这项工作是由美国国立卫生研究院授予R01 AI073783光Teter。我们感谢援助，发展批判性阅读的手稿中的亚细胞分馏协议和海伦布瑞斯博士谢恩梅西。

1。对毛地黄皂苷的制备<ol><li>离心管加入500μL100％的乙醇，并将其放置在在80℃10分钟的热块。 </li><li>溶解在加热乙醇250μL2.5毫克毛地黄皂苷产生的毛地黄皂苷1％原液。 </li><li>要产生0.04％毛地黄皂苷的工作液，添加40μL毛地黄皂苷原液HCN的缓冲液960μL（50 mM的HEPES pH值7.5，150 mM氯化钠，氯化钙2 2毫米，10毫米的N - ethylmaleimide，和一种蛋白酶抑制剂鸡尾酒）。 </li></ol> 2。细胞中毒和通透性我们的易位检测可应用于一系列的毒素和细胞株。下面，我们提供了一个详细的协议检测霍乱毒素（CT）。在图1提供程序概述。 <ol><li> HeLa细胞接种于6孔板，含有1毫升的DMEM，10％的补充小牛血清和抗生素antimycotic要达到1 × 10 6细胞/后孵育过夜，于37 ° C。为了产生足够的重复性检测胞浆内毒素，一式三份井要求每个条件。 </li><li>孵育过夜后，更换培养液，用DMEM培养液1毫升含有100毫微克/毫升神经节苷脂。这增加了CT结合位点的数量，神经节苷脂受体的CT插质膜进入细胞暴露到GM1的解决方案。 </li><li> 1小时孵化后在37 ° C，删除GM1介质中，用DMEM洗细胞两次。然后，将细胞在4 ° C为30分钟，在1毫升的DMEM含有1微克/毫升的CT。 </li><li>毒素介质中取出，用DMEM洗细胞两次，在1毫升的DMEM培养的细胞，在37 ° C为所需的时间间隔（S）。 </li><li>在每个程序期间结束，用PBS洗涤细胞，每孔250μL0.5 mM的EDTA的PBS孵育。让细胞坐在10<html> min，室温，然后从6孔板，他们积极与P1000 pipetman trituration。一个细胞以及已被收集后，结合第二，以及来自相同的条件下，然后第三。细胞刮刀也可用于收集细胞。 </li><li>从三个复制井在一个单一的离心管（750μL总体积），合并后的细胞悬液，并在桌面离心5分钟旋转5000小工规模大致相当于细胞沉淀应获得的所有条件。 </li><li>弃上清，重悬细胞沉淀在100μL0.04％毛地黄皂苷的解决方案。将细胞悬液，冰浴10分钟。 </li><li>旋转16,000桌面离心10分钟XG毛地黄皂苷透细胞。细胞质含有上清转移到一个新的离心管中，并保留的细胞器中的颗粒分数。 </li></ol>标题为“样品制备&gt; 3。 <ol><li>胞浆含上清组分HCN的缓冲区最终体积为1 mL稀释。至少有1毫升的样本量是必要的，以确保空气是从500μL样品环注射前清除。 </li><li>细胞器含颗粒组分悬浮于1毫升氢氰酸含有1％的Triton X - 100的缓冲。洗涤剂除了是要释放的毒素膜包裹池。 </li><li>毒素在浓度为100，10，1，和0.1毫微克/毫升的标准，是在HCN的缓冲区。 </li><li>细胞质和细胞器组分的并行准备对照实验，以确认的分馏过程中的保真度。分数产生重悬于20μL4倍样品缓冲液（细胞质分数）或120μL1X上样缓冲液（细胞器的一小部分），在第2节描述。每个分数的等效卷十二烷基硫酸钠聚丙烯酰胺凝胶电泳和探讨解决bŸ Western blot分析证明胞浆蛋白在上清分区和一个可溶性，居民中ER蛋白的颗粒分数16-19。 </li></ol> 4。 SPR幻灯片准备<ol><li>赖克特SR7000 SPR折射用于SPR的实验。 </li><li>设置在自组装膜的SPR仪器的镀金玻片上。要激活传感器的幻灯片，灌注一个1:1（V：V），0.4 M EDC和0.1 M的NHS的解决方案在41μL/ min的流速在10分钟的幻灯片。所有后续灌注将使用相同​​的流速。 </li><li>要删除的EDC：NHS的激活缓冲区，洗5分钟，用含0.05％吐温20（PBST）的板块。现在该板块包含活性酰胺系绳由于uncapping由EDC：NHS的解决方案。 </li><li>灌注反注册税务师抗体在20毫米醋酸钠（pH值5.5）15分钟的幻灯片在1:20,000稀释激活传感器。在屈光的初始下降DEX单位（区情报组）将被视为由于pH值的变化。这之后，将增加在区情报组的抗体捕获传感器幻灯片酰胺反应的系绳。 </li><li> PBST洗涤5分钟的传感器幻灯片中删除未结合抗体。 RIU信号捕获抗体的产生将高原和稳定，提供了一个新的基准信号。 </li><li>灌注1米乙醇胺（pH值8.5），超过5分钟的传感器幻灯片。这帽子和灭活任何未绑定的传感器幻灯片上留下的系绳。 </li></ol> 5。 SPR的分析样本<ol><li>要建立一个基线阅读，超过5分钟的抗体包被传感器灌注PBST。 </li><li> 300秒，灌注实验样品在传感器或毒素标准。删除从200秒的配体的缓冲区，并在传感器灌注PBST。 </li><li>每个灌注，势必毒素是从传感器，100秒，在pH值5.0 PBST洗涤。这将返回的信号其初始基准读数，从而使另一个样品相同的传感器处理。 </li><li>在加载一个新的样品，通过样品回路推了少量空气，驱逐从之前样品的任何残留液。使用在5.2-5.4所述的程序，我们已经运行12个样品上一张幻灯片没有重大损失的基准信号。 </li><li>搓擦2软件进行数据分析，和伊图编制软件。 </li></ol> 6。代表性的成果百日咳毒素（PT）是一种AB毒素从细胞表面移动到急诊室前一个链（PTS1）进入细胞质中，12。正如图2所示，我们基于SPR -易位检测可以检测PTS1陶醉CHO细胞的细胞质。无信号产生unintoxicated细胞的细胞质中，证实反PTS1抗体没有交叉反应与主机细胞质的一个组成部分。 “从陶醉中brefeldin A（BFA）存在的细胞的细胞质部分也未能产生一个积极的信号。博鳌亚洲论坛的ER易位网站6-8，12，20-25，因此，A链传递到细胞质防止毒素运输。在每次运行结束时，势必毒素去除传感器幻灯片。这允许多个样品上的一个传感器幻灯片进行筛选，从而提供了一个与不同实验条件下得到的结果之间的直接比较。 CT是另一个AB型，ER转位毒素4。图3A，CTA1检测从CT处理的HeLa细胞的细胞质部分。这强调我们的方法与多种细胞类型，并可以应用到任何毒素的抗- A链抗体可。检测无信号时从CT治疗的细胞胞质部分灌注对涂有反城巴抗体（图3B）的SPR传感器，从而表现出的CTA1亚基，而不是细胞结合城巴五聚体进入细胞质。图3C显示信号从细胞器的一小部分是规模比较弱信号从胞质分数。这是已知的CT运输效率低下的ER易位网站6，7，这反过来又限制的毒素量，可以达到细胞质一致。此外，细胞器的一小部分包含的CT holotoxin以及ER本地化CTA1，因此所产生的SPR信号的细胞器的一小部分的holotoxin相关城巴五聚体的附加质量充气。因此，这是不切合实际的情节上相同sensorgram的细胞器和细胞质组分数据。 我们的检测可以监测易位，胞浆内毒素（图4）随时间变化的的积累。细胞暴露于CT在4 ° C，温度，让毒素结合到质膜，但阻止细胞相关毒素的内化。经过的圣雷莫VAL未结合的毒素，细胞被加热到37 ° C这两种毒素运输ER和A链移位到细胞质可以在此温度下发生的。没有检测到毒素在细胞质15分钟后升温至37 ° C这反映滞后的（I）holotoxin贩运到急诊室所需的时间;（II）的A / B在急诊室亚基解离;及（iii）A链出口到细胞质。轻微池的胞浆内毒素检测30分钟后在37 ° C，胞浆内毒素的数量逐渐变大后，45和60分钟的追逐间隔检测。检测后5小时的追逐间隔 17更大的胞浆内毒素水平，从而表现出一个持续的，长期的细胞相关毒素主机细胞质交付。 我们的检测，还可以检测出毒素易位抑制细胞质（图5）。细胞治疗与10％二甲基亚砜（DMSO），化学伴侣，防止热disord化工e圈隔离CTA1亚基（T. Banerjee和K. Teter，未发表意见），在未经处理的对照细胞相比展示的细胞质CTA1低的水平。展开毒素A链移位到细胞质16-18的先决条件，因此CTA1二甲基亚砜诱导稳定相应阻止其从ER到细胞质的运动。 该协会的速率常数（K）从SPR的实验计算灌注缓冲区14，15，26配体浓度成正比。因此，它是可能的胞浆内毒素的浓度，以确定从图图的 K毒素毒素浓度的函数标准一个值。这一程序是用来量化在图5中的毒素易位二甲基亚砜诱导块：已知浓度的毒素产生的标准曲线来计算一个细胞质CTA1 concentrat离子0.3毫微克/毫升未经处理的细胞和二甲基亚砜处理细胞的0.1纳克/毫升（图6）。二甲基亚砜开展CTA1抑制，从而产生了在雌激素受体细胞质易位的CTA1的减少了3倍。 <img alt="图1" src="/files/ftp_upload/3686/3686fig1.jpg" /> 图1。协议概述。 （一）细胞培养与AB毒素在4 ° C时，允许毒素结合到细胞表面的温度，但会阻止毒素的内吞作用。毒素A和B亚基是由红色和蓝色圆圈，分别代表。 （二）不作承诺毒素是从介质中删除，并加热到37℃，以促进内吞作用和逆行holotoxin运输到ER细胞。 Holotoxin解离发生在ER，它使孤立的A链进入细胞质，通过一种蛋白质在ER膜的导电沟道（S）。 （三）细胞与毛地黄皂苷处理，以选择性通透血浆膜。 （四）离心用于对细胞进行分区成单独的细胞质和细胞器的分数。细胞质被挤压出细胞通过毛地黄皂苷产生毛孔位于上清中。完整的膜结合细胞器中发现的颗粒分数。 （五）检测毒素A链在宿主细胞质易位池，上清灌注对涂抗- A链抗体的SPR传感器。 <img alt="图2" src="/files/ftp_upload/3686/3686fig2.jpg" /> 图2。PTS1进入宿主细胞质易位的检测。 CHO细胞脉冲标记，在4 ° C为1微克/毫升的PT 30分钟。细胞，然后3小时的追逐，在37 ° C的毒素培养基中含有没有增加（陶醉）或5博鳌亚洲论坛微克/毫升（+博鳌亚洲论坛陶醉）。与毛地黄皂苷的细胞膜的通透性，使用分区细胞提取到单独的细胞器和胞质fracti项。一个涂有反PTS1抗体的SPR传感器是用来检测PTS1从未经处理或治疗的博鳌亚洲论坛的细胞的细胞质池。 PTS1标准灌流在传感器作为阳性对照，而从unintoxicated细胞的胞浆中的一小部分是在传感器作为阴性对照幻灯片灌注。在每次运行结束时，势必样品被剥离来自传感器的幻灯片。 <img alt="图3" src="/files/ftp_upload/3686/3686fig3.jpg" /> 图3。CTA1进入宿主细胞质易位的检测。脉冲标记HeLa细胞在4 ° C组与1微克/毫升的CT 2小时的追逐，在37 ° C的毒素培养基中含有没有增加（陶醉）或5博鳌亚洲论坛微克/毫升（+博鳌亚洲论坛陶醉）。与毛地黄皂苷的细胞膜的通透性，使用分区细胞提取到单独的细胞器和细胞质组分。 （一）细胞质组分灌注了涂有反注册税务师抗体的SPR传感器。已知注册税务师的数量被用来作为阳性对照，而从unintoxicated细胞的细胞质作为阴性对照。 （二）陶醉在博鳌亚洲论坛的情况下从细胞的细胞质部分灌注了涂有反城巴抗体的SPR传感器。一个纯化的CTB五聚体灌注在幻灯片作为阳性对照。 （三）细胞器的一小部分被溶解，用1％Triton X - 100的的前灌注超过SPR传感器与反注册税务师抗体涂。为便于比较，胞质部分（1毫升最终卷）从同一个细胞中提取和注册税务师标准也灌注在传感器。所有面板，势必样品被剥离传感器在每次运行结束。 <img alt="图4" src="/files/ftp_upload/3686/3686fig4.jpg" /> 图4。CTA1到细胞质进入动力学。 HeLa细胞在4 ° C组与1微克/毫升的CT追为15，30，45，在37分钟或60℃，无毒mediu脉冲标记米。要检测的毒素易位池，从毛地黄皂苷-透细胞的细胞质组分灌注了涂有反注册税务师抗体的SPR传感器。灌注在传感器以及注册税务师标准。在每次运行结束时，势必样品被剥离来自传感器的幻灯片。 <img alt="图5" src="/files/ftp_upload/3686/3686fig5.jpg" /> 图5。CTA1二甲基亚砜易位的抑制作用。脉冲标记HeLa细胞在4 ° C组与1微克/毫升的CT 2小时的追逐，在37 ° C的毒素培养基中含有没有增加（陶醉）或10％DMSO（+二甲基亚砜陶醉）。要检测的毒素易位池，从毛地黄皂苷-透细胞的细胞质组分灌注了涂有反注册税务师抗体的SPR传感器。 CTA的标准（100，10，1，和0.1毫微克/毫升）灌注在传感器作为阳性对照，只有1和0.1 ng / mL的标准缩放显示。从unintoxicate细胞质D细胞被用来作为阴性对照。在每次运行结束时，势必样品被剥离来自传感器的幻灯片。 <img alt="图6" src="/files/ftp_upload/3686/3686fig6.jpg" /> 图6。胞浆CTA1计算，K从图5中的一个注册税务师标准值绘制毒素浓度的函数。由此产生的标准曲线是用来判断的基础上 K从图5中，在未经处理和DMSO处理的细胞的细胞质CTA1浓度的实验样品的一个值。毒素类标准为实心圆;未经处理的细胞质是作为一个开放的广场;二甲基亚砜处理的细胞质是作为一个开放的圆圈。两个独立的实验范围的平均值±所示。

没有利益冲突的声明。

与现有方法的比较我们基于SPR -易位检测的一种快速，敏感，定量的方法来检测毒素进入宿主细胞质交付。该技术并不需要放射性标记或其他修改的毒素，它可以应用到任何毒素，抗毒素A链抗体可。监测到细胞质的毒素通过现有的方法还依赖于一个分区成单独的细胞质和膜组分11，23，27，28的细胞提取物的亚细胞分馏协议。这些方法使用免疫印迹或radiolabels检测胞浆内?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td>毛地黄皂苷</td><td>西格玛</td><td> D141 </td></tr><tr><td>乙醇</td><td>阿库罗斯福冈</td><td> 61509-0010 </td></tr><tr><td> DMEM培养液</td><td> Invitrogen公司</td><td> 11995065 </td></tr><tr><td>胎牛血清</td><td>亚特兰大生物</td><td> S11550 </td></tr><tr><td>神经节苷脂</td><td>西格玛</td><td> G7641 </td></tr><tr><td>注册税务师</td><td>西格玛</td><td> C2398 </td></tr><tr><td> PTS1 </td><td>名单</td><td> 182 </td></tr><tr><td> NHS（N -羟基） </td><td>刺穿</td><td> 24500 </td></tr><tr><td> EDC（1 - 乙基-3 - （3 - dimethylaminopropyl）亚胺） </td><td> Thermo Scientific的</td><td> 22981 </td></tr><tr><td>乙醇胺</td><td>泗GMA </td><td> E0135 </td></tr><tr><td> PBST </td><td>紫花苜蓿</td><td> 09-8903-100 </td></tr><tr><td>反CTA抗体</td><td>圣克鲁斯生物技术</td><td> SC - 80747 </td></tr><tr><td>反城巴抗体</td><td> Calbiochem </td><td> 227040 </td></tr><tr><td>抗PTS1抗体</td><td>圣克鲁斯生物技术</td><td> SC - 57639 </td></tr><tr><td>折射</td><td>赖克特</td><td> SR7000，SR7000DC </td></tr><tr><td> SPR传感器幻灯片</td><td>赖克特</td><td> 13206060 </td></tr><tr><td>注射泵</td><td>科尔帕尔默</td><td> 780200C </td></tr></tbody></table>

detection of toxin translocation into the host cytosol by surface plasmon resonance

AB毒素组成的酶的A亚单位和一个细胞结合B 亚基 1 。这些毒素分泌到细胞外的环境，但他们的行为于真核细胞质内的目标。有些AB毒素旅游载体囊泡从细胞表面的内质网（ER），才进入细胞质2-4。在急诊室，不赞成从其余的毒素和移动通过一种蛋白质，在进行渠道的催化A链，以达到其胞质目标 5 。易位，胞质链是难以察觉，因为毒素贩运到急诊室是一个非常低效的过程：路由到最内在毒素降解的溶酶体，所以只有一小部分的表面结合的毒素达到高尔基体和ER 6 -12。 为了监视从ER在培养细胞的细胞质中的毒素易位，我们结合highl亚细胞分馏协议y的表面等离子体共振（SPR）13-15敏感的检测方法。毒素处理细胞的细胞膜选择性通透与毛地黄皂苷，使收集的一个细胞质部分，这是对涂有抗毒素A链抗体的SPR传感器随后灌注。抗体包传感器，可以捕捉和检测pg / mL的胞浆内毒素的数量。有了这个协议，它是可以遵循的毒素进入到细胞质的动力学特征易位事件的抑制作用。胞浆内毒素的浓度也可以从已知数量的A链已灌注在传感器的标准生成标准曲线计算。我们的方法代表了一种快速，敏感，定量检测系统不需要放射性标记或其他修改目标毒素。

Watch this Scientific Journal Video about Detection of Toxin Translocation into the Host Cytosol by Surface Plasmon Resonance at JoVE.com

Detection of Toxin Translocation into the Host Cytosol by Surface Plasmon Resonance

在这份报告中，我们描述了表面等离子体共振是如何用来检测输入到宿主细胞质中的毒素。胞浆内毒素量，这个高度敏感的方法可以提供定量数据，并可以应用到一系列的毒素。

表面等离子体共振检测到宿主细胞质的毒素易位

AB toxins consist of an enzymatic A subunit and a cell-binding B subunit1.  These toxins are secreted into the extracellular milieu, but they act upon ...

detection-toxin-translocation-into-host-cytosol-surface-plasmon

Research

JoVE Journal

Immunology and Infection

13.2K 次观看.  University of Central Florida. 在这份报告中，我们描述了表面等离子体共振是如何用来检测输入到宿主细胞质中的毒素。胞浆内毒素量，这个高度敏感的方法可以提供定量数据，并可以应用到一系列的毒素。

视频: Detection of Toxin Translocation into the Host Cytosol by Surface Plasmon Resonance

Detection of Infectious Virus from Field-collected Mosquitoes by Vero Cell Culture Assay

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many of these viruses have intensified in their endemic ranges and expanded to new territories, necessitating effective surveillance and control programs to respond to these threats. One strategy to monitor virus activity involves collecting large numbers of mosquitoes from endemic sites and testing them for viral infection. In this article, we describe how to handle, process, and screen field-collected mosquitoes for infectious virus by Vero cell culture assay. Mosquitoes are sorted by trap location and species, and grouped into pools containing &le;50 individuals. Pooled specimens are homogenized in buffered saline using a mixer-mill and the aqueous phase is inoculated onto confluent Vero cell cultures (Clone E6). Cell cultures are monitored for cytopathic effect from days 3-7 post-inoculation and any viruses grown in cell culture are identified by the appropriate diagnostic assays. By utilizing this approach, we have isolated 9 different viruses from mosquitoes collected in Connecticut, USA, and among these, 5 are known to cause human disease. Three of these viruses (West Nile virus, Potosi virus, and La Crosse virus) represent new records for North America or the New England region since 1999. The ability to detect a wide diversity of viruses is critical to monitoring both established and newly emerging viruses in the mosquito population.

We describe a method to process and screen field-collected mosquitoes for a diversity of viruses by Vero cell culture assay. By employing this technique, we have detected 9 different viruses from 4 taxonomic families in mosquitoes collected in Connecticut.

从现场收集到的蚊子传染病毒Vero细胞培养法检测

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many ...

科研

免疫与感染

Detection of Invasive Pulmonary Aspergillosis in Haematological Malignancy Patients by using Lateral-flow Technology

Invasive pulmonary aspergillosis (IPA) is a leading cause of morbidity and mortality in haematological malignancy patients and hematopoietic stem cell transplant recipients1. Detection of IPA represents a formidable diagnostic challenge and, in the absence of a 'gold standard', relies on a combination of clinical data and microbiology and histopathology where feasible. Diagnosis of IPA must conform to the European Organization for Research and Treatment of Cancer and the National Institute of Allergy and Infectious Diseases Mycology Study Group (EORTC/MSG) consensus defining "proven", "probable", and "possible" invasive fungal diseases2. Currently, no nucleic acid-based tests have been externally validated for IPA detection and so polymerase chain reaction (PCR) is not included in current EORTC/MSG diagnostic criteria.
Identification of Aspergillus in histological sections is problematic because of similarities in hyphal morphologies with other invasive fungal pathogens3, and proven identification requires isolation of the etiologic agent in pure culture. Culture-based approaches rely on the availability of biopsy samples, but these are not always accessible in sick patients, and do not always yield viable propagules for culture when obtained.
An important feature in the pathogenesis of Aspergillus is angio-invasion, a trait that provides opportunities to track the fungus immunologically using tests that detect characteristic antigenic signatures molecules in serum and bronchoalveolar lavage (BAL) fluids. This has led to the development of the Platelia enzyme immunoassay (GM-EIA) that detects Aspergillus galactomannan and a 'pan-fungal' assay (Fungitell test) that detects the conserved fungal cell wall component (1 &rarr;3)-&beta;-D-glucan, but not in the mucorales that lack this component in their cell walls1,4. Issues surrounding the accuracy of these tests1,4-6 has led to the recent development of next-generation monoclonal antibody (MAb)-based assays that detect surrogate markers of infection1,5.
Thornton5 recently described the generation of an Aspergillus-specific MAb (JF5) using hybridoma technology and its use to develop an immuno-chromatographic lateral-flow device (LFD) for the point-of-care (POC) diagnosis of IPA. A major advantage of the LFD is its ability to detect activity since MAb JF5 binds to an extracellular glycoprotein antigen that is secreted during active growth of the fungus only5. This is an important consideration when using fluids such as lung BAL for diagnosing IPA since Aspergillus spores are a common component of inhaled air. The utility of the device in diagnosing IPA has been demonstrated using an animal model of infection, where the LFD displayed improved sensitivity and specificity compared to the Platelia GM and Fungitell (1 &rarr; 3)-&beta;-D-glucan assays7.
Here, we present a simple LFD procedure to detect Aspergillus antigen in human serum and BAL fluids. Its speed and accuracy provides a novel adjunct point-of-care test for diagnosis of IPA in haematological malignancy patients.

A rapid and accurate point-of-care test for invasive pulmonary aspergillosis is presented. It takes advantage of lateral-flow technology using a specific monoclonal antibody that binds to an Aspergillus antigen secreted during pulmonary infections. The assay is compatible with serum and brochoalveolar lavage and represents a novel adjunct test for disease diagnosis.

通过横向流技术检测在血液系统恶性肿瘤患者侵袭性肺曲菌病

Invasive pulmonary aspergillosis (IPA) is a leading cause of morbidity and mortality in haematological malignancy patients and hematopoietic stem cell ...

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target cells. Inside the host cell the effector proteins manipulate cellular functions to the benefit of the bacteria. To better understand the control of type III secretion during host cell interaction, sensitive and accurate assays to measure translocation are required. We here describe the application of an assay based on the fusion of a Yersinia enterocolitica effector protein fragment (Yersinia outer protein; YopE) with TEM-1 beta-lactamase for quantitative analysis of translocation. The assay relies on cleavage of a cell permeant FRET dye (CCF4/AM) by translocated beta-lactamase fusion. After cleavage of the cephalosporin core of CCF4 by the beta-lactamase, FRET from coumarin to fluorescein is disrupted and excitation of the coumarin moiety leads to blue fluorescence emission. Different applications of this method have been described in the literature highlighting its versatility. The method allows for analysis of translocation in vitro and also in in vivo, e.g., in a mouse model. Detection of the fluorescence signals can be performed using plate readers, FACS analysis or fluorescence microscopy. In the setup described here, in vitro translocation of effector fusions into HeLa cells by different Yersinia mutants is monitored by laser scanning microscopy. Recording intracellular conversion of the FRET reporter by the beta-lactamase effector fusion in real-time provides robust quantitative results. We here show exemplary data, demonstrating increased translocation by a Y. enterocolitica YopE mutant compared to the wild type strain.

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Effector translocation into host cells via a type III secretion system is a common virulence strategy among gram-negative bacteria. A beta-lactamase effector fusion based assay for quantitative analysis of translocation was applied. In Yersinia infected cells, conversion of a FRET reporter by the beta-lactamase is monitored using laser scanning microscopy.

分析<em&gt;小肠结肠炎耶尔森菌</em&gt;效应易位到宿主细胞中使用β-内酰胺酶效应融合体

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target ...

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 ...

Detection of Trypanosoma brucei Variant Surface Glycoprotein Switching by Magnetic Activated Cell Sorting and Flow Cytometry

Trypanosoma brucei, a protozoan parasite that causes both Human and Animal African Trypanosomiasis (known as sleeping sickness and nagana, respectively) cycles between a tsetse vector and a mammalian host. It evades the mammalian host immune system by periodically switching the dense, variant surface glycoprotein (VSG) that covers its surface. The detection of antigenic variation in Trypanosoma brucei can be both cumbersome and labor intensive. Here, we present a method for quantifying the number of parasites that have 'switched' to express a new VSG in a given population. The parasites are first stained with an antibody against the starting VSG, and then stained with a secondary antibody attached to a magnetic bead. Parasites expressing the starting VSG are then separated from the rest of the population by running the parasites over a column attached to a magnet. Parasites expressing the dominant, starting VSG are retained on the column, while the flow-through contains parasites that express a new VSG as well as some contaminants expressing the starting VSG. This flow-through population is stained again with a fluorescently labeled antibody against the starting VSG to label contaminants, and propidium iodide (PI), which labels dead cells. A known number of absolute counting beads that are visible by flow cytometry are added to the flow-through population. The ratio of beads to number of cells collected can then be used to extrapolate the number of cells in the entire sample. Flow cytometry is used to quantify the population of switchers by counting the number of PI negative cells that do not stain positively for the starting, dominant VSG. The proportion of switchers in the population can then be calculated using the flow cytometry data.

Detection of Trypanosoma brucei Variant Surface Glycoprotein Switching by Magnetic Activated Cell Sorting and Flow Cytometry

African trypanosomes grown in vitro undergo antigenic variation at a low rate, such that populations are made up of parasites expressing a dominant variant surface glycoprotein (VSG) type and a small population of "switched" variants. This protocol describes a fast method for detecting and quantifying these populations.

检测<em&gt;布氏锥虫</em&gt;变体表面糖蛋白通过磁性细胞分选和流式细胞仪开关

Trypanosoma brucei, a protozoan parasite that causes both Human and Animal African Trypanosomiasis (known as sleeping sickness and nagana, respectively) ...

Protocols for Investigating the Host-tissue Distribution, Transmission-mode, and Effect on the Host Fitness of a Densovirus in the Cotton Bollworm

Many novel viruses have been discovered in animal hosts using next-generation sequencing technologies. Previously, we reported a mutualistic virus, Helicoverpa armigera densovirus (HaDV2), in a lepidopteran species, the cotton bollworm, Helicoverpa armigera (Hubner). Here, we describe the protocols that are currently used to study the effect of HaDV2 on its host. First, we establish a HaDV2-free cotton bollworm colony from a single breeding pair. Then, we orally inoculate some neonate larval offspring with HaDV2-containing filtered liquid to produce two colonies with the same genetic background: one HaDV2-infected, the other uninfected. A protocol to compare life table parameters (e.g., larval, pupal, and adult periods and fecundity) between the HaDV2-infected and -uninfected individuals is also presented, as are the protocols for determining the host-tissue distribution and transmission efficiency of HaDV2. These protocols would also be suitable for investigating the effects of other orally transmitted viruses on their insect hosts, lepidopteran hosts in particular.

Here, we present a protocol to investigate the host-tissue distribution, transmission mode, and effect on the host fitness of a densovirus within a lepidopteran species, the cotton bollworm. This protocol can also be used for studying the interaction between other orally-transmitted viruses and their insect hosts.

协议侦办主机组织分布，传播模式和效果上浓核病毒的主机健身的棉铃虫

Many novel viruses have been discovered in animal hosts using next-generation sequencing technologies. Previously, we reported a mutualistic virus, ...

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome (HUS), colonize to the intestinal tract of humans. To study the detailed mechanism of EHEC colonization in vivo, it is essential to have animal models to monitor and quantify EHEC colonization. We demonstrate here a mouse-EHEC colonization model by transforming the bioluminescent expressing plasmid to EHEC to monitor and quantify EHEC colonization in living hosts. Animals inoculated with bioluminescence-labeled EHEC show intense bioluminescent signals in mice by detection with a non-invasive in vivo imaging system. After 1 and 2 days post infection, bioluminescent signals could still be detected in infected animals, which suggests that EHEC colonize in hosts for at least 2 days. We also demonstrate that these bioluminescent EHEC locate to mouse intestine, specifically in the cecum and colon, from ex vivo images. This mouse-EHEC colonization model may serve as a tool to advance the current knowledge of the EHEC colonization mechanism.

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

A detailed protocol of a mouse model for enterohemorrhagic E. coli (EHEC) colonization by using bioluminescence-labeled bacteria is presented. The detection of these bioluminescent bacteria by a non-invasive in vivo imaging system in live animals can advance our current understanding of EHEC colonization.

非侵入性 体内生物发光系统检测小鼠宿主肠出血性大肠杆菌的定植

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome ...

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella spp./Shigella spp. The gold standard method for the detection of Salmonella spp./Shigella spp. is direct culture but this is labor-intensive and time-consuming. Here, we describe a high-throughput platform for Salmonella spp./Shigella spp. screening, using real-time polymerase chain reaction (PCR) combined with guided culture. There are two major stages: real-time PCR and the guided culture. For the first stage (real-time PCR), we explain each step of the method: sample collection, pre-enrichment, DNA extraction and real-time PCR. If the real-time PCR result is positive, then the second stage (guided culture) is performed: selective culture, biochemical identification and serological characterization. We also illustrate representative results generated from it. The protocol described here would be a valuable platform for the rapid, specific, sensitive and high-throughput screening of Salmonella spp./Shigella spp.

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Salmonella spp./Shigella spp. are common pathogens attributed to diarrhea. Here, we describe a high-throughput platform for the screening of Salmonella spp./Shigella spp. using real-time PCR combined with guided culture.

沙门氏菌筛选的高通量平台.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...

Temporal Analysis of the Nuclear-to-cytoplasmic Translocation of a Herpes Simplex Virus 1 Protein by Immunofluorescent Confocal Microscopy

Infected cell protein 0 (ICP0) of herpes simplex virus 1 (HSV-1) is an immediate early protein containing a RING-type E3 ubiquitin ligase. It is responsible for the proteasomal degradation of host restrictive factors and the subsequent viral gene activation. ICP0 contains a canonical nuclear localization sequence (NLS). It enters the nucleus immediately after de novo synthesis and executes its anti-host defense functions mainly in the nucleus. However, later in infection, ICP0 is found solely in the cytoplasm, suggesting the occurrence of a nuclear-to-cytoplasmic translocation during HSV-1 infection. Presumably ICP0 translocation enables ICP0 to modulate its functions according to its subcellular locations at different infection phases. In order to delineate the biological function and regulatory mechanism of ICP0 nuclear-to-cytoplasmic translocation, we modified an immunofluorescent microscopy method to monitor ICP0 trafficking during HSV-1 infection. This protocol involves immunofluorescent staining, confocal microscope imaging, and nuclear vs. cytoplasmic distribution analysis. The goal of this protocol is to adapt the steady state confocal images taken in a time course into a quantitative documentation of ICP0 movement throughout the lytic infection. We propose that this method can be generalized to quantitatively analyze nuclear vs. cytoplasmic localization of other viral or cellular proteins without involving live imaging technology.

ICP0 undergoes nuclear-to-cytoplasmic translocation during HSV-1 infection. The molecular mechanism of this event is not known. Here we describe the use of confocal microscope as a tool to quantify ICP0 movement in HSV-1 infection, which lays the groundwork for quantitatively analyzing ICP0 translocation in future mechanistic studies.

免疫荧光共聚焦显微镜对单纯疱疹病毒1蛋白核细胞质转移的时间分析

Infected cell protein 0 (ICP0) of herpes simplex virus 1 (HSV-1) is an immediate early protein containing a RING-type E3 ubiquitin ligase. It is ...

Evaluation of Host-Pathogen Responses and Vaccine Efficacy in Mice

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a striking increase in life expectancy around the globe. Nonetheless, determining vaccine efficacy remains a challenge. Emerging evidence suggests that the current acellular vaccine (aPV) for Bordetella pertussis (B. pertussis) induces suboptimal immunity. Therefore, a major challenge is designing a next-generation vaccine that induces protective immunity without the adverse side effects of a whole-cell vaccine (wPV). Here we describe a protocol that we used to test the efficacy of a promising, novel adjuvant that skews immune responses to a protective Th1/Th17 phenotype and promotes a better clearance of a B. pertussis challenge from the murine respiratory tract. This article describes the protocol for mouse immunization, bacterial inoculation, tissue harvesting, and analysis of immune responses. Using this method, within our model, we have successfully elucidated crucial mechanisms elicited by a promising, next-generation acellular pertussis vaccine. This method can be applied to any infectious disease model in order to determine vaccine efficacy.

Here we present an elegant protocol for in vivo evaluation of vaccine effectiveness and host immune responses. This protocol can be adapted for vaccine models that study viral, bacterial, or parasitic pathogens.

宿主病原学反应及疫苗效果评价

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a ...