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 DME...

<ol>
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G., Combs, T. A., D'Orazio, S. E. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophils+from+Both+Susceptible+and+Resistant+Mice+Efficiently+Kill+Opsonized+Listeria+monocytogenes.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+depletion+reveals+a+novel+role+for+neutrophil-mediated+protection+in+the+liver+during+Listeria+monocytogenes+infection.">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>Bl&#233;riot, C., 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=Liver-resident+macrophage+necroptosis+orchestrates+type+1+microbicidal+inflammation+and+type-2-mediated+tissue+repair+during+bacterial+infection.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monocytes+are+the+predominant+cell+type+associated+with+Listeria+monocytogenes+in+the+gut,+but+they+do+not+serve+as+an+intracellular+growth+niche.">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/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., 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=Use+of+RNA+interference+in+Drosophila+S2+cells+to+identify+host+pathways+controlling+compartmentalization+of+an+intracellular+pathogen.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GILT+is+a+critical+host+factor+for+Listeria+monocytogenes+infection.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perturbation+of+vacuolar+maturation+promotes+listeriolysin+O-independent+vacuolar+escape+during+Listeria+monocytogenes+infection+of+human+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+host-encoded+Heme+Regulated+Inhibitor+(HRI)+facilitates+virulence-associated+activities+of+bacterial+pathogens.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+eIF2&#945;+Kinase+Heme-Regulated+Inhibitor+Protects+the+Host+from+Infection+by+Regulating+Intracellular+Pathogen+Trafficking.">The eIF2&#945; 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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Listeria+monocytogenes+switches+from+dissemination+to+persistence+by+adopting+a+vacuolar+lifestyle+in+epithelial+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+Gentamicin+Concentration+and+Exposure+Time+on+Intracellular+Yersinia+pestis.">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.

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		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">ACK Lysing Buffer</td>
			<td style="width:128px;">Gibco&#160;</td>
			<td style="width:119px;">A1049201</td>
			<td style="width:313px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">ArC Amine Reactive Compensation Beads&#160;</td>
			<td style="width:128px;">Life Technologies</td>
			<td style="width:119px;">A10346</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BHI (Brain Heart Infusion) broth</td>
			<td>EMD Milipore</td>
			<td>110493</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cell Strainer, 70 &#181;m</td>
			<td style="width:128px;">VWR</td>
			<td style="width:119px;">10199-656</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Collagenase D</td>
			<td style="width:128px;">Roche</td>
			<td>11088858001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMEM media&#160;</td>
			<td style="width:128px;">Gibco&#160;</td>
			<td>11965-092</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FACS tubes&#160;</td>
			<td style="width:128px;">BD Falcon</td>
			<td style="width:119px;">352054</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FBS - Heat Inactivated&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>F4135-500ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hanks&#8217; Balanced Salt solution</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td>H6648-6X500ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">LB agar</td>
			<td style="width:128px;">Grow Cells MS</td>
			<td style="width:119px;">MBPE-4040</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">LIVE/DEAD Fixable Yellow Dead Cell Stain kit&#160;</td>
			<td style="width:128px;">Life Technologies</td>
			<td style="width:119px;">L349S9</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rhodamine Phalloidin&#160;</td>
			<td style="width:128px;">Thermo Fischer</td>
			<td>R415</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">SP6800 Spectral Analyzer</td>
			<td style="width:128px;">Sony</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Syringe 28G 1/2&#34; 1cc</td>
			<td style="width:128px;">BD</td>
			<td style="width:119px;">329461</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TPP Tissue Culture 48 Well Plates&#160;</td>
			<td style="width:128px;">MIDSCI</td>
			<td>TP92048</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TPP Tissue Culture 6 Well Plates&#160;</td>
			<td style="width:128px;">MIDSCI</td>
			<td>TP92406</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">UltraComp eBeads</td>
			<td style="width:128px;">eBioscience</td>
			<td style="width:119px;">01-2222-42</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Antigen</td>
			<td style="width:128px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CD11b&#160;</td>
			<td style="width:128px;">Biolegend</td>
			<td style="width:119px;"></td>
			<td>Flurochrome = PE Cy5, Dilution = 1/100, Clone = M1/70</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CD11c</td>
			<td style="width:128px;">Biolegend</td>
			<td style="width:119px;"></td>
			<td>Flurochrome = AF 647, Dilution = 1/100, Clone = N418</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CD45</td>
			<td style="width:128px;">Biolegend</td>
			<td style="width:119px;"></td>
			<td>Flurochrome = APC Cy7, Dilution = 1/100, Clone = 30-F11</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">F4/80</td>
			<td style="width:128px;">Biolegend</td>
			<td style="width:119px;"></td>
			<td>Flurochrome = PE, Dilution = 1/100, Clone = BM8</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Live/Dead</td>
			<td style="width:128px;">Invitrogen</td>
			<td style="width:119px;"></td>
			<td>Flurochrome = AmCyN, Dilution = 1/100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ly6C</td>
			<td style="width:128px;">Biolegend</td>
			<td style="width:119px;"></td>
			<td>Flurochrome = PacBlue, Dilution = 1/200, Clone = HK1.4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MHC II</td>
			<td style="width:128px;">Biolegend</td>
			<td style="width:119px;"></td>
			<td>Flurochrome = AF 700, Dilution = 1/200, Clone = M5/114.15.2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NK 1.1</td>
			<td style="width:128px;">Biolegend</td>
			<td style="width:119px;"></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...

Watch this Scientific Journal Video about Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses at JoVE.com

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' ...

Microbial Pathogens that replicate intracellularly must eventually exit the infected cell. Which, when compared to other aspects of host pathogen interactions is relatively understudied. Here, we describe techniques to analyze pathogen efflux.We show how relatively simply assays can be used to either evaluate efflux efficiency or characterize post-efflux pathogens The general techniques described here are broadly applicable although specific pathogen host cell models may differ in kinetics, dosages and possibly read-outs. As in setting up any experimental system, it is essential that clear controls are established that will ensure that genuine, biological processes are observed in experimental samples or cohorts. Demonstrating the procedure will be Petoria Gayle, a graduate student from my laboratory.To begin with the infection experiment, use a pipette to add 20 microliters of the freshly prepared Lm inoculum to wells, with the dish slightly tilted and the pipette tip carefully placed in the overlying media. Avoid touching the walls of the well with the pipette tip. Gently pipette the contents of each well to ensure complete mixing.Do not shake or significantly tilt the plate to avoid spreading LM over the walls of the well. Return cell culture dish to the 37 degree Celsius, five percent carbon dioxide incubator. Use conditioned media from uninfected cells as diluent to prepare a 10 micrograms per milliliter gentamicin solution.At 30 minutes post-infection, carefully add 30 microliters of the gentamicin solution into the dish to reach a final concentration of two-point-five micrograms per milliliter. Gently pipette the contents of the well to mix and return the cell culture dish to the incubator. To harvest, slightly tilt the dish and carefully use a pipette to remove the entire media from the well.Add point-five milliliters of distilled, sterile water to the well. After 30 seconds, transfer the resulting water lysate to a one-point-five milliliter microcentrifuge tube and vortex vigorously for 10 seconds. Add four-point-five microliters and 50 microliters of the water lysate to 450 microliters of distilled, sterile water to prepare the tenfold and one-hundredfold dilutions.Briefly vortex to ensure thorough mixing. Add 50 microliters of the original tenfold diluted and one-hundredfold diluted samples onto lysogeny broth agar plates, and spread using a plate spreader. To assay for emerging LM, extract 10 microliters of the overlaying media from the dish and divide it into two five-microliter aliquots and micro centrifuge tubes.Add five-microliters of DMEM/FCS to one aliquot and add five-microliters of DMEM/FCS containing five-microliters per milliliter gentamicin to the second aliquot as control. Wait five minutes at room temperature. Add 90 microliters of distilled sterile water to each aliquot and vortex the two vigorously for 10 seconds.Then add 50 microliters of the sample on LB agar plates and spread using a plate spreader. Store the plates in 37 degrees celsius incubator for two days before enumerating Lm colonies. Using an automated cell counter, adjust the prepared, raw two-six-four-point-seven cells to the concentration of one times 10 to the sixth cells-per-milliliter.Add one milliliter of the sample to wells of a six-well tissue culture plate. Infect the cells with a prepared Lm inoculum, corresponding to a multiplicity of infection of 50. At one-point-five hours post-infection, in a one-point-five milliliter microcentrifuge tube filled with 500 microliters of four percent PFA, collect media from the first set of wells and place the tube on ice.To collect intracellular Lm, add one milliliter of distilled, sterile water to each well. After 60 seconds, transfer the resulting water lysate to a one-point-five milliliter microcentrifuge tube with 500 microliters of four-percent PFA. Vortex the tube vigorously for 10 seconds and place it on ice.For remaining wells, remove media and replace with one-milliliter of DMEM/FCS with five-micrograms per milliliter gentamicin to kill extracellular Lm.Promptly return the dish to the incubator. After 30 minutes, remove media and replace with DMEM/FCS without gentamicin. After another another two and four hours, collect media in lysates for the second and third time points into the tubes and place them on ice to chill.Centrifuge tubes at 10, 000 times G for seven minutes. Using a pipette, remove supernatant without disturbing the pellet. Add staining cocktail containing 50 microliters of four-percent PFA and 15 microliters of phalloidin into the tube and mix to suspend each pellet.Incubate tubes in the dark at four degrees Celsius for 20 minutes. After incubation, add one milliliter of FACS buffer to each tube and pipette up and down to mix. Spin tubes in the centrifuge at 10, 000 times G for seven minutes.Remove supernatant without disturbing the pellet. For immediate use, suspend each pellet in 400 microliters of FAX buffer. If storing for later analysis, add 200 microliters of FAX buffer and 200 microliters of four percent PFA into the tube and mix to suspend.Using the infection conditions in the brief treatment with the antibiotic gentamicin to eliminate non-internalized Lm, zero-point-one-five percent of the wild-type Lm is recovered after one-point-five hours of co-incubation with cultured macrophages. In the subsequent co-incubation, there was a fourfold and seven-point-fivefold increase in the recovery of viable Lm, which exclusively represent intracellular proliferation. Comparable profile was observed for the Lm strain possessing a hypervirulent prfA during initial infection but not between three and six HPI.When the prfA was deleted from the Lm strain, there is a subsequent reduction in the recovery of the strain after prolonged co-incubation. When gentamicin is removed from the wells at six HPI, extracellular Lm can be detected one hour later for both wild-type and hypervirulent prfA strains, but not for the strain with prfA deleted. Media overlaying both uninfected and infected macrophages contained light-scattering, bacterium-sized particulate matter.However, only media from infected macrophages possessed GFP positive signals within the size gating. A time-dependent increase in the appearance of phalloidin-positive Lm was observed with infected macrophages. When plating out the macrophages in the 48-well tissue culture dish, leave some wells without cells.These no-cell control wells are then treated exactly the same as the cell-containing wells, in terms of the addition of the pathogen, antibiotics, etc. to ensure that experimental signals are dependent on the presence of host cells. Similar to the experiments shown in figures one and three, pathogen or host factors and or small molecules can be tested as to their impact on pathogen cellular efflux.

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Research

JoVE Journal

Immunology and Infection

5.6K visualizações.  University of Miami Miller School of Medicine. Patógenos microbianos que se replicam intracelularmente devem eventualmente sair da célula infectada. Que, quando comparado com outros aspectos das interações de patógenos hospedeiros é relativamente subestudado. Aqui, descrevemos técnicas para analisar o efflux patógeno.Mostramos como ensaios relativamente simples podem ser usados para avaliar a eficiência efflux ou caracterizar patógenos pós-efflux As técnicas gerais descritas aqui são amplamente aplicáveis, embora mode...