Esta investigación fue financiada por una subvención del Instituto Nacional de Salud y la Universidad de Texas en Arlington crear fondos de MM.

1. Las plantas que crecen las bacterias y Preparación <ol><li> Para iniciar este procedimiento de sembrar las semillas de Arabidopsis en un v 01:01:01: v: v mezcla de medio de cultivo (Redi-plug y mezclar la tierra de plántulas, Sun Gro), vermiculita fina, y perlita. </li><li> Cultivar las plantas en una cámara de crecimiento (22 ° C, 12 horas de luz de 100 mmol / mm 2 / s al día y 65 ± 5% de humedad) y el agua según sea necesario. Las plantas están listas para su uso en 4-6 semanas cuando tienen las hojas jóvenes totalmente expandidas antes de la floración. </li><li> Dos días antes de comenzar el experimento de preparar a la cultura de la bacteria. Racha de Pseudomonas syringae en stock de glicerol en el medio LB modificado (10 g / L de triptona, 5 g / L de extracto de levadura, 5 g / L NaCl pH 7,0, y 1,5% de agar) e incubar durante 24 horas a 28 ° C. Uso de cultivo fresco para preparar el inóculo que comienzan siempre las placas de cultivo de las existencias de glicerol como las bacterias es menos virulenta después de subcultivos. </li><li> Use las bacterias que crecían en la placa para iniciar un cultivo de 10 ml de líquido de P. syringae, en un matraz Erlenmeyer de 50 ml. Incubar el cultivo de una noche a 28 ° C con agitación vigorosa hasta que se alcanza la densidad óptica o la DO a 600 nm entre 0,8 y 1. </li><li> Medir la DO a 600 y la cosecha de las células por centrifugación a 1360 xg durante 10 minutos. Resuspender las células en agua destilada para que el OD final es 0,2. Esta DO se corresponde con 10 8 unidades formadoras de colonias (UFC) / mL. Con la bacteria listo, proceder a la preparación de las hojas y realizar el ensayo. </li></ol> 2. Hoja de tinción e incubación con bacterias <ol><li> Con el fin de manchar las primeras hojas preparar 20 M de yoduro de propidio o la solución PI en el agua. 10 ml de solución es suficiente para teñir tres pequeñas hojas a la vez. </li><li> Recuperar las plantas de la cámara de crecimiento y con una pinza separar tres jóvenes, totalmente ampliado hojas. Sumerja las hojas enteras en la solución de PI durante 5 minutos. A continuación, retire las hojas y aclare a fondo con agua destilada. </li><li> Lugar el próximo una hoja con la cara inferior hacia abajo sobre un portaobjetos de microscopio. Cortar la hoja con una cuchilla afilada en cuatro partes con exclusión de la vena media, para que la hoja está plano en la diapositiva. </li><li> Para la incubación de las bacterias con las hojas, añadir 300 l de la suspensión bacteriana en las piezas de la hoja en la diapositiva. Asegúrese de que la superficie inferior de la hoja está en contacto con el inóculo. </li><li> Incubar las muestras en las mismas condiciones ambientales que fueron utilizados para cultivar las plantas. En el momento de observar las hojas bajo el microscopio, la transferencia de las piezas de la hoja de una nueva diapositiva con la superficie inferior hacia arriba. Tenga en cuenta que cubreobjetos no se utiliza en el montaje de las diapositivas. La muestra misma hoja se pueden visualizar varias veces durante la incubación y un curso de tiempo puede ser configurado de acuerdo con los objetivos de investigación. </li></ol> 3. Microscopía, Medición y Análisis de Datos <ol><li> Aquí un microscopio confocal de barrido láser (LSM 510 Meta, Carl Zeiss Inc.) se utiliza para examinar las hojas. Observar la superficie inferior de las hojas para detectar la fluorescencia de la PI (excitación 453 nm, emisión 543 y 620 nm). </li><li> Usando las muestras de hoja misma capturar imágenes de los estomas en el tiempo. Guardar las imágenes para medir la anchura de la apertura de los estomas. </li><li> Mida el ancho de la apertura de los estomas de al menos 60 estomas para cada tratamiento en cada momento mediante el navegador de LSM imagen. </li><li> Calcular el error promedio y estándar para el ancho de la abertura estomática. La significación estadística de los resultados se puede calcular utilizando dos colas, Student para datos apareados sabio t-test. </li></ol> 4. Imágenes representativas de la respuesta de los estomas de la incubación con bacterias <ol><li> Esta es una imagen típica microscópica (usando un objetivo 20x) de una superficie de la hoja de Arabidopsis bajo microscopio confocal de barrido láser de la vista de campo claro (Figura 1). </li><li> Propidio yoduro de manchas de la pared celular de las células viables aumentar la visibilidad de las células de guardia, además de permitir la identificación de las células en buen estado para la recolección de datos (Figura 2). Una serie de aberturas de los poros de estomas se puede ver en estas micrografías, de estomas completamente cerrado a los estomas abiertos. </li><li> Estomas totalmente cerrados se identifican por la forma de las células de guarda (Figura 3). El ancho de la abertura se considera 0 micras. </li><li> De estomas abiertos (Figura 4) la luz de malla que se muestra es medido mediante una línea recta trazada a través de la más amplia área del poro estomático. </li><li> Estos son típicos los resultados experimentales de este ensayo los estomas. Arabidopis hojas se incubaron en la oscuridad con tres cepas diferentes de Pseudomonas syringae. Sólo la bacteria Pst DC3000 fue capaz de abrir los estomas cerrados oscuro, medida por el ancho de la abertura de seleccionados al azar 60 estomas por la cepa bacteriana (Figura 5). </li></ol&gt; <img src="/files/ftp_upload/2185/2185fig1.jpg" alt="Figura 1" /> Figura 1. Micrografía de la superficie de una hoja de Arabidopsis. Un campo de visión de la superficie de las hojas bajo microscopio confocal de barrido láser (LSCM) con el objetivo de 20x. Tenga en cuenta que la apertura de estomas no es tan evidente cuando se compara con el punto de vista fluorescentes con la ayuda de tinción con yoduro de propidio (Figura 2). <img src="/files/ftp_upload/2185/2185fig2.jpg" alt="Figura 2" /> Figura 2. Micrografía de la superficie de una hoja de Arabidopsis de yoduro de propidio manchado. Un campo de visión de la superficie de las hojas bajo microscopio confocal de barrido láser (LSCM) con el objetivo de 20x. Tenga en cuenta una serie de apertura del poro estomático. Las flechas amarillas se señaló para cerrar completamente los estomas y flechas verdes apuntan a los estomas abiertos. <img src="/files/ftp_upload/2185/2185fig3.jpg" alt="Figura 3" /> Figura 3. Estomas se cierran completamente identificados por la forma de la abertura entre las células de guarda. El ancho de la abertura se considera 0 micras. <img src="/files/ftp_upload/2185/2185fig4.jpg" alt="Figura 4" /> Figura 4. Estomas se abren mostrando la luz de malla en micras. Las mediciones se realizaron utilizando el navegador LSCM sobre la base de una línea recta trazada a través de la amplia área del poro estomático. <img src="/files/ftp_upload/2185/2185fig5.jpg" alt="Figura 5" /> Figura 5. Apertura de los estomas de las hojas de Arabidopsis se incubaron con tres cepas de Pseudomonas syringae pv tomate:.. DC3118, DB29, y DC3000 Las plantas se mantuvieron en la oscuridad durante el tiempo de experimentación. Los resultados se muestran como la media (n = 50-70) ± error estándar. La significación estadística fue detectado con dos colas de la t-test. El experimento se repitió tres veces con resultados similares.

<ol>
	<li>Brooks, D. M., Hernandez-Guzman, G., Kloek, A. P., Alarcon-Chaidez, F., Sreedharan, A., Rangaswamy, V., Penaloza-Vazquez, A., Bender, C. L., Kunkel, B. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+a+well-defined+series+of+coronatine+biosynthetic+mutants+of+Pseudomonas+syringae+pv.+tomato+DC3000.">Identification and characterization of a well-defined series of coronatine biosynthetic mutants of Pseudomonas syringae pv. tomato DC3000.</a> Mol Plant-Microbe Interact. 17, 162-174 (2004).</li><li>Katagiri, F., Thilmony, R., He, S. Y., Somerville, C. R., Meyerowitz, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Arabidopsis+thaliana-Pseudomonas+syringae+interaction.">The Arabidopsis thaliana-Pseudomonas syringae interaction.</a> The Arabidopsis Book. , (2002).</li><li>Ma, S. W., Morris, V. L., Cuppels, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+DNA+region+required+for+production+of+the+phytotoxin+coronatine+by+Pseudomonas+syringae+pv.+tomato.">Characterization of a DNA region required for production of the phytotoxin coronatine by Pseudomonas syringae pv. tomato.</a> Mol Plant-Microbe Interact. 4, 69-74 (1991).</li><li>Melotto, M., Underwood, W., Koczan, J., Nomura, K., He, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+stomata+function+in+innate+immunity+against+bacterial+invasion.">Plant stomata function in innate immunity against bacterial invasion.</a> Cell. 126, 969-980 (2006).</li></ol>

Hemos presentado un procedimiento sencillo para medir la abertura de los estomas en el tejido foliar intacto lo que permite una fácil evaluación de la respuesta de los estomas a diferentes tratamientos. A pesar de que han presentado los resultados utilizando el sistema de modelo Arabidopsis / Pseudomonas syringae, el ensayo de la hoja intacta estomas pueden ser potencialmente a cabo con cualquier combinación planta-bacteria. El protocolo se puede modificar fácilmente para adaptarse a las necesidades de crecimiento de otras plantas y bacterias patógenas. El principio general y el procedimiento son los mismos. Además, este método puede ser beneficioso para los investigadores que deseen estudiar la producción funcional de las células de guardia no sólo a vivir los microbios, sino también a otros estímulos y agentes químicos bajo condiciones que mantienen entorno natural de la hoja. Hoja entera puede ser difícil de imagen debido a su espesor y la topografía irregular. Este problema puede aliviarse mediante la eliminación de la vena media de la hoja para que quede plana en la diapositiva y el uso de la microscopía confocal. Sin embargo, otro tipo de microscopio de fluorescencia se puede utilizar para obtener imágenes de alta resolución de la superficie de la hoja.

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<td>Propidium iodide</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>P4864</td>
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<td>Tryptone</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>BP1421-1</td>
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<td>0127-01-7</td>
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<td>&nbsp;</td>
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<td>7647-14-5</td>
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<td>Agar</td>
<td>&nbsp;</td>
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<td>J637-2500G</td>
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<td>Plant growth chamber</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Shaker incubator</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Laser scanning confocal microscope</td>
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assessing stomatal response to live bacterial cells using whole leaf imaging

Los estomas son aberturas naturales de la epidermis de la planta responsables del intercambio de gases entre el interior de plantas y el medio ambiente. Están formados por un par de células de guarda, que son capaces de cerrar el poro estomático en respuesta a una serie de factores externos como la intensidad de la luz, la concentración de dióxido de carbono, y la humedad relativa (RH). El poro estomático es también la principal vía de entrada de patógenos en las hojas, un paso crucial para el desarrollo de la enfermedad. Estudios recientes han revelado que el cierre de los poros es eficaz para reducir el desarrollo de enfermedades bacterianas en plantas de Arabidopsis, una parte integral de la inmunidad innata de plantas. Anteriormente, hemos utilizado las cáscaras de la epidermis para evaluar la respuesta de los estomas de bacterias vivas (Melotto et al 2006.), Sin embargo el mantenimiento de las condiciones ambientales favorables para las exfoliaciones de plantas, tanto la epidermis y las células bacterianas ha sido un desafío. Epidermis de la hoja puede ser mantenido con vida y saludable con tampón MES (10 mM KCl, 25 mM MES-KOH, pH 6,15) para los experimentos electrofisiológicos de las células de guardia. Sin embargo, este búfer no es adecuado para obtener la suspensión bacteriana. Por otro lado, las células bacterianas pueden mantenerse vivas en el agua que no es adecuada para mantener las cáscaras epidérmica por un largo período de tiempo. Cuando una flota cáscara de la epidermis en el agua, las células de la piel que están expuestas a aire seco en 4 horas el tiempo límite para realizar el experimento. Un método ideal para evaluar el efecto de un estímulo particular en las células de guardia debe presentar un mínimo de interferencia con la fisiología estomática y el medio ambiente natural de la planta tanto como sea posible. Por lo tanto, ha desarrollado un nuevo método para evaluar la respuesta de los estomas de bacterias vivas en el que está muy herida y la manipulación de la hoja de minimizar el objetivo de proporcionar una prueba de estomas fácilmente reproducible y confiable. El protocolo se basa en la tinción de la hoja intacta con yoduro de propidio (PI), la incubación de la coloración de la hoja con la suspensión bacteriana, y la observación de las hojas bajo microscopio confocal de barrido láser. Finalmente, este método permite la observación de la muestra misma hoja vivir durante largos períodos de tiempo con las condiciones que imitan las condiciones naturales en que las plantas son atacadas por patógenos.

Watch this Scientific Journal Video about Assessing Stomatal Response to Live Bacterial Cells using Whole Leaf Imaging at JoVE.com

Assessing Stomatal Response to Live Bacterial Cells using Whole Leaf Imaging

Hemos desarrollado un protocolo simple y reproducible para acceder a la respuesta estomática de bacterias vivas. Este método minimiza las lesiones y la manipulación de la hoja, en comparación con el uso de cáscaras de la epidermis se informó anteriormente.

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 ...

assessing-stomatal-response-to-live-bacterial-cells-using-whole-leaf

Research

JoVE Journal

Immunology and Infection

18.2K vistas.  University of Texas at Arlington. Hemos desarrollado un protocolo simple y reproducible para acceder a la respuesta estomática de bacterias vivas. Este método minimiza las lesiones y la manipulación de la hoja, en comparación con el uso de cáscaras de la epidermis se informó anteriormente.

Video: Assessing Stomatal Response to Live Bacterial Cells using Whole Leaf Imaging

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

Visualization of Bacterial Toxin Induced Responses Using Live Cell Fluorescence Microscopy

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external environment. The cell can either recover from this insult, which requires active membrane repair processes, or else die depending on the amount of toxin exposure and cell type1. In addition, these toxins induce strong inflammatory responses in infected hosts through activation of immune cells, including macrophages, which produce an array of pro-inflammatory cytokines2. Many Gram positive bacteria produce cholesterol binding toxins which have been shown to contribute to their virulence through largely uncharacterized mechanisms.
Morphologic changes in the plasma membrane of cells exposed to these toxins include their sequestration into cholesterol-enriched surface protrusions, which can be shed into the extracellular space, suggesting an intrinsic cellular defense mechanism3,4. This process occurs on all cells in the absence of metabolic activity, and can be visualized using EM after chemical fixation4. In immune cells such as macrophages that mediate inflammation in response to toxin exposure, induced membrane vesicles are suggested to contain cytokines of the IL-1 family and may be responsible both for shedding toxin and disseminating these pro-inflammatory cytokines5,6,7. A link between IL-1&beta; release and a specific type of cell death, termed pyroptosis has been suggested, as both are caspase-1 dependent processes8. To sort out the complexities of this macrophage response, which includes toxin binding, shedding of membrane vesicles, cytokine release, and potentially cell death, we have developed labeling techniques and fluorescence microscopy methods that allow for real time visualization of toxin-cell interactions, including measurements of dysfunction and death (Figure 1). Use of live cell imaging is necessary due to limitations in other techniques. Biochemical approaches cannot resolve effects occurring in individual cells, while flow cytometry does not offer high resolution, real-time visualization of individual cells. The methods described here can be applied to kinetic analysis of responses induced by other stimuli involving complex phenotypic changes in cells.

Methods for purifying the cholesterol binding toxin streptolysin O from recombinant E. coli and visualization of toxin binding to live eukaryotic cells are described. Localized delivery of toxin induces rapid and complex changes in targeted cells revealing novel aspects of toxin biology.

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 ...

Bioluminescent Bacterial Imaging In Vivo

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of bacteria in gene and cell therapy for cancer. Bacteria present an attractive class of vector for cancer therapy, possessing a natural ability to grow preferentially within tumors following systemic administration. Bacteria engineered to express the lux gene cassette permit BLI detection of the bacteria and concurrently tumor sites. The location and levels of bacteria within tumors over time can be readily examined, visualized in two or three dimensions. The method is applicable to a wide range of bacterial species and tumor xenograft types. This article describes the protocol for analysis of bioluminescent bacteria within subcutaneous tumor bearing mice. Visualization of commensal bacteria in the Gastrointestinal tract (GIT) by BLI is also described. This powerful, and cheap, real-time imaging strategy represents an ideal method for the study of bacteria in vivo in the context of cancer research, in particular gene therapy, and infectious disease. This video outlines the procedure for studying lux-tagged E. coli in live mice, demonstrating the spatial and temporal readout achievable utilizing BLI with the IVIS system.

Bioluminescent Bacterial Imaging In Vivo

This article describes the administration of lux-tagged bacteria to mice and subsequent in vivo analysis using IVIS bioluminescence imaging.

Imágenes bacteriana Bioluminiscente<em&gt; En Vivo</em

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of ...

Visualizing Protein-DNA Interactions in Live Bacterial Cells Using Photoactivated Single-molecule Tracking

Protein-DNA interactions are at the heart of many fundamental cellular processes. For example, DNA replication, transcription, repair, and chromosome organization are governed by DNA-binding proteins that recognize specific DNA structures or sequences. In vitro experiments have helped to generate detailed models for the function of many types of DNA-binding proteins, yet, the exact mechanisms of these processes and their organization in the complex environment of the living cell remain far less understood. We recently introduced a method for quantifying DNA-repair activities in live Escherichia coli cells using Photoactivated Localization Microscopy (PALM) combined with single-molecule tracking. Our general approach identifies individual DNA-binding events by the change in the mobility of a single protein upon association with the chromosome. The fraction of bound molecules provides a direct quantitative measure for the protein activity and abundance of substrates or binding sites at the single-cell level. Here, we describe the concept of the method and demonstrate sample preparation, data acquisition, and data analysis procedures.

Photoactivated localization microscopy (PALM) combined with single-molecule tracking allows direct observation and quantification of protein-DNA interactions in live Escherichia coli cells.

Visualización de la proteína-DNA en células bacterianas en directo mediante fotoactivado Tracking Single-molécula

Protein-DNA interactions are at the heart of many fundamental cellular processes. For example, DNA replication, transcription, repair, and chromosome ...

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised patients, the elderly, and pregnant women. The most common manifestations of listeriosis in humans include meningitis, encephalitis, and fetal abortion. A significant but much less documented sequelae of invasive L. monocytogenes infection involves the heart. The death rate from cardiac illness can be up to 35% despite treatment, however very little is known regarding L. monocytogenes colonization of cardiac tissue and its resultant pathologies. In addition, it has recently become apparent that subpopulations of L. monocytogenes have an enhanced capacity to invade and grow within cardiac tissue. This protocol describes in detail in vitro and in vivo methods that can be used for assessing cardiotropism of L. monocytogenes isolates. Methods are presented for the infection of H9c2 rat cardiac myoblasts in tissue culture as well as for the determination of bacterial colonization of the hearts of infected mice. These methods are useful not only for identifying strains with the potential to colonize cardiac tissue in infected animals, but may also facilitate the identification of bacterial gene products that serve to enhance cardiac cell invasion and/or drive changes in heart pathology. These methods also provide for the direct comparison of cardiotropism between multiple L. monocytogenes strains.

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Listeria monocytogenes causes fetal infections in pregnant women and meningitis in susceptible populations. Subpopulations of bacteria can colonize cardiac tissue, causing myocarditis in patients and laboratory animals. Here we present a protocol that describes how to assess L. monocytogenes cardiac cell invasion in vitro and cardiac colonization in infected animals.

Evaluación De La Invasión Bacteriana De Las Células Cardíacas En Cultivo Y Colonización Del Corazón En Ratones Infectados Usando Listeria monocytogenes

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised ...

Bacterial Leaf Infiltration Assay for Fine Characterization of Plant Defense Responses using the Arabidopsis thaliana-Pseudomonas syringae Pathosystem

In the absence of specialized mobile immune cells, plants utilize their localized programmed cell death and Systemic Acquired Resistance to defend themselves against pathogen attack. The contribution of a specific Arabidopsis gene to the overall plant immune response can be specifically and quantitatively assessed by assaying the pathogen growth within the infected tissue. For over three decades, the hemibiotrophic bacterium Pseudomonas syringae pv. maculicola ES4326 (Psm ES4326) has been widely applied as the model pathogen to investigate the molecular mechanisms underlying the Arabidopsis immune response. To deliver pathogens into the leaf tissue, multiple inoculation methods have been established, e.g., syringe infiltration, dip inoculation, spray, vacuum infiltration, and flood inoculation. The following protocol describes an optimized syringe infiltration method to deliver virulent Psm ES4326 into leaves of adult soil-grown Arabidopsis plants and accurately screen for enhanced disease susceptibility (EDS) towards this pathogen. In addition, this protocol can be supplemented with multiple pre-treatments to further dissect specific immune defects within different layers of plant defense, including Salicylic Acid (SA)-Triggered Immunity (STI) and MAMP-Triggered Immunity (MTI).

Bacterial Leaf Infiltration Assay for Fine Characterization of Plant Defense Responses using the Arabidopsis thaliana-Pseudomonas syringae Pathosystem

Quantification of pathogen growth is a powerful tool to characterize various Arabidopsis thaliana (hereafter: Arabidopsis) immune responses. The method described here presents an optimized syringe infiltration assay to quantify the Pseudomonas syringae pv. maculicola ES4326 growth in adult Arabidopsis leaves.

Ensayo de infiltración bacteriana de la hoja por Fine Caracterización de las respuestas de defensa de la planta utilizando el<em&gt; Thaliana-Pseudomonas syringae Arabidopsis</em&gt; Patosistema

In the absence of specialized mobile immune cells, plants utilize their localized programmed cell death and Systemic Acquired Resistance to defend ...

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 ...

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research investigating immunological responses against A. fumigatus has been limited by the lack of consistent and reliable assays for measuring the antifungal activity of specific immune cells in vitro. A new method is described to assess the antifungal activity of primary monocytes and neutrophils from human donors against A. fumigatus using FLuorescent Aspergillus REporter (FLARE) conidia. These conidia contain a genetically encoded dsRed reporter, which is constitutively expressed by live FLARE conidia, and are externally labeled with Alexa Fluor 633, which is resistant to degradation within the phagolysosome, thus allowing a distinction between live and dead A. fumigatus conidia. Video microscopy and flow cytometry are subsequently used to visualize the interaction between conidia and innate immune cells, assessing fungicidal activity whilst also providing a wealth of information on phagocyte migration, phagocytosis and the inhibition of fungal growth. This novel technique has already provided exciting new insights into the host-pathogen interaction of primary immune cells against A. fumigatus. It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.
It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Here, we describe a protocol to assess antifungal activity of primary human immune cells in real-time using fluorescent Aspergillus reporter conidia in conjunction with live-cell video microscopy and flow cytometry. Generated data provide insight into host cell-Aspergillus interactions such as fungicidal activity, phagocytosis, cell migration and inhibition of fungal growth.

Imágenes en vivo de la actividad antifúngica por los neutrófilos humanos primarios y los monocitos en respuesta a las<em&gt; A. fumigatus</em

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research ...

Assessing the Cellular Immune Response of the Fruit Fly, Drosophila melanogaster, Using an In Vivo Phagocytosis Assay

In all animals, innate immunity provides an immediate and robust defense against a broad spectrum of pathogens. Humoral and cellular immune responses are the main branches of innate immunity, and many of the factors regulating these responses are evolutionarily conserved between invertebrates and mammals. Phagocytosis, the central component of cellular innate immunity, is carried out by specialized blood cells of the immune system. The fruit fly, Drosophila melanogaster, has emerged as a powerful genetic model to investigate the molecular mechanisms and physiological impacts of phagocytosis in whole animals. Here we demonstrate an injection-based in vivo phagocytosis assay to quantify the particle uptake and destruction by Drosophila blood cells, hemocytes. The procedure allows researchers to precisely control the particle concentration and dose, making it possible to obtain highly reproducible results in a short amount of time. The experiment is quantitative, easy to perform, and can be applied to screen for host factors that influence pathogen recognition, uptake, and clearance.

Assessing the Cellular Immune Response of the Fruit Fly, Drosophila melanogaster, Using an In Vivo Phagocytosis Assay

This protocol describes an in vivo phagocytosis assay in adult Drosophila melanogaster to quantify phagocyte recognition and clearance of microbial infections.

Evaluar el Cellular la respuesta inmune de la mosca de la fruta, Drosophila melanogasterutilizando un ensayo de fagocitosis en Vivo

In all animals, innate immunity provides an immediate and robust defense against a broad spectrum of pathogens. Humoral and cellular immune responses are ...

Three-dimensional Imaging of Bacterial Cells for Accurate Cellular Representations and Precise Protein Localization

The shape of a bacterium is important for its physiology. Many aspects of cell physiology such as cell motility, predation, and biofilm production can be affected by cell shape. Bacterial cells are three-dimensional (3D) objects, although they are rarely treated as such. Most microscopy techniques result in two-dimensional (2D) images leading to the loss of data pertaining to the actual 3D cell shape and localization of proteins. Certain shape parameters, such as Gaussian curvature (the product of the two principal curvatures), can only be measured in 3D because 2D images do not measure both principal curvatures. Additionally, not all cells lie flat when mounting and 2D imaging of curved cells may not accurately represent the shapes of these cells. Accurately measuring protein localization in 3D can help determine the spatial regulation and function of proteins. A forward convolution technique has been developed that uses the blurring function of the microscope to reconstruct 3D cell shapes and to accurately localize proteins. Here, a protocol for preparing and mounting samples for live cell imaging of bacteria in 3D both to reconstruct an accurate cell shape and to localize proteins is described. The method is based on simple sample preparation, fluorescent image acquisition, and MATLAB-based image processing. Many high-quality fluorescent microscopes can be simply modified to take these measurements. These cell reconstructions are computationally intensive and access to high-throughput computational resources is recommended, although not necessary. This method has been successfully applied to multiple bacterial species and mutants, fluorescent imaging modalities, and microscope manufacturers.

This protocol explains how to prepare and mount bacterial samples for live three-dimensional imaging and how to reconstruct the three-dimensional shape of E. coli from those images.

Imagen tridimensional de células bacterianas para representaciones celulares precisas y localización precisa de proteínas

The shape of a bacterium is important for its physiology. Many aspects of cell physiology such as cell motility, predation, and biofilm production can be ...