Name: rna purification from intracellularly grown listeria monocytogenes in macrophage cells
Uploaded: 2016-06-04T14:00:00.000+00:00
Duration: 10 min 42 s
Description: Watch this Scientific Journal Video about RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells at JoVE.com

The research in the Herskovits lab is supported by 335400 ERC and R01A/109048 NIH grants.

Nota: Durante toda a experiência, as células de macrófagos foram incubadas a 37 ° C numa incubadora de CO2-ar forçado 5% e levado para fora da incubadora apenas para manipulações experimentais, que são realizadas numa câmara de segurança biológica de Classe II. Trabalhando com L. bactérias monocytogenes é de acordo com o nível de segurança biológica 2 regulamentos. 1. Preparação de células e infecção bacteriana (Dia 1 e 2) <ol><li> Dia 1 <ol><li> Semente de medula 2,0 x 10 7 células de osso derivadas de macrófagos (BMDM) sobre um prato de 145 milímetros em 30 ml BMDM + meios Pen-Strep (Tabela 2). Seed 3 placas para cada estirpe bacteriana a ser analisado. Incubar durante a noite a 37 ° C numa incubadora de CO2-ar forçado 5%. </li><li> Iniciar uma cultura bacteriana durante a noite de tipo selvagem (WT) L. monocytogenes 10403S estirpe em 10 ml de meio de infusão de cérebro e coração (BHI), a 30 ° C numa incubadora padrão. Colocar os tubos de cultura inclinada sem agitação. Nota:Estas condições de crescimento sobre-regular a expressão dos genes de flagelos, que promove a infecção eficiente. </li></ol></li><li> Dia 2 <ol><li> Pré-forma quente BMDM (sem antibióticos Pen-Strep) (Tabela 2) e fosfato tamponado salino (PBS) a 37 ° C. </li><li> Lavar monocamada de macrófagos duas vezes com 25 ml de PBS pré-aquecido para remover os antibióticos. Adicionar 30 ml de meio BMDM fresco. </li><li> Lava-se 1,5 ml de cultura bacteriana durante a noite com PBS, as bactérias por centrifugação a&gt; 14.000 xg durante 1 min, descartando o sobrenadante, e ressuspensão suavemente bactérias em 1,5 ml de PBS por pipetagem. Repita duas vezes. </li><li> Infect cada placa de macrófagos com 0,5 ml de uma cultura durante a noite lavou-se de tipo selvagem L. monocytogenes. Se o uso de vários pratos, infectar cada placa 15 min de intervalo. Este intervalo de tempo irá permitir a colheita cada placa individualmente no final da infecção. Nota: A multiplicidade de infecção (MOI) é ensaiado por diluição em séries da cultura bacteriana utilizada para a infecção em placas de agar BHI e contagem de unidades formadoras de colónias (CFU) após 24 horas de incubação das placas a 37 ° C. Utilizando-se 0,5 ml de WT L. monocytogenes estirpe resultados 10403S em um MOI de 100 ~ (100 cfu por bactérias celular de macrófagos). Ao analisar bactérias mutantes defeituosos no crescimento intracelular, uma MOI superior deve ser considerada. </li><li> Após 0,5 horas de incubação a 37 ° C, lavar as células infectadas duas vezes com PBS, para remover as bactérias não ligadas, e adicionar 30 ml de meio pré-aquecido BMDM. bactérias aderidas vai internalizar durante a próxima 0,5 hr. </li><li> À 1 h após a infecção, adicionar 30 uL de gentamicina (1: 1000 para atingir uma concentração final de 50 ug / ml) para matar bactérias extracelulares. </li><li> Montar o aparelho de filtro, colocando uma cabeça do filtro sobre um balão de recolha de líquido com um orifício de saída de vácuo. Em seguida, colocar um filtro (0,45 mm) sobre uma cabeça do filtro, seguido de um funil e cilindro de garantir as diferentes partes com um metal clâmpada. Prepare água livre de RNase gelada. </li><li> Às 6 h pós-infecção, as bactérias de colheita a partir de células infectadas como se segue. Trate cada placa individualmente. Nota: Normalmente, L. monocytogenes termina 1-log de ​​crescimento durante 6 hr de infecção em células de macrófagos. incubações mais longas poderia resultar numa proliferação bacteriana e a morte celular dos macrófagos, enquanto que períodos de incubação mais curtos pode resultar em cargas bacterianas consideravelmente inferiores. A MOI e o tempo de infecção pode ser modificado para se alcançar as condições óptimas. <ol><li> Lavar as células infectadas com PBS uma vez. Adicionar 20 ml de água livre de RNase gelada para lisar células de macrófagos. Usando um raspador de células, raspar as células da placa rapidamente, mas com cuidado. </li><li> Recolha de células lisadas para um tubo cónico de 50 ml. Vortex durante 30 seg. Centrifuga-se a 800 xg durante 3 min a 4 ° C. </li><li> Passar o sobrenadante através do aparelho de filtro, utilizando um sistema de vácuo. Com uma pinça, rolar o filtro e rapidamente transferi-lo para a 15 ml CONItubo de cal. Snap-congelar os tubos com os filtros em azoto líquido. </li><li> Montar o aparelho de filtração com um filtro novo para a amostra seguinte, como descrito no 1.2.7. </li><li> Armazenar os filtros congelados a -80 ° C durante o dia seguinte. Alternativamente, vá diretamente para a extração de ácidos nucléicos. </li></ol></li></ol></li></ol> 2. Ácidos Nucleicos Extraction (Dia 3) Nota: Executar todas as manipulações com soluções de fenol e clorofórmio em um exaustor. <ol><li> Prepara-se uma mistura 1: 1 de fenol acidificado: clorofórmio, 400 ul de cada amostra. Misturar em tubos separados, e em seguida, retirar a camada aquosa resultante por aspiração. Adicionar 40 ul de SDS a 10%. </li><li> Descongelar os tubos contendo filtro no gelo para mantê-los frio. Para cada tubo contendo o filtro de adicionar 650 ul de tampão de acetato-EDTA (AE) (Tabela 2). </li><li> Execute os seguintes passos tão rapidamente quanto possível. <ol><li> Vortex vigorosamente o tubo contendo o filtro paraque leva o filtro para a periferia do tubo e o tampão de lava completamente o filtro. Sempre mantenha o frio tubo, colocando-o de volta no gelo. Nota: Pode ser necessário Além disso vortex do tubo enquanto invertido para lavar completamente as bactérias fora do filtro. </li><li> Repita o procedimento para todos os tubos. Pode ser útil para girar para baixo a suspensão breve (1 min a 120 x g), no final do processo. </li></ol></li><li> Transferir o tampão de bactérias contendo a um tubo de microcentrífuga de 1,5 ml contendo SDS e a mistura de fenol / clorofórmio (como foi preparada no ponto 2.1). Repita o procedimento para todos os tubos. Pode ser necessário para rodar os tubos de filtragem contendo (1 min a 120 x g) novamente para obter tampão residual do filtro. </li><li> Colocar todos os tubos de 1,5 ml em um dispositivo de vórtice multi-tubos, e agitar com vortex à velocidade máxima durante 10 min. </li><li> Incubar os tubos num bloco de calor a 65 ° C durante 10 min. Centrifuga-se a velocidade máxima (&gt; 14.000 x g) durante 5 min. </li><li> Transferir a fase aquosa (cerca de 400 ul) de cada tUbe para um novo tubo de 1.5 ml contendo 40 ul de acetato de sódio 3 M (pH 5,2) e 1,0 ml de etanol a 100%. Vortex cada tubo completamente. Nota: O glicogénio podem ser adicionados nesta etapa para melhorar a visualização do sedimento precipitado. </li><li> Incubar as amostras a -80 ° C durante 1 h. Alternativamente, incubar as amostras a -20 ° C durante a noite. Em seguida, centrifugar as amostras a 4 ° C durante 20 minutos à velocidade máxima (&gt; 14.000 x g). </li><li> Aspirar cuidadosamente o etanol (camada superior) de cada tubo. Adicionar 500 ul de etanol frio a 70% a cada amostra e vórtice completamente. Centrifugar a 4 ° C durante 20 minutos à velocidade máxima (&gt; 1 4000 xg). </li><li> Aspirar cuidadosamente o etanol a partir de cada tubo. Secam-se as amostras durante cerca de 2 min, utilizando um evaporador sob vácuo sem aquecimento. Não excesso de secar as amostras. </li><li> Adicionar 25 mL de água livre de RNase para cada amostra. Incubar à temperatura ambiente durante 20 min. Cuidadosamente vórtice e spin-down. </li><li> Medir a concentração de ARN em tele amostras usando um microvolume UV-Vis. Esperar para extrair cerca de 0,5-1 mg de ácidos nucleicos por placa. Nota: repetições técnicas podem ser combinadas nesta fase. </li></ol> 3. DNase Tratamento <ol><li> Defina-se a reacção de acordo com o Quadro 1 num tubo de microcentrífuga. Incubar a 37 ° C durante 45 min. Adicionar 450 uL de água livre de RNase-e 500 ul de fenol-clorofórmio-IAA mistura (Tabela 2), as fases separadas por centrifugação durante 2 min a velocidade máxima (&gt; 14.000 x g). </li><li> Transferir a fase aquosa para um novo tubo e adicionar 500 mL de clorofórmio-IAA mistura (Tabela 2), e as fases separadas vórtice por 2 minutos de centrifugação a velocidade máxima (&gt; 14.000 x g). </li><li> Transferir a fase aquosa para um novo tubo e adicionar 1 ml de etanol e 50 uL de acetato de sódio 3 M (pH 5,2). Vórtice. Nota: O glicogénio podem ser adicionados nesta etapa para melhorar a visualização do sedimento precipitado. </li><li&gt; Incubar durante 1 hora a -80 ° C. Alternativamente, incubar as amostras a -20 ° C durante a noite. Centrifugar a 4 ° C durante 20 min, a velocidade máxima. </li><li> Aspirar cuidadosamente o etanol a partir de cada tubo. Adicionar 500 ul de etanol frio a 70% a cada amostra e vórtice completamente. Centrifugar a 4 ° C durante 20 minutos à velocidade máxima. Aspirar cuidadosamente o etanol a partir de cada tubo. </li><li> Secam-se as amostras durante cerca de 2 min, utilizando um evaporador sob vácuo sem aquecimento. Não excesso de secar as amostras. </li><li> Adicionar 12 ul de água livre de RNase, incubar durante 2 minutos à temperatura ambiente, vórtice, e girar para baixo. As amostras contêm RNA purificado, mantê-los no gelo. Em alternativa, dissolve-se o ARN em RNase H 2 O com 0,1 mM de EDTA ou tampão TE (Tris 10 mM, EDTA 1 mM). </li><li> Medir as concentrações de ARN utilizando um microvolume UV-Vis. Esperar ~ 100 ng de RNA por amostra (se repetições técnicas são combinadas). Nota: RNA extraído de acordo com este protocolo é adequado foanálise de transcrição do gene r por várias técnicas. Nós geralmente empregam análise de RT-qPCR para estudar L. monocytogenes transcrição de genes durante a infecção de células de macrófagos 1-4. </li></ol>

<ol>
	<li>Lobel, L., 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+metabolic+regulator+CodY+links+Listeria+monocytogenes+metabolism+to+virulence+by+directly+activating+the+virulence+regulatory+gene+prfA.">The metabolic regulator CodY links Listeria monocytogenes metabolism to virulence by directly activating the virulence regulatory gene prfA.</a> Mol Microbiol. , (2014).</li><li>Lobel, L., Sigal, N., Borovok, I., Ruppin, E., Herskovits, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrative+genomic+analysis+identifies+isoleucine+and+CodY+as+regulators+of+Listeria+monocytogenes+virulence.">Integrative genomic analysis identifies isoleucine and CodY as regulators of Listeria monocytogenes virulence.</a> PLoS Genet. 8 , e1002887 (2012).</li><li>Kaplan Zeevi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Listeria+monocytogenes+multidrug+resistance+transporters+and+cyclic+di-AMP,+which+contribute+to+type+I+interferon+induction,+play+a+role+in+cell+wall+stress.">Listeria monocytogenes multidrug resistance transporters and cyclic di-AMP, which contribute to type I interferon induction, play a role in cell wall stress.</a> J Bacteriol. 195, 5250-5261 (2013).</li><li>Rabinovich, L., Sigal, N., Borovok, I., Nir-Paz, R., Herskovits, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prophage+excision+activates+Listeria+competence+genes+that+promote+phagosomal+escape+and+virulence.">Prophage excision activates Listeria competence genes that promote phagosomal escape and virulence.</a> Cell. 150, 792-802 (2012).</li><li>Swaminathan, B., Gerner-Smidt, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+epidemiology+of+human+listeriosis.">The epidemiology of human listeriosis.</a> Microbes Infect. 9, 1236-1243 (2007).</li><li>Hamon, M., Bierne, H., Cossart, P. <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+multifaceted+model.">Listeria monocytogenes: a multifaceted model.</a> Nat Rev Microbiol. 4, 423-434 (2006).</li><li>Dussurget, O., Pizarro-Cerda, J., Cossart, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+determinants+of+listeria+monocytogenes+virulence.">Molecular determinants of listeria monocytogenes virulence.</a> Ann Rev Microbiol. 58, 587-610 (2004).</li><li>Portnoy, D. A., Auerbuch, V., Glomski, I. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cell+biology+of+Listeria+monocytogenes+infection:+the+intersection+of+bacterial+pathogenesis+and+cell-mediated+immunity.">The cell biology of Listeria monocytogenes infection: the intersection of bacterial pathogenesis and cell-mediated immunity.</a> J Cell Biol. 158, 409-414 (2002).</li><li>Freitag, N. E., Port, G. C., Miner, M. D. <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+-+from+saprophyte+to+intracellular+pathogen.">Listeria monocytogenes - from saprophyte to intracellular pathogen.</a> Nat Rev Microbiol. 7, 623-628 (2009).</li><li>Graham, J. E., Clark-Curtiss, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+Mycobacterium+tuberculosis+RNAs+synthesized+in+response+to+phagocytosis+by+human+macrophages+by+selective+capture+of+transcribed+sequences+(SCOTS).">Identification of Mycobacterium tuberculosis RNAs synthesized in response to phagocytosis by human macrophages by selective capture of transcribed sequences (SCOTS).</a> Proc Natl Acad Sci U S A. 96, 11554-11559 (1999).</li><li>Toledo-Arana, 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=The+Listeria+transcriptional+landscape+from+saprophytism+to+virulence.">The Listeria transcriptional landscape from saprophytism to virulence.</a> Nature. 459, 950-956 (2009).</li><li>Schnappinger, 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=Transcriptional+Adaptation+of+Mycobacterium+tuberculosis+within+Macrophages:+Insights+into+the+Phagosomal+Environment.">Transcriptional Adaptation of Mycobacterium tuberculosis within Macrophages: Insights into the Phagosomal Environment.</a> J Exp Med. 198, 693-704 (2003).</li><li>Albrecht, M., Sharma, C. M., Reinhardt, R., Vogel, J., Rudel, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deep+sequencing-based+discovery+of+the+Chlamydia+trachomatis+transcriptome.">Deep sequencing-based discovery of the Chlamydia trachomatis transcriptome.</a> Nucleic Acids Res. 38, 868-877 (2010).</li><li>La, M. V., Raoult, D., Renesto, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+whole+bacterial+pathogen+transcription+within+infected+hosts.">Regulation of whole bacterial pathogen transcription within infected hosts.</a> FEMS Microbiol Rev. 32, 440-460 (2008).</li><li>Westermann, A. J., Gorski, S. A., Vogel, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual+RNA-seq+of+pathogen+and+host.">Dual RNA-seq of pathogen and host.</a> Nat Rev Microbiol. 10, 618-630 (2012).</li><li>Rienksma, R. 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=Comprehensive+insights+into+transcriptional+adaptation+of+intracellular+mycobacteria+by+microbe-enriched+dual+RNA+sequencing.">Comprehensive insights into transcriptional adaptation of intracellular mycobacteria by microbe-enriched dual RNA sequencing.</a> BMC Genomics. 16, 34 (2015).</li><li>Afonso-Grunz, F., 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=Dual+3'Seq+using+deepSuperSAGE+uncovers+transcriptomes+of+interacting+Salmonella+enterica+Typhimurium+and+human+host+cells.">Dual 3'Seq using deepSuperSAGE uncovers transcriptomes of interacting Salmonella enterica Typhimurium and human host cells.</a> BMC Genomics. 16, 323 (2015).</li><li>Englen, M. D., Valdez, Y. E., Lehnert, N. M., Lehnert, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Granulocyte/macrophage+colony-stimulating+factor+is+expressed+and+secreted+in+cultures+of+murine+L929+cells.">Granulocyte/macrophage colony-stimulating factor is expressed and secreted in cultures of murine L929 cells.</a> J Immunol Methods. 184, 281-283 (1995).</li><li>Becavin, 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=Comparison+of+widely+used+Listeria+monocytogenes+strains+EGD,+10403S,+and+EGD-e+highlights+genomic+variations+underlying+differences+in+pathogenicity.">Comparison of widely used Listeria monocytogenes strains EGD, 10403S, and EGD-e highlights genomic variations underlying differences in pathogenicity.</a> MBio. 5, e00969-e900914 (2014).</li><li>Celada, A., Gray, P. W., Rinderknecht, E., Schreiber, R. D. <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+a+gamma-interferon+receptor+that+regulates+macrophage+tumoricidal+activity.">Evidence for a gamma-interferon receptor that regulates macrophage tumoricidal activity.</a> J Exp Med. 160, 55-74 (1984).</li></ol>

The authors have nothing to disclose.

O protocolo aqui descrito representa um método aperfeiçoado para o isolamento de ARN a partir de bactérias L. monocytogenes bactérias que crescem intracelularmente em células de macrófagos. Este protocolo baseia-se na lise diferencial de células e inclui duas etapas principais para enriquecimento de RNA bacteriana: sedimentação núcleos de macrófagos usando centrifugação e uma recolha rápida de bactérias por filtração. Estes passos são seguidos por um processo de extracção de ARN padrão. Emb...

Patógenos bacterianos intracelulares ─ bactérias que causam doenças infecciosas e capaz de crescer e reproduzir no interior das células de hospedeiros humanos ─ são um importante problema de saúde em todo o mundo 5. Para invadir e replicar dentro de uma célula de mamífero, patogénios intracelulares adquiriram sofisticados mecanismos de virulência e factores. Embora estes mecanismos são fundamentais para a capacidade de causar uma doença, pouco sabemos sobre a sua regulação e dinâmica. Uma vez que os perfis de bactérias cultivadas em meio líquido de expressão de genes não reflectem o ambiente real dentro de células hospedeiras, existe uma necessidade crescente de transcriptoma análises de bactérias cultivadas nos seus nichos intracelulares. Tais análises permitirá a decifração de adaptações bacterianas específicas desencadeadas pelo anfitrião e vai ajudar a identificar novos alvos para o projeto terapêutico. A análise do transcriptoma de bactérias cultivadas intracelularmente é altamente desafiador, uma vez ARN de mamífero superam ARN bacteriana por pelomenos dez vezes. Neste artigo, nós descrevemos um método experimental para isolar RNA bacteriana de Listeria monocytogenes bactérias que crescem no interior das células de macrófagos de murino. O ARN extraído pode ser utilizado para estudar adaptações intracelulares e mecanismos de virulência de bactérias patogénicas através de várias técnicas de análise de transcrição, tais como RT-PCR, o RNA-Seq, microarray e outras tecnologias de hibridação baseada. L. monocytogenes é o agente causador de listeriose em humanos, uma doença com manifestações clínicas dirigidas principalmente indivíduos imunocomprometidos, idosos e mulheres grávidas 6. isto é um agente patogénico intracelular facultativo Gram-positiva que invade uma grande variedade de células de mamíferos que tenham sido utilizados durante décadas como um modelo em interacções hospedeiro-patogénio estuda 7. Após a invasão, ele reside inicialmente em um vacúolo ou fagossoma (no caso de células fagocíticas), a partir do qual ele deve escapar para tele hospedar citosol da célula, a fim de se replicar. Vários factores de virulência foram mostrados para mediar o processo de escape, principalmente a hemolisina de formação de poros, listeriolisina O (LLO) e duas fosfolipases 8 adicionais. No citosol das bactérias utilizam a maquinaria a polimerização de actina hospedeiro para impulsionar-se em filamentos de actina dentro da célula e para espalhar a partir de uma célula para outra (Figura 1). Todos os principais fatores de virulência de L. monocytogenes envolvidas na invasão, a sobrevivência e a replicação intracelular, são activados pelo regulador da transcrição mestre virulência, PrfA. 8-10. Na última década, vários estudos realizados por nós e outros métodos para análise de transcriptoma de bactérias cultivadas intracelularmente aplicado com sucesso dentro das células hospedeiras 2,11-15. Duas abordagens principais são usadas para separar o RNA de bactérias de hospedeiro-ARN que são baseadas em: 1) enriquecimento selectivo de ARN bacteriana e 2) de isolamento de ARN por diferential lise celular. A primeira abordagem depende de hibridização subtrativa de extratos de RNA total de moléculas de RNA de mamíferos (por exemplo, utilizando kits comercialmente disponíveis) ou a captura seletiva de sequências transcritas bacterianas (SCOTS) 11. A segunda abordagem baseia-se na lise diferencial de bactérias e células hospedeiras, em que as células hospedeiras são lisadas, enquanto as células bacterianas permanecem intactos. As células bacterianas são em seguida separados a partir do lisado da célula hospedeira, geralmente por centrifugação, e o ARN é extraído por intermédio de técnicas convencionais. O principal problema com esta abordagem é que, juntamente com as bactérias intactas, as células hospedeiras núcleos também são isoladas, assim, as preparações de ARN ainda contêm ARN de mamífero. Uma maneira de ultrapassar este problema é separar as bactérias intactas a partir de células hospedeiras núcleos utilizando centrifugação diferencial, embora este processo normalmente leva tempo aumentar a preocupação de alterações no perfil de expressão de genes durante a extracção. Neste artigo apresentamos uma ba melhorada e rápidacteria protocolo de extracção de ARN, que é baseado no método de lise celular diferencial. Em primeiro lugar, L. monocytogenes infectados macrófagos são lisados ​​com água fria. Em seguida, os núcleos de macrófagos são removidos por uma breve centrifugação e bactérias intactas são rapidamente recolhidas em filtros, a partir do qual o ARN é isolado usando extracção com fenol quente SDS-de ácidos nucleicos bacterianos.

<table><tbody><tr><td>Listeria monocytogenes 10403S</td><td></td><td></td><td>&lt;sup&gt;20&lt;/sup&gt;</td></tr><tr><td>Bone marrow derived macrophages prepared from C57B/6 female mice</td><td></td><td></td><td>&lt;sup&gt;21&lt;/sup&gt;</td></tr><tr><td>H&lt;sub&gt;2&lt;/sub&gt;O, RNAse free</td><td>Thermo Scientific</td><td>10977-015</td><td>DEPC-treated water can be used</td></tr><tr><td>DMEM</td><td>Gibco</td><td>41965039</td><td></td></tr><tr><td>Glutamine</td><td>Gibco</td><td>25030081</td><td></td></tr><tr><td>Sodium pyruvate</td><td>Gibco</td><td>11360-088</td><td></td></tr><tr><td>&amp;#946;-Mercaptoethanol</td><td>Gibco</td><td>31350010</td><td></td></tr><tr><td>Pen/Strep</td><td>Gibco</td><td>15140-122</td><td></td></tr><tr><td>Gentamicin</td><td>Sigma-Aldrich</td><td>G1397</td><td></td></tr><tr><td>FBS</td><td>Gibco</td><td>10270106</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Phosphate Buffered Saline-PBS</td><td>Sigma-Aldrich</td><td>D8537</td><td></td></tr><tr><td>Brain heart infusion (BHI)</td><td>Merckmillipore</td><td>1104930500</td><td></td></tr><tr><td>Phenol saturated pH 4.3</td><td>Fisher</td><td>BP1751I-400</td><td></td></tr><tr><td>Chloroform</td><td>Fisher</td><td>BP1145-1</td><td></td></tr><tr><td>Iso-amyl alcohol</td><td>Sigma-Aldrich</td><td>W205702</td><td></td></tr><tr><td>Sodium acetate</td><td>Sigma-Aldrich</td><td>W302406</td><td></td></tr><tr><td>EDTA</td><td>Sigma-Aldrich</td><td>EDS</td><td></td></tr><tr><td>DNaseI</td><td>Fermentas</td><td>EN0521</td><td></td></tr><tr><td>SDS 10%</td><td>Sigma-Aldrich</td><td>L4522</td><td></td></tr><tr><td>Ethanol absolute</td><td>Merck Millipore</td><td>1070174000</td><td></td></tr><tr><td>37 &amp;deg;C, 5% CO&lt;sub&gt;2&lt;/sub&gt; forced-air incubator</td><td>Thermo Scientific</td><td>Model 3111</td><td></td></tr><tr><td>Cell scrapers</td><td>Nunc</td><td>179693</td><td></td></tr><tr><td>Kontes glass holder for 45 mm filters</td><td>Fisher</td><td>K953755-0045</td><td></td></tr><tr><td>MF-Millipore filters 45&amp;nbsp;mm, 0.45&amp;nbsp;&amp;micro;m</td><td>Merck Millipore</td><td>HAWP04700</td><td></td></tr><tr><td>SpeedVac system</td><td>Thermo Scientific</td><td>SPD131DDA</td><td></td></tr><tr><td>Vortex-Genie 2</td><td>Scientific Industries</td><td>Model G560E</td><td></td></tr><tr><td>NanoDrop</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>145 mm cell culture dishes</td><td>Greiner</td><td>639 160</td><td></td></tr><tr><td>1.7 ml tubes, RNase-free</td><td>Axygen</td><td>MCT-175-C</td><td></td></tr><tr><td>30 &amp;deg;C incubator</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>65 &amp;deg;C heat block</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>4 &amp;deg;C table centrifuge</td><td>Eppendorf</td><td>5417R</td><td></td></tr><tr><td>Sterile pipettes, 25 ml</td><td>Greiner</td><td></td><td></td></tr><tr><td>Falcon tubes, 50 ml</td><td>Greiner</td><td></td><td></td></tr><tr><td>Liquid nitrogen</td><td></td><td></td><td></td></tr></tbody></table>

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.

O sistema de modelo é mostrado na Figura 1 e inclui células de macrófagos infectados com L. monocytogenes bactérias, que se replicam no citoplasma de macrófagos. A Figura 2 representa o esquema experimental. A Figura 3 representa os resultados típicos de tal análise de RT-qPCR de genes de virulência durante WT L. monocytogenes crescimento em macrófagos em comparação com o crescimento em meio laboratorial rico...

Watch this Scientific Journal Video about RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells at JoVE.com

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.

A purificação a partir de ARN intracelularmente Grown<em&gt; Listeria monocytogenes</em&gt; Em macrófagos

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

rna-purification-from-intracellularly-grown-listeria-monocytogenes

Research

JoVE Journal

Immunology and Infection

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

9.1K visualizações.  Tel Aviv University. 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.

Vídeo: RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

A Simple and Efficient Method to Isolate Macrophages from Mixed Primary Cultures of Adult Liver Cells

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although the isolation methods of liver macrophages have been well-described4-6, most of these methods require sophisticated equipment, such as a centrifugal elutriator and technical skills. Here, we provide a novel method to obtain liver macrophages in sufficient number and purity from mixed primary cultures of adult rat liver cells, as schematically illustrated in Figure 1.
After dissociation of the liver cells by two-step perfusion method7,8,a fraction mostly composed of parenchymal hepatocytes is prepared and seeded into T75 tissue culture flasks with culture medium composed of DMEM and 10% FCS.Parenchymal hepatocytes lose the epithelial cell morphology within a few days in culture, degenerate or transform into fibroblast-like cells (Figure 2). As the culture proceeds, around day 6, phase contrast-bright, round macrophage-like cells start to proliferate on the fibroblastic cell sheet (Figure 2). The growth of the macrophage-like cells continue and reach to maximum levels around day 12, covering the cell sheet on the flask surface. By shaking of the culture flasks, macrophages are readily suspended into the culture medium. Subsequent transfer and short incubation in plastic dishes result in selective adhesion of macrophages(Figure 3), where as other contaminating cells remain suspended. After several rinses with PBS, attached macrophages are harvested. More than 106 cells can be harvested repeatedly from the same T75 tissue culture flask at two to three day intervals for more than two weeks(Figure 3).The purities of the isolated macrophages were 95 to 99%, as evaluated by flow cytometry or immunocytochemistry with rat macrophage-specific antibodies (Figure 4).The isolated cells show active phagocytosis of polystylene beads (Figure 5), proliferative response to recombinant GM-CSF, secretion of inflammatory/anti-inflammatory cytokines upon stimulation with LPS, and formation of multinucleated giant cells9.
In conclusion, we provide a simple and efficient method to obtain liver macrophages in sufficient number and purity without complex equipment and skills.This method might be applicable to other mammalian species.

A novel method to obtain macrophages from primary culture of rat liver cells is described. This method utilizes the proliferation of macrophages in the culture, followed by shaking of culture flasks and purification by selective attachment to plastic dishes. This technique efficiently provides liver macrophages without complex equipment and skills.

Um método simples e eficiente para isolar Macrófagos de Mixed culturas primárias de células do fígado Adulto

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although ...

Imunologia e Infecção

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.

Medir a carga bacteriana e resposta imune em camundongos infectados com Listeria monocytogenes</em

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

Purification of Pathogen Vacuoles from Legionella-infected Phagocytes

The opportunistic pathogen Legionella pneumophila is an amoeba-resistant bacterium, which also replicates in alveolar macrophages thus causing the severe pneumonia "Legionnaires' disease"1. In protozoan and mammalian phagocytes, L. pneumophila employs a conserved mechanism to form a specific, replication-permissive compartment, the "Legionella-containing vacuole" (LCV). LCV formation requires the bacterial Icm/Dot type IV secretion system (T4SS), which translocates as many as 275 "effector" proteins into host cells. The effectors manipulate host proteins as well as lipids and communicate with secretory, endosomal and mitochondrial organelles2-4.
The formation of LCVs represents a complex, robust and redundant process, which is difficult to grasp in a reductionist manner. An integrative approach is required to comprehensively understand LCV formation, including a global analysis of pathogen-host factor interactions and their temporal and spatial dynamics. As a first step towards this goal, intact LCVs are purified and analyzed by proteomics and lipidomics.
The composition and formation of pathogen-containing vacuoles has been investigated by proteomic analysis using liquid chromatography or 2-D gel electrophoresis coupled to mass-spectrometry. Vacuoles isolated from either the social soil amoeba Dictyostelium discoideum or mammalian phagocytes harboured Leishmania5, Listeria6, Mycobacterium7, Rhodococcus8, Salmonella9 or Legionella spp.10. However, the purification protocols employed in these studies are time-consuming and tedious, as they require e.g. electron microscopy to analyse LCV morphology, integrity and purity. Additionally, these protocols do not exploit specific features of the pathogen vacuole for enrichment.
The method presented here overcomes these limitations by employing D. discoideum producing a fluorescent LCV marker and by targeting the bacterial effector protein SidC, which selectively anchors to the LCV membrane by binding to phosphatidylinositol 4-phosphate (PtdIns(4)P)3,11 . LCVs are enriched in a first step by immuno-magnetic separation using an affinity-purified primary antibody against SidC and a secondary antibody coupled to magnetic beads, followed in a second step by a classical Histodenz density gradient centrifugation12,13 (Fig. 1).
A proteome study of isolated LCVs from D. discoideum revealed more than 560 host cell proteins, including proteins associated with phagocytic vesicles, mitochondria, ER and Golgi, as well as several GTPases, which have not been implicated in LCV formation before13. LCVs enriched and purified with the protocol outlined here can be further analyzed by microscopy (immunofluorescence, electron microscopy), biochemical methods (Western blot) and proteomic or lipidomic approaches.

Purification of Pathogen Vacuoles from Legionella-infected Phagocytes

This article describes a method for the isolation and purification of intact Legionella-containing vacuoles (LCVs) from amoeba and macrophages. The two-step protocol comprises LCV enrichment by immuno-magnetic separation using an antibody against a bacterial LCV marker and further purification by density gradient centrifugation.

Purificação de vacúolos patógeno de<em&gt; Legionella</em&gt; Infectados Fagócitos

The opportunistic pathogen Legionella pneumophila is an amoeba-resistant bacterium, which also replicates in alveolar macrophages thus causing the severe ...

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.

Transmissão Oral de<em&gt; Listeria monocytogenes</em&gt; Em camundongos através de ingestão 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.

Imagiologia inLC Secreção para investigar infecção celular pelo 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 ...

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

Macrophages are key phagocytic innate immune cells. When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the pathogen, produce various cytokines and chemokines to recruit and stimulate other immune cells, and present antigens to stimulate the adaptive immune response. Thus, being able to efficiently manipulate macrophages with techniques such as RNA-interference (RNAi) is critical to our ability to investigate this important innate immune cell. However, macrophages can be technically challenging to transfect and can exhibit inefficient RNAi-induced gene knockdown. In this protocol, we describe methods to efficiently transfect two mouse macrophage cell lines (RAW264.7 and J774A.1) with siRNA using the Amaxa Nucleofector 96-well Shuttle System and describe procedures to maximize the effect of siRNA on gene knockdown. Moreover, the described methods are adapted to work in 96-well format, allowing for medium and high-throughput studies. To demonstrate the utility of this approach, we describe experiments that utilize RNAi to inhibit genes that regulate lipopolysaccharide (LPS)-induced cytokine production.

In this protocol, we describe methods to efficiently transfect murine macrophage cell lines with siRNAs using the Amaxa Nucleofector 96-well Shuttle System, stimulate the macrophages with lipopolysaccharide, and monitor the effects on inflammatory cytokine production.

Usando RNA-interferência para investigar a imunitário inatas de resposta em macrófagos

Macrophages are key phagocytic innate immune cells.  When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the ...

Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages

Bacteria are able to secrete a variety of molecules via various secretory systems. Besides the secretion of molecules into the extracellular space or directly into another cell, Gram-negative bacteria can also form outer membrane vesicles (OMVs). These membrane vesicles can deliver their cargo over long distances, and the cargo is protected from degradation by proteases and nucleases. 
Legionella pneumophila (L. pneumophila) is an intracellular, Gram-negative pathogen that causes a severe form of pneumonia. In humans, it infects alveolar macrophages, where it blocks lysosomal degradation and forms a specialized replication vacuole. Moreover, L. pneumophila produces OMVs under various growth conditions. To understand the role of OMVs in the infection process of human macrophages, we set up a protocol to purify bacterial membrane vesicles from liquid culture. The method is based on differential ultracentrifugation. The enriched OMVs were subsequently analyzed with regard to their protein and lipopolysaccharide (LPS) amount and were then used for the treatment of a human monocytic cell line or murine bone marrow-derived macrophages. The pro-inflammatory responses of those cells were analyzed by enzyme-linked immunosorbent assay. Furthermore, alterations in a subsequent infection were analyzed. To this end, the bacterial replication of L. pneumophila in macrophages was studied by colony-forming unit assays. 
Here, we describe a detailed protocol for the purification of L. pneumophila OMVs from liquid culture by ultracentrifugation and for the downstream analysis of their pro-inflammatory potential on macrophages.

Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages

Here, we describe the purification of Legionella pneumophila (L. pneumophila) outer membrane vesicles (OMVs) from liquid cultures. These purified vesicles are then used for the treatment of macrophages to analyze their pro-inflammatory potential.

Legionella pneumophila Outer Membrane vesículas: Isolamento e Análise do seu potencial pró-inflamatórios em macrófagos

Bacteria are able to secrete a variety of molecules via various secretory systems. Besides the secretion of molecules into the extracellular space or ...

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 um patógeno bacteriano para sonda para celular e organísmica-nível Host respostas

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

Label-Free Quantitative Proteomics Workflow for Discovery-Driven Host-Pathogen Interactions

The technological achievements of mass spectrometry (MS)-based quantitative proteomics opens many undiscovered avenues for analyzing an organism&#8217;s global proteome under varying conditions. This powerful strategy applied to the interactions of microbial pathogens with the desired host comprehensively characterizes both perspectives towards infection. Herein, the workflow describes label-free quantification (LFQ) of the infectome of Cryptococcus neoformans, a fungal facultative intracellular pathogen that is the causative agent of the deadly disease cryptococcosis, in the presence of immortalized macrophage cells. The protocol details the proper protein preparation techniques for both pathogen and mammalian cells within a single experiment, resulting in appropriate peptide submission for liquid-chromatography (LC)-MS/MS analysis. The high throughput generic nature of LFQ allows a wide dynamic range of protein identification and quantification, as well as transferability to any host-pathogen infection setting, maintaining extreme sensitivity. The method is optimized to catalogue extensive, unbiased protein abundance profiles of a pathogen within infection-mimicking conditions. Specifically, the method demonstrated here provides essential information on C. neoformans pathogenesis, such as protein production necessary for virulence and identifies critical host proteins responding to microbial invasion.

Here, we present a protocol to profile the interplay between host and pathogen during infection by mass spectrometry-based proteomics. This protocol uses label-free quantification to measure changes in protein abundance of both host (e.g., macrophages) and pathogen (e.g., Cryptococcus neoformans) in a single experiment.

Fluxo de trabalho de proteômica quantitativa sem rótulo para interações de patógenos de host-pathogen orientadas para a descoberta

The technological achievements of mass spectrometry (MS)-based quantitative proteomics opens many undiscovered avenues for analyzing an organism&#8217;s ...

Flow Cytometry Analysis of Intracellular and Emergent Extracellular Listeria monocytogenes In Vitro

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> J. Vis. Exp. (2019)
In this video, we quantify intracellular and extracellular Listeria monocytogenes, an intracellular pathogenic bacteria, following their infection of cultured macrophages in vitro using flow cytometry.

Análise de citometria de fluxo de Listeria monocytogenes intracelular e extracelular emergente in vitro

Source: Gayle, P. et al., Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses. J. Vis. Exp. (2019)
In this video, we ...

A purificação a partir de ARN intracelularmente Grown

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Capítulos neste vídeo

Um método simples e eficiente para isolar Macrófagos de Mixed culturas primárias de células do fígado Adulto

Medir a carga bacteriana e resposta imune em camundongos infectados com Listeria monocytogenes

Purificação de vacúolos patógeno de

Transmissão Oral de

Imagiologia inLC Secreção para investigar infecção celular pelo patógeno bacteriano

Usando RNA-interferência para investigar a imunitário inatas de resposta em macrófagos

Legionella pneumophila Outer Membrane vesículas: Isolamento e Análise do seu potencial pró-inflamatórios em macrófagos

Usando um patógeno bacteriano para sonda para celular e organísmica-nível Host respostas

Fluxo de trabalho de proteômica quantitativa sem rótulo para interações de patógenos de host-pathogen orientadas para a descoberta

Análise de citometria de fluxo de Listeria monocytogenes intracelular e extracelular emergente in vitro