Name: purification of pathogen vacuoles from legionella-infected phagocytes
Uploaded: 2012-06-19T13:00:00
Description: Watch this Scientific Journal Video about Purification of Pathogen Vacuoles from Legionella-infected Phagocytes at JoVE.com

在我们的实验室研究是由马克斯·冯·Pettenkofer研究所，慕尼黑路德维希 - 马克西米利安大学德国研究基金会（DFG，您好1511/3-1）的Bundesministerium献给教化和Forschung（BMBF）的“医疗感染基因组学”的倡议（ 0315834C）。

1。轻型商用车分离的筹备工作<ol><li>连续出嗜肺军团菌生产甘油股票CYE平板DsRed的，快13,14轻型商用车隔离之前的5微克/毫升氯霉素（CAM）四天。细菌在37°C。 </li><li>种子出1×10 7 D。 discoideum生产GFP-calnexin或75厘米2组织培养的RAW264.7巨噬细胞瓶前一天的感染。使用10毫升20微克/毫升为四 G418的HL5中等discoideum和孵化的变形虫在23°C对于RAW264.7巨噬细胞...

<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>

在此前公布的方法相比，该协议是基于两个步骤，首先通过免疫磁性分离的LCVs，和第二，通过密度梯度离心LCVs净化。轻型商用车的隔离，可以很容易地后，荧光显微镜，要么荧光标记的细菌和细胞产生的细胞或抗体染色时使用。这里描述的协议是一个简单的，直接的方法，该方法在高纯度浓缩铀的LCVs结果。重要的是，在同质化缓冲区的蛋白酶抑制剂是必需的RAW264.7巨噬细胞，但D. discoideum ...

purification of pathogen vacuoles from legionella-infected phagocytes

条件致病菌嗜肺军团菌是一种抗阿米巴的细菌，这也是在肺泡巨噬细胞，从而导致重症肺炎“退伍军人病”1复制。在原生动物和哺乳动物吞噬，L。肺采用了保守的机制，以形成特定的许可，复制，车厢，“含军团菌的空泡”（轻型商用车）。轻型商用车的形成需要ICM / DOT细菌IV型分泌系统（T4SS）“275效应”的蛋白质，其中的translocates进入宿主细胞。效应操纵宿主蛋白以及血脂和分泌，内体和线粒体的细胞器2-4沟通。 的LCVs形成一个复杂的，强大的和冗余的过程，这是很难把握，在还原的方式。一个综合性的做法需要全面了解轻型商用车的形成，包括一个全球性的路径分析ogen宿主因子的相互作用及其时空动态。为实现这一目标的第一步，完整LCVs纯化和蛋白质组学和脂类组学分析。 已被调查的蛋白质组学分析，使用液相色谱或2-D电泳质谱仪耦合的组成和形成的含病原体的空泡。 9， 沙门氏菌 ， 球菌 8，7 分枝杆菌 ， 李斯特菌 6 利什曼 5 军团菌分离的社会土壤变形虫粘菌或哺乳动物吞噬细胞空泡窝藏10。然而，在这些研究中采用的协议是净化费时和繁琐，因为他们需要如电子显微镜分析轻型商用车的形态，完整性和纯度。此外，这些协议不利用EN病原体液泡的具体特点富集。 这里介绍的方法克服了这些限制，由用人四discoideum生产轻型商用车荧光标记和针对细菌效应蛋白SIDC，选择性地结合到磷酸肌醇4 -磷酸（PtdIns（4）P）的3,11轻型商用车膜锚。第一步由SIDC和二级抗体，加上磁珠古典Histodenz密度梯度离心12,13（ 图1），其次是第二步，对主要抗体亲和纯化的免疫磁性分离富集LCVs 。 一个蛋白质孤立LCVs的研究从 D discoideum发现560多宿主细胞包括吞噬囊泡，线粒体，ER和高尔基，以及一些GTP酶，它没有被牵连13日之前在轻型商用车形成相关蛋白的蛋白质，。丰富和纯化LCVs协议概述，这里还可以进一步分析显微镜（免疫荧光法，电子显微镜），生化方法（免疫印迹）和蛋白质或lipidomic办法。

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

本文介绍了一个完整的隔离和净化的方法<em&gt;军团</em&gt;从变形虫和巨噬细胞含有空泡（LCVs）。两步的协议，包括轻型商用车通过使用对细菌的轻型商用车标记和密度梯度离心进一步纯化抗体的免疫磁性分离富集。

病原液泡的分离纯化<em&gt;军团</em&gt;感染吞噬细胞

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

Research

JoVE Journal

Immunology and Infection

Purification of Pathogen Vacuoles from Legionella-infected Phagocytes

Invasion of Human Cells by a Bacterial Pathogen

由细菌病原体入侵人体细胞

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

科研

免疫与感染

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

为期一天的血培养的细菌病原体检测和耐药性检测的工作流程计划

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

热酚水提取纯化及革兰氏阴性菌脂多糖的可视化

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

流感病毒从感染细胞的染色质的核糖核蛋白复合物的亲和纯化

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

活细胞视频显微镜病病原菌的吞噬作用

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

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

环介导等温扩增（LAMP）法进行的物种特异性检测<em&gt;艾美耳球虫</em&gt;可感染鸡

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

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

RNA纯化在细胞内生长<em&gt;李斯特菌</em&gt;巨噬细胞

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

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

受肥胖相关炎症组织的巨噬细胞的分离、鉴定和纯化

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. ...

Identification of Virulence Markers of Mycobacterium abscessus for Intracellular Replication in Phagocytes

Identification of Virulence Markers of Mycobacterium abscessus for Intracellular Replication in Phagocytes

吞噬细胞内复制脓肿分枝杆菌毒力标志物的鉴定

What differentiates Mycobacterium abscessus from other saprophytic mycobacteria is the ability to resist phagocytosis by human macrophages and the ability ...

Purification of Extracellular Trypanosomes, Including African, from Blood by Anion-Exchangers (Diethylaminoethyl-cellulose Columns)

Purification of Extracellular Trypanosomes, Including African, from Blood by Anion-Exchangers Diethylaminoethyl-cellulose Columns

阴离子交换剂 (二乙基氨基乙基纤维素柱) 纯化包括非洲在内的细胞外锥虫体

This method allows the separation of trypanosomes, parasites responsible for animal and human African trypanosomiasis (HAT), from infected blood. This is ...