This work was supported by grants 51RT 0_126008 and 51RTP0_151029 for the Research and Technology Development (RTD) project InfectX and TargetInfectX, respectively, in the frame of SystemsX.ch, the Swiss Initiative for Systems Biology. We acknowledge grant 310030B_149886 from the Swiss National Science Foundation (SNSF). Work of S.H.L and A.C. was supported by the International PhD Program "Fellowships for Excellence" of the Biozentrum. Simone Muntwiler is acknowledged for technical assistance. We would like to thank Dirk Bumann for providing pNF106 and Jean Celli for pJC43 and pJC44.

注意:用活流产布鲁氏菌菌株的所有工作必须考虑到所有必要的法规和安全防范生物安全3级（BSL3）实验室内进行。 1.筛选板的制备和细菌细胞的培养<ol><li> 流产布鲁氏菌发酵剂的制备<ol><li>条纹流产布鲁氏菌在含有50μg/ ml卡那霉素（TSA /公里）一个胰酶大豆琼脂（TSA）平板2308（B.流产 ）-80℃的牛奶库存。在37℃下孵育板3-4天。采用应变流产布鲁氏菌 2308 pJC43（apHT :: GFP）13端点检测和应变流产布鲁氏菌 pAC042.08（apht ::红色荧光蛋白，TETO ::四面体-GFP）的入口检测。 注意: 流产布鲁氏菌的光滑脂多糖（LPS）是一个重要的毒力决定，但粗糙突变体可以在相对 ​​高的频率14发生。 W¯¯Ë因此建议用这个实验启动前测试应变文化的地位。 </li><li>四个TSA /公里板Restreak细菌覆盖全板，并在37°C孵育2-3天。 </li><li>使用从TSA /公里板在10毫升10％灭菌脱脂牛奶一次性塑料环，转让和悬浮细菌。确保细菌以及悬浮以确保在所有发酵剂培养物一致的细菌浓度。 </li><li>等分试样将250μl细菌悬浮液中于2ml螺旋盖试管并冷冻在-80℃。 </li><li>解冻起始培养物的等分试样，并转移25微升，50微升，100微升细菌起子培养分开加入250ml螺旋盖瓶用50ml胰蛋白酶大豆肉汤（TSB）中含有50μg/ ml卡那霉素（TSB /公里） 。密封用封口膜的瓶子。 </li><li>过夜孵育以100rpm和37℃的轨道振荡器的瓶子。 </li><li>测量在600 n中的光密度米（OD 600），以确定过夜培养后达到的OD 600 = 0.8-1.1所需起始培养的体积。 </li></ol></li><li> HeLa细胞的制备<ol><li>生长的HeLa细胞在Dulbecco改进的在37℃的潮湿培养箱中在补充有10％胎牛血清（FCS）（DMEM / 10％FCS）中，用5％CO 2生长细胞至80％汇合的Eagle培养基（DMEM）中。 </li></ol></li><li>准备与siRNA的隔离板。 <ol><li>稀释在无RNase水的siRNA到0.32μM的终浓度。转印5微升/孔黑色透明孔平底384-孔板。 <ol><li>使用，列1 2，23和24的标准控件。对于阴性对照，使用非靶向siRNA（加扰）和模拟井无siRNA（即仅转染试剂）。用于转染的控制，可使用能诱导细胞死亡的siRNA（KIF11），以及涉及在布鲁氏菌感染（ 例如 ，ARPC3已知宿主因素的阳性对照，参与肌动蛋白聚合的Arp2 / 3复合）的一个组成部分。转移市售的siRNA库或自定义的siRNA到其余的孔中。 </li><li>密封板可剥离的铝箔​​。在-20℃储存板。 注意:板可以储存在-20℃至少6个月，至根据制造商的建议年。 </li></ol></li></ol></li></ol> 2.反向siRNA转染<ol><li>在300 XG解冻离心黑色384孔板，在室温下20分钟。 注意:这将降低所有可能粘附到盖子或孔的一侧的液体。 </li><li>在DMEM 200无FCS在室温下:通过稀释转染试剂1准备转染培养基。使用比现有20分钟不再准备转染培养基。仔细混合使用前解决方案。 </li><li>添加25微升转染培养基到每个使用试剂分配器井并且通过移动该板来回混合溶液。 </li><li>孵育1小时的板在室温下，以允许的siRNA /转染试剂复合物的形成。 </li><li>在此期间，通过用2.5ml的0.05％胰蛋白酶-EDTA的PBS（胰蛋白酶）洗涤一75cm 2的烧瓶中的子融合细胞一旦制备HeLa细胞。 </li><li>添加1.5毫升新鲜胰蛋白酶，将烧瓶转移到37℃2-3分钟，直到细胞围捕。 </li><li>悬浮细胞在10ml预热的DMEM / 16％FCS中。 </li><li>计数细胞使用自动细胞计数器和制备的在DMEM / 16％FCS中10,000个细胞/ ml的细胞悬浮液。 </li><li>加入50μl细胞悬液，每孔用导致500个细胞/孔的试剂分配器。 </li><li>移动板来回实现甚至细胞的分布。留在室温下将板5-10分钟，以允许细胞定居下来。 </li><li>用封口膜密封该板孵育它72小时上的预热铝板在5％的CO 2中的37℃湿润培养箱中。 注意:预热铝板允许在整个384孔板相等的温度分布。 </li></ol> 3.感染与固定<ol><li>感染前一天，接种在B的1.1.7确定的量在250毫升50毫升TSB /公里介质流产起子培养螺丝帽瓶中。密封该瓶用Parafilm和在37℃和100rpm的轨道摇床过夜生长的细菌OD 600 = 0.8-1.1。 </li><li>测量细菌培养的外径600和通过稀释细菌培养在DMEM / 10％FCS中，以达到感染复数（MOI）的所需多样性制备感染培养基。 注意:要获得在HeLa细胞5-10％的感染率，10000的MOI使用。一个MOI滴定曲线应为每种细胞类型，以获得最佳的感染率来执行。 </li><li>交换384孔板的转染培养基用50微升感染培养基的使用自动板洗涤器。 </li><li>离心384孔板在400 xg离心4℃20分钟。 注意:这有助于同步感染过程。 </li><li>用封口膜密封该板，并用5％的CO 2孵育其放在预热的铝板在37℃的潮湿培养箱中培养4小时。 </li><li>用含有100微克/毫升了Gm使用自动洗板灭活胞菌的DMEM / 10％FCS中洗涤细胞（使用200μl/孔溢出清洗）。 注:在溢出清洗，自动洗板机同时免除和抽吸介质。 </li><li>有关条目测定法中，洗涤细胞用含有100微克/毫升Gm与100纳克/毫升的aTc使用自动洗板机的DMEM / 10％FCS中的第二时间（使用200μl/孔溢出清洗）。 </li><li>用封口膜密封该板，并在37℃湿润培养箱中，用5％CO 2的另一个它返回到预热的铝板40小时或4小时，分别在端点检测和入口测定。 </li><li>为固定，使用自动洗板（使用200μl/孔溢出洗涤）洗涤各孔用PBS。 </li><li>交换的PBS用自动洗板机中的0.2M HEPES 50微升3.7％多聚甲醛在pH7.4（PFA）。 注:警告:PFA是吸入有毒的，与皮肤接触及吞食。 </li><li>孵育在室温下20分钟。 </li><li>使用自动洗板机交换用50μlPBS固定网上平台。注:样品现已准备在需要时从BSL3取出。 注意:从BSL3设施任何样品的去除受程序的风险评估和验证，并取决于对生物安全的有关法规。 </li></ol> 4.染色<ol><li>用50μl的PBS洗涤细胞两次。 </li><li>透于50μl0.1％的Triton-X-100的PBS细胞10分钟。 </li><li> W¯¯灰细胞三次，用PBS。加入20μl的PBS含有1微克/毫升的DAPI（4&#39;，6-二脒基-2-苯基）孵育在室温下30分钟。 </li><li>洗涤细胞三次在PBS避光染色用铝箔。 </li></ol> 5.成像<ol><li>设置用于图像采集的自动化宽视场显微镜。 注意:用于使用MetaXpress软件分子器件ImageXpress显微镜的设置如下所述。适当的硬件规格和图像处理软件替代2D宽视场显微镜设备可以使用（详见材料/设备表）。 <ol><li>选择10X客观，相机分级= 1，增益= 1。 </li><li>选择对应于该384孔板的板形式。 </li><li>获得每孔9点。 </li><li>使用"启用基于激光焦点"和"聚焦板井底"。 </li><li>设置"第一井"作为初始为好发现样品和站点自动对焦"所有网站"。 </li><li>使用DAPI通道手动设置Z-偏移焦点。 </li><li>手动选择Z-从偏移量为DAPI所有其他通道。 </li><li>手动修正的曝光时间的所有信道，以确保一个较宽的动态范围，低曝光过度。 <ol><li>获取使用自动曝光功能的代表部位的图像。使用此曝光时间图像3-5遗迹遍布板及以上的所有站点手动调整曝光时间，直到亮的像素达到了〜最大亮度的80％。 </li></ol></li><li>图片使用端点检测的DAPI和GFP渠道全板。对于进入试验选择另外RFP通道。 </li></ol></li></ol> 6.自动图像分析注:CellProfiler 2 月15日被用作图像分析软件来分割细胞和细菌的对象并执行自动测量电所确定的对象中对此语句。该软件提供了单独的模块，其可以被组合成一个管道，将连续执行上的所有图像的模块图像分析算法，以自动执行特定的图像分析的任务。按照该协议，安装CellProfiler 2.1.1或更高版本。然后，加载提供管道，并按照以下说明调整模块中所需的参数。所有管线的各个模块的描述可以在补充文件中找到。 注意:两个单独的管道被用于每个测定。第一管线计算阴影模型，其用于通过所述第二管道以校正之前分析图像。阴影校正被施加到图像以减少从显微镜不均匀光路的影响。计算在所述第一管道中的阴影模型是一个漫长的过程，但结果会更高的准确性。 <ol> &lt;LI&gt;端点检测<ol><li>计算着色模型<ol><li>进入"文件"菜单，选择"导入"→"从文件管道..."，然后选择所提供的管道"BrucellaShadingCorrectionPart1-Ver007"。当被问及是否将传统的管道进行转换，回答"不转换"。 </li><li>进入"输出"→"查看输出设置"，然后选择与图像输入文件夹和所产生的着色模型输出文件夹。 </li><li>在模块（1）"LoadImages"，调整的名称为"文字，这些图像具有在共同的"相匹配的图像的名称。 </li><li>确保没有模块取消选中旁边模块的所有符号眼球运行完整分析前展示自己的显示器。注意:此显著减小所需的计算资源，同时处理管道。 </li><li>按"分析图像"开始着色模型的计算。 </LI&gt; </ol></li><li>运行图像分析<ol><li>进入"文件"菜单，选择"导入"→"从文件管道..."，然后选择所提供的管道"BrucellaEndpointWithShadingCorrectionPart2-Ver007"。当被问及是否将传统的管道进行转换，回答"不转换"。 </li><li>进入"输出"→"查看输出设置"，然后选择与图像输入文件夹和生成的电子表格输出文件夹。 </li><li>在模块（1）"LoadImages"调节"的文本，这些图像具有在共同的"相匹配的图像的名称的名称。 </li><li>在模块（2）"LoadSingleImage"，通过选择的位置，并在两个着色模型的文件名加载6.1.1下计算出的着色模型。 </li><li>在模块（4） - （5）"ImageMath"，调整参数相匹配的显微摄像机的位深度"通过乘以第一图像"。为8位和16位图片参数设置为"1.0"，12位图像参数设置为"16.0"。 </li><li>在模块（9）"ImageMath"调整参数"通过第二图像正片叠底"的值（开始与0.25），这将抑制DAPI染色布鲁氏菌信号，而抑制细胞核。 <ol><li>点击"开始测试模式"，然后激活通过点击模块的相应的图标暂停符号和模块（9）显示功能。 注:暂停符号会显示为黄色，而如果激活眼部符号会显示一个开放的眼睛。 </li><li>点击"运行"运行分析到与主动暂停符号模块（9）。要为模块，按"步"测试值。 </li><li>右键单击新开张的形象，并设置"图像对比度"到"日志归一化"。多次回踩模块（9），调整参数"乘以第二形象"，以及步过Ť他再次模块，直至而在指示过减法的同时黑色区域，结果图像中最小的布鲁氏菌信号被完全抑制。 </li></ol></li><li>在模块（10）"IdentifyPrimaryObjects"，设置参数"下限门槛"足够高（从零开始），使原子核是分段和背景被忽略。 <ol><li>以在模块为模块（10），步骤识别物有所值并如步骤6.1.2.6.1和6.1.2.6.2说明显示输入图像。 </li><li>右键单击输入图像和显示直方图。 注意:直方图的峰通常表示背景。 </li><li>从背景强度开始增加参数，直到核的良好分割为显示的输出图像中实现。 注:设置超过背景强度更高的阈值是空的网站尤其重要。 </li></ol></li><li>在模块（15）4; FilterObjects"，调整参数"最小值"，以便在核细胞清晰可见布鲁保持，和所有其他被滤除在此步骤中，忽略布鲁细胞核外。 <ol><li>要确定一个很好的价值，步过该模块，如步骤6.1.2.6.1和6.1.2.6.2说明显示输出图像。从零，其中标识为受感染的所有单元格开始，并增加小步骤的值，直到仅在核细胞清晰可见布鲁氏菌被保留。 </li></ol></li><li>在模块（16），"FilterObjects"，调整参数"最小值"，使细胞清晰可见布鲁氏菌在核周被保持，并且过滤掉所有其他细胞。使用类似的方法与先前模块中。 </li><li>在模块（17）"FilterObjects"，调整参数"最小值"，使细胞清晰可见布鲁氏菌在涡onoi胞体被保留，并过滤掉所有其他细胞。使用类似的方法与先前模块中。 注意:Voronoi单元体是核的径向延伸由25个像素与相邻的Voronoi细胞体没有重叠。 </li><li>退出测试模式，并确保没有模块取消选中旁边模块的所有符号眼球运行完整分析前展示自己的显示器。 注意:此显著减小所需的计算资源，同时处理管道。 </li><li>按"分析图像"开始分析。 </li><li>当分析完成后，检查所得到的PNG图像以及该CSV片，以确保分析整个板可靠地工作。 </li></ol></li></ol></li><li>进入分析<ol><li>计算着色模型<ol><li>进入"文件"菜单，选择"导入"→"管道从文件..."，然后选择提供Ð管道"BrucellaShadingCorrectionPart1-Ver007"。当被问及是否将传统的管道进行转换，回答"不转换"。 </li><li>进入"输出"→"查看输出设置"，然后选择与图像输入文件夹和所产生的着色模型输出文件夹。 </li><li>在模块（1）"LoadImages"调节"的文本，这些图像具有在共同的"相匹配的图像的名称的名称。 </li><li>确保没有模块取消选中旁边模块的所有符号眼球运行完整分析前展示自己的显示器。 注意:此显著减小所需的计算资源，同时处理管道。 </li><li>按"分析图像"开始着色模型的计算。 </li></ol></li><li>运行图像分析<ol><li>进入"文件"菜单，选择"导入"→"管道从文件..."，然后选择providED管道"BrucellaEntryWithShadingCorrectionPart2-Ver007"。当被问及是否将传统的管道进行转换，回答"不转换"。 </li><li>进入"输出"→"查看输出设置"，然后选择与图像输入文件夹和生成的电子表格输出文件夹。 </li><li>在模块（1）"LoadImages"调节"的文本，这些图像具有在共同的"相匹配的图像的名称的名称。 </li><li>在模块（2）"LoadSingleImage"，通过选择的位置，并在两个着色模型的文件名加载6.2.1下计算出的着色模型。 </li><li>在模块（4） - （5）"ImageMath"，调整参数相匹配的显微摄像机的位深度"通过乘以第一图像"。对于8位和16位的图像参数设置为"1.0"，12位图像参数设置为"16.0"。 </li><li>在模块（6）"IdentifyPrimaryObjects"，设置参数"罗WER阈值"不够高的约束，只有原子核是分段和背景被忽略。 <ol><li>以识别模块（6），步骤在模块良好的值，并在步骤6.1.2.6.1和6.1.2.6.2说明显示输入图像。 </li><li>右键单击输入图像和显示直方图。 注意:直方图的峰通常表示背景。 </li><li>从背景强度开始增加参数，直到核的良好分割为显示的输出图像中实现。 注意:设置上述背景的阈值是空的网站很重要的。 </li></ol></li><li>在模块（8）"IdentifyPrimaryObjects"，设置参数"下限阈值"不够高，只有布鲁氏菌是分段和背景被忽略。 <ol><li>以识别模块（8），步骤在模块良好的值，并在步骤6.1.2.6.1和6.1.2.6.2说明显示输入图像。 </li><li>右键单击输入图像和显示直方图。 注意:直方图的峰通常表示背景。 </li><li>从背景强度开始增加参数，直到所显示的输出图像中的布鲁氏菌的良好的分割实现。 注意:设置上述背景的阈值是空的网站很重要的。 </li></ol></li><li>在模块（11）"FilterObjects"，调整参数"最小值"，使背景和文物被过滤掉的，只有殖民地布鲁氏菌保持。 <ol><li>要确定一个很好的价值，步过该模块，如步骤6.1.2.6.1和6.1.2.6.2说明显示输出图像。在6.2.2.7和逐步选择的值开始增加，直到仅布鲁氏菌的对象保持。 </li></ol></li><li>退出测试模式，通过unche运行完整分析之前，确保没有模块的展示自己的显示器盛泰旁边模块的所有眼球的符号。 注意:此显著减小所需的计算资源，同时处理管道。 </li><li>按"分析图像"开始分析。 </li><li>当分析完成后，检查所得到的PNG图像以及该CSV片，以确保分析整个板行​​之有效。 </li></ol></li></ol></li></ol> 7.感染评分注意:这对细胞活力显著影响的siRNA必须谨慎考虑，因为这可以促进假阳性发现。一个改变的细胞数会影响实际MOI和必需基因靶向可以对病原体感染多效性影响。而通过的siRNA不完全耗尽允许必需基因的研究中，这些目标必须被通过其他方法（ 例如，药物干扰），以证实它们作为宿主因素的作用进行验证感染期间。 <ol><li>端点分析<ol><li>打开步骤6.1.2.12产生该CSV片和除以感染的细胞的数目（CellProfiler读出:"Count_InfectedCells"）计算出每孔的感染速率由细胞的总数（CellProfiler读出:"CountNuclei"）。如果每口井多个站点已经成像，首先总结所有站点每口井，建成了每孔数感染的细胞和细胞每孔总数。 </li><li>执行ž得分正常化占板与板的变化，如果板含有足够的非击中的siRNA。假设有足够数量每盘不打siRNA的中频饱满库筛选。 <img alt="式（1）" src="/files/ftp_upload/54263/54263eq1.jpg" /></li></ol></li><li>进入分析<ol><li>打开步骤6.2.2.10产生该CSV片和除以感染的细胞的数目计算出每孔的感染率（CellProfiler读出:"Count_InfectedCeLLS"）的细胞总数（CellProfiler读出:"CountNuclei"）如果每口井多个站点已经成像，首先总结所有站点每口井，建成了每孔数感染细胞和每孔总数量的细胞。 </li><li>量化感染细胞的细菌载荷，估计由每个感染细胞内细菌所占据的平均面积。为此，除所有细胞内细菌与细胞重叠的累计面积（CellProfiler读出:"AreaOccupied_AreaOccupied_TrueIntPathogenInCells"）由被感染的细胞在此站点的数目。考虑到伪影，使用该读出作为阱读出的所有地点的中位数。 注意:如果该试验被用作含大量参与布鲁氏菌感染基因的跟进检测，不适用Z分数正常化。相反，如果使用模拟井作为参考，以占板与板的变化进行正常化。 </li></ol></li></ol>

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The authors declare no conflicts of interest.

Bacterial pathogens have evolved numerous strategies to manipulate eukaryotic host cells to their benefit. Pathogens causing acute infections often show rapid proliferation which is accompanied by significant alarming of the immune system and loss of viability of infected cells. In contrast, Brucella and other pathogens that cause chronic infections manage to establish long-lasting interactions within host cells. Therefore, bacteria need to fine tune host cell functions to their benefit without disrupting cellular homeostasis. A common strategy to identify the host cell factors involved in an infection process on a genome scale is the use of systematic gene depletion for example by siRNAs coupled to a functional readout of infection. Here we present protocols suitable for high-content, high-throughput screening based on detection of fluorescent bacteria.
An important aspect of successful large-scale screening involves a robust automated image analysis and infection scoring. The methods described here rely on the detection of a cell nucleus by DAPI staining which is generally very robust, and the definition of an area of interest where the pathogen signal is measured. The endpoint assay makes use of the detection of proliferating bacteria in the peri-nucleus, nucleus, and Voronoi cell body. This allows reliable identification of the infected cells. Furthermore, it allows defining different thresholds for the individual compartments. While the thresholds can be set relatively low in the nucleus and peri-nucleus, higher thresholds should be employed for the Voronoi cell body. This helps to avoid the detection of GFP signal from neighboring cells which is important since the endpoint assay lasts for five days during which HeLa cells have proliferated and grown to confluency.
The entry assay in contrast only lasts for three days and HeLa cells have thus not grown to confluency. Here, only a Voronoi cell body is chosen as cellular object for three main reasons. First, early during infection some bacteria might not yet have reached the nuclear or peri-nuclear area and may not be detected by measurements within these objects. Second, in contrast to the endpoint assay, bacteria have not started intracellular replication. Thus, using the Voronoi cell body object avoids missing individual bacteria which are located further away from the nucleus. Third, the entry assay makes use of quantification of the bacterial load which makes it desirable to cover as many bacteria as possible, which is more reliably achieved by using a Voronoi cell body compared to a peri-nucleus.
In the entry assay, the dsRed fluorescent marker which is constitutively expressed in all bacteria is not directly used for infection scoring. However, it serves as important quality control. Visual inspection of images gives a good indication whether bacteria that are distant from any nucleus and thus outside cells show no or only very weak GFP signal compared to bacteria in close proximity of a nucleus, indicative of an intracellular location. This is an important control to ensure that induction of GFP by aTc as well as inactivation of extracellular bacteria by Gm was efficient. Here, it has to be considered that in the absence of aTc bacteria express very low levels of GFP. In addition, extracellular bacteria (red signal and no/weak GFP signal) that overlap with a HeLa cell body can provide further information. In this case, bacteria could either be bound to the cell surface or they were internalized but failed to induce the GFP signal, indicative of very rapid killing of bacteria inside a host cell. These hypotheses can then be further tested with alternative assays such as counting of colony forming units (CFU) on TSA plates.
A further advantage of using a tetracycline inducible system over differential antibody staining to visualize intracellular bacteria is its applicability for live cell imaging. Depletion of host factors potentially affects various stages of the infection process which can be resolved by quantifying the dynamics of intracellular stages. To this end, the tetracycline inducible system can be used with live cell imaging to measure the onset of replication or to provide the information of the percentage of intracellular bacteria that initiate replication. 
While the protocol described here is tailored for the use of siRNAs it can be easily adapted to other screening strategies, such as CRISPR-Cas9, small hairpin RNA (shRNAs), and chemical library screening. This would only require basic experimental modifications whereas the image analysis pipeline can be used directly regardless of the screening strategy. Moreover, the assay design is not restricted to Brucella and is readily adapted to other intracellular pathogens10. As such, this protocol is widely applicable for screening host factors involved in pathogen mediated-infection of host cells.

布鲁氏菌物种是属于类α-变形菌的革兰氏阴性，兼性细胞内病原体。它们引起流产和不孕在其自然宿主如牛，山羊，绵羊或导致在流行地区严重的经济损失。布鲁氏菌病是最重要的人畜共患病引起全球每年超过五十万新的人类感染1之一。传输给人类最常用的是与受感染动物的直接接触或通过污染的食物摄入引起的，如未经高温消毒的牛奶。发热疾病的症状是非特异性的，这会导致困难布鲁氏菌病诊断。如果不治疗，患者可发展为慢性感染更严重的症状，如关节炎，心内膜炎，和神经病2。 细胞水平上的布鲁氏菌能够侵入吞噬细胞和非吞噬细胞和细胞内内复制车厢被称为布鲁氏菌含泡（BCV）。细菌的内化外消旋，卢，和Cdc42 3直接激活需要肌动蛋白骨架重排。真核宿主细胞内部，沿BCV内吞途径贩卖，尽管与溶酶体的相互作用，细菌设法避免降解4。由囊泡ATP酶的BCV酸化时需要引起细菌IV型分泌系统（T4SS）5的表达。据认为，由T4SS分泌细菌效应是必不可少的布鲁氏菌 ，以确立其复制利基，由于T4SS 6的缺失或囊泡ATP酶引线抑制在建立细胞小生7的缺陷。细菌中，直到他们贩卖的阶段不复制 达成ER衍生的液泡车厢8。一旦细胞内增殖时，BCV在CL发现与ER标志物如钙联接蛋白和葡萄糖-6-磷酸酶6 OSE关联。 由布鲁氏菌进入细胞，避免溶酶体降解，最后复制在ER-状隔间的分子机制仍是未知。参与感染的不同步骤宿主因素主要被确定的目标的方法或小规模小干扰RNA（siRNA）在果蝇细胞9执行屏幕。这些都揭示个人的宿主因素的布鲁氏菌感染过程中的贡献，但我们仍远未整个过程有全面的了解。 这里，协议，允许使用与自动宽视场荧光显微镜和自动图像分析相结合的大型RNA干扰（RNAi）筛选人类宿主因子的鉴定呈现。 HeLa细胞反向siRNA转染作为describ进行ED早期10,11稍作修改。端点测定覆盖除了出口和感染邻近细胞的布鲁氏菌细胞内的生命周期的很大一部分。为了进一步表征在端点测定法鉴定的命中，则使用一个修改协议，以确定所涉及的感染的早期步骤的因素。 组成型表达GFP一个流产布鲁氏菌菌株用于细菌的地方允许感染细胞两天端点检测。在此期间，细菌进入细胞，投放到ER衍生的复制利基，并在周围核空间复制。高水平的GFP信号然后可用于可靠地检测含复制的细菌的单个细胞。 为了研究在高通量检测布鲁项，它是能够细胞内外细菌区分重要。该方法在这里circumv介绍经济需求测试的细胞内和细胞外的细菌差速器抗体染色。它是基于一个布氏杆菌菌株表达结合的四环素诱导的GFP与DsRed的组成型表达。组成型DsRed的标志物的存在允许在样品中存在的所有的细菌的鉴定。 GFP表达是由庆大霉素加入无毒四环素类似物无水四环素（ATC）与细胞外细菌的灭活同时的（GM）诱导。而细胞不透抗生素了Gm杀死胞外细菌，的aTc可以进入宿主细胞并在细胞内细菌选择性诱导GFP表达。此双报告允许使用宽视场显微镜从胞菌（仅DsRed的信号）单胞菌（GFP和DsRed的信号）的健壮的分离。为了通过细胞内布鲁氏菌达到可检测的GFP表达，我们已经发现，通过的aTc 4小时的诱导导致RELI能信号。类似的诱导方案在胞内细菌选择性地表达GFP先前已用于研究细胞内的志贺氏菌 12。

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tryptic Soy Agar (TSA)</td>
			<td style="width:71px;">BD</td>
			<td style="width:119px;">236950</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tryptic Soy Broth (TSB)</td>
			<td style="width:71px;">Fluka</td>
			<td style="width:119px;">22092</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Kanamycin sulfate</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">60615</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Skim milk</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">250 ml screw cap bottle</td>
			<td style="width:71px;">Corning</td>
			<td style="width:119px;">8396</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">DMEM</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">D5796</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fetal Calf Serum (FCS)</td>
			<td style="width:71px;">Gibco</td>
			<td style="width:119px;">10270</td>
			<td style="width:167px;">Heat-inactivated</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Trypsin-EDTA (0.5%)</td>
			<td style="width:71px;">Gibco</td>
			<td style="width:119px;">15400-054</td>
			<td style="width:167px;">10x stock solution, dilute 1:10 in PBS</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Scepte 2.0 Cell Counter</td>
			<td style="width:71px;">Merck Milipore</td>
			<td style="width:119px;">PHCC20060</td>
			<td style="width:167px;">Alternative cell counting devices can be used.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Greiner CELLSTAR 384-well plate</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">M2062</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Peelable aluminum foil</td>
			<td style="width:71px;">Costar</td>
			<td style="width:119px;">6570</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Reagent dispenser: &#34;Multidrop 384 Reagent Dispenser&#34;</td>
			<td style="width:71px;">Thermo Scientific</td>
			<td style="width:119px;">5840150</td>
			<td style="width:167px;">Alternative reagent dispenser can be used.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Transfection reagent: &#34;Lipofectamine RNAiMAX</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:119px;">13778-150</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="252">
			<td height="252" style="height:252px;width:216px;">Automated plate washer: &#34;Plate washer ELx50-16&#34;</td>
			<td style="width:71px;">BioTek</td>
			<td style="width:119px;">ELX5016</td>
			<td style="width:167px;">This plate washer contains a 16-channel manifold suitable for 384-well plates. It fits into a biosafety cabinet and has a lid covering the plate during washing which reduces the risk of aerosol production. Alternative plate washers with similar features could be used.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Gentamicin</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">G1397</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Anhydrotetracycline hydrochloride</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">37919</td>
			<td style="width:167px;">100 ug/ml solution in 100% ethanol is kept at -20&#176;C protected from light in aluminum-foil</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PBS</td>
			<td style="width:71px;">Gibco</td>
			<td style="width:119px;">20012</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="168">
			<td height="168" style="height:168px;width:216px;">Paraformaldehyde</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">P6148</td>
			<td style="width:167px;">Dissolve in 0.2 M HEPES buffer, pH 7.4. Store at -20&#176;C and thaw freshly the day before use. Caution, PFA is toxic by inhalation, in contact with skin and if swallowed.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">HEPES</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">H3375</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Triton X-100</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">T9284</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DAPI</td>
			<td style="width:71px;">Roche</td>
			<td style="width:119px;">10236276001</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Scrambled siRNA</td>
			<td style="width:71px;">Dharmacon</td>
			<td style="width:119px;">D-001810-10</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Kif11 siRNA</td>
			<td style="width:71px;">Dharmacon</td>
			<td style="width:119px;">L-003317-00</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">ARPC3 siRNA</td>
			<td style="width:71px;">Dharmacon</td>
			<td style="width:119px;">L-005284-00</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Brucella abortus 2308</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">pJC43 (apHT::GFP)</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Celli et al.12</td>
		</tr>
		<tr height="277">
			<td height="277" style="height:277px;width:216px;">pAC042.08 (apht::dsRed, tetO::tetR-GFP)</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Construction: pJC44 4 was digested with EcoRI followed by generation of blunt ends with Klenov enzyme and subsequent digestion with SalI. TetR-GFP was amplified from pNF106 using primer prAC090 and prAC092. Following digestion with SalI, the TetR-GFP product was ligated to the digested pJC44 vector.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Primer prAC090</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">TTTTTGAATTCTGGCAATTCCGACGTCTAAGAAACC</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Primer prAC092</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">TTTTTGTCGACTTTGTCCTACTCAGGAGAGCGTTC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HeLa CCL-2</td>
			<td style="width:71px;">ATCC</td>
			<td style="width:119px;">CCL-2</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="231">
			<td height="231" style="height:231px;width:216px;">ImageXpress Micro</td>
			<td style="width:71px;">Molecular Devices&#160;</td>
			<td style="width:119px;">IXM IMAGING MSCOPE</td>
			<td style="width:167px;">Automated cellular imaging microscope equipped with a precision motorized Z-stage. Alternative systems for automated microscopy and alternative components for hard- and software specified below can be employed.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">High-Speed Laser Auto-Focus</td>
			<td style="width:71px;">Molecular Devices&#160;</td>
			<td style="width:119px;">1-2300-1037</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">CFI Super Fluor 10x objective</td>
			<td style="width:71px;">Nikon&#160;</td>
			<td style="width:119px;">MRF00100</td>
			<td style="width:167px;">N.A 0.50, W.D 1.20mm, DIC Prism: 10x, Spring loaded</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Photometrics CoolSNAP HQ Monochrome CCD Camera</td>
			<td style="width:71px;">Molecular Devices&#160;</td>
			<td style="width:119px;">1-2300-1060</td>
			<td style="width:167px;">1392 x 1040 imaging pixels, 6.45 x 6.45-&#181;m pixels, 12 bits digitization</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">MetaXpress software</td>
			<td style="width:71px;">Molecular Devices&#160;</td>
			<td style="width:119px;">9500-0100</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:216px;">LUI-Spectra-X-7</td>
			<td style="width:71px;">Lumencor</td>
			<td style="width:119px;">SPECTRA X V-XXX-YZ</td>
			<td style="width:167px;">Light engine. The following light sources are used: violet (DAPI), cyan (GFP), green/yellow (RFP)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Single Band Exciter for DAPI</td>
			<td style="width:71px;">Semrock</td>
			<td style="width:119px;">FF01-377/50-25</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Single Band Emitter for DAPI</td>
			<td style="width:71px;">Semrock</td>
			<td style="width:119px;">FF02-447/60-25</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Single Band Dichroic for DAPI</td>
			<td style="width:71px;">Semrock</td>
			<td style="width:119px;">FF409-Di03-25x36</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Single Band Exciter for GFP</td>
			<td style="width:71px;">Semrock</td>
			<td style="width:119px;">FF02-472/30-25</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Single Band Emitter for GFP</td>
			<td style="width:71px;">Semrock</td>
			<td style="width:119px;">FF01-520/35-25</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Single Band Dichroic for GFP</td>
			<td style="width:71px;">Semrock</td>
			<td style="width:119px;">FF495-Di03-25x36</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Single Band Exciter for RFP</td>
			<td style="width:71px;">Semrock</td>
			<td style="width:119px;">FF01-562/40-25</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Single Band Emitter for RFP</td>
			<td style="width:71px;">Semrock</td>
			<td style="width:119px;">FF01-624/40-25</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Single Band Dichroic for RFP</td>
			<td style="width:71px;">Semrock</td>
			<td style="width:119px;">FF593-Di03-25x36</td>
			<td></td>
		</tr>
	</tbody>
</table>

microscopy-based assays for high-throughput screening of host factors involved in brucella infection of hela cells

布鲁氏菌的物种是感染的动物作为天然宿主兼细胞内病原体。传播给人类最常通过与受感染的动物的直接接触或通过污染的食物摄入引起的，并可能导致严重的慢性感染。 布鲁氏菌可以侵入的专业和非专业的吞噬细胞和内质网（ER）衍生空泡内复制。 布鲁氏菌对进入宿主细胞，溶酶体降解的回避和复制在ER形腔室所需的宿主因素仍是未知。在这里，我们描述两个测定以鉴定参与在HeLa细胞布鲁氏条目和复制宿主因素。该协议描述了使用RNA干扰的同时，可应用替代的筛选方法。该测定法基于检测荧光标记的细菌使用自动化在荧光标记的宿主细胞宽视野显微镜。荧光图像用CellProfiler标准化图像分析管道，允许单细胞为基础的感染得分进行分析。 在端点检测，细胞内的复制感染两天后进行测量。这使得细菌交通到增殖的细菌进入。 布鲁氏菌已成功建立了一种细胞内的利基将因此已在宿主细胞内增殖强烈围绕后12小时开始他们的复制利基。因为细胞内的细菌会的数量大大超过个别的细胞外或细胞内非复制性的细菌，应变组成型表达绿色荧光蛋白可以使用。然后强GFP信号是用来识别被感染的细胞。 与此相反，对于条目测定是必不可少的细胞内和细胞外的细菌区分开来。这里，被用于四环素诱导的GFP的菌株的编码。感应与由庆大霉素胞菌的同时失活的GFP使细胞内和细胞基于所述GFP信号外细菌之间的分化，与仅细胞内细菌能够表达GFP。这允许鲁棒检测单胞菌的启动细胞内增殖之前。

图1A示出用于自动识别在端点检测感染细胞的图像分析的一个例子。用DAPI染色的HeLa细胞的细胞核被确定，包围核8像素宽度的周围核，以及由25个像素的核的扩展Voronoi单元体进行了计算。因为细菌主要增殖在围核的空间，在这个范围内的小区的GFP强度是最稳健的测​​定感染的和非感染的细胞之间进行区分。在某些情况下，细菌被发现围核或覆盖的外扩散与核很大程度上。因此，这两个附加的对象也被认为是感染的细胞的鉴定。分割以及GFP强度测量用图像分析软件CellProfiler 2进行。 布鲁氏菌 <str翁&gt;要求对宿主细胞3成功入侵肌动蛋白重排。因此，肌动蛋白重塑部件的消耗是用于siRNA筛选合适的阳性对照。我们已经发现，Arp2 / 3复合部件，其参与肌动蛋白聚合的的siRNA介导的枯竭，强烈抑制HeLa细胞的布鲁氏菌感染（未公布的数据）。因此，ARPC3击倒用作阳性对照。如在图1B中看到的，ARPC3耗竭减少了展示感染后增殖细菌2天细胞的数量。施加自动图像分析管道将这些图像允许的观察到的效果（ 图1C）的定量。这是这里示出的数据从一个全基因组的siRNA屏幕的控制孔产生。 ž得分加到占板到板的变化。 图2A示出了第i法师分析用于识别被感染的细胞，并测量在输入测定细胞内的细菌负荷。 与此相反的端点测定中，条目测定使用细菌分割和Voronoi单元体作为蜂窝对象。只有布鲁氏菌细胞内的GFP信号被用来段在这种图像分析管道的病原体。胞内细菌通过2个像素的最小尺寸和超过胞外细菌的昏暗的GFP背景强度的GFP强度限定。的细胞被认为是感染的，如果至少有一个细胞内的细菌的对象，其Voronoi单元体重叠。此外，该细菌负载通过计算由细胞内细菌覆盖感染细胞的平均面积估计。 至于端点检测，ARPC3枯竭显示， 布鲁氏菌感染的进入试验的减少相比，有一个反面处理的细胞控制的siRNA（ 图2B）。感染率的定量证实降低了ARPC3耗竭感染的细胞（ 图2C）以及细胞内细菌在被感染的细胞（ 图2D）的数的数量。 <img alt="图1" src="/files/ftp_upload/54263/54263fig1.jpg" /> 图1:HeLa细胞分析感染了 B.流产 在端点测定表达GFP（A）的端点测定:荧光图像示出了端点测定（绿色= B.流产表达用DAPI，比例尺=50μm的染色绿色荧光蛋白，蓝色=核）的一个例子。 GFP表达B.流产被允许进入HeLa细胞4小时，随后由通用汽车杀灭细菌外的。 40小时允许细菌进一步温育的HeLa细胞内复制。自动图像analysi<html> s是CellProfiler管道被用来段DAPI染色的细胞核随后围核的计算（非重叠的核周围8个像素圈）和Voronoi单元体（不重叠的25个像素的核放射状延伸）无论是在红色显示。的细胞被认为是感染的，如果在至少一细胞区室（核，周围核，Voronoi单元体）集成的GFP强度超过相应的阈值。 （B）感染与B的HeLa细胞的代表性图像流产表达GFP 44小时感染后，细胞或者用炒（非定向）控制的siRNA或靶向的siRNA转染ARPC3。比例尺= 50微米。贫为ARPC3 HeLa细胞的感染的（C）的定量。数据被表示为条形图（栏=平均值; Z得分正常化;误差棒=平均值的标准误差; *** P &lt;0.001; Mann-Whitney检验; N = 7）。ES / ftp_upload / 54263 / 54263fig1large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图2" src="/files/ftp_upload/54263/54263fig2.jpg" /> 图2:由 B HeLa细胞感染的定量 。 流产 在条目测定表达四面体-GFP（A）的条目测定:荧光图像示出流产表示流产示出红色荧光蛋白信号DsRed的和GFP信号，红色=胞外B.（黄色=胞内B.条目测定的一个例子，蓝色=细胞核用DAPI，比例尺= 50微米）染色。B.流产表达红色荧光蛋白和四面体-GFP被允许进入HeLa细胞4小时，随后外细菌和胞内细菌带ATC GFP的同时诱导4小时杀死自动图像分析:使用了CellProfiler管道检测DAPI染色的细胞核，随后通过由25个像素的核的径向延伸一Voronoi单元体的计算（在白色显示）。基于GFP信号细菌分割。的细胞被认为是感染如果其Voronoi单元体重叠足够的尺寸和GFP强度中的至少一方的分割细菌的对象。在感染的细胞中的细菌负荷是通过用其Voronoi单元体的所有分段细菌的对象的区域的积分所示（单位=像素; NAN =无编号在非感染的细胞计算）。 （B）的实施例的图像表示内B的下降流产在与siRNA的靶向ARPC3转染的细胞（由黄色的菌数示出）相比较以控制与加扰的siRNA处理的细胞。感染的细胞的数目以及细胞内细菌在被感染细胞的平均数量在ARPC3减少击倒。在HeLa细胞感染率（ 三 ）定量耗尽为ARPC3。数据被表示为条形图（栏=均值;正常化至模拟;误差棒=平均值的标准误差* P &lt;0.05; Mann-Whitney检验; N = 4）。贫为ARPC3感染的HeLa细胞中的细菌负荷的（D）的定量。该数据被表示为条形图（巴=均值;正常化模拟;误差条=平均值的标准误差; * P &lt;0.05; Mann-Whitney检验; N = 4）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54263/54263fig2large.jpg" target="_blank">请点击此处查看这个大版本数字。</a> 补充文件1:CP2 管道-阴影校正 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54263/CP2_pipeline-shading_correction.cppipe" target="_blank">请点击此处下载此文件。</a> 补充文件2:CP2 管道-端点检测。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54263/CP2_pipeline-Endpoint_assay.cppipe" target="_blank">请点击此处下载此文件。</a> 补充文件3:CP2 管道-输入法 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54263/CP2_pipeline-Entry_assay.cppipe" target="_blank">请点击此处下载此文件。</a> 补充文件4:模块的说明 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54263/Description_of_Modules.docx" target="_blank">请点击这里下载此文件。</a>

Watch this Scientific Journal Video about Microscopy-based Assays for High-throughput Screening of Host Factors Involved in Brucella Infection of Hela Cells at JoVE.com

Microscopy-based Assays for High-throughput Screening of Host Factors Involved in Brucella Infection of Hela Cells

Two assays for microscopy-based high-throughput screening of host factors involved in Brucella infection are described. The entry assay detects host factors required for Brucella entry and the endpoint assay those required for intracellular replication. While applicable for alternative approaches, siRNA screening in HeLa cells is used to illustrate the protocols.

在参与宿主因素的高通量筛选基于显微镜的检测方法<em&gt;布鲁氏菌</em&gt; Hela细胞感染

Brucella species are facultative intracellular pathogens that infect animals as their natural hosts. Transmission to humans is most commonly caused by ...

microscopy-based-assays-for-high-throughput-screening-host-factors

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

Microscopy-based Assays for High-throughput Screening of Host Factors Involved in Brucella Infection of Hela Cells

8.2K 次观看.  University of Basel. Two assays for microscopy-based high-throughput screening of host factors involved in Brucella infection are described. The entry assay detects host factors required for Brucella entry and the endpoint assay those required for intracellular replication. While applicable for alternative approaches, siRNA screening in HeLa cells is used to illustrate the protocols.

视频: Microscopy-based Assays for High-throughput Screening of Host Factors Involved in Brucella Infection of Hela Cells

RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells

Viral pathogens represent a significant public health threat; not only can viruses cause natural epidemics of human disease, but their potential use in bioterrorism is also a concern. A better understanding of the cellular factors that impact infection would facilitate the development of much-needed therapeutics. Recent advances in RNA interference (RNAi) technology coupled with complete genome sequencing of several organisms has led to the optimization of genome-wide, cell-based loss-of-function screens. Drosophila cells are particularly amenable to genome-scale screens because of the ease and efficiency of RNAi in this system 1. Importantly, a wide variety of viruses can infect Drosophila cells, including a number of mammalian viruses of medical and agricultural importance 2,3,4. Previous RNAi screens in Drosophila have identified host factors that are required for various steps in virus infection including entry, translation and RNA replication 5. Moreover, many of the cellular factors required for viral replication in Drosophila cell culture are also limiting in human cells infected with these viruses 4,6,7,8, 9. Therefore, the identification of host factors co-opted during viral infection presents novel targets for antiviral therapeutics. Here we present a generalized protocol for a high-throughput RNAi screen to identify cellular factors involved in viral infection, using vaccinia virus as an example.

RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells

Novel host factors involved in viral infection can be identified through cell-based genome-wide loss of function RNAi screening. A Drosophila cell culture model is particularly amenable to this approach due to the ease and efficiency of RNAi. Here we demonstrate this technique using vaccinia virus as an example.

RNAi筛选参与宿主因素牛痘病毒感染使用果蝇细胞

Viral pathogens represent a significant public health threat; not only can viruses cause natural epidemics of human disease, but their potential use in ...

科研

免疫与感染

VIGS-Mediated Forward Genetics Screening for Identification of Genes Involved in Nonhost Resistance

Nonhost disease resistance of plants against bacterial pathogens is controlled by complex defense pathways. Understanding this mechanism is important for developing durable disease-resistant plants against wide range of pathogens. Virus-induced gene silencing (VIGS)-based forward genetics screening is a useful approach for identification of plant defense genes imparting nonhost resistance. Tobacco rattle virus (TRV)-based VIGS vector is the most efficient VIGS vector to date and has been efficiently used to silence endogenous target genes in Nicotiana benthamiana.

	In this manuscript, we demonstrate a forward genetics screening approach for silencing of individual clones from a cDNA library in N. benthamiana and assessing the response of gene silenced plants for compromised nonhost resistance against nonhost pathogens, Pseudomonas syringae pv. tomato T1, P. syringae pv. glycinea, and X. campestris pv. vesicatoria. These bacterial pathogens are engineered to express GFPuv protein and their green fluorescing colonies can be seen by naked eye under UV light in the nonhost pathogen inoculated plants if the silenced target gene is involved in imparting nonhost resistance. This facilitates reliable and faster identification of gene silenced plants susceptible to nonhost pathogens. Further, promising candidate gene information can be known by sequencing the plant gene insert in TRV vector. Here we demonstrate the high throughput capability of VIGS-mediated forward genetics to identify genes involved in nonhost resistance. Approximately, 100 cDNAs can be individually silenced in about two to three weeks and their relevance in nonhost resistance against several nonhost bacterial pathogens can be studied in a week thereafter. In this manuscript, we enumerate the detailed steps involved in this screening. VIGS-mediated forward genetics screening approach can be extended not only to identifying genes involved in nonhost resistance but also to studying genes imparting several biotic and abiotic stress tolerances in various plant species.

Virus-induced gene silencing is an useful tool for identifying genes involved in nonhost resistance of plants. We demonstrate the use of bacterial pathogens expressing GFPuv in identifying gene silenced plants susceptible to nonhost pathogens. This approach is easy, fast and facilitates large scale screening and similar protocol can be applied to studying various other plant-microbe interactions.

VIGS介导的正向遗传学筛选参与非寄主抗性基因鉴定


	Nonhost disease resistance of plants against bacterial pathogens is controlled by complex defense pathways. Understanding this mechanism is important ...

A Microscopic Phenotypic Assay for the Quantification of Intracellular Mycobacteria Adapted for High-throughput/High-content Screening

Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since several decades, the sudden burst of multi- and extensively-drug resistant strains is a serious threat for the control of tuberculosis. Therefore, it is essential to identify new targets and pathways critical for the causative agent of the tuberculosis, Mycobacterium tuberculosis (Mtb) and to search for novel chemicals that could become TB drugs. One approach is to set&#160;up methods suitable for the genetic and chemical screens of large scale libraries enabling the search of a needle in a haystack. To this end, we developed a phenotypic assay relying on the detection of fluorescently labeled Mtb within fluorescently labeled host&#160;cells using automated confocal microscopy. This in vitro assay allows an image based quantification of the colonization process of Mtb&#160;into the host and was optimized for the 384-well microplate format, which is proper for screens of siRNA-, chemical compound- or Mtb mutant-libraries. The images are then processed for multiparametric analysis, which provides read&#160;out inferring on the pathogenesis of Mtb&#160;within host cells.

Here, we describe a phenotypic assay applicable to the High-throughput/High-content screens of small-interfering synthetic RNA (siRNA), chemical compound, and Mycobacterium tuberculosis mutant libraries. This method relies on the detection of fluorescently labeled Mycobacterium tuberculosis within fluorescently labeled host&#160;cell using automated confocal microscopy.

用于适应高通量/高含量筛查的细胞内分枝杆菌定量的微观表型检测

Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since ...

High-throughput Screening for Broad-spectrum Chemical Inhibitors of RNA Viruses

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat because of increased worldwide exchanges and human populations penetrating more and more natural ecosystems. A good example of such an emerging situation is chikungunya virus epidemics of 2005-2006 in the Indian Ocean. Recent progresses in our understanding of cellular pathways controlling viral replication suggest that compounds targeting host cell functions, rather than the virus itself, could inhibit a large panel of RNA viruses. Some broad-spectrum antiviral compounds have been identified with host target-oriented assays. However, measuring the inhibition of viral replication in cell cultures using reduction of cytopathic effects as a readout still represents a paramount screening strategy. Such functional screens have been greatly improved by the development of recombinant viruses expressing reporter enzymes capable of bioluminescence such as luciferase. In the present report, we detail a high-throughput screening pipeline, which combines recombinant measles and chikungunya viruses with cellular viability assays, to identify compounds with a broad-spectrum antiviral profile.

In vitro assays to measure virus replication have been greatly improved by the development of recombinant RNA viruses expressing luciferase or other enzymes capable of bioluminescence. Here we detail a high-throughput screening pipeline that combines such recombinant strains of measles and chikungunya viruses to isolate broad-spectrum antivirals from chemical libraries.

高通量筛选RNA病毒的广谱化学抑制剂

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat ...

A Fluorescence-based Lymphocyte Assay Suitable for High-throughput Screening of Small Molecules

High-throughput screening (HTS) is currently the mainstay for the identification of chemical entities capable of modulating biochemical reactions or cellular processes. With the advancement of biotechnologies and the high translational potential of small molecules, a number of innovative approaches in drug discovery have evolved, which explains the resurgent interest in the use of HTS. The oncology field is currently the most active research area for drug screening, with no major breakthrough made for the identification of new immunomodulatory compounds targeting transplantation-related complications or autoimmune ailments. Here, we present a novel in vitro murine fluorescent-based lymphocyte assay easily adapted for the identification of new immunomodulatory compounds. This assay uses T or B cells derived from a transgenic mouse, in which the Nur77 promoter drives GFP expression upon T- or B-cell receptor stimulation. As the GFP intensity reflects the activation/transcriptional activity of the target cell, our assay defines a novel tool to study the effect of given compound(s) on cellular/biological responses. For instance, a primary screening was performed using 4,398 compounds in the absence of a &#34;target hypothesis&#34;, which led to the identification of 160 potential hits displaying immunomodulatory activities. Thus, the use of this assay is suitable for drug discovery programs exploring large chemical libraries prior to further in vitro/in vivo validation studies.

We present in the current study a novel fluorescence-based assay using lymphocytes derived from a transgenic mouse. This assay is suitable for high-throughput screening (HTS) of small molecules endowed with the capacity of either inhibiting or promoting lymphocyte activation.

基于荧光的淋巴细胞检测适用于小分子的高通量筛选

High-throughput screening (HTS) is currently the mainstay for the identification of chemical entities capable of modulating biochemical reactions or ...

Screening Assays to Characterize Novel Endothelial Regulators Involved in the Inflammatory Response

The endothelial layer is essential for maintaining homeostasis in the body by controlling many different functions. Regulation of the inflammatory response by the endothelial layer is crucial to efficiently fight against harmful inputs and aid in the recovery of damaged areas. When the endothelial cells are exposed to an inflammatory environment, such as the outer component of gram-negative bacteria membrane, lipopolysaccharide (LPS), they express soluble pro-inflammatory cytokines, such as Ccl5, Cxcl1 and Cxcl10, and trigger the activation of circulating leukocytes. In addition, the expression of adhesion molecules E-selectin, VCAM-1 and ICAM-1 on the endothelial surface enables the interaction and adhesion of the activated leukocytes to the endothelial layer, and eventually the extravasation towards the inflamed tissue. In this scenario, the endothelial function must be tightly regulated because excessive or defective activation in the leukocyte recruitment could lead to inflammatory-related disorders. Since many of these disorders do not have an effective treatment, novel strategies with a focus on the vascular layer must be investigated. We propose comprehensive assays that are useful to the search of novel endothelial regulators that modify leukocyte function. We analyze endothelial activation by using specific expression targets involved in leukocyte recruitment (such as, cytokines, chemokines, and adhesion molecules) with several techniques, including: real-time quantitative polymerase chain reaction (RT-qPCR), western-blot, flow cytometry and adhesion assays. These approaches determine endothelial function in the inflammatory context and are very useful to perform screening assays to characterize novel endothelial inflammatory regulators that are potentially valuable for designing new therapeutic strategies.

Vascular endothelium tightly controls leukocyte recruitment. Inadequate leukocyte extravasation contributes to human inflammatory diseases. Therefore, searching for novel regulatory elements of endothelial activation is necessary to design improved therapies for inflammatory disorders. Here, we describe a comprehensive methodology to characterize novel endothelial regulators that can modify leukocyte trafficking during inflammation.

用于炎症反应的新内皮调节因子的筛选测定

The endothelial layer is essential for maintaining homeostasis in the body by controlling many different functions. Regulation of the inflammatory ...

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 ...

Arbovirus Infections As Screening Tools for the Identification of Viral Immunomodulators and Host Antiviral Factors

RNA interference- and genome editing-based screening platforms have been widely used to identify host cell factors that restrict virus replication. However, these screens are typically conducted in cells that are naturally permissive to the viral pathogen under study. Therefore, the robust replication of viruses in control conditions may limit the dynamic range of these screens. Furthermore, these screens may be unable to easily identify cellular defense pathways that restrict virus replication if the virus is well-adapted to the host and capable of countering antiviral defenses. In this article, we describe a new paradigm for exploring virus-host interactions through the use of screens that center on naturally abortive infections by arboviruses such as vesicular stomatitis virus (VSV). Despite the ability of VSV to replicate in a wide range of dipteran insect and mammalian hosts, VSV undergoes a post-entry, abortive infection in a variety of cell lines derived from lepidopteran insects, such as the gypsy moth (Lymantria dispar). However, these abortive VSV infections can be &#34;rescued&#34; when host cell antiviral defenses are compromised. We describe how VSV strains encoding convenient reporter genes and restrictive L. dispar cell lines can be paired to set-up screens to identify host factors involved in arbovirus restriction. Furthermore, we also show the utility of these screening tools in the identification of virally encoded factors that rescue VSV replication during coinfection or through ectopic expression, including those encoded by mammalian viruses. The natural restriction of VSV replication in L. dispar cells provides a high signal-to-noise ratio when screening for the conditions that promote VSV rescue, thus enabling the use of simplistic luminescence- and fluorescence-based assays to monitor the changes in VSV replication. These methodologies are valuable for understanding the interplay between host antiviral responses and viral immune evasion factors.

Here, we present the protocols to identify 1) virus-encoded immunomodulators that promote arbovirus replication and 2) eukaryotic host factors that restrict arbovirus replication. These fluorescence- and luminescence-based methods allow researchers to rapidly obtain quantitative readouts of arbovirus replication in simplistic assays with low signal-to-noise ratios.

虫媒病毒感染作为鉴定病毒免疫调节剂和宿主抗病毒因子的筛选工具

RNA interference- and genome editing-based screening platforms have been widely used to identify host cell factors that restrict virus replication. ...

High-throughput Measurement of Plasma Membrane Resealing Efficiency in Mammalian Cells

In their physiological environment, mammalian cells are often subjected to mechanical and biochemical stresses that result in plasma membrane damage. In response to these damages, complex molecular machineries rapidly reseal the plasma membrane to restore its barrier function and maintain cell survival. Despite 60 years of research in this field, we still lack a thorough understanding of the cell resealing machinery. With the goal of identifying cellular components that control plasma membrane resealing or drugs that can improve resealing, we have developed a fluorescence-based high-throughput assay that measures the plasma membrane resealing efficiency in mammalian cells cultured in microplates. As a model system for plasma membrane damage, cells are exposed to the bacterial pore-forming toxin listeriolysin O (LLO), which forms large 30-50 nm diameter proteinaceous pores in cholesterol-containing membranes. The use of a temperature-controlled multi-mode microplate reader allows for rapid and sensitive spectrofluorometric measurements in combination with brightfield and fluorescence microscopy imaging of living cells. Kinetic analysis of the fluorescence intensity emitted by a membrane impermeant nucleic acid-binding fluorochrome reflects the extent of membrane wounding and resealing at the cell population level, allowing for the calculation of the cell resealing efficiency. Fluorescence microscopy imaging allows for the enumeration of cells, which constitutively express a fluorescent chimera of the nuclear protein histone 2B, in each well of the microplate to account for potential variations in their number and allows for eventual identification of distinct cell populations. This high-throughput assay is a powerful tool expected to expand our understanding of membrane repair mechanisms via screening for host genes or exogenously added compounds that control plasma membrane resealing.

Here we describe a high-throughput fluorescence-based assay that measures the plasma membrane resealing efficiency through fluorometric and imaging analyses in living cells. This assay can be used for screening drugs or target genes that regulate plasma membrane resealing in mammalian cells.

哺乳动物细胞血浆膜检测效率的高通量测量

In their physiological environment, mammalian cells are often subjected to mechanical and biochemical stresses that result in plasma membrane damage. In ...

High Throughput Co-culture Assays for the Investigation of Microbial Interactions

The study of interactions between microorganisms has led to numerous discoveries, from novel antimicrobials to insights in microbial ecology. Many approaches used for the study of microbial interactions require specialized equipment and are expensive and time intensive. This paper presents a protocol for co-culture interaction assays that are inexpensive, scalable to large sample numbers, and easily adaptable to numerous experimental designs. Microorganisms are cultured together, with each well representing one pairwise combination of microorganisms. A test organism is cultured on one side of each well and first incubated in monoculture. Subsequently, target organisms are simultaneously inoculated onto the opposite side of each well using a 3D-printed inoculation stamp. After co-culture, the completed assays are scored for visual phenotypes, such as growth or inhibition. These assays can be used to confirm phenotypes or identify patterns among isolates of interest. Using this simple and effective method, users can analyze combinations of microorganisms rapidly and efficiently. This co-culture approach is applicable to antibiotic discovery as well as culture-based microbiome research and has already been successfully applied to both applications.

The co-culture interaction assays presented in this protocol are inexpensive, high throughput, and simple. These assays can be used to observe microbial interactions in co-culture, identify interaction patterns, and characterize the inhibitory potential of a microbial strain of interest against human and environmental pathogens.