Name: tractable mammalian cell infections with protozoan-primed bacteria
Uploaded: 2013-04-02T12:00:00
Description: Watch this Scientific Journal Video about Tractable Mammalian Cell Infections with Protozoan-primed Bacteria at JoVE.com

我们感谢克雷格·罗伊博士和博士达里奥·赞博尼提供的模板原生动物细胞感染。我们感谢贾格迪普Obhrai博士，博士乔治安娜·珀迪，弗雷德·赫夫龙博士和托德·威斯纳设备和试剂;露露罗纳博士的手稿严格审查。流式细胞术在OHSU的流式细胞仪共享资源设施。这项工作是支持的，由俄勒冈州医学研究基金会和美国国立卫生研究院授予R21 AI088275（EDC）的资助。

1。引发阶段感染嗜肺军团菌文化制备<ol><li>将所有L.肺用质粒pAM239在测定中使用的菌株，编码的异丙基-β-D-1-硫代吡喃半乳糖苷（IPTG）诱导的绿色荧光蛋白（GFP）28。条纹到铁和半胱氨酸的细菌菌株补充缓冲木炭酵母提取物琼脂（实业家）含有6.25微克/毫升氯霉素（CM）（用于质粒维护），并孵育72 N-（2 -乙酰氨基）-2 -氨基乙磺酸（ACES）小时，在37°C。 </li><li>转...

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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survival+of+coliforms+and+bacterial+pathogens+within+protozoa+during+chlorination.">Survival of coliforms and bacterial pathogens within protozoa during chlorination.</a> Appl. Environ. Microbiol. 54, 3023-3033 (1988).</li><li>Horwitz, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+a+novel+phagosome+by+the+Legionnaires'+disease+bacterium+(Legionella+pneumophila)+in+human+monocytes.">Formation of a novel phagosome by the Legionnaires' disease bacterium (Legionella pneumophila) in human monocytes.</a> J. Exp. Med. 158, 1319-1331 (1983).</li><li>Horwitz, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phagocytosis+of+the+Legionnaires'+disease+bacterium+(Legionella+pneumophila)+occurs+by+a+novel+mechanism:+engulfment+within+a+pseudopod+coil.">Phagocytosis of the Legionnaires' disease bacterium (Legionella pneumophila) occurs by a novel mechanism: engulfment within a pseudopod coil.</a> Cell. 36, 27-33 (1984).</li><li>Horwitz, M. A., Silverstein, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Legionnaires'+disease+bacterium+(Legionella+pneumophila)+multiples+intracellularly+in+human+monocytes.">Legionnaires' disease bacterium (Legionella pneumophila) multiples intracellularly in human monocytes.</a> J. Clin. 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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+distinct+defects+in+intracellular+growth+complemented+by+a+single+genetic+locus+in+Legionella+pneumophila.">Two distinct defects in intracellular growth complemented by a single genetic locus in Legionella pneumophila.</a> Mol. Microbiol. 7, 7-19 (1993).</li><li>Marra, A., Blander, S. J., Horwitz, M. A., Shuman, H. A. <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+a+Legionella+pneumophila+locus+required+for+intracellular+multiplication+in+human+macrophages.">Identification of a Legionella pneumophila locus required for intracellular multiplication in human macrophages.</a> Proc. Natl. Acad. Sci. U.S.A. 89, 9607-9611 (1992).</li><li>Segal, G., Purcell, M., Shuman, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host+cell+killing+and+bacterial+conjugation+require+overlapping+sets+of+genes+within+a+22-kb+region+of+the+Legionella+pneumophila+genome.">Host cell killing and bacterial conjugation require overlapping sets of genes within a 22-kb region of the Legionella pneumophila genome.</a> Proc. Natl. Acad. Sci. U.S.A. 95, 1669-1674 (1998).</li><li>Segal, G., Shuman, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+new+region+required+for+macrophage+killing+by+Legionella+pneumophila.">Characterization of a new region required for macrophage killing by Legionella pneumophila.</a> Infect Immun. 65, 5057-5066 (1997).</li><li>Vogel, J. P., Andrews, H. L., Wong, S. K., Isberg, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conjugative+transfer+by+the+virulence+system+of+Legionella+pneumophila.">Conjugative transfer by the virulence system of Legionella pneumophila.</a> Science. 279, 873-876 (1998).</li><li>Haghjoo, E., Galan, 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+a+transcriptional+regulator+that+controls+intracellular+gene+expression+in+Salmonella+Typhi.">Identification of a transcriptional regulator that controls intracellular gene expression in Salmonella Typhi.</a> Mol. Microbiol. 64, 1549-1561 (2007).</li><li>Molofsky, A. B., Swanson, M. 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细菌基因表达被严密控制的生命周期的进展和响应周围的微环境中的信号通过一个组合。液泡的病原体，如L.肺应对多种宿主细胞衍生的线索条块中的吞噬体时。由于在宿主细胞中的营养耗竭集体结果，细菌随后的宿主细胞，25表示成功传播所需的因素进行补偿。L。肺适于有效地寄生原生动物细胞各种各样的，因此窝藏效应蛋白的一个广泛的目录来驱动此过程。这些效应蛋白...

大量的细菌病原体已经适应了广义的策略，利用宿主细胞，在细胞内生存和复制。在许多情况下，致病机制原生动物和后生动物的细胞之间的相似。然而，这两个微环境是非常不同的，并可能导致在毒力因子的差异表达1-4。退伍军人症细菌嗜肺军团菌广泛存在与世界各地的淡水环境5。更重要的是，L.原生动物细胞培养在肺感染的人类单核细胞中获得的致病优势之前，表明全局基因表达谱的退出原生动物细胞的细菌在体外培育生物体6-8比是不同的。在自然界中，淡水变形虫快速扩增细菌入侵提供营养丰富的局限。人类收购L. pneumophILA是最常见的吸入受污染的水含有细菌的水滴，。这很可能是这些水滴港口原生动物细胞相关的细菌，原生动物细胞更耐常规水处理的做法9,10。感染的肺泡巨噬细胞原生动物宿主细胞11-13中的细菌的细胞内的生命周期几乎相同的方式进行。 为了生存，并在真核细胞中复制，L.肺使用一个专门的IVB型分泌系统，称为点/ ICM提供近300&#39;效应蛋白进入宿主细胞的细胞质中14-16。这些效应蛋白统称颠覆细胞过程中，为了产生一个复制许可为细菌的隔室17,18的功能。在任意的26个基因，包括在细胞内的多个缺陷的菌株的Dot / Icm的转运结果删除iplication 19日至23日 。从历史上看，删除的个体效应编码基因很少株弱毒细胞内生长。这种现象被归结为几种假说的效应，包括冗余的功能和同源的副本。 有些毒力因子只表示的上下文中的宿主细胞相关的细胞内生长24。我们合理化，如果一个特定的效应，只表示的上下文中的原虫感染，然后的效应的贡献可能不能的野生型菌株相比，当两个进行体外培养。嗜肺军团菌的复制转换透射阶段进入稳定期文化25。相开关型过程中遇到的细胞内生长的养分消耗，，通过组装鞭毛活力26的例子。由于L.肺是INVA西伯和原生动物细胞致命的收获时，我们寻求发展的分析，更忠实地代表了致病状态，当它遇到的细菌宿主巨噬细胞。 为此，我们开发了一种多用途的的原生动物吸试验，可以适应任何合适的宿主同时为第一（涂刷细胞）和第二阶段（靶细胞）感染。通过使用稳定表达绿色荧光蛋白（GFP）的细菌感染的过程中易于处理。原生动物棘阿米巴castellanii感染模型如下的字段27中广泛使用的一种方法。对于吸步骤，L.杆菌菌株在体外培养，以在液体介质中的固定相，以产生标记为“透射”细菌（ 图1A）。接下来，细菌感染单层A. castellanii 18小时来实现的细胞内的生命周期的后期阶段。大液泡包含ING细菌可以被可视化，在该时间点，使用荧光显微镜（ 图1A）。原生动物细胞，然后裂解，从裂解物中回收的细菌测定使用荧光板读数器在512 nm的发射。荧光光密度与多重性感染（MOI）感染的靶细胞（ 图1，*的相关性曲线）来计算。入侵后（T 18）入侵（T 0），18小时后，靶细胞的荧光定量，代表细胞内的细菌。可以监视荧光显微镜和流式细胞仪，活菌数可通过殖民地电镀测量。吸分析总是伴随着感染与野生型L.肺的Dot / Icm的IV型分泌系统（ΔDOTA）（ 图1A）中的缺陷的菌株。重要的是提供内部控制之间的直接比较野生型aND任何等位基因突变株在感染过程中使用。在起动阶段期间的无毒ΔDOTA应变列入设置观察衰减等基因突变株， 在体外培养生长的表型相关的一个阈值。

<table border="1">
 <tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Reagent</td>
 </tr>
 <tr>
 <td>chloramphenicol</td>
 <td>Fisher Scientific</td>
 <td>BP904-100</td>
 <td>antibiotic</td>
 </tr>
 <tr>
 <td>IPTG</td>
 <td>Fisher Scientific</td>
 <td>BP1755-10</td>
 <td></td>
 </tr>
 <tr>
 <td>ACES</td>
 <td>Sigma</td>
 <td>A9758-1KG</td>
 <td>media component</td>
 </tr>
 <tr>
 <td>ATCC medium: 712 PYG</td>
 <td>ATCC</td>
 <td></td>
 <td>growth media for protozoans</td>
 </tr>
 <tr>
 <td>1X PBS</td>
 <td>Fisher Scientific</td>
 <td>SH30256FS</td>
 <td>phosphate buffered saline</td>
 </tr>
 <tr>
 <td>activated charcoal</td>
 <td>Fisher Scientific</td>
 <td>C272-212</td>
 <td>media component</td>
 </tr>
 <tr>
 <td>yeast extract</td>
 <td>Fisher Scientific</td>
 <td>BP1422-500</td>
 <td>media component</td>
 </tr>
 <tr>
 <td>peptone</td>
 <td>BD Diagnostics</td>
 <td>211677</td>
 <td>media component</td>
 </tr>
 <tr>
 <td>agar</td>
 <td>Fisher Scientific</td>
 <td>BP1423-2</td>
 <td>media component</td>
 </tr>
 <tr>
 <td>L-cysteine, 99%+</td>
 <td>Acros organics</td>
 <td>173601000</td>
 <td>media supplement</td>
 </tr>
 <tr>
 <td>Ferric nitrate nonahydrate</td>
 <td>Fisher Scientific</td>
 <td>I110-100</td>
 <td>media supplement</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Equipment</td>
 </tr>
 <tr>
 <td>EVOS fl</td>
 <td>AMG</td>
 <td>EVOS fl</td>
 <td>fluorescence microscope</td>
 </tr>
 <tr>
 <td>Smart Spec Plus</td>
 <td>Bio-Rad</td>
 <td>170-2525</td>
 <td>spectrophotometer</td>
 </tr>
 <tr>
 <td>5810 R centrifuge</td>
 <td>Eppendorf</td>
 <td>22627023</td>
 <td>bench top centrifuge</td>
 </tr>
 <tr>
 <td>Repeater plus</td>
 <td>Eppendorf</td>
 <td>22230201</td>
 <td>repeating pipette</td>
 </tr>
 <tr>
 <td>SpectraMax Gemini EM</td>
 <td>Molecular Devices</td>
 <td></td>
 <td>microplate reader</td>
 </tr>
 <tr>
 <td>Softmax Pro 5.3</td>
 <td>Molecular Devices</td>
 <td>0200-310</td>
 <td>microplate reader software</td>
 </tr>
 <tr>
 <td>5424 microfuge</td>
 <td>Eppendorf</td>
 <td>22620401</td>
 <td>table top microcentrifuge</td>
 </tr>
 <tr>
 <td>Fast-release pipette pump II</td>
 <td>Scienceware</td>
 <td>379111010</td>
 <td>pipette aid</td>
 </tr>
 <tr>
 <td>FACS Calibur</td>
 <td>BD Bioscience</td>
 <td></td>
 <td>flow cytometer</td>
 </tr>
 <tr>
 <td>FlowJo 7.6.1</td>
 <td>FlowJo</td>
 <td>license</td>
 <td>flow cytometery software</td>
 </tr>
 <tr>
 <td>15 ml tube</td>
 <td>BD Falcon</td>
 <td>352096</td>
 <td>polypropylene conical tube</td>
 </tr>
 <tr>
 <td>1.6 ml microfuge tube</td>
 <td>Neptune</td>
 <td>3745.X</td>
 <td>microcentrifuge tubes</td>
 </tr>
 </tbody>
</table>

tractable mammalian cell infections with protozoan-primed bacteria

许多细胞内的细菌病原体使用淡水原生动物作为一种天然水库增殖的环境中。退伍军人肺炎的病原体，获得了嗜肺军团菌 ，当第一次收获之前感染的哺乳动物巨噬细胞的原生动物细胞在体外培养的细菌的致病性优势。这表明可能无法正确地在体外表达，重要的毒力因子。我们已经开发出一种易于处理系统吸L.肺通过其自然的原生主机棘阿米巴castellanii之前哺乳动物细胞的感染。任何毒力因子的贡献可以被检查原生动物吸后，通过比较的突变株的细胞内生长的野生型细菌。表达GFP的野生型和突变型L。杆菌菌株用于感染原生动物的单分子膜在一个起动步骤，并允许达到的细胞内生长的后期。荧光的细菌从这些受感染的细胞，然后收获归一分光光度法产生相当数量的细菌到哺乳动物的巨噬细胞的继发感染。对于定量分析进行监测，活菌感染后，用荧光显微镜，流式细胞术，通过菌落镀敷。这种技术强调和依赖于宿主细胞相关的基因表达，通过模仿的环境，会遇到一个自然的收购路线的贡献。这种方法可以被修改，以适应使用媒介主机的任何细菌，作为一个装置，用于获得的致病优势。

为整个感染过程的一个典型的结果是在图1中概述。活细胞的荧光显微照片描绘单层A.castellanii感染野生型L。肺在起动阶段，在图1A中所示。一个成功的措施的起动步骤会导致人口的约90％的宿主细胞中含有大量空泡人口与绿色荧光蛋白标记的细菌在此MOI。在感染后18小时，A. castellanii细胞，细菌负荷达到最大，但裂解的人口是最少的。在光学显微镜下，?...

Watch this Scientific Journal Video about Tractable Mammalian Cell Infections with Protozoan-primed Bacteria at JoVE.com

Tractable Mammalian Cell Infections with Protozoan-primed Bacteria

这种技术提供了一种方法来收集，标准化和量化，自然原虫的宿主细胞培养的哺乳动物细胞感染前细胞内生长的细菌病原体。此方法可以被修改，以适应各种各样的起动阶段，以及靶细胞类型的宿主细胞。

听话的哺乳动物细胞原生动物引发的细菌感染

Many intracellular bacterial pathogens use freshwater protozoans as a natural reservoir for proliferation in the environment. Legionella pneumophila, the ...

Research

JoVE Journal

Immunology and Infection

Using Luciferase to Image Bacterial Infections in Mice

用荧光素酶在小鼠细菌性感染的形象

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

科研

免疫与感染

Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant

Multivirus特异性T细胞的生成，预防/治疗异基因造血干细胞移植后的病毒感染

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully ...

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

4D多模态成像<em&gt;柠檬酸rodentium</em在小鼠感染

This protocol outlines the steps required to  longitudinally monitor a bioluminescent bacterial infection using composite 3D diffuse light imaging ...

Fluorescence Microscopy Methods for Determining the Viability of Bacteria in Association with Mammalian Cells

荧光显微镜方法测定细菌在协会的可行性与哺乳动物细胞

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

生成和多表型高内涵筛选<em&gt;贝氏柯克斯体</em&gt;转座子突变体

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells

Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells

牙龈卟啉单作为模式生物的评价厌氧细菌的相互作用与宿主细胞

Anaerobic bacteria far outnumber aerobes in many human niches such as the gut, mouth, and vagina. Furthermore, anaerobic infections are common and ...

Using Zebrafish Models of Human Influenza A Virus Infections to Screen Antiviral Drugs and Characterize Host Immune Cell Responses

使用人类流感病毒感染的斑马鱼模型到屏幕抗病毒药物和表征宿主免疫细胞反应

Each year, seasonal influenza outbreaks profoundly affect societies worldwide. In spite of global efforts, influenza remains an intractable healthcare ...

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

使用的淋巴细胞外渗分析<em&gt;体外</em&gt;人类血脑屏障模型

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

The WinCF Model - An Inexpensive and Tractable Microcosm of a Mucus Plugged Bronchiole to Study the Microbiology of Lung Infections

该WinCF模式 - 粘液的廉价和易于处理的缩影细支气管堵塞，研究肺部感染的微生物

Many chronic airway diseases result in mucus plugging of the airways. Lungs of an individual with cystic fibrosis are an exemplary case where their ...

Confocal Imaging of Double-Stranded RNA and Pattern Recognition Receptors in Negative-Sense RNA Virus Infection

微链 rna 的共聚焦成像和模式识别受体在负感 rna 病毒感染中的应用

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors ...