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>转移的种菌的菌株成补充ACES缓冲与6.25微克/毫升CM和1mM IPTG的酵母提取物肉汤（AYE），培养在37℃下固定相（O / N）在轨道摇床</li><li>确认在体外培养物中，使用荧光显微镜的GFP表达。盖下培养10微升转移到载玻片上单和图像，使用60X与GFP激发/发射立方体（AMG EVOS佛罗里达州）的目标。 </li><li>稀释100微升等分每个体外培养至1:10使用无菌H 2 O。用1mM IPTG和6.25微克/毫升厘米和水作空白用100μlAYE的媒体。以OD600值测量的稀释液用分光光度计（Bio-Rad公司智能规格加）。必要计算其体积，以MOI = 20感染的以及为每个在体外培养中:V = [（阿米巴种子）×MOI] / [OD 600×（稀释倍数）×（常数）] = [（1×10 6个 ）×（20）] / [OD 600×（10）×（1×10 6）] = 2/OD 600。浓度的细菌被确定为OD 600 = 1.0 = 1×10 9 CFU /毫升。 </li></ol> 2。感染棘阿米巴castellanii的引发阶段<ol><li>维护和ATCC 712 PYG培养基中的变形虫在175厘米2瓶中，在室温下培养。 </li><li>更换介质变形虫尽头Tures的前24小时开始的细菌液体培养。就在同一天液细菌培养，收集和使用血球计数仪，光镜下细胞计数。 </li><li>变形虫用新鲜培养基稀释至终浓度为1×10 6细胞/ ml。种子的12孔细胞培养板中，用1​​毫升等分的阿米巴使用一个repeat移液器并孵育在RT下的O / N。 </li><li>孵育后，洗3倍与1毫升无菌PBS中，使用了10毫升血清移液管和一个手动移液管辅助的12孔细胞培养板的孔中。 </li><li>抽吸的PBS后，加1ml的感染介质（ATCC 712 PYG媒体减去葡萄糖，蛋白胨，酵母提取物），用1mM IPTG和6.25微克/毫升cm到每个孔中。在RT下孵育板1小时。 </li><li> MOI = 20感染井。离心板400×g离心5分钟（Eppendorf公司5810R）和漂浮在37℃水浴5分钟。转移板在37℃，5％CO 2的培养箱中培养18小时。 </li><li>使用活细胞成像在荧光显微镜下的细菌宿主细胞裂解和收获之前确认的感染。 </li></ol> 3。播种THP-1细胞为靶细胞阶段感染<ol><li> （这个过程开始前24小时，液细菌培养）。 75 cm 2的培养瓶中近会合在与10％热灭活的小牛血清（FBS）的RPMI 1640培养基中培养THP-1细胞。 </li><li>使用血细胞计数器计数悬浮细胞，含10％FBS和100 ng / ml的佛波醇-12 -肉豆蔻酸酯-13 -乙酸酯（PMA）的浓度为1×10 6细胞/ ml THP-1细胞在RPMI 1640培养基稀释，并在1毫升的等分试样在12孔细胞培养板的板。孵育48小时的板在37℃下，5％CO 2。 </li></ol> 4。目标细胞阶段感染，细菌感染的起动阶段后处理<ol><li>吸取媒体的引物变形虫。 </li><li>裂解一moebae使用500μl的冰冷，无菌超滤（UF）H 2 O和孵育在室温下10分钟。 </li><li>普尔的裂解物，根据应变类型和采取的E 512 nm的测量为每个池的溶胞产物用荧光板读数器（Molecular Devices公司酶标仪双子座EM）。 </li><li>计算OD 600项测试的汇集裂解calcOD 600 = 0.0008（E 512 -裂解背景）+ 0.0019。预先确定的公式，通过图形的直接比较的OD 600和E 512 nm的测量稀释的L.肺野生型表达绿色荧光蛋白， 在体外培养固定相（ 图1）。裂解物的未感染的变形虫，受到相同的实验条件下，受感染的变形虫，作为空白，并提供了一个修正值（ 例如，溶胞产物的背景），将其纳入方程。所需的体积吨ò感染在阱的MOI = 20时为每个溶解物游泳池计算:V = [（THP-1种子）所述MOI] / [calcOD 600×（常数）] = [（1×10 6）×（20）] ，/ [calcOD 600×（1×10 6）] = 20 / calcOD 600。 </li><li>在THP-1细胞的3倍，用PBS洗净，向每孔加入1毫升的新鲜RPMI 1640培养基（10％FBS）。孵育THP-1细胞，在37℃下1小时，5％CO 2。 </li><li>感染的THP-1细胞，在所计算出的MOI使用汇集的溶胞产物。允许一组井保持感染，作为流式细胞分析的阴性对照。应在不到30分钟内完成处理的起动阶段。裂解液置于冰上，感染前，以限制任何细菌基因表达的变化。 </li><li>离心板，浮动，以提高温度和孵化，在起动阶段。 </li><li>感染后一小时，取出媒体的愿望和3次PBS删除胞外细菌，洗井。 </li><li>加入1毫升频率sh的RPMI 1640培养液（10％FBS），井和返回板的培养箱。后立即更换媒体的时间作为时间零点（T 0）。孵育14-16小时的THP-1细胞在37℃下，5％CO 2。 </li></ol> 5。实验分析 5.1活细胞成像图像受感染的井用荧光显微镜（AMG EVOS佛罗里达州）10X或20X放大倍数（ 图1A-D）。这些图像可以被定量或定性的比较，以确定在起动阶段和靶细胞阶段感染感染水平。 5.2流式细胞仪<ol><li>可以比较流式细胞仪（BD FACS刀魂）的发射峰不同的感染。 Trypsinize受感染的THP-1细胞，并从孔中通过在PBS中混合，用移液管轻轻地清洗。 </li><li>游泳池的应变类型的细胞15毫升Falcon管和球团在1800 XG 2分钟在台式离心机。 </li><li>暂停颗粒在1毫升PBS，并转移到1.5 ml离心管中。 </li><li>为1800 X G（Eppendorf公司5424）和离心悬浮液，重新悬浮于1ml PBS颗粒。如果所得到的悬浮液高度浑浊（≥1.0的OD 600），他们必须额外PBS（1:3）稀释，在流式细胞仪中的线，以防止堵塞。 </li><li>使用无菌过滤PBS平衡的流式细胞仪和洗涤步骤之间的样品进样。绘制的正向/侧向散射未受感染的靶细胞的靶细胞感染样品注射之前。 </li><li>收集20000事件总数为每个条件使用488 nm激光激发和FL1通道。 </li><li>栅极捕获后的细胞群，以排除未感染的细胞和情节在直方图中的荧光强度（FlowJo）（ 图1E）。 </li></ol> 5.3 CFU电镀<ol><li>检查的效率of感染CFU电镀流式细胞收获细胞。准备样品的系列稀释液在超过滤的H 2 O在10 -1，10 -2，10 -3。 </li><li>板20μl的每个稀释到1/3的实业家板与6.25微克/毫升CM。 </li><li>板在37℃下孵育72小时。 </li><li>用牙签和细胞计数器计数的殖民地。 </li></ol>

<ol>
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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=Amoebae+promote+persistence+of+epidemic+strains+of+MRSA.">Amoebae promote persistence of epidemic strains of MRSA.</a> Environ. Microbiol. 8, 1130-1133 (2006).</li></ol>

作者宣称，他们没有竞争的金融利益。

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 ...

tractable-mammalian-cell-infections-with-protozoan-primed-bacteria

Research

JoVE Journal

Immunology and Infection

10.4K 次观看.  Oregon Health & Science University. 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。

视频: Tractable Mammalian Cell Infections with Protozoan-primed Bacteria

Using Luciferase to Image Bacterial Infections in Mice

Imaging is a valuable technique that can be used to monitor biological processes. In particular, the presence of cancer cells, stem cells, specific immune cell types, viral pathogens, parasites and bacteria can be followed in real-time within living animals 1-2. Application of bioluminescence imaging to the study of pathogens has advantages as compared to conventional strategies for analysis of infections in animal models3-4. Infections can be visualized within individual animals over time, without requiring euthanasia to determine the location and quantity of the pathogen. Optical imaging allows comprehensive examination of all tissues and organs, rather than sampling of sites previously known to be infected. In addition, the accuracy of inoculation into specific tissues can be directly determined prior to carrying forward animals that were unsuccessfully inoculated throughout the entire experiment. Variability between animals can be controlled for, since imaging allows each animal to be followed individually. Imaging has the potential to greatly reduce animal numbers needed because of the ability to obtain data from numerous time points without having to sample tissues to determine pathogen load3-4.
This protocol describes methods to visualize infections in live animals using bioluminescence imaging for recombinant strains of bacteria expressing luciferase. The click beetle (CBRLuc) and firefly luciferases (FFluc) utilize luciferin as a substrate5-6. The light produced by both CBRluc and FFluc has a broad wavelength from 500 nm to 700 nm, making these luciferases excellent reporters for the optical imaging in living animal models7-9. This is primarily because wavelengths of light greater than 600 nm are required to avoid absorption by hemoglobin and, thus, travel through mammalian tissue efficiently. Luciferase is genetically introduced into the bacteria to produce light signal10. Mice are pulmonary inoculated with bioluminescent bacteria intratracheally to allow monitoring of infections in real time. After luciferin injection, images are acquired using the IVIS Imaging System. During imaging, mice are anesthetized with isoflurane using an XGI-8 Gas Anethesia System. Images can be analyzed to localize and quantify the signal source, which represents the bacterial infection site(s) and number, respectively. After imaging, CFU determination is carried out on homogenized tissue to confirm the presence of bacteria. Several doses of bacteria are used to correlate bacterial numbers with luminescence. Imaging can be applied to study of pathogenesis and evaluation of the efficacy of antibacterial compounds and vaccines.

Methods for bioluminescence imaging of bacterial infections in living animals are decribed. Pathogens are modified to express luciferase allowing optical whole body imaging of infections in live animals. Animal models can be infected with luciferase expressing pathogens and the resulting course of disease visualized in real-time by bioluminescence imaging.

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

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully generated and infused T-cells specific for Epstein Barr virus (EBV), cytomegalovirus (CMV) and Adenovirus (Adv) using monocytes and EBV-transformed lymphoblastoid cell (EBV-LCL) gene-modified with an adenovirus vector as antigen presenting cells (APCs). As few as 2x105/kg trivirus-specific cytotoxic T lymphocytes (CTL) proliferated by several logs after infusion and appeared to prevent and treat even severe viral disease resistant to other available therapies. The broader implementation of this encouraging approach is limited by high production costs, complexity of manufacture and the prolonged time (4-6 weeks for EBV-LCL generation, and 4-8 weeks for CTL manufacture &#x2013; total 10-14 weeks) for preparation. To overcome these limitations we have developed a new, GMP-compliant CTL production protocol. First, in place of adenovectors to stimulate T-cells we use dendritic cells (DCs) nucleofected with DNA plasmids encoding LMP2, EBNA1 and BZLF1 (EBV), Hexon and Penton (Adv), and pp65 and IE1 (CMV) as antigen-presenting cells. These APCs reactivate T cells specific for all the stimulating antigens. Second, culture of activated T-cells in the presence of IL-4 (1,000U/ml) and IL-7 (10ng/ml) increases and sustains the repertoire and frequency of specific T cells in our lines. Third, we have used a new, gas permeable culture device (G-Rex) that promotes the expansion and survival of large cell numbers after a single stimulation, thus removing the requirement for EBV-LCLs and reducing technician intervention. By implementing these changes we can now produce multispecific CTL targeting EBV, CMV, and Adv at a cost per 106 cells that is reduced by &gt;90%, and in just 10 days rather than 10 weeks using an approach that may be extended to additional protective viral antigens. Our FDA-approved approach should be of value for prophylactic and treatment applications for high risk allogeneic HSCT recipients.

A rapid, simple and cost-effective protocol for the generation of donor-derived multivirus-specific CTLs (rCTL) for infusion to allogeneic hematopoietic stem cell transplant (HSCT) recipients at risk of developing CMV, Adv or EBV infections. This manufacturing process is GMP-compliant and should ensure the broader implementation of T-cell immunotherapy beyond specialized centers.

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

This protocol outlines the steps required to longitudinally monitor a bioluminescent bacterial infection using composite 3D diffuse light imaging tomography with integrated &mu;CT (DLIT-&mu;CT) and the subsequent use of this data to generate a four dimensional (4D) movie of the infection cycle. To develop the 4D infection movies and to validate the DLIT-&mu;CT imaging for bacterial infection studies using an IVIS Spectrum CT, we used infection with bioluminescent C. rodentium, which causes self-limiting colitis in mice. In this protocol, we outline the infection of mice with bioluminescent C. rodentium and non-invasive monitoring of colonization by daily DLIT-&mu;CT imaging and bacterial enumeration from feces for 8 days.
The use of the IVIS Spectrum CT facilitates seamless co-registration of optical and &mu;CT scans using a single imaging platform. The low dose &mu;CT modality enables the imaging of mice at multiple time points during infection, providing detailed anatomical localization of bioluminescent bacterial foci in 3D without causing artifacts from the cumulative radiation. Importantly, the 4D movies of infected mice provide a powerful analytical tool to monitor bacterial colonization dynamics in vivo.

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

Multi-modality imaging is a valuable approach for studying bacterial colonization in small animal models. This protocol outlines infection of mice with bioluminescent Citrobacter rodentium and the longitudinal monitoring of bacterial colonization using composite 3D diffuse light imaging tomography with &mu;CT imaging to create a 4D movie of C. rodentium infection.

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

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols to address these questions include colony count assays, gentamicin protection assays, and electron microscopy. Colony count and gentamicin protection assays only assess the viability of the entire bacterial population and are unable to determine individual bacterial viability. Electron microscopy can be used to determine the viability of individual bacteria and provide information regarding their localization in host cells. However, bacteria often display a range of electron densities, making assessment of viability difficult. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells. These assays were developed originally to assess survival of Neisseria gonorrhoeae in primary human neutrophils, but should be applicable to any bacterium-host cell interaction. These protocols combine membrane-permeable fluorescent dyes (SYTO9 and 4&#39;,6-diamidino-2-phenylindole [DAPI]), which stain all bacteria, with membrane-impermeable fluorescent dyes (propidium iodide and SYTOX Green), which are only accessible to nonviable bacteria. Prior to eukaryotic cell permeabilization, an antibody or fluorescent reagent is added to identify extracellular bacteria. Thus these assays discriminate the viability of bacteria adherent to and inside eukaryotic cells. A protocol is also provided for using the viability dyes in combination with fluorescent antibodies to eukaryotic cell markers, in order to determine the subcellular localization of individual bacteria. The bacterial viability dyes discussed in this article are a sensitive complement and/or alternative to traditional microbiology techniques to evaluate the viability of individual bacteria and provide information regarding where bacteria survive in host cells.

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host 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

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

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

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

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

Anaerobic bacteria far outnumber aerobes in many human niches such as the gut, mouth, and vagina. Furthermore, anaerobic infections are common and frequently of indigenous origin. The ability of some anaerobic pathogens to invade human cells gives them adaptive measures to escape innate immunity as well as to modulate host cell behavior. However, ensuring that the anaerobic bacteria are live during experimental investigation of the events may pose challenges. Porphyromonas gingivalis, a Gram-negative anaerobe, is capable of invading a variety of eukaryotic non-phagocytic cells. This article outlines how to successfully culture and assess the ability of P. gingivalis to invade human umbilical vein endothelial cells (HUVECs). Two protocols were developed: one to measure bacteria that can successfully invade and survive within the host, and the other to visualize bacteria interacting with host cells. These techniques necessitate the use of an anaerobic chamber to supply P. gingivalis with an anaerobic environment for optimal growth.
The first protocol is based on the antibiotic protection assay, which is largely used to study the invasion of host cells by bacteria. However, the antibiotic protection assay is limited; only intracellular bacteria that are culturable following antibiotic treatment and host cell lysis are measured. To assess all bacteria interacting with host cells, both live and dead, we developed a protocol that uses fluorescent microscopy to examine host-pathogen interaction. Bacteria are fluorescently labeled with 2&#39;,7&#39;-Bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) and used to infect eukaryotic cells under anaerobic conditions. Following fixing with paraformaldehyde and permeabilization with 0.2% Triton X-100, host cells are labeled with TRITC phalloidin and DAPI to label the cell cytoskeleton and nucleus, respectively. Multiple images taken at different focal points (Z-stack) are obtained for temporal-spatial visualization of bacteria. Methods used in this study can be applied to any cultivable anaerobe and any eukaryotic cell type.

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

This article presents two protocols: one to measure anaerobic bacteria that can successfully invade and survive within the host, and the other to visualize anaerobic bacteria interacting with host cells. This study can be applied to any cultivable anaerobe and any eukaryotic cell type.

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

Each year, seasonal influenza outbreaks profoundly affect societies worldwide. In spite of global efforts, influenza remains an intractable healthcare burden. The principle strategy to curtail infections is yearly vaccination. In individuals who have contracted influenza, antiviral drugs can mitigate symptoms. There is a clear and unmet need to develop alternative strategies to combat influenza. Several animal models have been created to model host-influenza interactions. Here, protocols for generating zebrafish models for systemic and localized human influenza A virus (IAV) infection are described. Using a systemic IAV infection model, small molecules with potential antiviral activity can be screened. As a proof-of-principle, a protocol that demonstrates the efficacy of the antiviral drug Zanamivir in IAV-infected zebrafish is described. It shows how disease phenotypes can be quantified to score the relative efficacy of potential antivirals in IAV-infected zebrafish. In recent years, there has been increased appreciation for the critical role neutrophils play in the human host response to influenza infection. The zebrafish has proven to be an indispensable model for the study of neutrophil biology, with direct impacts on human medicine. A protocol to generate a localized IAV infection in the Tg(mpx:mCherry) zebrafish line to study neutrophil biology in the context of a localized viral infection is described. Neutrophil recruitment to localized infection sites provides an additional quantifiable phenotype for assessing experimental manipulations that may have therapeutic applications. Both zebrafish protocols described faithfully recapitulate aspects of human IAV infection. The zebrafish model possesses numerous inherent advantages, including high fecundity, optical clarity, amenability to drug screening, and availability of transgenic lines, including those in which immune cells such as neutrophils are labeled with fluorescent proteins. The protocols detailed here exploit these advantages and have the potential to reveal critical insights into host-IAV interactions that may ultimately translate into the clinic.

Systemic and localized zebrafish infection models for human influenza A virus are demonstrated. Using a systemic infection model, zebrafish can be used to screen antiviral drugs. Using a localized infection model, zebrafish can be used to 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

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte extravasation might result in pathophysiological changes in the CNS. Thus, investigation of lymphocyte migration into the CNS is important to understand inflammatory CNS diseases and to develop new therapy approaches. Here we present an in vitro model of the human blood-brain barrier to study lymphocyte extravasation. Human brain microvascular endothelial cells (HBMEC) are confluently grown on a porous polyethylene terephthalate transwell insert to mimic the endothelium of the blood-brain barrier. Barrier function is validated by zonula occludens immunohistochemistry, transendothelial electrical resistance (TEER) measurements as well as analysis of evans blue permeation. This model allows investigation of the diapedesis of rare lymphocyte subsets such as CD56brightCD16dim/- NK cells. Furthermore, the effects of other cells, cytokines and chemokines, disease-related alterations, and distinct treatment regimens on the migratory capacity of lymphocytes can be studied. Finally, the impact of inflammatory stimuli as well as different treatment regimens on the endothelial barrier can be analyzed.

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

Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro.

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

Many chronic airway diseases result in mucus plugging of the airways. Lungs of an individual with cystic fibrosis are an exemplary case where their mucus-plugged bronchioles create a favorable habitat for microbial colonization. Various pathogens thrive in this environment interacting with each other and driving many of the symptoms associated with CF disease. Like any microbial community, the chemical conditions of their habitat have a significant impact on the community structure and dynamics. For example, different microorganisms thrive in differing levels of oxygen or other solute concentrations. This is also true in the CF lung, where oxygen concentrations are believed to drive community physiology and structure. The methods described here are designed to mimic the lung environment and grow pathogens in a manner more similar to that from which they cause disease. Manipulation of the chemical surroundings of these microbes is then used to study how the chemistry of lung infections governs its microbial ecology. The method, called the WinCF system, is based on artificial sputum medium and narrow capillary tubes meant to provide an oxygen gradient similar to that which exists in mucus-plugged bronchioles. Manipulating chemical conditions, such as the media pH of the sputum or antibiotics pressure, allows for visualization of the microbiological differences in those samples using colored indicators, watching for gas or biofilm production, or extracting and sequencing the nucleic acid contents of each sample.

The mucus plugged airways of cystic fibrosis (CF) patients are an ideal environment for microbial pathogens to thrive. The manuscript describes a novel method for studying the CF lung microbiome in an environment that mimics where they cause disease and how alterations of chemical conditions can drive microbial dynamics.

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

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors (PRRs) such as the retinoic acid (RIG-I) like receptors (RLRs) RIG-I and melanoma differentiation-associated protein 5 (MDA-5) leads to the induction of the innate immune response. The formation and intracellular distribution of dsRNA in positive-sense RNA virus infection has been well characterized by microscopy. Many negative-sense RNA viruses, including some arenaviruses, trigger the innate immune response during infection. However, negative-sense RNA viruses were thought to produce low levels of dsRNA, which hinders the imaging study of PRR recognition of viral dsRNA. Additionally, infection experiments with highly pathogenic arenaviruses must be performed in high containment biosafety level facilities (BSL-4). The interaction between viral RNA and PRRs for highly pathogenic RNA virus is largely unknown due to the additional technical challenges that researchers need to face in the BSL-4 facilities. Recently, a monoclonal antibody (Mab) (clone 9D5) originally used for pan-enterovirus detection has been found to specifically detect dsRNA with a higher sensitivity than the traditional J2 or K1 anti-dsRNA antibodies. Herein, by utilizing the 9D5 antibody, we describe a confocal microscopy protocol that has been used successfully to visualize dsRNA, viral protein and PRR simultaneously in individual cells infected by arenavirus. The protocol is also suitable for imaging studies of dsRNA and PRR distribution in pathogenic arenavirus infected cells in BSL4 facilities.

Double-stranded RNA produced during RNA virus replication can be recognized by pattern recognition receptors to induce an innate immune response. For negative-sense RNA viruses, the interaction between the low-level dsRNA and PRRs remains unclear. We have developed a confocal microscopy method to visualize arenavirus dsRNA and PRR in individual cells.

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 ...