This work was supported by NIGMS, National Institutes of Health (GM094623). Significant portions of this work were performed in the Environmental Molecular Sciences Laboratory, a DOE/BER national scientific user facility located at the Pacific Northwest National Laboratory (PNNL). PNNL is operated for the DOE by Battelle under Contract DE-AC05-76RLO1830.

1.提前准备<ol><li>温暖的无菌磷酸盐缓冲盐水（PBS），以37℃。 </li><li>温暖的Dulbecco改性的Eagle培养基（DMEM）和极限必需培养基（MEM），补充有10％胎牛血清（FBS），100国际单位/毫升青霉素和100微克/毫升链霉素至37℃。注：分别为这些代表正常生长介质（NGM）为RAW和HeLa细胞。 </li></ol> 2.准备细胞<ol><li>生长的RAW 264.7细胞中的NGM 70-90％汇合。 <ol><li>保持在湿润的95％空气/ 5％CO 2的气氛中的细胞在37℃。 </li></ol></li><li>生长的HeLa细胞中的NGM 70-90％汇合。 <ol><li>保持在湿润的95％空气/ 5％CO 2的气氛中的细胞在37℃。 </li></ol></li><li>收集前，用无菌PBS洗涤细胞单层一次。 </li><li>收集在无菌锥形管中预汇合的细胞。 <ol><li>轻轻刮去RAW细胞用橡皮警察在PBS，反复吹打分散细胞聚集。 </li><li>分离HeLa细胞与0.25％胰蛋白酶溶液，直到视力检查显示了从培养表面解离。例如，使用2〜3毫升为T-75烧瓶中。相应地调整音量，保证胰蛋白酶溶液覆盖整个生长表面。 <ol><li>淬火用含有10％FBS的NGM离解反应。 </li><li>使用NGM的至少两倍的量，以胰蛋白酶，反复移液以分散细胞聚集。 </li></ol></li></ol></li><li>轻轻离心沉淀细胞，在900×g离心4分钟。 </li><li>重悬在相同体积用无菌PBS胰蛋白酶/骤冷溶液。 </li><li>计数使用血球或粒子计数器细胞。 </li><li>轻轻离心沉淀细胞，在900×g离心4分钟。 </li><li>重悬在PBS中的适当体积为5.5×10 6〜6.0×10 6个细胞/ ml。注：例如，一个T-75瓶将产生逼近的ately 7.5×10 6个细胞29。 </li><li>保持细胞悬液在冰上直到电。 </li></ol> 3.准备电穿孔<ol><li>预冷电试管（0.7厘米的缝隙）冰上。 </li><li>打开电设备和设置电压0.3千伏。 注：电容和电阻并不在我们的电穿孔（由制造商设定在10μF和600Ω）可调设置。 </li><li>填回收板的NGM，并在37℃下平衡的湿润的95％空气/ 5％CO 2的气氛中</li></ol> 4.电穿孔<ol><li>放置400微升细胞悬浮液中预先冷却的反应杯，并添加20微克选择蛋白与样本池（50微克/毫升）。 </li><li>弗里克试管轻轻〜10次混合而不损伤细胞。注意：反应杯也可以倒置几次以调匀，但不要吸管上下或涡流，以避免损坏电池。 </li><l我&gt;干外实验皿用纸巾或其它抽头的，以避免在所述电穿孔的电弧。 </li><li>在0.3千伏为1.5-1.7毫秒电​​穿孔的样品。注意：这是典型的用于本研究。 </li><li>紧接着电轻弹试管轻轻〜10次调匀。 </li></ol> 5.电镀细胞<ol><li>商店试管在冰上，直到准备电穿孔细胞放置在复苏板块。 </li><li>对于大多数下游分析，洗涤细胞1×预热的NGM去除多余的效应蛋白。 <ol><li>去除细胞进行分析，并暂停在3-5毫升NGM的。 </li><li>通过离心沉淀细胞，在900×g离心4分钟。 </li><li>悬浮在NGM足够的音量所需的板尺寸（ 如 2毫升35毫米盘）。 </li></ol></li><li>对于下游分析去除细胞的适当的量。 <ol><li>例1：板到玻璃底菜显微镜分析。 </li><li>例2：板INTØ细胞培养塑料蛋白质分析等亲和纯化。 </li></ol></li><li>允许细胞在平衡板在加湿的95％空气/ 5％CO 2的气氛中以回收在37℃下至少4小时。 </li></ol> 6.显微镜分析 6.1）固定/免疫染色<ol><li>洗涤细胞后4小时恢复期1×用无菌PBS。 </li><li>固定的细胞在100％甲醇中，在室温下2分钟。使用足够的甲醇，以完全覆盖细胞（ 如 2毫升35毫米盘）。 </li><li>洗3次，用无菌PBS洗涤。 </li><li>通透细胞用0.4％的Triton X-100的PBS 15分钟。例如，使用方法1毫升35毫米直径板。 <ol><li>根据表位与目标蛋白的位置调整通透的Triton X-100和实力的长度。注意：最好的结果将需要凭经验为每个目标确定的，但是在上述条件应足以大多数Cytosolic目标。 </li></ol></li><li>用5％的牛血清白蛋白（BSA）的PBS阻断1小时，在室温。例如，使用方法1毫升35毫米直径板。 </li><li>洗涤3次，用PBS。 </li><li>孵育在抗体结合解第一抗体（在PBS中0.1％的Triton X-100和1％BSA）中过夜，在4 &#160; °下与温和的摇摆。例如，使用0.5毫升为35毫米直径板。 <ol><li>按照制造商的建议抗体稀释。注意：例如，链霉亲和结合肽标签（SBP-标签）抗体被用于以1：1000稀释，而PKN1抗体被用于以1：200的稀释。 </li></ol></li><li>洗涤4X，用PBS。 </li><li>孵育在抗体结合解适当荧光标记的二抗1小时，在室温下，避光。 <ol><li>例如，使用的Alexa 488或Alexa 647进行1小时，在室温下进行。 <ol><li>按照制造商的建议蚂蚁ibody稀释。 （ 例如，约1：1000本研究）。 </li></ol></li><li>添加其它污渍根据需要，例如，5微米的小麦胚芽agglutinnin（WGA）缀合的Alexa 647或DAPI根据制造商的建议，进行1小时，在室温下进行。 </li></ol></li><li>洗涤5次，用PBS，并在4℃储存，避光，直到准备图像。 </li></ol> 6.2）共聚焦显微镜和图像分析<ol><li>在使用63X油浸物镜倒置共聚焦显微镜图像样本。 <ol><li>图像的绿色通道使用氩激光的488nm处线，具有492-542纳米之间的带宽发射。 </li><li>图像的红色通道用一个633纳米二极管激光器，具有640-718纳米之间的带宽发射。 </li><li>图像蓝色通道使用一个405nm的二极管激光器，具有407-453纳米之间的带宽发射。 </li><li>确保多通道的z堆叠覆盖细胞体积的全部。 </li></ol></li><li>处理图像与适当的图像处理软件。 </li></ol> 7.亲和纯化<ol><li>后4小时恢复期间用4℃的PBS洗两次电穿孔的细胞。 </li><li>裂解用〜加入1.0ml裂解缓冲液（1％的Triton X-100与蛋白酶抑制剂混合物和磷酸酶抑制剂的PBS）在冰上。根据制造商的说明，使用抑制剂。注意：例如，无论是在本研究中使用的磷酸酶和蛋白酶抑制剂在100倍供给的，但其他的制剂应该工作得同样好。 </li><li>及时刮掉细胞橡胶警察并收集到锥形管。 </li><li>裂解剧烈涡旋和超声。注：其它裂解方法可工作得同样为好，但将需要效率来凭经验确定作为对测定规定的适用性。 <ol><li>超声处理3×30秒，间歇漩涡。 </li></ol></li><li>离心机以10,000×g离心10分钟，在4℃以收集细胞碎片和不溶性聚集体;保存上清液。 </li><li>结合电穿孔细胞裂解物等体积（〜1.0ml）中，用50微升链霉亲和琼脂糖树脂悬浮液在4℃下过夜终过端旋转。注意：此树脂捕获经由其链霉结合肽的亲和标签的电穿孔蛋白（和相关的复合物）。 </li><li>离心在2500 xg离心2分钟，弃上清。 </li><li>用40柱床体积（约1毫升）的PBS洗涤两次。 </li><li>添加30微升4×LDS加样缓冲液（141毫摩尔Tris碱，2％的LDS，10％甘油，0.51毫摩尔EDTA，0.22毫SERVA蓝G，0.175毫酚红，pH值8.5），20微升DIH 2 O和1微升0.5M的三（2-羧乙基）膦（TCEP），允许一些量留在珠。 </li><li>热，在95℃下进行10分钟，并在冰上冷却。 </li><li>自旋&gt; 10,000 xg离心在4℃下5分钟。 </li><li>收集上清。 </li><li>使用合适的抗体注意进行免疫印迹：他重，抗PKN1初级抗体被使用。 </li></ol>

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D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+GtgE,+a+novel+virulence+factor+encoded+on+the+Gifsy-2+bacteriophage+of+Salmonella+enterica+serovar+Typhimurium.">Identification of GtgE, a novel virulence factor encoded on the Gifsy-2 bacteriophage of Salmonella enterica serovar Typhimurium.</a> Journal of Bacteriology. 184, 5234-5239 (2002).</li><li>Niemann, G. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discovery+of+novel+secreted+virulence+factors+from+Salmonella+enterica+serovar+Typhimurium+by+proteomic+analysis+of+culture+supernatants.">Discovery of novel secreted virulence factors from Salmonella enterica serovar Typhimurium by proteomic analysis of culture supernatants.</a> Infection and Immunity. 79, 33-43 (2011).</li><li>Spano, S., Liu, X., 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=Proteolytic+targeting+of+Rab29+by+an+effector+protein+distinguishes+the+intracellular+compartments+of+human-adapted+and+broad-host+Salmonella.">Proteolytic targeting of Rab29 by an effector protein distinguishes the intracellular compartments of human-adapted and broad-host Salmonella.</a> Proceedings of the National Academy of Sciences of the United States of America. 108, 18418-18423 (2011).</li><li>Haraga, A., Miller, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Salmonella+type+III+secretion+effector+interacts+with+the+mammalian+serine/threonine+protein+kinase+PKN1.">A Salmonella type III secretion effector interacts with the mammalian serine/threonine protein kinase PKN1.</a> Cellular Microbiology. 8, 837-846 (2006).</li><li>Zinchuk, V., Grossenbacher-Zinchuk, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+colocalization+analysis+of+confocal+fluorescence+microscopy+images.">Quantitative colocalization analysis of confocal fluorescence microscopy images.</a> Current Protocols in Cell Biology / Editorial Board, Juan S. Bonifacino ... [et al]. 4 (Unit 4.19), (2011).</li><li>Kimple, M. E., Sondek, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overview+of+affinity+tags+for+protein+purification.">Overview of affinity tags for protein purification.</a> Current Protocols in Protein Science / Editorial Board, John E. Coligan ... [et al]. 9 (Unit 9.9), (2004).</li></ol>

作者什么都没有透露。

从致病细菌分泌的效应已经发展到在宿主细胞内环境的作用，因此它是有帮助的主机内研究它们在原位 。引入感兴趣的特定效应物到宿主细胞中允许相关病原体 - 宿主相互作用进行研究孤立，而不与其他细菌蛋白质的干扰。的目标是探讨电穿孔以引入细菌效应蛋白进入真核宿主细胞，从而避免了与转染或转导相关的挑战的一种手段。绿色荧光蛋白作为对照和沙门氏菌效应蛋白进行了...

许多革兰氏阴性菌采用专门的分泌系统注入毒性相关蛋白（称为效应器）直接导入宿主细胞1-5。这些效应物具有广泛的生物学功能，包括：抑制宿主免疫的，细胞骨架的变化，细胞内运输和信号传导，转录变化的修饰，和宿主蛋白酶的改变6-9。某些效应的功能是已知的，但是在主机的目标和其他许多人的生化作用（多个）仍有待确定。而比较野生型和重组体细菌感染是研究细胞内效应器毒力机制的有效的方法，它通常是有利的引入个体效应入​​宿主细胞。因此，简单的方法，用于在宿主细胞中的上下文中引入和表征细菌效应蛋白是高度期望的。 简化了实验分析的Wi第一个效应是至关重要的其它效应可能有相反的或多余的功能。要做到这一点简化，研究人员先前推出的大分子物质进入细胞的许多不同的方法，包括病毒转导10，显微注射11，刮装载12,13，细胞融合与化学诱导注射14，专有蛋白“转”试剂15，磷酸钙沉淀16，和电穿孔17-20。所引入的分子范围从核酸包括DNA，RNA和RNA干扰物种的蛋白质，细胞不透染料和抗体的细胞内目标21,22。一些方法都有局限性，包括大分子的可以引入的类型，以及特定下游分析可由于高的细胞毒性，作用破坏机制，低功效，或导入效率的限制。转染，一个ofte正使用的表达方法在哺乳动物细胞中的细菌的基因，也受到限制，一些有关的宿主细胞类型，例如巨噬细胞和原代细胞，是朝着转染特别耐。除此之外，它难以控制导入外源DNA后产生的细菌蛋白的水平。 许多工作已建立的核酸引入细菌和哺乳动物细胞作为通用实验室技术电穿孔;然而，人们一直在研究转化为在生理条件下提供的蛋白质进入细胞的最佳方法。对蛋白质的转染报告是有希望的，但需要昂贵的试剂和优化。引入潜在的有毒细菌效应成各种各样的以最小的成本细胞靶的愿望促使我们考虑电穿孔作为用于体内研究这些蛋白质的方法。 蛋白质电23-25 ​​是一个满足HOD引入蛋白质成经由electropermeabilization活细胞，也被称为电-转染或电注入26。这种技术使用高强度的电脉冲以在细胞膜孔。这些可逆孔隙允许通常由细胞内的空间中排除大分子进入细胞。在去除外部电场，膜可以重新密封，使细胞能留存穿过孔27,28的分子。 一个标准的电穿孔被用于本研究中一致地引入细菌效应入两个小鼠巨噬细胞样细胞和人上皮细胞。该方法是快速的，有效的，廉价的，在细胞生存力没有明显的下降。引入的蛋白可通过免疫荧光显微镜进行可视化，或用于功能测定法。这已被证明使用绿色荧光蛋白（GFP），为无毒的标准，以及2 沙门氏菌效应蛋白，SspH1和GtgE。我们建议蛋白电作为剧目的细菌毒力的蛋白质及其功能的真核宿主细胞研究的一个额外的工具。

<table border="0" cellpadding="0" cellspacing="0" width="1132">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:532px;">Material or Equipment</td>
			<td style="width:177px;">Company</td>
			<td style="width:108px;">Catalog Number</td>
			<td style="width:315px;">Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.25% Trypsin-EDTA Solution</td>
			<td>Cellgro</td>
			<td>25-050-Cl</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.4cm Gap-disposable electroporation cuvettes</td>
			<td>Bio-Rad</td>
			<td>165-2088</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">100% Methanol</td>
			<td>Any</td>
			<td>N/A</td>
			<td>Flammable, Toxic</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bovine Serum Albumin (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>A4919</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell Counting Apparatus - Hemocytometer or Coulter Counter</td>
			<td>Beckman Coulter</td>
			<td>Model Z1</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Cell Culture Incubator</td>
			<td>Any</td>
			<td>N/A</td>
			<td>Humidified 95% air/5% CO2 atmosphere at 37 &#176;C&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell Culture Plastic</td>
			<td>Any</td>
			<td>N/A</td>
			<td>Cell culture flasks/plates, pipets, tubes, rubber policeman</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#39;s Modification of Eagle&#8217;s Medium (DMEM)</td>
			<td>Cellgro</td>
			<td>10-013</td>
			<td>Warm to 37 &#176;C&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Electroporator</td>
			<td>Bio-Rad</td>
			<td>E. coli Pulser</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal Bovine Serum (FBS)</td>
			<td>Cellgro</td>
			<td>35-016-CV</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fluorescent confocal microscope&#160;</td>
			<td>Ziess</td>
			<td>Model&#160; LSM 710&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glass Bottom Dishes for Microscopy</td>
			<td>Wilco Wells</td>
			<td>HBSt-3522</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HALT Protease Inhibitor Cocktail</td>
			<td>Pierce</td>
			<td align="right">78430</td>
			<td>Corrosive, Toxic</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HeLa Cell Line</td>
			<td>ATCC</td>
			<td>ATCC CCL-2</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">LDS 4X Loading Buffer</td>
			<td>Invitrogen</td>
			<td>NP0007</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Minimal Essential Medium (MEM)&#160;</td>
			<td>Cellgro</td>
			<td>10-010</td>
			<td>Warm to 37 &#176;C&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Other fluorescent stains (WGA, DAPI) in conjunction with anti-fade reagent</td>
			<td>Any</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin/Streptomycin</td>
			<td>Cellgro</td>
			<td>30-002-Cl</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RAW 264.7 Cell line</td>
			<td>ATCC</td>
			<td>TIB-71</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Primary Antibody Against Target of Interest</td>
			<td>Any</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Secondary Antibody Conjugated to Fluorophore</td>
			<td>Any</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phosphate Buffered Saline</td>
			<td>Any</td>
			<td>N/A</td>
			<td>Chill to 4 &#176;C&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sterile Phosphate Buffered Saline</td>
			<td>Any</td>
			<td>N/A</td>
			<td>Warm to 37 &#176;C&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Streptavidin Agarose Resin Suspension&#160;</td>
			<td>Pierce</td>
			<td align="right">20353</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Table Top Centrifuge Capable of Accepting Conical Tubes (swinging bucket preferred)</td>
			<td>Any</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TCEP</td>
			<td>Sigma-Aldrich</td>
			<td align="right">646547</td>
			<td>Corrosive, Toxic</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T8585</td>
			<td>Irritant, Toxic</td>
		</tr>
	</tbody>
</table>

electroporation of functional bacterial effectors into mammalian cells

蛋白质相互作用的活细胞中的上下文中的研究可以产生大约本地化，动力学和相互作用伙伴的关键信息。这些信息是在宿主 - 病原体相互作用的背景下尤为可贵。许多病原体的蛋白质的宿主细胞内的多种方式，如功能，从而使宿主的免疫系统和存活的逃避细胞内环境中。研究这些病原体蛋白宿主-细胞相互作用，几种方法是常用的，其中包括： 在体内感染用表达一个标记或突变蛋白，或引入病原体的基因通过转染或转导的菌株。每一种方法都有优点和缺点。我们试图直接引入外源蛋白进入细胞的一种手段。电穿孔通常用于将核酸引入细胞中，但已被更很少应用于蛋白质虽然生物物理基础是完全一样的。一个标准的电穿孔被用于引入亲和标签效应细菌到哺乳动物细胞。人类上皮细胞和小鼠巨噬细胞用传统方法进行培养，分离，并置于0.4厘米的空隙电试管与感兴趣的外源细菌病原体蛋白（ 例如鼠伤寒沙门氏菌 GtgE）。电穿孔（0.3千伏）和短（4小时）的恢复期后，细胞内蛋白质是由通过其亲和标记物的荧光标记的蛋白，并检查空间和时间分布通过共聚焦显微镜证实。电穿孔的蛋白质也被证明是功能性细胞内，并能正确的亚细胞贩卖和蛋白质 - 蛋白质相互作用的。而该外源蛋白倾向于积聚在细胞的表面上，将电穿孔样品具有大量增加的细胞内效应器浓度相对单独孵育。该协议是简单，速度不够快，在做AParallel时尚，允许病原体蛋白在宿主细胞中的高通量特征包括亚细胞靶向和毒性蛋白的功能。

作为概念的初步证明，纯化的绿色荧光蛋白被成功地引入到使用电穿孔哺乳动物细胞。绿色荧光蛋白，近似27 kD的分子量蛋白质通常引入哺乳动物细胞（通常为质粒DNA表​​达），为无显著细胞毒性的分子生物学工具。 HeLa细胞孵育（ 图1A）或电穿孔（ 图1B）与25微克/毫升的GFP，随后通过免疫荧光共聚焦显微镜检查荧光GFP信号。以证明GFP是蜂窝胞质溶胶内，将细胞也沾上?...

Watch this Scientific Journal Video about Electroporation of Functional Bacterial Effectors into Mammalian Cells at JoVE.com

Electroporation of Functional Bacterial Effectors into Mammalian Cells

Electroporation was used to insert purified bacterial virulence effector proteins directly into living eukaryotic cells. Protein localization was monitored by confocal immunofluorescence microscopy. This method allows for studies on trafficking, function, and protein-protein interactions using active exogenous proteins, avoiding the need for heterologous expression in eukaryotic cells.

电功能性细菌效应器到哺乳动物细胞

The study of protein interactions in the context of living cells can generate critical information about localization, dynamics, and interacting partners. ...

electroporation-of-functional-bacterial-effectors-into-mammalian-cells

Research

JoVE Journal

Immunology and Infection

10.0K 次观看.  Pacific Northwest National Laboratory. Electroporation was used to insert purified bacterial virulence effector proteins directly into living eukaryotic cells. Protein localization was monitored by confocal immunofluorescence microscopy. This method allows for studies on trafficking, function, and protein-protein interactions using active exogenous proteins, avoiding the need for heterologous expression in eukaryotic cells. 

视频: Electroporation of Functional Bacterial Effectors into Mammalian Cells

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.

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

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host cells1. These adhesins rely on interactions with host cell surface receptors or soluble proteins acting as a bridge between bacteria and host. Adhesion is a critical first step prior to invasion and/or secretion of toxins, thus it is a key event to be studied in bacterial pathogenesis. Furthermore, adhered bacteria often induce exquisitely fine-tuned cellular responses, the studies of which have given birth to the field of 'cellular microbiology'2. Robust assays for bacterial adhesion on host cells and their invasion therefore play key roles in bacterial pathogenesis studies and have long been used in many pioneer laboratories3,4. These assays are now practiced by most laboratories working on bacterial pathogenesis.
Here, we describe a standard adherence assay illustrating the contribution of a specific adhesin. We use the Escherichia coli strain 27875, a human pathogenic strain expressing the autotransporter Adhesin Involved in Diffuse Adherence (AIDA). As a control, we use a mutant strain lacking the aidA gene, 2787&Delta;aidA (F. Berthiaume and M. Mourez, unpublished), and a commercial laboratory strain of E. coli, C600 (New England Biolabs). The bacteria are left to adhere to the cells from the commonly used HEp-2 human epithelial cell line. This assay has been less extensively described before6.

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

This protocol is a simple bacterial adhesion assay consisting in counting the numbers of bacterial colony forming units that are adhered onto cultured cells. The assay is robust, independent of the adhesin studied, and numerous variations are used in most laboratories working on bacterial pathogenesis.

到哺乳动物的上皮细胞在体外细菌粘附的含量

To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host ...

Isolation of Functional Cardiac Immune Cells

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5 These immune cells, which include mast cells, T-cells and macrophages; store and release a variety of biologically active mediators including cytokines and proteases such as tryptase.6-8 These mediators have been shown to be key players in extracellular matrix metabolism by activating matrix metalloproteinases or causing collagen accumulation by modulating the cardiac fibroblasts' function.9-11 However, available techniques for isolating cardiac immune cells have been problematic because they use bacterial collagenase to digest the myocardial tissue. This technique causes activation of the immune cells and thus a loss of function. For example, cardiac mast cells become significantly less responsive to compounds that cause degranulation.12 Therefore, we developed a technique that allows for the isolation of functional cardiac immune cells which would lead to a better understanding of the role of these cells in cardiac disease.13, 14
This method requires a familiarity with the anatomical location of the rat's xiphoid process, axilla and falciform ligament, and pericardium of the heart. These landmarks are important to increase success of the procedure and to ensure a higher yield of cardiac immune cells. These isolated cardiac immune cells can then be used for characterization of functionality, phenotype, maturity, and co-culture experiments with other cardiac cells to gain a better understanding of their interactions.

This method for isolating functional immune cells from the heart provides an alternative to the conventional methods of collagenase digestion, which causes unwanted immune cell activation, resulting in a decreased responsiveness of these cells. Our method of isolation yields functional cardiac immune cells by avoiding problems associated with enzymatic digestion.

心脏功能免疫细胞的分离

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5  These immune cells, which ...

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques enable the observation of live cellular behavior inside the organ but can be challenging in some types of cancer due to organ and tumor accessibility such as intestine. Direct observation of intestinal tumors has not been previously reported. A surgical procedure described here allows direct observation of myeloid cell dynamics within the intestinal tumors in live mice by using transgenic fluorescent reporter mice and injectable tracers or antibodies. For this purpose, a four-color, multi-region, micro-lensed spinning disk confocal microscope that allows long-term continuous imaging with rapid image acquisition has been used. ApcMin/+ mice that develop multiple adenomas in the small intestine are crossed with c-fms-EGFP mice to visualize myeloid cells and with ACTB-ECFP mice to visualize intestinal epithelial cells of the crypts. Procedures for labeling different tumor components, such as blood vessels and neutrophils, and the procedure for positioning the tumor for imaging through the serosal surface are also described. Time-lapse movies compiled from several hours of imaging allow the analysis of myeloid cell behavior in situ in the intestinal microenvironment.

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

By using transgenic reporter mice and injectable fluorescent labels, long-term intravital spinning disk confocal microscopy enables direct visualization of myeloid cell behavior into intestinal adenoma in the ApcMin/+ colorectal cancer model.

在髓系细胞动态实时成像<em&gt;装甲运兵车<sup&gt;最小/ +</sup</em&gt;肠道肿瘤通过旋转盘共聚焦显微镜

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques ...

Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. Polysaccharides such as glycogen, glycoproteins, and glycolipids stain bright magenta making it easy to enumerate positive and negative cells within the tissue. In muscle cells PAS staining is used to determine the glycogen content in different types of muscle cells, while in blood cell samples PAS staining has been explored as a diagnostic tool for a variety of conditions. Blood contains a proportion of white blood cells that belong to the immune system. The notion that cells of the immune system possess glycogen and use it as an energy source has not been widely explored. Here, we describe an adapted version of the PAS staining protocol that can be applied on peripheral blood mononuclear immune cells from human venous blood. Small cells with PAS-positive granules and larger cells with diffuse PAS staining were observed. Treatment of samples with amylase abrogates these patterns confirming the specificity of the stain. An alternate technique based on enzymatic digestion confirmed the presence and amount of glycogen in the samples. This protocol is useful for hematologists or immunologists studying polysaccharide content in blood-derived lymphocytes.

Periodic acid Schiff staining is a technique that visualizes the polysaccharide content of tissues. This article demonstrates periodic acid Schiff staining protocol adapted for use on peripheral blood mononuclear cells purified from human venous blood. Such samples are enriched for lymphocytes and other white blood cells of the immune system.

与碘酸雪夫染色检测糖原的外周血单个核细胞

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. ...

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 ...

Bacterial Artificial Chromosomes: A Functional Genomics Tool for the Study of Positive-strand RNA Viruses

Reverse genetics, an approach to rescue infectious virus entirely from a cloned cDNA, has revolutionized the field of positive-strand RNA viruses, whose genomes have the same polarity as cellular mRNA. The cDNA-based reverse genetics system is a seminal method that enables direct manipulation of the viral genomic RNA, thereby generating recombinant viruses for molecular and genetic studies of both viral RNA elements and gene products in viral replication and pathogenesis. It also provides a valuable platform that allows the development of genetically defined vaccines and viral vectors for the delivery of foreign genes. For many positive-strand RNA viruses such as Japanese encephalitis virus (JEV), however, the cloned cDNAs are unstable, posing a major obstacle to the construction and propagation of the functional cDNA. Here, the present report describes the strategic considerations in creating and amplifying a genetically stable full-length infectious JEV cDNA as a bacterial artificial chromosome (BAC) using the following general experimental procedures: viral RNA isolation, cDNA synthesis, cDNA subcloning and modification, assembly of a full-length cDNA, cDNA linearization, in vitro RNA synthesis, and virus recovery. This protocol provides a general methodology applicable to cloning full-length cDNA for a range of positive-strand RNA viruses, particularly those with a genome of &gt;10 kb in length, into a BAC vector, from which infectious RNAs can be transcribed in vitro with a bacteriophage RNA polymerase.

Here, a protocol is presented for creating an infectious bacterial artificial chromosome containing a full-length cDNA of the positive-strand genomic RNA of Japanese encephalitis virus. This protocol can be used to construct a functional cDNA of other positive-strand RNA viruses, making it a powerful genomic tool for studying virus biology.

细菌人工染色体：对于正链RNA病毒研究的功能基因组学工具

Reverse genetics, an approach to rescue infectious virus entirely from a cloned cDNA, has revolutionized the field of positive-strand RNA viruses, whose ...

Implementation of a Permeable Membrane Insert-based Infection System to Study the Effects of Secreted Bacterial Toxins on Mammalian Host Cells

Many bacterial pathogens secrete potent toxins to aid in the destruction of host tissue, to initiate signaling changes in host cells or to manipulate immune system responses during the course of infection. Though methods have been developed to successfully purify and produce many of these important virulence factors, there are still many bacterial toxins whose unique structure or extensive post-translational modifications make them difficult to purify and study in in vitro systems. Furthermore, even when pure toxin can be obtained, there are many challenges associated with studying the specific effects of a toxin under relevant physiological conditions. Most in vitro cell culture models designed to assess the effects of secreted bacterial toxins on host cells involve incubating host cells with a one-time dose of toxin. Such methods poorly approximate what host cells actually experience during an infection, where toxin is continually produced by bacterial cells and allowed to accumulate gradually during the course of infection. This protocol describes the design of a permeable membrane insert-based bacterial infection system to study the effects of Streptolysin S, a potent toxin produced by Group A Streptococcus, on human epithelial keratinocytes. This system more closely mimics the natural physiological environment during an infection than methods where pure toxin or bacterial supernatants are directly applied to host cells. Importantly, this method also eliminates the bias of host responses that are due to direct contact between the bacteria and host cells. This system has been utilized to effectively assess the effects of Streptolysin S (SLS) on host membrane integrity, cellular viability, and cellular signaling responses. This technique can be readily applied to the study of other secreted virulence factors on a variety of mammalian host cell types to investigate the specific role of a secreted bacterial factor during the course of infection.

Here, a method using a permeable membrane insert-based infection system to study the effects of Streptolysin S, a secreted toxin produced by Group A Streptococcus, on keratinocytes is described. This system can be readily applied to the study of other secreted bacterial proteins on various host cell types during infection.

基于插入渗透膜感染系统的实现研究分泌细菌病毒对哺乳动物宿主细胞的作用

Many bacterial pathogens secrete potent toxins to aid in the destruction of host tissue, to initiate signaling changes in host cells or to manipulate ...

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.

从非耕种植物病原菌利用酵母双杂交筛选Unravelling一个细菌效应中的作用

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

Immunofluorescence Analysis of Stress Granule Formation After Bacterial Challenge of Mammalian Cells

Fluorescent imaging of cellular components is an effective tool to investigate host-pathogen interactions. Pathogens can affect many different features of infected cells, including organelle ultrastructure, cytoskeletal network organization, as well as cellular processes such as Stress Granule (SG) formation. The characterization of how pathogens subvert host processes is an important and integral part of the field of pathogenesis. While variable phenotypes may be readily visible, the precise analysis of the qualitative and quantitative differences in the cellular structures induced by pathogen challenge is essential for defining statistically significant differences between experimental and control samples. SG formation is an evolutionarily conserved stress response that leads to antiviral responses and has long been investigated using viral infections1. SG formation also affects signaling cascades and may have other still unknown consequences2. The characterization of this stress response to pathogens other than viruses, such as bacterial pathogens, is currently an emerging area of research3. For now, quantitative and qualitative analysis of SG formation is not yet routinely used, even in the viral systems. Here we describe a simple method for inducing and characterizing SG formation in uninfected cells and in cells infected with a cytosolic bacterial pathogen, which affects the formation of SGs in response to various exogenous stresses. Analysis of SG formation and composition is achieved by using a number of different SG markers and the spot detector plug-in of ICY, an open source image analysis tool.

We describe a method for the qualitative and quantitative analysis of stress granule formation in mammalian cells after the cells are challenged with bacteria and a number of different stresses. This protocol can be applied to investigate the cellular stress granule response in a wide range of host-bacterial interactions.

哺乳动物细胞细菌挑战后应激颗粒形成的免疫荧光分析

Fluorescent imaging of cellular components is an effective tool to investigate host-pathogen interactions. Pathogens can affect many different features of ...