Name: assessing bacterial invasion of cardiac cells in culture and heart colonization in infected mice using listeria monocytogenes
Uploaded: 2015-05-27T14:00:00.000+00:00
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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

Este trabajo fue apoyado por las subvenciones del Servicio de Salud Pública AI41816 y AI099339 (N.E.F.) y por F31AI094886-01 (P.D.M.) del NIAID. Los contenidos son responsabilidad exclusiva de los autores y no representan necesariamente los puntos de vista oficiales de las fuentes de financiación.

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	<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. 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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>

Los autores no reportan intereses financieros en competencia.

L. monocytogenes es un patógeno humano extendido y bien caracterizado, capaz de causar una serie de manifestaciones diferentes de la enfermedad15. La bacteria se ha descrito previamente por su capacidad de translocar a través de barreras, como la barrera hematoencefálica y las barreras placentario-fetales, con el fin de alcanzar y colonizar el sistema nervioso central y el feto en desarrollo, respectivamente. La capacidad in vivo del organismo para colonizar estos tejidos a menudo se compl...

Listeria monocytogenes es un patógeno intracelular Gram-positivo capaz de causar enfermedades graves en poblaciones susceptibles, incluyendo ancianos, mujeres embarazadas, personas con VIH/SIDA y personas que reciben quimioterapia1. La infección en estas poblaciones es frecuentemente el resultado de la ingestión de productos alimenticios contaminados, y la mayoría de las infecciones están asociadas con brotes alimentarios a gran escala2,3. En humanos y otros mamíferos, L. monocytogenes es capaz de translocar a través de la frontera epitelial del intestino delgado, siendo posteriormente transportado al hígado4,5. Los modelos animales sugieren que las bacterias ingeridas se repliquen dentro de las vellosidades intestinales y transiten al hígado a través de la vena porta o se propaguen a través de los ganglios linfáticos mesentéricos hacia el torrente sanguíneo, lo que lleva a la diseminación hematógena al hígado y al bazo6,7. En el hígado y el bazo, la bacteria es capaz de mediar en fagocitos profesionales, así como en las células parenquimatosas residentes, y establece rápidamente infecciones dentro de estos órganos. A medida que aumenta la carga bacteriana, numerosas bacterias se dispersan de nuevo en la sangre, donde son capaces de colonizar aún más los tejidos susceptibles, incluyendo el sistema nervioso central y la placenta (donde esté presente). La colonización de estos sitios excluye las manifestaciones más comunes de listeriosis en humanos, incluyendo meningitis, encefalitis y aborto fetal2.
Recientemente se ha demostrado que determinadas subpoblaciones de L. monocytogenes tienen una mayor capacidad de invadir y replicarse dentro del tejido cardíaco8. Las manifestaciones de afectación cardíaca son variadas, y van desde la endocarditis y la pericarditis, hasta la miocarditis fulminante completa con anomalías de conducción9-13. El número total de casos cardiacos de los monocytogenes del L. por año es bajo pero puede debajo estimado pues esta faceta de la infección no se reconoce generalmente bien. La colonización del corazón por patógenos a menudo requiere predisposiciones del huésped, como daño valvular preexistente o válvulas cardíacas artificiales. Existen, sin embargo, aislados de L. monocytogenes que han sido identificados que son notables por su capacidad de colonizar los corazones de animales infectados en ausencia de cualquier daño cardíaco y/o anormalidades8.
Adjunto se describen in vitro e in vivo los métodos para evaluar la colonización bacteriana del tejido cardiaco dentro de animales infectados usando análisis de la invasión en cultura del tejido así como infecciones animales vivas. Estos métodos han demostrado ser útiles no sólo para identificar cepas con el potencial de colonizar el tejido cardíaco en animales infectados, sino que también deben ser útiles para la identificación de productos de genes bacterianos que sirven para mejorar la invasión de células cardíacas y / o dar lugar a cambios en la patología del corazón. Estos métodos facilitan la comparación del cardiotropismo entre múltiples cepas. Para los métodos descritos aquí, L. monocytogenes 10403S se utiliza como un representante bien estudiado de una cepa no cardiotrópica y el aislado clínico 07PF0776 se utiliza como un ejemplo representativo de una cepa cardiotrópica. Estas dos cepas se eligieron para proporcionar una comparación para la invasión bacteriana de células cardíacas in vitro y la colonización de corazones de ratones infectados in vivo. El aislado 07PF0776 es un aislado clínico recuperado de un absceso interventricular que causó una arritmia fatal en un paciente VIH+8. Los aislados de L. monocytogenes pueden variar en su potencial de virulencia, y dada la propensión de Listeria a infectar a personas con inmunosupresión y mujeres embarazadas, las personas dentro de estas poblaciones deben tener cuidado al evaluar diferentes aislados clínicos.

<table><tbody><tr><td>Difco</td><td>BHI Broth Base</td><td>237200</td><td>2 kg of Brain Heart Infusion powder (without agar)</td></tr><tr><td>Difco</td><td>Agar</td><td>214510</td><td>2 kg of Granulated Agar</td></tr><tr><td>Difco</td><td>BHI Agar</td><td>241810</td><td>2 kg 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>2 kg of Luria Broth Base Powder (without agar)</td></tr><tr><td>Fisher</td><td>Glass Coverslips</td><td>12-545-80</td><td>12 mm 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>14 ml 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>50 mg/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>75 cm&lt;sup&gt;2&lt;/sup&gt; tissue culture treated flask</td></tr><tr><td>Denville</td><td>1.5mL microcentrifuge tubes</td><td>2013004</td><td>1.5 ml 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

Los monocytogenes de la listeriería son un patógeno intracelular facultativo Gram-positivo que es capaz de causar infecciones invasores serias en pacientes immunocompromised, los ancianos, y las mujeres embarazadas. Las manifestaciones mas comunes de la listeriosis en seres humanos incluyen meningitis, encefalitis, y aborto fetal. Una secuela significativa pero mucho menos documentada de la infección invasor de los monocytogenes del L. implica el corazón. La tasa de mortalidad por enfermedad cardíaca puede ser de hasta el 35% a pesar del tratamiento, sin embargo se sabe muy poco sobre la colonización de L. monocytogenes del tejido cardíaco y sus patologías resultantes. Además, recientemente se ha hecho evidente que las subpoblaciones de L. monocytogenes tienen una mayor capacidad para invadir y crecer dentro del tejido cardíaco. Este protocolo describe detalladamente los métodos ines vitro e in vivo que se pueden utilizar para determinar cardiotropism de los aislantes de los monocytogenes del L. Los métodos se presentan para la infección de los mioblastos cardiacos de la rata H9c2 en cultura del tejido así como para la determinación de la colonización bacteriana de los corazones de ratones infectados. Estos métodos son útiles no sólo para identificar tensiones con el potencial de colonizar el tejido cardiaco en animales infectados, pero pueden también facilitar la identificación de los productos bacterianos del gen que sirven para aumentar la invasión de la célula cardiaca y/o para conducir cambios en patología del corazón. Estos métodos también proporcionan la comparación directa del cardiotropismo entre múltiples cepas de L. monocytogenes.

Los aislantes seleccionados de los monocytogenes del L. exhiben la invasión aumentada de células cardiacas en cultivo celular y en modelos del ratón de la infección. La Figura 1 muestra un ejemplo de cómo pueden aparecer colonias bacterianas después del revestimiento puntual de suspensiones en medios de agar. Este método permite una evaluación precisa de las UFC dentro de una muestra sin utilizar un gran número de placas de medios de agar. La Figura 2 muestra un ejemplo...

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 causa infecciones fetales en mujeres embarazadas y meningitis en poblaciones susceptibles. Las subpoblaciones de bacterias pueden colonizar el tejido cardíaco, causando miocarditis en pacientes y animales de laboratorio. Aquí presentamos un protocolo que describe cómo evaluar la invasión de células cardíacas de L. monocytogenes in vitro y la colonización cardíaca en animales infectados.

Evaluación De La Invasión Bacteriana De Las Células Cardíacas En Cultivo Y Colonización Del Corazón En Ratones Infectados Usando 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 vistas.  College of Medicine, University of Illinois at Chicago. Listeria monocytogenes causa infecciones fetales en mujeres embarazadas y meningitis en poblaciones susceptibles. Las subpoblaciones de bacterias pueden colonizar el tejido cardíaco, causando miocarditis en pacientes y animales de laboratorio. Aquí presentamos un protocolo que describe cómo evaluar la invasión de células cardíacas de L. monocytogenes in vitro y la colonización cardíaca en animales infectados. 

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

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.

La medición de la carga bacteriana y la respuesta inmune en ratones infectados con Listeria monocytogenes</em

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

Investigación

Inmunología e Infección

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.

La transmisión oral de<em&gt; Listeria monocytogenes</em&gt; En ratones a través de la ingestión de alimentos contaminados

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

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 secreción de investigar la infección celular por el patógeno bacteriano<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 ...

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.

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.

La infección experimental con<em&gt; Listeria monocytogenes</em&gt; Como un modelo para el estudio de host Respuestas interferón gamma

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 ...

Visualization of Streptococcus pneumoniae within Cardiac Microlesions and Subsequent Cardiac Remodeling

During bacteremia Streptococcus pneumoniae can translocate across the vascular endothelium into the myocardium and form discrete bacteria-filled microscopic lesions (microlesions) that are remarkable due to the absence of infiltrating immune cells. Due to their release of cardiotoxic products, S. pneumoniae within microlesions are thought to contribute to the heart failure that is frequently observed during fulminate invasive pneumococcal disease in adults. Herein is demonstrated a protocol for experimental mouse infection that leads to reproducible cardiac microlesion formation within 30 hr. Instruction is provided on microlesion identification in hematoxylin &#38; eosin stained heart sections and the morphological distinctions between early and late microlesions are highlighted. Instruction is provided on a protocol for verification of S. pneumoniae within microlesions using antibodies against pneumococcal capsular polysaccharide and immunofluorescent microscopy. Last, a protocol for antibiotic intervention that rescues infected mice and for the detection and assessment of scar formation in the hearts of convalescent mice is provided. Together, these protocols will facilitate the investigation of the molecular mechanisms underlying pneumococcal cardiac invasion, cardiomyocyte death, cardiac remodeling as a result of exposure to S. pneumoniae, and the immune response to the pneumococci in the heart.

Visualization of Streptococcus pneumoniae within Cardiac Microlesions and Subsequent Cardiac Remodeling

Streptococcus pneumoniae forms discrete non-purulent microscopic lesions in the heart. Outlined is the protocol for a murine model of cardiac microlesion formation. Instruction is provided on microlesion visualization using microscopy, discrimination between early and late microlesions, and methods to detect cardiac remodeling in hearts of convalescent animals.

Visualización de<em&gt; Streptococcus pneumoniae</em&gt; Dentro cardíacos microlesiones y la remodelación cardiaca posterior

During bacteremia Streptococcus pneumoniae can translocate across the vascular endothelium into the myocardium and form discrete bacteria-filled ...

Medicina

Listeria monocytogenes Infection of the Brain

Listeria monocytogenes is an intracellular bacterial pathogen that is frequently associated with food-borne infection. The ability of&#160;L. monocytogenes to cross the blood-brain barrier (BBB) is concerning as it can lead to life-threatening meningitis and encephalitis. The BBB protects the brain microenvironment from various toxic metabolites and microbial pathogens found in the blood following infection, and therefore supports brain homeostasis. The mechanisms by which L. monocytogenes present in the bloodstream cross the BBB to cause brain infections are not fully understood and there is also a lack of a robust model system to study brain infections by L. monocytogenes. Here, we present a simple mouse infection model to determine whether bacteria have crossed the BBB and to quantitate the burden of bacteria that have colonized the brain in vivo. In this method, animals were infected intravenously with L. monocytogenes and were humanely euthanized by exposure to CO2 followed by cervical dislocation. Cardiac perfusion of the animals was performed prior to harvesting infected organs. Blood was collected before perfusion and the number of bacteria per organ or mL of blood was determined by plating dilutions of the blood or organ homogenates on agar plates and counting the number of colonies formed. This method can be used to study novel receptor-ligand interactions that enhance infection of the brain by L. monocytogenes and can be easily adapted for the study of multiple bacterial pathogens.

Listeria monocytogenes Infection of the Brain

During infection, Listeria monocytogenes is capable of crossing the blood-brain barrier to colonize the brain. In this protocol, we demonstrate how to assess bacterial colonization of organs following infection of mice. A procedure to perform whole organ perfusion for specific determination of bacterial numbers in the brain parenchyma is provided.

Listeria monocytogenes Infección del cerebro

Listeria monocytogenes is an intracellular bacterial pathogen that is frequently associated with food-borne infection. The ability of&#160;L. ...

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.

We describe both in vitro and in vivo infection assays that can be used to analyze the activities of host-encoding factors.

Usando un patógeno bacteriano a punta de prueba para las respuestas celular y organísmica nivel Host

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' ...

A Technique to Establish a Bacterial Infection Model in an Ex Vivo Organ Culture

Source: Rizzuto, G. A., et al. <a href="https://app.jove.com/v/54237">Human Placental and Decidual Organ Cultures to Study Infections at the Maternal-fetal Interface.</a> J. Vis. Exp. (2016)
This video demonstrates a method to establish an infection model in placental tissues to study infection propagation. An ex vivo placental organ culture is infected with Listeria monocytogenes, a facultative intracellular parasite. The bacteria invade extravillous trophoblasts (EVTs) to enter the placental tissue and then propagate to other cells via cell-to-cell spread.

Una técnica para establecer un modelo de infección bacteriana en un cultivo de órganos ex vivo

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human ...

JoVE Encyclopedia of Experiments

Immunology

Immunopathology

In Vitro Models

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

Source: Gayle, P. et al., <a href="https://app.jove.com/v/58775">Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses.</a>&#160;J. Vis. Exp.&#160; (2019).
This video demonstrates an in vitro quantification technique to determine Listeria monocytogenes infection of macrophages by the colony-forming unit assay. The bacterial colonies on the agar plate represent the intracellular bacterial population that escapes the host-defense mechanism and survives within the macrophages.

Cuantificación de la infección bacteriana en las células huésped mediante el ensayo de la unidad formadora de colonias

Source: Gayle, P. et al., Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses.&#160;J. Vis. Exp.&#160; (2019).
This video ...

Immune Response

Host-Pathogen Interaction

Assessing Bacterial Colonization in a Mouse Brain following Listeria monocytogenes Infection

Source: Ghosh, P., et al., <a href="https://app.jove.com/v/58723">Listeria monocytogenes Infection of the Brain.</a> J. Vis. Exp. (2018)
This video demonstrates the quantification of the infective bacterial load from the perfused mouse brain infected with Listeria monocytogenes.

Evaluación de la colonización bacteriana en el cerebro de un ratón después de una infección por Listeria monocytogenes

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.&#160;
1. Organ Harvesting and ...

Evaluación De La Invasión Bacteriana De Las Células Cardíacas En Cultivo Y Colonización Del Corazón En Ratones Infectados Usando Listeria monocytogenes

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La medición de la carga bacteriana y la respuesta inmune en ratones infectados con Listeria monocytogenes

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Visualización de

Listeria monocytogenes Infección del cerebro

Usando un patógeno bacteriano a punta de prueba para las respuestas celular y organísmica nivel Host

Una técnica para establecer un modelo de infección bacteriana en un cultivo de órganos ex vivo

Cuantificación de la infección bacteriana en las células huésped mediante el ensayo de la unidad formadora de colonias

Evaluación de la colonización bacteriana en el cerebro de un ratón después de una infección por Listeria monocytogenes