Name: the nematode caenorhabditis elegans - a versatile in vivo model to study host-microbe interactions
Uploaded: 2017-10-18T14:00:00.000+00:00
Duration: 11 min 58 s
Description: Watch this Scientific Journal Video about The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions at JoVE.com

这项工作是在伍斯特理工学院的支持下进行的。

1. 制备线虫生长培养基 (NGM) <ol> <li> 用于 1 l 介质, 结合 20 g 琼脂、2.5 g 有机氮源 ( 如 、bacto-蛋白胨) 和3克氯化钠在 2 l 烧瓶中。加入975毫升的无菌水. 
	<ol> <li> 在无菌搅拌栏中添加。如果使用自动介质倒, 高压釜管和介质为15分钟;如果有更高的音量, 介质应更长的时间进行蒸压. </li>
	</ol> </li> <li> 在搅拌板上设置介质, 并允许冷却。
	<ol> <li> 一旦媒体被热触碰 (大约60和 #176; C) 菌增加1毫升的1米 MgSO 4 和 #9679; H 2 O, 1 毫升的 1M CaCl 2 , 25 毫升的 KPO 4 , 和1毫升的0.5% 的胆固醇在乙醇中. </li>
		<li> 将介质 (手动或自动泵) 倒入 35 mm x 10 mm 无菌培养皿中。允许在使用前1-2 天晾干引擎盖. 
		注: 板材应存储在4和 #176; C, 以防止污染. </li>
	</ol> </li> </ol> 2。制作蠕虫选择 <ol> <li> 将一根1.5 厘米的铝线切下。小心地将大约0.5 厘米的长度插入到巴斯德吸管的尖端. 
	<ol> <li> 使用本生燃烧器或酒精灯, 将玻璃吸管尖端慢慢地旋转到火焰中, 直到导线牢固地附着在吸管上, 而不会损害吸管尖端的原始形状. </li>
	</ol> </li> </ol> 3。 大肠杆菌 培养基的制备 <ol> <li> 使用无菌循环, 在200毫升的 Luria 汤中接种单一菌落的 大肠杆菌 菌株 OP50。在37和 #176 摇晃过夜; C. </li>
	<li> 液体养殖已准备就绪, 可在次日使用, 并可存储在4和 #176; C 时不使用. 
	注意: OP50 的 大肠杆菌 菌株被用作 C. 线虫的食物来源 此区域性可在4和 #176 存储时使用长达几个月; C. </li>
</ol> 4。基本的 线虫 维护 <ol> <li> 种子准备 NGM 琼脂板与 大肠杆菌 , 通过发现大约 50-100 和 #181; L 准备 OP50 液体文化到板材的中心. </li>
	<li> 盖板, 使其在室温下干燥24小时。一旦干燥, 放置在37和 #176 的盘子; C 隔夜为迅速成长或保持在室温下, 如果增长是渴望的. </li>
	<li> 通过任意和 #34 将蠕虫添加到板中; 分块、#34; (请参阅协议步骤5和 #34; 将蠕虫和 #34;), 逐个选取蠕虫, 或将蠕虫卵直接放到板上 (参见协议步骤6和 #34; 蛋准备和 #34;). </li>
</ol> 5。分块和拾取蠕虫 注意: 和 #34; #34. 是指在 C. 线虫 维护中常见的一种做法。这涉及切割含有蠕虫的 NGM 琼脂板的一部分, 并将其转移到一个新的板块, 从而也转移了大量的蠕虫在这个过程中。污染也可以用这种方式从盘子里切出来。当采摘蠕虫时, 重要的是要保持琼脂的完整性, 因为蠕虫可以在琼脂的孔中丢失。此外, 重要的是, 让采摘冷却前触摸板, 以防止融化的琼脂或燃烧的蠕虫. 
<ol> <li> 为了快速将大量的蠕虫转移到新的板上, 请用刮刀将含有所选蠕虫的一块 NGM 琼脂切开。删除切割件, 并将其面朝下的 OP50 草坪的边缘上的一个新的板块. </li>
	<li> 在协议步骤8.2、#34、生存分析和 #34 中分别传输蠕虫; 使用蠕虫拾取。在蠕虫的选择中火焰, 直到它是红色的。从火焰中取出, 让它冷却几秒钟. </li>
	<li> 用铁丝在镐上, 轻轻刮去 OP50 文化的草坪上生长的新盘子, 盖上铁丝上的镐. </li>
	<li> 在导线尖端的粘性培养物会使蠕虫粘在拾取的尖端, 从而用滑动运动轻轻地拾取选定的蠕虫. </li>
	<li> 一旦选定的蠕虫被收集, 将它们放到一个新的盘子上, 轻轻地在琼脂上滑动区域性。将导线放入火焰中, 以去除拾取中的剩余区域性. </li>
</ol> 6。鸡蛋准备 <ol> <li> 将大约20成年动物 (大约 2-3 天后孵化时, 生长在20和 #176; C) 按协议步骤 5, #34; 分块和采摘蠕虫, 和 #34; 在一个有50个种子的盘子上和 #181 大肠杆菌菌株, OP50. </li>
	<li> 收集鸡蛋, 在1-2 天后用10毫升的 M9 缓冲液淹没盘子。使用玻璃血清吸管, 旋流, 并轻轻地搅动琼脂板上的内容, 以释放鸡蛋粘在 OP50 或成年动物。收集所有含有卵和成虫的液体. </li>
	<li> 将 M9 和 OP50 溶液转换为15毫升锥形管。离心机15毫升锥管在 2100 x g 2 min. </li>
	<li> 吸气约9毫升的上清, 不干扰 OP50 颗粒. </li>
	<li> 重2毫升无菌水中的颗粒, 1 毫升的漂白剂和1毫升的0.25 米氢氧化钠. </li>
	<li> 通过反转轻轻混合, 直到成年动物的大多数 (大约 70%) 出现裂解; 通常情况下, 由于蛋壳的原因, 鸡蛋在漂白过程中保持完好。离心锥管在 990 x g 为 2 min. </li>
	<li> 吸入上清液而不干扰颗粒。重新悬浮在10毫升的 M9 缓冲颗粒. </li>
	<li> 离心锥管 990 x g, 2 分钟, 无干扰颗粒。200、#181 的 M9 缓冲颗粒重新悬浮. 
	注: 卵子准备可能需要多次试验。如果鸡蛋暴露在漂白剂中太久, 它们可能会被破坏。如果鸡蛋不孵化, 要么减少溶液中漂白剂的浓度, 要么减少接触漂白剂的时间. </li>
</ol> 7。感染板设置 <ol> <li> 将3-5 毫升的 YPD 分配到一个无菌试管中, 并使用一个无菌循环将它接种到一个单一菌落的白色念珠菌 中. 
	<ol> <li> 将导管放置在旋转鼓上大约16-18 小时, 在30和 #176; C. </li>
	</ol> </li> <li> 标记一个1.5 毫升离心管, 以包含 C. 白色念珠菌 , 另一个包含 OP50。记录每个空管的重量. 
	<ol> <li> 将500和 #181; 过夜的 c. 白色念珠菌 培养成管标为 白色念珠菌 和1500和 #181; 夜间 OP50 培养 (从步骤 3.1) 到标有 OP50 的管子. </li>
		<li> 离心10分钟, 16100 x g. 吸上清液而不干扰颗粒. </li>
		<li> 重新挂起500和 #181 的每个颗粒; 无菌水的 L。离心机为5分钟, 在 16100 x g. </li>
		<li> 吸入上清液而不干扰颗粒。记录离心管的最终重量. </li><li> 通过从最终重量中减去离心管的初始重量来确定每个颗粒的重量. </li>
	</ol> </li> <li> 使用无菌水, 将 re-suspend 为10毫克/毫升, 并将 OP50 颗粒至200毫克/毫升。通过结合10和 #181; 50 毫克/毫升链霉素, 2.5 和 #181; l 的 OP50 培养, 0.5 和 #181; l. 白色念珠菌 培养, 7 和 #181; 无菌水的 l. 
	<ol> <li> 对于控制板, 通过用无菌水替换 C. 白色念珠菌 的体积来创建主混合。种子20和 #181; L 感染混合或 OP50 控制解决方案, NGM 板的中心使用微. 
		注: 在分析过程中, 需要大约100蠕虫来确定统计意义。这种化验通常是三份的。因此, 一个3.2x 的感染混合以及3.2x 控制解决方案作出。这些混合被制成略超过3x 的原始, 以确保一个完整的20和 #181; 我被播种在每一个盘子上, 允许有错误的空间。此主组合是创建的, 因为在初始幼体阶段 (L1-L3) 中, 蠕虫的咽太小, 无法使用 C. 白念珠菌 。对于接触药物, 如氟康唑 ( 图 3B ), 该剂被引入到感染混合物。药剂的集中是经验主义地坚定的。例如, 在 图 3B 中, 50 和 #181; m 氟康唑的代表, 但 0, 12.5, 25, 50 和100和 #181; m 氟康唑也使用相同的协议进行了测试. </li>
	</ol> </li> </ol> 8。变形肛门区 (dar) 表型与生存试验分析 <ol> <li> 可视化 dar 表型 注: dar 在 L3 阶段和更远处是最好的可视化。Dar 在蠕虫的后肛门区域 ( 图 2A ) 中显示为一个小突起, 随着时间的推移, 它变得更加突出。
	<ol> <li> 通过在解剖显微镜的舞台上快速敲击板来确认动物是否有 dar; 没有 dar 的动物将立即向后移动, 而与 dar 的动物将需要反复的敲击来反转它们的方向, 或者不会这样做. </li>
	</ol> </li> <li> 生存分析 <ol> <li> 完整的蛋准备, 如协议步骤6中所述, #34; 鸡蛋准备和 #34;。计算下解剖范围内的卵, 用 M9 稀释卵子溶液, 直到每 #181 约5-6 健康卵的浓度; 我成功了使用吸管, 免除20和 #181; 样品中含有约30鸡蛋在准备的 NGM 板上, 在细菌培养和侧板之间的区域 (卵和蠕虫食物, OP50, 是在两个不同的斑点). </li>
		<li> 在20和 #176 孵育板; C 为48小时。在解剖显微镜下, 统计每个板块上的活的和死的成虫数, 以及与 Dar 的蠕虫数量。用 Dar 记录百分比. </li>
		<li> 如果它不响应由铝线敲击头部或在板上敲击, 则认为是动物死亡. </li>
		<li> 每隔一天, 按协议步骤5中所述的方法选择转移剩余的成虫, 并 #34; 分块和拾取蠕虫、#34; 进入新的感染或控制板。在这一点上, 如果需要, 通过跟踪每天的活、死和缺失蠕虫的数量来进行生存试验. 
		注: 此化验可运行两周, 从准备卵子开始。此外, 卵子的解决方案不应被分配到细菌培养, 因为解决方案可能含有漂白剂的痕迹, 可以杀死 OP50。该解决方案也应免除约0.5 厘米远离板块的边缘, 因为鸡蛋可以成为卡在两侧的板块和琼脂。此外, 由于蠕虫的迅速扩散, 将成人转移到新的板块上是将其与后代分离的关键. </li>
	</ol> </li> <li> 分析生存 <ol> <li> 使用生存曲线分析软件分析结果 (参阅材料列表). </li>
		<li> 使用 Grehan-Breslow-魏氏方法比较生存曲线 (假设早期生存时间的数据比以后的时间更准确, 相应地对数据进行加权) 以及 Logrank 统计工具 45 . 
		注意: 例如, 在 图 4B -c 中, 使用变种人 c. 白色念珠菌治疗的蠕虫的生存率与基因野生型控制或 complementarystrains 治疗相比. 
	</ol>
	</li>
</ol>

作者没有什么可透露的。

用于检测线虫感染和生存期的方法对我们所描述的c. 白色念珠菌的影响可以通过修改来测试另一种病原体。另一种细菌或真菌的液体培养物可以用类似的方式制成并喂给C. 线虫。此外, 串行感染可以通过首先暴露幼虫到一个病原体, 然后转移到一个新的板块, 包含一个独立的病原体后, 成年。
在进行这种化验时, 注意时间是很重要的。首先, 当完成鸡蛋准备, 以收获鸡蛋的化验, 这是至关重要的限制时间的数量, 鸡蛋暴露在漂白剂。在漂白剂溶液中溶解成虫以释放卵所需的时间长短不一。在管理漂白溶液后, 必须在解剖显微镜下密切观察蠕虫的解决方案。一旦大约70% 的蠕虫已经解体, 那么解决方法应该是离心的。如果卵子的制备不产生可行的卵, 则减少漂白剂的浓度或接触漂白剂的时间。在完成这项化验时, 同样重要的是要注意将成年蠕虫转移到新的盘子上, 以避免混淆研究对象与他们的后代。限制鸡蛋的数量, 在开始的化验, 以25-30 鸡蛋也应该防止这一点。最后, 将成虫转移到新的盘子上, 也会减少蠕虫爬行在培养皿两侧并死亡的几率。
我们利用了线虫线虫作为研究微生物或微生物及其宿主之间致病或共生关系的多功能模型宿主。我们的系统可以用来作为一种工具, 研究微生物或宿主-微生物相互作用的细胞和分子水平, 利用前和反向遗传方法, 但也探索新的分子治疗干预。我们已经展示了这个系统的多功能性, 通过进行遗传屏幕来识别致病基因40, 那些与微生物适应性相关的病毒41, 那些介导宿主对微生物的反应17,18, 以及使用用于药物发现的高吞吐量应用程序的模型24.这是一个很好的模式, 供本科生使用, 因为它不需要昂贵的基础设施来维持殖民地, 考虑到动物可以被冷冻保存的未来的应用。最后, 这为体外研究和小鼠模型提供了一个完美的 "go-between"。

线虫已被用作一个强大的模型有机体超过50年。在二十世纪六十年代, 南非生物学家悉尼. 博纳率先使用了线虫来研究神经元的发育, 为长期的科学家们研究线虫的细胞和动物生物学的各个方面铺平了道路。这一血统包括诺贝尔奖得主克雷格. 梅洛和安德鲁火为他们的 rna 干扰工作1, 罗伯特 Horvitz 并且约翰 Sulston 为他们的工作器官发展和细胞凋亡2,3,4, 和马丁查尔菲为他的工作绿色荧光蛋白5。尽管这种模型生物体已经被传统用于研究分子和发育生物学, 但在过去15年中, 研究人员已经开始使用线虫来调查各种人类病原体的生物学, 包括假单胞菌铜绿假单胞菌,金黄色葡萄球菌,沙门氏菌 enterica, 和沙雷菌6,7,89,10。这些研究表明, 许多涉及人与病原体相互作用的机制都是在线虫中保存的, 但也有一些免疫机制是独特的, 这种模式生物体11,12。在自然界中,线虫遇到了来自土壤中的受感染病原体的各种威胁, 这为进化和维持一个复杂的先天免疫系统在其肠道腔内提供了强烈的选择性压力。许多涉及保护肠道腔的基因和机制都是由高度保守的元素组成的, 它们也存在于高哺乳动物中11,13。因此,线虫是研究胃肠病原体的一个很好的模型, 如沙门氏菌 enterica14,痢疾 boydii15, 或霍乱弧菌16。
在这里, 我们强调了显着的多功能性的c. 线虫作为一个模型的宿主, 以研究传染性药物, 如C. 白色念珠菌。C. 线虫作为模型宿主, 可以对毒性进行高通量筛选, 这比小鼠模型更便宜、更耗时, 通常用于研究念珠菌病42。
在这项研究中, 我们表明, 该模型和 assosiated 生存分析可以可靠地用于研究宿主先天免疫效应的重要性, 以抵消感染, 病原体的决定因素驱动毒力, 和药理化合物, 可以干预发病机制。与先前所描述的化验方法不同, 此法提供了一种研究动物生命周期中的病原体, 从幼虫阶段到成年, 而不是仅成年到死亡的手段43,44。总之, 我们的线虫--白色念珠菌模型是一种多功能、功能强大的工具, 不仅可以用于研究驱动感染和免疫的遗传基础, 还可用于识别治疗干预的新化合物。

<table><tbody><tr><td>Agar (granulated, bacterilogical grade)</td><td>Apex BioResearch Products</td><td>20-248</td><td></td></tr><tr><td>Aluminum Wire (95% Pt, 32 Gauge)</td><td>Genesee Scientific</td><td>59-1M32P</td><td></td></tr><tr><td>Axiovision Zeiss Inverted Microscope</td><td>Axiovision Zeiss</td><td></td><td></td></tr><tr><td>Bacto-Peptone</td><td>Fisher BioReagants</td><td>BP1420-500</td><td></td></tr><tr><td>C. elegans strain Bli-3</td><td>Caenorhabditis Genetics Center</td><td>Bli-3(e767) CB767</td><td></td></tr><tr><td>Calcium Chloride</td><td>Fisher Scientific</td><td>BP51-250</td><td></td></tr><tr><td>Cholesterol, Sigma Grade, minimum 99%</td><td>Sigma</td><td>C8667-25G</td><td></td></tr><tr><td>Disposable Culture Tubes (20 x 150 mm)</td><td>FIsherBrand</td><td>14-961-33</td><td></td></tr><tr><td>Dissection Microscope (NI-150 High Intensity Illuminator)</td><td>Nikon Instrument Inc.</td><td></td><td></td></tr><tr><td>E. coli</td><td>Caenorhabditis Genetics Center</td><td>OP50</td><td></td></tr><tr><td>GraphPad Prism (Survival Curve Analysis Software)</td><td>GraphPad Software</td><td></td><td></td></tr><tr><td>LB Broth (Miller&#039;s)</td><td>Apex BioResearch Products</td><td>11-120</td><td></td></tr><tr><td>Magnesium Sulfate</td><td>Fisher Scientific</td><td>10034-99-8</td><td></td></tr><tr><td>Medium Petri Dishes (35 X 10 mm)</td><td>Falcon</td><td>353001</td><td></td></tr><tr><td>Potassium Phosphate monobasic</td><td>Sigma</td><td>P0662-500G</td><td></td></tr><tr><td>Sodium Chloride</td><td>Fisher Scientific</td><td>BP358-1</td><td></td></tr><tr><td>Sodium Phosphate</td><td>Fisher Scientific</td><td>BP332-500</td><td></td></tr><tr><td>Wildtype C. albicans SC5314</td><td>ATCC</td><td>SC5314</td><td></td></tr><tr><td>Wildtype C. elegans</td><td>Caenorhabditis Genetics Center</td><td>N2</td><td></td></tr></tbody></table>

the nematode caenorhabditis elegans - a versatile in vivo model to study host-microbe interactions

我们演示了一种使用线虫线虫作为模型宿主来研究微生物相互作用的方法。通过饮食引入微生物, 使肠道成为疾病的主要部位。线虫肠道结构和功能模仿哺乳动物的肠道, 是透明的, 使其适于对殖民的微观研究。在这里, 我们表明病原体可以导致疾病和死亡。我们能够识别出具有改变毒性的微生物突变体。它对生物胁迫的固有反应使线虫成为一个极好的系统, 可以探测宿主先天免疫相互作用的方方面面。我们发现, 双氧化酶基因突变的寄主不能产生活性氧, 无法抵抗微生物的侮辱。我们进一步证明了所提出的生存试验的通用性, 表明它可以用于研究微生物生长抑制剂的影响。这种方法也可用于发现真菌致病因子作为发展新的抗真菌药物的目标, 以及提供一个机会, 进一步揭示宿主-微生物相互作用。这种检测方法的设计很好地适应了高通量全基因组的屏幕, 而低温保存蠕虫以备将来使用的能力使得它成为一个 cost-effective 和有吸引力的整体动物模型来研究。

病机检测 (图 1) 使用c. 白色念珠菌和c. 线虫以前曾被我们的实验室描述17,18和其他实验室19,,。我们演示了使用可研究 白色念珠菌的毒力, 该病毒显示了c. 白念珠菌的细胞迅速被蠕虫吸收并积聚在肠道内, 导致运动迟缓, 肛门畸形区域 (Dar) (图 2A), 外阴肿胀 (图 2B), 肚腑膨胀 (图 3A) 和杀伤力 (图3,图 4)。我们还测量了受感染蠕虫的寿命, 作为一种定量的健身方法。例如, 感染了c. 白色念珠菌的c. 线虫活10-12 天, 而未感染的对照为20-22。在c. 线虫中感染了c. 白念珠菌双敲出变种, efg1/efg1 cph1/cph1, 这是两个关键的毒力因子的感染所需的鼠标, 大鼠和人类上皮模型21, 23, 线虫存活时间明显长于控件 (图 3B)。这些实验表明, 我们在这个简单的关于白念珠菌毒力模型中所学到的一些经验教训在高等哺乳动物中可能仍然有效, 反之亦然。
我们还表明, 线虫模型系统可以药理调制 (图 3B)。在服用最常用的抗真菌药物氟康唑存在的情况下, 与对照组相比, 与C. 白色念珠菌有关的蠕虫存活时间明显延长。实验证明, 该线虫模型可用于小分子筛查。事实上, 我们的线虫模型有助于识别 filastatin, 一个小分子抑制真菌毒力的各个方面24。
疾病表型和显微分析C. 线虫感染 
c. 线虫在六天内暴露于c. 白色念珠菌, 并观察到感染、疾病进展和死亡的迹象。Dar 表型是最明显的4天的生存化验, 指出一个突出的肛门区域, 是不可见的未受感染的动物 (图 2A)。感染了C. 白念珠菌的蠕虫也会在外阴区域出现肿胀 (图 2B)。在这两种情况下, 蠕虫都无法在到达此阶段后清除感染。为了使白色白念珠菌在肠道内的定植可视化, 蠕虫被喂食带有 RFP 标签的野生型的c. 白念珠菌, 这会导致荧光红 (图 3A) 的定植区域。为了产生这些图像, 蠕虫被麻醉在一个2% 琼脂糖垫含有0.01 米叠氮化钠。蠕虫被暴露在野生类型的c. 白色白念珠菌或 RFP 标记的c. 白念珠菌, 并转移到琼脂糖垫上的5µM M9 缓冲液中。琼脂糖垫, 然后覆盖了一个片。蠕虫在200X 和400X 放大使用荧光显微镜能力倒置显微镜。图像是在差分干涉对比 (Nomarski) 和荧光光学下创建的。使用软件增强了荧光图像 (图 3A)。受感染蠕虫的时间序列显微确定, 在检测的第三天, C. 白色念珠菌定植于肠道腔内17。感染的蠕虫在未感染的控制中表现出比观察到的更严重的肠道扩张。
研究感染的遗传学和药理学工具 接下来, 我们使用遗传和药理调节来测试模型。我们还测试了以前记录的毒力因子, 以调节菌丝的C. 型白色念珠菌的过渡25 , 并已显示在体内的小鼠和线虫的感染是重要的20,26.我们测试了蠕虫的能力, 以生存感染引起的C. 白色 efg1/efg1 cph1/cph1双突变体.Efg1 是一个高度保守的转录因子和一个重要组成部分的循环 AMP/蛋白激酶 a (PKA) 代谢途径。在C. 白念珠菌此通路调节菌丝形态发生的27, 白色不透明开关28, 以及关键毒力因子的武库。这些毒力因素包括 Hwp1 和 Hwp2, 两个酵母特异性细胞壁蛋白参与粘附和生物膜的形成, Eap1, 一个细胞壁黏附参与绑定到人类上皮细胞29, 和 Sap5, 一个水解酶参与上皮组织侵入30。Cph1 是一个转录因子, 调节许多代谢过程, 包括交配, 丝, 和生物膜的形成, 并已被证明是发挥关键作用的破坏性上皮细胞31和人类重建上皮21.这两种基因的破坏都对cph1/cph1efg1/efg1中的毒力和同时中断产生重大影响, 包括小鼠25、果蝇的各种动物模型中的剧烈毒力降低。 32, 斑马鱼33,34和飞蛾34。cph1/cph1efg1/efg1双突变体是由C. 白念珠菌社区视为无毒应变的定义和黄金标准的验证新的模型系统。与同源野生类型相比, efg1δ和cph1δ单突变体显示的 dar (10% 和 ~ 50% 分别) 下降, 而efg1δ cph1δ双变种无法引出 dar 响应17。这些结果概括了小鼠的毒力模式, 其中cph1∆突变体略有衰减, efg1∆突变体为 significantly 衰减, 双突变体完全无毒25。感染了cph1/cph1 efg1/efg1C. 白念珠菌的蠕虫在统计学上的存活时间比对照组长 (p 和 #60; 0.01 用于日志秩和 Breslow 统计测试, n=60) 表明这两种基因对于 C 是必需的。白色念珠菌对线虫的毒力 (图 3B)。
为了探讨使用我们的线虫模型进行潜在药物筛选的可能性, 我们测试了添加氟康唑对感染的最终结果的影响。我们使用了各种不同浓度的氟康唑, 发现50µM 给我们的最重要的结果。感染了C. 白念珠菌和50µM 氟康唑 (图 3B) 的蠕虫在统计学上的寿命比对照组长 (p 和 #60; 0.01 在日志级别和 Breslow 统计测试, n=60)。这集中是经验主义地确定的 (参见笔记在协议步7的结尾, "传染板材设置了)。这些原理实验证明, 我们的线虫模型可用于小分子抗真菌筛选。该模型实际上是在发现 filastatin, 一个小分子, 抑制真菌毒力的各个方面, 目前正在进行进一步的临床前研究24。因此, 我们的检测方法适合于探讨白念珠菌的毒力策略和药理作用。
先天宿主反应研究 接下来, 我们想要研究对病原体的相互宿主防御, 因为这先天免疫的方面在哺乳动物中被保存11,35。众所周知, C. 线虫在细菌和真菌感染时都产生 ROS18、36、37 , 作为其防御机制的一部分。ROS 对入侵的生物体有生物的影响, 在先天免疫方面起着重要的作用。zcf15/zcf15对 ros 的超易感性在体外导致我们假设其在线虫中的毒性降低是由于抵御宿主产生的 ROS 的能力降低。为了测试我们的假设, 我们决定了zcf15/zcf15的能力, 以杀死任何野生类型的蠕虫或蠕虫的能力, 产生 ROS。具有代表性的白念珠菌突变体zcf15对活性氧种类 (图 4A) 的毒性在野生型的线虫中表现为减少。C. 线虫通过由基因bli-3编码的 Ce-Duox1, 通过在肠道腔内产生胞外活性氧来响应病原体的摄取。在 ros 生产过程中, 肠道细胞还通过DAF-16产生胞内抗氧化剂, 以保护其自身的组织免受活性氧损害的影响3638。Ce-Duox1 是一种具有 N 端过氧化物酶域的蛋白质, 一个 c-末端超氧化物生成的氧化酶域和两个中央钙调素结合位点39。在微生物感染, 这种蛋白质使用胞浆细胞生成胞外毒性 ROS 的肠道腔内, 以抵消感染。在2009的一项研究中的一系列生化检测中, 耆那教的et al.显示, ROS 在酵母菌感染时大量产生, 而通过 bli-3 (e767) bli-3 功能的丧失会显著降低线虫产生 ros 的能力。如图 4B所示, 与zcf15/zcf15相匹配的蠕虫比野生类型或补充菌株存活的时间要长得多 (p & #60; 0.01 由日志秩测试), 表明ZCF15对于毒力是必需的。然而, 当我们挑战 ROS 缺乏的C. elegansbli-3 (e767) 时, 我们获得了在zcf15/zcf15、野生类型和补充应变 (图 4C) 之间可比较的杀戮动力学。这一证据表明, 除非宿主的生产活性氧的能力受到损害, 否则zcf15/zcf15将无法杀死线虫。在一起, 这些结果表明, ZCF15是完全毒力所必需的, 而且这个基因很可能涉及病原体抵抗宿主产生的 ROS 的能力。据我们所知, 这是第一次ZCF15被证明参与了致病性和 ROS 抵抗。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56487/56487fig1.jpg"> 
图 1: C. 线虫感染的示意图表示。线虫暴露于混合的大肠杆菌菌株 OP50 和病原体, 并在成年后4天内观察到感染和疾病进展的迹象。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56487/56487fig2.jpg"> 
图 2: 疾病表型。(A) 变形肛门区域 (Dar) 是可见的 (用箭头表示) 作为感染蠕虫后肛门区域 (右面板) 四天后暴露的肿胀。左侧面板上的蠕虫未受感染, 并作为一种控制。(B) 感染蠕虫的一个子集 (约 15%) 显示了外阴肿胀 (右面板, 用箭头表示) 与未感染的控制蠕虫 (左面板) 相比, 这表示传播感染导致 matricidal 死亡。所有图像都按照代表性结果18中的描述进行了拍摄。
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56487/56487fig3.jpg"> 
图 3:白念珠菌-- c. 线虫模型适于显微操作, 并可药理和基因调控。() C. 白色念珠菌在线虫肠道内堆积, 3 后感染。酵母细胞很快被蠕虫摄取, 并在肠道内完全完整地堆积, 这表明它们能够在咽部的机械粉碎中存活。图象在代表性结果18中所描述的那样。(B) 的卡普兰-梅尔线虫生存曲线与野生型的c. 白色念珠菌、 cph1/cph1efg1/efg1双突变体或野生类型的白念珠菌+ 50 µM 氟康唑相比, 未感染的控制蠕虫。
<img alt="Figure 4" class="xfigimg" src="/files/ftp_u alt="Figure 4" class="xfigimg" src=""></img alt="Figure 4" class="xfigimg" src="/files/ftp_u>pload/56487/56487fig4 
图4。ZCF15是必需的, 以抵御C. 线虫生成的 ROS。() ZCF15负责对百草枯进行野生类型的抗性, 这是已知的产生活性氧的物种。野生型、敲除或补充菌株在液体培养中生长过夜, 悬浮 OD=1。文化被稀释1:5 和镀在 YPD 或 YPD 包含1毫米百草枯。C. 线虫通过bli-3生成 ROS, 以抗击侵入肠道管腔的病原体38。(B) 卡普兰-梅尔的生存曲线显示, ZCF15删除限制了对野生类型蠕虫的捕杀。(C) 卡普兰-梅尔的生存曲线显示了在zcf15/zcf15、野生类型和补充应变之间的相似生存率, 当 ROS 缺陷的C. elegansbli-3 (e767)被感染。

Watch this Scientific Journal Video about The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions at JoVE.com

The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions

线虫线虫线虫-一个多用途的体内模型研究宿主-微生物相互作用

在这里, 我们提出线虫线虫线虫作为一个多用途的寄主模型来研究微生物的相互作用。

We demonstrate a method using Caenorhabditis elegans as a model host to study microbial interaction. Microbes are introduced via the diet making the ...

the-nematode-caenorhabditis-elegans-versatile-vivo-model-to-study

Nanyang Technological University

Research

JoVE Journal

Genetics

The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions

9.3K 次观看.  Worcester Polytechnic Institute. 在这里, 我们提出线虫线虫线虫作为一个多用途的寄主模型来研究微生物的相互作用。

视频: The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions

Two Infection Assays to Study Non-Lethal Virulence Phenotypes in C. Albicans using C. Elegans

While pathogens can be deadly to humans, many of them cause a range of infection types with non-lethal phenotypes. Candida albicans, an opportunistic fungal pathogen of humans, is the fourth most common cause of nosocomial infections which results in ~40% mortality. However, other C. albicans infections are less severe and rarely lethal and include vulvovaginal candidiasis, impacting ~75% of women, as well as oropharyngeal candidiasis, predominantly impacting infants, AIDS patients and cancer patients. While murine models are most frequently used to study C. albicans pathogenesis, these models predominantly assess host survival and are costly, time consuming, and limited in replication. Therefore, several mini-model systems, including Drosophila melanogaster, Danio rerio, Galleria mellonella, and Caenorhabditis elegans, have been developed to study C. albicans. These mini-models are well-suited for screening mutant libraries or diverse genetic backgrounds of C. albicans. Here we describe two approaches to study C. albicans infection using C. elegans. The first is a fecundity assay which measures host reproduction and monitors survival of individual hosts. The second is a lineage expansion assay which measures how C. albicans infection affects host population growth over multiple generations. Together, these assays provide a simple, cost-effective way to quickly assess C. albicans virulence.

<meta charset="utf8"></head><body>使用秀丽隐杆线虫研究白色念珠菌非致命毒力表型的两种感染试验

Fungal opportunist pathogens can cause life-threatening as well as minor infections, but non-lethal phenotypes are frequently ignored when studying virulence. Therefore, we developed a nematode model that monitors both the survival and reproduction aspects of host to investigate fungal virulence.

使用秀丽隐杆线虫研究白色念珠菌非致命毒力表型的两种感染试验

While pathogens can be deadly to humans, many of them cause a range of infection types with non-lethal phenotypes. Candida albicans, an opportunistic ...

科研

遗传学

Automated Separation of C. elegans Variably Colonized by a Bacterial Pathogen

The wormsorter is an instrument analogous to a FACS machine that is used in studies of Caenorhabditis elegans, typically to sort worms based on expression of a fluorescent reporter. Here, we highlight an alternative usage of this instrument, for sorting worms according to their degree of colonization by a GFP-expressing pathogen. This new usage allowed us to address the relationship between colonization of the worm intestine and induction of immune responses. While C. elegans immune responses to different pathogens have been documented, it is still unknown what initiates them. The two main possibilities (which are not mutually exclusive) are recognition of pathogen-associated molecular patterns, and detection of damage caused by infection. To differentiate between the two possibilities, exposure to the pathogen must be dissociated from the damage it causes. The wormsorter enabled separation of worms that were extensively-colonized by the Gram-negative pathogen Pseudomonas aeruginosa, with the damage likely caused by pathogen load, from worms that were similarly exposed, but not, or marginally, colonized. These distinct populations were used to assess the relationship between pathogen load and the induction of transcriptional immune responses. The results suggest that the two are dissociated, supporting the possibility of pathogen recognition.

Automated Separation of C. elegans Variably Colonized by a Bacterial Pathogen

The wormsorter facilitates genetic screens in Caenorhabditis elegans by sorting worms according to expression of fluorescent reporters. Here, we describe a new usage:&#160;sorting according to colonization by a GFP-expressing pathogen, and we employ it to examine the poorly understood role of pathogen recognition in initiating immune responses.

自动分离<em&gt; C。线虫</em&gt;可多段由细菌病原体殖民者

The wormsorter is an instrument analogous to a FACS machine that is used in studies of Caenorhabditis elegans, typically to sort worms based on expression ...

免疫与感染

Evaluating Virulence and Pathogenesis of Aeromonas Infection in a Caenorhabditis elegans Model

The human pathogen Aeromonas has been clinically shown to cause gastroenteritis, wound infections, septicemia, and urinary tract infections. Most human diseases have been reported to be associated with four species of bacteria: Aeromonas dhakensis, Aeromonas hydrophila, Aeromonas veronii, and Aeromonas caviae. The model organism Caenorhabditis elegans is a bacterivore that provides an excellent infection model by which to study the bacterial pathogenesis of Aeromonas. Here, we introduce three different experiments to study Aeromonas infection using a C. elegans model, including survival, liquid toxicity, and muscle necrosis assays. The results of the three methods determining the virulence of Aeromonas were consistent. A. dhakensis was shown to be the most toxic among the 4 major Aeromonas species causing clinical infections. These methods are shown to be a convenient way to evaluate the toxicity among and within Aeromonas species and contribute to our understanding of the pathogenesis of Aeromonas infection.

Evaluating Virulence and Pathogenesis of Aeromonas Infection in a Caenorhabditis elegans Model

Here, we introduce three different experiments to study Aeromonas infection in C. elegans. Using these convenient methods, it is easy to evaluate the toxicity among and within Aeromonas species.

线虫模型中气单胞菌感染的毒力及发病机制评价

The human pathogen Aeromonas has been clinically shown to cause gastroenteritis, wound infections, septicemia, and urinary tract infections. Most human ...

Selective Cleaning of Wild Caenorhabditis Nematodes to Enrich for Intestinal Microbiome Bacteria

Caenorhabditis elegans (C. elegans)&#160;has proven to be an excellent model for studying host-microbe interactions and the microbiome, especially in the context of the intestines. Recently, ecological sampling of wild Caenorhabditis nematodes has discovered a diverse array of associated microbes, including bacteria, viruses, fungi, and microsporidia. Many of these microbes have interesting colonization or infection phenotypes that warrant further study, but they are often unculturable. This protocol presents a method to enrich the desired intestinal microbes in C. elegans and related nematodes and reduce the presence of the many contaminating microbes adhering to the cuticle. This protocol involves forcing animals into the dauer stage of development and using a series of antibiotic and detergent washes to remove external contamination. As dauer animals have physiological changes that protect nematodes from harsh environmental conditions, any intestinal microbes will be protected from these conditions. But, for enrichment to work, the microbe of interest must be maintained when animals develop into dauers. When the animals leave the dauer stage, they are singly propagated into individual lines. F1 populations are then selected for desired microbes or infection phenotypes and against visible contamination. These methods will allow researchers to enrich unculturable microbes in the intestinal lumen, which make up part of the natural microbiome of C. elegans and intracellular intestinal pathogens. These microbes can then be studied for colonization or infection phenotypes and their effects on the host fitness.

Selective Cleaning of Wild Caenorhabditis Nematodes to Enrich for Intestinal Microbiome Bacteria

Wild Caenorhabditis nematodes are associated with many microbes, often in the gut lumen or infecting the intestine. This protocol details a method to enrich unculturable microbes colonizing the intestine, taking advantage of the resistance of the dauer cuticle.

选择性清洁野生 卡诺菌线 虫以富集肠道微生物组细菌

Caenorhabditis elegans (C. elegans)&#160;has proven to be an excellent model for studying host-microbe interactions and the microbiome, especially in the ...

Studying Inherited Immunity in a Caenorhabditis elegans Model of Microsporidia Infection

Inherited immunity describes how some animals can pass on the "memory" of a previous infection to their offspring. This can boost pathogen resistance in their progeny and promote survival. While inherited immunity has been reported in many invertebrates, the mechanisms underlying this epigenetic phenomenon are largely unknown. The infection of Caenorhabditis elegans by the natural microsporidian pathogen Nematocida parisii results in the worms producing offspring that are robustly resistant to microsporidia. The present protocol describes the study of intergenerational immunity in the simple and genetically tractable N. parisii -C. elegans infection model. The current article describes methods for infecting C. elegans and generating immune-primed offspring. Methods are also given for assaying resistance to microsporidia infection by staining for microsporidia and visualizing infection by microscopy. In particular, inherited immunity prevents host cell invasion by microsporidia, and fluorescence in situ hybridization (FISH) can be used to quantify invasion events. The relative amount of microsporidia spores produced in the immune-primed offspring can be quantified by staining the spores with a chitin-binding dye. To date, these methods have shed light on the kinetics and pathogen specificity of inherited immunity, as well as the molecular mechanisms underlying it. These techniques, alongside the extensive tools available for C. elegans research, will enable important discoveries in the field of inherited immunity.

Studying Inherited Immunity in a Caenorhabditis elegans Model of Microsporidia Infection

The infection of Caenorhabditis elegans by the microsporidian parasite Nematocida parisii enables the worms to produce offspring that are highly resistant to the same pathogen. This is an example of inherited immunity, a poorly understood epigenetic phenomenon. The present protocol describes the study of inherited immunity in a genetically tractable worm&#160;model.

秀 丽隐杆线虫 感染模型中的遗传免疫研究

Inherited immunity describes how some animals can pass on the "memory" of a previous infection to their offspring. This can boost pathogen resistance in ...

RNA Fluorescence in situ Hybridization (FISH) to Visualize Microbial Colonization and Infection in Caenorhabditis elegans Intestines

The intestines of wild Caenorhabditis nematodes are inhabited by a variety of microorganisms, including gut microbiome bacteria and pathogens, such as microsporidia and viruses. Because of the similarities between Caenorhabditis elegans and mammalian intestinal cells, as well as the power of the C. elegans system, this host has emerged as a model system to study host intestine-microbe interactions in vivo. While it is possible to observe some aspects of these interactions with bright-field microscopy, it is difficult to accurately classify microbes and characterize the extent of colonization or infection without more precise tools. RNA fluorescence in situ hybridization (FISH) can be used as a tool to identify and visualize microbes in nematodes from the wild or to experimentally characterize and quantify infection in nematodes infected with microbes in the lab. FISH probes, labeling the highly abundant small subunit ribosomal RNA, produce a bright signal for bacteria and microsporidian cells. Probes designed to target conserved regions of ribosomal RNA common to many species can detect a broad range of microbes, whereas targeting divergent regions of the ribosomal RNA is useful for narrower detection. Similarly, probes can be designed to label viral RNA. A protocol for RNA FISH staining with either paraformaldehyde (PFA) or acetone fixation is presented. PFA fixation is ideal for nematodes associated with bacteria, microsporidia, and viruses, whereas acetone fixation is necessary for the visualization of microsporida spores. Animals were first washed and fixed in paraformaldehyde or acetone. After fixation, FISH probes were incubated with samples to allow for the hybridization of probes to the desired target. The animals were again washed and then examined on microscope slides or using automated approaches. Overall, this FISH protocol enables detection, identification, and quantification of the microbes that inhabit the C. elegans intestine, including microbes for which there are no genetic tools available.

RNA Fluorescence in situ Hybridization FISH to Visualize Microbial Colonization and Infection in Caenorhabditis elegans Intestines

Intestinal microbes, including extracellular bacteria and intracellular pathogens like the Orsay virus and microsporidia (fungi), are often associated with wild Caenorhabditis nematodes. This article presents a protocol for detecting and quantifying microbes that colonize and/or infect C. elegans nematodes, and for measuring pathogen load after controlled infections in the lab.

RNA荧光 原位 杂交（FISH）可视化 秀丽隐杆线虫 肠道中的微生物定植和感染

The intestines of wild Caenorhabditis nematodes are inhabited by a variety of microorganisms, including gut microbiome bacteria and pathogens, such as ...

High-Throughput Screening of Microbial Isolates with Impact on Caenorhabditis elegans Health

With its small size, short lifespan, and easy genetics, Caenorhabditis elegans offers a convenient platform to study the impact of microbial isolates on host physiology. It also fluoresces in blue when dying, providing a convenient means of pinpointing death. This property has been exploited to develop high-throughput label-free C. elegans survival assays (LFASS). These involve time-lapse fluorescence recording of worm populations set in multiwell plates, from which population median time of death can be derived. The present study adopts the LFASS approach to screen multiple microbial isolates at once for the effects on C. elegans susceptibility to severe heat and oxidative stresses. Such microbial screening pipeline, which can notably be used to prescreen probiotics, using severe stress resistance as a proxy for host health is reported here. The protocol describes how to grow both C. elegans gut microbiota isolate collections and synchronous worm populations in multiwell arrays before combining them for the assays. The example provided covers the testing of 47 bacterial isolates and one control strain on two worm strains, in two stress assays in parallel. However, the approach pipeline is readily scalable and applicable to the screening of many other modalities. Thus, it provides a versatile setup to rapidly survey a multiparametric landscape of biological and biochemical conditions that impact C. elegans health.

High-Throughput Screening of Microbial Isolates with Impact on Caenorhabditis elegans Health

Gut microbes may positively or negatively impact the health of their host via specific or conserved mechanisms. Caenorhabditis elegans is a convenient platform to screen for such microbes. The present protocol describes high-throughput screening of 48 bacterial isolates for impact on nematode stress resistance, used as a proxy for worm health.

微生物分离株的高通量筛选对 秀丽隐杆线虫 健康的影响

With its small size, short lifespan, and easy genetics, Caenorhabditis elegans offers a convenient platform to study the impact of microbial isolates on ...

生物学

Using Single-Worm Data to Quantify Heterogeneity in Caenorhabditis elegans-Bacterial Interactions

The nematode Caenorhabditis elegans is a model system for host-microbe and host-microbiome interactions. Many studies to date use batch digests rather than individual worm samples to quantify bacterial load in this organism. Here it is argued that the large inter-individual variability seen in bacterial colonization of the C. elegans intestine is informative, and that batch digest methods discard information that is important for accurate comparison across conditions. As describing the variation inherent to these samples requires large numbers of individuals, a convenient 96-well plate protocol for disruption and colony plating of individual worms is established.

Using Single-Worm Data to Quantify Heterogeneity in Caenorhabditis elegans-Bacterial Interactions

This protocol describes a 96-well disruption of individual bacterially colonized Caenorhabditis elegans following cold paralysis and surface bleaching&#160;to remove external bacteria. The resulting suspension is plated on agar plates to allow accurate, medium-throughput quantification of bacterial load in large numbers of individual worms.

使用单蠕虫数据量化 秀丽隐杆线虫的异质性 - 细菌相互作用

The nematode Caenorhabditis elegans is a model system for host-microbe and host-microbiome interactions. Many studies to date use batch digests rather ...

Isolation and Characterization of the Natural Microbiota of the Model Nematode Caenorhabditis elegans

The nematode Caenorhabditis elegans interacts with a large diversity of microorganisms in nature. In general, C. elegans is commonly found in rotten plant matter, especially rotten fruits like apples or on compost heaps. It is also associated with certain invertebrate hosts such as slugs and woodlice. These habitats are rich in microbes, which serve as food for C. elegans and which can also persistently colonize the nematode gut. To date, the exact diversity and consistency of the native C. elegans microbiota across habitats and geographic locations is not fully understood. Here, we describe a suitable approach for isolating C. elegans from nature and characterizing the microbiota of worms. Nematodes can be easily isolated from compost material, rotting apples, slugs, or attracted by placing apples on compost heaps. The prime time for finding C. elegans in the Northern Hemisphere is from September until November. Worms can be washed out of collected substrate material by immersing the substrate in buffer solution, followed by the collection of nematodes and their transfer onto nematode growth medium or PCR buffer for subsequent analysis. We further illustrate how the samples can be used to isolate and purify the worm-associated microorganisms and to process worms for 16S ribosomal RNA analysis of microbiota community composition. Overall, the described methods may stimulate new research on the characterization of the C. elegans microbiota across habitats and geographic locations, thereby helping to obtain a comprehensive understanding of the diversity and stability of the nematode's microbiota as a basis for future functional research.

Isolation and Characterization of the Natural Microbiota of the Model Nematode Caenorhabditis elegans

Caenorhabditis elegans is one of the main model species in biology, yet almost all research is performed in the absence of its naturally associated microbes. The methods described here will help to improve our understanding of the diversity of associated microbes as a basis for future functional C. elegans research.

模型线虫秀丽隐杆线虫天然微生物群的分离和表征

The nematode Caenorhabditis elegans interacts with a large diversity of microorganisms in nature. In general, C. elegans is commonly found in rotten plant ...

线虫线虫线虫-一个多用途的体内模型研究宿主-微生物相互作用

摘要

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此视频中的章节

使用秀丽隐杆线虫研究白色念珠菌非致命毒力表型的两种感染试验

使用秀丽隐杆线虫研究白色念珠菌非致命毒力表型的两种感染试验

自动分离

线虫模型中气单胞菌感染的毒力及发病机制评价

选择性清洁野生卡诺菌线虫以富集肠道微生物组细菌

秀丽隐杆线虫感染模型中的遗传免疫研究

RNA荧光原位杂交（FISH）可视化秀丽隐杆线虫肠道中的微生物定植和感染

微生物分离株的高通量筛选对秀丽隐杆线虫健康的影响

使用单蠕虫数据量化秀丽隐杆线虫的异质性 - 细菌相互作用

模型线虫秀丽隐杆线虫天然微生物群的分离和表征

线虫线虫线虫-一个多用途的体内模型研究宿主-微生物相互作用

摘要

探索更多视频

此视频中的章节

使用秀丽隐杆线虫研究白色念珠菌非致命毒力表型的两种感染试验

使用秀丽隐杆线虫研究白色念珠菌非致命毒力表型的两种感染试验

自动分离

线虫模型中气单胞菌感染的毒力及发病机制评价

选择性清洁野生 卡诺菌线 虫以富集肠道微生物组细菌

秀 丽隐杆线虫 感染模型中的遗传免疫研究

RNA荧光 原位 杂交（FISH）可视化 秀丽隐杆线虫 肠道中的微生物定植和感染

微生物分离株的高通量筛选对 秀丽隐杆线虫 健康的影响

使用单蠕虫数据量化 秀丽隐杆线虫的异质性 - 细菌相互作用

模型线虫秀丽隐杆线虫天然微生物群的分离和表征

选择性清洁野生卡诺菌线虫以富集肠道微生物组细菌

秀丽隐杆线虫感染模型中的遗传免疫研究

RNA荧光原位杂交（FISH）可视化秀丽隐杆线虫肠道中的微生物定植和感染

微生物分离株的高通量筛选对秀丽隐杆线虫健康的影响

使用单蠕虫数据量化秀丽隐杆线虫的异质性 - 细菌相互作用