Questo lavoro è stato supportato da sovvenzioni del Servizio Sanitario Pubblico AI41816 e AI099339 (N.E.F.) e da F31AI094886-01 (P.D.M.) di NIAID. I contenuti sono di esclusiva responsabilità degli autori e non rappresentano necessariamente le opinioni ufficiali delle fonti di finanziamento.

<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>

Gli autori non segnalano interessi finanziari concorrenti.

L. monocytogenes è un agente patogeno umano diffuso e ben caratterizzato, in grado di causare una serie di diverse manifestazioni di malattia15. Il batterio è stato precedentemente descritto per la sua capacità di trasporre attraverso barriere, come la barriera ematico-encefalica e le barriere placentare-fetali, al fine di raggiungere e colonizzare il sistema nervoso centrale e sviluppare il feto, rispettivamente. La capacità in vivo dell'organismo di colonizzare questi tessuti è spesso ...

La Listeria monocytogenes è un agente patogeno intracellulare gram-positivo in grado di causare gravi malattie nelle popolazioni sensibili, tra cui anziani, donne incinte, persone con HIV / AIDS e persone che ricevono la chemioterapia1. L'infezione in queste popolazioni è spesso il risultato dell'ingestione di prodotti alimentari contaminati e la maggior parte delle infezioni sono associate a focolai di origine alimentare su largascala 2,3. Nell'uomo e in altri mammiferi, L. monocytogenes è in grado di trasporre attraverso il confine epiteliale dell'intestino tenue, per poi essere trasportato al fegato4,5. I modelli animali suggeriscono che i batteri ingeriti si replicano all'interno dei villi intestinali e transitano al fegato attraverso la vena portale o si diffondono attraverso i linfonodi mesenterici nel flusso sanguigno, portando alla diffusione ematogena al fegato e alla milza6,7. Nel fegato e nella milza, il batterio è in grado di mediare l'assorbimento sia nei fagociti professionisti che nelle cellule parenchimali residenti e stabilisce rapidamente infezioni all'interno di questi organi. Con l'aumentare della carica batterica, numerosi batteri vengono dispersi nel sangue, dove sono in grado di colonizzare ulteriormente i tessuti sensibili tra cui il sistema nervoso centrale e la placenta (dove presente). La colonizzazione di questi siti preclude le manifestazioni più comuni di listeriosi negli esseri umani, tra cui meningite, encefalite e aborto fetale2.
Alcune sottopopolazioni di L. monocitogene hanno recentemente dimostrato di avere una maggiore capacità di invadere e replicare all'interno del tessuto cardiaco8. Le manifestazioni di coinvolgimento cardiaco sono varie e vanno dall'endocardite e pericardite, alla miocardite fulminante completa di anomalie di conduzione9-13. Il numero complessivo di casi cardiaci L. monocytogenes all'anno è basso, ma può essere sottovalutato in quanto questo aspetto dell'infezione non è generalmente ben riconosciuto. La colonizzazione del cuore da parte di agenti patogeni richiede spesso predisposizioni ospiti come danni valvulari preesiste o valvole cardiache artificiali. Esistono, tuttavia, isolati di L. monocytogenes che sono stati identificati che si notano per la loro capacità di colonizzare il cuore degli animali infetti in assenza di danni cardiaci e/o anomalie8.
Nel presente documento sono descritti metodi in vitro e in vivo per valutare la colonizzazione batterica del tessuto cardiaco all'interno di animali infetti utilizzando test di invasione nella coltura tissutale e infezioni animali vive. Questi metodi si sono dimostrati utili non solo per identificare ceppi con il potenziale di colonizzare il tessuto cardiaco negli animali infetti, ma dovrebbero anche essere utili per l'identificazione di prodotti genetici batterici che servono a migliorare l'invasione delle cellule cardiache e / o provocare cambiamenti nella patologia cardiaca. Questi metodi facilitano il confronto del cardiotropismo tra più ceppi. Per i metodi qui descritti, L. monocytogenes 10403S è usato come rappresentante ben studiato di un ceppo non cardiotropico e l'isolato clinico 07PF0776 è usato come esempio rappresentativo di un ceppo cardiotropico. Questi due ceppi sono stati scelti per fornire un confronto per l'invasione batterica di cellule cardiache in vitro e la colonizzazione dei cuori dei topi infetti in vivo. L'isolato 07PF0776 è un isolato clinico recuperato da un ascesso interventricolare che ha causato un'aritmia fatale in un paziente HIV+8. L. gli isolati monocitogeni possono variare nel loro potenziale di virulenza e, data la propensione della Listeria a infettare le persone con immunosoppressione e le donne incinte, le persone all'interno di queste popolazioni dovrebbero prestare attenzione nel valutare diversi isolati clinici.

<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 è un agente patogeno intracellulare facultativo Gram-positivo in grado di causare gravi infezioni invasive in pazienti immunocompro compromessi, anziani e donne in gravidanza. Le manifestazioni più comuni di listeriosi negli esseri umani includono meningite, encefalite e aborto fetale. Una sequela significativa ma molto meno documentata dell'infezione invasiva di L. monocytogenes coinvolge il cuore. Il tasso di mortalità per malattia cardiaca può essere fino al 35% nonostante il trattamento, tuttavia si sa molto poco per quanto riguarda la colonizzazione L. monocytogenes del tessuto cardiaco e le sue patologie risultanti. Inoltre, è recentemente diventato evidente che le sottopopolazioni di L. monocytogenes hanno una maggiore capacità di invadere e crescere all'interno del tessuto cardiaco. Questo protocollo descrive in dettaglio i metodi in vitro e in vivo che possono essere utilizzati per valutare il cardiotropismo degli isolati L. monocytogenes. Sono presentati metodi per l'infezione dei mioblasti cardiaci del ratto H9c2 nella coltura tissutale e per la determinazione della colonizzazione batterica dei cuori dei topi infetti. Questi metodi sono utili non solo per identificare i ceppi con il potenziale di colonizzare il tessuto cardiaco negli animali infetti, ma possono anche facilitare l'identificazione di prodotti genetici batterici che servono a migliorare l'invasione delle cellule cardiache e / o guidare cambiamenti nella patologia cardiaca. Questi metodi prevedono anche il confronto diretto del cardiotropismo tra più ceppi di L. monocytogenes.

Isolati selezionati di L. monocytogenes mostrano una maggiore invasione delle cellule cardiache nella coltura cellulare e nei modelli di infezione dei topi. La figura 1 mostra un esempio di come le colonie batteriche possano apparire a seguito della placcatura spot delle sospensioni sui mezzi agar. Questo metodo consente una valutazione accurata dei CFC all'interno di un campione senza utilizzare un gran numero di piastre di supporto agar. La figura 2 mostra un esempio di saggio...

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

La listeria monocytogenes causa infezioni fetali nelle donne in gravidanza e meningite nelle popolazioni sensibili. Le sottopopolazioni di batteri possono colonizzare il tessuto cardiaco, causando miocardite in pazienti e animali da laboratorio. Qui presentiamo un protocollo che descrive come valutare l'invasione delle cellule cardiache di L. monocytogenes in vitro e la colonizzazione cardiaca negli animali infetti.

Valutazione dell'invasione batterica delle cellule cardiache in coltura e colonizzazione cardiaca nei topi infetti utilizzando listeria monocytogenes

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

assessing-bacterial-invasion-cardiac-cells-culture-heart-colonization

Research

JoVE Journal

Immunology and Infection

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

5.4K Views.  College of Medicine, University of Illinois at Chicago. La listeria monocytogenes causa infezioni fetali nelle donne in gravidanza e meningite nelle popolazioni sensibili. Le sottopopolazioni di batteri possono colonizzare il tessuto cardiaco, causando miocardite in pazienti e animali da laboratorio. Qui presentiamo un protocollo che descrive come valutare l'invasione delle cellule cardiache di L. monocytogenes in vitro e la colonizzazione cardiaca negli animali infetti. 

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

Assessing Stomatal Response to Live Bacterial Cells using Whole Leaf Imaging

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of guard cells, which are able to close the stomatal pore in response to a number of external factors including light intensity, carbon dioxide concentration, and relative humidity (RH). The stomatal pore is also the main route for pathogen entry into leaves, a crucial step for disease development. Recent studies have unveiled that closure of the pore is effective in minimizing bacterial disease development in Arabidopsis plants; an integral part of plant innate immunity. Previously, we have used epidermal peels to assess stomatal response to live bacteria (Melotto et al. 2006); however maintaining favorable environmental conditions for both plant epidermal peels and bacterial cells has been challenging. Leaf epidermis can be kept alive and healthy with MES buffer (10 mM KCl, 25 mM MES-KOH, pH 6.15) for electrophysiological experiments of guard cells. However, this buffer is not appropriate for obtaining bacterial suspension. On the other hand, bacterial cells can be kept alive in water which is not proper to maintain epidermal peels for long period of times. When an epidermal peel floats on water, the cells in the peel that are exposed to air dry within 4 hours limiting the timing to conduct the experiment. An ideal method for assessing the effect of a particular stimulus on guard cells should present minimal interference to stomatal physiology and to the natural environment of the plant as much as possible. We, therefore, developed a new method to assess stomatal response to live bacteria in which leaf wounding and manipulation is greatly minimized aiming to provide an easily reproducible and reliable stomatal assay. The protocol is based on staining of intact leaf with propidium iodide (PI), incubation of staining leaf with bacterial suspension, and observation of leaves under laser scanning confocal microscope. Finally, this method allows for the observation of the same live leaf sample over extended periods of time using conditions that closely mimic the natural conditions under which plants are attacked by pathogens.

We have developed a simple and reproducible protocol to access stomatal response to live bacteria. This method minimizes wounding and manipulation of the leaf as compared to the use of epidermal peels reported previously.

Valutazione della risposta stomatica a Live cellule batteriche utilizzando Imaging Whole Leaf

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of ...

Ricerca

Immunologia e infezioni

Using Luciferase to Image Bacterial Infections in Mice

Imaging is a valuable technique that can be used to monitor biological processes. In particular, the presence of cancer cells, stem cells, specific immune cell types, viral pathogens, parasites and bacteria can be followed in real-time within living animals 1-2. Application of bioluminescence imaging to the study of pathogens has advantages as compared to conventional strategies for analysis of infections in animal models3-4. Infections can be visualized within individual animals over time, without requiring euthanasia to determine the location and quantity of the pathogen. Optical imaging allows comprehensive examination of all tissues and organs, rather than sampling of sites previously known to be infected. In addition, the accuracy of inoculation into specific tissues can be directly determined prior to carrying forward animals that were unsuccessfully inoculated throughout the entire experiment. Variability between animals can be controlled for, since imaging allows each animal to be followed individually. Imaging has the potential to greatly reduce animal numbers needed because of the ability to obtain data from numerous time points without having to sample tissues to determine pathogen load3-4.
This protocol describes methods to visualize infections in live animals using bioluminescence imaging for recombinant strains of bacteria expressing luciferase. The click beetle (CBRLuc) and firefly luciferases (FFluc) utilize luciferin as a substrate5-6. The light produced by both CBRluc and FFluc has a broad wavelength from 500 nm to 700 nm, making these luciferases excellent reporters for the optical imaging in living animal models7-9. This is primarily because wavelengths of light greater than 600 nm are required to avoid absorption by hemoglobin and, thus, travel through mammalian tissue efficiently. Luciferase is genetically introduced into the bacteria to produce light signal10. Mice are pulmonary inoculated with bioluminescent bacteria intratracheally to allow monitoring of infections in real time. After luciferin injection, images are acquired using the IVIS Imaging System. During imaging, mice are anesthetized with isoflurane using an XGI-8 Gas Anethesia System. Images can be analyzed to localize and quantify the signal source, which represents the bacterial infection site(s) and number, respectively. After imaging, CFU determination is carried out on homogenized tissue to confirm the presence of bacteria. Several doses of bacteria are used to correlate bacterial numbers with luminescence. Imaging can be applied to study of pathogenesis and evaluation of the efficacy of antibacterial compounds and vaccines.

Methods for bioluminescence imaging of bacterial infections in living animals are decribed. Pathogens are modified to express luciferase allowing optical whole body imaging of infections in live animals. Animal models can be infected with luciferase expressing pathogens and the resulting course of disease visualized in real-time by bioluminescence imaging.

Utilizzando luciferasi di infezioni batteriche immagine in topi

Imaging is a valuable technique that can be used to monitor biological processes.  In particular, the presence of cancer cells, stem cells, specific ...

Invasion of Human Cells by a Bacterial Pathogen

Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus . The general protocol can be applied to the study of cell invasion by virtually any culturable bacterium. The stages at which specific aspects of invasion can be studied, such as the role of actin rearrangement or caveolae, will be highlighted. Host cells are grown in flasks and when ready for use are seeded into 24-well plates containing Thermanox coverslips. Using coverslips allows subsequent removal of the cells from the wells to reduce interference from serum proteins deposited onto the sides of the wells (to which S. aureus would attach). Bacteria are grown to the required density and washed to remove any secreted proteins (e.g. toxins). Coverslips with confluent layers of endothelial cells are transferred to new 24-well plates containing fresh culture medium before the addition of bacteria. Bacteria and cells are then incubated together for the required amount of time in 5% CO2 at 37&deg;C. For S. aureus this is typically between 15-90 minutes. Thermanox coverslips are removed from each well and dip-washed in PBS to remove unattached bacteria. If total associated bacteria (adherent and internalised) are to be quantified, coverslips are then placed in a fresh well containing 0.5% Triton X-100 in PBS. Gentle pipetting leads to complete cell lysis and bacteria are enumerated by serial dilution and plating onto agar. If the number of bacteria that have invaded the cells is needed, coverslips are added to wells containing 500 &mu;l tissue culture medium supplemented with gentamicin and incubation continued for 1 h, which will kill all external bacteria. Coverslips can then be washed, cells lysed and bacteria enumerated by plating onto agar as described above. If the experiment requires direct visualisation, coverslips can be fixed and stained for light, fluorescence or confocal microscopy or prepared for electron microscopy.

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

Invasione di cellule umane da un agente patogeno batterico

Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus . The general protocol can be ...

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of Listeria, which results in systemic infection2. After injection, Listeria quickly disseminates to the spleen and liver due to uptake by CD8&alpha;+ dendritic cells and Kupffer cells3,4. Once phagocytosed, various bacterial proteins enable Listeria to escape the phagosome, survive within the cytosol, and infect neighboring cells5. During the first three days of infection, different innate immune cells (e.g. monocytes, neutrophils, NK cells, dendritic cells) mediate bactericidal mechanisms that minimize Listeria proliferation. CD8+ T cells are subsequently recruited and responsible for the eventual clearance of Listeria from the host, typically within 10 days of infection6.
Successful clearance of Listeria from infected mice depends on the appropriate onset of host immune responses6	. There is a broad range of sensitivities amongst inbred mouse strains7,8. Generally, mice with increased susceptibility to Listeria infection are less able to control bacterial proliferation, demonstrating increased bacterial load and/or delayed clearance compared to resistant mice. Genetic studies, including linkage analyses and knockout mouse strains, have identified various genes for which sequence variation affects host responses to Listeria infection6,8-14. Determination and comparison of infection kinetics between different mouse strains is therefore an important method for identifying host genetic factors that contribute to immune responses against Listeria. Comparison of host responses to different Listeria strains is also an effective way to identify bacterial virulence factors that may serve as potential targets for antibiotic therapy or vaccine design.
We describe here a straightforward method for measuring bacterial load (colony forming units [CFU] per tissue) and preparing single-cell suspensions of the liver and spleen for FACS analysis of immune responses in Listeria-infected mice. This method is particularly useful for initial characterization of Listeria infection in novel mouse strains, as well as comparison of immune responses between different mouse strains infected with Listeria. We use the Listeria monocytogenes EGD strain15 that, when cultured on blood agar, exhibits a characteristic halo zone around each colony due to &beta;-hemolysis1 (Figure 1). Bacterial load and immune responses can be determined at any time-point after infection by culturing tissue homogenate on blood agar plates and preparing tissue cell suspensions for FACS analysis using the protocols described below. We would note that individuals who are immunocompromised or pregnant should not handle Listeria, and the relevant institutional biosafety committee and animal facility management should be consulted before work commences.

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes is a model organism for studying immune responses and genetic susceptibility to intracellular bacteria in mice. This method enables one to measure bacterial load and generate single-cell suspensions of the liver and spleen from mice for FACS analysis to determine changes in immune cells due to Listeria infection.

Misurazione della carica batterica e Risposte immunitarie in topi infettati con Listeria monocytogenes</em

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of ...

Oral Transmission of Listeria monocytogenes in Mice via Ingestion of Contaminated Food

L. monocytogenes are facultative intracellular bacterial pathogens that cause food borne infections in humans. Very little is known about the gastrointestinal phase of listeriosis due to the lack of a small animal model that closely mimics human disease. This paper describes a novel mouse model for oral transmission of L. monocytogenes. Using this model, mice fed L. monocytogenes-contaminated bread have a discrete phase of gastrointestinal infection, followed by varying degrees of systemic spread in susceptible (BALB/c/By/J) or resistant (C57BL/6) mouse strains. During the later stages of the infection, dissemination to the gall bladder and brain is observed. The food borne model of listeriosis is highly reproducible, does not require specialized skills, and can be used with a wide variety of bacterial isolates and laboratory mouse strains. As such, it is the ideal model to study both virulence strategies used by L. monocytogenes to promote intestinal colonization, as well as the host response to invasive food borne bacterial infection.

Oral Transmission of Listeria monocytogenes in Mice via Ingestion of Contaminated Food

This paper describes a novel method for oral infection of mice using Listeria monocytogenes-contaminated food. The protocol can readily be adapted for use with other food borne bacterial pathogens.

Trasmissione orale di<em&gt; Listeria monocytogenes</em&gt; Nei topi tramite ingestione di alimenti contaminati

L. monocytogenes are facultative intracellular bacterial pathogens that cause food borne infections in humans. Very little is known about the ...

Enteric Bacterial Invasion Of Intestinal Epithelial Cells In Vitro Is Dramatically Enhanced Using a Vertical Diffusion Chamber Model

The interactions of bacterial pathogens with host cells have been investigated extensively using in vitro cell culture methods. However as such cell culture assays are performed under aerobic conditions, these in vitro models may not accurately represent the in vivo environment in which the host-pathogen interactions take place. We have developed an in vitro model of infection that permits the coculture of bacteria and host cells under different medium and gas conditions. The Vertical Diffusion Chamber (VDC) model mimics the conditions in the human intestine where bacteria will be under conditions of very low oxygen whilst tissue will be supplied with oxygen from the blood stream. Placing polarized intestinal epithelial cell (IEC) monolayers grown in Snapwell inserts into a VDC creates separate apical and basolateral compartments. The basolateral compartment is filled with cell culture medium, sealed and perfused with oxygen whilst the apical compartment is filled with broth, kept open and incubated under microaerobic conditions. Both Caco-2 and T84 IECs can be maintained in the VDC under these conditions without any apparent detrimental effects on cell survival or monolayer integrity. Coculturing experiments performed with different C. jejuni wild-type strains and different IEC lines in the VDC model with microaerobic conditions in the apical compartment reproducibly result in an increase in the number of interacting (almost 10-fold) and intracellular (almost 100-fold) bacteria compared to aerobic culture conditions1. The environment created in the VDC model more closely mimics the environment encountered by C. jejuni in the human intestine and highlights the importance of performing in vitro infection assays under conditions that more closely mimic the in vivo reality. We propose that use of the VDC model will allow new interpretations of the interactions between bacterial pathogens and host cells.

Enteric Bacterial Invasion Of Intestinal Epithelial Cells In Vitro Is Dramatically Enhanced Using a Vertical Diffusion Chamber Model

Performing cell culture assays for investigating bacterial adhesion and invasion under aerobic conditions is usually unrepresentative of the in vivo environment. A Vertical Diffusion Chamber model allows study of interactions of the human pathogen Campylobacter jejuni with intestinal epithelial cells under more in vivo-like conditions, resulting in enhanced bacterial invasion.

Enterico invasione batterica della cellule epiteliali intestinali<em&gt; In Vitro</em&gt; È drammaticamente migliorata utilizzando una camera di diffusione verticale Modello

The interactions of bacterial pathogens with host cells have been investigated extensively using in vitro cell culture methods. However as such cell ...

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.

Imaging InlC secrezione per Indagare infezione Cellular dalla batterica patogeni<em&gt; Listeria monocytogenes</em

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

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4+ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-&#945;/&#946; cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Unlike cell-free HIV-1 particles, infected CD4+ T cells are effectively sensed by plasmacytoid dendritic cells (pDCs). This manuscript describes a method where peripheral blood mononuclear cells (PBMCs) or isolated pDCs are co-cultured with HIV-1 infected T cells to evaluate innate sensing by pDCs as assessed by release of type-I IFN.

Valutare l&#39;innato rilevamento dell&#39;infezione da HIV-1 CD4 infetti<sup&gt; +</sup&gt; Cellule T dalle cellule dendritiche plasmacitoidi Utilizzando un<em&gt; Ex vivo</em&gt; Sistema di co-coltura.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

Analysis of the transcriptome of bacterial pathogens during mammalian infection is a valuable tool for studying genes and factors that mediate infection. However, isolating bacterial RNA from infected cells or tissues is a challenging task, since mammalian RNA mostly dominates the lysates of infected cells. Here we describe an optimized method for RNA isolation of Listeria monocytogenes bacteria growing within bone marrow derived macrophage cells. Upon infection, cells are mildly lysed and rapidly filtered to discard most of the host proteins and RNA, while retaining intact bacteria. Next, bacterial RNA is isolated using hot phenol-SDS extraction followed by DNase treatment. The extracted RNA is suitable for gene transcription analysis by multiple techniques. This method is successfully employed in our studies of Listeria monocytogenes gene regulation during infection of macrophage cells 1-4. The protocol can be easily modified to study other bacterial pathogens and cell types.

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

Here we describe a method for bacterial RNA isolation from Listeria monocytogenes bacteria growing inside murine macrophages. This technique can be used with other intracellular pathogens and mammalian host cells.

RNA Purificazione da nella cellula Grown<em&gt; Listeria monocytogenes</em&gt; In macrofagi

Analysis of the transcriptome of bacterial pathogens during mammalian infection is a valuable tool for studying genes and factors that mediate infection. ...

Experimental Infection with Listeria monocytogenes as a Model for Studying Host Interferon-&#947; Responses

L. monocytogenes is a gram-positive bacterium that is a cause of food borne disease in humans. Experimental infection of mice with this pathogen has been highly informative on the role of innate and adaptive immune cells and specific cytokines in host immunity against intracellular pathogens. Production of IFN-&#947; by innate cells during sublethal infection with L. monocytogenes is important for activating macrophages and early control of the pathogen1-3. In addition, IFN-&#947; production by adaptive memory lymphocytes is important for priming the activation of innate cells upon reinfection4. The L. monocytogenes infection model thus serves as a great tool for investigating whether new therapies that are designed to increase IFN-&#947; production have an impact on IFN-&#947; responses in vivo and have productive biological effects such as increasing bacterial clearance or improving mouse survival from infection. Described here is a basic protocol for how to conduct intraperitoneal infections of C57BL/6J mice with the EGD strain of L. monocytogenes and to measure IFN-&#947; production by NK cells, NKT cells, and adaptive lymphocytes by flow cytometry. In addition, procedures are described to: (1) grow and prepare the bacteria for inoculation, (2) measure bacterial load in the spleen and liver, and (3) measure animal survival to endpoints. Representative data are also provided to illustrate how this infection model can be used to test the effect of specific agents on IFN-&#947; responses to L. monocytogenes and survival of mice from this infection.

Experimental Infection with &lt;em&gt;Listeria monocytogenes&lt;/em&gt; as a Model for Studying Host Interferon-&amp;#947; Responses

This protocol describes how to inoculate C57BL/6J mice with the EGD strain of Listeria monocytogenes (L. monocytogenes) and to measure interferon-&#947; (IFN-&#947;) responses by natural killer (NK) cells, natural killer T (NKT) cells, and adaptive T lymphocytes post-infection. This protocol also describes how to conduct survival studies in mice after infection with a modified LD50 dose of the pathogen.

Infezione sperimentale con<em&gt; Listeria monocytogenes</em&gt; Come modello per lo studio Host Interferone-gamma Risposte

L. monocytogenes is a gram-positive bacterium that is a cause of food borne disease in humans. Experimental infection of mice with this pathogen has been ...