of specialized cells 11-17. These subpopulations coexist and often show spatial and temporal organization within the biofilm 18-21.
Biofilm formation in the model organism Bacillus subtilis requires the differentiation of distinct subpopulations of specialized cells. Among them, the subpopulation of matrix producers, responsible to produce and secrete the extracellular matrix of the biofilm is essential for biofilm formation 11,19. Hence, differentiation of matrix producers is a hallmark of biofilm formation in B. subtilis.
We have used fluorescent reporters to visualize and quantify the subpopulation of matrix producers in biofilms of B. subtilis 15,19,22-24. Concretely, we have observed that the subpopulation of matrix producers differentiates in response to the presence of self-produced extracellular signal surfactin 25. Interestingly, surfactin is produced by a subpopulation of specialized cells different from the subpopulation of matrix producers 15.
We have detailed in this report the technical approach necessary to visualize and quantify the subpopulation of matrix producers and surfactin producers within the biofilms of B. subtilis. To do this, fluorescent reporters of genes required for matrix production and surfactin production are inserted into the chromosome of B. subtilis. Reporters are expressed only in a subpopulation of specialized cells. Then, the subpopulations can be monitored using fluorescence microscopy and flow cytometry (See Fig 1).
The fact that different subpopulations of specialized cells coexist within multicellular communities of bacteria gives us a different perspective about the regulation of gene expression in prokaryotes. This protocol addresses this phenomenon experimentally and it can be easily adapted to any other working model, to elucidate the molecular mechanisms underlying phenotypic heterogeneity within a microbial community.

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Microbial biofilms are generally constituted by distinct subpopulations of specialized cells. Single-cell analysis of these subpopulations requires the use of fluorescent reporters. Here we describe a protocol to visualize and monitor several subpopulationswithin B. subtilis biofilms using fluorescence microscopy and flow cytometry.

Sola célula de análisis de Bacillus subtilis Las biopelículas mediante microscopía de fluorescencia y citometría de flujo

Biofilm formation is a general attribute to almost all bacteria 1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly ...

Live-cell Video Microscopy of Fungal Pathogen Phagocytosis

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

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

Vídeo en vivo de células Microscopía de fagocitosis hongo patógeno

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

Application of Long-term cultured Interferon-&#947; Enzyme-linked Immunospot Assay for Assessing Effector and Memory T Cell Responses in Cattle

Effector and memory T cells are generated through developmental programing of na&#239;ve cells following antigen recognition. If the infection is controlled up to 95 % of the T cells generated during the expansion phase are eliminated (i.e., contraction phase) and memory T cells remain, sometimes for a lifetime. In humans, two functionally distinct subsets of memory T cells have been described based on the expression of lymph node homing receptors. Central memory T cells express C-C chemokine receptor 7 and CD45RO and are mainly located in T-cell areas of secondary lymphoid organs. Effector memory T cells express CD45RO, lack CCR7 and display receptors associated with lymphocyte homing to peripheral or inflamed tissues. Effector T cells do not express either CCR7 or CD45RO but upon encounter with antigen produce effector cytokines, such as interferon-&#947;. Interferon-&#947; release assays are used for the diagnosis of bovine and human tuberculosis and detect primarily effector and effector memory T cell responses. Central memory T cell responses by CD4+ T cells to vaccination, on the other hand, may be used to predict vaccine efficacy, as demonstrated with simian immunodeficiency virus infection of non-human primates, tuberculosis in mice, and malaria in humans. Several studies with mice and humans as well as unpublished data on cattle, have demonstrated that interferon-&#947; ELISPOT assays measure central memory T cell responses. With this assay, peripheral blood mononuclear cells are cultured in decreasing concentration of antigen for 10 to 14 days (long-term culture), allowing effector responses to peak and wane; facilitating central memory T cells to differentiate and expand within the culture.

Application of Long-term cultured Interferon-&amp;#947; Enzyme-linked Immunospot Assay for Assessing Effector and Memory T Cell Responses in Cattle

Long-term cultured interferon-&#947; enzyme-linked immunospot assay is used as a measure of central memory responses and correlates with protective anti-mycobacterial vaccine responses. With this assay, peripheral blood mononuclear cells are stimulated with mycobacterial antigens and interleukin-2 for 14 days, enabling differentiation and expansion of central memory T cells.

Aplicación de Ensayo inmunoensayo de largo plazo culta Interferón-γ ligado a enzimas para la evaluación efectoras y de memoria T respuestas celulares en el ganado

Effector and memory T cells are generated through developmental programing of na&#239;ve cells following antigen recognition. If the infection is ...

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 ...

Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging

Unlike mammals, insects such as Drosophila have multiple taste organs. The chemosensory neurons on the legs, proboscis, wings and ovipositor of Drosophila express gustatory receptors1,2, ion channels3-6, and ionotropic receptors7 that are involved in the detection of volatile and non-volatile sensory cues. These neurons directly contact tastants such as food, noxious substances and pheromones and therefore influence many complex behaviors such as feeding, egg-laying and mating. Electrode recordings and calcium imaging have been widely used in insects to quantify the neuronal responses evoked by these tastants. However, electrophysiology requires specialized equipment and obtaining measurements from a single taste sensillum can be technically challenging depending on the cell-type, size, and position. In addition, single neuron resolution in Drosophila can be difficult to achieve since taste sensilla house more than one type of chemosensory neuron. The live calcium imaging method described here allows responses of single gustatory neurons in live flies to be measured. This method is especially suitable for imaging neuronal responses to lipid pheromones and other ligand types that have low solubility in water-based solvents.

Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging

The forelegs and proboscis of Drosophila contain a rich repertoire of gustatory sensory neurons. Here, we present a method using calcium imaging to measure physiological responses from sensory neurons in the foreleg and proboscis of live flies upon exogenous application of a gustatory pheromone.

La medición de las respuestas fisiológicas de<em&gt; Drosophila</em&gt; Las neuronas sensoriales a los lípidos feromonas Uso de Imagen de calcio en Vivo

Unlike mammals, insects such as Drosophila have multiple taste organs. The chemosensory neurons on the legs, proboscis, wings and ovipositor of Drosophila ...

Quantification of Efferocytosis by Single-cell Fluorescence Microscopy

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling and cellular processes that control efferocytosis. This quantification can be difficult to perform as apoptotic cells are often efferocytosed piecemeal, thus necessitating methods which can accurately delineate between the efferocytosed portion of an apoptotic target versus residual unengulfed cellular fragments. The approach outlined herein utilizes dual-labeling approaches to accurately quantify the dynamics of efferocytosis and efferocytic capacity of efferocytes such as macrophages. The cytosol of the apoptotic cell is labeled with a cell-tracking dye to enable monitoring of all apoptotic cell-derived materials, while surface biotinylation of the apoptotic cell allows for differentiation between internalized and non-internalized apoptotic cell fractions. The efferocytic capacity of efferocytes is determined by taking fluorescent images of live or fixed cells and quantifying the amount of bound versus internalized targets, as differentiated by streptavidin staining. This approach offers several advantages over methods such as flow cytometry, namely the accurate delineation of non-efferocytosed versus efferocytosed apoptotic cell fractions, the ability to measure efferocytic dynamics by live-cell microscopy, and the capacity to perform studies of cellular signaling in cells expressing fluorescently-labeled transgenes. Combined, the methods outlined in this protocol serve as the basis for a flexible experimental approach that can be used to accurately quantify efferocytic activity and interrogate cellular signaling pathways active during efferocytosis.

Efferocytosis, the phagocytic removal of apoptotic cells, is required to maintain homeostasis and is facilitated by receptors and signaling pathways that allow for the recognition, engulfment, and internalization of apoptotic cells. Herein, we present a fluorescence microscopy protocol for the quantification of efferocytosis and the activity of efferocytic signaling pathways.

Cuantificación de Efferocytosis por microscopía de fluorescencia unicelular

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling ...

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

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

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