Name: imaging inlc secretion to investigate cellular infection by the bacterial pathogen listeria monocytogenes
Uploaded: 2013-09-19T22:00:00
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 er støttet af Institut Pasteur, Institut National de la Santé et de la Recherche Médicale, Institut National de la Recherche Agronomique, ERC Advanced Grant (233.348), Agence Nationale de la Recherche (Grant MIE- SignRupVac), Louis-Jeantet Foundation og Fondation Le Roch Les Mousquetaires. AK er en modtager af et stipendium fra Pasteur-Paris University internationale ph.d. Program / Institut Carnot Maladies Infectieuses. Vi takker Jason Mercer for at optimere den cellulære transfektionsprotokol.

1.. Forberedelse for Cellulær og bakteriekulturer Transfektionsfremgangsmåder Værktøj og primære antistoffer <ol><li> Forbered en frisk agarplade at isolere enkelte L. monocytogenes kolonier fra en bakteriel glycerol stock (50% glycerol/50% mættet bakteriel flydende kultur natten over), som holdes ved -80 ° C. <ol><li> Ved hjælp af en aluminium rack (opbevaret ved -80 ° C) til at transportere en frossen bakteriel glycerol stock afsætter bakterier på en Brain Heart Infusion (BHI) agar. </li><li> Ink...

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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 parametre er afgørende for den succes, vores inlc-afsløring protokol, herunder brug af sunde cellelinjer viser en tilstrækkelig stor cytoplasma til at tillade en utvetydig påvisning af inlc signal. I analysen præsenterer vi i denne artikel vil vi foreslå at anvende HeLa CCL2 celler, som er særligt velegnet til vores analyse på grund af udvidelse af deres cytosol rum, andre HeLa-kloner, såsom HeLa Kyoto celler vise et mindre cytoplasma, men kan bruges med vores infektion protokol (HeLa Kyoto celler er sær...

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

Gram positive bakterie Listeria monocytogenes er en fødevare-patogen, der invaderer værtsceller forstyrrer dets internalisering vacuole og replikerer i cytoplasma værtsceller 1. L. monocytogenes nem manipulation i laboratoriet sammenhæng (hurtig vækst, lav toksicitet for raske individer) i forbindelse med den fortsatte bakterielle virulens træk observeret i cellulære og dyremodeller (hæmolytisk aktivitet, leukocytose) tilladt sin oprindelige brug i 1960&#39;erne som en vigtig model for studiet af intracellulære parasitisme og til etablering af det teoretiske grundlag for cellulær immunitet mod infektion 2. I slutningen af 1980&#39;erne og begyndelsen af 1990&#39;erne, dissektion af den bakterielle intracellulære cyklus 3 samt molekylær karakterisering af de vigtigste bakterielle virulensfaktorer 4-7 foretrak brugen af L. monocytogenes som en vigtig molekylær værktøj til manipulation og study værts cellefunktioner. Tilstedeværelsen af avirulente (L. innocua) og virulente (L. monocytogenes) arter i Listeria slægten banede vejen for komparativ genomisk undersøgelser 8, som sammen med den nylige oprettelse af komplet V. monocytogenes transkriptom 9, har øget vores forståelse af udviklingen af L. monocytogenes som et humant patogen og som et modelsystem for infektion studerer 10. L. monocytogenes inducerer sin internalisering i værtsceller ved interaktion af de bakterielle overfladeproteiner inla og INLB med deres værtscellereceptorer E-cadherin og mødte henholdsvis 11-12. Indledende kandidat-baserede studier førte til identifikation af α / β catenins-actin link som en væsentlig komponent i den inla-invasion vej 13 og phosphoinositid 3-kinase (PI 3-K) som en kritisk effektor af INLB- afhængig invasion cascade 14-15. Proteom og funktionelle assays efterfølgende tillod identifikation af nye cytoskeletelementer 16 og lipid second messengers 17 kræves for værtscelle invasion. Transkriptionelle undersøgelser 18 og massespektrometri-baserede kvantitative proteomics 19 har for nylig kastet nyt lys om aktivering af værten signalering kaskader og undertrykkelse af immunreaktioner i løbet af L. monocytogenes infektion. Systembiologi tilgang baseret på inaktivering af store sæt af gener (kinomes, komplette genomer) af små interfererende RNA (siRNA) lyddæmpning har for nylig åbnet nye muligheder for analyse af globale vært signalering kaskader i forbindelse med specifikke cellulære funktioner, herunder fagocytose og patogen internalisering 20. Genome-wide siRNA-skærme er tidligere blevet udført for at undersøge cellulære kaskader kræves for infektion af L. monocytogenes i fagocyterende Drosophila </em&gt; S2-celler 21-22, men denne type analyse er ikke udført i ikke-fagocytceller, der repræsenterer kritiske mål for infektion in vivo. Vi har optimeret en protokol for mikroskopisk påvisning af sene stadier af infektion med L. monocytogenes, der er velegnet til high-throughput siRNA studier af bakteriel indrejse inden epitelceller. Vores analyse tager fordel af en meget invasiv L. monocytogenes stamme, der præsenterer et punkt mutation i PrfA, de store transkriptionelle regulator af L. monocytogenes virulensfaktorer 6: denne mutation (opkaldt PrfA *) gør PrfA konstitutivt aktiv 23 og fører til en øget ekspression af invasionen proteinerne inla og INLB, derfor begunstige bakteriel post i ellers dårligt inficerede ikke-fagocytceller. Vores udlæsning for infektion er baseret på påvisning af den cytosoliske akkumulering af det secernerede bakterielt protein inlc: dette molekyle eren pleiotrop effektor der udtrykkes fortrinsvis ved intra-cytoplasmatisk L. monocytogenes 9, og som deltager ikke kun i den bakterielle celle-til-celle-spredning 24, men som også modulerer værtens immunresponser 25. Den fluorescerende mærkning af inlc sekretion af intracellulære bakterier ikke kun gør det muligt klart at skelne inficerede fra ikke-inficerede celler, men repræsenterer også et slutpunkt udlæsning, der kan bruges til efterfølgende at dissekere infektion i de forskellige faser: indrejse, vakuolær flugt, cytosol bakteriel spredning og celle-til-celle-spredning. Denne mikroskopi-baseret protokol kan kobles til siRNA skærme derfor at studere cellulære veje, der er involveret i infektion af værtsceller ved 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

Bakterielle intracellulære patogener kan opfattes som molekylære værktøjer til at dissekere cellulære signalleringskaskader på grund af deres evne til at udsøgt manipulere og undergrave cellefunktioner, som er nødvendige for infektion af værten målvæv. Blandt disse bakterielle patogener, Listeria monocytogenes er en grampositiv mikroorganisme, der er blevet anvendt som et paradigme for intracellulær parasitisme i karakteriseringen af cellulære immunreaktioner, og som har spillet instrumentale roller i opdagelsen af molekylære mekanismer, der styrer dynamik trafficking cytoskeletale og membran. I denne artikel beskriver vi en robust mikroskopisk analyse til påvisning af sene cellulær infektion stadier af L. monocytogenes er baseret på fluorescerende mærkning af inlc, et udskilt bakterielt protein, som akkumuleres i cytoplasmaet af inficerede celler, og dette assay kan kobles til automatiserede high-throughput små interfererende RNA skærme for at forkulleacterize cellulære signalveje, der er involveret i op-eller nedregulering af infektion.

Fluorescerende mærkning af cytoplasmatisk inlc giver en robust udlæsning for celle infektion med L. monocytogenes, som illustreret i Figur 1: den centrale celle i mikrografiet er stærkt inficeret med stammen P14.PrfA * 23, som det kan observeres i fasekontrast billede (pilespidser, figur 1A), og det bekræftes af DAPI signal, hvor enkelte bakterier kan klart skelnes (figur 1B). Den inlc farvning (overlejret til DAPI farvning i figur 1C) viser, hvo...

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 grampositiv bakteriel patogen, der ofte anvendes som en vigtig model til undersøgelse af intracellulære parasitter. Imaging sent L. monocytogenes infektion faser i forbindelse med små interfererende RNA skærme giver mulighed for global undersøgelse af cellulære veje er nødvendig for bakteriel infektion af target værtsceller.

Imaging inlc Sekretion at undersøge Cellular Infektion med bakteriepatogen<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 ...

Research

JoVE Journal

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

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

The Insect<em&gt; Galleria mellonella</em&gt; Som en kraftfuld Infektion modellen til bakteriel 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

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

Oral transmission af<em&gt; Listeria monocytogenes</em&gt; I Mus via indtagelse af forurenet mad

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

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

Automatiseret Adskillelse af<em&gt; C. elegans</em&gt; Variabel koloniseret af et bakterielt 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 bakteriel invasion af hjerteceller i kultur og hjerte kolonisering i inficerede mus ved hjælp af 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 Oprensning 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

Eksperimentel infektion med<em&gt; Listeria monocytogenes</em&gt; Som en model til at 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 ...