La recherche en laboratoire P. Cossart est soutenu par l&#39;Institut Pasteur, l&#39;Institut National de la Santé et de la Recherche Médicale, l&#39;Institut National de la Recherche Agronomique, ERC avancée Grant (233348), l&#39;Agence Nationale de la Recherche (Grant MIE- SignRupVac), la Fondation Louis-Jeantet et la Fondation Le Roch Les Mousquetaires. AK est récipiendaire d&#39;une bourse de l&#39;Université Paris-Pasteur Programme Doctoral International / Institut Carnot Maladies Infectieuses. Nous remercions Jason Mercer pour optimiser le protocole de transfection cellulaire.

Une. Préparation des cultures cellulaires et bactériennes, transfection Outils et anticorps primaires <ol><li> Préparer une plaque de gélose frais d&#39;isoler individuelle L. monocytogenes colonies à partir d&#39;un stock de glycérol bactérienne (50% glycerol/50% saturé culture de la nuit liquide bactérienne) conservé à -80 ° C. <ol><li> L&#39;utilisation d&#39;un rack en aluminium (conservé à -80 ° C) pour transporter un bactérienne glycérol d&#39;surgelés, les bactéries à balayage sur un coeur cervelle (BHI) de plaque de gélose. </li><li> Incuber la plaque à 37 ° C pendant 48 h (ou jusqu&#39;à ce que des colonies bactériennes individuelles peuvent être isolés). </li><li> Conservez cette plaque travailler ensuite à 4 ° C pendant une durée maximale de 1 mois (elle sera utilisée pour ensemencer des cultures liquides). </li><li> Dans notre protocole, nous utilisons le L. monocytogenes sérotype 1/2a EGDe.PrfA * souche, mais comme cela est illustré sur la figure 1, le L. monocytogenes sérotype 4b P14.PrfA * souche donne des résultats similaires. </li></ol></li><li> IlCellules de CCL2 La ATCC sont cultivées dans du milieu Eagle modifié par Dulbecco (DMEM) supplémenté avec 10% de sérum fœtal bovin (FBS) en l&#39;absence d&#39;antibiotiques. </li><li> Pools indépendants de brouillés (contrôle) et des anti-Met siRNAs sont chargés dans 384 puits de plaques de culture cellulaire par microscopie noirs. <ol><li> Diluer 160 pmol de siRNA dans 500 pi d&#39;eau sans RNase et ajouter 5 ul de cette solution par puits (concentration finale: 1,6 pmol). </li><li> Conservez cette plaque à -20 ° C pour une période maximale de 1 an. </li></ol></li><li> Des anticorps polyclonaux de lapin dirigés contre la protéine bactérienne RICL ont été obtenus par immunisation à l&#39;aide d&#39;une protéine recombinante RICL-GST comme décrit précédemment 25. </li></ol> 2. Inverser siRNA Transfection cellulaire <ol><li> 72 heures avant l&#39;infection amener à la température ambiante, une plaque 384 puits de culture cellulaire de microscopie noir contenant 1,6 pmol siRNA dans 5 pi d&#39;eau sans RNase dans chaque puits. </li><li> Centrifuger la plaque pendant 3 min à 300rcf à la température ambiante pour faire baisser siRNA qui aurait pu être déposé dans les parois des puits. </li><li> Préparer à manger DMEM de température complété avec 0,4% Lipofectamine RNAiMAX. </li><li> Ajouter 25 ul du milieu de transfection DMEM / RNAiMAX à chaque puits (ne pas laisser incuber le milieu de transfection plus de 20 minutes avant de l&#39;ajouter à la siRNA). </li><li> Déplacer la plaque avant et en arrière de mélanger le siRNA avec la solution de transfection, puis garder la plaque pendant 1 heure à température ambiante pour permettre complexes siARN-Lipofectamine pour former. </li><li> Laver les cellules HeLa à partir d&#39;un flacon-confluente ou une culture de cellules sous-confluentes une fois avec 10 ml 37 ° C préchauffé PBS. </li><li> Détacher les cellules HeLa par addition de 1 ml à 37 ° C préchauffé trypsine dans la fiole de culture de cellules et incuber pendant 3 à 5 min à 37 ° C. </li><li> Re-suspendre les cellules dans 10 ml 37 ° C préchauffé DMEM supplémenté avec 16% de FBS. </li><li> Compter les cellules et à préparer une suspension cellulaire de 12 000 cellules par ml dans du DMEM supplémenté avec16% de FBS. </li><li> Ajouter 50 ul de la suspension cellulaire dans chaque puits de la plaque de 384 puits. </li><li> Déplacer la plaque rapidement d&#39;avant en arrière pour répartir les cellules et les cellules laisser s&#39;installer pendant 10 min à température ambiante. </li><li> Sceller la plaque avec du Parafilm et garder pendant 72 heures dans un contenant du CO 2 atmosphère humidifiée de 5% à 37 ° C. </li></ol> 3. Infection cellulaire et coloration <ol><li> Le jour avant l&#39;infection, prendre une seule colonie de L. monocytogenes dans la plaque de gélose BHI et remettre en suspension dans 5 ml de milieu de BHI de liquide dans un tube de 15 ml en polystyrène. </li><li> Incuber pendant une nuit à 37 ° C dans un dispositif de Shacking pour permettre la croissance bactérienne. </li><li> Le jour de l&#39;infection, se laver 1 ml de la L. nuit monocytogenes culture par centrifugation 2 min à 10600 rcf sur une centrifugeuse de table. </li><li> Jeter le surnageant (qui contient le sécrétée cytotoxine listériolysine O) et remettre en suspension le culot dans 1 ml de PBS (REPEAt l&#39;étape de lavage 3 fois plus). </li><li> Lire la densité optique à 600 nm bactérienne et estimer le nombre de bactéries (OD = 1 est équivalent à 1E9 bactéries / ml). </li><li> Préparer le L. adéquate monocytogenes dilution dans du DMEM supplémenté avec 1% de FBS: en utilisant des souches hautement invasives comme EGDe.PrfA *, nous suggérons l&#39;utilisation de bactéries 5E4 dans 30 pi de milieu par puits (la multiplicité d&#39;infection est estimé à 25 pour 2000 cellules). </li><li> Retirer le milieu de culture de cellules dans chaque puits (80 ul) et de la remplacer par l&#39;ajout de 30 ul de la L. monocytogenes milieu contenant. </li><li> Centrifuger la plaque à 200 rcf pendant 5 min à température ambiante pour synchroniser le processus d&#39;infection. </li><li> Incuber la plaque pendant 1 heure dans un CO 2-atmosphère humidifiée contenant 5% à 37 ° C sur un bloc d&#39;aluminium préchauffé. </li><li> Retirez le L. monocytogenes contenant du milieu de chaque puits et ajouter 30 ul de DMEM préchauffé additionné de 10% de FBS et40 pg / ml de gentamicine de tuer extracellulaire L. monocytogenes. </li><li> Incuber pendant 4 heures dans un CO 2-atmosphère contenant 5% à 37 ° C sur un bloc de métal préchauffée. </li><li> Préparer une solution de PBS supplémenté avec 8% de formaldehyde (cette solution doit être préparé de telle sorte que les monomères de formaldéhyde, de préférence aux polymères de paraformaldéhyde, sont utilisés). </li><li> Sans écarter le milieu de culture de cellules, ajouter 30 ul de PBS supplémenté avec 8% de formaldéhyde (concentration finale: 4%) et incuber pendant 15 min à température ambiante. </li><li> Retirer le fixateur et de lavage des cellules trois fois avec 80 ul de PBS par puits (maintenir les cellules dans un volume final de 80 ul de PBS par puits). </li><li> Préparer une dilution 1:250 du lapin anti-RICL sérum dans du PBS additionné de 0,2% de saponine. </li><li> Ajouter 10 ul de la solution d&#39;anticorps primaire à chaque puits après avoir enlevé le PBS et incuber pendant 30 min à température ambiante. </li><li> Jeter le primairesolution d&#39;anticorps et laver quatre fois avec 40 pl de PBS par puits. </li><li> Diluer l&#39;anticorps secondaire Alexa Fluor 546 à couplage anti-lapin (1:250), la solution de DAPI (1:1500) et phalloïdine-DY647 (1:150) dans du PBS supplémenté avec 0,2% de saponine. </li><li> Ajouter 10 ul de cette solution de coloration secondaire pour chaque puits et incuber pendant 30 min à température ambiante. </li><li> Jeter la solution de coloration secondaire et laver quatre fois avec 40 pl de PBS par puits (laisser un volume final de 40 ul dans chaque puits et sceller la plaque). </li><li> La plaque peut être reproduite immédiatement ou peut être conservé à 4 ° C à l&#39;abri de la lumière (couvrir de papier d&#39;aluminium) pour une analyse ultérieure. </li></ol> 4. Acquisition et analyse d&#39;images <ol><li> Acquérir des images en trois canaux différents (350 nm, 546 nm et 647 nm) en utilisant un objectif 10X monté sur un microscope automatisé (acquérir préférentiellement 9 images par puits). </li><li> Le signal RICL peut être mesurée en utilisant l&#39;imagelogiciel d&#39;analyse comme CellProfiler qui permet la segmentation automatique des noyaux et des corps cellulaires utilisant le DAPI et la coloration de phalloidin, respectivement. </li></ol>

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Nous n&#39;avons rien à communiquer.

Plusieurs paramètres sont essentiels pour le succès de notre protocole RICL de détection, y compris l&#39;utilisation de lignées de cellules saines présentant un assez grand cytoplasme pour permettre une détection sans ambiguïté du signal RICL. Dans l&#39;analyse que nous présentons dans cet article, nous proposons l&#39;utilisation de cellules HeLa CCL2, qui sont particulièrement bien adapté à notre analyse en raison de l&#39;extension de leur espace cytosolique; autres clones HeLa comme les cellules HeLa K...

A correction was made to <a href="http://www.jove.com/video/51043">Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes</a>. Three authors were omitted from the article at the time of publication. The acknowledgments section was also updated. The author list has been updated from:
Andreas K&#252;hbacher1,2,3, Edith Gouin1,2,3, Pascale Cossart1,2,3, Javier Pizarro-Cerd&#225;1,2,3 
1Unit&#233; des Interactions Bact&#233;ries Cellules, Pasteur Institute, 2INSERM U604, 3Institut National de la Recherche Agronomique (INRA), USC2020
to
Andreas K&#252;hbacher1,2,3, Edith Gouin1,2,3, Jason Mercer4, Mario Emmenlauer5, Christoph Dehio5, Pascale Cossart1,2,3, Javier Pizarro-Cerd&#225;1,2,3 
1Unit&#233; des Interactions Bact&#233;ries Cellules, Pasteur Institute,2INSERM U604, 3Institut National de la Recherche Agronomique (INRA), USC2020, 4Institute of Biochemistry, ETH Z&#252;rich 5Focal Area Infection Biology, Biozentrum, University of Basel
The acknowledgments where update from:
Research in P. Cossart laboratory is supported by the Pasteur Institute, the Institut National de la Sant&eacute; et de la Recherche M&eacute;dicale, the Institut National de la Recherche Agronomique, ERC Advanced Grant (233348), the Agence Nationale de la Recherche (Grant MIE-SignRupVac), the Louis-Jeantet Foundation and the Fondation Le Roch Les Mousquetaires. A.K. is a recipient of a scholarship from the Pasteur-Paris University International Doctoral Program/Institut Carnot Maladies Infectieuses. We thank Jason Mercer for optimizing the cellular transfection protocol.
to
Research in P. Cossart laboratory is supported by the Pasteur Institute, the Institut National de la Sant&eacute; et de la Recherche M&eacute;dicale, the Institut National de la Recherche Agronomique, ERC Advanced Grant (233348), the Agence Nationale de la Recherche (Grant MIE-SignRupVac), the Louis-Jeantet Foundation and the Fondation Le Roch Les Mousquetaires. A.K. is a recipient of a scholarship from the Pasteur-Paris University International Doctoral Program/Institut Carnot Maladies Infectieuses. We acknowledge support by grant 51RT 0_126008 for the Research and Technology Development (RTD) project InfectX in the frame of SystemsX.ch, the Swiss Initiative for Systems Biology (to C.D.).

A correction to author list has been made for the article Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes. Three authors were omitted from the article at the time of publication.

Erratum: Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

La bactérie à Gram positif Listeria monocytogenes est un agent pathogène d&#39;origine alimentaire qui envahit les cellules de l&#39;hôte, perturbe sa vacuole d&#39;internalisation et se réplique dans le cytoplasme des cellules hôtes 1. L. monocytogenes facilité de manipulation dans le contexte de laboratoire (d&#39;une croissance rapide, une faible toxicité pour les individus en bonne santé) associée à la persistance de caractéristiques de virulence bactériennes observées dans des modèles cellulaires et animaux (l&#39;activité hémolytique, leucocytose) a permis son utilisation initiale dans les années 1960 comme un modèle majeur pour l&#39;étude du parasitisme intracellulaire et pour la mise en place des fondements théoriques de l&#39;immunité cellulaire contre l&#39;infection 2. À la fin des années 1980 et au début des années 1990, la dissection du cycle intracellulaire bactérienne 3 ainsi que la caractérisation moléculaire de la virulence bactérienne la plus importante facteurs 4-7 favorisé l&#39;utilisation de L. monocytogenes comme un outil moléculaire clé pour la manipulation et le goujony des fonctions de la cellule hôte. La présence de non virulente (L. innocua) et (L. monocytogenes) espèces virulentes dans le genre Listeria ont ouvert la voie à des études génomiques comparatives 8 qui, avec la création récente de la L. complète monocytogenes transcriptome 9, ont accru notre compréhension de l&#39;évolution de L. monocytogenes comme un agent pathogène humain et en tant que système modèle pour l&#39;infection études de 10. L. monocytogenes induit son internalisation dans les cellules hôtes lors de l&#39;interaction des protéines de surface bactériennes INLA et INLB avec leurs récepteurs de la cellule hôte E-cadhérine et Met, respectivement 11-12. Études sur la base de candidats initiaux ont conduit à l&#39;identification de l&#39;α / β caténines-actine lien comme une composante importante de la voie InlA-invasion 13 et de la phosphoinositide 3-kinase (PI 3-K) comme un effecteur critique de la InlB- dépendant invasion cascade 14 à 15. Analyses protéomiques et base fonctionnelle suite ont permis d&#39;identifier de nouveaux éléments du cytosquelette et 16 seconds messagers lipidiques 17 requis pour l&#39;invasion de la cellule hôte. Études de la transcription 18 et protéomique de masse basé spectrométrie de 19 quantitatives ont récemment jeté un nouvel éclairage sur l&#39;activation de la signalisation accueil cascades et la répression de la réponse immunitaire au cours de L. monocytogenes infection. Biologie systémique basée sur l&#39;inactivation des grands ensembles de gènes (kinomes, génomes complets) par petits ARN interférents (siRNA) silencieux ont récemment ouvert de nouvelles voies pour l&#39;analyse de l&#39;hôte mondiale cascades de signalisation dans le cadre des fonctions cellulaires spécifiques, y compris la phagocytose et pathogène internalisation 20. Écrans de siRNA du génome entier ont déjà été menées pour examiner cascades cellulaires nécessaires pour l&#39;infection de L. monocytogenes dans phagocytaire Drosophila </em&gt; cellules S2 21-22, mais ce type d&#39;analyse n&#39;a pas été réalisée dans des cellules non-phagocytaires, qui représentent des cibles importantes pour l&#39;infection in vivo. Nous avons optimisé un protocole pour la détection microscopique des stades tardifs de l&#39;infection par L. monocytogenes qui est adapté pour les études à haut débit siARN d&#39;entrée bactérienne dans les cellules épithéliales. Notre essai tire parti d&#39;un L. hautement invasive monocytogenes souche qui présente une mutation ponctuelle dans PrfA, le régulateur transcriptionnel majeur de L. monocytogenes facteurs de virulence 6: cette mutation (nommé PrfA *) rend PrfA constitutivement active 23 et conduit à une augmentation de l&#39;expression des protéines d&#39;invasion de l&#39;INLA et INLB, favorisant ainsi l&#39;entrée des bactéries dans les cellules non-phagocytaires sinon mal infectés. Notre lecture de l&#39;infection est basé sur la détection de l&#39;accumulation cytosolique de la sécrétée de la protéine bactérienne RICL: cette molécule estun effecteur pleiotrope qui s&#39;exprime préférentiellement par voie intra-cytoplasmique L. 9 monocytogenes et qui participe non seulement à la cellule-cellule au-bactérien réparties 24 mais qui modulent également les réponses immunitaires de l&#39;hôte 25. Le marquage fluorescent des RICL la sécrétion par les bactéries intracellulaires non seulement permet de distinguer clairement infectée à partir de cellules non infectées, mais représente également une lecture du point final qui peut être utilisé par la suite pour disséquer infection dans ses différentes étapes: d&#39;entrée, évasion vacuolaire, bactérienne cytosolique la prolifération et la propagation de cellule à cellule. Ce protocole basé sur la microscopie peut être couplé à écrans siARN donc d&#39;étudier les voies cellulaires impliquées dans l&#39;infection des cellules hôtes par L. monocytogenes.

<table border="1">
		<tbody>
		 <tr>
			<td>Name of Material/ Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments/Description</td>
		 </tr>
		 <tr>
			<td>Bacto Brain Heart Infusion</td>
			<td>BD</td>
			<td>237500</td>
			<td>For liquid BHI preparation</td>
		 </tr>
		 <tr>
			<td>Bacto Agar</td>
			<td>BD</td>
			<td>214010</td>
			<td>Supplement to liquid BHI for BHI agar plates </td>
		 </tr>
		 <tr>
			<td>Heat-Inactivated Fetal Bovine Serum</td>
			<td>Biowest</td>
			<td>51830-500</td>
			<td></td>
		 </tr>
		 <tr>
			<td>DMEM</td>
			<td>Invitrogen</td>
			<td>61965-026</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Lipofectamine RNAiMax</td>
			<td>Invitrogen</td>
			<td>13778-100</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Gentamicin</td>
			<td>Sigma </td>
			<td>G1397-10ML</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Formaldehyde (16%)</td>
			<td>EMS</td>
			<td>15710</td>
			<td>Prepare fresh before each experiment</td>
		 </tr>
		 <tr>
			<td>Anti-Rabbit Alexa Fluor 546</td>
			<td>Invitrogen</td>
			<td>A-11035</td>
			<td></td>
		 </tr>
		 <tr>
			<td>DAPI</td>
			<td>Invitrogen</td>
			<td>D-1306</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Phalloidin Dy647</td>
			<td>Dyomics</td>
			<td>647-33</td>
			<td></td>
		 </tr>
		 <tr>
			<td>siRNA Scramble</td>
			<td>Dharmacon</td>
			<td>D-001810-10</td>
			<td></td>
		 </tr>
		 <tr>
			<td>siRNA Met</td>
			<td>Dharmacon</td>
			<td>L-003156-00-0005</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Black 384-well microscopy cell culture plate</td>
			<td>Corning</td>
			<td>3985</td>
			<td></td>
		 </tr>
		 <tr>
			<td>AxioObserver Z1 microscope</td>
			<td>Zeiss</td>
			<td>431007 9901</td>
			<td></td>
		 </tr>
		 <tr>
			<td>sCMOS camera</td>
			<td>Andor</td>
			<td>Neo</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Metamorph analysis software</td>
			<td>Molecular Devices</td>
			<td>4000</td>
			<td></td>
		 </tr>
		 <tr>
			<td>CellProfiler analysis software</td>
			<td>Broad Institute</td>
			<td>Public software available at <a href="http://www.cellprofiler.org/" target="_blank">http://www.cellprofiler.org/</a></td>
			<td></td>
		 </tr>
		</tbody>
 </table>

imaging inlc secretion to investigate cellular infection by the bacterial pathogen listeria monocytogenes

Pathogènes intracellulaires bactériennes peuvent être conçus comme des outils moléculaires pour disséquer les cascades de signalisation cellulaire en raison de leur capacité à manipuler exquise et subvertir les fonctions cellulaires qui sont nécessaires pour l&#39;infection des tissus cibles de l&#39;hôte. Parmi ces bactéries pathogènes, Listeria monocytogenes est un micro-organisme à Gram positif qui a été utilisé comme un paradigme pour parasitisme intracellulaire dans la caractérisation des réponses immunitaires cellulaires, et qui a joué un rôle instrumental dans la découverte des voies moléculaires contrôlant cytosquelette et la membrane dynamique de la traite. Dans cet article, on décrit un test microscopique robuste pour la détection des stades de l&#39;infection cellulaire L. fin monocytogenes basées sur le marquage fluorescent de RICL, une protéine bactérienne sécrétée qui s&#39;accumule dans le cytoplasme des cellules infectées, ce dosage peut être couplé à de petits écrans d&#39;ARN automatisées à haut débit interférant de manière à carboniseracterize voies de signalisation cellulaires impliqués dans la hausse ou à la régulation à la baisse de l&#39;infection.

Marquage fluorescent cytoplasmique RICL fournit une lecture robuste pour l&#39;infection des cellules par L. monocytogenes, comme illustré sur la figure 1: la cellule centrale dans la micrographie est fortement infectées par la souche P14.PrfA * 23 comme cela peut être observé dans l&#39;image à contraste de phase (pointes de flèches, figure 1A) et il est confirmé par le signal DAPI où bactéries individuelles peuvent être clairement distingués (fig...

Watch this Scientific Journal Video about Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes at JoVE.com

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

Listeria monocytogenes est une bactérie pathogène à Gram positif fréquemment utilisé comme un modèle important pour l&#39;étude du parasitisme intracellulaire. Imagerie fin L. monocytogenes stades d&#39;infection dans le contexte de petits écrans ARN interférant permet l&#39;étude globale des voies cellulaires nécessaires à l&#39;infection bactérienne de cellules hôtes cibles.

Imagerie RICL sécrétion d&#39;enquêter sur l&#39;infection cellulaire par le pathogène bactérien<em&gt; Listeria monocytogenes</em

Bacterial intracellular  pathogens can be conceived as molecular tools to dissect cellular signaling  cascades due to their capacity to exquisitely ...

imaging-inlc-secretion-to-investigate-cellular-infection-bacterial

Research

JoVE Journal

Immunology and Infection

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

9.8K vues.  Pasteur Institute.  Listeria monocytogenes est une bactérie pathogène à Gram positif fréquemment utilisé comme un modèle important pour l'étude du parasitisme intracellulaire. Imagerie fin L. monocytogenes stades d'infection dans le contexte de petits écrans ARN interférant permet l'étude globale des voies cellulaires nécessaires à l'infection bactérienne de cellules hôtes cibles. 

Vidéo: Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of infection with influenza virus alone. Instead, most individuals are thought to have succumbed to a secondary bacterial infection, predominately caused by the bacterium Streptococcus pneumoniae (the pneumococcus). The synergistic relationship between infections caused by influenza virus and the pneumococcus has subsequently been observed during the 1957 Asian influenza virus pandemic, as well as during seasonal outbreaks of the virus (reviewed in 1, 2). Here, we describe a protocol used to investigate the mechanism(s) that may be involved in increased morbidity as a result of concurrent influenza A virus and S. pneumoniae infection. We have developed an infant murine model to reliably and reproducibly demonstrate the effects of influenza virus infection of mice colonised with S. pneumoniae. Using this protocol, we have provided the first insight into the kinetics of pneumococcal transmission between co-housed, neonatal mice using in vivo imaging 3.

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.

Utiliser l&#39;imagerie Bioluminescent chargé d&#39;enquêter sur une synergie entre Streptococcus pneumoniae Et virus Influenza A de souriceaux

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

Recherche

Immunologie et infection

Invasion of Human Cells by a Bacterial Pathogen

Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus . The general protocol can be applied to the study of cell invasion by virtually any culturable bacterium. The stages at which specific aspects of invasion can be studied, such as the role of actin rearrangement or caveolae, will be highlighted. Host cells are grown in flasks and when ready for use are seeded into 24-well plates containing Thermanox coverslips. Using coverslips allows subsequent removal of the cells from the wells to reduce interference from serum proteins deposited onto the sides of the wells (to which S. aureus would attach). Bacteria are grown to the required density and washed to remove any secreted proteins (e.g. toxins). Coverslips with confluent layers of endothelial cells are transferred to new 24-well plates containing fresh culture medium before the addition of bacteria. Bacteria and cells are then incubated together for the required amount of time in 5% CO2 at 37&deg;C. For S. aureus this is typically between 15-90 minutes. Thermanox coverslips are removed from each well and dip-washed in PBS to remove unattached bacteria. If total associated bacteria (adherent and internalised) are to be quantified, coverslips are then placed in a fresh well containing 0.5% Triton X-100 in PBS. Gentle pipetting leads to complete cell lysis and bacteria are enumerated by serial dilution and plating onto agar. If the number of bacteria that have invaded the cells is needed, coverslips are added to wells containing 500 &mu;l tissue culture medium supplemented with gentamicin and incubation continued for 1 h, which will kill all external bacteria. Coverslips can then be washed, cells lysed and bacteria enumerated by plating onto agar as described above. If the experiment requires direct visualisation, coverslips can be fixed and stained for light, fluorescence or confocal microscopy or prepared for electron microscopy.

A general protocol for the study of invasion of host cells by a bacterial pathogen, focusing on Staphylococcus aureus and human endothelial cells.

L&#39;invasion des cellules humaines par une bactérie pathogène

Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus . The general protocol can be ...

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 ...

The Insect Galleria mellonella as a Powerful Infection Model to Investigate Bacterial Pathogenesis

The study of bacterial virulence often requires a suitable animal model. Mammalian models of infection are costly and may raise ethical issues. The use of insects as infection models provides a valuable alternative. Compared to other non-vertebrate model hosts such as nematodes, insects have a relatively advanced system of antimicrobial defenses and are thus more likely to produce information relevant to the mammalian infection process. Like mammals, insects possess a complex innate immune system1. Cells in the hemolymph are capable of phagocytosing or encapsulating microbial invaders, and humoral responses include the inducible production of lysozyme and small antibacterial peptides2,3. In addition, analogies are found between the epithelial cells of insect larval midguts and intestinal cells of mammalian digestive systems. Finally, several basic components essential for the bacterial infection process such as cell adhesion, resistance to antimicrobial peptides, tissue degradation and adaptation to oxidative stress are likely to be important in both insects and mammals1. Thus, insects are polyvalent tools for the identification and characterization of microbial virulence factors involved in mammalian infections.
Larvae of the greater wax moth Galleria mellonella have been shown to provide a useful insight into the pathogenesis of a wide range of microbial infections including mammalian fungal (Fusarium oxysporum, Aspergillus fumigatus, Candida albicans) and bacterial pathogens, such as Staphylococcus aureus, Proteus vulgaris, Serratia marcescens Pseudomonas aeruginosa, Listeria monocytogenes or Enterococcus faecalis4-7. Regardless of the bacterial species, results obtained with Galleria larvae infected by direct injection through the cuticle consistently correlate with those of similar mammalian studies: bacterial strains that are attenuated in mammalian models demonstrate lower virulence in Galleria, and strains causing severe human infections are also highly virulent in the Galleria model8-11. Oral infection of Galleria is much less used and additional compounds, like specific toxins, are needed to reach mortality.
G. mellonella larvae present several technical advantages: they are relatively large (last instar larvae before pupation are about 2 cm long and weight 250 mg), thus enabling the injection of defined doses of bacteria; they can be reared at various temperatures (20 &deg;C to 30 &deg;C) and infection studies can be conducted between 15 &deg;C to above 37 &deg;C12,13, allowing experiments that mimic a mammalian environment. In addition, insect rearing is easy and relatively cheap. Infection of the larvae allows monitoring bacterial virulence by several means, including calculation of LD5014, measurement of bacterial survival15,16 and examination of the infection process17. Here, we describe the rearing of the insects, covering all life stages of G. mellonella. We provide a detailed protocol of infection by two routes of inoculation: oral and intra haemocoelic. The bacterial model used in this protocol is Bacillus cereus, a Gram positive pathogen implicated in gastrointestinal as well as in other severe local or systemic opportunistic infections18,19.

The Insect Galleria mellonella as a Powerful Infection Model to Investigate Bacterial Pathogenesis

Oral and intra haemocolic infection of larvae of the greater wax moth Galleria mellonella is described. This insect can be used to study virulence factors of entomopathogenic as well as mammalian opportunistic bacteria. Rearing of the insects, methods of infection and examples of in vivo analysis are described.

L&#39;insecte<em&gt; Galleria mellonella</em&gt; Comme un modèle d&#39;infection puissant pour étudier la pathogenèse bactérienne

The study of bacterial virulence often requires a suitable animal model. Mammalian models of infection are costly and may raise ethical issues. The use of ...

Oral Transmission of Listeria monocytogenes in Mice via Ingestion of Contaminated Food

L. monocytogenes are facultative intracellular bacterial pathogens that cause food borne infections in humans. Very little is known about the gastrointestinal phase of listeriosis due to the lack of a small animal model that closely mimics human disease. This paper describes a novel mouse model for oral transmission of L. monocytogenes. Using this model, mice fed L. monocytogenes-contaminated bread have a discrete phase of gastrointestinal infection, followed by varying degrees of systemic spread in susceptible (BALB/c/By/J) or resistant (C57BL/6) mouse strains. During the later stages of the infection, dissemination to the gall bladder and brain is observed. The food borne model of listeriosis is highly reproducible, does not require specialized skills, and can be used with a wide variety of bacterial isolates and laboratory mouse strains. As such, it is the ideal model to study both virulence strategies used by L. monocytogenes to promote intestinal colonization, as well as the host response to invasive food borne bacterial infection.

Oral Transmission of Listeria monocytogenes in Mice via Ingestion of Contaminated Food

This paper describes a novel method for oral infection of mice using Listeria monocytogenes-contaminated food. The protocol can readily be adapted for use with other food borne bacterial pathogens.

La transmission orale de<em&gt; Listeria monocytogenes</em&gt; Chez la souris par l&#39;ingestion d&#39;aliments contaminés

L. monocytogenes are facultative intracellular bacterial pathogens that cause food borne infections in humans. Very little is known about the ...

Automated Separation of C. elegans Variably Colonized by a Bacterial Pathogen

The wormsorter is an instrument analogous to a FACS machine that is used in studies of Caenorhabditis elegans, typically to sort worms based on expression of a fluorescent reporter. Here, we highlight an alternative usage of this instrument, for sorting worms according to their degree of colonization by a GFP-expressing pathogen. This new usage allowed us to address the relationship between colonization of the worm intestine and induction of immune responses. While C. elegans immune responses to different pathogens have been documented, it is still unknown what initiates them. The two main possibilities (which are not mutually exclusive) are recognition of pathogen-associated molecular patterns, and detection of damage caused by infection. To differentiate between the two possibilities, exposure to the pathogen must be dissociated from the damage it causes. The wormsorter enabled separation of worms that were extensively-colonized by the Gram-negative pathogen Pseudomonas aeruginosa, with the damage likely caused by pathogen load, from worms that were similarly exposed, but not, or marginally, colonized. These distinct populations were used to assess the relationship between pathogen load and the induction of transcriptional immune responses. The results suggest that the two are dissociated, supporting the possibility of pathogen recognition.

Automated Separation of C. elegans Variably Colonized by a Bacterial Pathogen

The wormsorter facilitates genetic screens in Caenorhabditis elegans by sorting worms according to expression of fluorescent reporters. Here, we describe a new usage:&#160;sorting according to colonization by a GFP-expressing pathogen, and we employ it to examine the poorly understood role of pathogen recognition in initiating immune responses.

Séparation automatique des<em&gt; C. elegans</em&gt; Façon variable colonisé par une bactérie pathogène

The wormsorter is an instrument analogous to a FACS machine that is used in studies of Caenorhabditis elegans, typically to sort worms based on expression ...

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 ...

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

Analysis of the transcriptome of bacterial pathogens during mammalian infection is a valuable tool for studying genes and factors that mediate infection. However, isolating bacterial RNA from infected cells or tissues is a challenging task, since mammalian RNA mostly dominates the lysates of infected cells. Here we describe an optimized method for RNA isolation of Listeria monocytogenes bacteria growing within bone marrow derived macrophage cells. Upon infection, cells are mildly lysed and rapidly filtered to discard most of the host proteins and RNA, while retaining intact bacteria. Next, bacterial RNA is isolated using hot phenol-SDS extraction followed by DNase treatment. The extracted RNA is suitable for gene transcription analysis by multiple techniques. This method is successfully employed in our studies of Listeria monocytogenes gene regulation during infection of macrophage cells 1-4. The protocol can be easily modified to study other bacterial pathogens and cell types.

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

Here we describe a method for bacterial RNA isolation from Listeria monocytogenes bacteria growing inside murine macrophages. This technique can be used with other intracellular pathogens and mammalian host cells.

ARN Purification de Intracellulairement Grown<em&gt; Listeria monocytogenes</em&gt; Dans les cellules macrophages

Analysis of the transcriptome of bacterial pathogens during mammalian infection is a valuable tool for studying genes and factors that mediate infection. ...

Experimental Infection with Listeria monocytogenes as a Model for Studying Host Interferon-&#947; Responses

L. monocytogenes is a gram-positive bacterium that is a cause of food borne disease in humans. Experimental infection of mice with this pathogen has been highly informative on the role of innate and adaptive immune cells and specific cytokines in host immunity against intracellular pathogens. Production of IFN-&#947; by innate cells during sublethal infection with L. monocytogenes is important for activating macrophages and early control of the pathogen1-3. In addition, IFN-&#947; production by adaptive memory lymphocytes is important for priming the activation of innate cells upon reinfection4. The L. monocytogenes infection model thus serves as a great tool for investigating whether new therapies that are designed to increase IFN-&#947; production have an impact on IFN-&#947; responses in vivo and have productive biological effects such as increasing bacterial clearance or improving mouse survival from infection. Described here is a basic protocol for how to conduct intraperitoneal infections of C57BL/6J mice with the EGD strain of L. monocytogenes and to measure IFN-&#947; production by NK cells, NKT cells, and adaptive lymphocytes by flow cytometry. In addition, procedures are described to: (1) grow and prepare the bacteria for inoculation, (2) measure bacterial load in the spleen and liver, and (3) measure animal survival to endpoints. Representative data are also provided to illustrate how this infection model can be used to test the effect of specific agents on IFN-&#947; responses to L. monocytogenes and survival of mice from this infection.

Experimental Infection with &lt;em&gt;Listeria monocytogenes&lt;/em&gt; as a Model for Studying Host Interferon-&amp;#947; Responses

This protocol describes how to inoculate C57BL/6J mice with the EGD strain of Listeria monocytogenes (L. monocytogenes) and to measure interferon-&#947; (IFN-&#947;) responses by natural killer (NK) cells, natural killer T (NKT) cells, and adaptive T lymphocytes post-infection. This protocol also describes how to conduct survival studies in mice after infection with a modified LD50 dose of the pathogen.

Infection expérimentale avec<em&gt; Listeria monocytogenes</em&gt; En tant que modèle pour l&#39;étude de l&#39;hôte Réponses Interféron-y

L. monocytogenes is a gram-positive bacterium that is a cause of food borne disease in humans. Experimental infection of mice with this pathogen has been ...

Listeria monocytogenes Infection of the Brain

Listeria monocytogenes is an intracellular bacterial pathogen that is frequently associated with food-borne infection. The ability of&#160;L. monocytogenes to cross the blood-brain barrier (BBB) is concerning as it can lead to life-threatening meningitis and encephalitis. The BBB protects the brain microenvironment from various toxic metabolites and microbial pathogens found in the blood following infection, and therefore supports brain homeostasis. The mechanisms by which L. monocytogenes present in the bloodstream cross the BBB to cause brain infections are not fully understood and there is also a lack of a robust model system to study brain infections by L. monocytogenes. Here, we present a simple mouse infection model to determine whether bacteria have crossed the BBB and to quantitate the burden of bacteria that have colonized the brain in vivo. In this method, animals were infected intravenously with L. monocytogenes and were humanely euthanized by exposure to CO2 followed by cervical dislocation. Cardiac perfusion of the animals was performed prior to harvesting infected organs. Blood was collected before perfusion and the number of bacteria per organ or mL of blood was determined by plating dilutions of the blood or organ homogenates on agar plates and counting the number of colonies formed. This method can be used to study novel receptor-ligand interactions that enhance infection of the brain by L. monocytogenes and can be easily adapted for the study of multiple bacterial pathogens.

Listeria monocytogenes Infection of the Brain

During infection, Listeria monocytogenes is capable of crossing the blood-brain barrier to colonize the brain. In this protocol, we demonstrate how to assess bacterial colonization of organs following infection of mice. A procedure to perform whole organ perfusion for specific determination of bacterial numbers in the brain parenchyma is provided.