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养物）的最大时间。 </li><li>在我们的协议中，我们使用了L。菌血清型1/2A EGDe.PrfA *应变，但如图1所示 ，L。菌血清型4b的P14.PrfA *应变给出了类似的结果。 </li></ol></li><li>他拉ATCC CCL2细胞生长在Dulbecco改良的Eagle培养基（DMEM）中在没有抗生素的补充有10％胎牛血清（FBS）。 </li><li>加扰（对照）和抗-Met的siRNA被在黑色384孔显微镜细胞培养板中加载的独立池。 <ol><li>稀释160皮摩尔的siRNA在500微升不含RNA酶的水中，加入5微升该溶液以每孔（终浓度：1.6皮摩尔）。 </li><li>保持这种板在-20℃下1年的最长期限。 </li></ol></li><li>多克隆兔抗InlC的已通过免疫使用InlC的-GST重组蛋白如前面25所描述获得的细菌蛋白。 </li></ol> 2。反向的siRNA转染细胞<ol><li>感染前72小时，使至室温含有1.6皮摩尔的siRNA在5微升不含RNA酶的水，每孔一个黑色384孔显微镜细胞培养板。 </li><li>离心板3分钟，在300相对离心力在室温下，以降低的siRNA可能已经沉积在孔的壁上。 </li><li>制备室温下的DMEM补充有0.4％的Lipofectamine RNAiMAX。 </li><li>加25微升DMEM / RNAiMAX转染培养基到每个孔中（将其添加到siRNA的前不孵育转染培养基中时间超过20分钟）。 </li><li>移动板来回以混合的siRNA与转染溶液，然后保持该板1小时，在室温下，以允许的siRNA-脂质体复合物的形成。 </li><li>洗涤HeLa细胞从汇合或亚汇合细胞培养烧瓶一次，用10ml 37℃预热的PBS中。 </li><li>分离HeLa细胞中加入1 ml 37℃预热的胰蛋白酶的细胞培养瓶中，孵育3至5分钟，在37℃下</li><li>重悬细胞于10毫升37℃预热的DMEM培养液，16％胎牛血清。 </li><li>计数细胞并制备每毫升12000的细胞在DMEM中的细胞悬浮液添加有16％FBS。 </li><li>添加50微升的细胞悬液以每孔中的384孔板中。 </li><li>快速移动板来回分发细胞，让细胞沉淀下来10分钟，室温下。 </li><li>密封该板用封口膜，并保持它为72小时，在湿润的5％CO 2气氛中含有在37℃下</li></ol> 3。细胞感染与染色<ol><li>感染前一天，取L的单菌落从BHI琼脂平板上的单核细胞 ，重悬它在5ml液体BHI培养基的15毫升的聚苯乙烯管中。 </li><li>孵育过夜，在37℃下在甩歌甩屏设备以允许细菌生长。 </li><li>在感染当天，洗将1ml过夜L的通过在台式离心机离心2分钟，10,600 RCF 菌文化。 </li><li>弃去上清液（其中包含分泌毒素李斯特菌O），并重新悬浮于1ml的PBS（repea颗粒吨洗涤步骤3次以上）。 </li><li>读出的细菌的光密度在600nm处，并估计细菌的数量（OD = 1相当于1E9细菌/ ml）。 </li><li>准备充足的L。菌稀释于DMEM补充有1％FBS的：使用高度侵袭性菌株像EGDe.PrfA *，我们建议在30μl的培养基，每孔使用5E4细菌（感染复数被估计为25对2000个细胞）。 </li><li>除去各孔中的细胞培养基（80微升），并通过加入30微升的L的更换单核细胞增生含中。 </li><li>离心板上于200相对离心力为5分钟，在室温下以同步的感染过程。 </li><li>在湿润的5％CO 2气氛中含有在37℃的预热铝块孵育板1小时。 </li><li>取出L。单核细胞的含介质从各孔中，加入30微升预热的DMEM补充有10％FBS和40微克/毫升的庆大霉素杀死细胞外L。菌等 。 </li><li>孵育在5％CO 2气氛中含有在37℃的预热的金属块上4小时。 </li><li>制备PBS中的溶液添加有8％的甲醛（该溶液应新鲜制备，使得甲醛的单体，而不是低聚甲醛的聚合物，被使用）。 </li><li>而不丢弃，细胞培养基，加入30微升PBS补充有8％的甲醛（最终浓度：4％），孵育15分钟，在室温下进行。 </li><li>除去固定液，用80μl的PBS中，每孔洗涤细胞三次（保持细胞在80微升PBS中，每孔的终体积）。 </li><li>准备一个1:250稀释在PBS中的兔抗血清InlC的补充有0.2％皂苷。 </li><li>加10μl的第一抗体溶液以每井后除去PBS中孵育30分钟，在室温下进行。 </li><li>丢弃的主抗体溶液和洗涤4次，每孔40微升PBS。 </li><li>稀释的二抗Alexa Fluor 546 - 偶联的抗兔抗体（1:250），在DAPI溶液（1:1,500）和鬼笔环肽-Dy647（1:150）在PBS中添加了0.2％皂苷。 </li><li>加10微升该二次染色溶液的各孔中，孵育30分钟，在室温下进行。 </li><li>丢弃二次染色溶液中，用每孔40微升PBS洗4次（留下40微升在每个终体积以及与密封板）。 </li><li>该板可以立即进行成像，或者可以被存储在4℃下避光保存（用铝箔覆盖），用于随后的分析。 </li></ol> 4。图像采集与分析<ol><li>在使用10X物镜安装在自动显微镜（每获得优先以及9张影像）三种不同的通道（350纳米，546纳米和647纳米）获取图像。 </li><li>该InlC的信号可以用图像来测量分析软件，如CellProfiler，允许使用的DAPI和鬼笔环肽染色，分别细胞核和细胞体的自动分割。 </li></ol>

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我们什么都没有透露。

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 ...

imaging-inlc-secretion-to-investigate-cellular-infection-bacterial

Research

JoVE Journal

Immunology and Infection

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

9.8K 次观看.  Pasteur Institute.  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。 

视频: 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

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of infection with influenza virus alone. Instead, most individuals are thought to have succumbed to a secondary bacterial infection, predominately caused by the bacterium Streptococcus pneumoniae (the pneumococcus). The synergistic relationship between infections caused by influenza virus and the pneumococcus has subsequently been observed during the 1957 Asian influenza virus pandemic, as well as during seasonal outbreaks of the virus (reviewed in 1, 2). Here, we describe a protocol used to investigate the mechanism(s) that may be involved in increased morbidity as a result of concurrent influenza A virus and S. pneumoniae infection. We have developed an infant murine model to reliably and reproducibly demonstrate the effects of influenza virus infection of mice colonised with S. pneumoniae. Using this protocol, we have provided the first insight into the kinetics of pneumococcal transmission between co-housed, neonatal mice using in vivo imaging 3.

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

A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.

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

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 ...

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

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of Listeria, which results in systemic infection2. After injection, Listeria quickly disseminates to the spleen and liver due to uptake by CD8&alpha;+ dendritic cells and Kupffer cells3,4. Once phagocytosed, various bacterial proteins enable Listeria to escape the phagosome, survive within the cytosol, and infect neighboring cells5. During the first three days of infection, different innate immune cells (e.g. monocytes, neutrophils, NK cells, dendritic cells) mediate bactericidal mechanisms that minimize Listeria proliferation. CD8+ T cells are subsequently recruited and responsible for the eventual clearance of Listeria from the host, typically within 10 days of infection6.
Successful clearance of Listeria from infected mice depends on the appropriate onset of host immune responses6	. There is a broad range of sensitivities amongst inbred mouse strains7,8. Generally, mice with increased susceptibility to Listeria infection are less able to control bacterial proliferation, demonstrating increased bacterial load and/or delayed clearance compared to resistant mice. Genetic studies, including linkage analyses and knockout mouse strains, have identified various genes for which sequence variation affects host responses to Listeria infection6,8-14. Determination and comparison of infection kinetics between different mouse strains is therefore an important method for identifying host genetic factors that contribute to immune responses against Listeria. Comparison of host responses to different Listeria strains is also an effective way to identify bacterial virulence factors that may serve as potential targets for antibiotic therapy or vaccine design.
We describe here a straightforward method for measuring bacterial load (colony forming units [CFU] per tissue) and preparing single-cell suspensions of the liver and spleen for FACS analysis of immune responses in Listeria-infected mice. This method is particularly useful for initial characterization of Listeria infection in novel mouse strains, as well as comparison of immune responses between different mouse strains infected with Listeria. We use the Listeria monocytogenes EGD strain15 that, when cultured on blood agar, exhibits a characteristic halo zone around each colony due to &beta;-hemolysis1 (Figure 1). Bacterial load and immune responses can be determined at any time-point after infection by culturing tissue homogenate on blood agar plates and preparing tissue cell suspensions for FACS analysis using the protocols described below. We would note that individuals who are immunocompromised or pregnant should not handle Listeria, and the relevant institutional biosafety committee and animal facility management should be consulted before work commences.

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

Listeria monocytogenes is a model organism for studying immune responses and genetic susceptibility to intracellular bacteria in mice. This method enables one to measure bacterial load and generate single-cell suspensions of the liver and spleen from mice for FACS analysis to determine changes in immune cells due to Listeria infection.

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 study of bacterial virulence often requires a suitable animal model. Mammalian models of infection are costly and may raise ethical issues. The use of insects as infection models provides a valuable alternative. Compared to other non-vertebrate model hosts such as nematodes, insects have a relatively advanced system of antimicrobial defenses and are thus more likely to produce information relevant to the mammalian infection process. Like mammals, insects possess a complex innate immune system1. Cells in the hemolymph are capable of phagocytosing or encapsulating microbial invaders, and humoral responses include the inducible production of lysozyme and small antibacterial peptides2,3. In addition, analogies are found between the epithelial cells of insect larval midguts and intestinal cells of mammalian digestive systems. Finally, several basic components essential for the bacterial infection process such as cell adhesion, resistance to antimicrobial peptides, tissue degradation and adaptation to oxidative stress are likely to be important in both insects and mammals1. Thus, insects are polyvalent tools for the identification and characterization of microbial virulence factors involved in mammalian infections.
Larvae of the greater wax moth Galleria mellonella have been shown to provide a useful insight into the pathogenesis of a wide range of microbial infections including mammalian fungal (Fusarium oxysporum, Aspergillus fumigatus, Candida albicans) and bacterial pathogens, such as Staphylococcus aureus, Proteus vulgaris, Serratia marcescens Pseudomonas aeruginosa, Listeria monocytogenes or Enterococcus faecalis4-7. Regardless of the bacterial species, results obtained with Galleria larvae infected by direct injection through the cuticle consistently correlate with those of similar mammalian studies: bacterial strains that are attenuated in mammalian models demonstrate lower virulence in Galleria, and strains causing severe human infections are also highly virulent in the Galleria model8-11. Oral infection of Galleria is much less used and additional compounds, like specific toxins, are needed to reach mortality.
G. mellonella larvae present several technical advantages: they are relatively large (last instar larvae before pupation are about 2 cm long and weight 250 mg), thus enabling the injection of defined doses of bacteria; they can be reared at various temperatures (20 &deg;C to 30 &deg;C) and infection studies can be conducted between 15 &deg;C to above 37 &deg;C12,13, allowing experiments that mimic a mammalian environment. In addition, insect rearing is easy and relatively cheap. Infection of the larvae allows monitoring bacterial virulence by several means, including calculation of LD5014, measurement of bacterial survival15,16 and examination of the infection process17. Here, we describe the rearing of the insects, covering all life stages of G. mellonella. We provide a detailed protocol of infection by two routes of inoculation: oral and intra haemocoelic. The bacterial model used in this protocol is Bacillus cereus, a Gram positive pathogen implicated in gastrointestinal as well as in other severe local or systemic opportunistic infections18,19.

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

Oral and intra haemocolic infection of larvae of the greater wax moth Galleria mellonella is described. This insect can be used to study virulence factors of entomopathogenic as well as mammalian opportunistic bacteria. Rearing of the insects, methods of infection and examples of in vivo analysis are described.

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

L. monocytogenes are facultative intracellular bacterial pathogens that cause food borne infections in humans. Very little is known about the gastrointestinal phase of listeriosis due to the lack of a small animal model that closely mimics human disease. This paper describes a novel mouse model for oral transmission of L. monocytogenes. Using this model, mice fed L. monocytogenes-contaminated bread have a discrete phase of gastrointestinal infection, followed by varying degrees of systemic spread in susceptible (BALB/c/By/J) or resistant (C57BL/6) mouse strains. During the later stages of the infection, dissemination to the gall bladder and brain is observed. The food borne model of listeriosis is highly reproducible, does not require specialized skills, and can be used with a wide variety of bacterial isolates and laboratory mouse strains. As such, it is the ideal model to study both virulence strategies used by L. monocytogenes to promote intestinal colonization, as well as the host response to invasive food borne bacterial infection.

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

This paper describes a novel method for oral infection of mice using Listeria monocytogenes-contaminated food. The protocol can readily be adapted for use with other food borne bacterial pathogens.

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

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 of a fluorescent reporter. Here, we highlight an alternative usage of this instrument, for sorting worms according to their degree of colonization by a GFP-expressing pathogen. This new usage allowed us to address the relationship between colonization of the worm intestine and induction of immune responses. While C. elegans immune responses to different pathogens have been documented, it is still unknown what initiates them. The two main possibilities (which are not mutually exclusive) are recognition of pathogen-associated molecular patterns, and detection of damage caused by infection. To differentiate between the two possibilities, exposure to the pathogen must be dissociated from the damage it causes. The wormsorter enabled separation of worms that were extensively-colonized by the Gram-negative pathogen Pseudomonas aeruginosa, with the damage likely caused by pathogen load, from worms that were similarly exposed, but not, or marginally, colonized. These distinct populations were used to assess the relationship between pathogen load and the induction of transcriptional immune responses. The results suggest that the two are dissociated, supporting the possibility of pathogen recognition.

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

The wormsorter facilitates genetic screens in Caenorhabditis elegans by sorting worms according to expression of fluorescent reporters. Here, we describe a new usage:&#160;sorting according to colonization by a GFP-expressing pathogen, and we employ it to examine the poorly understood role of pathogen recognition in initiating immune responses.

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

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised patients, the elderly, and pregnant women. The most common manifestations of listeriosis in humans include meningitis, encephalitis, and fetal abortion. A significant but much less documented sequelae of invasive L. monocytogenes infection involves the heart. The death rate from cardiac illness can be up to 35% despite treatment, however very little is known regarding L. monocytogenes colonization of cardiac tissue and its resultant pathologies. In addition, it has recently become apparent that subpopulations of L. monocytogenes have an enhanced capacity to invade and grow within cardiac tissue. This protocol describes in detail in vitro and in vivo methods that can be used for assessing cardiotropism of L. monocytogenes isolates. Methods are presented for the infection of H9c2 rat cardiac myoblasts in tissue culture as well as for the determination of bacterial colonization of the hearts of infected mice. These methods are useful not only for identifying strains with the potential to colonize cardiac tissue in infected animals, but may also facilitate the identification of bacterial gene products that serve to enhance cardiac cell invasion and/or drive changes in heart pathology. These methods also provide for the direct comparison of cardiotropism between multiple L. monocytogenes strains.

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

Listeria monocytogenes causes fetal infections in pregnant women and meningitis in susceptible populations. Subpopulations of bacteria can colonize cardiac tissue, causing myocarditis in patients and laboratory animals. Here we present a protocol that describes how to assess L. monocytogenes cardiac cell invasion in vitro and cardiac colonization in infected animals.

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

Analysis of the transcriptome of bacterial pathogens during mammalian infection is a valuable tool for studying genes and factors that mediate infection. However, isolating bacterial RNA from infected cells or tissues is a challenging task, since mammalian RNA mostly dominates the lysates of infected cells. Here we describe an optimized method for RNA isolation of Listeria monocytogenes bacteria growing within bone marrow derived macrophage cells. Upon infection, cells are mildly lysed and rapidly filtered to discard most of the host proteins and RNA, while retaining intact bacteria. Next, bacterial RNA is isolated using hot phenol-SDS extraction followed by DNase treatment. The extracted RNA is suitable for gene transcription analysis by multiple techniques. This method is successfully employed in our studies of Listeria monocytogenes gene regulation during infection of macrophage cells 1-4. The protocol can be easily modified to study other bacterial pathogens and cell types.

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

Here we describe a method for bacterial RNA isolation from Listeria monocytogenes bacteria growing inside murine macrophages. This technique can be used with other intracellular pathogens and mammalian host 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

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 highly informative on the role of innate and adaptive immune cells and specific cytokines in host immunity against intracellular pathogens. Production of IFN-&#947; by innate cells during sublethal infection with L. monocytogenes is important for activating macrophages and early control of the pathogen1-3. In addition, IFN-&#947; production by adaptive memory lymphocytes is important for priming the activation of innate cells upon reinfection4. The L. monocytogenes infection model thus serves as a great tool for investigating whether new therapies that are designed to increase IFN-&#947; production have an impact on IFN-&#947; responses in vivo and have productive biological effects such as increasing bacterial clearance or improving mouse survival from infection. Described here is a basic protocol for how to conduct intraperitoneal infections of C57BL/6J mice with the EGD strain of L. monocytogenes and to measure IFN-&#947; production by NK cells, NKT cells, and adaptive lymphocytes by flow cytometry. In addition, procedures are described to: (1) grow and prepare the bacteria for inoculation, (2) measure bacterial load in the spleen and liver, and (3) measure animal survival to endpoints. Representative data are also provided to illustrate how this infection model can be used to test the effect of specific agents on IFN-&#947; responses to L. monocytogenes and survival of mice from this infection.

Experimental Infection with &lt;em&gt;Listeria monocytogenes&lt;/em&gt; as a Model for Studying Host Interferon-&amp;#947; Responses

This protocol describes how to inoculate C57BL/6J mice with the EGD strain of Listeria monocytogenes (L. monocytogenes) and to measure interferon-&#947; (IFN-&#947;) responses by natural killer (NK) cells, natural killer T (NKT) cells, and adaptive T lymphocytes post-infection. This protocol also describes how to conduct survival studies in mice after infection with a modified LD50 dose of the pathogen.

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 ...

Listeria monocytogenes Infection of the Brain

Listeria monocytogenes is an intracellular bacterial pathogen that is frequently associated with food-borne infection. The ability of&#160;L. monocytogenes to cross the blood-brain barrier (BBB) is concerning as it can lead to life-threatening meningitis and encephalitis. The BBB protects the brain microenvironment from various toxic metabolites and microbial pathogens found in the blood following infection, and therefore supports brain homeostasis. The mechanisms by which L. monocytogenes present in the bloodstream cross the BBB to cause brain infections are not fully understood and there is also a lack of a robust model system to study brain infections by L. monocytogenes. Here, we present a simple mouse infection model to determine whether bacteria have crossed the BBB and to quantitate the burden of bacteria that have colonized the brain in vivo. In this method, animals were infected intravenously with L. monocytogenes and were humanely euthanized by exposure to CO2 followed by cervical dislocation. Cardiac perfusion of the animals was performed prior to harvesting infected organs. Blood was collected before perfusion and the number of bacteria per organ or mL of blood was determined by plating dilutions of the blood or organ homogenates on agar plates and counting the number of colonies formed. This method can be used to study novel receptor-ligand interactions that enhance infection of the brain by L. monocytogenes and can be easily adapted for the study of multiple bacterial pathogens.

Listeria monocytogenes Infection of the Brain

During infection, Listeria monocytogenes is capable of crossing the blood-brain barrier to colonize the brain. In this protocol, we demonstrate how to assess bacterial colonization of organs following infection of mice. A procedure to perform whole organ perfusion for specific determination of bacterial numbers in the brain parenchyma is provided.