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

Dette arbejde blev støttet af Public Health Service tilskud AI41816 og AI099339 (N.E.F.) og af F31AI094886-01 (P.D.M.) fra NIAID. Indholdet er udelukkende forfatternes ansvar og repræsenterer ikke nødvendigvis finansieringskildernes officielle synspunkter.

1. Opbevarings- og kulturforhold for L. monocytogenes stammer
<ol><li>Forbered solide medier ved autoklavering hjerne-hjerte infusion agar (BHI) og hælde smeltet medier i petriskåle. Lad pladerne tørre natten over ved stuetemperatur, og overnatter derefter igen ved 37 °C.</li>
	<li>Ved hjælp af en steril løkke udtages en lille prøve L. monocytogenes fra enten tidligere fremstillede fryselagre (bakterier suspenderet i 20% glycerol i BHI flydende medier, opbevaret ved -80 °C) eller fra en agarpl...

<ol>
	<li>Freitag, N. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+hot+dogs+to+host+cells:+how+the+bacterial+pathogen+Listeria+monocytogenes+regulates+virulence+gene+expression.">From hot dogs to host cells: how the bacterial pathogen Listeria monocytogenes regulates virulence gene expression.</a> Future Microbiol. 1, 89-101 (2006).</li><li>Drevets, D. A., Bronze, M. S. <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:+epidemiology,+human+disease,+and+mechanisms+of+brain+invasion.">Listeria monocytogenes: epidemiology, human disease, and mechanisms of brain invasion.</a> FEMS Immunol Med Microbiol. 53, 151-165 (2008).</li><li>Farber, J. M., Peterkin, P. I. <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.+a+food-borne+pathogen.">Listeria monocytogenes. a food-borne pathogen.</a> Microbiol. Rev. 55, 476-511 (1991).</li><li>Lecuit, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+how+Listeria+monocytogenes+targets+and+crosses+host+barriers.">Understanding how Listeria monocytogenes targets and crosses host barriers.</a> Clin Microbiol Infect. 11, 430-436 (2005).</li><li>Lecuit, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+listeriosis+and+animal+models.">Human listeriosis and animal models.</a> Microbes Infect. 9, 1216-1225 (2007).</li><li>Bou Ghanem, E. 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=InlA+promotes+dissemination+of+Listeria+monocytogenes+to+the+mesenteric+lymph+nodes+during+food+borne+infection+of+mice.">InlA promotes dissemination of Listeria monocytogenes to the mesenteric lymph nodes during food borne infection of mice.</a> PLoS Pathog. 8, e1003015 (2012).</li><li>Melton-Witt, J. A., Rafelski, S. M., Portnoy, D. A., 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=Oral+infection+with+signature-tagged+Listeria+monocytogenes+reveals+organ-specific+growth+and+dissemination+routes+in+guinea+pigs.">Oral infection with signature-tagged Listeria monocytogenes reveals organ-specific growth and dissemination routes in guinea pigs.</a> Infect Immun. 80, 720-732 (2012).</li><li>Alonzo, F., Bobo, L. D., Skiest, D. J., Freitag, N. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+subpopulations+of+Listeria+monocytogenes+with+enhanced+invasion+of+cardiac+cells.">Evidence for subpopulations of Listeria monocytogenes with enhanced invasion of cardiac cells.</a> J Med Microbiol. , (2011).</li><li>Adler, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammatory+pseudotumor+of+the+heart+caused+by+Listeria+monocytogenes+infection.">Inflammatory pseudotumor of the heart caused by Listeria monocytogenes infection.</a> J Infect. 58, 161-163 (2009).</li><li>Antolin, J., Gutierrez, A., Segoviano, R., Lopez, R., Ciguenza, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endocarditis+due+to+Listeria:+description+of+two+cases+and+review+of+the+literature.">Endocarditis due to Listeria: description of two cases and review of the literature.</a> Eur J Intern Med. 19, 295-296 (2008).</li><li>Brouqui, P., Raoult, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endocarditis+due+to+rare+and+fastidious+bacteria.">Endocarditis due to rare and fastidious bacteria.</a> Clin Microbiol Rev. 14, 177-207 (2001).</li><li>Brusch, J. 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. 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=Genome+sequence+of+Listeria+monocytogenes+07PF0776,+a+cardiotropic+serovar+4b+strain.">Genome sequence of Listeria monocytogenes 07PF0776, a cardiotropic serovar 4b strain.</a> J Bacteriol. 194, 3552 (2012).</li></ol>

L. monocytogenes er et udbredt og velkaraktereret menneskeligt patogen, der er i stand til at forårsage en række forskellige sygdoms manifestationer15. Bakterien er tidligere blevet beskrevet for sin evne til at translokere på tværs af barrierer, såsom blod-hjerne-barriere og placenta-foster barrierer, for at nå og kolonisere centralnervesystemet og udvikle fosteret, henholdsvis. Organismens in vivo-evne til at kolonisere disse væv suppleres ofte af en in vitro-evne til at inv...

Listeria monocytogenes er et Gram-positivt intracellulært patogen, der kan forårsage alvorlig sygdom hos modtagelige befolkningsgrupper, herunder ældre, gravide kvinder, hiv/aids-personer og personer, der får kemoterapi1. Infektion i disse populationer er ofte et resultat af indtagelse af forurenede fødevarer, og de fleste infektioner er forbundet med store fødevarebårne udbrud2,3. Hos mennesker og andre pattedyr er L. monocytogenes i stand til at translokere over tyndtarmens epitelkant og derefter transporteres til leveren4,5. Dyremodeller tyder på, at indtagede bakterier replikeres i tarmens villi og transit til leveren gennem portalvenen eller spredes via de mesenteriske lymfeknuder i blodet, hvilket fører til hæmatotogen formidling til leveren og milt6,7. I leveren og milten er bakterien i stand til at formidle optagelse i både professionelle fagocytter såvel som bosiddende parenchymale celler og etablerer hurtigt infektioner i disse organer. Efterhånden som bakteriebelastningen øges, spredes mange bakterier tilbage i blodet, hvor de er i stand til yderligere at kolonisere modtagelige væv, herunder centralnervesystemet og moderkagen (hvor de er til stede). Kolonisering af disse steder udelukker de mest almindelige manifestationer af listeriose hos mennesker, herunder meningitis, encephalitis og fosterabort2.
Udvalgte delpopulationer af L. monocytogenes har for nylig vist sig at have en øget kapacitet til at invadere og replikere i hjertevæv8. Manifestationer af hjerte involvering er varierede, og spænder fra endocarditis og pericarditis, til fulminant myocarditis komplet med ledning abnormiteter9-13. Det samlede antal L. monocytogenes hjertetilfælde om året er lavt, men kan være undervurderet, da denne facet af infektion er ikke generelt anerkendt. Kolonisering af hjertet af patogener kræver ofte værtsdispositioner såsom allerede eksisterende valvular skader eller kunstige hjerteklapper. Der er dog isolater af L. monocytogenes, der er blevet identificeret, der er bemærkelsesværdige for deres evne til at kolonisere hjerter af inficerede dyr i mangel af hjerteskader og / eller abnormiteter8.
Heri er beskrevet in vitro og in vivo metoder til vurdering af bakteriel kolonisering af hjertevæv i inficerede dyr ved hjælp af invasionsanalyser i vævskultur samt levende dyreinfektioner. Disse metoder har vist sig nyttige til ikke kun at identificere stammer med potentiale til at kolonisere hjertevæv hos inficerede dyr, men bør også være nyttige til identifikation af bakterielle genprodukter, der tjener til at forbedre hjertecelleinvasion og / eller resultere i ændringer i hjertepatologi. Disse metoder letter sammenligningen af kardiotropisme mellem flere stammer. For de metoder, der er beskrevet her, anvendes L. monocytogenes 10403S som en velunderprøvet repræsentant for en ikke-kardiotropisk stamme, og det kliniske isolate 07PF0776 bruges som et repræsentativt eksempel på en kardiotropisk stamme. Disse to stammer blev valgt til at give en sammenligning for bakteriel invasion af hjerteceller in vitro og kolonisering af hjerter af inficerede mus in vivo. Den isolere 07PF0776 er en klinisk isolere inddrives fra en mellemliggende byld, der forårsagede en dødelig arytmi i en HIV + patient8. L. monocytogenes isolater kan variere i deres virulens potentiale, og i betragtning af tilbøjeligheden til Listeria at inficere personer med immunosuppression og gravide kvinder, personer i disse populationer bør udvise forsigtighed, mens vurderingen af forskellige kliniske isolater.

<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 er et Gram-positivt fakultets intracellulært patogen, der er i stand til at forårsage alvorlige invasive infektioner hos immunkompromitterede patienter, ældre og gravide kvinder. De mest almindelige manifestationer af listeriose hos mennesker omfatter meningitis, encephalitis og fosterabort. En betydelig, men langt mindre dokumenteret sequelae af invasive L. monocytogenes infektion involverer hjertet. Dødeligheden fra hjertesygdomme kan være op til 35% på trods af behandling, men meget lidt er kendt med hensyn til L. monocytogenes kolonisering af hjertevæv og dets deraf følgende patologier. Derudover er det for nylig blevet klart, at delpopulationer af L. monocytogenes har en øget kapacitet til at invadere og vokse inden for hjertevæv. Denne protokol beskriver i detaljer in vitro- og in vivo-metoder, der kan bruges til vurdering af kardiotropisme af L. monocytogenes isolater. Metoder præsenteres for infektion af H9c2 rotte hjerte myoblaster i vævskultur samt til bestemmelse af bakteriel kolonisering af hjerter af inficerede mus. Disse metoder er nyttige ikke kun til at identificere stammer med potentiale til at kolonisere hjertevæv hos inficerede dyr, men kan også lette identifikationen af bakterielle genprodukter, der tjener til at forbedre hjertecelleinvasion og / eller drive ændringer i hjertepatologi. Disse metoder giver også mulighed for direkte sammenligning af kardiotropisme mellem flere L. monocytogenes stammer.

Udvalgte isolater af L. monocytogenes udviser øget invasion af hjerteceller i cellekultur og i musemodeller af infektion. Figur 1 viser et eksempel på, hvordan bakteriekolonier kan forekomme efter spot plating af suspensioner på agar medier. Denne metode giver mulighed for nøjagtig vurdering af CFU'er i en prøve uden brug af et stort antal agarmedieplader. Figur 2 viser et eksempel på en vævskulturbaseret analyse, der sammenligner belastningens evne til at invadere hjerte...

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 forårsager fosterinfektioner hos gravide kvinder og meningitis hos modtagelige befolkningsgrupper. Subpopulationer af bakterier kan kolonisere hjertevæv, forårsager myocarditis hos patienter og forsøgsdyr. Her præsenterer vi en protokol, der beskriver, hvordan man vurderer L. monocytogenes hjertecelleinvasion in vitro og hjertekolonisering hos inficerede dyr.

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

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