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细胞1的生物学。然而，研究单个效应蛋白的生化活性已被证明是复杂的，原因如下:效应蛋白中1）的功能冗余，效应蛋白的接合2）的时间调节，以及3）短跑运动员器蛋白在环境中工作与其他效应蛋白，其功能的干扰。因此，效应蛋白质的活性，通常研究了过表达的研究。不幸的是，该效应蛋白的瞬时表达的使用也有几个缺点。因此，只有单细胞分析测定可以使用，并且如果效应蛋白的活性依赖于另一个效应蛋白的活性的效应蛋白的活性不能被分析。为了克服至少第一缺点，稳定的细胞系都可以使用。在情况下，效应器蛋白的表达是​​有毒的细胞，这是不适合的。在这种情况下，稳定的和可诱导的细胞系可以使用类似的系统被建立为我们在这里描述为细胞凋亡诱导蛋白（见下文）的瞬时表达。然而，我们的结果（ 图5）表明，CAEB的表达可以防止发生凋亡的细胞，因此不有毒的。有解剖中，在其中的凋亡级联CAEB干扰凋亡诱导内加强几个选项。一种可能性是，以确定CAEB的宿主细胞的结合伴侣。然而，这是非常具有挑战性的结合配偶的身份可能不利于凋亡级联中解释CAEB的作用点。另一种可能性是使用不同的细胞凋亡诱导剂，但CAEB抑制细胞凋亡的诱导通过两种不同的细胞凋亡诱导剂，紫外光和星形孢菌素7，这表明该方法可能也不会导致CAEB的作用点的识别。在缩小CAEB，将其在这里利用的作用点的另一种可能性，是使用一个系统，其中所述细胞凋亡信号传导级联可诱发在定义的步骤。 在被选作感兴趣的蛋白质已经与细胞生理学，其expre干扰细菌效应蛋白的表达情况裂变也可以放置在控制之下的诱导表达系统的， 例如 ，如用于表达靶蛋白之一。然后，感兴趣和靶蛋白上的两个蛋白质的表达将伴随地，当配位体加入到细胞培养物中允许研究者来监视细胞不同影响对细胞生理学和信令系统输出诱导。 显然，表达感兴趣的蛋白质，并试图通过选择途径蛋白的诱导表达，以确定在一个特定的细胞内通路的作用点的效果最好，如果感兴趣的蛋白质干扰所选途径的生理活性。在这种情况下，读出仅仅是通常与通路相关联，如细胞死亡的细胞凋亡信号或活化转录的Wnt信号的情况下的情况下，缺乏的信号。原则上，蛋白激活特定途径也可以与这件T探测YPE检测系统。它仅需要建立不同​​的，例如，使用Si / sh的诱导型表达/ miRNA的针对一个途径蛋白和测试，如果所关注的蛋白质可以拯救途径信号。可替代地，特定的蛋白质的小分子抑制剂可用于中断通路中的信号转导，然后由感兴趣的蛋白质的诱导表达，以检查途径拯救。然而，这种类型的方法将实验比上面给出的协议更有挑战性。 靶基因的选择要探测在干扰测定法是，在原则上，直接。最好是测试尽可能多的在所选择的信号通路尽可能蛋白质微调与通路相互作用感兴趣的网站上的蛋白质的鉴定。这应当以迭代方式来完成，因为它并不总是监视所有潜在INTERAC实验可行或甚至可能化的合作伙伴在一次实验。因此，最好是首先测试隔开均匀沿着整个通路几种蛋白，然后细化使用获得的第一轮的信息的实验。在信号通路，许多蛋白质​​被翻译后修饰，无论是通过一个共价修饰，如磷酸化或蛋白修饰，或由处理事件，例如蛋白水解切割。因此，各蛋白质或者是在一个基态或在活化形式。如果可能的话，最好是同时测试形式的蛋白质的原因有两个。之一是该活化形式提供了一个更高的输出信号31，其呈现了更严格的挑战为感兴趣的蛋白质。在我们的验证性原则，例如，凋亡信号，胱天蛋白酶3被蛋白水解裂解和二聚化激活。此激活事件可以人为通过重新排列未处理亲蛋白酶基因进行模拟。小亚基是人为地放置在大亚基的前部和两个由一个接头连接。这被称为"反向蛋白酶"和代表酶的活化形式表达时35。它被选择，由于其程度的活性更高。第二个原因，用于探测一个途径蛋白的两个基态和活化形式，是因为它允许一个区分如果感兴趣的蛋白干扰活化靶蛋白的，或者如果它作用于通路的蛋白的下游。在第一种情况下，细胞死亡将是禁止，如果基态形式被表达，但如果该活化形式存在。在第二种情况下，既不蛋白质会导致细胞凋亡。因此，测试这两种形式的改性蛋白质的提高了分析系统的分辨率。 克隆靶基因的诱导表达载体需要几个决定。首先，应当将靶基因的表达仅发生瞬时或是更​​好，如果该载体为stably整合入宿主细胞系的基因组？如果仅瞬时表达是必需的，一个简单的载体如pUHC13-3 24，其中包含一个四环素-依赖的启动子和polyA位点，或一个双向载体如基因的PBI-2或PBI-3 36，其耦合表达感兴趣的报道基因更容易监测靶基因表达的表达，就足够了。如果稳定的表达是优选的，应该使用一个载体如pWHE655 33（ 图3）。它包含侧翼的转基因表达单元，以防止从具有可诱导转基因表达的干扰在基因组整合位点的位置效应绝缘体。它还包含一个链接的，但独立地表示，抗性盒用于G418以促进载体整合事件的选择。这些修改的最终结果是增加了的克隆具有优良调节特性33,37,38的数目。本身的cDNA靶基因的quences可以通过使用独特的限制性位点EcoRI和EcoRV位33被克隆到该载体中。表达反Caspase-3的cDNA的载体的克隆进行了详细的参考33.克隆表达人Bax蛋白被类似地进行载体的描述。人类bax表达的cDNA从质粒pVenus-Bax蛋白39使用寡核苷酸引物引入限制性位点EcoRI和EcoRV位通过PCR扩增。 PCR片段与限制用两种酶的纯化后，将片段与同样受限pWHE655，得到pWHE655TREtight-Bax表达。第二，该最小启动子是最适合的转基因表达？原始最小启动子P 和hCMV * -1 24显示一些细胞类型特异性泄漏30，部分原因是由于功能性干扰素α诱导应答元件29的存在。这导致了第二代的发展四环素依赖启动子，称为ΔMtetO，TREtight和SG-TRE，与TETO元件之间并与不同的最小启动子序列33,40-42改变的间距和序列。他们显示效果大打折扣泄漏在没有强力霉素。在这些第二代子，ΔMtetO具有强力霉素33的存在下，最低的转基因表达水平。 TREtight介导的转基因表达33的中间和SG-TRE的最高水平。进一步的修改已经最小化的四环素应答启动子，甚至更43,44的背景表达。因此，四环素反应性启动子的选择可根据各个转基因的要求或限制进行。 独立的实验方法的类型，靶蛋白的表达的紧密度是很重要的。例如，细胞凋亡的诱导与活化形式的促凋亡蛋白导致细胞死亡，即使当各自靶蛋白的仅极少量表达31-33。所需的严格性通过添加四环素依赖transsilencer到条件监管系统30来实现的。它确保从诱导型启动子的转录积极抑制，这是特别重要的，当瞬时转染中进行的，因为它们通常是相当泄漏在其转基因表达30。如果信号通路不诱导细胞死亡进行探测，对于靶基因表达的严格调控的要求可以放宽，并诱导系统未经transsilencer可替代地43,44使用。 用于探测的信号传导途径的诱导型调节系统不必是四环素系统。其主要优点是，它已被广泛用于在哺乳动物细胞中超过20年了，它是最发达和最通道aracterized诱导型控制系统。然而，至少有25个其他的条件调节系统45,46，可以采用可替代地，或除四环素系统。两个或三个不同的诱导系统可能在探测会聚信号通路或用于测试任一靶基因或目的蛋白质的冗余是有用的。 这里提出的步骤应与该导致从被探测的各信号传导途径是绝对可靠的输出信号的转染效率。因为稳定的细胞系表达的均匀方式（ 图4B）感兴趣的蛋白质，被瞬时转染可诱导的质粒表达目的蛋白的所有细胞被探查与途径信号感兴趣的蛋白质的干扰。

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><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&amp;nbsp;</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 ，将细胞在两种细胞系中的90％以上的表达绿色荧光蛋白。因此，这些稳定的细胞系可用于进一步分析CAEB的功能。在接下来的步骤中，CAEB的抗凋亡活性通过免疫印迹分析使用抗切割的PARP抗体检测。核的PARP蛋白裂解灭活DNA修复活性并且是TERMI的典型标志物细胞凋亡的最终阶段。在HEK293-GFP或HEK293-GFP-CAEB细胞中未检测PARP的裂解，这表明GFP既不GFP-CAEB的表达，也不是对细胞有毒。然而，当细胞用星形孢菌素处理过的，内在凋亡的有效诱导，PARP切割检测（ 图5）。重要的是，将HEK293-GFP-CAEB细胞表现出减少的PARP裂解，表明贝氏柯克斯体的IV型分泌系统效应蛋白CAEB 7的抗凋亡活性。 为了分析在该步骤CAEB干扰凋亡途径，在TET-在系统采用。详细地说，将HEK293-GFP或HEK293-GFP-CAEB细胞用一个调节器的质粒组成型表达一个强力霉素受控双阻遏/活化剂设置（ 图2）和一个pTRE响应质粒编码或者全长人Bax蛋白或活化形式人caspase 3，被称为revCasp 3（图3）。这个系统是被严格调控，这表现在在非诱导条件（ 图6）缺乏PARP切割的。除强力霉素对转染细胞的导致Bax蛋白的表达或revCasp 3.如果CAEB干扰凋亡途径Bax的下游，然后通过表达和Bax的随后活化产生的细胞凋亡信号应阻止由CAEB表达。事实上，HEK293稳定表达GFP-CAEB深刻地抑制细胞凋亡诱导强力霉素控制Bax的表达，而HEK293-GFP的细胞显示明显的PARP切割。这一结果表明，CAEB块诱导凋亡Bax的激活的下游。接着，进行了分析CAEB是否可与胱天蛋白酶干扰-3激活 - 在凋亡途径晚期事件。 HEK293-GFP细胞，以及将HEK293-GFP-CAEB细胞，表现出PARP切割（ 图6），这表明一旦执行胱天蛋白酶3 ACTIVated，CAEB不能防止细胞凋亡的发生。总之，这些数据表明，CAEB Bax和胱天蛋白酶3的活化之间起作用。 <img alt="图1" src="/files/ftp_upload/52903/52903fig1.jpg" /> 图中的四环素调控系统1.示意图概览，四环素-亮系统是由两个不同的质粒，监管和响应质粒。监管质粒编码TET-响应transsilencer（TTS;实心圆）和四环素响应反式激活（rtTA，空心圆）。这两种蛋白质的强组成伸长因子-1α启动子（P EF-1a）的控制下组成型表达。 TTS和rtTA是嵌合转录因子选自由真核转录调控结构域（消音器或活化剂，分别）和细菌四环素阻遏（四面体）。四面体介导DNA结合到七TET </的EM&gt;对响应质粒符（TETO）序列。TETO位于可诱导的最小巨细胞病毒启动子（P CMV）上游的，一起形成的四环素响应元件（TRE）。在非诱导条件下，TTS势必TRE防止表达。另外强力霉素（阿霉素;实心菱形）后，rtTA交互霉素导致构象变化和激活。激活rtTA取代TTS上的反应质粒，使各自的目标基因Bax及caspase 3的转录，从而导致细胞凋亡诱导和细胞死亡。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52903/52903fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/52903/52903fig2.jpg" /> 监管质粒pWHE125图2.示意图。必不可少的元素在BAC传播特里亚，包括一个复制起点（ori PBR）和抗生素抗性盒针对氨苄青霉素（安培R）和对四环素-transregulators，如强组成立即早期启动子/人类巨细胞病毒的增强子的表达（P / ËHCMV），内部核糖体结合的脊髓灰质炎病毒（PV-IRES）和聚腺苷酸现场从猿猴病毒40（SV40多聚腺苷酸）显示的网站。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52903/52903fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/52903/52903fig3.jpg" /> 响应质粒的图3示意图概述。元素必需为在细菌中，包括一个复制起点（ori PBR）和抗生素抗性盒（安培R）的传播，以及用于诱导表达真核生物，如转基因expressi录像带上与春节相关的最小启动TREtight（P TREtight），感兴趣的人Bax蛋白（hBax）的基因和多聚腺苷酸的网站从猿猴病毒40（SV40聚A），被描绘。转基因表达盒是由鸡HS4绝缘体核心序列（HS4核心）的两个副本串联重复直接两侧。此外，G418抗性盒，包括鼠磷酸甘油酸激酶1启动子（P mPGK），新霉素抗性基因（G418 R）和人胸苷激酶聚A（TK多聚A）是存在的。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52903/52903fig3large.jpg" target="_blank">请点击此处查看大图这个数字。</a> <img alt="图4" src="/files/ftp_upload/52903/52903fig4.jpg" /> 图4.将HEK293-GFP和HEK293-GFP-CAEB稳定表达绿色荧光蛋白。（A）的 HEK293-GFP和的全细胞裂解物将HEK293-GFP-CAEB细胞进行免疫印迹的GFP显示稳定表达。如图（B）代表流式细胞仪（FACS）分析HEK293-GFP和HEK293-GFP-CAEB细胞表现出了GFP表达强度。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52903/52903fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/52903/52903fig5.jpg" /> 图5.影响GFP-CAEB对星形孢菌素诱导的细胞凋亡。将HEK293-GFP和HEK293-GFP-CAEB细胞中的表达被与4μM的星形孢菌素处理过的6小时以诱导固有凋亡。细胞凋亡是通过剪切的PARP免疫印迹分析分析。 PARP的裂解减少在HEK293-GFP-CAEB细胞相比，HEK293-GFP的细胞。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52903/52903fig5large.jpg" target="_blank">请点击此处查看大图</a>这个数字。 <img alt="图6" src="/files/ftp_upload/52903/52903fig6.jpg" /> 图6.用法顿体系来缩小CAEB的作用点。（A）凋亡诱导Bax蛋白在HEK293-GFP和HEK293-GFP-CAEB细胞中的表达。细胞凋亡是通过剪切的PARP免疫印迹分析分析。 PARP切割减少在HEK293-GFP-CAEB细胞相比，HEK293-GFP细胞。 （B）的凋亡诱导revCasp 3在HEK293-GFP和HEK293-GFP-CAEB细胞中的表达。细胞凋亡是通过剪切的PARP免疫印迹分析分析。 PARP的裂解是可比的HEK293-GFP-CAEB和HEK293-GFP的细胞。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52903/52903fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

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

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

9.2K 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

A Fluorescence-based Method to Study Bacterial Gene Regulation in Infected Tissues

Bacterial virulence genes are often regulated at the transcriptional level by multiple factors that respond to different environmental signals. Some factors act directly on virulence genes; others control pathogenesis by adjusting the expression of downstream regulators or the accumulation of signals that affect regulator activity. While regulation has been studied extensively during in vitro growth, relatively little is known about how gene expression is adjusted during infection. Such information is important when a particular gene product is a candidate for therapeutic intervention. Transcriptional approaches like quantitative, real-time RT-PCR and RNA-Seq are powerful ways to examine gene expression on a global level but suffer from many technical challenges including low abundance of bacterial RNA compared to host RNA, and sample degradation by RNases. Evaluating regulation using fluorescent reporters is relatively easy and can be multiplexed with fluorescent proteins with unique spectral properties. The method allows for single-cell, spatiotemporal analysis of gene expression in tissues that exhibit complex three-dimensional architecture and physiochemical gradients that affect bacterial regulatory networks. Such information is lost when data are averaged over the bulk population. Herein, we describe a method for quantifying gene expression in bacterial pathogens in situ. The method is based on simple tissue processing and direct observation of fluorescence from reporter proteins. We demonstrate the utility of this system by examining the expression of Staphylococcus aureus thermonuclease (nuc), whose gene product is required for immune evasion and full virulence ex vivo and in vivo. We show that nuc-gfp is strongly expressed in renal abscesses and reveal heterogeneous gene expression due in part to apparent spatial regulation of nuc promoter activity in abscesses fully engaged with the immune response. The method can be applied to any bacterium with a manipulatable genetic system and any infection model, providing valuable information for preclinical studies and drug development.

Described here is a method for analyzing bacterial gene expression in animal tissues at a cellular level. This method provides a resource for studying the phenotypic diversity occurring within a bacterial population in response to the tissue environment during an infection.

基于荧光的感染组织细菌基因调控方法

Bacterial virulence genes are often regulated at the transcriptional level by multiple factors that respond to different environmental signals. Some ...

科研

遗传学

Tractable Mammalian Cell Infections with Protozoan-primed Bacteria

Many intracellular bacterial pathogens use freshwater protozoans as a natural reservoir for proliferation in the environment. Legionella pneumophila, the causative agent of Legionnaires' pneumonia, gains a pathogenic advantage over in vitro cultured bacteria when first harvested from protozoan cells prior to infection of mammalian macrophages. This suggests that important virulence factors may not be properly expressed in vitro. We have developed a tractable system for priming L. pneumophila through its natural protozoan host Acanthamoeba castellanii prior to mammalian cell infection. The contribution of any virulence factor can be examined by comparing intracellular growth of a mutant strain to wild-type bacteria after protozoan priming. GFP-expressing wild-type and mutant L. pneumophila strains are used to infect protozoan monolayers in a priming step and allowed to reach late stages of intracellular growth. Fluorescent bacteria are then harvested from these infected cells and normalized by spectrophotometry to generate comparable numbers of bacteria for a subsequent infection into mammalian macrophages. For quantification, live bacteria are monitored after infection using fluorescence microscopy, flow cytometry, and by colony plating. This technique highlights and relies on the contribution of host cell-dependent gene expression by mimicking the environment that would be encountered in a natural acquisition route. This approach can be modified to accommodate any bacterium that uses an intermediary host as a means for gaining a pathogenic advantage.

This technique provides a method to harvest, normalize and quantify intracellular growth of bacterial pathogens that are pre-cultivated in natural protozoan host cells prior to infections of mammalian cells. This method can be modified to accommodate a wide variety of host cells for the priming stage as well as target cell types.

听话的哺乳动物细胞原生动物引发的细菌感染

Many intracellular bacterial pathogens use freshwater protozoans as a natural reservoir for proliferation in the environment. Legionella pneumophila, the ...

免疫与感染

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

Bacterial intracellular pathogens can be conceived as molecular tools to dissect cellular signaling cascades due to their capacity to exquisitely manipulate and subvert cell functions which are required for the infection of host target tissues. Among these bacterial pathogens, Listeria monocytogenes is a Gram positive microorganism that has been used as a paradigm for intracellular parasitism in the characterization of cellular immune responses, and which has played instrumental roles in the discovery of molecular pathways controlling cytoskeletal and membrane trafficking dynamics. In this article, we describe a robust microscopical assay for the detection of late cellular infection stages of L. monocytogenes based on the fluorescent labeling of InlC, a secreted bacterial protein which accumulates in the cytoplasm of infected cells; this assay can be coupled to automated high-throughput small interfering RNA screens in order to characterize cellular signaling pathways involved in the up- or down-regulation of infection.

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

Listeria monocytogenes is a Gram positive bacterial pathogen frequently used as a major model for the study of intracellular parasitism. Imaging late L. monocytogenes infection stages within the context of small-interfering RNA screens allows for the global study of cellular pathways required for bacterial infection of target host cells.

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 ...

Dissecting Innate Immune Signaling in Viral Evasion of Cytokine Production

In response to a viral infection, the host innate immune response is activated to up-regulate gene expression and production of antiviral cytokines. Conversely, viruses have evolved intricate strategies to evade and exploit host immune signaling for survival and propagation. Viral immune evasion, entailing host defense and viral evasion, provides one of the most fascinating and dynamic interfaces to discern the host-virus interaction. These studies advance our understanding in innate immune regulation and pave our way to develop novel antiviral therapies.
Murine &#947;HV68 is a natural pathogen of murine rodents. &#947;HV68 infection of mice provides a tractable small animal model to examine the antiviral response to human KSHV and EBV of which perturbation of in vivo virus-host interactions is not applicable. Here we describe a protocol to determine the antiviral cytokine production. This protocol can be adapted to other viruses and signaling pathways.
Recently, we have discovered that &#947;HV68 hijacks MAVS and IKK&#946;, key innate immune signaling components downstream of the cytosolic RIG-I and MDA5, to abrogate NF&#922;B activation and antiviral cytokine production. Specifically, &#947;HV68 infection activates IKK&#946; and that activated IKK&#946; phosphorylates RelA to accelerate RelA degradation. As such, &#947;HV68 efficiently uncouples NF&#922;B activation from its upstream activated IKK&#946;, negating antiviral cytokine gene expression. This study elucidates an intricate strategy whereby the upstream innate immune activation is intercepted by a viral pathogen to nullify the immediate downstream transcriptional activation and evade antiviral cytokine production.

We describe a protocol to measure the antiviral cytokine production in mice infected with a model herpesvirus, murine gamma herpesvirus 68 (&#947;HV68) that is closely-related to human Kaposi&#8217;s sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV). Utilizing genetically modified mouse strains and mouse embryonic fibroblasts (MEFs), we assessed the antiviral cytokine production both in vivo and ex vivo. &#8220;Reconstituting&#8221; the expression of innate immune components in knockout embryonic fibroblasts by lentiviral transduction, we further pinpoint specific innate immune molecules and dissect the key signaling events that differentially regulate the antiviral cytokine production.

细胞因子产生的病毒逃避解剖先天免疫信号

In response to a viral infection, the host innate immune response is activated to up-regulate gene expression and production of antiviral cytokines. ...

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 ...

Applying Fluorescence Resonance Energy Transfer (FRET) to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing

Coxiella burnetii, the causative agent of Q fever, is an intracellular pathogen that relies on a Type IV Dot/Icm Secretion System to establish a replicative niche. A cohort of effectors are translocated through this system into the host cell to manipulate host processes and allow the establishment of a unique lysosome-derived vacuole for replication. The method presented here involves the combination of two well-established techniques: specific gene silencing using siRNA and measurement of effector translocation using a FRET-based substrate that relies on &#946;-lactamase activity. Applying these two approaches, we can begin to understand the role of host factors in bacterial secretion system function and effector translocation. In this study we examined the role of Rab5A and Rab7A, both important regulators of the endocytic trafficking pathway. We demonstrate that silencing the expression of either protein results in a decrease in effector translocation efficiency. These methods can be easily modified to examine other intracellular and extracellular pathogens that also utilize secretion systems. In this way, a global picture of host factors involved in bacterial effector translocation may be revealed.

Applying Fluorescence Resonance Energy Transfer FRET to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing

Investigating the interactions between bacterial pathogens and their hosts is an important area of biological research. Here, we describe the necessary techniques to measure effector translocation by Coxiella burnetii during siRNA gene silencing using BlaM substrate.

应用荧光共振能量转移（FRET）审查效应转运效率由<em&gt;贝氏柯克斯体</em&gt; siRNA沉默中

Coxiella burnetii, the causative agent of Q fever, is an intracellular pathogen that relies on a Type IV Dot/Icm Secretion System to establish a ...

设计用于利什曼病治疗的四环素对照合成回路

Immunometabolic Circuits in Infection for Advancing Host Directed Therapies

Immuno-metabolism is a pivotal determinant in the progression of leishmaniasis. Synthetic biology-based approach has garnered significant attention as a step toward therapeutic intervention targeting host-associated factors that drive leishmaniasis. Synthetic biology entails the engineering of genetic components in an orthogonal and modular manner to precisely modulate biological systems, imparting novel functions to cells. In the presented study, elucidation of a systematic pipeline for the development of an inducible tetracycline-controlled (TetON)-based synthetic circuit was aimed at delivering succinate dehydrogenase as a therapeutic agent to facilitate the elimination of intracellular Leishmania parasites. The outlined protocol describes the designing of a synthetic circuit and its subsequent validation. The proposed strategy also concentrates on the incorporation of synthetic circuits in the plasmid backbone as a delivery vehicle. Additionally, delivery machinery employing polyplexes-based nano-particles for the delivery of synthetic circuits was used in murine macrophage cell lines without compromising the cellular morphology. Standardization of the method was conducted for selecting transfected cells and determining optimal induction concentration for synthetic circuit expression. Observations reveal a distinct reduction in intracellular parasite burden in transfected cells compared to infected cells. Pro-inflammatory cytokines were expressed post-infection in synthetic circuit transfected and induced cells as a mechanism to promote parasite elimination. This underscores the synthetic biology-based method as a potent approach in leishmaniasis by targeting host factors associated with disease progression.

作者聚焦:利用系统和合成生物学在利 什曼原虫 重症感染中创新疗法

Here, a protocol for designing, constructing, and delivering tetracycline-controlled based inducible synthetic circuit is presented. The effect of the synthetic circuits on parasite elimination and cytokine expression can be studied by channelizing the presented pipeline. This protocol is relevant for researchers studying synthetic circuit-based therapeutics in infectious diseases.

感染中的免疫代谢回路用于推进宿主定向治疗

Immuno-metabolism is a pivotal determinant in the progression of leishmaniasis. Synthetic biology-based approach has garnered significant attention as a ...

生物工程

A Hydroponic Co-cultivation System for Simultaneous and Systematic Analysis of Plant/Microbe Molecular Interactions and Signaling

An experimental design mimicking natural plant-microbe interactions is very important to delineate the complex plant-microbe signaling processes. Arabidopsis thaliana-Agrobacterium tumefaciens provides an excellent model system to study bacterial pathogenesis and plant interactions. Previous studies of plant-Agrobacterium interactions have largely relied on plant cell suspension cultures, the artificial wounding of plants, or the artificial induction of microbial virulence factors or plant defenses by synthetic chemicals. However, these methods are distinct from the natural signaling in planta, where plants and microbes recognize and respond in spatial and temporal manners. This work presents a hydroponic cocultivation system where intact plants are supported by metal mesh screens and cocultivated with Agrobacterium. In this cocultivation system, no synthetic phytohormone or chemical that induces microbial virulence or plant defense is supplemented. The hydroponic cocultivation system closely resembles natural plant-microbe interactions and signaling homeostasis in planta. Plant roots can be separated from the medium containing Agrobacterium, and the signaling and responses of both the plant hosts and the interacting microbes can be investigated simultaneously and systematically. At any given timepoint/interval, plant tissues or bacteria can be harvested separately for various &#34;omics&#34; analyses, demonstrating the power and efficacy of this system. The hydroponic cocultivation system can be easily adapted to study: 1) the reciprocal signaling of diverse plant-microbe systems, 2) signaling between a plant host and multiple microbial species (i.e.&#160;microbial consortia or microbiomes), 3) how nutrients and chemicals are implicated in plant-microbe signaling, and 4) how microbes interact with plant hosts and contribute to plant tolerance to biotic or abiotic stresses.

The described hydroponic cocultivation system supports intact plants with metal mesh screens and cocultivates them with bacteria. Plant tissue, bacteria, and secreted molecules can then be separately harvested for downstream analyses, simultaneously allowing for the molecular responses of both plant hosts and interacting microbes or microbiomes to be investigated.

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

摘要

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基于荧光的感染组织细菌基因调控方法

听话的哺乳动物细胞原生动物引发的细菌感染

成像InlC的分泌由细菌病原体调查感染蜂窝

细胞因子产生的病毒逃避解剖先天免疫信号

监视分泌细菌毒力因子的组装使用站点特定的交联

应用荧光共振能量转移（FRET）审查效应转运效率由

感染中的免疫代谢回路用于推进宿主定向治疗

感染中的免疫代谢回路用于推进宿主定向治疗

感染中的免疫代谢回路用于推进宿主定向治疗

用于植物/微生物分子相互作用和信号的同时系统分析的水培共培养系统