Diese Arbeit wurde durch ein Stipendium der National Institutes of Health und der University of Texas at Arlington einrichten Mittel MM unterstützt.

1. Wachsenden Pflanzen und Bakterien vorbereiten <ol><li> Zu Beginn dieses Verfahrens zu säen Arabidopsis Samen auf einem 1:1:1 v: v: v Mischung aus Kultursubstrat (Redi-Earth-Plug-und Pflanzgut-Mix, Sun Gro), feine Vermiculit und Perlit. </li><li> Wachsen die Pflanzen in einer Wachstumskammer (22 ° C, 12 Stunden von 100 mmol / mm 2 / sec Tageslicht und 65 ± 5% Luftfeuchtigkeit) und Wasser als nötig. Die Pflanzen sind bereit, in 4-6 Wochen zu verwenden, wenn sie jungen, voll entfalteten Blätter vor Verschraubung haben. </li><li> Zwei Tage vor dem Beginn der Vorbereitung des Experiments das Bakterium Kultur. Streak Pseudomonas syringae aus Glycerin Lager auf modifizierten LB-Medium (10 g / l Trypton, 5 g / L Hefeextrakt, 5 g / L NaCl pH 7,0 und 1,5% Agar) und Inkubation für 24 Stunden bei 28 ° C. Verwenden Sie frische Kultur des Inokulums vorbereiten und beginnen immer Kulturplatten aus Glycerin Aktien wie Bakterien wird weniger virulent nach Subkultivieren. </li><li> Verwenden Bakterien, die auf der Platte wuchs zu einem 10 mL Flüssigkultur von P. starten syringae in einer 50 ml-Erlenmeyerkolben. Inkubieren Sie die Kultur über Nacht bei 28 ° C unter kräftigem Schütteln, bis es optische Dichte oder OD bei 600 nm zwischen 0,8 und 1 erreicht. </li><li> Messen Sie die OD bei 600 und Ernte der Zellen durch Zentrifugation bei 1360 xg für 10 Minuten. Resuspendieren der Zellen in destilliertem Wasser, so dass die endgültige OD beträgt 0,2. Das OD entspricht 10 8 koloniebildende Einheiten (KBE) / ml. Mit den Bakterien fertig ist, fahren Sie die Blätter vorbereiten und durchführen der Test. </li></ol> 2. Blatt Färbung und Inkubation mit Bakterien <ol><li> Um Flecken auf den Blättern zunächst vorbereiten 20 uM Propidiumiodid oder PI-Lösung in Wasser. 10 mL der Lösung ist ausreichend, um 3 kleine Blätter zu einem Zeitpunkt, zu färben. </li><li> Rufen Sie die Pflanzen aus der Klimakammer und mit einer Pinzette lösen 3 junge, voll entwickelte Blätter. Tauchen Sie die ganze Blätter in der PI-Lösung für 5 Minuten. Dann die Blätter entfernen und spülen Sie sie kurz mit destilliertem Wasser. </li><li> Nächster Ort ein Blatt mit der Unterseite nach unten auf einen Objektträger. Schneiden Sie das Blatt mit einer scharfen Rasierklinge in vier Teile mit Ausnahme der Mitte Vene, so dass das Blatt legt auf der Folie flach. </li><li> Um inkubieren die Bakterien mit den Blättern, fügen Sie 300 ul Bakteriensuspension unter die Blattstücke auf der Folie. Stellen Sie sicher, dass die untere Fläche des Blattes in Kontakt mit dem Inokulum wird. </li><li> Inkubieren Sie die Proben unter den gleichen Umweltbedingungen, mit denen die Pflanzen wachsen konnten. Zu der Zeit, um Blätter unter dem Mikroskop beobachten, übertragen Sie die Blattstücke auf eine neue Folie mit geringer Oberfläche nach oben zeigt. Beachten Sie, dass Deckglas nicht verwendet wird bei der Montage der Folien. Das gleiche Blatt Probe kann mehrere Male während der Inkubation abgebildet werden und einen zeitlichen Verlauf kann bis nach der Forschungsziele festgelegt werden. </li></ol> 3. Mikroskopie, Messtechnik und Datenanalyse <ol><li> Hier wird ein Laser-Scanning-konfokalen Mikroskop (LSM 510 Meta, Carl Zeiss Inc.) wird verwendet, um die Blätter zu untersuchen. Beachten Sie die untere Fläche der Blätter, um die Fluoreszenz von PI (Anregung 453 nm, Emission 543 und 620 nm) zu erkennen. </li><li> Mit der gleichen Blattproben Aufnahmen von Spaltöffnungen im Lauf der Zeit. Speichern Sie die Bilder für die Messung der Breite der Stomata Blende. </li><li> Messen Stomata Maschenweite von mindestens 60 Spaltöffnungen für jede Behandlung zu jedem Zeitpunkt mit LSM-Bild-Browser. </li><li> Berechnen Sie die durchschnittliche und Standardfehler für die Stomata Maschenweite. Die statistische Signifikanz der Ergebnisse kann anhand 2-seitig, gepaart kluge Student-t-Test. </li></ol> 4. Repräsentative Bilder stomatäre Response to Inkubation mit Bakterien <ol><li> Hier ist eine typische mikroskopische Bild (mit einem 20x-Objektiv) eines Arabidopsis Blattoberfläche unter Laser-Scanning-konfokalen Mikroskop in Hellfeld-Ansicht (Abbildung 1). </li><li> Propidiumiodid färbt die Zellwand von lebensfähigen Zellen eine Erhöhung der Sichtbarkeit der Schließzellen neben der Identifizierung von unbeschädigten Zellen für die Datenerfassung (Abbildung 2) ermöglicht. Eine Reihe von Stomata Porenöffnungen kann in diesen Aufnahmen zu sehen ist, von ganz geschlossen Stomata weit geöffnet Spaltöffnungen. </li><li> Komplett geschlossen Spaltöffnungen werden durch die Form der Schließzellen (Abbildung 3) identifiziert. Die Maschenweite ist als 0 um betragen. </li><li> Für offene Stomata (Abbildung 4) die Maschenweite dargestellt wird durch die Verwendung einer geraden Linie quer über den breitesten Bereich der Stomata Pore gezogen gemessen. </li><li> Dies sind typische experimentelle Ergebnisse dieser Stomata Assay. Arabidopis Blätter wurden im Dunkeln mit drei verschiedenen Stämmen von Pseudomonas syringae inkubiert. Nur das Bakterium Pst DC3000 konnte dunklen geschlossenen Spaltöffnungen, durch die Maschenweite von zufällig wählte 60 Spaltöffnungen pro Bakterienstamm (Abbildung 5) gemessen öffnen. </li></ol&gt; <img src="/files/ftp_upload/2185/2185fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Mikroskopische Aufnahme der Oberfläche eines Arabidopsis Blatt. One Blickfeld der Blattoberfläche unter Laser-Scanning-konfokalen Mikroskop (LSCM) mit dem 20x-Objektiv. Beachten Sie, dass Stomata Blende ist nicht so offensichtlich, wenn die fluoreszierende Ansicht Propidiumiodid-Färbung (Abbildung 2) gestützte verglichen. <img src="/files/ftp_upload/2185/2185fig2.jpg" alt="Abbildung 2" /> Abbildung 2. Mikroskopische Aufnahme der Oberfläche einer Propidiumiodid-gefärbten Arabidopsis Blatt. One Blickfeld der Blattoberfläche unter Laser-Scanning-konfokalen Mikroskop (LSCM) mit dem 20x-Objektiv. Beachten Sie eine Reihe von Stomata Porenöffnung. Gelbe Pfeile sind spitz, um vollständig zu schließen Spaltöffnungen und grünen Pfeile sind weit offen Spaltöffnungen hingewiesen. <img src="/files/ftp_upload/2185/2185fig3.jpg" alt="Abbildung 3" /> Abbildung 3. Ganz in der Nähe Spaltöffnungen durch die Form und die Öffnung zwischen den Wächter-Zellen identifiziert. Die Maschenweite ist als 0 um betragen. <img src="/files/ftp_upload/2185/2185fig4.jpg" alt="Abbildung 4" /> Abbildung 4. Offene Spaltöffnungen, welche die Maschenweite in um. Die Messungen wurden mit Hilfe der LSCM Browser auf einer geraden Linie quer über den breitesten Bereich der Stomata Pore gezogen Basis genommen. <img src="/files/ftp_upload/2185/2185fig5.jpg" alt="Abbildung 5" /> Abbildung 5. Stomata Apertur von Arabidopsis Blättern mit drei Stämmen von Pseudomonas syringae pv tomato inkubiert:.. DC3118, DB29 und DC3000 Die Pflanzen wurden im Dunkeln während der Versuchszeit gehalten. Die Ergebnisse werden als Mittelwert dargestellt (n = 50-70) ± Standardfehler. Statistische Signifikanz wurde mit zweiseitigen Student-t-Test nachgewiesen. Das Experiment wurde dreimal mit ähnlichen Ergebnissen wiederholt.

<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>

Wir haben ein straight forward Verfahren Stomata Öffnung in intakten Blattgewebe ermöglicht eine einfache Beurteilung der Stomata Reaktion auf unterschiedliche Behandlungen Maßnahme. Obwohl wir die Ergebnisse mit dem Modellsystem Arabidopsis / Pseudomonas syringae vorgestellt haben, kann der intakten Blattes Stomata Assay potenziell mit jedem Werk-Bakterium Kombination durchgeführt werden. Das Protokoll kann leicht modifiziert werden, um notwendige Wachstum von anderen Pflanzen und bakterielle Erreger angepasst werden. Das übergeordnete Prinzip und Verfahren bleiben gleich. Darüber hinaus kann diese Methode nützlich sein, um Forscher, die funktionale Leistung von Schließzellen nicht nur Mikroben leben studieren wollen, sondern auch auf andere Reize und chemische Kampfstoffe unter Bedingungen, die das Blatt die natürliche Umwelt zu erhalten. Whole Blatt kann schwierig sein Bild aufgrund seiner Dicke und unebenen Topographie. Dieses Problem kann durch das Entfernen der Vene Mitte des Blattes so dass es flach auf den Schlitten und mit Hilfe der konfokalen Mikroskopie gelindert werden. Jedoch können auch andere Arten von Fluoreszenz-Mikroskop verwendet, um Bilder mit hoher Auflösung von der Blattoberfläche zu erhalten.

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<td>Propidium iodide</td>
<td>&nbsp;</td>
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<td>P4864</td>
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<td>Tryptone</td>
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<td>Plant growth chamber</td>
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assessing stomatal response to live bacterial cells using whole leaf imaging

Stomata sind natürliche Öffnungen in der Anlage Epidermis verantwortlich für den Gasaustausch zwischen Pflanze Innen-und Umwelt. Sie sind durch ein Paar von Schließzellen, die in der Lage, die Stomata Pore in Reaktion auf eine Reihe von externen Faktoren wie Lichtintensität, Kohlendioxid-Konzentration, und die relative Luftfeuchtigkeit (RH) in der Nähe entstehen. Die Stomata Pore ist auch die Hauptroute für den Erregernachweis Eintrag in die Blätter, ein entscheidender Schritt für die Entwicklung der Krankheit. Jüngste Studien haben enthüllt, dass die Schließung der Poren wirksam minimiert bakterielle Erkrankung Entwicklung in Arabidopsis-Pflanzen ist, ein integraler Bestandteil der Anlage angeborenen Immunität. Bisher haben wir epidermalen Schalen verwendet, um Stomata Antwort zu bewerten, um Bakterien (Melotto et al 2006.) Leben, aber die Aufrechterhaltung günstiger Umweltbedingungen für beide Anlagen epidermalen Peelings und bakteriellen Zellen war eine echte Herausforderung. Blattepidermis gehalten lebendig und gesund mit MES-Puffer (10 mM KCl, 25 mM MES-KOH, pH 6,15) für elektrophysiologische Experimente von Schließzellen werden. Allerdings ist dieser Puffer nicht geeignet für den Erhalt Bakteriensuspension. Auf der anderen Seite, können bakterielle Zellen am Leben erhalten in Wasser, das ist nicht richtig, epidermal Peelings für längere Zeit aufrecht zu erhalten. Wenn eine epidermale peel schwimmt auf dem Wasser, wodurch die Zellen in der Schale, die an der Luft trocknen innerhalb von 4 Stunden ausgesetzt sind, das Timing, das Experiment durchzuführen. Eine ideale Methode zur Beurteilung der Wirkung eines bestimmten Reizes auf der Hut Zellen sollten minimal Störungen Stomata Physiologie und der natürlichen Umgebung der Pflanze so viel wie möglich zu präsentieren. Wir haben deshalb eine neue Methode entwickelt, um Stomata Antwort zu bewerten, um Bakterien, in denen Blatt Verwundung und Manipulation stark minimiert zielt darauf ab, eine leicht reproduzierbare und zuverlässige Stomata Assay bieten zu leben. Das Protokoll basiert auf Färbung der intakten Blattes mit Propidiumiodid (PI), die Inkubation Färbung Blatt mit Bakteriensuspension und Beobachtung der Blätter unter Laser-Scanning-konfokalen Mikroskop. Schließlich ermöglicht dieses Verfahren für die Beobachtung der gleichen Live-Blatt Probe über längere Zeit mit Bedingungen, die genau imitieren die natürlichen Bedingungen, unter denen Pflanzen mit Krankheitserregern angegriffen werden.

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

Wir haben eine einfache und reproduzierbare Protokoll Stomata Reaktion auf lebende Bakterien Zugang entwickelt. Diese Methode minimiert Verwundung und Manipulation des Blattes über die Nutzung von epidermalen schält Vergleich bereits berichtet.

Die Beurteilung stomatäre Response to Bakterienzellen Live mit 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 ...

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

Research

JoVE Journal

Immunology and Infection

18.2K Aufrufe.  University of Texas at Arlington. Wir haben eine einfache und reproduzierbare Protokoll Stomata Reaktion auf lebende Bakterien Zugang entwickelt. Diese Methode minimiert Verwundung und Manipulation des Blattes über die Nutzung von epidermalen schält Vergleich bereits berichtet.

Serie: 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.

Mit Luciferase to Image Bakterielle Infektionen bei Mäusen

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

Forschung

Immunologie und Infektion

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.

Visualisierung der bakteriellen Toxin induzierten Reaktionen mit Live Cell Fluoreszenzmikroskopie

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 Bakterielle Imaging<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.

Visualisierung von Protein-DNA-Wechselwirkungen in der lebenden Bakterienzellen Verwendung Photoactivated Einzelmolekül-Tracking

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.

Beurteilung der bakteriellen Invasion von Herzzellen in Kultur und Herzkolonisation bei infizierten Mäusen mit 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.

Bakterielle Blatt Infiltration Assay für Bildende Charakterisierung pflanzlicher Abwehrreaktionen mit Hilfe der<em&gt; Arabidopsis thaliana-Pseudomonas syringae</em&gt; Pathosystem

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.

Mess physiologischen Reaktionen von<em&gt; Drosophila</emNeurone&gt; Sensory zu Lipid Pheromone Verwendung von 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 ...

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.

Live-Imaging von Anti-Pilz-Aktivität von Human Primary Neutrophilen und Monozyten in Reaktion auf<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.

Beurteilung der zellulären Immunantwort der Fruchtfliege Drosophila Melanogaster, mit einem In-Vivo-Phagozytose-Assay

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.

Dreidimensionale Bildgebung von Bakterienzellen für genaue zelluläre Darstellungen und präzise Proteinlokalisierung

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 ...