Pesquisa em nosso laboratório foi suportado pelo Max von Pettenkofer Institute, da Universidade Ludwig-Maximilians de Munique, a Fundação Alemã de Pesquisa (DFG, HI 1511/3-1) eo Bundesministerium für Bildung und Forschung (BMBF) &quot;Genomics Infecção médicos&quot; iniciativa ( 0315834C).

1. Preparativos para isolamento LCV <ol><li> Streak a Legionella pneumophila produzir DsRed-Express 13,14 a partir de ações de glicerol em placas de ágar CYE com 5 mg / ml de cloranfenicol (Cam) quatro dias antes do isolamento LCV. Incubar as bactérias a 37 ° C. </li><li> Semente out 1 x 10 7 D. discoideum produzindo GFP-calnexin ou RAW264.7 macrófagos murinos em cultura de tecidos 75 cm 2 balões uma infecção dias antes. Use 10 ml HL5 médio com 20 mcg / mL para D. G418 discoideum e incubar a amebas a 23 ° C. Para RAW264.7 macrófagos usar 10 ml de meio RPMI 1640 suplementado com FCS 10% (inactivado pelo calor) e 1% de glutamina, e cultivar as células a 37 ° C e 5% de CO 2. Use pelo menos três frascos de 75 cm 2 por infecção e amostra (mínimo 6 x 10 7 células). </li><li> Inocule uma cultura durante a noite com L. pneumophila a partir da placa CYE. Pegue um tubo de ensaio 15 mL com 3 mL EJA médioe 5 ug / mL Cam. Inocular com 100 uL de uma suspensão bacteriana para se obter um 600nm OD de 0,1. Incubar a cultura durante a noite sobre uma roda de rotação sobrecarga a 37 ° C durante 21-22 hr. </li></ol> 2. LCV isolamento <ol><li> Alterar o meio de D. células discoideum para remover o antibiótico, que iriam interferir com a infecção a seguir. </li><li> Medir a OD da cultura durante a noite. As bactérias devem ter atingido a sua infectividade do pico a uma OD de 600 nm ≥ 3, o que corresponde a 2 x 10 9 bactérias / ml. </li><li> Infectar as células por adição de aproximadamente 500 ul da L. pneumophila cultura durante a noite para as células que crescem em 10 ml HL5 médio (D. discoideum) ou 10 ml de RPMI 1640 (macrófagos). Isto corresponde a uma multiplicidade de infecção (MOI) de 50. Subsequentemente, a infecção é sincronizado por centrifugação das bactérias sobre as células a 500 × g durante 10 min. A centrifugação é seguidopor uma incubação do D. células discoideum a 25 ° C e os macrófagos RAW264.7 a 37 ° C e 5% de CO 2, respectivamente. O tempo de infecção de 1 hr inclui o passo de centrifugação. </li><li> Após a infecção remover o meio e lavar as células uma vez para remover bactérias extracelulares. Use tampão gelado Sorc para D. discoideum e PBS gelado para RAW264.7 macrófagos. Adicionar 3 ml de tampão SH, suplementado com inibidores da protease (Roche) a cada frasco e recolher as células, usando um raspador de células. Reunir as amostras correspondentes em um tubo de ensaio de 15 mL. </li><li> Para a homogeneização da amostra de uso 3 mL de plástico Luer-Lock seringas e da bola de aço inoxidável homogeneizador. Certifique-se de que você trabalha em gelo. Antes de começar, lave o homogeneizador bola com água destilada, para evitar qualquer contaminação detergente e lave-o com gelo tampão HS frio para se livrar de bolhas de ar. Use a bola 8 apuramento mM. </li><li> Encha a 3 mL primeiro no und seringa montá-lo no diae homogeneizador. Pressione a amostra e para trás nove vezes através do homogeneizador. Trocar as seringas em seguida para evitar a contaminação com material não-homogeneizado. Recolha e reunir a amostra homogeneizada num tubo de ensaio de 15 mL e levar uma amostra 150 ul para análise microscópica. Antes de prosseguir com uma amostra diferente desmontar e lavar o homogeneizador de bola. </li><li> Bloquear o homogeneizado com CS 2% ou FCS durante 30 min num agitador em gelo ou em uma roda de sobrecarga de fiação (10-20 rpm) a 4 ° C. </li><li> Após o bloqueio utilizar um anticorpo de afinidade purificada primária contra SIDC (diluição 1:3000) ou contra qualquer outro marcador bacteriana exclusivamente de ligação à membrana LCV. Solução vórtice do anticorpo antes de o adicionar ao homogeneizado. Incubar a amostra durante 1 hora num agitador em gelo ou em uma roda de sobrecarga de fiação (10-20 rpm) a 4 ° C. </li><li> Enquanto isso, prepare o gradiente Histodenz. Use um tubo de ensaio 15 mL por gradiente e três frascos de 75 cm 2 de uma amostra. Abetor, adicionar 5,75 mL de Histodenz 35% para o tubo, e em segundo lugar, cuidadosamente adicionar 5,75 mL de solução a 10% Histodenz no topo. Depois cuidadosamente colocar os tubos na horizontal para baixo por 1 hora. </li><li> Após a incubação do homogeneizado com o reagente de bloqueio e do anticorpo primário, de centrifugação a 600 × g durante 15 min a 4 ° C para sedimentar a amostra. Descartar o sobrenadante e ressuspender o pellet em 1,5 mL de tampão SH. Transferir as amostras a um tubo de ensaio fresco 15 mL e levar uma amostra de 40 ul para análise microscópica mais tarde. </li><li> Incubar a pelete com o anticorpo secundário - MACS cabra anti-coelho IgG grânulos micro (diluição 1:25) - para 30 min num agitador a 4 ° C. Enquanto isso, colocar as colunas no suporte magnético MACS e equilibrar-los com 0,5 mL de tampão HS. </li><li> Subsequentemente, aplicam-se as amostras à coluna. Collect 150 uL do fluxo através de análise microscópica subsequente. Lavar a coluna três vezes com 0,5 mL de tampão SH. </li><li> Remova as colunas de tele ímã e eluato os LCVs com 0,5 mL de tampão HS de imediato squirting. Tomar uma amostra de 20 l para análise microscópica. O passo de purificação por imuno-magnético separação Espera-se que o rendimento por 1 × 10 7 infectado D. células discoideum aproximadamente 1 × 10 6 LCVs intactas, que são cerca de sete vezes enriquecidos no eluído em comparação com o 13 flow-through. </li><li> Colocar os tubos de gradiente Histodenz de volta para uma posição vertical e cuidadosamente carregar o eluato em cima. Centrifugar os tubos a 3350 × g durante 1 hora a 4 ° C. </li><li> Após centrifugação tomar 1,5 mL fracções a partir da parte inferior do tubo de ensaio, utilizando uma pipeta de Pasteur de vidro longo. No total recolher oito fracções, cada um a partir do fundo do tubo. Sem camadas visíveis são observados no gradiente Histodenz contínua. O rendimento mais elevado de LCVs é tipicamente esperado na fracção 4, à razão de 2 × 10 ~ 5 LCVs intactas por 1 × 10 7 emtadas amebas 13. Pequenas quantidades de LCVs também estão presentes nas fracções 3 e 5, respectivamente. Assim, o rendimento global de LCVs purificadas é ~ 1 × 10 6 LCVs intactos por 6 × 10 7 infectado D. células discoideum 13. A recuperação de VCL podem ser realizados à temperatura ambiente (RT), e os LCVs aparecem estável durante várias horas. </li></ol> 3. A análise microscópica das amostras coletadas <ol><li> Prepara-se uma de 24 poços de fundo plano placa de cultura de tecido com poli-L-lisina lamelas revestidas. </li><li> Pipetar cada amostra colhida durante a purificação LCV mais 150 uL de cada fracção de gradiente de densidade para a placa de 24 cavidades. Adicionar 0,5 mL de tampão de HS a cada poço para diluir a concentração Histodenz elevada. Depois, centrifugar a placa a 600 × g durante 10 min a 4 ° C. Cuidadosamente remover o sobrenadante e fixar as amostras com paraformaldeído a 4% (PFA) durante 20 min à TA. Após a fixação lavar as amostras duas vezes. Nose Sorc para D. discoideum e PBS para RAW264.7 macrófagos. </li><li><ol class="lalpha"><li> Montar as amostras de GFP-calnexin que expressam D. discoideum, tomadas em diferentes etapas durante LCV isolamento, diretamente no vidro de slides usando Vectashield por exemplo. </li><li> Incubar as amostras de RAW264.7 macrófagos, tomadas em diferentes etapas durante o isolamento VCL, com solução de bloqueio (1% BSA em PBS) durante 10 min à TA. Use um anticorpo anti-SIDC primário para corar as LCVs e incubar durante 1 hora numa câmara húmida à RT (purificado por afinidade de coelho anti-SIDC; 1:1000 em tampão de bloqueio). Subsequentemente, lavar as amostras três vezes com PBS. Incubar com um anticorpo secundário (por exemplo anti-coelho Cy5; 1:200 em solução de bloqueio) durante 30 min numa câmara húmida e escuro à temperatura ambiente. Por fim, lave as amostras três vezes com PBS e montá-los em vidro de slides usando Vectashield por exemplo. </li></ol></li><li> Integridade, Morfologia e quantidade dos LCVs isoladas de GFP-calnexin expressar D. discoideum ou a partir de RAW264.7 macrófagos são analisados ​​por um microscópio de fluorescência equipado com os filtros adequados. </li></ol> 4. Os resultados representativos A qualidade e rendimento da purificação LCV por imuno-afinidade de separação e centrifugação em gradiente de densidade pode ser seguido por microscopia de fluorescência (Fig. 1). Uma visão geral das amostras coletadas durante o isolamento LCV de D. discoideu m ou macrófagos é mostrado (Fig. 2). O homogenato de L. pneumophila infectadas fagócitos mostra LCVs intactos, mas também uma grande quantidade de detritos de células e as bactérias extracelulares. Depois de granulação a amostra, a imagem é semelhante ao homogenato, por vezes, uma mais densa de bits. Uma vez que LCVs intactas deve manter a coluna, o fluxo através contém principalmente bactérias extracelulares e restos celulares. Após eluição da amostra a partir da coluna, o eluato contém LCVs intactas em grandes quantidades. Em nossas mãos, o purificati LCV<html> em parece ser mais eficaz para a D. discoideum do que para macrófagos. Em D. discoideum dos vacúolos de agentes patogénicos aparecem redondo eo rendimento de LCVs isoladas é de mais de 10 vezes maior em comparação com os macrófagos (Fig. 3). Em macrófagos intactos SCBV corados com diferentes anticorpos também aparecem mais de forma irregular. <img alt="A Figura 1" src="/files/ftp_upload/4118/4118fig1.jpg" /> Figura 1. Visão esquemática do isolamento LCV. A) Isolamento de LCVs por imuno-magnético de separação utilizando um anticorpo de afinidade purificada contra a proteína bacteriana SIDC efectora, localizando exclusivamente para a membrana LCV. A secundários MACS micro anticorpo grânulo acoplado e um íman são usadas para isolar LCVs a partir de fragmentos de células. B) Após LCVs imuno-magnéticos de separação são ainda purificado por Histodenz centrifugação em gradiente de densidade. LCVs são enriquecidos na fração de 4. upload/4118/4118fig2.jpg &quot;/&gt; Amostras Figura 2. Coletados durante a purificação LCV. Imagens de homogeneizado, pellet, flow-through e eluato de D. discoideum ou macrófagos são representados. As amostras mostram L. pneumophila produzir DsRed-Express (vermelho) em D. discoideum produzindo GFP-calnexin sinal (painel, verde superior) ou em macrófagos corados com um anticorpo anti-SIDC (ciano, painel inferior). Bares, 10 mM. <img alt="A Figura 3" src="/files/ftp_upload/4118/4118fig3.jpg" /> Figura 3. Purificada a partir de LCVs D. discoideum ou macrófagos. As amostras mostram fracção 4, após Histodenz centrifugação em gradiente de densidade de L. pneumophila produzindo DsRed-expresso (vermelho) em (A) D. discoideum produzindo GFP-calnexin sinal (verde) ou (B) em macrófagos corados com um anticorpo anti-SIDC (ciano). Bares, 10 mM.

<ol>
	<li>Hilbi, H., Hoffmann, C., Harrison, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Legionella+spp.+outdoors:+colonization,+communication+and+persistence.">Legionella spp. outdoors: colonization, communication and persistence.</a> Environ. Microbiol. Rep. 3, 286-296 (2011).</li><li>Zhu, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+identification+of+protein+substrates+of+the+Dot/Icm+type+IV+transporter+of+Legionella+pneumophila.">Comprehensive identification of protein substrates of the Dot/Icm type IV transporter of Legionella pneumophila.</a> PLoS ONE. 6, e17638 (2011).</li><li>Hilbi, H., Weber, S., Finsel, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anchors+for+effectors:+subversion+of+phospho-inositide+lipids+by+Legionella.">Anchors for effectors: subversion of phospho-inositide lipids by Legionella.</a> Front Microbiol. 2, 91 (2011).</li><li>Hubber, A., Roy, C. R. <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+host+cell+function+by+Legionella+pneumophila+type+IV+effectors.">Modulation of host cell function by Legionella pneumophila type IV effectors.</a> Annu. Rev. Cell Dev. Biol. 26, 261-283 (2010).</li><li>Kima, P. E., Dunn, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploiting+calnexin+expression+on+phagosomes+to+isolate+Leishmania+parasitophorous+vacuoles.">Exploiting calnexin expression on phagosomes to isolate Leishmania parasitophorous vacuoles.</a> Microb. Pathog. 38, 139-145 (2005).</li><li>L&#252;hrmann, A., Haas, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+to+purify+bacteria-containing+phagosomes+from+infected+macrophages.">A method to purify bacteria-containing phagosomes from infected macrophages.</a> Methods Cell Sci. 22, 329-341 (2000).</li><li>Sturgill-Koszycki, S., Haddix, P. L., Russell, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+interaction+between+Mycobacterium+and+the+macrophage+analyzed+by+two-dimensional+polyacrylamide+gel+electrophoresis.">The interaction between Mycobacterium and the macrophage analyzed by two-dimensional polyacrylamide gel electrophoresis.</a> Electrophoresis. 18, 2558-2565 (1997).</li><li>Fernandez-Mora, E., Polidori, M., L&#252;hrmann, A., Schaible, U. E., Haas, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maturation+of+Rhodococcus+equi-containing+vacuoles+is+arrested+after+completion+of+the+early+endosome+stage.">Maturation of Rhodococcus equi-containing vacuoles is arrested after completion of the early endosome stage.</a> Traffic. 6, 635-653 (2005).</li><li>Mills, S. D., Finlay, B. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+Salmonella+typhimurium+and+Yersinia+pseudotuberculosis-containing+phagosomes+from+infected+mouse+macrophages:+Y.+pseudotuberculosis+traffics+to+terminal+lysosomes+where+they+are+degraded.">Isolation and characterization of Salmonella typhimurium and Yersinia pseudotuberculosis-containing phagosomes from infected mouse macrophages: Y. pseudotuberculosis traffics to terminal lysosomes where they are degraded.</a> Eur. J. Cell Biol. 77, 35-47 (1998).</li><li>Shevchuk, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+analysis+of+Legionella-containing+phagosomes+isolated+from+Dictyostelium.">Proteomic analysis of Legionella-containing phagosomes isolated from Dictyostelium.</a> Int. J. Med. Microbiol. 299, 489-508 (2009).</li><li>Brombacher, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rab1+guanine+nucleotide+exchange+factor+SidM+is+a+major+phosphatidylinositol+4-phosphate-binding+effector+protein+of+Legionella+pneumophila.">Rab1 guanine nucleotide exchange factor SidM is a major phosphatidylinositol 4-phosphate-binding effector protein of Legionella pneumophila.</a> J. Biol. Chem. 284, 4846-4856 (2009).</li><li>Urwyler, S., Finsel, I., Ragaz, C., Hilbi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+Legionella-containing+vacuoles+by+immuno-magnetic+separation.">Isolation of Legionella-containing vacuoles by immuno-magnetic separation.</a> Curr. Protoc. Cell Biol. Chapter 3, Unit 3 (2010).</li><li>Urwyler, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteome+analysis+of+Legionella+vacuoles+purified+by+magnetic+immunoseparation+reveals+secretory+and+endosomal+GTPases.">Proteome analysis of Legionella vacuoles purified by magnetic immunoseparation reveals secretory and endosomal GTPases.</a> Traffic. 10, 76-87 (2009).</li><li>Mampel, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Planktonic+replication+is+essential+for+biofilm+formation+by+Legionella+pneumophila+in+a+complex+medium+under+static+and+dynamic+flow+conditions.">Planktonic replication is essential for biofilm formation by Legionella pneumophila in a complex medium under static and dynamic flow conditions.</a> Appl. Environ. Microbiol. 72, 2885-2895 (2006).</li></ol>

Não há conflitos de interesse declarados.

Em contraste com os métodos publicados anteriormente, este protocolo é baseada em dois passos, primeiro a separação dos LCVs por uma abordagem imuno-magnética, e em segundo lugar, a purificação de LCVs por centrifugação em gradiente de densidade. O isolamento LCV pode ser facilmente seguido por microscopia de fluorescência, quando tanto as bactérias fluorescente etiquetado e GFP-produtoras de células ou de coloração de anticorpo é usado. O protocolo aqui descrito é um método simples, hetero-a frente, o ...

purification of pathogen vacuoles from legionella-infected phagocytes

O patógeno oportunista Legionella pneumophila é uma bactéria resistente à ameba, que também replica em macrófagos alveolares, assim causando o grave pneumonia &quot;Legionários doença &#39;&quot; 1. Em fagócitos protozoários e de mamíferos, L. pneumophila emprega um mecanismo conservado para formar um específico, compartimento de replicação permissiva, a &quot;Legionella contendo vacúolo&quot; (LCV). Formação requer a LCV bacteriana Icm / Dot tipo de sistema de secreção IV (T4SS), que transloca tantos como 275 &quot;&quot; efectoras proteínas em células hospedeiras. Os efetores manipular proteínas do hospedeiro, bem como lipídios e se comunicar com secreção, organelas endossômicas e mitocondrial 2-4. A formação de LCVs representa um processo complexo, robusto e redundante, que é difícil de agarrar de uma maneira redutora. Uma abordagem integrativa é necessária para compreender a formação abrangente LCV, incluindo uma análise global do caminhoOgen-host as interações e sua dinâmica temporal e espacial. Como um primeiro passo para este objetivo, LCVs intactos são purificados e analisados ​​por proteômica e lipidomics. A composição e formação de vacúolos contendo agente patogénico foi investigado por análise proteômica utilizando cromatografia líquida ou 2-D de electroforese em gel acoplado a espectrometria de massa. Vacúolos isolados, quer social discoideum a ameba Dictyostelium solo ou fagócitos mamíferos abrigou Leishmania 5, 6 Listeria, Mycobacterium 7, Rhodococcus 8, 9 ou Salmonella Legionella spp. 10. No entanto, os protocolos de purificação utilizados nestes estudos são demorados e tedioso, em que requerem microscopia electrónica por exemplo, para analisar LCV morfologia, integridade e pureza. Além disso, estes protocolos não exploram características específicas do vacúolo patógeno para enrichment. O método aqui apresentado supera essas limitações, empregando D. discoideum produzir um marcador fluorescente e LCV por alvo a proteína bacteriana SIDC efectora, que selectivamente âncoras para a membrana LCV ligando-se à fosfatidilinositol 4-fosfato (PtdIns (4) P) 3,11. LCVs são enriquecidos num primeiro passo por imuno-magnético de separação utilizando um anticorpo de afinidade purificada primária contra SIDC e um anticorpo secundário acoplado a esferas magnéticas, seguido de uma segunda etapa por uma centrifugação de gradiente de densidade clássica Histodenz 12,13 (Fig. 1) . Um estudo do proteoma de VCL isolados de D. discoideum revelou mais de 560 proteínas da célula hospedeira, incluindo proteínas associadas com vesículas fagocíticas, mitocôndrias, ER e de Golgi, bem como vários GTPases, que não têm sido implicados na formação LCV antes de 13. LCVs enriquecido e purificado com oprotocolo descrito aqui pode ser ainda analisada por microscopia (imunofluorescência, microscopia electrónica), métodos bioquímicos (Western blot) e abordagens proteómicos ou lipidomic.

Watch this Scientific Journal Video about Purification of Pathogen Vacuoles from Legionella-infected Phagocytes at JoVE.com

Purification of Pathogen Vacuoles from Legionella-infected Phagocytes

Este artigo descreve um método para o isolamento e purificação de intacta<em&gt; Legionella</em&gt; Contendo vacúolos (VCL) da ameba e macrófagos. O protocolo de dois passos compreende enriquecimento LCV por imuno-magnético de separação utilizando um anticorpo contra um marcador LCV bacteriana e mais purificação por centrifugação em gradiente de densidade.

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 ...

purification-of-pathogen-vacuoles-from-legionella-infected-phagocytes

Research

JoVE Journal

Immunology and Infection

Purification of Pathogen Vacuoles from Legionella-infected Phagocytes

12.4K visualizações.  Ludwig-Maximilians-Universität. Este artigo descreve um método para o isolamento e purificação de intacta Legionella Contendo vacúolos (VCL) da ameba e macrófagos. O protocolo de dois passos compreende enriquecimento LCV por imuno-magnético de separação utilizando um anticorpo contra um marcador LCV bacteriana e mais purificação por centrifugação em gradiente de densidade.

Vídeo: Purification of Pathogen Vacuoles from Legionella-infected Phagocytes

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.

Invasão das células humanas por um agente patogénico

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

Imunologia e Infecção

One-day Workflow Scheme for Bacterial Pathogen Detection and Antimicrobial Resistance Testing from Blood Cultures

Bloodstream infections are associated with high mortality rates because of the probable manifestation of sepsis, severe sepsis and septic shock1. Therefore, rapid administration of adequate antibiotic therapy is of foremost importance in the treatment of bloodstream infections. The critical element in this process is timing, heavily dependent on the results of bacterial identification and antibiotic susceptibility testing. Both of these parameters are routinely obtained by culture-based testing, which is time-consuming and takes on average 24-48 hours2, 4. The aim of the study was to develop DNA-based assays for rapid identification of bloodstream infections, as well as rapid antimicrobial susceptibility testing. The first assay is a eubacterial 16S rDNA-based real-time PCR assay complemented with species- or genus-specific probes5. Using these probes, Gram-negative bacteria including Pseudomonas spp., Pseudomonas aeruginosa and Escherichia coli as well as Gram-positive bacteria including Staphylococcus spp., Staphylococcus aureus, Enterococcus spp., Streptococcus spp., and Streptococcus pneumoniae could be distinguished. Using this multiprobe assay, a first identification of the causative micro-organism was given after 2 h.
Secondly, we developed a semi-molecular assay for antibiotic susceptibility testing of S. aureus, Enterococcus spp. and (facultative) aerobe Gram-negative rods6. This assay was based on a study in which PCR was used to measure the growth of bacteria7. Bacteria harvested directly from blood cultures are incubated for 6 h with a selection of antibiotics, and following a Sybr Green-based real-time PCR assay determines inhibition of growth. The combination of these two methods could direct the choice of a suitable antibiotic therapy on the same day (Figure 1). In conclusion, molecular analysis of both identification and antibiotic susceptibility offers a faster alternative for pathogen detection and could improve the diagnosis of bloodstream infections.

The design of a straightforward one-day workflow scheme for bacterial pathogen diagnostics enables the rapid recognition of bloodstream infections. The inclusion of eight clinically relevant bacterial targets and their antibiotic resistance profiles offers the clinician an initial insight on the same day, which can lead to more adequate therapy.

Um dia-Esquema de fluxo de trabalho para detecção de patógenos de bactérias e de resistência antimicrobiana de hemoculturas

Bloodstream infections are associated with high mortality rates because of the probable manifestation of sepsis, severe sepsis and septic shock1. ...

Purification and Visualization of Lipopolysaccharide from Gram-negative Bacteria by Hot Aqueous-phenol Extraction

Lipopolysaccharide (LPS) is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of lipid A, which is embedded in the outer membrane, a core oligosaccharide and repeating O-antigen units that extend outward from the surface of the cell1, 2. LPS is an immunodominant molecule that is important for the virulence and pathogenesis of many bacterial species, including Pseudomonas aeruginosa, Salmonella species, and Escherichia coli3-5, and differences in LPS O-antigen composition form the basis for serotyping of strains. LPS is involved in attachment to host cells at the initiation of infection and provides protection from complement-mediated killing; strains that lack LPS can be attenuated for virulence6-8. For these reasons, it is important to visualize LPS, particularly from clinical isolates. Visualizing LPS banding patterns and recognition by specific antibodies can be useful tools to identify strain lineages and to characterize various mutants. 
In this report, we describe a hot aqueous-phenol method for the isolation and purification of LPS from Gram-negative bacterial cells. This protocol allows for the extraction of LPS away from nucleic acids and proteins that can interfere with visualization of LPS that occurs with shorter, less intensive extraction methods9. LPS prepared this way can be separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and directly stained using carbohydrate/glycoprotein stains or standard silver staining methods. Many anti-sera to LPS contain antibodies that cross-react with outer membrane proteins or other antigenic targets that can hinder reactivity observed following Western immunoblot of SDS-PAGE-separated crude cell lysates. Protease treatment of crude cell lysates alone is not always an effective way of removing this background using this or other visualization methods. Further, extensive protease treatment in an attempt to remove this background can lead to poor quality LPS that is not well resolved by any of the aforementioned methods. For these reasons, we believe that the following protocol, adapted from Westpahl and Jann10, is ideal for LPS extraction.

We describe a modified hot aqueous-phenol extraction method for purifying lipopolysaccharide (LPS) from Gram-negative bacteria. Once extracted, the LPS can be subsequently analyzed by SDS-PAGE and visualized by direct staining or Western immunoblot.

Purificação e visualização de lipopolissacarídeo de bactérias Gram-negativas por extracção aquosa de fenol-Hot

Lipopolysaccharide (LPS)  is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of  lipid A, which is ...

Affinity Purification of Influenza Virus Ribonucleoprotein Complexes from the Chromatin of Infected Cells

Like all negative-strand RNA viruses, the genome of influenza viruses is packaged in the form of viral ribonucleoprotein complexes (vRNP), in which the single-stranded genome is encapsidated by the nucleoprotein (NP), and associated with the trimeric polymerase complex consisting of the PA, PB1, and PB2 subunits. However, in contrast to most RNA viruses, influenza viruses perform viral RNA synthesis in the nuclei of infected cells. Interestingly, viral mRNA synthesis uses cellular pre-mRNAs as primers, and it has been proposed that this process takes place on chromatin1. Interactions between the viral polymerase and the host RNA polymerase II, as well as between NP and host nucleosomes have also been characterized1,2.
Recently, the generation of recombinant influenza viruses encoding a One-Strep-Tag genetically fused to the C-terminus of the PB2 subunit of the viral polymerase (rWSN-PB2-Strep3) has been described. These recombinant viruses allow the purification of PB2-containing complexes, including vRNPs, from infected cells. To obtain purified vRNPs, cell cultures are infected, and vRNPs are affinity purified from lysates derived from these cells. However, the lysis procedures used to date have been based on one-step detergent lysis, which, despite the presence of a general nuclease, often extract chromatin-bound material only inefficiently. 
Our preliminary work suggested that a large portion of nuclear vRNPs were not extracted during traditional cell lysis, and therefore could not be affinity purified. To increase this extraction efficiency, and to separate chromatin-bound from non-chromatin-bound nuclear vRNPs, we adapted a step-wise subcellular extraction protocol to influenza virus-infected cells. Briefly, this procedure first separates the nuclei from the cell and then extracts soluble nuclear proteins (here termed the "nucleoplasmic" fraction). The remaining insoluble nuclear material is then digested with Benzonase, an unspecific DNA/RNA nuclease, followed by two salt extraction steps: first using 150 mM NaCl (termed "ch150"), then 500 mM NaCl ("ch500") (Fig. 1). These salt extraction steps were chosen based on our observation that 500 mM NaCl was sufficient to solubilize over 85% of nuclear vRNPs yet still allow binding of tagged vRNPs to the affinity matrix.
After subcellular fractionation of infected cells, it is possible to affinity purify PB2-tagged vRNPs from each individual fraction and analyze their protein and RNA components using Western Blot and primer extension, respectively. Recently, we utilized this method to discover that vRNP export complexes form during late points after infection on the chromatin fraction extracted with 500 mM NaCl (ch500)3.

Influenza viruses replicate their RNA genome in association with host-cell chromatin. Here, we present a method to purify intact viral ribonucleoprotein complexes from the chromatin of infected cells. Purified viral complexes can be analyzed by both Western blot and primer extension of protein and RNA content, respectively.

Purificação por afinidade de Complexos de Influenza Virus ribonucleoproteína da cromatina de células infectadas

Like all negative-strand RNA viruses, the genome of influenza viruses is packaged in the form of viral ribonucleoprotein complexes (vRNP), in which the ...

Live-cell Video Microscopy of Fungal Pathogen Phagocytosis

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of phagocytes to the site where pathogens are located; (ii) recognition of pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRRs); (iii) engulfment of microorganisms bound to the phagocyte cell membrane, and (iv) processing of engulfed cells within maturing phagosomes and digestion of the ingested particle. Studies that assess phagocytosis in its entirety are informative1, 2, 3, 4, 5 but are limited in that they do not normally break the process down into migration, engulfment and phagosome maturation, which may be affected differentially. Furthermore, such studies assess uptake as a single event, rather than as a continuous dynamic process. We have recently developed advanced live-cell imaging technologies, and have combined these with genetic functional analysis of both pathogen and host cells to create a cross-disciplinary platform for the analysis of innate immune cell function and fungal pathogenesis. These studies have revealed novel aspects of phagocytosis that could only be observed using systematic temporal analysis of the molecular and cellular interactions between human phagocytes and fungal pathogens and infectious microorganisms more generally. For example, we have begun to define the following: (a) the components of the cell surface required for each stage of the process of recognition, engulfment and killing of fungal cells1, 6, 7, 8; (b) how surface geometry influences the efficiency of macrophage uptake and killing of yeast and hyphal cells7; and (c) how engulfment leads to alteration of the cell cycle and behavior of macrophages 9, 10.
In contrast to single time point snapshots, live-cell video microscopy enables a wide variety of host cells and pathogens to be studied as continuous sequences over lengthy time periods, providing spatial and temporal information on a broad range of dynamic processes, including cell migration, replication and vesicular trafficking. Here we describe in detail how to prepare host and fungal cells, and to conduct the video microscopy experiments. These methods can provide a user-guide for future studies with other phagocytes and microorganisms.

We describe methods for live-cell video microscopy of Candida albicans phagocytosis by macrophages. These methods enable stage-specific analysis of macrophage migration, recognition, engulfment and phagosome maturation and reveal novel aspects of phagocytosis.

Live-célula Microscopia vídeo de Fagocitose patógeno fúngico

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of ...

Loop-mediated Isothermal Amplification (LAMP) Assays for the Species-specific Detection of Eimeria that Infect Chickens

Eimeria species parasites, protozoa which cause the enteric disease coccidiosis, pose a serious threat to the production and welfare of chickens. In the absence of effective control clinical coccidiosis can be devastating. Resistance to the chemoprophylactics frequently used to control Eimeria is common and sub-clinical infection is widespread, influencing feed conversion ratios and susceptibility to other pathogens such as Clostridium perfringens. Despite the availability of polymerase chain reaction (PCR)-based tools, diagnosis of Eimeria infection still relies almost entirely on traditional approaches such as lesion scoring and oocyst morphology, but neither is straightforward. Limitations of the existing molecular tools include the requirement for specialist equipment and difficulties accessing DNA as template. In response a simple field DNA preparation protocol and a panel of species-specific loop-mediated isothermal amplification (LAMP) assays have been developed for the seven Eimeria recognised to infect the chicken. We now provide a detailed protocol describing the preparation of genomic DNA from intestinal tissue collected post-mortem, followed by setup and readout of the LAMP assays. Eimeria species-specific LAMP can be used to monitor parasite occurrence, assessing the efficacy of a farm&#8217;s anticoccidial strategy, and to diagnose sub-clinical infection or clinical disease with particular value when expert surveillance is unavailable.

Loop-mediated Isothermal Amplification LAMP Assays for the Species-specific Detection of Eimeria that Infect Chickens

Diagnosis of Eimeria infection in chickens remains demanding. Parasite morphology- and host pathology-led approaches are commonly inconclusive, while molecular approaches based on PCR have proven demanding in cost and expertise. The aim of this protocol is to establish loop-mediated isothermal amplification (LAMP) as a straightforward molecular diagnostic for eimerian infection.

Isothermal amplificação (lâmpada), Ensaios mediada por loop para detecção específica da espécie os de<em&gt; Eimeria</em&gt; Que infectam Galinhas

Eimeria species parasites, protozoa which cause the enteric disease coccidiosis, pose a serious threat to the production and welfare of chickens. In 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.

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. ...

Development and Identification of a Novel Subpopulation of Human Neutrophil-derived Giant Phagocytes In Vitro

Neutrophils (PMN) are best known for their phagocytic functions against invading pathogens and microorganisms. They have the shortest half-life amongst leukocytes and in their non-activated state are constitutively committed to apoptosis. When recruited to inflammatory sites to resolve inflammation, they produce an array of cytotoxic molecules with potent antimicrobial killing. Yet, when these powerful cytotoxic molecules are released in an uncontrolled manner they can damage surrounding tissues. In recent years however, neutrophil versatility is increasingly evidenced, by demonstrating plasticity and immunoregulatory functions. We have recently identified a new neutrophil-derived subpopulation, which develops spontaneously in standard culture conditions without the addition of cytokines/growth factors such as granulocyte colony-stimulating factor (GM-CSF)/interleukin (IL)-4. Their phagocytic abilities of neutrophil remnants largely contribute to increase their size immensely; therefore they were termed giant phagocytes (G&#981;). Unlike neutrophils, G&#981; are long lived in culture. They express the cluster of differentiation (CD) neutrophil markers CD66b/CD63/CD15/CD11b/myeloperoxidase (MPO)/neutrophil elastase (NE), and are devoid of the monocytic lineage markers CD14/CD16/CD163 and the dendritic CD1c/CD141 markers. They also take-up latex and zymosan, and respond by oxidative burst to stimulation with opsonized-zymosan and PMA. G&#981; also express the scavenger receptors CD68/CD36, and unlike neutrophils, internalize oxidized-low density lipoprotein (oxLDL). Moreover, unlike fresh neutrophils, or cultured monocytes, they respond to oxLDL uptake by increased reactive oxygen species (ROS) production. Additionally, these phagocytes contain microtubule-associated protein-1 light chain 3B (LC3B) coated vacuoles, indicating the activation of autophagy. Using specific inhibitors it is evident that both phagocytosis and autophagy are prerequisites for their development and likely NADPH oxidase dependent ROS. We describe here a method for the preparation of this new subpopulation of long-lived, neutrophil-derived phagocytic cells in culture, their identification and their currently known characteristics. This protocol is essential for obtaining and characterizing G&#981; in order to further investigate their significance and functions.

Development and Identification of a Novel Subpopulation of Human Neutrophil-derived Giant Phagocytes In Vitro

We describe here a method for obtaining and identifying a newly characterized subpopulation of neutrophil-derived giant phagocytes. These cells develop in culture from fresh human blood neutrophils, and are characterized by phagocytosis, autophagy, immensely large size, and extended lifespan. This method is essential to further investigate this unique neutrophil-derived subpopulation.

Desenvolvimento e Identificação de uma subpopulação Novel dos fagócitos Human Giant neutrófilos derivado<em&gt; In Vitro</em

Neutrophils (PMN) are best known for their phagocytic functions against invading pathogens and microorganisms. They have the shortest half-life amongst ...

Unravelling the Function of a Bacterial Effector from a Non-cultivable Plant Pathogen Using a Yeast Two-hybrid Screen

Unravelling the molecular mechanisms of disease manifestations is important to understand pathologies and symptom development in plant science. Bacteria have evolved different strategies to manipulate their host metabolism for their own benefit. This bacterial manipulation is often coupled with severe symptom development or the death of the affected plants. Determining the specific bacterial molecules responsible for the host manipulation has become an important field in microbiological research. After the identification of these bacterial molecules, called &#34;effectors,&#34; it is important to elucidate their function. A straightforward approach to determine the function of an effector is to identify its proteinaceous binding partner in its natural host via a yeast two-hybrid (Y2H) screen. Normally the host harbors numerous potential binding partners that cannot be predicted sufficiently by any in silico algorithm. It is thus the best choice to perform a screen with the hypothetical effector against a whole library of expressed host proteins. It is especially challenging if the causative agent is uncultivable like phytoplasma. This protocol provides step-by-step instructions for DNA purification from a phytoplasma-infected woody host plant, the amplification of the potential effector, and the subsequent identification of the plant&#39;s molecular interaction partner with a Y2H screen. Even though Y2H screens are commonly used, there is a trend to outsource this technique to biotech companies that offer the Y2H service at a cost. This protocol provides instructions on how to perform a Y2H in any decently equipped molecular biology laboratory using standard lab techniques.

Bacterial effector proteins are important for establishing successful infections. This protocol describes the experimental identification of proteinaceous binding partners of a bacterial effector protein in its natural plant host. Identifying these effector interactions via yeast two-hybrid screens has become an important tool in unravelling molecular pathogenicity strategies.

Desvendando a função de uma bacteriana Effector de um patógeno não-cultiváveis ​​meio de uma levedura Screen Two-hybrid

Unravelling the molecular mechanisms of disease manifestations is important to understand pathologies and symptom development in plant science. Bacteria ...

Isolation, Characterization, and Purification of Macrophages from Tissues Affected by Obesity-related Inflammation

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. Diet-induced obesity (DIO) is a valuable model in studying the role of macrophage heterogeneity; however, adequate macrophage isolations are difficult to acquire from inflamed tissues. In this protocol, we outline the isolation steps and necessary troubleshooting guidelines derived from our studies for obtaining a suitable population of tissue-resident macrophages from mice following 18 weeks of high-fat (HFD) or high-fat/high-cholesterol (HFHCD) diet intervention. This protocol focuses on three hallmark tissues studied in obesity and atherosclerosis including the liver, white adipose tissues (WAT), and the aorta. We highlight how dualistic usage of flow cytometry can achieve a new dimension of isolation and characterization of tissue-resident macrophages. A fundamental section of this protocol addresses the intricacies underlying tissue-specific enzymatic digestions and macrophage isolation, and subsequent cell-surface antibody staining for flow cytometric analysis. This protocol addresses existing complexities underlying fluorescent-activated cell sorting (FACS) and presents clarifications to these complexities so as to obtain broad range characterization from adequately sorted cell populations. Alternate enrichment methods are included for sorting cells, such as the dense liver, allowing for flexibility and time management when working with FACS. In brief, this protocol aids the researcher to evaluate macrophage heterogeneity from a multitude of inflamed tissues in a given study and provides insightful troubleshooting tips that have been successful for favorable cellular isolation and characterization of immune cells in DIO-mediated inflammation.

This protocol allows a researcher to isolate and characterize tissue-resident macrophages in various hallmark inflamed tissues extracted from diet-induced models of metabolic disorders.