Mice were treated humanely in accordance with all appropriate government guidelines for the Care and Use of Laboratory Animals of the National Institutes of Health and their use was approved for this entire study by the University of Miami Institutional Animal Care and Use Committee (protocol 16-053).
1. Preparing Cells for Infection
<ol>
	<li>Propagate mouse macrophage-like cell line RAW 264.7 in DMEM tissue culture media supplemented with 10% fetal calf serum (henceforth referred to as DMEM/FCS) using a 125 mL flask and a tissue culture incubator maintained at 37 &#176;C/5% CO2.</li>
	<li>The day before the infection, use a cell scraper to remove cells from the expansion flask and collect in a 15 mL screw cap conical tube. Centrifuge cells at 800 x g for 10 min, decant supernatant and suspend the cell pellet in 1 mL of DMEM/FCS (without antibiotics) and determine the titer using a hemocytometer or an automated cell counter.</li>
	<li>Adjust titer to 2 x 106 cells/mL and add 0.10 mL (2 x 105 cells) to wells of a 48-well tissue culture dish. Ensure that the cell suspension covers the entire surface of the well.
	<ol>
		<li>Plate cells in additional wells for a later source of conditioned media. Also place 0.10 mL of DMEM/FCS in three additional wells to serve as a &#39;no cell control&#39;.</li>
	</ol>
	</li>
	<li>Incubate overnight in a tissue culture incubator at 37 &#176;C/5% CO2. The next day, do not remove from incubator until the bacterium inoculum is prepared and ready to use.</li>
</ol>
2. Preparing Listeria monocytogenes (Lm) for infection
<ol>
	<li>Inoculate 2 mL of brain-heart infusion (BHI) with a single colony from a freshly-streaked agar plate (&#60;2 days) of Lm and incubate overnight at 37 &#176;C with shaking at 130 rotations/min.</li>
	<li>The next day, add 0.10 mL of overnight culture to 2 mL pre-warmed BHI and continue incubating at 37 &#176;C with shaking until the optical density (OD600) is between 0.4 and 0.6.</li>
	<li>Determine approximate bacterial titer using an empirically-derived conversion formula. In our lab, an exponentially growing Lm culture in BHI is approximately 1.3 x 109 colony forming units (CFU)/OD600. Minimize the amount of time bacterial cultures are exposed to room temperature.</li>
	<li>Just prior to infection adjust the titer to 5 x 107 CFU/mL using pre-warmed DMEM/FCS as diluent.</li>
</ol>
3. Infection
<ol>
	<li>Working quickly and precisely, add 20 &#181;L (1 x 106 CFU; multiplicity of infection, MOI = 5) of the freshly-prepared Lm inoculum (step 2.4 above) to wells by slightly tilting the dish and carefully placing the pipet tip in the overlying media; 
	NOTE:&#160;avoid touching the walls of the well with the pipet tip.</li>
	<li>Gently pipet the contents of each well to ensure complete mixing; do not shake or significantly tilt the plate as this will spread Lm over the walls of the well. Return cell culture dish to 37 &#176;C/5% CO2 incubator.</li>
	<li>Prepare a 10 &#181;g/mL gentamicin solution using conditioned media from uninfected wells as diluent.</li>
	<li>At 30 minutes post infection (mpi), carefully add 30 &#181;L of the gentamicin solution (final concentration 2.5 &#181;g/mL) and gently pipet the contents of the well as before and return cell culture dish to 37 &#176;C/5% CO2 incubator.</li>
	<li>At 60 mpi, harvest appropriate wells for either CFU assay (60 mpi time point) or FCM analysis (as described below in sections 4 and 5, respectively).</li>
	<li>For remaining wells, at various times thereafter either harvest cells as before or, for the Lm emergence assay, entirely remove media from well and replace with 150 &#181;L of gentamicin-free conditioned media.</li>
</ol>
4. Sampling Processing and Analysis of Intracellular and Emergent Lm by CFU Assay
<ol>
	<li>To harvest, slightly tilt the dish and carefully remove the entire media from the well. Add 0.50 mL of distilled sterile water (dsH2O) to the well. After 30 s, transfer the resulting water lysate (100) to a 1.5 mL microcentrifuge tube and vortex vigorously for 10 s.</li>
	<li>Prepare 10-1 and 10-2 dilutions by adding either 4.5 &#181;L or 50 &#181;L of the water lysate to 450 &#181;L of dsH2O and briefly vortex to ensure thorough mixing.</li>
	<li>Spread 50 &#181;L of the 100, 10-1 and/or 10-2 samples onto lysogeny broth (LB) agar plates.</li>
	<li>To assay for emergent Lm (step 3.6 above), remove 10 &#181;L of the overlaying media and divide it into two 5 &#181;L aliquots: to one aliquot add 5 &#181;L of DMEM/FCS and to the second aliquot add 5 &#181;L of DMEM/FCS containing 5 &#181;g/mL gentamicin. The latter control distinguishes between extracellular Lm (gentamicin sensitive) and intracellular Lm (gentamicin resistant) in the subsequent CFU assay.</li>
	<li>After 5 min at room temperature, add 90 &#181;L of dsH2O, vortex vigorously for 10 s, and spread 50 &#181;L on LB agar plates.</li>
	<li>Enumerate Lm colonies on LB agar plates following 2 days of incubation at 37 &#176;C.</li>
</ol>
5. Sampling Processing and Analysis of Intracellular and Emergent Lm by Flow Cytometry (FCM)
<ol>
	<li>Prepare RAW 264.7 cells as described in steps 1.1-4. Adjust cells to 1 x 106 cells/mL and add 1 mL (1 x 106 cells) to wells of a 6-well tissue culture dish.</li>
	<li>Prepare bacterium inoculum as described in step 2.1-2.3 and infect cells with volume of inoculum corresponding to a MOI of 50 (5 x 107 CFU).</li>
	<li>At 1.5 hours post infection (hpi), collect media from first set of wells and place in a 1.5 mL microcentrifuge tube with 500 &#181;L of 4% paraformaldehyde (PFA) and place on ice until all wells have been processed.</li>
	<li>To collect intracellular Lm, add 1 mL of dsH2O to the well and after 60 s, transfer the resulting water lysate to a 1.5 mL microcentrifuge tube with 500 &#181;L of 4% PFA. Vortex vigorously for 10 s and place on ice until all wells have been processed. (Sample processing is described in step 5.8 below.)</li>
	<li>For remaining wells, remove media and replace with 1 mL of DMEM/FCS with 5 &#181;g/mL gentamicin to kill extracellular Lm and promptly return cells to incubator.</li>
	<li>After 30 min (2 hpi) remove media and replace with DMEM/FCS without gentamicin.</li>
	<li>After 2 and 4 h (4 and 6 hpi) take second and third time points by collecting media and lysates as steps 5.3 and 5.4.</li>
	<li>Centrifuge tubes at 10,000 x g for 7 min. Remove supernatant without disturbing pellet.</li>
	<li>Suspend each pellet in staining cocktail of 50 &#181;L of 4% PFA and 15 &#181;L of phalloidin. Incubate tubes in the dark at 4 &#176;C for 20 min.</li>
	<li>After incubation add 1 mL of FACS buffer to each tube and pipette up and down to mix.</li>
	<li>Spin tubes at 10,000 x g for 7 min. Remove supernatant without disturbing pellet.</li>
	<li>Suspend each pellet in 400 &#181;L of FACS buffer for immediate use, or 200 &#181;L of FACS buffer and 200 &#181;L of 4% PFA if storing for later analysis.</li>
	<li>Run samples on a flow cytometry and analyze the collected data.</li>
</ol>
6. Sampling Processing and Analysis of Lm Colonization and Host Responses in the Liver
<ol>
	<li>Prepare Lm for infection as described in step 2.1-2.3.</li>
	<li>Calculate the necessary amount of subculture for desired titer of 3 x 106 CFU/mL.</li>
	<li>Just prior to infection dilute the amount of calculated bacteria with pre-warmed Hank&#39;s Balanced Salt Solution (HBSS) in a final volume of 1 mL. This is the input inoculum that is injected into the mouse.</li>
	<li>Using a 28 G&#189; 100U insulin syringe, draw 100 &#181;L (3 x 105 CFU) of input inoculum and remove any visible air bubbles.</li>
	<li>Place the mouse (strain C57BL/6; males between 6 and 12 weeks old) into the holding restraint and use warm water (no more than 45 &#176;C) to dilate the tail vein, then inject the input inoculum into the vein.</li>
	<li>Plate 10-2 and 10-3 dilutions of the input inoculum onto LB agar 10bcm plates and incubate overnight at 37 &#176;C to determine the actual input dosage.</li>
	<li>At 18 hpi, humanely euthanize mouse (CO2 asphyxiation followed by cervical dislocation) and using sterile scissors cut open mouse peritoneum to collect frontal lobe of the liver using separate pairs of tweezers and scissors for the outside and inside of the mouse. Place the liver in a 6 cm Petri dish on ice.</li>
	<li>Cut the liver into small cubes and place into a glass vial with a magnetic stir bar. Add 2 mL of 2 mg/mL collagenase D reconstituted in DMEM without FCS. Incubate vials at 37 &#176;C on a magnetic stirrer for 30&#8722;45 min. Once the tissue is homogenized, add 3 mL of DMEM/FCS to inactivate the collagenase.</li>
	<li>Filter the cells through a 70 &#181;m cell strainer into a conical tube, adding more DMEM/FCS if necessary.</li>
	<li>Centrifuge cells at 800 x g to pellet at 4 &#176;C for 10 min, remove supernatant and suspend cell pellet in 500 &#181;L of ACK buffer to lyse red blood cells.</li>
	<li>Incubate cells at room temperature for 5 min in ACK buffer. Suspend cells in 1 mL of DMEM/FCS.</li>
	<li>Centrifuge cells at 800 x g to pellet at 4 &#176;C for 10 min, remove supernatant and suspend in 1 mL of DMEM/FCS. Then, count the cells using a hemocytometer or an automated cell counter.</li>
	<li>Transfer 1 x 106 cells to 1.5 mL microcentrifuge tube and spin to pellet. Then suspend in 1 mL of dsH2O and vortex to lyse the cells. Plate 50 &#181;L on LB plates at undiluted and 10-2 dilutions and incubate at 37 &#176;C to enumerate Lm for CFU analysis.</li>
	<li>Transfer 1 x 106 cells into FACS tubes for each tissue sample according to the cell counts and wash with 1 mL of FACS buffer.</li>
	<li>Centrifuge cells at 800 x g to pellet at 4 &#176;C for 10 min and remove supernatant.</li>
	<li>Prepare an antibody cocktail containing the antibodies of interest based on the recommended concentration for each antibody and FACS buffer. See Table of Materials for recommended amounts used in the analysis.</li>
	<li>Suspend pellet in 100 &#181;L of antibody cocktail and incubate tubes in the dark at 4 &#176;C for 30 min.</li>
	<li>After incubation, add 2 mL of FACS buffer to each tube and suspend cells.</li>
	<li>Centrifuge cells at 800 x g to pellet at 4 &#176;C and discard the supernatant. Then suspend the pellet in 400 &#181;L of FACS buffer for immediate use, or 200 &#181;L of FACS buffer and 200 &#181;L of 4% PFA if storing for later.</li>
	<li>Analyze cells by flow cytometry (see step 5.13).</li>
</ol>

<ol><li>Freitag, N. E., Port, G. C., Miner, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Listeria+monocytogenes+-+from+saprophyte+to+intracellular+pathogen%5BTitle%5D)+AND+%22Nature+Review+Microbiology%22%5BJournal%5D)">Listeria monocytogenes - from saprophyte to intracellular pathogen.</a> Nature Review Microbiology. 7 (9), 623-628 (2009).</li>
<li>Ray, K., Marteyn, B., Sansonetti, P. J., Tang, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Life+on+the+inside%3A+the+intracellular+lifestyle+of+cytosolic+bacteria%5BTitle%5D)+AND+%22Nature+Review+Microbiology%22%5BJournal%5D)">Life on the inside: the intracellular lifestyle of cytosolic bacteria.</a> Nature Review Microbiology. 7 (5), 333-340 (2009).</li>
<li>Miner, M. D., Port, G. C., Freitag, N. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Functional+impact+of+mutational+activation+on+the+Listeria+monocytogenes+central+virulence+regulator+PrfA%5BTitle%5D)+AND+%22Microbiology%22%5BJournal%5D)">Functional impact of mutational activation on the Listeria monocytogenes central virulence regulator PrfA.</a> Microbiology. 154, 3579-3589 (2008).</li>
<li>McCormack, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Perforin-2+Protects+Host+Cells+and+Mice+by+Restricting+the+Vacuole+to+Cytosol+Transitioning+of+a+Bacterial+Pathogen%5BTitle%5D)+AND+%22Infection+and+Immunity%22%5BJournal%5D)">Perforin-2 Protects Host Cells and Mice by Restricting the Vacuole to Cytosol Transitioning of a Bacterial Pathogen.</a> Infection and Immunity. 84, 1083-1091 (2016).</li>
<li>Gregory, S. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Complementary+adhesion+molecules+promote+neutrophil-Kupffer+cell+interaction+and+the+elimination+of+bacteria+taken+up+by+the+liver%5BTitle%5D)+AND+%22Journal+of+Immunology%22%5BJournal%5D)">Complementary adhesion molecules promote neutrophil-Kupffer cell interaction and the elimination of bacteria taken up by the liver.</a> Journal of Immunology. 168, 308-315 (2002).</li>
<li>Shi, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Monocyte+trafficking+to+hepatic+sites+of+bacterial+infection+is+chemokine+independent+and+directed+by+focal+intercellular+adhesion+molecule-1+expression%5BTitle%5D)+AND+%22Journal+of+Immunology%22%5BJournal%5D)">Monocyte trafficking to hepatic sites of bacterial infection is chemokine independent and directed by focal intercellular adhesion molecule-1 expression.</a> Journal of Immunology. 184, 6266-6274 (2010).</li>
<li>Pitts, M. G., Combs, T. A., D'Orazio, S. E. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neutrophils+from+Both+Susceptible+and+Resistant+Mice+Efficiently+Kill+Opsonized+Listeria+monocytogenes%5BTitle%5D)+AND+%22Infection+and+Immunity%22%5BJournal%5D)">Neutrophils from Both Susceptible and Resistant Mice Efficiently Kill Opsonized Listeria monocytogenes.</a> Infection and Immunity. 86, 00085(2018).</li>
<li>Carr, K. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Specific+depletion+reveals+a+novel+role+for+neutrophil-mediated+protection+in+the+liver+during+Listeria+monocytogenes+infection%5BTitle%5D)+AND+%22European+Journal+of+Immunology%22%5BJournal%5D)">Specific depletion reveals a novel role for neutrophil-mediated protection in the liver during Listeria monocytogenes infection.</a> European Journal of Immunology. 41 (9), 2666-2676 (2011).</li>
<li>Blériot, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Liver-resident+macrophage+necroptosis+orchestrates+type+1+microbicidal+inflammation+and+type-2-mediated+tissue+repair+during+bacterial+infection%5BTitle%5D)+AND+%22Immunity%22%5BJournal%5D)">Liver-resident macrophage necroptosis orchestrates type 1 microbicidal inflammation and type-2-mediated tissue repair during bacterial infection.</a> Immunity. 42 (1), 145-158 (2015).</li>
<li>Jones, G. S., D'Orazio, S. E. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Monocytes+are+the+predominant+cell+type+associated+with+Listeria+monocytogenes+in+the+gut%2C+but+they+do+not+serve+as+an+intracellular+growth+niche%5BTitle%5D)+AND+%22Journal+of+Immunology%22%5BJournal%5D)">Monocytes are the predominant cell type associated with Listeria monocytogenes in the gut, but they do not serve as an intracellular growth niche.</a> Journal of Immunology. 198, 2796-2804 (2017).</li>
<li>Agaisse, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genome-wide+RNAi+screen+for+host+factors+required+for+intracellular+bacterial+infection%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Genome-wide RNAi screen for host factors required for intracellular bacterial infection.</a> Science. 309, 1248-1251 (2005).</li>
<li>Cheng, L. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Use+of+RNA+interference+in+Drosophila+S2+cells+to+identify+host+pathways+controlling+compartmentalization+of+an+intracellular+pathogen%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Science+U+S+A%22%5BJournal%5D)">Use of RNA interference in Drosophila S2 cells to identify host pathways controlling compartmentalization of an intracellular pathogen.</a> Proceedings of the National Academy of Science U S A. 102, 13646-13651 (2005).</li>
<li>Singh, R., Jamieson, A., Cresswell, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((GILT+is+a+critical+host+factor+for+Listeria+monocytogenes+infection%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">GILT is a critical host factor for Listeria monocytogenes infection.</a> Nature. 455, 1244-1247 (2008).</li>
<li>Burrack, L. S., Harper, J. W., Higgins, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Perturbation+of+vacuolar+maturation+promotes+listeriolysin+O-independent+vacuolar+escape+during+Listeria+monocytogenes+infection+of+human+cells%5BTitle%5D)+AND+%22Cellular+Microbiology%22%5BJournal%5D)">Perturbation of vacuolar maturation promotes listeriolysin O-independent vacuolar escape during Listeria monocytogenes infection of human cells.</a> Cellular Microbiology. 11, 1382-1398 (2009).</li>
<li>Shrestha, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+host-encoded+Heme+Regulated+Inhibitor+%28HRI%29+facilitates+virulence-associated+activities+of+bacterial+pathogens%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">The host-encoded Heme Regulated Inhibitor (HRI) facilitates virulence-associated activities of bacterial pathogens.</a> PLoS One. 8, 68754(2013).</li>
<li>Bahnan, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+eIF2%CE%B1+Kinase+Heme-Regulated+Inhibitor+Protects+the+Host+from+Infection+by+Regulating+Intracellular+Pathogen+Trafficking%5BTitle%5D)+AND+%22Infection+and+Immunity%22%5BJournal%5D)">The eIF2α Kinase Heme-Regulated Inhibitor Protects the Host from Infection by Regulating Intracellular Pathogen Trafficking.</a> Infection and Immunity. 20, 00707(2018).</li>
<li>Kortebi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Listeria+monocytogenes+switches+from+dissemination+to+persistence+by+adopting+a+vacuolar+lifestyle+in+epithelial+cells%5BTitle%5D)+AND+%22PLoS+Pathogen%22%5BJournal%5D)">Listeria monocytogenes switches from dissemination to persistence by adopting a vacuolar lifestyle in epithelial cells.</a> PLoS Pathogen. 13, 1006734(2017).</li>
<li>VanCleave, T. T., Pulsifer, A. R., Connor, M. G., Warawa, J. M., Lawrenz, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Impact+of+Gentamicin+Concentration+and+Exposure+Time+on+Intracellular+Yersinia+pestis%5BTitle%5D)+AND+%22Frontiers+in+Cellular+and+Infection+Microbiology%22%5BJournal%5D)">Impact of Gentamicin Concentration and Exposure Time on Intracellular Yersinia pestis.</a> Frontiers in Cellular and Infection Microbiology. 7, 505(2017).</li>
</ol>

The authors have nothing to disclose.

Due to its rapid and processive cellular infection program, Lm is an ideal pathogen to probe cellular activities that impact infection. A number of host factors have been identified that either positively or negatively affect Lm cellular infection11,12,13,14. Two such host factors characterized in our laboratory, Perforin-2 and the Heme Regulated Inhibitor (P2 and hI), regulat...

Infection-based experimental models are inherently challenging due to their dependence on the starting state conditions of the host and pathogen, the various pathogen infection strategies, and the difficulty of attributing pathogen- and host-driven processes based on outcomes. The bacterium Listeria monocytogenes (Lm) has become an ideal pathogen to probe host defense responses because of its genetic and microbiological tractability, its rapid and processive cellular infection strategy, and the relatively clear relationship between its cellular- and organismic-level infection phenotypes. The cellular infection of Lm proceeds through four distinct phases1: (i) cellular invasion that concludes with Lm being enclosed within a vacuole; (ii) Lm-directed dissolution of the vacuole membrane and release of Lm into the cytosol; (iii) intracytosolic replication; and (iv) physical penetration of the plasma membrane that results in either the infection of directly adjacent cells (such as in an epithelial sheet) or, in solitary cells, release of Lm into the extracellular milieu. Each of these phases are promoted by specific Lm-encoded factors (referred to as &#39;virulence factors&#39;) that, when deleted, cause infection defects in both cellular and animal models. This general infection strategy has been independently evolved by a number of the so-called &#39;cytosolic&#39; pathogens2.
Colony-forming unit (CFU) assays are widely employed to evaluate both in vitro (i.e., cellular) as well as in vivo (i.e., organismic) infection outcomes. In addition to their high sensitivity, particularly for in vivo infections, CFU assays provide an unambiguous readout for pathogen invasion and intracellular survival/proliferation. CFU assays have been extensively used to analyze both Lm and host cell determinants that impact infection. As informative as these prior studies have been to analyze cellular invasion and intracytosolic replication, CFU assays have not, to the best of our knowledge, been used to track the fourth phase of the Lm infection process: cellular escape. Here, we describe relatively simple means of how cellular escape (hereafter referred to as &#39;emergence&#39;) can be monitored by CFU assay (as well as by flow cytometry) and show examples of how both pathogen- and host-encoded factors regulate this phase of the Lm infection cycle. The analysis of the terminal phase of the cellular Lm infection cycle may make it possible to identify additional pathogen and host cell infection-specific factors and activities.

<table><tbody><tr><td>ACK Lysing Buffer</td><td>Gibco&amp;nbsp;</td><td>A1049201</td><td></td></tr><tr><td>ArC Amine Reactive Compensation Beads&amp;nbsp;</td><td>Life Technologies</td><td>A10346</td><td></td></tr><tr><td>BHI (Brain Heart Infusion) broth</td><td>EMD Milipore</td><td>110493</td><td></td></tr><tr><td>Cell Strainer, 70 &amp;micro;m</td><td>VWR</td><td>10199-656</td><td></td></tr><tr><td>Collagenase D</td><td>Roche</td><td>11088858001</td><td></td></tr><tr><td>DMEM media&amp;nbsp;</td><td>Gibco&amp;nbsp;</td><td>11965-092</td><td></td></tr><tr><td>FACS tubes&amp;nbsp;</td><td>BD Falcon</td><td>352054</td><td></td></tr><tr><td>FBS - Heat Inactivated&amp;nbsp;</td><td>Sigma-Aldrich</td><td>F4135-500ML</td><td></td></tr><tr><td>Hanks&amp;rsquo; Balanced Salt solution</td><td>Sigma-Aldrich</td><td>H6648-6X500ML</td><td></td></tr><tr><td>LB agar</td><td>Grow Cells MS</td><td>MBPE-4040</td><td></td></tr><tr><td>LIVE/DEAD Fixable Yellow Dead Cell Stain kit&amp;nbsp;</td><td>Life Technologies</td><td>L349S9</td><td></td></tr><tr><td>Rhodamine Phalloidin&amp;nbsp;</td><td>Thermo Fischer</td><td>R415</td><td></td></tr><tr><td>SP6800 Spectral Analyzer</td><td>Sony</td><td></td><td></td></tr><tr><td>Syringe 28 G 1/2&quot; 1cc</td><td>BD</td><td>329461</td><td></td></tr><tr><td>TPP Tissue Culture 48 Well Plates&amp;nbsp;</td><td>MIDSCI</td><td>TP92048</td><td></td></tr><tr><td>TPP Tissue Culture 6 Well Plates&amp;nbsp;</td><td>MIDSCI</td><td>TP92406</td><td></td></tr><tr><td>UltraComp eBeads</td><td>eBioscience</td><td>01-2222-42</td><td></td></tr><tr><td>&lt;strong&gt;Antigen&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>CD11b&amp;nbsp;</td><td>Biolegend</td><td></td><td>Flurochrome = PE Cy5, Dilution = 1/100, Clone = M1/70</td></tr><tr><td>CD11c</td><td>Biolegend</td><td></td><td>Flurochrome = AF 647, Dilution = 1/100, Clone = N418</td></tr><tr><td>CD45</td><td>Biolegend</td><td></td><td>Flurochrome = APC Cy7, Dilution = 1/100, Clone = 30-F11</td></tr><tr><td>F4/80</td><td>Biolegend</td><td></td><td>Flurochrome = PE, Dilution = 1/100, Clone = BM8</td></tr><tr><td>Live/Dead</td><td>Invitrogen</td><td></td><td>Flurochrome = AmCyN, Dilution = 1/100</td></tr><tr><td>Ly6C</td><td>Biolegend</td><td></td><td>Flurochrome = PacBlue, Dilution = 1/200, Clone = HK1.4</td></tr><tr><td>MHC II</td><td>Biolegend</td><td></td><td>Flurochrome = AF 700, Dilution = 1/200, Clone = M5/114.15.2</td></tr><tr><td>NK 1.1</td><td>Biolegend</td><td></td><td>Flurochrome = BV 605, Dilution = 1/100, Clone = PK136</td></tr></tbody></table>

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.

Assessing the role of pathogen and host-encoded factors impacting cellular infection 
Using the infection conditions described above, 0.15% of the input wild-type Lm is recovered after 1.5 h of co-incubation with cultured macrophages (Figure 1A). In the subsequent 1.5 h of co-incubation (3 h post infection, hpi), there was a 4-fold increase in recovery of viable Lm and from 3 to 6 hpi there was an addit...

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

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' ...

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5.9K Views.  University of Miami Miller School of Medicine. We describe both in vitro and in vivo infection assays that can be used to analyze the activities of host-encoding factors.

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

Tractable Mammalian Cell Infections with Protozoan-primed Bacteria

Many intracellular bacterial pathogens use freshwater protozoans as a natural reservoir for proliferation in the environment. Legionella pneumophila, the causative agent of Legionnaires' pneumonia, gains a pathogenic advantage over in vitro cultured bacteria when first harvested from protozoan cells prior to infection of mammalian macrophages. This suggests that important virulence factors may not be properly expressed in vitro. We have developed a tractable system for priming L. pneumophila through its natural protozoan host Acanthamoeba castellanii prior to mammalian cell infection. The contribution of any virulence factor can be examined by comparing intracellular growth of a mutant strain to wild-type bacteria after protozoan priming. GFP-expressing wild-type and mutant L. pneumophila strains are used to infect protozoan monolayers in a priming step and allowed to reach late stages of intracellular growth. Fluorescent bacteria are then harvested from these infected cells and normalized by spectrophotometry to generate comparable numbers of bacteria for a subsequent infection into mammalian macrophages. For quantification, live bacteria are monitored after infection using fluorescence microscopy, flow cytometry, and by colony plating. This technique highlights and relies on the contribution of host cell-dependent gene expression by mimicking the environment that would be encountered in a natural acquisition route. This approach can be modified to accommodate any bacterium that uses an intermediary host as a means for gaining a pathogenic advantage.

This technique provides a method to harvest, normalize and quantify intracellular growth of bacterial pathogens that are pre-cultivated in natural protozoan host cells prior to infections of mammalian cells. This method can be modified to accommodate a wide variety of host cells for the priming stage as well as target cell types.

Many intracellular bacterial pathogens use freshwater protozoans as a natural reservoir for proliferation in the environment. Legionella pneumophila, the ...

A Comparative Approach to Characterize the Landscape of Host-Pathogen Protein-Protein Interactions

Significant efforts were gathered to generate large-scale comprehensive protein-protein interaction network maps. This is instrumental to understand the pathogen-host relationships and was essentially performed by genetic screenings in yeast two-hybrid systems. The recent improvement of protein-protein interaction detection by a Gaussia luciferase-based fragment complementation assay now offers the opportunity to develop integrative comparative interactomic approaches necessary to rigorously compare interaction profiles of proteins from different pathogen strain variants against a common set of cellular factors.
This paper specifically focuses on the utility of combining two orthogonal methods to generate protein-protein interaction datasets: yeast two-hybrid (Y2H) and a new assay, high-throughput Gaussia princeps protein complementation assay (HT-GPCA) performed in mammalian cells.
A large-scale identification of cellular partners of a pathogen protein is performed by mating-based yeast two-hybrid screenings of cDNA libraries using multiple pathogen strain variants. A subset of interacting partners selected on a high-confidence statistical scoring is further validated in mammalian cells for pair-wise interactions with the whole set of pathogen variants proteins using HT-GPCA. This combination of two complementary methods improves the robustness of the interaction dataset, and allows the performance of a stringent comparative interaction analysis. Such comparative interactomics constitute a reliable and powerful strategy to decipher any pathogen-host interplays.

This article focuses on the identification of high-confident interaction datasets between host and pathogen proteins using a combination of two orthogonal methods: yeast two-hybrid followed by a high-throughput interaction assay in mammalian cells called HT-GPCA.

Significant efforts were gathered to generate  large-scale comprehensive protein-protein interaction network maps. This is  instrumental to understand the ...

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing the mucosal surface of the nasopharynx, where it can reside, multiply and eventually overcome host defences to invade to other tissues of the host. Establishment of an infection in the normally lower respiratory tract results in pneumonia. Alternatively, the bacteria can disseminate into the bloodstream causing bacteraemia, which is associated with high mortality rates2, or else lead directly to the development of pneumococcal meningitis. Understanding the kinetics of, and immune responses to, nasopharyngeal colonization is an important aspect of S. pneumoniae infection models.
Our mouse model of intranasal colonization is adapted from human models3 and has been used by multiple research groups in the study of host-pathogen responses in the nasopharynx4-7. In the first part of the model, we use a clinical isolate of S. pneumoniae to establish a self-limiting bacterial colonization that is similar to carriage events in human adults. The procedure detailed herein involves preparation of a bacterial inoculum, followed by the establishment of a colonization event through delivery of the inoculum via an intranasal route of administration. Resident macrophages are the predominant cell type in the nasopharynx during the steady state. Typically, there are few lymphocytes present in uninfected mice8, however mucosal colonization will lead to low- to high-grade inflammation (depending on the virulence of the bacterial species and strain) that will result in an immune response and the subsequent recruitment of host immune cells. These cells can be isolated by a lavage of the tracheal contents through the nares, and correlated to the density of colonization bacteria to better understand the kinetics of the infection.

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Colonization of the murine nasopharynx with Streptococcus pneumoniae and the subsequent extraction of adherent or recruited cells is described. This technique involves flushing the nasopharynx and collection of the fluid through the nares and is adaptable for various readouts, including differential cell quantification and analysis of mRNA expression in situ.

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing ...

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.

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.

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

Gene Expression Profiling of Infecting Microbes Using a Digital Bar-coding Platform

For most mammalian pathogens, gene expression profiling studies have been limited by technical difficulties to accurately quantify pathogen gene transcripts from infected tissues. Pathogen RNA constitutes a tiny portion of the total RNA isolated from infected tissue samples. Both microarray and RNAseq technologies have difficulties in generating reliable reads for weakly expressed pathogen genes. Mutant pathogen strains with reduced in vivo proliferation pose an even bigger challenge. Here we describe an in vivo gene expression profiling protocol that is very fast, extremely sensitive and highly reproducible. We developed this protocol during our investigation of the fungal pathogen Candida albicans in a murine model of hematogenously disseminated candidiasis. Using this protocol, we have documented time courses of dynamically regulated C. albicans gene expression during kidney infection, and discovered unexpected features of gene expression responses to antifungal drug treatment in vivo.

We developed a protocol that is fast, sensitive and reproducible for pathogen gene expression profiling during an infection.

For most mammalian pathogens, gene expression profiling studies have been limited by technical difficulties to accurately quantify pathogen gene ...

Microscopy-based Assays for High-throughput Screening of Host Factors Involved in Brucella Infection of Hela Cells

Brucella species are facultative intracellular pathogens that infect animals as their natural hosts. Transmission to humans is most commonly caused by direct contact with infected animals or by ingestion of contaminated food and can lead to severe chronic infections.
Brucella can invade professional and non-professional phagocytic cells and replicates within endoplasmic reticulum (ER)-derived vacuoles. The host factors required for Brucella entry into host cells, avoidance of lysosomal degradation, and replication in the ER-like compartment remain largely unknown. Here we describe two assays to identify host factors involved in Brucella entry and replication in HeLa cells. The protocols describe the use of RNA interference, while alternative screening methods could be applied. The assays are based on the detection of fluorescently labeled bacteria in fluorescently labeled host cells using automated wide-field microscopy. The fluorescent images are analyzed using a standardized image analysis pipeline in CellProfiler which allows single cell-based infection scoring.
In the endpoint assay, intracellular replication is measured two days after infection. This allows bacteria to traffic to their replicative niche where proliferation is initiated around 12 hr after bacterial entry. Brucella which have successfully established an intracellular niche will thus have strongly proliferated inside host cells. Since intracellular bacteria will greatly outnumber individual extracellular or intracellular non-replicative bacteria, a strain constitutively expressing GFP can be used. The strong GFP signal is then used to identify infected cells.
In contrast, for the entry assay it is essential to differentiate between intracellular and extracellular bacteria. Here, a strain encoding for a tetracycline-inducible GFP is used. Induction of GFP with simultaneous inactivation of extracellular bacteria by gentamicin enables the differentiation between intracellular and extracellular bacteria based on the GFP signal, with only intracellular bacteria being able to express GFP. This allows the robust detection of single intracellular bacteria before intracellular proliferation is initiated.

Microscopy-based Assays for High-throughput Screening of Host Factors Involved in Brucella Infection of Hela Cells

Two assays for microscopy-based high-throughput screening of host factors involved in Brucella infection are described. The entry assay detects host factors required for Brucella entry and the endpoint assay those required for intracellular replication. While applicable for alternative approaches, siRNA screening in HeLa cells is used to illustrate the protocols.

Brucella species are facultative intracellular pathogens that infect animals as their natural hosts. Transmission to humans is most commonly caused by ...

Evaluation of Host-Pathogen Responses and Vaccine Efficacy in Mice

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a striking increase in life expectancy around the globe. Nonetheless, determining vaccine efficacy remains a challenge. Emerging evidence suggests that the current acellular vaccine (aPV) for Bordetella pertussis (B. pertussis) induces suboptimal immunity. Therefore, a major challenge is designing a next-generation vaccine that induces protective immunity without the adverse side effects of a whole-cell vaccine (wPV). Here we describe a protocol that we used to test the efficacy of a promising, novel adjuvant that skews immune responses to a protective Th1/Th17 phenotype and promotes a better clearance of a B. pertussis challenge from the murine respiratory tract. This article describes the protocol for mouse immunization, bacterial inoculation, tissue harvesting, and analysis of immune responses. Using this method, within our model, we have successfully elucidated crucial mechanisms elicited by a promising, next-generation acellular pertussis vaccine. This method can be applied to any infectious disease model in order to determine vaccine efficacy.

Here we present an elegant protocol for in vivo evaluation of vaccine effectiveness and host immune responses. This protocol can be adapted for vaccine models that study viral, bacterial, or parasitic pathogens.

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a ...

Label-Free Quantitative Proteomics Workflow for Discovery-Driven Host-Pathogen Interactions

The technological achievements of mass spectrometry (MS)-based quantitative proteomics opens many undiscovered avenues for analyzing an organism&#8217;s global proteome under varying conditions. This powerful strategy applied to the interactions of microbial pathogens with the desired host comprehensively characterizes both perspectives towards infection. Herein, the workflow describes label-free quantification (LFQ) of the infectome of Cryptococcus neoformans, a fungal facultative intracellular pathogen that is the causative agent of the deadly disease cryptococcosis, in the presence of immortalized macrophage cells. The protocol details the proper protein preparation techniques for both pathogen and mammalian cells within a single experiment, resulting in appropriate peptide submission for liquid-chromatography (LC)-MS/MS analysis. The high throughput generic nature of LFQ allows a wide dynamic range of protein identification and quantification, as well as transferability to any host-pathogen infection setting, maintaining extreme sensitivity. The method is optimized to catalogue extensive, unbiased protein abundance profiles of a pathogen within infection-mimicking conditions. Specifically, the method demonstrated here provides essential information on C. neoformans pathogenesis, such as protein production necessary for virulence and identifies critical host proteins responding to microbial invasion.

Here, we present a protocol to profile the interplay between host and pathogen during infection by mass spectrometry-based proteomics. This protocol uses label-free quantification to measure changes in protein abundance of both host (e.g., macrophages) and pathogen (e.g., Cryptococcus neoformans) in a single experiment.

The technological achievements of mass spectrometry (MS)-based quantitative proteomics opens many undiscovered avenues for analyzing an organism&#8217;s ...

High-Throughput Transcriptome Analysis for Investigating Host-Pathogen Interactions

Pathogens can cause a wide variety of infectious diseases. The biological processes induced by the host in response to infection determine the severity of the disease.&#160;To study such processes, researchers can use high-throughput sequencing techniques (RNA-seq) that measure the dynamic changes of the host transcriptome at different stages of infection, clinical outcomes, or disease severity.This investigation can lead to a better understanding of the diseases, as well as uncovering potential drug targets and treatments. The protocol presented here describes a complete pipeline to analyze RNA-sequencing data from raw reads to functional analysis. The pipeline is divided into five steps: (1) quality control of the data; (2) mapping and annotation of genes; (3) statistical analysis to identify differentially expressed genes and co-expressed genes; (4) determination of the molecular degree of the perturbation of samples; and (5) functional analysis. Step 1 removes technical artifacts that may impact the quality of downstream analyses. In step 2, genes are mapped and annotated according to standard library protocols.&#160;The statistical analysis in step 3 identifies genes that are differentially expressed or co-expressed in infected samples, in comparison with non-infected ones. Sample variability and the presence of potential biological outliers are verified using the molecular degree of perturbation approach in step 4. Finally, the functional analysis in step 5 reveals the pathways associated with the disease phenotype. The presented pipeline aims to support researchers through the RNA-seq data analysis from host-pathogen interaction studies and drive future&#160;in vitro or in vivo experiments, that are essential to understand the molecular mechanism of infections.

The protocol presented here describes a complete pipeline to analyze RNA-sequencing transcriptome data from raw reads to functional analysis, including quality control and preprocessing steps to advanced statistical analytical approaches.

Pathogens can cause a wide variety of infectious diseases. The biological processes induced by the host in response to infection determine the severity of ...

Quantification of Bacterial Infection in Host Cells by the Colony-Forming Unit Assay

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>&#160;J. Vis. Exp.&#160; (2019).
This video demonstrates an in vitro quantification technique to determine Listeria monocytogenes infection of macrophages by the colony-forming unit assay. The bacterial colonies on the agar plate represent the intracellular bacterial population that escapes the host-defense mechanism and survives within the macrophages.

Source: Gayle, P. et al., Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses.&#160;J. Vis. Exp.&#160; (2019).
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Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses

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Explore More Videos

Tractable Mammalian Cell Infections with Protozoan-primed Bacteria

A Comparative Approach to Characterize the Landscape of Host-Pathogen Protein-Protein Interactions

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

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

Gene Expression Profiling of Infecting Microbes Using a Digital Bar-coding Platform

Microscopy-based Assays for High-throughput Screening of Host Factors Involved in Brucella Infection of Hela Cells

Evaluation of Host-Pathogen Responses and Vaccine Efficacy in Mice

Label-Free Quantitative Proteomics Workflow for Discovery-Driven Host-Pathogen Interactions

High-Throughput Transcriptome Analysis for Investigating Host-Pathogen Interactions

Quantification of Bacterial Infection in Host Cells by the Colony-Forming Unit Assay