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.).

1. Preparation of Cellular and Bacterial Cultures, Transfection Tools and Primary Antibodies
<ol>
	<li>Prepare a fresh agar plate to isolate individual L. monocytogenes colonies from a bacterial glycerol stock (50% glycerol/50% saturated bacterial liquid overnight culture) kept at -80 &#176;C.
	<ol>
		<li>Using an aluminum rack (kept at -80 &#176;C) to transport a frozen bacterial glycerol stock, streak bacteria on a Brain Heart Infusion (BHI) agar plate.</li>
		<li>Incubate the plate at 37 &#176;C during 48 hr (or until individual bacterial colonies can be isolated).</li>
		<li>Keep this working plate afterwards at 4 &#176;C during a maximum time of 1 month (it will be used to seed liquid cultures).</li>
		<li>In our protocol, we use the L. monocytogenes serotype 1/2a EGDe.PrfA* strain, but as illustrated in Figure 1, the L. monocytogenes serotype 4b P14.PrfA* strain gives similar results.</li>
	</ol>
	</li>
	<li>HeLa ATCC CCL2 cells are grown in Dulbecco Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) in the absence of antibiotics.</li>
	<li>Independent pools of scrambled (control) and anti-Met siRNAs are loaded in black 384-well microscopy cell culture plates.
	<ol>
		<li>Dilute 160 pmol of siRNA in 500 &#956;l of RNase-free water and add 5 &#956;l of this solution per well (final concentration: 1.6 pmol).</li>
		<li>Keep this plate at -20 &#176;C for a maximum period of 1 year.</li>
	</ol>
	</li>
	<li>Polyclonal rabbit antibodies against the bacterial protein InlC have been obtained by immunization using an InlC-GST recombinant protein as described previously25.</li>
</ol>
2. Reverse siRNA Cell Transfection
<ol>
	<li>72 hr before infection bring to room temperature a black 384-well microscopy cell culture plate containing 1.6 pmol siRNA in 5 &#956;l of RNase-free water in each well.</li>
	<li>Centrifuge the plate for 3 min at 300 rcf at room temperature to bring down siRNA which could have been deposited in the walls of the wells.</li>
	<li>Prepare room temperature DMEM supplemented with 0.4% Lipofectamine RNAiMAX.</li>
	<li>Add 25 &#956;l of the DMEM/RNAiMAX transfection medium to each well (do not incubate the transfection medium longer than 20 min before adding it to the siRNA).</li>
	<li>Move the plate back and forth to mix the siRNA with the transfection solution, then keep the plate for 1 hr at room temperature to allow siRNA-Lipofectamine complexes to form.</li>
	<li>Wash HeLa cells from a confluent- or sub-confluent cell culture flask once with 10 ml 37 &#176;C pre-warmed PBS.</li>
	<li>Detach HeLa cells by adding 1 ml 37 &#176;C prewarmed trypsin to the cell culture flask and incubate for 3 to 5 min at 37 &#176;C.</li>
	<li>Re-suspend cells in 10 ml 37 &#176;C prewarmed DMEM supplemented with 16% FBS.</li>
	<li>Count cells and prepare a cell suspension of 12,000 cells per ml in DMEM supplemented with 16% FBS.</li>
	<li>Add 50 &#956;l of the cell suspension to each well in the 384-well plate.</li>
	<li>Move the plate quickly back and forth to distribute cells and let cells settle down for 10 min at room temperature.</li>
	<li>Seal the plate with Parafilm and keep it for 72 hr in a humidified 5% CO2-containing atmosphere at 37 &#176;C.</li>
</ol>
3. Cellular Infection and Staining
<ol>
	<li>The day before infection, take a single colony of L. monocytogenes from the BHI agar plate and resuspend it in 5 ml of liquid BHI medium in a 15 ml polystyrene tube.</li>
	<li>Incubate overnight at 37 &#176;C in a shacking device to allow for bacterial growth.</li>
	<li>The day of the infection, wash 1 ml of the overnight L. monocytogenes culture by centrifuging 2 min at 10,600 rcf on a table-top centrifuge.</li>
	<li>Discard the supernatant (which contains the secreted cytotoxin listeriolysin O) and re-suspend the pellet in 1 ml of PBS (repeat the washing step 3 more times).</li>
	<li>Read the bacterial optical density at 600 nm and estimate the number of bacteria (OD=1 is equivalent to 1E9 bacteria/ml).</li>
	<li>Prepare the adequate L. monocytogenes dilution in DMEM supplemented with 1% FBS: using highly invasive strains like EGDe.PrfA*, we suggest the use of 5E4 bacteria in 30 &#956;l of medium per well (the multiplicity of infection is estimated as 25 for 2,000 cells).</li>
	<li>Remove the cell culture medium in each well (80 &#956;l) and replace it by adding 30 &#956;l of the L. monocytogenes-containing medium.</li>
	<li>Centrifuge the plate at 200 rcf for 5 min at room temperature to synchronize the infection process.</li>
	<li>Incubate the plate for 1 hr in a humidified 5% CO2-containing atmosphere at 37 &#176;C on a prewarmed aluminum block.</li>
	<li>Remove the L. monocytogenes-containing medium from each well and add 30 &#956;l of prewarmed DMEM supplemented with 10% FBS and 40 &#956;g/ml gentamicin to kill extracellular L. monocytogenes.</li>
	<li>Incubate for 4 hr in a 5% CO2-containing atmosphere at 37 &#176;C on a prewarmed metal block.</li>
	<li>Prepare a solution of PBS supplemented with 8% formaldehyde (this solution should be prepared fresh so that the formaldehyde monomers, rather than the paraformaldehyde polymers, are used).</li>
	<li>Without discarding the cell culture medium, add 30 &#956;l of PBS supplemented with 8% formaldehyde (final concentration: 4%) and incubate for 15 min at room temperature.</li>
	<li>Remove the fixative and wash the cells three times with 80 &#956;l of PBS per well (keep the cells in a final volume of 80 &#956;l of PBS per well).</li>
	<li>Prepare a 1:250 dilution of the rabbit anti-InlC serum in PBS supplemented with 0.2% saponin.</li>
	<li>Add 10 &#956;l of the primary antibody solution to each well after removing the PBS and incubate for 30 min at room temperature.</li>
	<li>Discard the primary antibody solution and wash four times with 40 &#956;l of PBS per well.</li>
	<li>Dilute the secondary Alexa Fluor 546-coupled anti-rabbit antibody (1:250), the DAPI solution (1:1,500) and phalloidin-Dy647 (1:150) in PBS supplemented with 0.2% saponin.</li>
	<li>Add 10 &#956;l of this secondary staining solution to each well and incubate for 30 min at room temperature.</li>
	<li>Discard the secondary staining solution and wash four times with 40 &#956;l of PBS per well (leave a final volume of 40 &#956;l in each well and seal the plate).</li>
	<li>The plate can be imaged immediately or can be stored at 4 &#176;C protected from light (cover with aluminum foil) for subsequent analysis.</li>
</ol>
4. Image Acquisition and Analysis
<ol>
	<li>Acquire images in three different channels (350 nm, 546 nm and 647 nm) using a 10X objective mounted on an automated microscope (acquire preferentially 9 images per well).</li>
	<li>The InlC signal can be measured using image analysis software like CellProfiler that allows automated segmentation of nuclei and cell bodies using the DAPI and the phalloidin staining, respectively.</li>
</ol>

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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. A., 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=Histone+modifications+induced+by+a+family+of+bacterial+toxins.">Histone modifications induced by a family of bacterial toxins.</a> Proceedings of the National Academy of Sciences USA. 104, 17555-17559 (2007).</li><li>Ribet, D., 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=Listeria+monocytogenes+impairs+SUMOylation+for+efficient+infection.">Listeria monocytogenes impairs SUMOylation for efficient infection.</a> Nature. 464, 1192-1195 (2010).</li><li>R&#228;met, M., Manfruelli, P., Pearson, A., Mathey-Prevot, B., Ezekowitz, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+genomic+analysis+of+phagocytosis+and+identification+of+a+Drosophila+receptor+for+E.+coli.">Functional genomic analysis of phagocytosis and identification of a Drosophila receptor for E. coli.</a> Nature. 416, 644-648 (2002).</li><li>Agaisse, H., Burrack, L. S., Philips, J. A., Rubin, E. J., Perrimon, N., Higgins, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+RNAi+screen+for+host+factors+required+for+intracellular+bacterial+infection.">Genome-wide RNAi screen for host factors required for intracellular bacterial infection.</a> Science. 309, 1248-1251 (2005).</li><li>Cheng, L. W., Viala, J. P., Stuurman, N., Wiedemann, U., Vale, R. D., Portnoy, D. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Gly145Ser+substitution+in+the+transcriptional+activator+PrfA+causes+constitutive+overexpression+of+virulence+factors+in+Listeria+monocytogenes.">A Gly145Ser substitution in the transcriptional activator PrfA causes constitutive overexpression of virulence factors in Listeria monocytogenes.</a> Journal of Bacteriology. 179, 1533-1540 (1997).</li><li>Rajabian, T., 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=The+bacterial+virulence+factor+InlC+perturbs+apical+cell+junctions+and+promotes+cell-to-cell+spread+of+Listeria.">The bacterial virulence factor InlC perturbs apical cell junctions and promotes cell-to-cell spread of Listeria.</a> Nature Cell Biology. 11, 1212-1218 (2009).</li><li>Gouin, E., 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=The+Listeria+monocytogenes+InlC+protein+interferes+with+innate+immune+responses+by+targeting+the+I&#954;B+kinase+subinit+IKK&#945;.">The Listeria monocytogenes InlC protein interferes with innate immune responses by targeting the I&#954;B kinase subinit IKK&#945;.</a> Proceedings of the National Academy of Sciences USA. 107, 17333-17338 (2010).</li><li>Pizarro-Cerd&#225;, J., Lecuit, 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=Measuring+and+analysing+invasion+of+mammalian+cells+by+bacterial+pathogens:+the+Listeria+monocytogenes+system.">Measuring and analysing invasion of mammalian cells by bacterial pathogens: the Listeria monocytogenes system.</a> Methods in Molecular Microbiology. 31, 161-177 (2002).</li><li>Snijder, B., Sacher, R., R&#228;m&#246;, P., Damm, E. 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We have nothing to disclose.

Several parameters are critical for the success of our InlC-detection protocol, including the use of healthy cell lines displaying a sufficiently large cytoplasm to allow an unambiguous detection of the InlC signal. In the assay we present in this article we propose the use of HeLa CCL2 cells, which are particularly well suited for our assay due to the extension of their cytosolic space; other HeLa clones such as HeLa Kyoto cells display a smaller cytoplasm but can be used with our infection protocol (HeLa Kyoto cells ar...

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

The Gram positive bacterium Listeria monocytogenes is a food-borne pathogen that invades host cells, disrupts its internalization vacuole and replicates in the cytoplasm of host cells1. L. monocytogenes ease of manipulation in the laboratory context (rapid growth, low toxicity for healthy individuals) associated with the persistence of bacterial virulence traits observed in cellular and animal models (hemolytic activity, leukocytosis) allowed its initial use in the 1960s as a major model for the study of intracellular parasitism and for the establishment of the theoretical foundations of cellular immunity against infection2. In the late 1980s and early 1990s, the dissection of the bacterial intracellular cycle3 as well as the molecular characterization of the most important bacterial virulence factors4-7 favored the use of L. monocytogenes as a key molecular tool for the manipulation and study of host cell functions. The presence of avirulent (L. innocua) and virulent (L. monocytogenes) species in the Listeria genus paved the way for comparative genomic studies8 that, together with the recent establishment of the complete L. monocytogenes transcriptome9, have increased our understanding of the evolution of L. monocytogenes as a human pathogen and as a model system for infection studies10.
L. monocytogenes induces its internalization into host cells upon interaction of the bacterial surface proteins InlA and InlB with their host cell receptors E-cadherin and Met, respectively11-12. Initial candidate-based studies led to the identification of the &#945;/&#946; catenins-actin link as a significant component of the InlA-invasion pathway13 and of the phosphoinositide 3-kinase (PI 3-K) as a critical effector of the InlB-dependent invasion cascade14-15. Proteomic and functional-based assays subsequently allowed the identification of novel cytoskeletal elements16 and lipid second messengers17 required for host cell invasion. Transcriptional studies18 and mass spectrometry-based quantitative proteomics19 have recently shed new light concerning the activation of host signaling cascades and the repression of immune responses during L. monocytogenes infection. Systems biology approaches based on the inactivation of large sets of genes (kinomes, complete genomes) by small interfering RNA (siRNA) silencing have recently opened new avenues for the analysis of global host signaling cascades in the context of specific cellular functions, including phagocytosis and pathogen internalization20. Genome-wide siRNA screens have been previously performed to investigate cellular cascades required for infection of L. monocytogenes in phagocytic Drosophila S2 cells21-22, but this type of analysis has not been performed in non-phagocytic cells, which represent critical targets for infection in vivo.
We have optimized a protocol for the microscopical detection of late stages of infection by L. monocytogenes that is suited for high-throughput siRNA studies of bacterial entry within epithelial cells. Our assay takes advantage of a highly invasive L. monocytogenes strain that presents a point mutation in PrfA, the major transcriptional regulator of L. monocytogenes virulence factors6: this mutation (named PrfA*) renders PrfA constitutively active23 and leads to an increased expression of the invasion proteins InlA and InlB, therefore favoring bacterial entry in otherwise poorly infected non-phagocytic cells. Our readout for infection is based on the detection of the cytosolic accumulation of the secreted bacterial protein InlC: this molecule is a pleiotropic effector that is expressed preferentially by intra-cytoplasmic L. monocytogenes9 and which participates not only in the bacterial cell-to-cell spread24 but which also modulates host immune responses25. The fluorescent labeling of InlC secretion by intracellular bacteria not only allows to clearly distinguish infected from non-infected cells, but also represents an end-point readout that can be used to subsequently dissect infection in its different steps: entry, vacuolar escape, cytosolic bacterial proliferation and cell-to-cell spread. This microscopy-based protocol can be coupled to siRNA screens therefore to study cellular pathways involved in the infection of host cells by L. monocytogenes.

<table><tbody><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 &lt;a href=&quot;http://www.cellprofiler.org/&quot; target=&quot;_blank&quot;&gt;http://www.cellprofiler.org/&lt;/a&gt;</td><td></td></tr></tbody></table>

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

Bacterial intracellular pathogens can be conceived as molecular tools to dissect cellular signaling cascades due to their capacity to exquisitely manipulate and subvert cell functions which are required for the infection of host target tissues. Among these bacterial pathogens, Listeria monocytogenes is a Gram positive microorganism that has been used as a paradigm for intracellular parasitism in the characterization of cellular immune responses, and which has played instrumental roles in the discovery of molecular pathways controlling cytoskeletal and membrane trafficking dynamics. In this article, we describe a robust microscopical assay for the detection of late cellular infection stages of L. monocytogenes based on the fluorescent labeling of InlC, a secreted bacterial protein which accumulates in the cytoplasm of infected cells; this assay can be coupled to automated high-throughput small interfering RNA screens in order to characterize cellular signaling pathways involved in the up- or down-regulation of infection.

Fluorescent labeling of cytoplasmic InlC provides a robust readout for cell infection by L. monocytogenes, as illustrated in Figure 1: the central cell in the micrograph is highly infected by the strain P14.PrfA*23 as it can be observed in the phase contrast image (arrowheads, Figure 1A) and it is confirmed by the DAPI signal where individual bacteria can be clearly distinguished (Figure 1B). The InlC staining (superposed to the DAPI staining in Figur...

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 is a Gram positive bacterial pathogen frequently used as a major model for the study of intracellular parasitism. Imaging late L. monocytogenes infection stages within the context of small-interfering RNA screens allows for the global study of cellular pathways required for bacterial infection of target host cells.

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

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

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9.8K Views.  Pasteur Institute. Listeria monocytogenes is a Gram positive bacterial pathogen frequently used as a major model for the study of intracellular parasitism. Imaging late L. monocytogenes infection stages within the context of small-interfering RNA screens allows for the global study of cellular pathways required for bacterial infection of target host cells.

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

Using Luciferase to Image Bacterial Infections in Mice

Imaging is a valuable technique that can be used to monitor biological processes. In particular, the presence of cancer cells, stem cells, specific immune cell types, viral pathogens, parasites and bacteria can be followed in real-time within living animals 1-2. Application of bioluminescence imaging to the study of pathogens has advantages as compared to conventional strategies for analysis of infections in animal models3-4. Infections can be visualized within individual animals over time, without requiring euthanasia to determine the location and quantity of the pathogen. Optical imaging allows comprehensive examination of all tissues and organs, rather than sampling of sites previously known to be infected. In addition, the accuracy of inoculation into specific tissues can be directly determined prior to carrying forward animals that were unsuccessfully inoculated throughout the entire experiment. Variability between animals can be controlled for, since imaging allows each animal to be followed individually. Imaging has the potential to greatly reduce animal numbers needed because of the ability to obtain data from numerous time points without having to sample tissues to determine pathogen load3-4.
This protocol describes methods to visualize infections in live animals using bioluminescence imaging for recombinant strains of bacteria expressing luciferase. The click beetle (CBRLuc) and firefly luciferases (FFluc) utilize luciferin as a substrate5-6. The light produced by both CBRluc and FFluc has a broad wavelength from 500 nm to 700 nm, making these luciferases excellent reporters for the optical imaging in living animal models7-9. This is primarily because wavelengths of light greater than 600 nm are required to avoid absorption by hemoglobin and, thus, travel through mammalian tissue efficiently. Luciferase is genetically introduced into the bacteria to produce light signal10. Mice are pulmonary inoculated with bioluminescent bacteria intratracheally to allow monitoring of infections in real time. After luciferin injection, images are acquired using the IVIS Imaging System. During imaging, mice are anesthetized with isoflurane using an XGI-8 Gas Anethesia System. Images can be analyzed to localize and quantify the signal source, which represents the bacterial infection site(s) and number, respectively. After imaging, CFU determination is carried out on homogenized tissue to confirm the presence of bacteria. Several doses of bacteria are used to correlate bacterial numbers with luminescence. Imaging can be applied to study of pathogenesis and evaluation of the efficacy of antibacterial compounds and vaccines.

Methods for bioluminescence imaging of bacterial infections in living animals are decribed. Pathogens are modified to express luciferase allowing optical whole body imaging of infections in live animals. Animal models can be infected with luciferase expressing pathogens and the resulting course of disease visualized in real-time by bioluminescence imaging.

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

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.

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

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

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.

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised ...

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.

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.

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.

Listeria monocytogenes is an intracellular bacterial pathogen that is frequently associated with food-borne infection. The ability of&#160;L. ...

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.

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

Imaging of In Situ Interferon Gamma Production in the Mouse Spleen following Listeria monocytogenes Infection

Cytokines are small proteins secreted by cells, mediating cell-cell communications that are crucial for effective immune responses. One characteristic of cytokines is their pleiotropism, as they are produced by and can affect a multitude of cell types. As such, it is important to understand not only which cells are producing cytokines, but also in which environment they do so, in order to define more specific therapeutics. Here, we describe a method to visualize cytokine production in situ following bacterial infection. This technique relies on imaging cytokine-producing cells in their native environment by confocal microscopy. To do so, tissue sections are stained for markers of multiple cell types together with a cytokine stain. Key to this method, cytokine secretion is blocked directly in vivo before harvesting the tissue of interest, allowing for detection of the cytokine that accumulated inside the producing cells. The advantages of this method are multiple. First, the microenvironment in which cytokines are produced is preserved, which could ultimately inform on the signals required for cytokine production and the cells affected by those cytokines. In addition, this method gives an indication of the location of the cytokine production in vivo, as it does not rely on artificial in vitro re-stimulation of the producing cells. However, it is not possible to simultaneously analyze cytokine downstream signaling in cells that receive the cytokine. Similarly, the cytokine signals observed correspond&#160;only to the time-window during which cytokine secretion was blocked. While we describe the visualization of the cytokine Interferon (IFN) gamma in the spleen following mouse infection by the intracellular bacteria Listeria monocytogenes, this method could potentially be adapted to the visualization of any cytokine in most organs.

Imaging of In Situ Interferon Gamma Production in the Mouse Spleen following Listeria monocytogenes Infection

Here, we describe a simple confocal imaging method to visualize the in situ localization of cells secreting the cytokine Interferon gamma in murine secondary lymphoid organs. This protocol can be extended for the visualization of other cytokines in diverse tissues.

Cytokines are small proteins secreted by cells, mediating cell-cell communications that are crucial for effective immune responses. One characteristic of ...

A Technique to Establish a Bacterial Infection Model in an Ex Vivo Organ Culture

Source: Rizzuto, G. A., et al. <a href="https://app.jove.com/v/54237">Human Placental and Decidual Organ Cultures to Study Infections at the Maternal-fetal Interface.</a> J. Vis. Exp. (2016)
This video demonstrates a method to establish an infection model in placental tissues to study infection propagation. An ex vivo placental organ culture is infected with Listeria monocytogenes, a facultative intracellular parasite. The bacteria invade extravillous trophoblasts (EVTs) to enter the placental tissue and then propagate to other cells via cell-to-cell spread.

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human ...

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Flow Cytometry Analysis of Intracellular and Emergent Extracellular Listeria monocytogenes In Vitro

Source: Gayle, P. et al., <a href="https://app.jove.com/v/58775">Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses.</a> J. Vis. Exp. (2019)
In this video, we quantify intracellular and extracellular Listeria monocytogenes, an intracellular pathogenic bacteria, following their infection of cultured macrophages in vitro using flow cytometry.

Source: Gayle, P. et al., Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses. J. Vis. Exp. (2019)
In this video, we ...

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

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Chapters in this video

Using Luciferase to Image Bacterial Infections in Mice

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

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

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

Experimental Infection with Listeria monocytogenes as a Model for Studying Host Interferon-γ Responses

Listeria monocytogenes Infection of the Brain

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

Imaging of In Situ Interferon Gamma Production in the Mouse Spleen following Listeria monocytogenes Infection

A Technique to Establish a Bacterial Infection Model in an Ex Vivo Organ Culture

Flow Cytometry Analysis of Intracellular and Emergent Extracellular Listeria monocytogenes In Vitro