This work was supported by the Deutsche Forschungsgemeinschaft (DFG) through the Collaborative Research Initiative 796 (SFB796) to A.L. and C.B., and through the ERA-NET PathoGenoMics 3rd call to A.L.

1.产生的稳定细胞系的利益表达的蛋白<ol><li>通过加入热灭活的FCS和1％青霉素/链霉素准备媒体市售Dulbecco&#39;s改良的Eagle培养基（DMEM），补充有含有GlutaMAX-I，丙酮酸，和4.5g / L的D-葡萄糖。 </li><li>在5％CO 2培养HEK293细胞中的媒体，在37℃。子培养细胞每三天。删除媒体和悬浮细胞15毫升新鲜培养基。吸管1毫升到一个新的75cm 2的细胞培养瓶中，并添加14毫升介质。 </li><li>通过使用血球分析细胞数量。 </li><li>种子HEK293细胞在6孔板中以每孔2×10 5个细胞的密度，并培育24小时。 </li><li>转染使用由转染试剂的制造商提供的协议。转染使用转染试剂的细胞与载体pEGFP-C2或质粒pEGFP-C2-CAEB。在使用之前，预热染REAG耳鼻喉科和需要RT媒体。使用3：1的比例转染试剂与DNA。 <ol><li>详细地说，吸移管1.5微升转染试剂直接加入50微升无血清DMEM培养基。为复合物形成，吸取0.5的DNA编码GFP或GFP-CAEB到转染试剂含有混合物的微克孵育15分钟，在室温。 </li><li>通过在逐滴方式加入，将反应混合物转染细胞并在5％CO 2孵育在37℃下24小时。 </li></ol></li><li>加入1毫克/毫升，在5％CO 2的遗传霉素进行选择的GFP阳性克隆在37℃。 </li><li> 6天后，分离单个细胞通过流式细胞术在一个96孔板窝藏介质和HEK293上清液在1排序：1的比例。产生从该均在4966×g下离心15分钟培养汇合HEK293细胞的HEK293上清液。 </li><li>在接下来的一天，更换培养基含有1.5培养基0毫克/ ml遗传霉素。更改每周一次的媒体。如果细胞形成汇合单层，将它们转移到一个24孔板中。 5在介质含有1.5毫克/ ml遗传：每周以1:1的比例分培养细胞两次。最后，分析GFP阳性细胞的免疫印迹分析的百分比和流式细胞术。 </li></ol>通过免疫分析稳定细胞系2.分析<ol><li>除去上清液，用温PBS洗涤一次附着的细胞。将细胞重悬于100微升的2×Laemmli样品缓冲液中，加热5分钟，在95℃，并储存在-20℃。 </li><li>加载在12％SDS-PAGE凝胶，每个样品约4×10 4个细胞。此外，负载6微升市售蛋白质梯作为分子量标记物。 180伏的恒定电压下运行的凝胶电泳系统凝胶45-60分钟，用1×Laemmli的运行缓冲液。 </li><li>吸干蛋白到PVDF膜上（0孔径。为45μm），在16伏特用于使用一台转印迹SD半干转移细胞60分钟的恒定电压。 </li><li>块膜在10ml的5％脱脂奶粉的PBS / 0.05％吐温20（MPT）中1小时，在室温。 </li><li>稀释4微升抗GFP抗体在4毫升MPT和孵化膜O / N在4℃。 </li><li>执行三个10分钟的洗涤步骤，用10毫升的PBS / 0.05％吐温20。 </li><li>孵育膜用0.8微升辣根过氧化物酶缀合的第二抗体在4毫升的MPT的。 </li><li> 1小时后在室温下孵育，洗膜三次，用PBS / 0.05％吐温20（如在2.6中描述），并根据制造商的方案检测辣根过氧化物酶活性用市售的试剂盒。适用标准装备片开发可视化蛋白质带。 </li></ol> 3.分析使用流式细胞仪的细胞。 <ol><li>重悬在PBS细胞。使用HEK293细胞作为阴性对照调整前进SC东北黑钙土（FSC）和侧向散射（SSC），以使细胞在规模。在排除细胞双峰，聚集和细胞碎片活细胞绘制一个门。 </li><li>调整光电倍增管（PMT）的增益，以使未染色细胞是在最左边的直方图的FL1通道（488纳米氩离子激光）的。 </li><li>为了分析的HEK293-GFP和HEK293-GFP-CAEB细胞的荧光强度打开FL1通道的直方图，并采集门控人口10,000个事件。 </li><li>分析使用商业FACS分析软件中的数据。 </li></ol> 4.转染的稳定细胞系的诱导表达系统的<ol><li>种子HEK293细胞稳定表达GFP或GFP-CAEB在12孔板中以1×10 5个细胞/孔的密度。 </li><li> 19小时后播种，共转染的细胞，调节质粒和反应质粒编码两种或Bax蛋白活化的caspase 3。 <ol><li>共转染的稳定的HEK293细胞用100ng pWHE125-P调节器质粒（ 图2）和100ng的响应质粒pWHE655-hBax或pWHE655-revCasp3（ 图3）和0.4微升的聚乙烯亚胺。 </li><li>制备的DNA和聚乙烯亚胺染试剂每75微升的Opti-MEM培养基中，孵育在室温，精确至5分钟。为复合物形成，孵育两种混合物一起进行15分钟，在室温。 </li></ol></li><li>添加聚乙烯亚胺/ DNA溶液滴至细胞并在5％CO 2孵育在37℃。 </li></ol> 5.诱导细胞凋亡<ol><li> 5小时转染后，通过加入1μg/ ml的多西环素的培养基中诱导的促凋亡蛋白的表达。在5％CO 2孵育细胞18小时，在37℃。 </li></ol>宿主细胞凋亡的免疫分析6.分析<ol><li>检查之前H细胞在光学显微镜下arvesting检查细胞死亡诱导。细胞凋亡是可检测由死的细胞周围的凋亡小体的存在。 </li><li>除去上清液，用温PBS洗涤一次附着的细胞。将细胞重悬于100微升的2×Laemmli样品缓冲液中，加热5分钟，在95℃，并储存在-20℃。 </li><li>加载在12％SDS-PAGE凝胶，每个样品约4×10 4个细胞。此外，负载6微升市售蛋白质梯作为分子量标记物。 180伏的恒定电压下运行的凝胶电泳系统凝胶45-60分钟，用1×Laemmli的运行缓冲液。 </li><li>吸干蛋白到PVDF膜（0.45微米孔径大小）以16伏特用于使用一台转印迹SD半干转移细胞60分钟的恒定电压。 </li><li>块膜在10ml的5％脱脂奶粉的PBS / 0.05％吐温20（MPT）中1小时，在室温。 </li><li>稀释4μ升反切割聚ADP-核糖聚合酶（PARP）抗体在4毫升MPT和孵化O / N在4℃。 </li><li>执行三个10分钟的洗涤步骤，用10毫升的PBS / 0.05％吐温20。 </li><li>孵育膜用0.8微升辣根过氧化物酶缀合的第二稀释在4ml MPT抗体。 </li><li> 1小时后在室温下孵育，洗膜三次，用PBS / 0.05％吐温20（如在6.7中描述），并检测辣根过氧化物酶的活性具有根据制造商的协议商业试剂盒。适用标准装备片开发可视化蛋白质带。 </li><li> 30分钟，在室温下孵育除去结合的抗体用7ml Western印迹溶出缓冲区。 </li><li>洗涤三次后，用PBS / 0.05％吐温20（如在6.7中描述），探针与4微升抗肌动蛋白抗体的4ml MPT O / N在4℃下的膜。 </li><li>经过三次洗涤步骤，MPT（如6.7所述），孵化膜与0.8微升豪rseradish过氧化物酶缀合的二抗稀释于4ml的MPT的。 </li><li> 1小时后在室温下孵育，洗膜三次，用PBS / 0.05％吐温20（如在6.7中描述和检测辣根过氧化物酶的活性通过根据制造商的协议商业试剂盒。应用的标准设备胶片显影可视化蛋白条带。 注意：在板振荡器上在指定的时间和温度进行所有的孵育步骤。 </li></ol>

The authors have nothing to disclose.

许多病原细菌怀有分泌系统分泌或易位细菌效应蛋白进入宿主细胞。这些效应蛋白具有调节在宿主细胞中的过程和途径，从而使细菌存活并在其各自的细胞内利基复制的能力。了解生化活动和效应蛋白的分子机制将有助于建立一个更好地了解致病性，并可能有助于开发新的治疗手段，以抗击疾病。另外，作为效应蛋白经常通过模仿宿主细胞活动发挥它们的功能，它们也可以被用来进一步了解真核?...

许多革兰氏阴性细菌病原体的毒力取决于专门的分泌系统劫持真核宿主细胞。细菌利用这些分泌系统注入细菌毒性蛋白（效应物）到宿主细胞来调节各种细胞和生化活性。效应蛋白的研究不仅提供了卓越的洞察宿主/病原体相互作用的基本方面，但也到真核细胞1的基础生物学。宿主细胞凋亡的调节已被证明是一种重要的毒力机制的许多细胞内病原体，和一些效应蛋白调节细胞凋亡已经确定2-9。然而，他们的活动的精确的分子机制仍然是难以捉摸的，在许多情况下。 细胞凋亡，程序性细胞死亡的一种形式，在免疫应答中起重要作用，以感染10。导致细胞凋亡的两个主要途径有已经确定：针对线粒体（内在凋亡）或信号的直接转导通过细胞死亡受体在质膜（外源性凋亡）。本征或线粒体介导的细胞死亡途径由细胞内信号所触发的，并涉及Bax和Bak的，所述的Bcl-2家族的两个促凋亡成员的活化。这家由控制细胞死亡11-14亲和抗凋亡调节蛋白。细胞凋亡的激活导致低聚的Bax和Bak的后跟线粒体外膜的随后通透性，导致细胞色素C释放到细胞质中。细胞色素c释放引发的凋亡小15激活效应半胱氨酸蛋白酶3和7通过激活caspase 9的。这导致选择底物，除其他外，导致磷脂酰丝氨酸在细胞表面16的暴露的蛋白水解，并释放一个专用的DNase该片段沟道romatin 17,18。 为了确定在凋亡级联的个体效应蛋白干扰内，可诱导的表达系统被雇用19。管制系统，转基因的条件式已在分析过程中发生，发展和维持疾病20-23的细胞内的蛋白质的功能或它的对组织，器官和生物体发展的重要性，以及一个宝贵的工具。典型地，可诱导的控制系统，如四环素系统24在这里使用的，形成人工转录单位（参见图1）。一种成分是一种叫做tTA的（四环素依赖性转录激活剂）人工工程转录因子，通过融合的细菌转录阻遏四面体25到介导转录激活或沉默24,26的哺乳动物蛋白结构域形成。第二组分是一种混合启动子，称为TRE（四环素响应元件），它由一个真核最小启动子的，含有至少一个TATA盒和转录起始位点，结合到同源DNA结合位点为四面体，TETO 24,25的多个重复。第三成分是四面体，四环素或其衍生物，如无水四环素或强力霉素25之一的天然配体。在配体加入到培养基中，四面体失去其亲和力TETO并从TRE解离。其结果，靶基因的转录被废除。转基因表达可以，因此，被严格控制在两个细胞培养物以时间和剂量依赖的方式和在动物20,23,24。与tTA的，转基因的表达发生组成型，除了在四环素存在下进行。这可以是在细胞毒性或致癌蛋白的研究一个缺点，因为四环素首先必须从体系中除去，转基因expressio前n，将会和可监控单元上的靶蛋白的影响。这可能是耗时的并且不总是完全，特别是在转基因动物27。为了解决此限制，一个四面体突变体具有逆响应于多西环素的存在被用来产生新的转录因子，rtTA（反向TTA）28。它仅结合于TRE和，伴随，激活在强力霉素存在下转录。的系统， 即残余泄漏，在不存在TRE结合转录因子的转基因表达，始发或者（i）从位置效应在基因组整合位点，（ⅱ）从TRE本身29，或（iii）来自非特异性tTA的/ rtTA 28的结合，是通过引入一个附加的转录消音处理，称为TTS（四环素依赖的转录的消音器）30到系统。它形成了一个双重调节网络一起rtTA（ 见图1）。在没有强力霉素，TTS结合TRE积极关闭任何剩余的转录。在强力霉素存在时，TTS解离TRE和rtTA具约束力同时诱导靶基因的表达。严紧的这个附加层往往是必要的，表达高活性细胞毒性蛋白质31-34。 使用这种严格控制的双调节器系统，细胞凋亡级联可以在限定的步骤，使给定的效应蛋白是否能够与细胞凋亡诱导干扰分析来启动。这种方法不仅可用于研究细菌效应蛋白的抗凋亡活性，但也可用于促凋亡或有毒的蛋白质的诱导表达，或用于解剖与其他信号传导途径的干扰。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>DMEM</td>
			<td>life technologies</td>
			<td>31966-021</td>
			<td></td>
		</tr>
		<tr>
			<td>FCS</td>
			<td>Biochrom</td>
			<td>S0115</td>
			<td></td>
		</tr>
		<tr>
			<td>Pen/Strep</td>
			<td>life technologies</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>OptiMEM</td>
			<td>life technologies</td>
			<td>51985</td>
			<td></td>
		</tr>
		<tr>
			<td>X-tremeGENE 9</td>
			<td>Roche</td>
			<td>6365752001</td>
			<td></td>
		</tr>
		<tr>
			<td>Geneticin</td>
			<td>Roth</td>
			<td>CP11.3</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylenimine</td>
			<td>Polyscienes</td>
			<td>23966</td>
			<td></td>
		</tr>
		<tr>
			<td>Doxycycline</td>
			<td>Sigma Aldrich</td>
			<td>D9891</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini-PROTEAN Tetra Cell</td>
			<td>Bio-Rad</td>
			<td>165-8000EDU</td>
			<td></td>
		</tr>
		<tr>
			<td>Trans-Blot SD Semi-Dry Transfer Cell</td>
			<td>Bio-Rad</td>
			<td>170-3940</td>
			<td></td>
		</tr>
		<tr>
			<td>PageRuler Prestained Protein Ladder</td>
			<td>Thermo Scientific</td>
			<td>26616</td>
			<td></td>
		</tr>
		<tr>
			<td>PVDF membrane</td>
			<td>Millipore</td>
			<td>IPVH00010</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-GFP&#160;</td>
			<td>life technologies</td>
			<td>A6455</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-cleaved PARP</td>
			<td>BD Bioscience</td>
			<td>611038</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-actin</td>
			<td>Sigma Aldrich</td>
			<td>A2066</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse IgG (H+L)-HRPO</td>
			<td>Dianova</td>
			<td>111-035-062</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit IgG (H+L)-HRPO</td>
			<td>Dianova</td>
			<td>111-035-045</td>
			<td></td>
		</tr>
		<tr>
			<td>ECL Western Blotting Substrate</td>
			<td>Thermo Scientific</td>
			<td>32106</td>
			<td></td>
		</tr>
		<tr>
			<td>Restore Plus Western Blot Stripping Buffer</td>
			<td>Thermo Scientific</td>
			<td>46428</td>
			<td></td>
		</tr>
	</tbody>
</table>

applying an inducible expression system to study interference of bacterial virulence factors with intracellular signaling

这里提出的技术允许一个来分析在该步骤目标蛋白质，或者小分子，与信号传导途径的组分相互作用。该方法是基于，一方面，在一个特定的蛋白质的诱导表达引发的信令事件在所选择的信号级联限定和预定的步骤。相伴表达，另一方面，目的基因的则允许调查者评估，如果表达靶蛋白的活性被位于上游或发起信令事件的下游，根据所获得的信号通路的读出。这里，凋亡级联被选为一个定义信号传导途径来证明协议的功能。致病细菌，如贝氏柯克斯体 ，易位效应蛋白，与在宿主细胞宿主细胞死亡诱导干扰，以确保细菌存活的细胞，并促进其传播的生物体。 C.该贝氏柯克斯效应蛋白CAEB诱导细胞凋亡与UV光或具有星形孢菌素后有效抑制宿主细胞死亡。来缩小在该步骤CAEB干扰凋亡信号的传播，选择蛋白与充分表征的促凋亡活性在多西环素可诱导的方式瞬时表达。如果CAEB充当这些蛋白质的上游，将细胞凋亡进行畅通无阻。如果CAEB行为下游，细胞死亡会被抑制。选择的试验蛋白质分别为Bax蛋白，其作用在线粒体水平，和caspase 3，这是主要的刽子手蛋白酶。 CAEB干扰诱导Bax表达的细胞死亡，而不是由胱天蛋白酶3的表达。 CAEB，由此，交互这两种蛋白质之间的凋亡级联。

首先，将HEK293细胞系稳定表达的兴趣（CAEB）的蛋白质作为GFP融合蛋白分别建立。作为对照，将HEK293细胞系稳定表达GFP的也产生。 GFP和GFP-CAEB的表达通过免疫印迹分析证实。代表性免疫印迹（ 图4A）显示GFP和GFP-CAEB的稳定和清晰地可检测的表达。然而，这种分析不能确定所有的细胞是否表达GFP或GFP-CAEB。因此，稳定地转染的HEK293细胞系还通过流式细胞仪分析。 如图4B所示 ，...

Watch this Scientific Journal Video about Applying an Inducible Expression System to Study Interference of Bacterial Virulence Factors with Intracellular Signaling at JoVE.com

Applying an Inducible Expression System to Study Interference of Bacterial Virulence Factors with Intracellular Signaling

The method described here is used to induce the apoptotic signaling cascade at defined steps in order to dissect the activity of an anti-apoptotic bacterial effector protein. This method can also be used for inducible expression of pro-apoptotic or toxic proteins, or for dissecting interference with other signaling pathways.

施加诱导表达系统研究与细胞内信号细菌毒力因子的干扰

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a ...

applying-an-inducible-expression-system-to-study-interference

Research

JoVE Journal

Immunology and Infection

9.1K 次观看.  Friedrich-Alexander-Universität. The method described here is used to induce the apoptotic signaling cascade at defined steps in order to dissect the activity of an anti-apoptotic bacterial effector protein. This method can also be used for inducible expression of pro-apoptotic or toxic proteins, or for dissecting interference with other signaling pathways.

视频: Applying an Inducible Expression System to Study Interference of Bacterial Virulence Factors with Intracellular Signaling

Infection of Zebrafish Embryos with Intracellular Bacterial Pathogens

Zebrafish (Danio rerio) embryos are increasingly used as a model for studying the function of the vertebrate innate immune system in host-pathogen interactions 1. The major cell types of the innate immune system, macrophages and neutrophils, develop during the first days of embryogenesis prior to the maturation of lymphocytes that are required for adaptive immune responses. The ease of obtaining large numbers of embryos, their accessibility due to external development, the optical transparency of embryonic and larval stages, a wide range of genetic tools, extensive mutant resources and collections of transgenic reporter lines, all add to the versatility of the zebrafish model. Salmonella enterica serovar Typhimurium (S. typhimurium) and Mycobacterium marinum can reside intracellularly in macrophages and are frequently used to study host-pathogen interactions in zebrafish embryos. The infection processes of these two bacterial pathogens are interesting to compare because S. typhimurium infection is acute and lethal within one day, whereas M. marinum infection is chronic and can be imaged up to the larval stage 2, 3. The site of micro-injection of bacteria into the embryo (Figure 1) determines whether the infection will rapidly become systemic or will initially remain localized. A rapid systemic infection can be established by micro-injecting bacteria directly into the blood circulation via the caudal vein at the posterior blood island or via the Duct of Cuvier, a wide circulation channel on the yolk sac connecting the heart to the trunk vasculature. At 1 dpf, when embryos at this stage have phagocytically active macrophages but neutrophils have not yet matured, injecting into the blood island is preferred. For injections at 2-3 dpf, when embryos also have developed functional (myeloperoxidase-producing) neutrophils, the Duct of Cuvier is preferred as the injection site. To study directed migration of myeloid cells towards local infections, bacteria can be injected into the tail muscle, otic vesicle, or hindbrain ventricle 4-6. In addition, the notochord, a structure that appears to be normally inaccessible to myeloid cells, is highly susceptible to local infection 7. A useful alternative for high-throughput applications is the injection of bacteria into the yolk of embryos within the first hours after fertilization 8. Combining fluorescent bacteria and transgenic zebrafish lines with fluorescent macrophages or neutrophils creates ideal circumstances for multi-color imaging of host-pathogen interactions. This video article will describe detailed protocols for intravenous and local infection of zebrafish embryos with S. typhimurium or M. marinum bacteria and for subsequent fluorescence imaging of the interaction with cells of the innate immune system.

Transparent zebrafish embryos have proved useful model hosts to visualize and functionally study interactions between innate immune cells and intracellular bacterial pathogens, such as Salmonella typhimurium and Mycobacterium marinum. Micro-injection of bacteria and multi-color fluorescence imaging are essential techniques involved in the application of zebrafish embryo infection models.

斑马鱼的胚胎细胞内细菌病原体感染

Zebrafish (Danio rerio) embryos are increasingly used as a model for studying the function of the vertebrate innate immune system in host-pathogen ...

科研

免疫与感染

Rearing and Injection of Manduca sexta Larvae to Assess Bacterial Virulence

Manduca sexta, commonly known as the tobacco hornworm, is considered a significant agricultural pest, feeding on solanaceous plants including tobacco and tomato. The susceptibility of M. sexta larvae to a variety of entomopathogenic bacterial species1-5, as well as the wealth of information available regarding the insect's immune system6-8, and the pending genome sequence9 make it a good model organism for use in studying host-microbe interactions during pathogenesis. In addition, M. sexta larvae are relatively large and easy to manipulate and maintain in the laboratory relative to other susceptible insect species. Their large size also facilitates efficient tissue/hemolymph extraction for analysis of the host response to infection.
The method presented here describes the direct injection of bacteria into the hemocoel (blood cavity) of M. sexta larvae. This approach can be used to analyze and compare the virulence characteristics of various bacterial species, strains, or mutants by simply monitoring the time to insect death after injection. This method was developed to study the pathogenicity of Xenorhabdus and Photorhabdus species, which typically associate with nematode vectors as a means to gain entry into the insect. Entomopathogenic nematodes typically infect larvae via natural digestive or respiratory openings, and release their symbiotic bacterial contents into the insect hemolymph (blood) shortly thereafter10. The injection method described here bypasses the need for a nematode vector, thus uncoupling the effects of bacteria and nematode on the insect. This method allows for accurate enumeration of infectious material (cells or protein) within the inoculum, which is not possible using other existing methods for analyzing entomopathogenesis, including nicking11 and oral toxicity assays12. Also, oral toxicity assays address the virulence of secreted toxins introduced into the digestive system of larvae, whereas the direct injection method addresses the virulence of whole-cell inocula.
The utility of the direct injection method as described here is to analyze bacterial pathogenesis by monitoring insect mortality. However, this method can easily be expanded for use in studying the effects of infection on the M. sexta immune system. The insect responds to infection via both humoral and cellular responses. The humoral response includes recognition of bacterial-associated patterns and subsequent production of various antimicrobial peptides7; the expression of genes encoding these peptides can be monitored subsequent to direct infection via RNA extraction and quantitative PCR13. The cellular response to infection involves nodulation, encapsulation, and phagocytosis of infectious agents by hemocytes6. To analyze these responses, injected insects can be dissected and visualized by microscopy13, 14.

Rearing and Injection of Manduca sexta Larvae to Assess Bacterial Virulence

The method described here utilizes direct injection of entomopathogenic bacteria into the hemocoel of Manduca sexta insect larvae. M. sexta is a commercially available and well-studied insect. Thus, this method represents a simple approach to analyzing host-bacterial interactions from the perspective of one or both partners.

饲养和注射<em&gt;烟草天蛾</em&gt;幼虫评估细菌的毒力

Manduca sexta, commonly known as the tobacco hornworm, is considered a significant agricultural pest, feeding on solanaceous plants including tobacco and ...

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable alternative to animal model systems for the study of a specific step of microbial pathogen infection. A human monocytic cell line THP-1 which, upon phorbol ester treatment, is differentiated into macrophages, has previously been used to study virulence strategies of many intracellular pathogens including Mycobacterium tuberculosis. Here, we discuss a protocol to enact an in vitro cell culture model system using THP-1 macrophages to delineate the interaction of an opportunistic human yeast pathogen Candida glabrata with host phagocytic cells. This model system is simple, fast, amenable to high-throughput mutant screens, and requires no sophisticated equipment. A typical THP-1 macrophage infection experiment takes approximately 24 hr with an additional 24-48 hr to allow recovered intracellular yeast to grow on rich medium for colony forming unit-based viability analysis. Like other in vitro model systems, a possible limitation of this approach is difficulty in extrapolating the results obtained to a highly complex immune cell circuitry existing in the human host. However, despite this, the current protocol is very useful to elucidate the strategies that a fungal pathogen may employ to evade/counteract antimicrobial response and survive, adapt, and proliferate in the nutrient-poor environment of host immune cells.

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

The current article outlines a protocol to establish an in vitro cell culture model system to study the interaction of a facultative intracellular human fungal pathogen Candida glabrata with human macrophages which will be a useful tool to advance our knowledge of fungal virulence mechanisms.

建立一个<em&gt;在体外</em&gt;系统来研究细胞内的行为<em&gt;光滑念珠菌</em&gt;在人THP-1巨噬细胞

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable ...

Following in Real Time the Impact of Pneumococcal Virulence Factors in an Acute Mouse Pneumonia Model Using Bioluminescent Bacteria

Pneumonia is one of the major health care problems in developing and industrialized countries and is associated with considerable morbidity and mortality. Despite advances in knowledge of this illness, the availability of intensive care units (ICU), and the use of potent antimicrobial agents and effective vaccines, the mortality rates remain high1. Streptococcus pneumoniae is the leading pathogen of community-acquired pneumonia (CAP) and one of the most common causes of bacteremia in humans. This pathogen is equipped with an armamentarium of surface-exposed adhesins and virulence factors contributing to pneumonia and invasive pneumococcal disease (IPD). The assessment of the in vivo role of bacterial fitness or virulence factors is of utmost importance to unravel S. pneumoniae pathogenicity mechanisms. Murine models of pneumonia, bacteremia, and meningitis are being used to determine the impact of pneumococcal factors at different stages of the infection. Here we describe a protocol to monitor in real-time pneumococcal dissemination in mice after intranasal or intraperitoneal infections with bioluminescent bacteria. The results show the multiplication and dissemination of pneumococci in the lower respiratory tract and blood, which can be visualized and evaluated using an imaging system and the accompanying analysis software.

Streptococcus pneumoniae is the leading pathogen causing severe community-acquired pneumonia and responsible for over 2&#160;million deaths worldwide. The impact of bacterial factors implicated in fitness or virulence can be monitored in real-time in an acute mouse pneumonia or bacteremia model using bioluminescent bacteria.

在实时以下的肺炎球菌毒力因子在小鼠急性肺炎模型的影响采用发光细菌

Pneumonia is one of the major health care problems in developing and industrialized countries and is associated with considerable morbidity and mortality. ...

Monitoring the Assembly of a Secreted Bacterial Virulence Factor Using Site-specific Crosslinking

This article describes a method to detect and analyze dynamic interactions between a protein of interest and other factors in vivo. Our method is based on the amber suppression technology that was originally developed by Peter Schultz and colleagues1. An amber mutation is first introduced at a specific codon of the gene encoding the protein of interest. The amber mutant is then expressed in E. coli together with genes encoding an amber suppressor tRNA and an amino acyl-tRNA synthetase derived from Methanococcus jannaschii. Using this system, the photo activatable amino acid analog p-benzoylphenylalanine (Bpa) is incorporated at the amber codon. Cells are then irradiated with ultraviolet light to covalently link the Bpa residue to proteins that are located within 3-8 &#197;. Photocrosslinking is performed in combination with pulse-chase labeling and immunoprecipitation of the protein of interest in order to monitor changes in protein-protein interactions that occur over a time scale of seconds to minutes. We optimized the procedure to study the assembly of a bacterial virulence factor that consists of two independent domains, a domain that is integrated into the outer membrane and a domain that is translocated into the extracellular space, but the method can be used to study many different assembly processes and biological pathways in both prokaryotic and eukaryotic cells. In principle interacting factors and even specific residues of interacting factors that bind to a protein of interest can be identified by mass spectrometry.

This article illustrates the use of pulse-chase radio labeling in combination with site-specific photocrosslinking to monitor interactions between a protein of interest and other factors in E. coli. Unlike traditional chemical cross-linking methods, this approach generates high resolution &#8220;snapshots&#8221; of an ordered assembly pathway in a living cell.

监视分泌细菌毒力因子的组装使用站点特定的交联

This article describes a method to detect and analyze dynamic interactions between a protein of interest and other factors in vivo. Our method is based on ...

Application of Fluorescent Nanoparticles to Study Remodeling of the Endo-lysosomal System by Intracellular Bacteria

Fluorescent nanoparticles (NPs) with desirable chemical, optical and mechanical properties are promising tools to label intracellular organelles. Here, we introduce a method using gold-BSA-rhodamine NPs to label the endo-lysosomal system of eukaryotic cells and monitor manipulations of host cellular pathways by the intracellular pathogen Salmonella enterica. The NPs were readily internalized by HeLa cells and localized in late endosomes/lysosomes. Salmonella infection induced rearrangement of the vesicles and accumulation of NPs in Salmonella-induced membrane structures. We deployed the Imaris software package for quantitative analyses of confocal microscopy images. The number of objects and their size distribution in non-infected cells were distinct from the ones in Salmonella-infected cells, indicating extremely remodeling of the endo-lysosomal system by WT Salmonella.

This article describes methods for the synthesis and fluorescent labeling of nanoparticles (NPs). The NPs were applied in pulse-chase experiments to label the endo-lysosomal system of eukaryotic cells. Manipulation of the endo-lysosomal system by activities of the intracellular pathogen Salmonella enterica were followed by live cell imaging and quantified.

通过胞内细菌，研究内溶酶体系统的改造荧光纳米粒子中的应用

Fluorescent nanoparticles (NPs) with desirable chemical, optical and mechanical properties are promising tools to label intracellular organelles. Here, we ...

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. Regulatory systems that utilize intracellular cyclic di-GMP (c-di-GMP) as a second messenger are one such class of target. c-di-GMP is a signaling molecule found in almost all bacteria that acts to regulate an extensive range of processes including antibiotic resistance, biofilm formation and virulence. The understanding of how c-di-GMP signaling controls aspects of antibiotic resistant biofilm development has suggested approaches whereby alteration of the cellular concentrations of the nucleotide or disruption of these signaling pathways may lead to reduced biofilm formation or increased susceptibility of the biofilms to antibiotics. We describe a simple high-throughput bioreporter protocol, based on green fluorescent protein (GFP), whose expression is under the control of the c-di-GMP responsive promoter cdrA, to rapidly screen for small molecules with the potential to modulate c-di-GMP cellular levels in Pseudomonas aeruginosa (P. aeruginosa). This simple protocol can screen upwards of 3,500 compounds within 48 hours and has the ability to be adapted to multiple microorganisms.

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

This article describes the high-throughput assay that has been successfully established to screen large libraries of small molecules for their potential ability to manipulate cellular levels of cyclic di-GMP in Pseudomonas aeruginosa, providing a new powerful tool for antibacterial drug discovery and compound testing.

高通量设置的建立筛选小分子能调节C-二GMP信令<em&gt;绿脓杆菌</em

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. ...

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 ...

Identification of Virulence Markers of Mycobacterium abscessus for Intracellular Replication in Phagocytes

What differentiates Mycobacterium abscessus from other saprophytic mycobacteria is the ability to resist phagocytosis by human macrophages and the ability to multiply inside such cells. These virulence traits render M. abscessus pathogenic, especially in vulnerable hosts with underlying structural lung disease, such as cystic fibrosis, bronchiectasis or tuberculosis. How patients become infected with M. abscessus remains unclear. Unlike many mycobacteria, M. abscessus is not found in the environment but might reside inside amoebae, environmental phagocytes that represent a potential reservoir for M. abscessus. Indeed, M. abscessus is resistant to amoebal phagocytosis and the intra-amoeba life seems to increase M. abscessus virulence in an experimental model of infection. However, little is known about M. abscessus virulence in itself. To decipher the genes conferring an advantage to M. abscessus intracellular life, a screening of a M. abscessus transposon mutant library was developed. In parallel, a method of RNA extraction from intracellular Mycobacteria after co-culture with amoebae was developed. This method was validated and allowed the sequencing of whole M. abscessus transcriptomes inside the cells; providing, for the first time, a global view on M. abscessus adaptation to intracellular life. Both approaches give us an insight into M. abscessus virulence factors that enable M. abscessus to colonize the airways in humans.

Identification of Virulence Markers of Mycobacterium abscessus for Intracellular Replication in Phagocytes

Here, we present two protocols to study Phagocyte-Mycobacterium abscessus interactions: the screening of a transposon mutant library for bacterial intracellular deficiency and the determination of bacterial intracellular transcriptome from RNA sequencing. Both approaches provide insight into the genomic advantages and transcriptomic adaptations enhancing intracellular bacteria fitness.

吞噬细胞内复制脓肿分枝杆菌毒力标志物的鉴定

What differentiates Mycobacterium abscessus from other saprophytic mycobacteria is the ability to resist phagocytosis by human macrophages and the ability ...

In Vivo Infection with Leishmania amazonensis to Evaluate Parasite Virulence in Mice

Leishmania spp. are protozoan parasites that cause leishmaniases, diseases that present a wide spectrum of clinical manifestations from cutaneous to visceral lesions. Currently, 12 million people are estimated to be infected with Leishmania worldwide and over 1 billion people live at the risk of infection. Leishmania amazonensis is endemic in Central and South America and usually leads to the cutaneous form of the disease, which can be directly visualized in an animal model. Therefore, L. amazonensis strains are good models for cutaneous leishmaniasis studies because they are also easily cultivated in vitro. C57BL/6 mice mimic the L. amazonensis-driven disease progression observed in humans and are considered one of the best mice strains model for cutaneous leishmaniasis. In the vertebrate host, these parasites inhabit macrophages despite the defense mechanisms of these cells. Several studies use in vitro macrophage infection assays to evaluate the parasite infectivity under different conditions. However, the in vitro approach is limited to an isolated cell system that disregards the organism&#39;s response. Here, we compile an in vivo murine infection method that provides a systemic physiological overview of the host-parasite interaction. The detailed protocol for the in vivo infection of C57BL/6 mice with L. amazonensis comprises parasite differentiation into infective amastigotes, mice footpad cutaneous inoculation, lesion development, and parasite load determination. We propose this well-established method as the most adequate method for physiological studies of the host immune and metabolic responses to cutaneous leishmaniasis.

In Vivo Infection with Leishmania amazonensis to Evaluate Parasite Virulence in Mice

Here, we present a compiled protocol to evaluate the cutaneous infection of mice with Leishmania amazonensis. This is a reliable method for studying parasite virulence, allowing a systemic view of the vertebrate host response to the infection.

在 Vivo 感染利什曼病亚马孙病评估小鼠的寄生虫毒性

Leishmania spp. are protozoan parasites that cause leishmaniases, diseases that present a wide spectrum of clinical manifestations from cutaneous to ...