Name: assessing bacterial invasion of cardiac cells in culture and heart colonization in infected mice using listeria monocytogenes
Uploaded: 2015-05-27T14:00:00
Description: Watch this Scientific Journal Video about Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes at JoVE.com

This work was supported by Public Health Service grants AI41816 and AI099339 (N.E.F.) and by F31AI094886-01 (P.D.M.) from NIAID. The contents are solely the responsibility of the authors and do not necessarily represent the official views of the funding sources.

1. Storage and Culture Conditions for L. monocytogenes Strains
<ol>
	<li>Prepare solid media by autoclaving brain-heart infusion agar (BHI) and pouring the molten media into petri plates. Allow the plates to dry overnight at room temperature, then overnight again at 37 &#176;C.</li>
	<li>Using a sterile loop, obtain a small sample of L. monocytogenes from either previously made freezer stocks (bacteria suspended in 20% glycerol in BHI liquid media, stored at -80 &#176;C) or from an agar plate containi...

<ol>
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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+infections+in+the+immunosuppressed+patient.">Cardiac infections in the immunosuppressed patient.</a> Infect Dis Clin North Am. 15, 613-638 (2001).</li><li>McCue, M. J., Moore, E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myocarditis+with+microabscess+formation+caused+by+Listeria+monocytogenes+associated+with+myocardial+infarct.">Myocarditis with microabscess formation caused by Listeria monocytogenes associated with myocardial infarct.</a> Hum Pathol. 10, 469-472 (1979).</li><li>Menard, 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=Modulation+of+L-type+calcium+channel+expression+during+retinoic+acid-induced+differentiation+of+H9C2+cardiac+cells.">Modulation of L-type calcium channel expression during retinoic acid-induced differentiation of H9C2 cardiac cells.</a> J Biol Chem. 274, 29063-29070 (1999).</li><li>Czuprynski, C. J. <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:+silage,+sandwiches+and+science.">Listeria monocytogenes: silage, sandwiches and science.</a> Anim Health Res Rev. 6, 211-217 (2005).</li><li>Grundler, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+surface+proteins+InlA+and+InlB+are+interdependently+required+for+polar+basolateral+invasion+by+Listeria+monocytogenes+in+a+human+model+of+the+blood-cerebrospinal+fluid+barrier.">The surface proteins InlA and InlB are interdependently required for polar basolateral invasion by Listeria monocytogenes in a human model of the blood-cerebrospinal fluid barrier.</a> Microbes Infect. 15, 291-301 (2013).</li><li>Zeldovich, V. B., Robbins, J. R., Kapidzic, M., Lauer, P., Bakardjiev, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invasive+extravillous+trophoblasts+restrict+intracellular+growth+and+spread+of+Listeria+monocytogenes.">Invasive extravillous trophoblasts restrict intracellular growth and spread of Listeria monocytogenes.</a> PLoS Pathog. 7, e1002005 (2011).</li><li>Kimes, B. W., Brandt, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Properties+of+a+clonal+muscle+cell+line+from+rat+heart.">Properties of a clonal muscle cell line from rat heart.</a> Experimental cell research. 98, 367-381 (1976).</li><li>McMullen, P. 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L. monocytogenes is a widespread and well-characterized human pathogen, capable of causing a number of different disease manifestations15. The bacterium has been previously described for its ability to translocate across barriers, such as the blood-brain-barrier and placental-fetal barriers, in order to reach and colonize the central nervous system and developing fetus, respectively. The in vivo ability of the organism to colonize these tissues is often complemented by an in vitro ab...

Listeria monocytogenes is a Gram-positive intracellular pathogen capable of causing severe disease in susceptible populations, including the elderly, pregnant women, people with HIV/AIDS, and persons receiving chemotherapy1. Infection in these populations is frequently the result of ingesting contaminated food products, and most infections are associated with large-scale food-borne outbreaks2,3. In humans and other mammals, L. monocytogenes is capable of translocating across the epithelial border of the small intestine, thereafter being transported to the liver4,5. Animal models suggest that ingested bacteria replicate within the intestinal villi and transit to the liver through the portal vein or spread via the mesenteric lymph nodes into the blood stream, leading to hematogenous dissemination to the liver and spleen6,7. In the liver and spleen, the bacterium is capable of mediating uptake into both professional phagocytes as well as resident parenchymal cells, and quickly establishes infections within these organs. As bacterial load increases, numerous bacteria are dispersed back into the blood, where they are capable of further colonizing susceptible tissues including the central nervous system and placenta (where present). Colonization of these sites precludes most common manifestations of listeriosis in humans, including meningitis, encephalitis, and fetal abortion2.
Selected subpopulations of L. monocytogenes have been recently shown to have an enhanced capacity to invade and replicate within cardiac tissue8. Manifestations of heart involvement are varied, and range from endocarditis and pericarditis, to fulminant myocarditis complete with conduction abnormalities9-13. The overall number of L. monocytogenes cardiac cases per year is low but may be under estimated as this facet of infection is not generally well recognized. Colonization of the heart by pathogens often requires host predispositions such as pre-existing valvular damage or artificial heart valves. There are, however, isolates of L. monocytogenes that have been identified that are notable for their capacity to colonize the hearts of infected animals in the absence of any cardiac damage and/or abnormalities8.
Herein are described in vitro and in vivo methods for assessing bacterial colonization of cardiac tissue within infected animals using invasion assays in tissue culture as well as live animal infections. These methods have proven useful in not only for identifying strains with the potential to colonize cardiac tissue in infected animals, but should also be useful for the identification of bacterial gene products that serve to enhance cardiac cell invasion and/or result in changes in heart pathology. These methods facilitate the comparison of cardiotropism between multiple strains. For the methods described here, L. monocytogenes 10403S is used as a well-studied representative of a non-cardiotropic strain and the clinical isolate 07PF0776 is used as a representative example of a cardiotropic strain. These two strains were chosen to provide a comparison for bacterial invasion of cardiac cells in vitro and colonization of hearts of infected mice in vivo. The isolate 07PF0776 is a clinical isolate recovered from an interventricular abscess that caused a fatal arrhythmia in an HIV+ patient8. L. monocytogenes isolates may vary in their virulence potential, and given the propensity for Listeria to infect persons with immunosuppression and pregnant women, persons within these populations should exercise caution while assessing different clinical isolates.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Difco</td>
			<td>BHI Broth Base</td>
			<td>237200</td>
			<td>2KG of Brain Heart Infusion powder (without agar)</td>
		</tr>
		<tr>
			<td>Difco</td>
			<td>Agar</td>
			<td>214510</td>
			<td>2KG of Granulated Agar</td>
		</tr>
		<tr>
			<td>Difco</td>
			<td>BHI Agar</td>
			<td>241810</td>
			<td>2KG of Brain Heart Infusion Powder with 7.5% Agar</td>
		</tr>
		<tr>
			<td>in vitrogen</td>
			<td>LB Broth Base</td>
			<td>12975-084</td>
			<td>2KG of Luria Broth Base Powder (without agar)</td>
		</tr>
		<tr>
			<td>Fisher</td>
			<td>Glass Coverslips</td>
			<td>12-545-80</td>
			<td>12mm glass coverslips</td>
		</tr>
		<tr>
			<td>Falcon</td>
			<td>24-well Tissue Culture Plate</td>
			<td>35-3047</td>
			<td>24 Well, Plasma-treated Tissue Culture Plate</td>
		</tr>
		<tr>
			<td>Falcon</td>
			<td>14mL Polystyrene tube</td>
			<td>352051</td>
			<td>14mL Polystyrene tubes</td>
		</tr>
		<tr>
			<td>Falcon</td>
			<td>96-well U-bottom plate</td>
			<td>35-3077</td>
			<td>Non-tissue culture treated 96-well plate</td>
		</tr>
		<tr>
			<td>Corning</td>
			<td>0.05% Trypsin, 0.53 mM EDTA (1X)</td>
			<td>25-052-CI</td>
			<td>Stock solution of Trypsin EDTA for tissue culture</td>
		</tr>
		<tr>
			<td>Corning</td>
			<td>DMEM (High glucose, high pyruvate)</td>
			<td>15-013-CU</td>
			<td>DMEM media without FBS, glutamine, or antibiotics</td>
		</tr>
		<tr>
			<td>Corning</td>
			<td>Pen/Strep/Glut Solution</td>
			<td>30-009-CI</td>
			<td>Stock mixture of penicillin, streptomycin, and L-glutamine for tissue culture</td>
		</tr>
		<tr>
			<td>Corning</td>
			<td>Gentamicin</td>
			<td>30-005-CR</td>
			<td>50mg/mL Stock solution of Gentamicin for tissue culture</td>
		</tr>
		<tr>
			<td>Cellgro (Corning)</td>
			<td>L-Glutamine</td>
			<td>25005197</td>
			<td>Stock L-glutamine solution without antibiotics</td>
		</tr>
		<tr>
			<td>Denville</td>
			<td>75cm^2 Flask</td>
			<td>T1225</td>
			<td>75cm^2 tissue culture treated flask</td>
		</tr>
		<tr>
			<td>Denville</td>
			<td>1.5mL microcentrifuge tubes</td>
			<td>2013004</td>
			<td>1.5mL microcentrifuge tubes</td>
		</tr>
		<tr>
			<td>ATCC</td>
			<td>H9c2 Cardiac Myoblasts</td>
			<td>CRL-1446</td>
			<td>Rat cardiac myoblasts</td>
		</tr>
		<tr>
			<td>Hyclone</td>
			<td>Fetal Bovine Serum</td>
			<td>SH30070.63</td>
			<td>Fetal Bovine Serum</td>
		</tr>
	</tbody>
</table>

assessing bacterial invasion of cardiac cells in culture and heart colonization in infected mice using listeria monocytogenes

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised patients, the elderly, and pregnant women. The most common manifestations of listeriosis in humans include meningitis, encephalitis, and fetal abortion. A significant but much less documented sequelae of invasive L. monocytogenes infection involves the heart. The death rate from cardiac illness can be up to 35% despite treatment, however very little is known regarding L. monocytogenes colonization of cardiac tissue and its resultant pathologies. In addition, it has recently become apparent that subpopulations of L. monocytogenes have an enhanced capacity to invade and grow within cardiac tissue. This protocol describes in detail in vitro and in vivo methods that can be used for assessing cardiotropism of L. monocytogenes isolates. Methods are presented for the infection of H9c2 rat cardiac myoblasts in tissue culture as well as for the determination of bacterial colonization of the hearts of infected mice. These methods are useful not only for identifying strains with the potential to colonize cardiac tissue in infected animals, but may also facilitate the identification of bacterial gene products that serve to enhance cardiac cell invasion and/or drive changes in heart pathology. These methods also provide for the direct comparison of cardiotropism between multiple L. monocytogenes strains.

Selected isolates of L. monocytogenes exhibit enhanced invasion of cardiac cells in cell culture and in mouse models of infection. Figure 1 shows an example of how bacterial colonies may appear following spot plating of suspensions on agar media. This method allows accurate assessment of CFUs within a sample without using large numbers of agar media plates. Figure 2 shows an example of a tissue culture-based assay comparing the ability of strain 10403S to invade heart cells with...

Watch this Scientific Journal Video about Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes at JoVE.com

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

Listeria monocytogenes causes fetal infections in pregnant women and meningitis in susceptible populations. Subpopulations of bacteria can colonize cardiac tissue, causing myocarditis in patients and laboratory animals. Here we present a protocol that describes how to assess L. monocytogenes cardiac cell invasion in vitro and cardiac colonization in infected animals.

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

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

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