Cette recherche a été soutenue par une subvention du National Institute of Health et l&#39;Université du Texas à Arlington mis en place des fonds à MM.

1. Culture de plantes et bactéries Préparation <ol><li> Pour commencer cette procédure semer des graines d&#39;Arabidopsis sur un v 01:01:01: v: v mélange de milieu de culture (terre, Redi-Plug et mélanger des semis, Sun Gro), la vermiculite fine et de perlite. </li><li> Cultiver les plantes dans une chambre de croissance (22 ° C, 12 heures de lumière de 100 pmol / mm 2 / sec par jour et 65 ± 5% d&#39;humidité) et de l&#39;eau au besoin. Les plantes sont prêtes à utiliser dans 4-6 semaines quand ils ont des jeunes feuilles complètement développées avant boulonnage. </li><li> Deux jours avant de commencer l&#39;expérience de préparer la culture bactérie. Streak Pseudomonas syringae du stock de glycérol sur du milieu LB modifié (10 g / L de tryptone, 5 g / L extrait de levure, 5 g / L de NaCl pH 7,0, et 1,5% de gélose) et incuber pendant 24 heures à 28 ° C. Utilisez culture fraîche pour préparer l&#39;inoculum et commencez toujours des plaques de culture de stocks de glycérol comme des bactéries devient moins virulente après l&#39;Afrique sub-culture. </li><li> Utiliser des bactéries qui poussaient sur ​​la plaque pour démarrer une culture de 10 ml de liquide de P. syringae dans un erlenmeyer de 50 ml. Incuber la culture de la nuit à 28 ° C avec une agitation vigoureuse jusqu&#39;à ce qu&#39;il atteigne la densité optique ou DO à 600 nm entre 0,8 et 1. </li><li> Mesurer la DO à 600 et récolte les cellules par centrifugation à 1360 xg pendant 10 minutes. Resuspendre les cellules dans l&#39;eau distillée de telle sorte que la DO finale est de 0,2. Cette OD correspond à 10 8 unités formant colonie (UFC) / ml. Avec les bactéries prêt, commencer à préparer les feuilles et réaliser le dosage. </li></ol> 2. Coloration Leaf et incubation avec des bactéries <ol><li> Afin de colorer les feuilles d&#39;abord préparer l&#39;iodure de propidium 20 uM ou de la solution PI dans l&#39;eau. 10 ml de solution est suffisante pour colorer les trois petites feuilles à la fois. </li><li> Récupérer les plantes de la chambre de croissance et avec une pince détache trois jeunes, entièrement développé feuilles. Plongez les feuilles entières dans la solution PI pendant 5 minutes. Ensuite, retirer les feuilles et les rincer rapidement à l&#39;eau distillée. </li><li> Ensuite placer une feuille avec la surface inférieure vers le bas sur une lame de microscope. Couper la feuille avec une lame de rasoir en quatre morceaux hors de la veine mi sorte que la feuille à plat sur la diapositive. </li><li> Pour incuber les bactéries avec les feuilles, ajouter 300 ul de suspension bactérienne sous les morceaux de feuilles sur la diapositive. Assurez-vous que la surface inférieure de la feuille est en contact avec l&#39;inoculum. </li><li> Incuber les échantillons dans les mêmes conditions environnementales qui ont été utilisées pour cultiver les plantes. Au moment d&#39;observer les feuilles sous le microscope, le transfert des morceaux de feuilles d&#39;une nouvelle diapositive avec surface inférieure vers le haut. Notez que lamelle n&#39;est pas utilisé lors du montage des lames. L&#39;échantillon même feuille peut être imagée plusieurs fois pendant l&#39;incubation et un cours à temps peut être mis en place en fonction des objectifs de recherche. </li></ol> 3. Analyse au microscope, de mesure et de données <ol><li> Voici un microscope confocal à balayage laser (LSM 510 Meta, Carl Zeiss Inc) est utilisée pour examiner les feuilles. Observez la surface inférieure des feuilles pour détecter la fluorescence du PI (excitation 453 nm, émission 543 et 620 nm). </li><li> En utilisant les échantillons de feuilles mêmes de capturer des images de stomates au fil du temps. Enregistrer les images pour mesurer la largeur de l&#39;ouverture des stomates. </li><li> Mesurez la largeur d&#39;ouverture des stomates d&#39;au moins 60 stomates pour chaque traitement à chaque point de l&#39;image en utilisant le navigateur LSM. </li><li> Calculer l&#39;erreur moyenne et standard pour la largeur d&#39;ouverture des stomates. La signification statistique des résultats peut être calculée en utilisant 2-tailed, Student apparié sages t-test. </li></ol> 4. Des images représentatives de réponse stomatique d&#39;incubation avec des bactéries <ol><li> Voici une image typique microscopiques (en utilisant un objectif 20x) d&#39;une surface de la feuille d&#39;Arabidopsis sous microscope confocal à balayage laser en vue champ clair (Figure 1). </li><li> Taches iodure de propidium la paroi cellulaire des cellules viables accroître la visibilité des cellules de garde en plus de permettre l&#39;identification des cellules intactes pour la collecte de données (figure 2). Une gamme d&#39;ouvertures de pores stomatiques peuvent être vus dans ces micrographies, de stomates complètement fermé à la stomates ouverts. </li><li> Stomates complètement fermé sont identifiés par la forme des cellules de garde (figure 3). La largeur d&#39;ouverture est considérée comme 0 um. </li><li> Pour stomates ouverts (figure 4) la largeur d&#39;ouverture affichée est mesurée en utilisant une ligne droite tracée à travers la plus vaste zone du pore stomatique. </li><li> Ces résultats expérimentaux sont typiques de ce dosage stomatique. Arabidopis feuilles ont été incubés dans le noir avec trois différentes souches de Pseudomonas syringae. Seule la bactérie Pst DC3000 a pu ouvrir foncé fermée stomates, telle que mesurée par la largeur d&#39;ouverture de hasard 60 stomates par souche bactérienne (figure 5). </li></ol&gt; <img src="/files/ftp_upload/2185/2185fig1.jpg" alt="Figure 1" /> Figure 1. Micrographie de la surface d&#39;une feuille d&#39;Arabidopsis. Un champ de vision de la surface des feuilles sous microscope confocal à balayage laser (LSCM) en utilisant l&#39;objectif 20x. Notez que l&#39;ouverture des stomates n&#39;est pas aussi évidente par rapport à la vue fluorescentes aidé par coloration iodure de propidium (figure 2). <img src="/files/ftp_upload/2185/2185fig2.jpg" alt="Figure 2" /> Figure 2. Micrographie de la surface d&#39;une feuille d&#39;Arabidopsis iodure de propidium teinté. Un champ de vision de la surface des feuilles sous microscope confocal à balayage laser (LSCM) en utilisant l&#39;objectif 20x. Remarque une gamme d&#39;ouverture des pores stomatiques. Les flèches jaunes sont pointés de fermer complètement les stomates et les flèches vertes sont pointés aux stomates ouverts. <img src="/files/ftp_upload/2185/2185fig3.jpg" alt="Figure 3" /> Figure 3. Complètement stomates se ferment identifié par la forme de l&#39;ouverture et entre les cellules de garde. La largeur d&#39;ouverture est considérée comme 0 um. <img src="/files/ftp_upload/2185/2185fig4.jpg" alt="Figure 4" /> Figure 4. Stomates ouverts montrant la largeur d&#39;ouverture en um. Les mesures ont été prises par l&#39;aide du navigateur LSCM basé sur une ligne droite tirée à travers la plus vaste zone du pore stomatique. <img src="/files/ftp_upload/2185/2185fig5.jpg" alt="Figure 5" /> Figure 5. L&#39;ouverture des stomates des feuilles d&#39;Arabidopsis incubées avec trois souches de Pseudomonas syringae pv tomate:.. DC3118, DB29, et DC3000 Les plantes ont été maintenus dans l&#39;obscurité pendant le temps de l&#39;expérimentation. Les résultats sont présentés comme la moyenne (n = 50-70) ± erreur standard. La signification statistique a été détecté avec deux queues de Student t-test. L&#39;expérience a été répétée trois fois avec des résultats similaires.

<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>

Nous avons présenté une procédure simple pour mesurer l&#39;ouverture des stomates dans les tissus foliaires intacte permettant une évaluation facile de la réponse stomatique à différents traitements. Bien que nous ayons présenté des résultats en utilisant le système modèle Arabidopsis / Pseudomonas syringae, le dosage des feuilles intactes stomatique peut être potentiellement réalisée avec n&#39;importe quelle combinaison de plantes bactérie. Le protocole peut être facilement modifié pour s&#39;adapter aux exigences de croissance des autres plantes et les bactéries pathogènes. Le principe général et la procédure reste la même. En outre, cette méthode peut être bénéfique pour les chercheurs qui désirent étudier sortie fonctionnelle des cellules de garde non seulement de vivre les microbes, mais aussi à d&#39;autres stimuli et des agents chimiques dans des conditions qui maintiennent l&#39;environnement naturel de la feuille. Feuilles entières peuvent être difficiles à l&#39;image due à son épaisseur et sa topographie irrégulière. Ce problème peut être atténué par la suppression de la nervure médiane de la feuille de sorte qu&#39;elle repose à plat sur la lame et en utilisant la microscopie confocale. Toutefois, d&#39;autres types de microscope à fluorescence peut être utilisée pour obtenir des images haute résolution de la surface des feuilles.

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

Les stomates sont des ouvertures naturelles dans l&#39;épiderme des plantes responsables de l&#39;échange gazeux entre l&#39;intérieur des plantes et environnement. Ils sont formés par une paire de cellules de garde, qui sont capables de fermer les pores stomatiques en réponse à un certain nombre de facteurs externes, y compris l&#39;intensité lumineuse, la concentration de dioxyde de carbone, et l&#39;humidité relative (HR). Les pores des stomates est aussi la principale voie d&#39;entrée des pathogènes dans les feuilles, une étape cruciale pour le développement de la maladie. De récentes études ont dévoilé que la fermeture du pore est efficace pour minimiser le développement de maladies bactériennes dans des plantes d&#39;Arabidopsis, une partie intégrante de l&#39;immunité innée des plantes. Auparavant, nous avons utilisé les peelings épiderme pour évaluer la réponse des stomates à vivre des bactéries (Melotto et al 2006.), Mais le maintien de conditions environnementales favorables pour les peelings plantes fois l&#39;épiderme et des cellules bactériennes a été difficile. Épiderme des feuilles peut être gardé en vie et en bonne santé avec du tampon MES (10 mM de KCl, 25 mM MES-KOH, pH 6,15) pour des expériences électrophysiologiques des cellules de garde. Cependant, ce buffer n&#39;est pas appropriée pour l&#39;obtention d&#39;une suspension bactérienne. D&#39;autre part, les cellules bactériennes peuvent être maintenues en vie dans l&#39;eau qui n&#39;est pas appropriée pour maintenir les peelings épiderme pour une longue période de temps. Quand une flotte Peel épidermiques sur l&#39;eau, les cellules de la peau qui sont exposées à l&#39;air sec dans les 4 heures en limitant le moment de mener l&#39;expérience. Une méthode idéale pour évaluer l&#39;effet d&#39;un stimulus particulier sur les cellules de garde doit présenter un minimum d&#39;interférence à la physiologie des stomates et à l&#39;environnement naturel de la plante autant que possible. Nous avons donc développé une nouvelle méthode pour évaluer la réponse des stomates à vivre dans laquelle les bactéries en blessant des feuilles et la manipulation sont grandement minimisés visant à fournir un test facilement reproductible et fiable des stomates. Le protocole est basé sur la coloration des feuilles intactes avec de l&#39;iodure de propidium (PI), l&#39;incubation de la coloration des feuilles avec une suspension bactérienne, et l&#39;observation des feuilles sous microscope confocal à balayage laser. Enfin, cette méthode permet l&#39;observation de l&#39;échantillon de feuille vivent même sur de longues périodes de temps en utilisant des conditions qui simulent étroitement les conditions naturelles dans lesquelles les plantes sont attaquées par des agents pathogènes.

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

Nous avons développé un protocole simple et reproductible pour accéder à la réponse stomatique bactéries vivantes. Cette méthode minimise blessant et la manipulation de la feuille par rapport à l&#39;utilisation des pelures épidermiques rapportés précédemment.

Évaluer la réponse stomatique à Live cellules bactériennes en utilisant l&#39;imagerie feuilles entières

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 vues.  University of Texas at Arlington. Nous avons développé un protocole simple et reproductible pour accéder à la réponse stomatique bactéries vivantes. Cette méthode minimise blessant et la manipulation de la feuille par rapport à l'utilisation des pelures épidermiques rapportés précédemment.

Vidéo: 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.

En utilisant la luciférase aux infections bactériennes chez les souris image

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

Recherche

Immunologie et infection

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.

Visualisation des réponses toxine bactérienne induite en utilisant direct microscopie de fluorescence cellulaire

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.

Bioluminescent imagerie bactérienne<em&gt; In 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.

Visualiser les interactions protéine-ADN dans des cellules bactériennes vivantes en utilisant photoactivés suivi unique molécule

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.

Évaluation de l’invasion bactérienne des cellules cardiaques en culture et colonisation cardiaque chez des souris infectées à l’aide de 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.

Bactérienne Feuille Infiltration Essai de caractérisation fine des réponses de défense des plantes à l&#39;aide du<em&gt; Arabidopsis thaliana-Pseudomonas syringae</em&gt; Pathosystème

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.

Mesurer les réponses physiologiques de<em&gt; Drosophila</em&gt; Neurones sensoriels à Lipid Phéromones Utilisation en direct d&#39;imagerie de calcium

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.

Imagerie en direct de l&#39;activité par Human Antifongique primaire neutrophiles et des monocytes en réponse à<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.

Évaluation de la réponse immunitaire cellulaire de la drosophile, Drosophila melanogaster, à l’aide d’un test In Vivo la phagocytose

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.

Imagerie tridimensionnelle des cellules bactériennes pour des représentations cellulaires précises et une localisation précise des protéines

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 ...