Name: imaging inlc secretion to investigate cellular infection by the bacterial pathogen listeria monocytogenes
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Description: Watch this Scientific Journal Video about Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes at JoVE.com

Forskning i P. COSSART laboratorium støttes av Pasteur Institute, Institut National de la Santé et de la Recherche MEDICALE, Institut National de la Recherche Agronomique, ERC Advanced Grant (233 348), den Agence Nationale de la Recherche (Grant MIE- SignRupVac), Louis-JEANTET Foundation og Fondation Le Roch Les Mousquetaires. AK er en mottaker av et stipend fra Pasteur-Paris University International Doctoral Program / Institut Carnot sykdommer Infectieuses. Vi takker Jason Mercer for optimalisering av mobilnettet transfeksjon protokollen.

En. Forberedelse av Cellular og bakteriekulturer, Transfeksjonspartikler Transfeksjonspartikler Verktøy og primære antistoffer <ol><li> Forbered en frisk agar plate til å isolere enkelte L. monocytogenes kolonier fra en bakteriell glycerol lager (50% glycerol/50% mettet bakteriell væske over natten kultur) holdt ved -80 ° C. <ol><li> Ved hjelp av en aluminiumbeholder (oppbevart ved -80 ° C) for å transportere en frosne bakterie glycerol arkiv, stripe bakterier på et hjerne-hjerte-infusjon (BHI) agar p...

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M., Cossart, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=E-cadherin+is+the+receptor+for+internalin,+a+surface+protein+required+for+entry+of+L.+monocytogenes+into+epithelial+cells.">E-cadherin is the receptor for internalin, a surface protein required for entry of L. monocytogenes into epithelial cells.</a> Cell. 84, 923-932 (1996).</li><li>Shen, Y., Naujokas, M., Park, M., Ireton, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=InIB-dependent+internalization+of+Listeria+is+mediated+by+the+Met+receptor+tyrosine+kinase.">InIB-dependent internalization of Listeria is mediated by the Met receptor tyrosine kinase.</a> Cell. 103, 501-510 (2000).</li><li>Lecuit, M., Hurme, R., Pizarro-Cerda, J., Ohayon, H., Geiger, B., Cossart, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+role+for+alpha-and+beta-catenins+in+bacterial+uptake.">A role for alpha-and beta-catenins in bacterial uptake.</a> Proceedings of the National Academy of Sciences USA. 97, 10008-10013 (2000).</li><li>Ireton, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+role+for+phosphoinositide+3-kinase+in+bacterial+invasion.">A role for phosphoinositide 3-kinase in bacterial invasion.</a> Science. 274, 780-782 (1996).</li><li>Ireton, K., Payrastre, B., Cossart, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Listeria+monocytogenes+protein+InlB+is+an+agonist+of+mammalian+phosphoinositide+3-kinase.">The Listeria monocytogenes protein InlB is an agonist of mammalian phosphoinositide 3-kinase.</a> Journal of Biological Chemistry. 274, 17025-17032 (1999).</li><li>Pizarro-Cerd&#225;, J., Jonqui&#232;res, R., Gouin, E., Vandekerckhove, J., Garin, J., Cossart, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+protein+patterns+associated+with+Listeria+monocytogenes+InlA-+or+InlB+phagosomes.">Distinct protein patterns associated with Listeria monocytogenes InlA- or InlB phagosomes.</a> Cellular Microbiology. 4, 101-115 (2002).</li><li>Pizarro-Cerd&#225;, J., Payrastre, B., Wang, Y. -. J., Veiga, E., Yin, H. L., Cossart, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Type+II+phosphatidylinositol+4-kinases+promote+Listeria+monocytogenes+entry+into+target+cells.">Type II phosphatidylinositol 4-kinases promote Listeria monocytogenes entry into target cells.</a> Cellular Microbiology. 9, 2381-2390 (2007).</li><li>Hamon, M. 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Flere parametere er kritiske for hvor vellykket inlc-deteksjon protokollen, herunder bruk av friske cellelinjer som viser en tilstrekkelig stor cytoplasma til å tillate en utvetydig påvisning av inlc signal. I analysen presenteres i denne artikkelen man foreslår derfor bruken av HeLa-celler CCL2, noe som er spesielt godt egnet for vår analyse på grunn av forlengelsen av de cytosoliske plass, andre HeLa-kloner som HeLa-celler Kyoto vise et mindre cytoplasma men kan benyttes med vår infeksjon protokollen (HeLa Kyoto...

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

Den Gram positive bakterien Listeria monocytogenes er en matbåren patogen som invaderer vertsceller, forstyrrer dens intern vacuole og replikerer i cytoplasma av vertsceller en. L. monocytogenes enkel manipulasjon i laboratoriet sammenheng (rask vekst, lav toksisitet for friske individer) forbundet med utholdenhet av bakterielle virulens trekk observert i cellulære og dyremodeller (hemolytisk aktivitet, leukocytose) tillot sin opprinnelige bruk i 1960 som en viktig modell for studiet av intracellulære snylting og for etableringen av det teoretiske grunnlaget for cellulær immunitet mot infeksjon to. I slutten av 1980 og begynnelsen av 1990-tallet, disseksjon av bakterie intracellulær syklus 3 samt molekylær karakterisering av de viktigste bakteriell virulens faktorer 4-7 favoriserte bruk av L. monocytogenes som en nøkkel molekylære verktøy for manipulasjon og study av vertcellefunksjonene. Tilstedeværelsen av avirulent (L. innocua) og virulente (L. monocytogenes) arter i Listeria slekten banet vei for komparative genomiske studiene åtte som sammen med den nylige etableringen av komplett V. monocytogenes transcriptome 9, har økt vår forståelse av utviklingen av L. monocytogenes som et menneskelig patogen og som et modellsystem for infeksjonsstudier 10. L. monocytogenes induserer sin internalisering i vertsceller på samspillet mellom bakterielle overflateproteiner INLA og InlB med sine vertscellereseptorer E-cadherin og møtte henholdsvis 11-12. Innledende kandidatbaserte undersøkelser førte til identifikasjon av α / β catenins-aktin kobling som en vesentlig komponent i INLA-invasjon veien 13 og for phosphoinositide 3-kinase (PI 3-K) som en kritisk effektor av InlB- avhengig invasjon cascade 14-15. Proteomic og funksjonell-baserte analyser senere tillot identifisering av nye cytoskeletal elementene 16 og lipid andre budbringere 17 som kreves for vertscellen invasjon. Transkripsjons studier 18 og massespektrometri-baserte kvantitative proteomikk 19 har nylig kaste nytt lys om aktivering av verts signaliserer kaskader og undertrykkelse av immunresponser under L. monocytogenes infeksjon. Systembiologi tilnærminger basert på inaktivering av store sett av gener (kinomes, komplette genomer) av små interfererende RNA (siRNA) stanse har nylig åpnet nye veier for analyse av globale vert signale kaskader i sammenheng med spesifikke cellulære funksjoner, inkludert fagocytose og patogen intern 20. Genome-wide siRNA-skjermer har tidligere blitt utført for å undersøke cellulære kaskader som kreves for infeksjon i L. monocytogenes i fagocyttisk Drosophila </em&gt; S2 celler 21-22, men denne typen analyser er ikke utført i ikke-fagocytiske celler, som representerer viktige mål for infeksjon in vivo. Vi har optimalisert en protokoll for den mikroskopiske påvisning av sene stadier av infeksjon med L. monocytogenes som er egnet for high-throughput siRNA studier av bakteriell oppføring i epitelceller. Vår analyse tar nytte av en meget inngripende L. monocytogenes belastningen som presenterer et punkt mutasjon i PrfA, de store transcriptional regulator av L. monocytogenes virulens faktorer 6: denne mutasjonen (oppkalt PrfA *) gjengir PrfA konstitutivt aktiv 23 og fører til økt uttrykk av invasjonen proteiner INLA og InlB, derfor favorisere bakterie oppføring i ellers dårlig infiserte ikke-fagocyttiske celler. Vår avlesning for infeksjon er basert på påvisning av den cytosoliske oppsamling av det utskilte bakterieprotein inlc: dette molekylet eren pleiotropisk effector som uttrykkes fortrinnsvis ved intra-cytoplasmatisk L. monocytogenes 9 og som deltar ikke bare i bakteriell celle-til-celle spre 24, men som også modulerer vert immunresponser 25. Den fluorescerende merking av inlc sekresjon av intracellulære bakterier ikke bare gjør det mulig å klart skille infiserte fra ikke-infiserte celler, men representerer også et ende-punkt avlesning som kan benyttes til senere å dissekere infeksjon i sine forskjellige trinn: registrering, vacuolar flukt, cytosoliske bakteriell spredning og celle-til-celle-spredning. Denne mikros-basert protokoll kan bli koblet til siRNA skjermer således å studere cellulære baner som er involvert i infeksjonen av vertsceller av 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

Bakteriell intracellulære patogener kan bli oppfattet som molekylære verktøy for å dissekere cellulære signal kaskader på grunn av deres evne til å utsøkt manipulere og styrte celle funksjoner som er nødvendige for infeksjon av verts målet vev. Blant disse bakterielle patogener, Listeria monocytogenes er en Gram-positiv mikroorganisme som har vært brukt som et paradigme for intracellulær parasitt i karakterisering av cellulære immunresponser, og som har spilt instrument roller i oppdagelsen av molekylære veier kontrollerende cytoskeletal og membransmugler dynamikk. I denne artikkelen beskriver vi en robust mikroskopiske analyse for påvisning av sen cellular infeksjon stadier av L. monocytogenes basert på det fluorescerende merking av inlc, en utskilt bakterielt protein som akkumuleres i cytoplasma i infiserte celler, og dette assay kan bli koblet til automatiserte high-throughput små interfererende RNA-skjermer for å forkulleacterize cellulære signalveier involvert i opp-eller nedregulering av infeksjon.

Fluorescerende merking av cytoplasmatisk inlc gir en robust avlesning for celle infeksjon av L. monocytogenes, som illustrert i Figur 1: den sentrale celle i mikrografi er sterkt infisert av belastningen P14.PrfA * 23 som det kan observeres i fasekontrastbilde (pilspisser, Figur 1A), og det bekreftes av DAPI signal hvor enkelte bakterier kan tydelig skilles (Figur 1B). Den inlc farging (overlagret på DAPI farging i figur 1C) viser hvordan dette utsk...

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 er en Gram-positiv bakteriell patogen ofte brukt som en stor modell, for studier av intracellulære parasitt. Imaging sent L. monocytogenes infeksjon stadier innenfor rammen av små-interfererende RNA skjermer muliggjør global studie av mobilnettet veier som kreves for bakteriell infeksjon av målet vertsceller.

Imaging inlc Secretion å etterforske Cellular Infeksjon av Bakteriell Patogen<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 ...

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Immunology and Infection

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

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 ...

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 ...

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

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 ...

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

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Insektet<em&gt; Galleria mellonella</em&gt; Som en kraftig infeksjon Model for å etterforske Bakteriell Patogenese

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

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Oral Overføring av<em&gt; Listeria monocytogenes</em&gt; I Mus via Inntak av forurenset mat

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

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Automatisert Separering av<em&gt; C. elegans</em&gt; Trinnløs kolonisert av en bakteriell Patogen

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

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

Vurdering av bakterieinvasjon av hjerteceller i kultur og hjertekolonisering hos infiserte mus ved hjelp av 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

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

RNA Purification fra intracellulært Grown<em&gt; Listeria monocytogenes</em&gt; I makrofagceller

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

Experimental Infection with Listeria monocytogenes as a Model for Studying Host Interferon-ÃƒÆ’Ã…Â½Ãƒâ€šÃ‚Â³ Responses

Eksperimentell infeksjon med<em&gt; Listeria monocytogenes</em&gt; Som en modell for å studere Host interferon-y 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 ...