Name: Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses (Video) | JoVE Journal
Uploaded: 2019-02-22T12:30:00.000+00:00
Duration: 8 min 38 s
Description: Learn techniques to analyze bacterial pathogen efflux using in vitro and in vivo infection assays. This method evaluates host-encoding factors and pathogen exit efficiency for studying host-pathogen interactions.

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5 minutes

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

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 additio...

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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Research

JoVE Journal

Immunology and Infection

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

Invasion of Human Cells by a Bacterial Pathogen

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.

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

This video demonstrates a technique for assessing the ability of Porphyromonas gingivalis, an anaerobic pathogenic bacterium, to adhere to and invade human umbilical vein endothelial cells (HUVECs). After infecting the host cells with the bacteria, the host cells are lysed to release both adhered and internalized bacteria. When the lysate is plated in an appropriate medium, the presence of colonies indicates successful bacteria-host cell interactions.

A Bacterial Infection Assay for Studying Anaerobic Bacteria-Host Cell Interactions

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.

Tractable Mammalian Cell Infections with Protozoan-primed Bacteria

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.

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

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

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

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

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.

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

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.

Evaluation of Host-Pathogen Responses and Vaccine Efficacy in Mice

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.

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

Detection of host-bacterial pathogen interactions based on phenotypic adherence using high-throughput fluorescence labeling imaging along with automated statistical analysis methods enables rapid evaluation of potential bacterial interactions with host cells.

Automated, High-Throughput Detection of Bacterial Adherence to Host Cells

Learn techniques to analyze bacterial pathogen efflux using in vitro and in vivo infection assays. This method evaluates host-encoding factors and pathogen exit efficiency for studying host-pathogen interactions.

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

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