Name: imaging inlc secretion to investigate cellular infection by the bacterial pathogen listeria monocytogenes
Uploaded: 2013-09-19T22:00:00
Description: Watch this Scientific Journal Video about Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes at JoVE.com

研究体育科萨特实验室是支持的巴斯德研究所，研究所国家德拉桑特等德拉RECHERCHEMédicale，研究所法国国家农艺，ERC高级格兰特（233348），该法新社国立德拉RECHERCHE（批准MIE- SignRupVac），路易 - Jeantet基金会和基金会乐罗克莱斯Mousquetaires酒店。 AK是从巴斯德 - 巴黎大学国际博士课程/学院卡诺弊端Infectieuses奖学金的获得者。我们感谢杰森 - 美世优化细胞转染协议。

1。细胞的制备和细菌培养，转染工具和一抗<ol><li>准备一个新鲜的琼脂平板上分离单个L。菌菌落从细菌甘油（50％饱和菌液过夜培养glycerol/50％）保持在-80°C。 <ol><li>使用铝框（保持在-80℃），以输送在脑心浸液（BHI）琼脂平板冻结的细菌甘油，条纹细菌。 </li><li>在48小时（或直到单个细菌菌落可被分离）将培养板在37℃。 </li><li>保持这种工作板后，在4℃在1个月（它将被用于接种液体培...

<ol>
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J., Veiga, E., Yin, H. L., Cossart, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Type+II+phosphatidylinositol+4-kinases+promote+Listeria+monocytogenes+entry+into+target+cells.">Type II phosphatidylinositol 4-kinases promote Listeria monocytogenes entry into target cells.</a> Cellular Microbiology. 9, 2381-2390 (2007).</li><li>Hamon, M. A., 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=Histone+modifications+induced+by+a+family+of+bacterial+toxins.">Histone modifications induced by a family of bacterial toxins.</a> Proceedings of the National Academy of Sciences USA. 104, 17555-17559 (2007).</li><li>Ribet, 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=Listeria+monocytogenes+impairs+SUMOylation+for+efficient+infection.">Listeria monocytogenes impairs SUMOylation for efficient infection.</a> Nature. 464, 1192-1195 (2010).</li><li>R&#228;met, M., Manfruelli, P., Pearson, A., Mathey-Prevot, B., Ezekowitz, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+genomic+analysis+of+phagocytosis+and+identification+of+a+Drosophila+receptor+for+E.+coli.">Functional genomic analysis of phagocytosis and identification of a Drosophila receptor for E. coli.</a> Nature. 416, 644-648 (2002).</li><li>Agaisse, H., Burrack, L. S., Philips, J. A., Rubin, E. J., Perrimon, N., Higgins, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+RNAi+screen+for+host+factors+required+for+intracellular+bacterial+infection.">Genome-wide RNAi screen for host factors required for intracellular bacterial infection.</a> Science. 309, 1248-1251 (2005).</li><li>Cheng, L. W., Viala, J. P., Stuurman, N., Wiedemann, U., Vale, R. D., Portnoy, D. 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几个参数是我们InlC的检测协议的成功，包括使用健康细胞系显示一个足够大的胞质，以允许一个明确的检测InlC的信号是至关重要的。在试验中，我们提出本文中，我们提议使用的HeLa细胞的CCL2的，这是特别适合于我们的测定法由于其细胞内空间的延伸中，其他的HeLa克隆诸如海拉京都细胞显示出更小的细胞质中，但可用于与我们的感染协议（京都的HeLa细胞，特别适合于分割的分析，因为细胞不与?...

A correction was made to <a href="http://www.jove.com/video/51043">Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes</a>. Three authors were omitted from the article at the time of publication. The acknowledgments section was also updated. The author list has been updated from:
Andreas K&#252;hbacher1,2,3, Edith Gouin1,2,3, Pascale Cossart1,2,3, Javier Pizarro-Cerd&#225;1,2,3 
1Unit&#233; des Interactions Bact&#233;ries Cellules, Pasteur Institute, 2INSERM U604, 3Institut National de la Recherche Agronomique (INRA), USC2020
to
Andreas K&#252;hbacher1,2,3, Edith Gouin1,2,3, Jason Mercer4, Mario Emmenlauer5, Christoph Dehio5, Pascale Cossart1,2,3, Javier Pizarro-Cerd&#225;1,2,3 
1Unit&#233; des Interactions Bact&#233;ries Cellules, Pasteur Institute,2INSERM U604, 3Institut National de la Recherche Agronomique (INRA), USC2020, 4Institute of Biochemistry, ETH Z&#252;rich 5Focal Area Infection Biology, Biozentrum, University of Basel
The acknowledgments where update from:
Research in P. Cossart laboratory is supported by the Pasteur Institute, the Institut National de la Sant&eacute; et de la Recherche M&eacute;dicale, the Institut National de la Recherche Agronomique, ERC Advanced Grant (233348), the Agence Nationale de la Recherche (Grant MIE-SignRupVac), the Louis-Jeantet Foundation and the Fondation Le Roch Les Mousquetaires. A.K. is a recipient of a scholarship from the Pasteur-Paris University International Doctoral Program/Institut Carnot Maladies Infectieuses. We thank Jason Mercer for optimizing the cellular transfection protocol.
to
Research in P. Cossart laboratory is supported by the Pasteur Institute, the Institut National de la Sant&eacute; et de la Recherche M&eacute;dicale, the Institut National de la Recherche Agronomique, ERC Advanced Grant (233348), the Agence Nationale de la Recherche (Grant MIE-SignRupVac), the Louis-Jeantet Foundation and the Fondation Le Roch Les Mousquetaires. A.K. is a recipient of a scholarship from the Pasteur-Paris University International Doctoral Program/Institut Carnot Maladies Infectieuses. We acknowledge support by grant 51RT 0_126008 for the Research and Technology Development (RTD) project InfectX in the frame of SystemsX.ch, the Swiss Initiative for Systems Biology (to C.D.).

A correction to author list has been made for the article Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes. Three authors were omitted from the article at the time of publication.

Erratum: Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

对革兰氏阳性细菌李斯特菌食源性病原体侵入宿主细胞，破坏其内在液泡和复制的宿主细胞1的细胞质。L。菌易于操纵与在细胞和动物模型中观察到细菌毒力性状的持久性相关的实验室环境（快速增长，对健康人低毒性）（溶血活性，白细胞增多）允许在上世纪60年代初期使用作为一个重要的模型研究细胞内寄生，并建立细胞抗感染2免疫力的理论基础。 20世纪80年代末90年代初，细菌细胞内循环3，以及最重要的细菌毒力的分子特性的解剖因素4-7赞成使用L的菌等作为操纵和螺柱的一个关键分子工具Ÿ的宿主细胞的功能。无毒的李斯特菌属的存在（L.英诺克 ）和毒力（ 单增李斯特菌 ）的物种铺平了道路，比较基因组研究8，与最近设立了完整的L.在一起单核细胞转录组9，增加了我们L的进化的理解菌作为一种人类病原体和感染模型系统研究10。 单增李斯特菌在细菌表面的蛋白质INLA和InlB与宿主细胞受体的E-钙粘蛋白和蛋氨酸，分别为11-12之间的相互作用诱导其内化进入宿主细胞。初始候选为基础的研究导致了α/β连环蛋白-肌动蛋白链路的标识作为磷脂酰肌醇3 -激酶（PI3-K）和INLA入侵通路13的作为InlB-关键效应一个显著成分依赖入侵卡斯卡德14-15。蛋白质组学和功能为基础的检测后允许新颖的骨架元素16和所需的宿主细胞侵袭脂质第二信使17的鉴定。转录研究18和质谱为基础的定量蛋白质组学19最近关于棚主信号通路的激活和免疫反应L.在镇压新的光菌感染。系统生物学的方法基础上，大型成套基因（kinomes，完整的基因组）的小干扰RNA失活（RNA干扰）沉默最近已经开始了全球主机在特定的细胞功能，包括细胞吞噬功能的背景下信号通路分析的新途径和病原体内在20。全基因组siRNA筛选此前已进行调查所需的L的感染细胞级联在果蝇吞噬单核细胞增生 </eM&gt; S2细胞21-22，但还没有在非吞噬细胞来进行这种类型的分析，这代表体内感染的关键目标。 我们已经进行了优化的协议由L.的显微镜检测感染的晚期阶段这适合于上皮细胞内细菌条目的高通量siRNA的研究菌等 。我们的实验需要一个高度侵袭性L的优势菌菌株呈现在PRFA，L的主要转录调节子的点突变菌毒力因子6：这种突变（名为PRFA *）呈现PRFA组成性激活23，并导致入侵的蛋白质INLA和InlB的表达增加，从而有利于在其他不佳感染非吞噬细胞的细菌进入。我们的读出对于感染是基于分泌性细菌蛋白InlC的的细胞质积累的检测：该分子是一个由胞质内L.优先表达一种多效性效应单核细胞增生 9和其参与不仅在细菌细胞至细胞传播24，但它也调节宿主的免疫反应25。 InlC的分泌的胞内细菌的荧光标记不仅可以明确区分感染与非感染的细胞，也代表一个端点的读出，可用于随后解剖感染在其不同的步骤：输入，空泡逃逸，胞浆菌增殖和细胞 - 细胞间传播。这个显微镜为基础的协议可以被耦合到siRNA筛选因此，研究涉及在宿主细胞中的由L.感染细胞途径菌等 。

<table border="1">
		<tbody>
		 <tr>
			<td>Name of Material/ Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments/Description</td>
		 </tr>
		 <tr>
			<td>Bacto Brain Heart Infusion</td>
			<td>BD</td>
			<td>237500</td>
			<td>For liquid BHI preparation</td>
		 </tr>
		 <tr>
			<td>Bacto Agar</td>
			<td>BD</td>
			<td>214010</td>
			<td>Supplement to liquid BHI for BHI agar plates </td>
		 </tr>
		 <tr>
			<td>Heat-Inactivated Fetal Bovine Serum</td>
			<td>Biowest</td>
			<td>51830-500</td>
			<td></td>
		 </tr>
		 <tr>
			<td>DMEM</td>
			<td>Invitrogen</td>
			<td>61965-026</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Lipofectamine RNAiMax</td>
			<td>Invitrogen</td>
			<td>13778-100</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Gentamicin</td>
			<td>Sigma </td>
			<td>G1397-10ML</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Formaldehyde (16%)</td>
			<td>EMS</td>
			<td>15710</td>
			<td>Prepare fresh before each experiment</td>
		 </tr>
		 <tr>
			<td>Anti-Rabbit Alexa Fluor 546</td>
			<td>Invitrogen</td>
			<td>A-11035</td>
			<td></td>
		 </tr>
		 <tr>
			<td>DAPI</td>
			<td>Invitrogen</td>
			<td>D-1306</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Phalloidin Dy647</td>
			<td>Dyomics</td>
			<td>647-33</td>
			<td></td>
		 </tr>
		 <tr>
			<td>siRNA Scramble</td>
			<td>Dharmacon</td>
			<td>D-001810-10</td>
			<td></td>
		 </tr>
		 <tr>
			<td>siRNA Met</td>
			<td>Dharmacon</td>
			<td>L-003156-00-0005</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Black 384-well microscopy cell culture plate</td>
			<td>Corning</td>
			<td>3985</td>
			<td></td>
		 </tr>
		 <tr>
			<td>AxioObserver Z1 microscope</td>
			<td>Zeiss</td>
			<td>431007 9901</td>
			<td></td>
		 </tr>
		 <tr>
			<td>sCMOS camera</td>
			<td>Andor</td>
			<td>Neo</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Metamorph analysis software</td>
			<td>Molecular Devices</td>
			<td>4000</td>
			<td></td>
		 </tr>
		 <tr>
			<td>CellProfiler analysis software</td>
			<td>Broad Institute</td>
			<td>Public software available at <a href="http://www.cellprofiler.org/" target="_blank">http://www.cellprofiler.org/</a></td>
			<td></td>
		 </tr>
		</tbody>
 </table>

imaging inlc secretion to investigate cellular infection by the bacterial pathogen listeria monocytogenes

细菌细胞内病原体可以设想作为分子工具来剖析，由于其能力，以精美的操纵和颠覆所必需的宿主靶组织感染细胞功能的细胞信号级联。在这些细菌病原体， 李斯特菌是，已被用来作为一个范例为细胞内寄生的细胞免疫应答的表征一种革兰氏阳性微生物，并且其在分子通路控制细胞骨架和膜运输动力学的发现发挥大的作用。在这篇文章中，我们描述了一个强大的显微镜测定法属晚期细胞感染阶段的检测根据InlC的，分泌的细菌蛋白，其存储在被感染细胞的细胞质中的荧光标记的单核细胞 ，该测定可耦合到自动化的高通量小分子干扰RNA的屏幕，以便为characterize涉及的向上或向下调节感染的细胞信号途径。

细胞质InlC的荧光标记由L.提供了一个强大的读出对细胞的感染菌 ，如示于图1：在显微照片的中心的细胞是高度感染菌株P14.PrfA * 23，因为它可以在相位对比图像（箭头， 图1A）中可以观察到，它是由DAPI信号确认其中个别的细菌可以清楚地分辨（ 图1B）。该InlC的染色（叠加到DAPI染色在图1C）示出了如何分泌蛋白密集堆积在受感染细胞的胞...

Watch this Scientific Journal Video about Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes at JoVE.com

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

李斯特菌是一种革兰氏阳性细菌病原体常作为细胞内寄生的研究的主要模型。后期成像L.菌的小干扰RNA的屏幕范围内感染阶段允许所需的靶宿主细胞的细菌感染的细胞通路的全球研究。

成像InlC的分泌由细菌病原体调查感染蜂窝<em&gt;单核细胞增生李斯特氏菌</em

Bacterial intracellular  pathogens can be conceived as molecular tools to dissect cellular signaling  cascades due to their capacity to exquisitely ...

Research

JoVE Journal

Immunology and Infection

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

利用生物发光成像之间调查的协同作用肺炎链球菌 A型流感病毒的婴儿小鼠

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

科研

免疫与感染

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 ...

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

测量细菌感染小鼠和免疫反应李斯特菌</em

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of ...

The Insect Galleria mellonella as a Powerful Infection Model to Investigate Bacterial Pathogenesis

The Insect Galleria mellonella as a Powerful Infection Model to Investigate Bacterial Pathogenesis

昆虫<em&gt;大蜡螟</em作为一个强大的的感染模型探讨细菌致病

The study of bacterial virulence often requires a suitable animal model. Mammalian models of infection are costly and may raise ethical issues. The use of ...

Oral Transmission of Listeria monocytogenes in Mice via Ingestion of Contaminated Food

Oral Transmission of Listeria monocytogenes in Mice via Ingestion of Contaminated Food

口头传播的<em&gt;单核细胞增生李斯特氏菌</em小鼠通过食入被污染的食物

L. monocytogenes are facultative intracellular bacterial pathogens that cause food borne infections in humans. Very little is known about the ...

Automated Separation of C. elegans Variably Colonized by a Bacterial Pathogen

Automated Separation of C. elegans Variably Colonized by a Bacterial Pathogen

自动分离<em&gt; C。线虫</em&gt;可多段由细菌病原体殖民者

The wormsorter is an instrument analogous to a FACS machine that is used in studies of Caenorhabditis elegans, typically to sort worms based on expression ...

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

使用李斯特菌单细胞基因评估受感染小鼠培养和心脏殖民化中心脏细胞的细菌入侵

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised ...

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. ...

Experimental Infection with Listeria monocytogenes as a Model for Studying Host Interferon-&#947; Responses

Experimental Infection with Listeria monocytogenes as a Model for Studying Host Interferon-ÃƒÆ’Ã…Â½Ãƒâ€šÃ‚Â³ Responses

实验性感染<em&gt;李斯特菌</em&gt;作为研究主机干扰素γ响应模型

L. monocytogenes is a gram-positive bacterium that is a cause of food borne disease in humans. Experimental infection of mice with this pathogen has been ...