Name: use of shigella flexneri to study autophagy-cytoskeleton interactions
Uploaded: 2014-09-09T18:00:00
Duration: 12 min 52 s
Description: Watch this Scientific Journal Video about Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions at JoVE.com

在SM实验室工作是由威康信托基金会的研究职业发展奖学金[WT097411MA]支持。

1，监测自噬和细胞骨架在体外用组织培养细胞<ol><li>准备学菌 <ol><li>板南菌 M90T（野生型），从-80℃甘油到刚果红的胰蛋白酶大豆酪蛋白（TCS）的琼脂平板。孵育过夜，在37℃。相同的板可以用于多种实验。 </li><li>选择一个单独的殖民地，在37℃生长于8ml TCS媒体在摇床过夜。 注：刚果红的结合表明，毒力质粒已被保留。 </li><li>继代培养细菌的指数级增长，接种新鲜TCS与隔夜细菌培养在1/80稀释，并在37℃生长在摇床至OD 600 = 0.3-0.6。 </li><li>旋细菌传代于1000×g离心5分钟。在1,000 XG洗用MEM和离心沉淀5分钟。重组沉淀于MEM至OD 600 = 0.3-0.6。 </li></ol></li><li>准备海拉策LLS的感染<ol><li>生长在“完全培养基”， 即 HeLa细胞，MEM加L-丙氨酰-L-谷氨酰胺补充有1mM丙酮酸钠，0.1mM非必需氨基酸溶液，和10％的胎牛血清。 </li><li>板状1-1.5×10 5个细胞在6孔板24或48小时的实验开始之前。盘上的玻璃盖玻片的6孔​​板的显微镜，或板在35 mm的玻璃底培养皿以制备用于实时成像。 注：（ 如肌动蛋白尾巴，septin笼）要按照自噬（ 例如 ，Atg8结合/ LC3 +已经自噬体）采用实时成像和细胞骨架动力学志贺氏菌感染过程中的实时性，组织培养细胞可以通过瞬时转染GFP的， RFP-或CFP标签的结构（见讨论）。 </li></ol></li><li>感染<ol><li>感染细胞100:1感染复数（MOI）的志贺氏菌 （OD 600 = 0.3 - 0.6）稀释于MEM;直接添加到HeLa细胞接种于6瓦特ELL板24 - 48小时前感染在2毫升的MEM（血清饥饿）（如1.2节所述）。 </li><li>为了最大限度地提高细菌粘附于宿主细胞，离心细菌，细胞在700×g离心10分钟，在室温下进行。离心后，温育30分钟，在37℃，5％CO 2和进行感染。 </li><li>用新鲜的MEM洗两次感染的细胞并用含有庆大霉素完全培养基中孵育（50微克/毫升，以消除细胞外细菌）为1-4小时，这取决于实验。 </li></ol></li><li>固定和标签感染HeLa细胞的显微镜<ol><li>用1×PBS洗两次感染的细胞，并固定15分钟在4％多聚甲醛中的1×PBS在室温下进行。要删除多聚甲醛，洗涤细胞2倍用1×PBS。 </li><li>孵育固定的细胞在50mM氯化铵，在室温下10分钟。用1×PBS洗涤一次，并透化细胞4分钟，用0.​​1％的辛基酚环氧乙烷缩合物，在室温吨emperature。 注：替代辛基酚环氧乙烷缩合物为透，如皂角苷或甲醇，可应用于不同的保存蜂窝结构20。 </li><li>在1X PBS洗涤细胞，培养在湿室中对自噬的关键组件30分钟一抗（ 如 P62 / SQSTM1）或septin细胞骨架（SEPT2，SEPT6，SEPT7，SEPT9和SEPT11表达在HeLa细胞）（在室温）〜过夜（4℃）。 </li><li>两次用1×PBS用鬼笔环肽（在4℃）洗涤细胞，并培育在湿室中与二次抗体，以及标记的丝状肌动蛋白（F-肌动蛋白），30分钟（在室温下）至过夜。为宿主的染色细胞核添加4“，6二脒-2 - 苯基吲哚（DAPI）。 </li><li>在1X PBS洗涤细胞，并安装在玻璃盖玻片上使用安装媒介的幻灯片。 </li></ol></li><li>感染HeLa细胞显微成像<ol><li>图像的INFected细胞使用或落射荧光共聚焦显微镜和63X或100X的目标识别DAPI标记的志贺氏菌 。 注意： 如图1A - 1C，septin笼可以被可视化为环样结构，〜0.6微米的直径，周围胞浆菌聚合肌动蛋白和招募自噬标记物（ 如 P62和LC3）7,8。相反，细菌聚合肌动蛋白尾部不会被septin笼条块，不会被靶向自噬7,8。 </li><li>使用落射荧光或共聚焦显微镜和63X或100X物镜，量化每显微镜视野内细菌的数量。同时量化细菌滞留在septin笼子里，有针对性地吞噬，或聚合肌动蛋白尾巴和逃避吞噬的数量。 </li><li>为了确定细菌的包埋在septin笼或聚合肌动蛋白尾巴的百分比，取Z堆栈的图像系列INFE的CTED细胞过程中的图像和来自至少3次独立实验计算每个实验至少500-1,000细菌。 </li></ol></li></ol>自噬和细胞骨架的体外 2。功能分析注意：两个遗传学和药理学方法可以用于扰乱自噬在受感染的组织培养细胞，并在感染过程中这些治疗方法可以监测的影响。 <ol><li>的siRNA介导的沉默<ol><li>镀在6孔板中的完全培养基0.8×10 5个HeLa细胞在玻璃盖玻片上。 </li><li>使用基于脂质的转染试剂用siRNA对自噬和/或细胞骨架标记物转染次日。 </li><li>孵化所需时间后，感染细胞， 志贺氏菌如1.3节所述。 </li><li>修复并按照1.4节所述标记的细胞。 </li></ol></li><li>药理操纵 如解聚肌动蛋白细胞骨架采用细胞松弛素D或latrunculin B，解聚微管采用噻氨酯哒唑，来阻止肌动球蛋白的活性使用II型肌球蛋白，或扰乱septin装配使用forchlorfeneuron。自噬作用可以通过使用雷帕霉素或刺激阻断通过巴弗洛霉素。 <ol><li>操纵志贺氏菌感染过程中细胞骨架，第一次感染的细胞， 志贺氏菌如1.3节所述，并允许有足够的时间为细菌进入细胞和吞噬体到细胞质逃避（ 如 ，&gt; 1.5小时后感染）。 </li><li>稀释药物从储液[贮存液悬浮在二甲亚砜（DMSO）]中的MEM至5 M（细胞松弛素D，latrunculin B，噻氨酯哒唑），20 M（forchlorfeneuron）或50 M（II型肌球蛋白）的终浓度，并处理细胞30分钟，在37℃。药物的总金额（二甲基亚砜/ D的量地毯混合物）每板加入/细胞的小瓶是每加入2ml介质1-5微升（根据原液）。治疗的细胞具有相似的剂量的DMSO稀释的MEM作为阴性对照。 </li><li>修复并按照1.4节所述标记的细胞。 注：对于操纵自噬通量（在感染或者未感染的细胞），用雷帕霉素（20纳米）或巴弗洛霉素（160纳米）的4至12小时的延长的药物治疗。 </li></ol></li><li>免疫印迹 注：自噬活性可以通过测定自噬标记如P62和LC3蛋白水平进行定量。 <ol><li>培养所需的时间后，收集并裂解细胞用于免疫印迹分析。运行蛋白提取物对8，10，或14％的丙烯酰胺凝胶。 </li><li>自噬通量， 即得。自噬作用的速率，可如21,22描述进行分析。 </li></ol></li><li>显微成像和定量<ol><li>使用落射荧光或共聚焦显微镜和63X或100X对象IVE，siRNA对志贺氏菌感染的作用或药物治疗可通过定量显微术进行评估（ 即 ，计数自噬体，septin笼和肌动蛋白的尾部），如第1.5节中描述的和在图2A和2B所示。 </li></ol></li></ol> 3， 在学活体成像菌相互作用与细胞自噬与细胞骨架注： 志贺氏菌感染的斑马鱼模型可用于研究septin笼养和自噬在体内 19。 <ol><li>准备学菌 <ol><li>文化学菌如​​1.1节所述。 </li><li>在OD 600 = 0.3-0.6，旋8毫升细菌传代培养以1000×g离心10分钟。在1,000 XG洗涤沉淀用1×PBS，离心10分钟。 </li><li>悬浮颗粒在80微升0.1％酚红PBS洗涤，得到〜2000个细菌/ NL。保持BACT冰里尔准备放慢增长。 注意：添加酚红将有助于注入幼虫时，以可视化的接种。 </li></ol></li><li>准备斑马鱼幼虫注射 注：斑马鱼被放置鸡蛋和被确定为胚胎，直到72小时受精后，当他们被称为幼虫。 <ol><li>滋生的成年斑马鱼作为放置男4例，女8例在韦斯特23描述（通常是2:1的比例）为一个独立的鱼缸布满大理石（这将防止大人吃了催生蛋）的底部。另外，前一天晚上把鸡蛋收集筐的繁殖缸中。 注：卵受精〜30分钟后，灯光在斑马鱼设施23去的，应该尽快进行收集，以防止霉菌生长。蛋收集筐用来收集卵子，这样他们可以很容易地收集并且还保护鸡蛋从成人。 </li><li>收集胚胎s和清洁它们通过洗涤在胚胎培养基（E2）与0.003％的漂白剂10分钟。用漂白剂除去E2，洗胚胎5倍于E2的培养基中，在28℃下生长在10cm培养皿（100个胚/50毫升E2介质）的胚胎。 </li><li>如果胚胎或幼虫，将用于显微镜研究，在24小时后受精添加0.003％的N-苯基硫脲的E2介质，以防止黑化。保持胚胎在28°C下正常发展。 注：斑马鱼幼虫准备在感染72小时后受精。 </li><li>对于感染和显微镜方法，麻醉斑马鱼幼虫在E2为200g /毫升三卡因。 </li></ol></li><li>对于静脉和局部感染制剂斑马鱼幼虫 注：为志贺氏菌感染过程中评估斑马鱼的生存，进行尾静脉注射。以可视化的septin和自噬标记招募志贺氏菌 ，进行感染，在局部部位，如尾肌。 <ol><li>对于尾静脉注射，用背面朝针横向放置麻醉幼虫。 如图3A所示 ，将针尖端附近的（后）到泌尿生殖开口，旨在为尾静脉，并刺穿皮肤和提供所需的细菌的剂量（注射体积1-5 NL）。 注意：静脉注射感染是具有挑战性的表演，将需要几个星期的培训，以获得舒适与此过程。培训注入酚红（无细菌），将有助于正确评估注射部位。 请注意：在志贺氏菌的情况下，剂量依赖性的实验表明，低剂量感染（&lt;千CFU）48小时内被清除，且高剂量的感染（&gt; 4000 CFU）导致在48小时19承载的死亡率。 </li><li>对于尾部肌肉感染，如在3.3.1节中描述的位置麻醉幼虫。 如图3A所示 ，将针当心Ÿ在体节的肌肉（骨骼肌即段），并注入少量（ 即 ，1 NL）菌制剂。 </li></ol></li><li>细菌的幼虫静脉注射<ol><li>在24所述拉硼硅玻璃microcapillaries。 </li><li>连接针的三维粗手工操纵器的保持器，并打破针尖细镊子。 </li><li>要加载的针，将一滴细菌培养到盖玻片。开关上的微量注射器和气体筒，略微淹没针尖进入降，并填满细菌制备所需量的针。 </li><li>要校准的注射量，将矿物油对盖玻片一滴注入细菌制剂。用千分尺测量的液滴的直径，并计算出注射体积[V =（4/3）πR3]。 注意：使用40磅注射的设置和50毫秒与bacteri人准备在3.1节中描述会给〜2,000 CFU / NL。 </li><li>准备使用的塑料模具中韦斯特23所述喷射板。 </li><li>幼虫转移到喷射板，并用细画笔它们对齐。东方和如第3.3.1节所述注入幼虫。 </li><li>为评估斑马鱼生存，转移感染幼虫分别在24孔板中的1ml的E2 /孔，孵育在28℃。每日对未来2-5天，情节生存监测感染的幼虫随时间（ 图3B）。 </li></ol></li><li>电镀斑马鱼幼虫的细菌定量 注意：在无菌罩工作，以避免污染。 <ol><li>来评估细菌注射到鱼（在时间0小时后的感染）和对细菌的定量在期望的时间点的数目，牺牲斑马鱼幼体与三卡因的过量（200-500毫克/升）。将个别幼虫在1.5ml POLypropylene微量离心管中加入200μl的0.1％辛基酚环氧乙烷缩合物的1×PBS和机械均化用杵。 注意：要确认细菌负荷的注射量，泵同等剂量到无菌1X PBS下​​降和板出来。 </li><li>使在无菌水中的斑马鱼幼体匀浆物的系列稀释，并镀到溶原肉汤（LB）琼脂平板上。幼虫也可以被镀在刚果红TCS板已经保持了致命的质粒或志贺氏菌未区分。 注：钢板3个或更多非感染鱼作为对照，以检查用于感染斑马鱼幼体的状态。 </li><li>板在37℃下过夜培养后，计数菌落。 如图3C所示 ，表示用对数标度。 注：斑马鱼幼体的感染过程中的细菌负荷，也可使用荧光标记的志贺氏菌和微观ı可视maging如第3.7或3.8（ 图3D）描述。 </li></ol></li><li>斑马鱼幼虫的免疫染色<ol><li>在期望的时间点，用牺牲的三卡因过量的幼虫。收集在1.5ml聚丙烯离心管（10〜20尾/管）的鱼。 </li><li>固定用4％的多聚甲醛，0.4％的辛基酚环氧乙烷缩合物的幼虫在1×PBS中孵育在轨道搅拌器（以避免幼虫聚类）2小时（在室温下）或过夜（4℃）。 注意：电子显微镜可用于感染斑马鱼幼体超微结构分析。在这种情况下，麻醉胚胎应根据Mostowy 等[19]是固定的，并进行处理。 </li><li>洗涤3次在1×PBS中0.4％的辛基酚环氧乙烷缩合物为5分钟，则块在封闭液（10％胎牛血清，1％DMSO，0.1％聚氧乙烯单月桂酸酯中的1×PBS）中1小时，在室温下进行。 </li><li>迪琵琶在阻断溶液中的一级抗体。添加的幼虫以稀释的第一抗体和在轨道搅拌器孵育过夜，在4℃。使用的主要抗体，如第1.4.3如上所述。 </li><li> 15分钟在0.1％聚氧乙烯单月桂酸酯1X PBS在室温下洗幼虫4倍。 </li><li>稀释的二级抗体在阻断溶液中。添加的幼虫以稀释的二级抗体，并在轨道搅拌器孵育过夜，在4℃。使用相同的二抗和毒伞素如在1.4.4节中描述。 </li><li>洗涤4倍，在室温下在0.1％的聚氧乙烯单月桂酸酯的1×PBS 15分钟。对宿主细胞的细胞核染色过程中的第一个这15分钟的洗涤的加DAPI（150纳米的最终浓度）。 </li><li>为保存荧光标记的幼虫，逐步培养他们在15，30，60甘油梯度，和80％的稀释在1×PBS和0.1％聚氧乙烯单月桂酸酯2小时（在室温回火ature）至过夜（4℃）。 注：彩绘幼虫可存放很长一段时间中的80％甘油在4℃下（ 例如 ，4个月。）。 </li></ol></li><li>的固定斑马鱼幼虫显微成像<ol><li>固定甘油嵌入式幼虫转移到一小滴80％甘油的35毫米培养皿（用于立体显微镜）或全玻璃底菜（共聚焦miscopy）。 </li><li>取Z堆叠图像系列感染的幼虫，并根据需要处理的图像。 </li><li>使用或落射荧光共聚焦显微镜和10X或20X客观上为整个生物体的成像。然后用激光共聚焦显微镜和40X，63X，100X或客观的可视化单个细胞和细胞自噬的招聘和细胞骨架的标记个别细菌在体内 19（图4A和4B）。 注：在尾肌杂志征稿鱼，沿着玻璃底菜，使EA的底部安装在甘油平SY焦点。 </li></ol></li><li>住的感染斑马鱼幼虫显微成像 注：幼虫是光学通道，从而在体内自噬体可以使用GFP-LC3转基因斑马鱼线25被可视化。感染斑马鱼幼虫的第3.3节和挂接在本节所描述的。 <ol><li>在E2的制备低熔点的1％琼脂糖（LMA），并允许其冷却至35-37℃，以避免损坏幼虫/杀。分发LMA滴在35毫米培养皿（用于立体显微镜）或全玻璃底菜（共聚焦显微镜）。 </li><li>转让麻醉斑马鱼幼虫单独（用尽可能少的水越好）向LMA下降。东方幼虫用画笔并等待琼脂糖固化所需的位置。 </li><li>覆盖整个盘表面与LMA，并且覆盖有含200微克/毫升三卡因，以避免从晒出制备和允许鱼从水交换氧气E2。 </li><li>使用或落射荧光共聚焦显微镜和10X或20X物镜成像整个斑马鱼幼虫。利用共聚焦显微镜和40X，63X，100X或客观的可视化细菌authopagosomes（ 如 GFP-LC3 +已经空泡周围的志贺氏菌 ） 的体内 。 </li><li>取Z堆叠感染幼虫随时间（ 例如 ，每2分钟在几个小时）来可视化自噬体，以及它们的动态，实时地。 </li></ol></li></ol>自噬和细胞骨架的体内 4功能分析注：自噬的药理学和遗传学扰动对感染过程的影响可以在整体动物水平进行监测，并在单细胞水平。 <ol><li>自噬被操纵吗啉注射液<ol><li>通过在65℃下进行10分钟预热重构于无菌水至1mM的储备溶液吗啉代寡核苷酸。商店在室温下进行。 注：吗啉代寡核苷酸注射必须在1-4细胞期胚胎进行。 </li><li>制备吗啉代寡核苷酸的工作，用无菌0.1％酚红的Dulbecco磷酸缓冲盐溶液。装入针在3.4.2节中描述。​​，吗啉代寡核苷酸注射量可以按照第3.4.3所述进行校准。 注意：酚红将有助于可视化中喷射出的体积。 </li><li>制备的胚胎定位腔室（ 即 ，与腈基丙烯酸酯胶在10cm培养皿盖朝向针边缘显微镜载玻片部分去除）。转移1-4细胞期胚胎的腔室用少量的水和用细画笔将它们对齐。 </li><li>穿透蛋壳和蛋黄顺利。一旦进入，踩下踏板的吗啉代寡核苷酸溶液所需的量注入。 注意：尽量减少注射至0.5的容积 - 2 NL;量较高的塔ñ5 NL可能会导致发育缺陷，增加蛋死亡率。 </li><li>显微注射后，（如第3.2节所描述的漂白），并培育他们在培养皿中与E2在28℃的清洁胚胎。 </li><li>感染72小时后受精控制（ 即 ，斑马鱼幼虫注射控制吗啉代寡核苷酸）或P62 morphants（ 即 ，斑马鱼幼虫注射P62吗啉代寡核苷酸）与志贺氏菌如3.4节所述。如3.5节所述评估的生存和细菌的负担在未来2-5天。如3.7节所述和图4C高亮或图像活斑马鱼幼虫在3.8节所描述的图像固定的斑马鱼幼虫。 注：有效吗啉代寡核苷酸的给药量可以根据它的效率来抑制转录剪接或蛋白质的翻译（见讨论）来评估。 </li></ol></li></ol>

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作者宣称，他们没有竞争的经济利益。

当使用组织培养细胞中监测自噬和细胞骨架在体外 ， 在部分1和2描述的方案可以适用于各种各样的组织培养物的细胞类型。此外，按照自噬（ 例如 ，Atg8结合/ LC3 +已经自噬体）和细胞骨架（ 例如 ，肌动蛋白尾，septin笼）在志贺氏菌在感染过程中实时使用实时成像动力学，组织培养细胞可短暂使用GFP-转染的， RFP-或CFP-标记的构建体如前所述7,8。以增加细胞感染痢疾杆菌的百分比（ 即 ，通常希望用于实时分析考虑到志贺氏菌可以在100侵入的HeLa细胞的5-30％：1 MOI），直接加志贺氏菌 400微升（OD 600 = 0.3 -0.6）细胞在2毫升的MEM（血清饥饿），并等待至少1.5小时，感染后有足够的细菌进入，从吞噬体，复制，AU逃生tophagy认可和septin隔离罩。或者，可以使用志贺氏菌 M90T英辉菌株表达的粘附AfaE并在上皮细胞高得多的侵袭能力相比M90T应变28。值得注意的是，M90T英辉应变尚未在体内使用斑马鱼试验。 志贺氏菌的菌落的平板，可以保持在4℃下2-3天，并用于多种实验。然而，随着时间的推移，已失去毒力质粒也可以吸收刚果红和出现痢疾杆菌的菌落，以保留了其毒性质粒。为此，我们建议在可能时使用新鲜的细菌种群。 当监测感染体内的细胞生物学，协议中的第3和第4使用野生型AB线的斑马鱼中描述。并监控志贺氏菌 -leukocyte相互作用，转基因斑马鱼线可被使用， 例如，MPX：GFP或LYZ：DsRed的视觉IZE中性粒细胞19,29,30或MPEG1：mcherry可视化的巨噬细胞19,31。为了形象地吞噬体内 ，将GFP-LC3转基因斑马鱼线19,24可以按照3.8节所述使用。 扰乱吞噬体内 ，有效吗啉代寡核苷酸的剂量具有实验是根据它的效率来抑制转录剪接或蛋白质翻译来进行评估。最好是进行滴定实验，并确认耗尽通过RT-PCR（用于拼接吗啉代寡核苷酸）或通过SDS-PAGE（对于平移吗啉代寡核苷酸）32。从斑马鱼胚胎或幼虫的RNA分离可使用异硫氰酸胍 - 酚 - 氯仿提取来进行。从斑马鱼幼虫（8〜15尾/管）中提取蛋白质，均质机械用200微升裂解液（1M，三，5M的NaCl，0.5M EDTA的杵，0.01％，辛基酚环氧乙烷condensate和蛋白酶抑制剂）。在19,000 XG在4℃下进行15分钟的离心管中，将上清液转移到新管中。加入Laemmli缓冲液并加热该样品在95℃下进行15分钟。溶胞产物可以贮存于-80℃，直到需要的，并且可以如在第2.3节中描述的通过Western印迹法来评估。 斑马鱼是体内药物应用的理想模型。分析使用吗啉代寡核苷酸可以补充建立药物操纵自噬（ 例如 ，雷帕霉素和巴弗洛霉素）。未感染和/或感染的幼虫可与雷帕霉素（1.5μM）或巴弗洛霉素（80 nm）的稀释中的E2和自噬通量可通过Western印迹法如在25,33中描述进行评估处理。自噬在操纵被感染的幼虫的​​感染和存活的结果的结果可以按照3.5节所述进行评估。 除了研究的宿主细胞决定因素， 在体外和体内的协议可以被应用到评估所需的自噬识别细菌的决定因素，使用细菌突变菌株中差异由自噬的认可， 例如 ， 志贺氏ΔicsA（志贺氏菌蛋白质ICSA募集的N- WASP然后ARP2 / 3肌动蛋白尾巴和septin笼形成;在缺席聆讯下，就没有肌动蛋白尾巴，没有septin笼）和ShigellaΔicsB（志贺氏菌通过避免细菌效应蛋白ICSB，从而防止细胞自噬机制，以ICSA招聘的自噬反应，在其缺席可以有更多的septin笼，更多的自噬）7,8。 志贺氏菌是不是斑马鱼的天然病原体，并在37℃，最佳生长。然而，工作显示所需志贺氏菌侵袭的毒力因子，从吞噬液泡和复制在CY逃脱tosol可以表示和有功能在斑马鱼幼体在28℃19。 28℃下是最常用的温度下，斑马鱼的饲养和标准温度，以确保正常的斑马鱼发育23。引人注目的是，在主要的致病事件导致志贺氏菌对人类（ 即巨噬细胞的细胞死亡，浸润和增殖的上皮细胞内，细胞至细胞的传播，炎性破坏宿主上皮细胞）被忠实地复制在志贺氏菌感染的斑马鱼模型19。 自噬和细胞骨架基因广泛表达，并具有广泛的生物学功能。小鼠的研究表明，基因敲除必需的自噬26或septin基因5顷胚胎致死，并且很可能是一些这些基因也将是必需的斑马鱼发育（尽管这个问题可以通过以下事实斑马鱼有多个减小旁系同源基因33）。如果是的话，也有几种选择来克服这个问题，其中包括（i）使用的药理学试剂，以调节自噬和细胞骨架，（ⅱ）吗啉，可滴定向下，（ⅲ）敲除的基因可以被设计为仅在特定的细胞类型，和/或（iv）的基因涉及自噬识别来说不是必需的动物发展（ 例如 ，P62）可以靶向。 而斑马鱼是一种理想的模型系统来研究自噬和志贺氏菌感染过程中细胞骨架，分子工具目前所缺乏的。领域需要产生新的工具和感兴趣的蛋白质的驱动细胞特异性表达。击倒的自噬/细胞骨架基因的表达，新啉序列是必需的，并且（ 例如 ，TALEN，CRISPR / Cas9）也可以使用新的方法用于基因工程。在此期间，先前针对人或小鼠的研究中产生了多种工具可在斑马鱼同样的工作。 胞内菌S.菌产生的一种特殊的模式生物，以解决关键问题在生物，包括细菌的要被免疫系统1,2识别的能力。宿主细胞采用近年来septin家族限制S的运动菌 ，并针对他们自噬，细胞免疫功能的自主7,8的重要组成部分。这些意见建议的新分子结构，研究自噬及其降解胞内细菌的能力。一个主要问题是，现在完全破译潜在的分子和细胞活动，并验证这些事件的细菌感染在体内体外分析使用相关的动物模型。为此，在斑马鱼已经被确立为S的分析的有价值的新主机菌感染19。细菌与宿主细胞之间的相互作用可以在高分辨率成像，并斑马鱼模型应该证明理解志贺氏菌感染的细胞生物体内有用的。斑马鱼幼体可以被用来研究细菌自噬在宿主防御中的作用，以及工作表明，自噬作用的扰动可能不利宿主存活响应于志贺氏菌感染19影响。 该意见从志贺氏菌的研究中产生的，利用组织培养细胞和体内利用斑马鱼幼虫可以提供理解宿主防御的基本进展septin笼养和自噬在体外 。他们也可以提出旨在打击传染病的新战略。 本报告的一个重要目标是使体外分析的分子和细胞发生的事情（ 即自噬，肌动蛋白尾巴，septin笼养）在耳鼻喉科的情况下细菌感染时体内愤怒的有机体，利用斑马鱼幼虫。如果不熟悉斑马鱼生物学和处理，可以指在深度协议进行适当的斑马鱼饲养23和在斑马鱼感染19,35的体内分析。

志贺氏菌 ，革兰氏阴性肠侵袭性细菌，可以从吞噬体的胞液中逸出，并聚合宿主肌动蛋白细胞骨架，以逃避细胞内免疫反应，促进细胞内和细胞间移动1,2。尽管在体外 3,4肌动蛋白为基础的运动的理解，限制细菌传播体内的机制尚未完全定义。这是先天免疫和宿主防御的更完整的理解是至关重要的。 近年来septin家族，蛋白质的后生动物之间高度保守的家族，是鸟苷三磷酸（GTP）结合该组装成的杂寡聚复合物，并形成了与细胞膜和细胞骨架的5,6相关联的非极性丝蛋白。最近的工作已经发现感染的宿主细胞可预防志贺氏菌肌动蛋白基蠕动通过靶向autoph compartmentalizing菌AGY内“septin笼”，揭示了肌动蛋白抵消基于蠕动7,8的第一个细胞机制。调查的开阔场现在已成了“septin生物学和感染”。 Septin组装，诱发多种病原体（ 如李斯特菌7,9,10，分枝杆菌7,8，白色念珠菌 11），可能会成为在宿主防御5,12的一个关键问题。 自噬，一个高度保守的细胞内降解过程中，被认为是因为它能够提供胞浆菌溶酶体13,14能力，细胞自主免疫力的重要组成部分。然而，细菌噬在体内的作用，以限制或促进细菌复制仍了解15,16很差。斑马鱼（ 斑马鱼 ）已成为脊椎动物模型感染的研究，因为它是光学访问在幼虫期时的先天免疫系统已经是功能性17,18。最近的工作特点斑马鱼幼虫S的易感性菌 ，一种范式细菌吞噬15，并采用了志贺氏菌 -zebrafish感染模型，研究自噬的操作进行抗菌治疗体内 19。 这份报告提供了新的工具和实验研究学菌相互作用与细胞自噬和细胞骨架。在第一步骤中，协议监测自噬细胞骨架相互作用所使用志贺氏菌感染的人上皮细胞系HeLa的说明。以评估的自噬细胞骨架相互作用的体外志贺氏菌感染过程中的作用，方法来操作的自噬和细胞骨架成分（使用的siRNA或药理学试剂）被提供。新的研究显示，通过使用志贺氏菌感染Ø˚F斑马鱼幼体，类似分析可应用于研究感染的细胞生物学体内 。协议的准备和感染斑马鱼幼虫的详细，并评估在体内的宿主反应志贺氏菌感染，协议确定提供主机的生存和幼虫感染细菌的负担。方法监测的septin和自噬标记招募到志贺氏菌 （使用固定的或活的斑马鱼幼体）和方法[用吗啉代寡核苷酸（在1-4细胞期胚胎中注射）或药理学试剂的体内测试这些过程中的作用（直接加入到斑马鱼的浴水）进行了讨论。本工作方案预计将提供深入了解所需感染的宿主细胞内反应的控制机制。

<table border="0" cellpadding="0" cellspacing="0" width="771">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:412px;">Name of Material/ Equipment</td>
			<td style="width:240px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:412px;">Bafilomycin A1</td>
			<td style="width:240px;">Sigma-Aldrich</td>
			<td>B1793&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:412px;">Blebbistatin</td>
			<td style="width:240px;">Sigma-Aldrich</td>
			<td>B0560</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Borosilicate glass microcapillars&#160;</td>
			<td>Harvard Apparatus</td>
			<td>30038</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Coarse manual manipulator&#160;</td>
			<td>Narishige</td>
			<td style="width:119px;">M-152</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cytochalasin D</td>
			<td style="width:240px;">Sigma-Aldrich</td>
			<td style="width:119px;">C6762</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4&#39;,6-diamidino-2-phenylindole (DAPI)</td>
			<td style="width:240px;">Molecular Probes</td>
			<td style="width:119px;">D1306</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:412px;">Dumont #5 fine tweezers</td>
			<td style="width:240px;">Fine Science Tools</td>
			<td style="width:119px;">11254-20</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Forchlorfeneuron&#160;</td>
			<td style="width:240px;">Sigma-Aldrich</td>
			<td style="width:119px;">32974</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:412px;">Goat anti-mouse IgG (H+L) antibody</td>
			<td style="width:240px;">Molecular Probes</td>
			<td style="width:119px;">N/A&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:412px;">Goat ant-rabbit IgG (H+L) antibody</td>
			<td style="width:240px;">Molecular Probes</td>
			<td style="width:119px;">N/A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:412px;">JetPEI transfection reagent</td>
			<td style="width:240px;">Polyplus transfection</td>
			<td style="width:119px;">101-01N</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:412px;">Latrunculin B</td>
			<td style="width:240px;">Sigma-Aldrich</td>
			<td style="width:119px;">L5288</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:412px;">LC3 antibody</td>
			<td style="width:240px;">Novus Biologicals</td>
			<td style="width:119px;">NB100-2220</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:412px;">Low melting agarose</td>
			<td style="width:240px;">Promega</td>
			<td style="width:119px;">V2111</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:412px;">MatTek glass bottom dish</td>
			<td style="width:240px;">MatTek corporation</td>
			<td style="width:119px;">P35G-1.0-14</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MEM plus L-alanyl-L-glutamine&#160;</td>
			<td style="width:240px;">GIBCO</td>
			<td style="width:119px;">41090028</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:412px;">MEM non-essential amino acids solution</td>
			<td style="width:240px;">GIBCO</td>
			<td style="width:119px;">11140-035</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microinjector&#160;</td>
			<td>Narishige</td>
			<td>IM-300</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micropipette puller device&#160;</td>
			<td>Sutter Instrument Co., Novato,</td>
			<td>P-87&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:412px;">Mineral oil</td>
			<td style="width:240px;">Sigma-Aldrich</td>
			<td style="width:119px;">P35G-1.0-14</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:412px;">Monoclonal anti-tubulin, acetylated antibody&#160;</td>
			<td style="width:240px;">Sigma-Aldrich</td>
			<td style="width:119px;">T6793</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nocodazole</td>
			<td style="width:240px;">Sigma-Aldrich</td>
			<td style="width:119px;">M1404</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N-phenylthiourea&#160;</td>
			<td>Sigma-Aldrich</td>
			<td style="width:119px;">P7629</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:412px;">Paraformaldehyde</td>
			<td style="width:240px;">Sigma-Aldrich</td>
			<td style="width:119px;">158127</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phalloidin&#160;</td>
			<td style="width:240px;">Molecular Probes</td>
			<td>A12379</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:412px;">Phenol red solution</td>
			<td style="width:240px;">Sigma-Aldrich</td>
			<td style="width:119px;">P0290</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:412px;">Protease inhibitor cocktail</td>
			<td style="width:240px;">Roche</td>
			<td style="width:119px;">4693116001</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:412px;">p62/SQSTM1 antibody</td>
			<td style="width:240px;">Cliniscience</td>
			<td style="width:119px;">PM045</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rapamycin</td>
			<td style="width:240px;">Sigma-Aldrich</td>
			<td style="width:119px;">R8781</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:412px;">Sodium pyruvate</td>
			<td style="width:240px;">GIBCO</td>
			<td style="width:119px;">11360039</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:412px;">Transfection reagent</td>
			<td style="width:240px;">Life Technologies</td>
			<td style="width:119px;">12252-011</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vectashield hard set mounting medium containing DAPI&#160;</td>
			<td style="width:240px;">Vector Laboratories</td>
			<td style="width:119px;">H-1500&#160;</td>
		</tr>
	</tbody>
</table>

use of shigella flexneri to study autophagy-cytoskeleton interactions

福氏志贺氏菌是一种细胞内的病原体可以从吞噬体逃脱到达细胞质中，并聚合主机肌动蛋白细胞骨架，以促进其蠕动和传播。新的工作表明，参与肌动蛋白为基础的运动蛋白也与自噬作用，细胞内降解过程的细胞中自主免疫至关重要。引人注目的是，宿主细胞可以防止S的肌动蛋白为基础的运动菌通过划分细菌内“septin笼”，并将其定位于自噬。这些观察结果表明，近年来septin家族的一个更完整的理解，一个家庭的丝状GTP结合蛋白，将提供新的见解自噬过程。这份报告描述的协议来监控造成南自噬细胞骨架的相互作用菌在体外利用组织培养细胞和体内使用斑马鱼幼体。这些协议使胞内机制及调查毫秒控制细菌传播的分子，细胞和整个生物体水平。

在组织培养细胞的体外，S.感染菌可从吞噬体逸出并侵入细胞溶胶。在胞质溶胶中，宿主细胞可以防止志贺氏菌的肌动蛋白为基础的运动通过划分细菌内septin笼（ 图1A）。细菌通过septin笼截留还可以通过自噬标记物p62蛋白（ 图1B）和LC3（ 图1C）进行标记。这些观察结果突出宿主防御的制约传播病原体侵入了一种新的机制，也​​揭示了细胞自噬与细胞骨架之间的新联系。引人注目的是，自噬标记物的耗尽显著减少细菌（ 图2A）septin笼中，并工作还表明，septin笼中的耗尽显著降低招募自噬标记8。因此，至少在志贺氏菌的情况下，septin保持架组件和自噬体formation可以被看作是相互依存的流程。 志贺氏菌的条块其他细胞需要由septin笼子包括肌动蛋白聚合和肌动球蛋白的活性（ 图2B）。 有志贺氏菌的不自然的小鼠模型中，与志贺氏菌病，septin生物学和细菌吞噬体内的调查可以从感染的新动物模型中，斑马鱼幼体19中受益。有可能通过注入细菌中的各种解剖部位的感染斑马鱼幼体如尾静脉注射对生存的实验，和尾部肌肉注射于体内显微术（ 图3A）。根据不同的剂量，S.菌注入斑马鱼幼虫既可以在48小时后感染清除，否则可能导致一个渐进的，并最终致命的感染（ 图3B - 3D）， 志贺氏菌毒力发构建函数来表示在28℃下，斑马鱼的最佳生长温度和斑马鱼的感染由志贺氏菌是严格依赖于它的III型分泌系统（T3SS）19，在人类疾病的一个重要毒力决定。总之，这些观察结果表明，斑马鱼幼虫代表体内分析， 志贺氏菌感染的有价值的新主机。 斑马鱼幼虫的光学辅助功能使体内 （ 图4A）septin笼养，即以前从未使用哺乳动物宿主模型来完成的成就的可视化。为了补充证据表明，septin笼诱捕针对性地吞噬体内的细菌，可以感染的转基因斑马鱼幼虫表达GFP-LC3，观察自噬标记招聘志贺氏菌 （ 图4B）。对于自噬体痢疾杆菌在体内 ，EL ultrastrucutral分析ectron显微镜可用于清楚地表明细菌通过双膜液泡19的胞质封存。自噬被认为是细胞自主免疫力的重要组成部分，对胞质菌14-16至关重要的防御机制。 在体内表征的自噬作用，p62的吗啉代处理斑马鱼幼体可以被使用。不同的是核心自噬机制[ 如 ，36自噬相关蛋白（ATGS）26]，P62是不是脊椎动物的发育27，因此斑马鱼幼虫必需能正常开发之前感染。引人注目的是，P62耗尽的幼虫接种S。菌导致显著死亡率增加，增加细菌的负担19。在体外的工作表明septin保持架组件的协议是相互依存与自噬体形成7,8，septin招聘痢疾杆菌明显减少，P62耗尽的幼虫（ <str翁&gt;图4C）。这些数据表明，斑马鱼生存依赖于p62的介导的自噬作用，以控制细胞内细菌感染。 <img alt="图1" fo:content-width="5in" src="/files/ftp_upload/51601/51601fig1highres.jpg" width="500" /> 图1的体外 septin笼（A）HeLa细胞感染S.菌为4小时40分钟，固定，标记抗体SEPT9和鬼笔环肽，并通过共聚焦显微术成像。比例尺为1μm（B）的HeLa细胞感染了S。菌为4小时40分钟，固定，标记抗体与p62蛋白及SEPT2，并通过荧光显微镜成像。比例尺，1微米（C）转染HeLa细胞用GFP-Atg8结合/ LC3，感染了S。菌为4小时40分钟，固定，通过荧光英里标记抗体SEPT2，和成像croscopy。比例尺，1微米。这些数字已经被修改Mostowy 等 7。 <img alt="图2" fo:content-width="5in" src="/files/ftp_upload/51601/51601fig2highres.jpg" width="500" /> 图2蜂窝要求志贺氏菌 -septin笼形成：（A）HeLa细胞，与对照处理（CTRL），p62蛋白，ATG5，ATG6或ATG7的siRNA。的siRNA处理的细胞的全细胞裂解物进行免疫印迹对GAPDH，P62，ATG5，ATG6，或ATG7显示的siRNA耗尽（顶部）的效率。的siRNA处理的细胞被感染S.菌为4小时40分钟，固定，并标示为定量显微镜。曲线图（下图），从每个处理Ñ≥3实验代表平均值％± 志贺氏菌的SEM内SEPT2笼（B）的HeLa细胞感染了S。 ˚Flexneri，用DMSO处理，细胞松弛素D（CytD），latrunculin B（LATB），噻氨酯哒唑（NOCO），或II型肌球蛋白（水泡）和4小时40分钟后固定及标示定量显微镜。图表代表平均值％± 志贺氏菌的SEM内SEPT2笼从每个处理两个独立的实验，这些数字已被修改从Mostowy 等 7。 <img alt="图3" fo:content-width="5in" src="/files/ftp_upload/51601/51601fig3highres.jpg" width="500" /> 图3。 志贺氏菌感染的斑马鱼模型。 （一）图片来说明体视显微镜下的斑马鱼幼虫的方向。 （左面板）斑马鱼幼虫72小时受精后进行横向定位在喷射板与他们的背侧朝向注射针。 （中图），血液感染由注入电流进行阿拉斯细菌（红色溶液）在尾静脉，后到泌尿生殖开口。 （右面板）的感染在尾部肌肉是通过在体节注入细菌（红色溶液）中进行。（B）的72小时受精后幼虫的存活曲线注射各种剂量的S。菌和培养在28℃培养48小时后的感染。有效的接种物被分类为低（&lt;10 3 CFU，空心圆），中期（〜4×10 3 CFU，空心三角形）或高（〜10 4 CFU，空心方块）。从每个接种物类n≥3实验意味着活细菌在从个别幼虫匀浆％±SEM（水平长条）（℃）枚举在不同时期感染后通过电镀到LB的测定请注意，只有幼虫幸存下来都包含在列举分析了感染。平均值±标准差（单杠）也显示（D）由志贺氏菌 GFP的分布确定实时成像用荧光立体显微镜在不同的时间使用低，中，高剂量接种（尾静脉注射）后感染。 。传输的图像（灰色）和GFP荧光（绿色）（B）的叠加- （D）这些数字已经被修改Mostowy 等[19]。 <img alt="图4" fo:content-width="5in" src="/files/ftp_upload/51601/51601fig4highres.jpg" width="500" /> 图4 志贺氏菌感染体内的细胞生物学（A）斑马鱼幼虫24小时，固定，标记抗体SEPT7（红色）和GFP（绿色感染的尾部肌肉与GFP- 志贺氏菌 （低剂量） ），并通过共聚焦显微镜成像。比例尺，5微米（B）的GFP-LC3斑马鱼幼虫感染mCherry- 志贺氏菌 （中剂量）为4小时后，固定的，标记有抗mCherry（红色）和GFP（绿色），和由共聚焦显微镜成像。或P62（右图）吗啉感染了志贺氏菌 GFP-4小时（中剂量），固定的，标有抗SEPT7（。用对照（左图CTRL）比例尺，1.5微米（三）斑马鱼幼虫处理红色）和GFP（绿色），和由共聚焦显微镜成像。箭头突出志贺氏菌的一些例子滞留在septin笼（CTRL）否（P62耗尽）4小时后感染。比例尺，5微米。这些数字已经被修改Mostowy 等[19]。

Watch this Scientific Journal Video about Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions at JoVE.com

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

为了对付病原体的传播，宿主细胞重组的细胞骨架，以区域化的细菌，诱导自噬。使用志贺氏菌感染的组织培养细胞，宿主与病原体的决定背后这个过程的识别和表征。使用志贺氏菌感染的斑马鱼模型，发现分子和机制中的作用进行了研究体内 。

使用<em&gt;福氏痢疾杆菌</em&gt;研究自噬，细胞骨架相互作用

Shigella flexneri is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote ...

use-of-shigella-flexneri-to-study-autophagy-cytoskeleton-interactions

Research

JoVE Journal

Immunology and Infection

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

Use of an Optical Trap for Study of Host-Pathogen Interactions for Dynamic Live Cell Imaging

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and temporal control between the phagocytic cell and microbe has rendered focused observations into the initial interactions of host response to pathogens difficult. Historically, intercellular contact events such as phagocytosis3 have been imaged by mixing two cell types, and then continuously scanning the field-of-view to find serendipitous intercellular contacts at the appropriate stage of interaction. The stochastic nature of these events renders this process tedious, and it is difficult to observe early or fleeting events in cell-cell contact by this approach. This method requires finding cell pairs that are on the verge of contact, and observing them until they consummate their contact, or do not. To address these limitations, we use optical trapping as a non-invasive, non-destructive, but fast and effective method to position cells in culture.
Optical traps, or optical tweezers, are increasingly utilized in biological research to capture and physically manipulate cells and other micron-sized particles in three dimensions4. Radiation pressure was first observed and applied to optical tweezer systems in 19705, 6, and was first used to control biological specimens in 19877. Since then, optical tweezers have matured into a technology to probe a variety of biological phenomena8-13.
We describe a method14 that advances live cell imaging by integrating an optical trap with spinning disk confocal microscopy with temperature and humidity control to provide exquisite spatial and temporal control of pathogenic organisms in a physiological environment to facilitate interactions with host cells, as determined by the operator. Live, pathogenic organisms like Candida albicans and Aspergillus fumigatus, which can cause potentially lethal, invasive infections in immunocompromised individuals15, 16 (e.g. AIDS, chemotherapy, and organ transplantation patients), were optically trapped using non-destructive laser intensities and moved adjacent to macrophages, which can phagocytose the pathogen. High resolution, transmitted light and fluorescence-based movies established the ability to observe early events of phagocytosis in living cells. To demonstrate the broad applicability in immunology, primary T-cells were also trapped and manipulated to form synapses with anti-CD3 coated microspheres in vivo, and time-lapse imaging of synapse formation was also obtained. By providing a method to exert fine spatial control of live pathogens with respect to immune cells, cellular interactions can be captured by fluorescence microscopy with minimal perturbation to cells and can yield powerful insight into early responses of innate and adaptive immunity.

A method is described to individually select, manipulate, and image live pathogens using an optical trap coupled to a spinning disk microscope. The optical trap provides spatial and temporal control of organisms and places them adjacent to host cells. Fluorescence microscopy captures dynamic intercellular interactions with minimal perturbation to cells.

动态活细胞成像的宿主 - 病原体相互作用的研究使用的光学陷阱

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and ...

科研

免疫与感染

Use of the EpiAirway Model for Characterizing Long-term Host-pathogen Interactions

Nontypeable Haemophilus influenzae (NTHi) are human-adapted Gram-negative bacteria that can cause recurrent and chronic infections of the respiratory mucosa 1; 2. To study the mechanisms by which these organisms survive on and inside respiratory tissues, a model in which successful long-term co-culture of bacteria and human cells can be performed is required. We use primary human respiratory epithelial tissues raised to the air-liquid interface, the EpiAirway model (MatTek, Ashland, MA). These are non-immortalized, well-differentiated, 3-dimensional tissues that contain tight junctions, ciliated and nonciliated cells, goblet cells that produce mucin, and retain the ability to produce cytokines in response to infection. 
This biologically relevant in vitro model of the human upper airway can be used in a number of ways; the overall goal of this method is to perform long-term co-culture of EpiAirway tissues with NTHi and quantitate cell-associated and internalized bacteria over time. As well, mucin production and the cytokine profile of the infected co-cultures can be determined. This approach improves upon existing methods in that many current protocols use submerged monolayer or Transwell cultures of human cells, which are not capable of supporting bacterial infections over extended periods3. For example, if an organism can replicate in the overlying media, this can result in unacceptable levels of cytotoxicity and loss of host cells, arresting the experiment. The EpiAirway model allows characterization of long-term host-pathogen interactions. Further, since the source for the EpiAirway is normal human tracheo-bronchial cells rather than an immortalized line, each is an excellent representation of actual human upper respiratory tract tissue, both in structure and in function4.
For this method, the EpiAirway tissues are weaned off of anti-microbial and anti-fungal compounds for 2 days prior to delivery, and all procedures are performed under antibiotic-free conditions. This necessitates special considerations, since both bacteria and primary human tissues are used in the same biosafety cabinet, and are co-cultured for extended periods.

This method allows characterization of extended bacterial co-culture with EpiAirways, primary human respiratory epithelial tissue grown at the air-liquid interface, a biologically relevant in vitro model. The approach can be used with any microbe that is amenable to long-term co-culture.

使用的EpiAirway模型，用于描述长期宿主 - 病原体相互作用

Nontypeable Haemophilus influenzae  (NTHi) are human-adapted Gram-negative bacteria that can cause recurrent and chronic infections of the respiratory ...

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires&#39; disease, is an important human pathogen that infects and replicates within alveolar macrophages. Its virulence depends on the Dot/Icm type IV secretion system (T4SS), which is essential to establish a replication permissive vacuole known as the Legionella containing vacuole (LCV). L. pneumophila infection can be modeled in mice however most mouse strains are not permissive, leading to the search for novel infection models. We have recently shown that the larvae of the wax moth Galleria mellonella are suitable for investigation of L. pneumophila infection. G. mellonella is increasingly used as an infection model for human pathogens and a good correlation exists between virulence of several bacterial species in the insect and in mammalian models. A key component of the larvae&#39;s immune defenses are hemocytes, professional phagocytes, which take up and destroy invaders. L. pneumophila is able to infect, form a LCV and replicate within these cells. Here we demonstrate protocols for analyzing L. pneumophila virulence in the G. mellonella model, including how to grow infectious L. pneumophila, pretreat the larvae with inhibitors, infect the larvae and how to extract infected cells for quantification and immunofluorescence microscopy. We also describe how to quantify bacterial replication and fitness in competition assays. These approaches allow for the rapid screening of mutants to determine factors important in L. pneumophila virulence, describing a new tool to aid our understanding of this complex pathogen.

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

The larva of the wax moth Galleria mellonella was recently established as an in vivo model to study Legionella pneumophila infection. Here, we demonstrate fundamental techniques to characterize the pathogenesis of Legionella in the larvae, including inoculation, measurement of bacterial virulence and replication as well as extraction and analysis of infected hemocytes.

使用<em&gt;大蜡螟</em&gt;作为模式生物来研究<em&gt;嗜肺军团菌</em&gt;感染

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires&#39; disease, is an important human pathogen that infects and ...

Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential to be used as drug therapies for HIV-1 infection. Post-integration inhibitors include ribozymes, short hairpin (sh) RNAs, small interfering (si) RNAs, U1 interference (U1i) RNAs and RNA aptamers. Many of these have been identified using transient co-transfection assays with an HIV-1 expression plasmid and some have advanced to clinical trials. In addition to measures of efficacy, small RNAs have been evaluated for their potential to affect the expression of human RNAs, alter cell growth and/or differentiation, and elicit innate immune responses. In the protocols described here, a set of transient transfection assays designed to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps in the HIV-1 replication cycle are described. We have used these assays to identify new ribozymes and optimize the format of shRNAs and siRNAs targeting HIV-1 RNA. The methods provide a quick set of assays that are useful for screening new anti-HIV-1 RNAs and could be adapted to screen other post-integration inhibitors of HIV-1 replication.

Methods to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps of the HIV-1 replication cycle are described. These methods are useful for screening new molecules and optimizing the format of existing ones.

RNA的疗效和毒性的评价基因或药物治疗靶向HIV-1的生产使用

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential ...

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

Transposon mutagenesis is a method that allows gene disruption via the random genomic insertion of a piece of DNA called a transposon. The protocol below outlines a method for high efficiency transfer between bacterial strains of a plasmid harboring a transposon containing a kanamycin resistance marker. The plasmid-borne transposase is encoded by a variant tnp gene that inserts the transposon into the genome of the recipient strain with very low insertional bias. This method thus allows the creation of large mutant libraries in which transposons have been inserted into unique genomic positions in a recipient strain of either Escherichia coli or Shigella flexneri bacteria. By using bacterial conjugation, as opposed to other methods such as electroporation or chemical transformation, large libraries with hundreds of thousands of unique clones can be created. This yields high-density insertion libraries, with insertions occurring as frequently as every 4-6 base pairs in non-essential genes. This method is superior to other methods as it allows for an inexpensive, easy to use, and high efficiency method for the creation of a dense transposon insertion library. The transposon library can be used in downstream applications such as transposon sequencing (Tn-Seq), to infer genetic interaction networks, or more simply, in mutational (forward genetic) screens.

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

Presented here is a simple method for creating a high-density transposon insertion library in Escherichia coli or Shigella flexneri using bacterial conjugation. This protocol allows the creation of a collection of hundreds of thousands of unique mutants in bacteria by the random genomic insertion of a transposon.

使用细菌共轭的 Enterobacterial 菌株 (如大肠杆菌或志贺菌福氏) 创建稠密的转插入库

Transposon mutagenesis is a method that allows gene disruption via the random genomic insertion of a piece of DNA called a transposon. The protocol below ...

Imaging Ca2+ Responses During Shigella Infection of Epithelial Cells

Ca2+ is a ubiquitous ion involved in all known cellular processes. While global Ca2+ responses may affect cell fate, local variations in free Ca2+ cytosolic concentrations, linked to release from internal stores or an influx through plasma membrane channels, regulate cortical cell processes. Pathogens that adhere to or invade host cells trigger a reorganization of the actin cytoskeleton underlying the host plasma membrane, which likely affects both global and local Ca2+ signaling. Because these events may occur at low frequencies in a pseudo-stochastic manner over extended kinetics, the analysis of Ca2+ signals induced by pathogens raises major technical challenges that need to be addressed.
Here, we report protocols for the detection of global and local Ca2+ signals upon a Shigella infection of epithelial cells. In these protocols, artefacts linked to a prolonged exposure and photodamage associated with the excitation of Ca2+ fluorescent probes are troubleshot by stringently controlling the acquisition parameters over defined time periods during a Shigella invasion. Procedures are implemented to rigorously analyze the amplitude and frequency of global cytosolic Ca2+ signals during extended infection kinetics using the chemical probe Fluo-4.

Imaging Ca2+ Responses During Shigella Infection of Epithelial Cells

Here, we present protocols to visualize calcium (Ca2+) responses elicited by HeLa cells infected by Shigella. By optimizing the parameters of bacterial infection and imaging with Ca2+ fluorescent probes, atypical global and local Ca2+ signals induced by bacteria over a large range of infection kinetics are characterized.

成像 Ca2 +在志贺菌感染上皮细胞期间的响应

Ca2+ is a ubiquitous ion involved in all known cellular processes. While global Ca2+ responses may affect cell fate, local variations in free Ca2+ ...

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 ...

Complementary Use of Microscopic Techniques and Fluorescence Reading in Studying Cryptococcus-Amoeba Interactions

To simulate Cryptococcus infection, amoeba, which is the natural predator of cryptococcal cells in the environment, can be used as a model for macrophages. This predatory organism, similar to macrophages, employs phagocytosis to kill internalized cells. With the aid of a confocal laser-scanning microscope, images depicting interactive moments between cryptococcal cells and amoeba are captured. The resolution power of the electron microscope also helps to reveal the ultrastructural detail of cryptococcal cells when trapped inside the amoeba food vacuole. Since phagocytosis is a continuous process, quantitative data is then integrated in the analysis to explain what happens at the timepoint when an image is captured. To be specific, relative fluorescence units are read in order to quantify the efficiency of amoeba in internalizing cryptococcal cells. For this purpose, cryptococcal cells are stained with a dye that makes them fluoresce once trapped inside the acidic environment of the food vacuole. When used together, information gathered through such techniques can provide critical information to help draw conclusions on the behavior and fate of cells when internalized by amoeba and, possibly, by other phagocytic cells.

Complementary Use of Microscopic Techniques and Fluorescence Reading in Studying Cryptococcus-Amoeba Interactions

This paper details a protocol for preparing a co-culture of cryptococcal cells and amoebae that is studied using still, fluorescent images and high-resolution transmission electron microscope images. Illustrated here is how quantitative data can complement such qualitative information.

在研究隐球菌-阿米巴相互作用中,显微技术和荧光读数的补充应用

To simulate Cryptococcus infection, amoeba, which is the natural predator of cryptococcal cells in the environment, can be used as a model for ...

A High-throughput Shigella-specific Bactericidal Assay

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing activity by assessing the ability of antibodies in serum to bind to bacteria and activate complement. This complement activation results in the lysis and killing of the target bacteria. These assays are valuable because they go beyond quantifying antibody production to elucidate the biological functions that these antibodies have, allowing researchers to study the role that antibodies may play in preventing infection. SBAs have been used to study immune responses for many human pathogens, but there is no widely accepted methodology for Shigella at present. Historically, SBAs have been very labor-intensive, requiring many time-consuming steps to accurately quantify surviving bacteria. This protocol describes a simple, robust, and high-throughput assay that measures functional antibodies specific for Shigella in serum in vitro. The method described here offers many advantages over traditional SBAs, including the use of frozen bacterial stocks, 96 well assay plates, a micro-culture system, and automated colony-counting. All of these modifications make this assay less labor-intensive and more high-throughput. This protocol is simpler and faster to perform than traditional SBAs while still using simple technologies and readily available reagents. The protocol has been successfully applied in multiple independent laboratories and the assay is robust and reproducible. The assay can be used to assess immune responses in pre-clinical as well as clinical studies. Quantifying shigellacidal antibody titers both before and after antigen exposure (either by immunization or infection) allows for a broader understanding of how functional antibody responses are generated and their contribution to protective immunity. The development of this standardized, well-characterized assay may greatly facilitate Shigella vaccine design.

A High-throughput Shigella-specific Bactericidal Assay

Here we present a protocol to measure Shigellacidal activity of antibodies in serum. Serum is mixed with bacteria and exogenous complement, incubated, and the reaction mixture is plated on agar plates. Viable bacteria form colonies which are counted, using an automated colony enumerator, and used to determine the bactericidal titer.

一种高通量志贺氏菌特异性杀菌试验

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing ...

Human Colonoid Monolayers to Study Interactions Between Pathogens, Commensals, and Host Intestinal Epithelium

Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal epithelium. Due to their closed structures and significant supporting extracellular matrix, 3D cultures are not ideal for host-pathogen studies. Enteroids or colonoids can be grown as epithelial monolayers on permeable tissue culture membranes to allow manipulation of both luminal and basolateral cell surfaces and accompanying fluids. This enhanced luminal surface accessibility facilitates modeling bacterial-host epithelial interactions such as the mucus-degrading ability of enterohemorrhagic E. coli (EHEC) on colonic epithelium. A method for 3D culture fragmentation, monolayer seeding, and transepithelial electrical resistance (TER) measurements to monitor the progress towards confluency and differentiation are described. Colonoid monolayer differentiation yields secreted mucus that can be studied by the immunofluorescence or immunoblotting techniques. More generally, enteroid or colonoid monolayers enable a physiologically-relevant platform to evaluate specific cell populations that may be targeted by pathogenic or commensal microbiota.

Here, we present a protocol to culture human enteroid or colonoid monolayers that have intact barrier function to study host epithelial-microbiota interactions at the cellular and biochemical level.

人类舱内单聚物研究病原体、共脉和宿主肠道上皮细胞之间的相互作用

Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal ...