We thank Dr. Shahid Mukhtar for critiquing the manuscript and Dr. Xinnian Dong for the sample data analysis file. This work is supported by a NSF-CAREER award (IOS-1350244) to KPM and the UAB Biology Department.

Les auteurs ont rien à révéler.

Avec la diminution des terres agricoles disponibles et augmentation de la population, les chercheurs du monde entier sont mis au défi avec des besoins urgents pour l&#39;amélioration des cultures. Le rendement peut être grandement influencée par divers stress biotiques et abiotiques. Parmi eux, une infection pathogène est l&#39;une des principales causes de la baisse de rendement des cultures, responsable d&#39;environ 12% de pertes dans les États-Unis seuls 45. Pour résoudre ce problème, la recherche...

En raison de leur nature sessiles, les plantes sont constamment menacés par une pléthore d&#39;agents pathogènes présentant différents modes de vie et des stratégies nutritionnelles 1. En première approximation, les agents pathogènes biotrophes maintiennent leur hôte vivante pour récupérer les nutriments, tandis que les agents pathogènes et les toxines nécrotrophes enzymes activement secrets pour tuer le tissu hôte et se nourrissent sur ​​les cellules mortes 1. Un autre groupe d&#39;agents pathogènes, hemibiotrophs appelés, commence leur cours de l&#39;infection par le stade biotrophe et les changements à l&#39;étape nécrotrophe après avoir atteint un certain seuil de pathogène accumulation 2. Afin de se défendre efficacement contre ces micro-organismes, les plantes ont développé un système immunitaire inné compliquée équipé de mécanismes de surveillance multiples pour détecter l&#39;attaque d&#39;agents pathogènes et de déclencher localisée mort cellulaire programmée 3 ainsi que la résistance systémique acquise (SAR) 4. La recherche actuelle est axée sur la caractérisation de la sig essentielcomposants naling et croisées pourparlers dans le système immunitaire de la plante 5. Comme proposé dans le modèle &quot;Zig-Zag&quot; 5, la première couche de l&#39;usine de la réponse immunitaire innée nécessite la présence de plasma membrane localisée Motif Recognition Receptors (PRR) pour détecter l&#39;invasion d&#39;un microbe. PRR sont capables de reconnaître des modèles Microbe-Associated Molecular (MAMPS) et d&#39;établir l&#39;immunité MAMP-Triggered (MTI) 6. Outre induire une régulation positive de la transcription des gènes codant pour des protéines PR antimicrobiens 7, MTI conduit à une variété d&#39;événements qui arrête la croissance des pathogènes, y compris la production d&#39;espèces réactives de l&#39;oxygène (ROS) et de l&#39;azote réactif espèces (RNS), le dépôt de callose à la paroi cellulaire ainsi que l&#39;activation de la signalisation de la kinase de multiples voies 8. Jusqu&#39;à présent, plusieurs MAMPS ont été identifiés pour déclencher MTI chez Arabidopsis, y compris Flg22 bactérienne 9 </sup&gt; (Un fragment d&#39;acide 22 aminés dérivée de la flagelline), 10 (18 elf18 acides aminés de la traduction bactérienne facteur d&#39;élongation Tu) et une pièce de structure de la paroi cellulaire 11 peptidoglycanes. Pour établir une infection réussie, certains agents pathogènes spécialisés ont évolué la capacité de protéines secrets virulence effectrices dans les espaces intracellulaires ou intercellulaires, et par conséquent réprimer MTI et de déclencher sensibilité effecteur-Triggered (ETS) 12,13. Par exemple, les effecteurs de virulence peuvent inactiver mitogen-activated protein kinase (MAPK) phosphorylation cascades de MTI pour induire le développement de la maladie dans le tissu infecté 14-16. Lors de la co-évolution dynamique entre hôtes et pathogènes, les plantes ont également développé la stratégie de contre-attaque de reconnaître les protéines effectrices et atténuer la virulence des agents pathogènes 17 molécules. Cette reconnaissance directe ou indirecte effecteur est médiée par la résistance aux maladies (R) 18 protéines. La plupart des tourlet sont membres de NB-LRR (Nucleotide Binding et riche en leucine répétitions) famille 19. La perception d&#39;un effecteur avirulent par une protéine R provoque une réponse immunitaire plus forte et plus large que l&#39;immunité caractérisé effecteur-Triggered (ETI) 20. Outre induire l&#39;expression de gènes de défense 21 et la production de métabolites de la défense 22, ETI conduit souvent à une mort rapide localisée cellulaire programmée connu comme réponse hypersensible (HR) pour restreindre l&#39;agent pathogène de se propager dans le tissu adjacent 3. En plus de l&#39;localisé mort cellulaire programmée 23, les plantes sont capables d&#39;initier une longue durée et la réponse immunitaire systémique appelée résistance systémique acquise (SAR) 4. Lors de l&#39;épreuve avec un agent pathogène biotrophe, les cellules végétales déclenchent la biosynthèse et l&#39;accumulation d&#39;un acide salicylique de phytohormone endogène (SA) et des protéines PR dans les deux tissus locaux et systémiques 24. Grâce à tsa démarche, un état ​​de préparation est réalisée dans les feuilles non infectées qui permet de monter des réponses plus rapides de la défense au cours d&#39;une infection ultérieure par un large spectre d&#39;agents pathogènes 24. SA et ses analogues synthétiques tels que le benzo (1,2,3) -thiadiazole-7-carbothioïque ester de l&#39;acide S-méthyl (BTH) et l&#39;acide 2,6-dichloro-isonicotinique (INA) sont capables d&#39;induire chimiquement l&#39;acide salicylique (SA) Immunité -Triggered (IST) à la demande externe 24. Nonexpressor de la pathogenèse-Les gènes associés (1 NPR1) est proposé pour être l&#39;un des récepteurs et des fonctions SA en tant que régulateur transcriptionnel majeur au cours de la réaction de défense SA-médiation dans les deux tissus locaux et systémiques 21,25,26. Il a été démontré de façon concluante que NPR1 est nécessaire à l&#39;établissement SAR et la perte de NPR1 mène à susceptibilité dramatique vers Pseudomonas syringae 25. Pour caractériser largement la contribution moléculaire de l&#39;usinecomposants dans les interactions plante-pathogène, de multiples essais biologiques ont été développés pour mesurer des événements spécifiques de la défense, y compris ROS éclatement 27, dépôt de callose 28, l&#39;expression des gènes de défense et l&#39;accumulation de leurs produits protéiques 21. Bien que ces dosages individuels peuvent fournir des indications sur une forme spécifique de la réponse immunitaire de la plante, aucun d&#39;entre eux, cependant, sont en mesure de représenter la réponse complète de la défense au niveau de la plante entière. A l&#39;inverse, la quantification de l&#39;évolution de l&#39;infection par l&#39;agent pathogène après fournit une estimation globale de la réponse immunitaire au niveau des organismal. Par conséquent, le développement et l&#39;optimisation d&#39;un test pathogène inoculation précis et hautement normalisé est essentiel pour alimenter la recherche et les découvertes sur les réponses immunitaires Arabidopsis. Pseudomonas syringae, une bactérie à Gram-négatif, a été identifié comme un phytopathogène capable de provoquer une maladie chez une gamme de plantes hôtes y compris Arabidopsest 29. Comme le système plante-pathogène modèle, Arabidopsis - P. interaction syringae a été largement appliqué à comprendre les mécanismes moléculaires sous-jacents réponses végétales défense 29. Jusqu&#39;à maintenant, plus de 50 P. pathovars syringae ont été identifiés sur la base de leur capacité à infecter les 30 espèces végétales différentes . P. syringae pv. DC3000 de tomate (Pst DC3000) 31 et P. syringae pv. maculicola ES4326 (PSM ES4326) 32 sont les deux souches virulentes plus largement utilisés et largement caractérisés. En plus d&#39;être reconnu par la plante et le déclenchement de la réponse MTI, Pst DC3000 et PSM ES4326 sont capables de sécréter des protéines effectrices virulentes de supprimer MTI et de déclencher ETS de favoriser la croissance de 31,33 pathogène. Pour disséquer fonctionnellement l&#39;interaction entre Arabidopsis et P. syringae, multiple0; méthodes d&#39;infection des agents pathogènes ont été développés sur la base de l&#39;approche de prestation des agents pathogènes. Pour les plantes des sols cultivés, agent pathogène peut être délivré par la seringue infiltration, infiltration sous vide, dip inoculation et inoculation par pulvérisation 29,34. Récemment, des semis dosage inondations inoculation a été développé pour effectuer des écrans de grande envergure sur la culture de tissus plaques cultivés jeunes plants d&#39;Arabidopsis 35. Seringue infiltration, comme l&#39;un de l&#39;approche la plus couramment utilisée, délivre manuellement l&#39;agent pathogène dans le apoplaste à travers les ouvertures de la feuille naturelle appelés stomates 29. Grâce à cette approche, des quantités égales de P. syringae peut être infiltré dans la feuille infectée et la force de la réponse immunitaire de la plante est inversement corrélée aux niveaux de croissance des agents pathogènes. Par conséquent, la quantification de la croissance de l&#39;agent pathogène sert une approche optimale pour évaluer la fonction immunitaire au niveau de la plante entière. En outre, une seringue infiltration peut distinguer le tissu local et systémique, qui peut be applicable à caractériser les mécanismes moléculaires sous-jacents SAR 36. Dans le protocole suivant, nous décrivons une seringue infiltration dosage optimisé avec Psm ES4326 pour cribler des mutants d&#39;Arabidopsis pour une meilleure sensibilité à la maladie (EDS). Ce protocole consiste à employer deux génotypes d&#39;Arabidopsis: une de type sauvage écotype Columbia-0 (Col-0) plantes (contrôle) et des mutants npr1-1 perte de fonction (hypersensible) qui seront infectés avec la souche bactérienne virulente Psm ES4326 37. Le mutant npr1-1 porte une mutation ponctuelle dans la séquence consensus de répétition ankyrine-NPR1 de la molécule, ce qui modifie l&#39;histidine hautement conservé de tyrosine et rend la protéine non fonctionnelle 25. En outre, un certain nombre de modifications de la seringue de dosage infiltration sont décrits qui permettent la quantification des défauts dans les couches spécifiques de la réponse immunitaire, y compris MTI et IST.

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bacterial leaf infiltration assay for fine characterization of plant defense responses using the arabidopsis thaliana-pseudomonas syringae pathosystem

En l&#39;absence de cellules immunitaires mobiles spécialisées, les plantes utilisent leur mort cellulaire programmée localisée et systémique résistance acquise à se défendre contre les attaques de pathogènes. La contribution d&#39;un gène d&#39;Arabidopsis spécifique à la réponse immunitaire de l&#39;installation globale peut être spécifiquement et évalué quantitativement par dosage de la croissance de l&#39;agent pathogène dans le tissu infecté. Pendant plus de trois décennies, la bactérie hémibiotrophe Pseudomonas syringae pv. Maculicola ES4326 (PSM ES4326) a été largement appliquée comme le modèle pathogène pour étudier les mécanismes moléculaires sous-jacents de la réponse immunitaire Arabidopsis. Afin de fournir des agents pathogènes dans les tissus foliaires, de multiples méthodes de vaccination ont été mis en place, par exemple, une seringue infiltration, dip inoculation, pulvérisation, infiltration sous vide, et l&#39;inoculation d&#39;inondation. Le protocole suivant décrit une méthode d&#39;infiltration seringue optimisé pour fournir virulente Psm ES4326 dans les feuilles de l&#39;adultesol cultivé des plants d&#39;Arabidopsis et l&#39;écran avec précision pour une meilleure sensibilité à la maladie (EDS) en direction de cet agent pathogène. En outre, ce protocole peut être complétée avec de multiples pré-traitements pour disséquer défauts immunitaires spécifiques au sein des différentes couches de défense de la plante, y compris l&#39;acide salicylique (SA) Immunité -Triggered (IST) et immunitaires MAMP-Triggered (MTI).

Le protocole que nous décrivons ici représente un P. optimisé syringae essai seringue d&#39;infiltration d&#39;évaluer quantitativement la réponse immunitaire dans des plantes Arabidopsis. Comme illustré sur la figure 1, l&#39;infiltration de la seringue de la MSP ES4326 est suivie par l&#39;extraction et la quantification de l&#39;agent pathogène par des dilutions en série et colonies énumération. Comme décrit dans l&#39;étape...

Watch this Scientific Journal Video about Bacterial Leaf Infiltration Assay for Fine Characterization of Plant Defense Responses using the Arabidopsis thaliana-Pseudomonas syringae Pathosystem at JoVE

Bacterial Leaf Infiltration Assay for Fine Characterization of Plant Defense Responses using the Arabidopsis thaliana-Pseudomonas syringae Pathosystem | Immunology and Infection | Video

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 ...

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

Plant Biology

Plant Responses to the Environment

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21.5K vues.  University of Alabama at Birmingham. 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.

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

Assay for Pathogen-Associated Molecular Pattern (PAMP)-Triggered Immunity (PTI) in Plants

To perceive potential pathogens in their environment, plants use pattern recognition receptors (PRRs) present on their plasma membranes. PRRs recognize conserved microbial features called pathogen-associated molecular patterns (PAMPs) and this detection leads to PAMP-triggered immunity (PTI), which effectively prevents colonization of plant tissues by non-pathogens1,2. The most well studied system in PTI is the FLS2-dependent pathway3. FLS2 recognizes the PAMP flg22 that is a component of bacterial flagellin. 
Successful pathogens possess virulence factors or effectors that can suppress PTI and allow the pathogen to cause disease1. Some plants in turn possess resistance genes that detect effectors or their activity, which leads to effector-triggered immunity (ETI)2.
We describe a cell death-based assay for PTI modified from Oh and Collmer4. The assay was standardized in N. benthamiana, which is being used increasingly as a model system for the study of plant-pathogen interactions5. PTI is induced by infiltration of a non-pathogenic bacterial strain into leaves. Seven hours later, a bacterial strain that either causes disease or which activates ETI is infiltrated into an area overlapping the original infiltration zone. PTI induced by the first infiltration is able to delay or prevent the appearance of cell death due to the second challenge infiltration. Conversely, the appearance of cell death in the overlapping area of inoculation indicates a breakdown of PTI. 
Four different combinations of inducers of PTI and challenge inoculations were standardized (Table 1). The assay was tested on non-silenced N. benthamiana plants that served as the control and plants silenced for FLS2 that were predicted to be compromised in their ability to develop PTI.

Assay for Pathogen-Associated Molecular Pattern PAMP-Triggered Immunity PTI in Plants

A cell death-based assay for PTI in Nicotiana benthamiana plants is described.

Dosage pour Pathogen-Associated Molecular Motif (PAMP)-Triggered Immunité (PTI) chez les plantes

To perceive potential pathogens in their environment, plants use pattern recognition receptors (PRRs) present on their plasma membranes. PRRs recognize ...

Recherche

Immunologie et infection

Assessing Stomatal Response to Live Bacterial Cells using Whole Leaf Imaging

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

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

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 ...

Microwave Assisted Rapid Diagnosis of Plant Virus Diseases by Transmission Electron Microscopy

Investigations of ultrastructural changes induced by viruses are often necessary to clearly identify viral diseases in plants. With conventional sample preparation for transmission electron microscopy (TEM) such investigations can take several days1,2 and are therefore not suited for a rapid diagnosis of plant virus diseases. Microwave fixation can be used to drastically reduce sample preparation time for TEM investigations with similar ultrastructural results as observed after conventionally sample preparation3-5. Many different custom made microwave devices are currently available which can be used for the successful fixation and embedding of biological samples for TEM investigations5-8. In this study we demonstrate on Tobacco Mosaic Virus (TMV) infected Nicotiana tabacum plants that it is possible to diagnose ultrastructural alterations in leaves in about half a day by using microwave assisted sample preparation for TEM. We have chosen to perform this study with a commercially available microwave device as it performs sample preparation almost fully automatically5 in contrast to the other available devices where many steps still have to be performed manually6-8 and are therefore more time and labor consuming. As sample preparation is performed fully automatically negative staining of viral particles in the sap of the remaining TMV-infected leaves and the following examination of ultrastructure and size can be performed during fixation and embedding.

This study describes a method that allows the rapid and clear diagnosis of plant virus diseases in about half a day by using a combination of microwave assisted plant sample preparation for transmission electron microscopy and negative staining methods.

Micro-ondes assistée diagnostic rapide des maladies virales des plantes par microscopie électronique à transmission

Investigations of ultrastructural changes induced by viruses are often necessary to clearly identify viral diseases in plants. With conventional sample ...

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of Listeria, which results in systemic infection2. After injection, Listeria quickly disseminates to the spleen and liver due to uptake by CD8&alpha;+ dendritic cells and Kupffer cells3,4. Once phagocytosed, various bacterial proteins enable Listeria to escape the phagosome, survive within the cytosol, and infect neighboring cells5. During the first three days of infection, different innate immune cells (e.g. monocytes, neutrophils, NK cells, dendritic cells) mediate bactericidal mechanisms that minimize Listeria proliferation. CD8+ T cells are subsequently recruited and responsible for the eventual clearance of Listeria from the host, typically within 10 days of infection6.
Successful clearance of Listeria from infected mice depends on the appropriate onset of host immune responses6	. There is a broad range of sensitivities amongst inbred mouse strains7,8. Generally, mice with increased susceptibility to Listeria infection are less able to control bacterial proliferation, demonstrating increased bacterial load and/or delayed clearance compared to resistant mice. Genetic studies, including linkage analyses and knockout mouse strains, have identified various genes for which sequence variation affects host responses to Listeria infection6,8-14. Determination and comparison of infection kinetics between different mouse strains is therefore an important method for identifying host genetic factors that contribute to immune responses against Listeria. Comparison of host responses to different Listeria strains is also an effective way to identify bacterial virulence factors that may serve as potential targets for antibiotic therapy or vaccine design.
We describe here a straightforward method for measuring bacterial load (colony forming units [CFU] per tissue) and preparing single-cell suspensions of the liver and spleen for FACS analysis of immune responses in Listeria-infected mice. This method is particularly useful for initial characterization of Listeria infection in novel mouse strains, as well as comparison of immune responses between different mouse strains infected with Listeria. We use the Listeria monocytogenes EGD strain15 that, when cultured on blood agar, exhibits a characteristic halo zone around each colony due to &beta;-hemolysis1 (Figure 1). Bacterial load and immune responses can be determined at any time-point after infection by culturing tissue homogenate on blood agar plates and preparing tissue cell suspensions for FACS analysis using the protocols described below. We would note that individuals who are immunocompromised or pregnant should not handle Listeria, and the relevant institutional biosafety committee and animal facility management should be consulted before work commences.

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes is a model organism for studying immune responses and genetic susceptibility to intracellular bacteria in mice. This method enables one to measure bacterial load and generate single-cell suspensions of the liver and spleen from mice for FACS analysis to determine changes in immune cells due to Listeria infection.

Mesure de la charge bactérienne et les réponses immunitaires chez des souris infectées avec des Listeria monocytogenes</em

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of ...

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 ...

Systemic Bacterial Infection and Immune Defense Phenotypes in Drosophila Melanogaster

The fruit fly Drosophila melanogaster is one of the premier model organisms for studying the function and evolution of immune defense. Many aspects of innate immunity are conserved between insects and mammals, and since Drosophila can readily be genetically and experimentally manipulated, they are powerful for studying immune system function and the physiological consequences of disease. The procedure demonstrated here allows infection of flies by introduction of bacteria directly into the body cavity, bypassing epithelial barriers and more passive forms of defense and allowing focus on systemic infection. The procedure includes protocols for the measuring rates of host mortality, systemic pathogen load, and degree of induction of the host immune system. This infection procedure is inexpensive, robust and quantitatively repeatable, and can be used in studies of functional genetics, evolutionary life history, and physiology.

Systemic Bacterial Infection and Immune Defense Phenotypes in Drosophila Melanogaster

Drosophila melanogaster is an outstanding model organism for studying innate immune systems and the physiological consequences of infection and disease. This protocol describes how to deliver robust and quantitatively repeatable bacterial infections to D. melanogaster, and how to subsequently measure infection severity and quantify the host immune response.

Infection bactérienne systémique et défense immunitaire phénotypes<em&gt; Drosophila melanogaster</em

The fruit fly Drosophila melanogaster is one of the premier model organisms for studying the function and evolution of immune defense. Many aspects of ...

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

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

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

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

Application de long terme de culture interféron-γ-enzymatique immunospot Essai d&#39;évaluation de mémoires et effectrices réponses des cellules T chez les bovins

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

Characterization of Thymus-dependent and Thymus-independent Immunoglobulin Isotype Responses in Mice Using Enzyme-linked Immunosorbent Assay

Antibodies, also termed as immunoglobulins (Ig), secreted by differentiated B lymphocytes, plasmablasts/plasma cells, in humoral immunity provide a formidable defense against invading pathogens via diverse mechanisms. One major goal of vaccination is to induce protective antigen-specific antibodies to prevent life-threatening infections. Both thymus-dependent (TD) and thymus-independent (TI) antigens can elicit robust antigen-specific IgM responses and can also induce the production of isotype-switched antibodies (IgG, IgA and IgE) as well as the generation of memory B cells with the help provided by antigen presenting cells (APCs). Here, we describe a protocol to characterize TD and TI Ig isotype responses in mice using enzyme-linked immunosorbent assay (ELISA). In this protocol, TD and TI Ig responses are elicited in mice by intraperitoneal (i.p.) immunization with hapten-conjugated model antigens TNP-KLH (in alum) and TNP-polysaccharide (in PBS), respectively. To induce TD memory response, a booster immunization of TNP-KLH in alum is given at 3 weeks after the first immunization with the same antigen/adjuvant. Mouse sera are harvested at different time points before and after immunization. Total serum Ig levels and TNP-specific antibodies are subsequently quantified using Ig isotype-specific Sandwich and indirect ELISA, respectively. In order to correctly quantify the serum concentration of each Ig isotype, the samples need to be appropriately diluted to fit within the linear range of the standard curves. Using this protocol, we have consistently obtained reliable results with high specificity and sensitivity. When used in combination with other complementary methods such as flow cytometry, in vitro culture of splenic B cells and immunohistochemical staining (IHC), this protocol will allow researchers to gain a comprehensive understanding of antibody responses in a given experimental setting.

In this paper, we describe a protocol to characterize T-dependent and T-independent immunoglobulin (Ig) isotype responses in mice using ELISA. This method used alone or in combination with flow cytometry will allow researchers to identify differences in B cell-mediated Ig isotype responses in mice following T-dependent and T-independent antigen immunization.

Caractérisation des Thymus-dépendants et indépendants de Thymus immunoglobuline Isotype réponses chez la souris à l’aide d’Enzyme-linked Immunosorbent Assay

Antibodies, also termed as immunoglobulins (Ig), secreted by differentiated B lymphocytes, plasmablasts/plasma cells, in humoral immunity provide a ...

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

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

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

À l’aide d’un bactérie pathogène à sonde pour réponses cellulaires et organismique au niveau de l’hôte

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' ...

Opsonophagocytic Killing Assay to Assess Immunological Responses Against Bacterial Pathogens

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental procedure in which phagocytic cells are co-cultured with bacterial units. The immune cells will phagocytose and kill the bacterial cultures in a complement-dependent manner. The efficiency of the immune-mediated cell killing is dependent on a number of factors and can be used to determine how different bacterial cultures compare with regard to resistance to cell death. In this way, the efficacy of potential immune-based therapeutics can be assessed against specific bacterial strains and/or serotypes. In this protocol, we describe a simplified OPKA that utilizes basic culture conditions and cell counting to determine bacterial cell viability after co-culture with treatment conditions and HL-60 immune cells. This method has been successfully utilized with a number of different pneumococcal serotypes, capsular and acapsular strains, and other bacterial species. The advantages of this OPKA protocol are its simplicity, versatility (as this assay is not limited to antibody treatments as opsonins), and minimization of time and reagents to assess basic experimental groups.

This opsonophagocytic killing assay is used to compare the ability of phagocytic immune cells to respond to and kill bacteria based on different treatments and/or conditions. Classically, this assay serves as the gold&#160;standard for assessing effector functions of antibodies raised against a bacterium as opsonin.

Opsonophagocytic meurtre dosage pour évaluer les réponses immunologiques contre les bactéries pathogènes

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental ...