Name: a rapid image-based bacterial virulence assay using amoeba
Uploaded: 2018-06-27T15:00:00.000+00:00
Duration: 5 min 22 s
Description: Watch this Scientific Journal Video about A Rapid Image-based Bacterial Virulence Assay Using Amoeba at JoVE.com

KP 和写和修订的手稿。KP 进行了实验和分析。这项工作得到了国家卫生研究院 (NIH) 职业过渡奖 (K22AI112816) 的支持。

所有实验程序都是在加利福尼亚大学欧文分校进行的。
1. 缓冲器和解决方案
<ol><li>在1升玻璃瓶中加入25克的溶源性汤 (磅), 在1升的双蒸馏水 (ddH2O) 中添加一个磅米勒混合物。对于培养皿, 添加额外的20克琼脂。灭菌釜。将25毫升的熔融琼脂培养基倒入10厘米直径的培养皿中, 并允许室温固化。将液体介质存放在室温和琼脂板上, 温度为4摄氏度。</li>
	<li>在1升玻璃瓶中混合1克葡萄糖, 制备葡萄糖酵母蛋白胨 (别扭) 板, 2 克的蛋白胨, 0.25 克酵母提取物, 4.2 克的2PO4, 5.1 克的 Na2HPO47H2O, 25 克的琼脂在1升 ddH2o. 高压釜消毒。将25毫升的熔融琼脂倒入10厘米直径的培养皿中, 并允许室温固化。存储在4摄氏度。</li>
	<li>用10 克蛋白胨、7克酵母提取物、15克 d-葡萄糖、0.12 克钠2HPO47H2O、1.4 g 的µg 2 4、40的维生素 B12的1升玻璃瓶制备蛋白胨 S (PS) 培养基, 80 的叶酸在800毫升的 ddH2o) 校准电子 ph 表使用 ph 值4和 ph 7 标准, 如果 ph 计没有在一天内使用。<ol>
<li>使用 ph 计测量 PS 介质的 ph 值。添加5米 KOH 或5米 H3PO4调整 pH 值为6.5。将最终音量调整为1升, 使用 ddH2o 过滤器消毒0.22 µm 真空过滤器, 并存储在4摄氏度。 注意: 通过佩戴防护设备, 包括手套、防护服、护眼和脸部防护, 谨慎地进行 pH 调整。</li>
	</ol>
</li>
	<li>在1升玻璃瓶中, 通过添加3.4 克2PO4、13.4 克 Na2HPO4 7H2o 和 ddH2o 至800毫升的总容积, 准备10x 开发缓冲器素数 (DB) 缓冲器。如果 ph 计没有在一天内使用, 请使用 ph 值4和 ph 值7标准校准电子 ph 值表。<ol>
<li>使用电子 pH 计测量缓冲器的 ph 值。根据需要轻轻添加5米或5米3PO4 , 将 pH 值调整为6.5。将最终音量调整为1升, 使用 ddH2O, 热处理消毒。在室温下贮存。 注意: 通过佩戴防护设备, 包括手套、防护服、护眼和脸部防护, 谨慎地进行 pH 调整。</li>
	</ol>
</li>
	<li>使1x 开发缓冲区 (DB) 混合100毫升 10x DB 的缓冲器与1毫升灭菌2米氯化镁2, 和1毫升灭菌 1 m CaCl2。使用蒸压灭菌 ddH2o 在室温下, 将最终音量调整为1升玻璃瓶中的1升。</li>
	<li>准备 PS: 1 升玻璃瓶中的 DB 介质, 混合100毫升灭菌 PS 培养基, 100 毫升灭菌的 10x DB 缓冲液, 并灭菌 ddH2O 到总容积 1 l 补充与1毫升灭菌 2 m 氯化镁2和1毫升灭菌 1 m CaCl2. 贮存在4摄氏度。</li>
	<li>在制造商提供的塑料容器中加入50µL 的亚砜, 在 calcein 的50µg 中, 准备 calcein 的库存解决方案。存储在-20 摄氏度。</li>
</ol>2. 阿米巴的生长和维持
<ol><li>生长大肠杆菌菌株17作为阿米巴的食物来源在最初的成长期。将大肠杆菌从贮存于-80 摄氏度的冰冻储存到 LB 琼脂板上, 在一夜之间孵化37摄氏度以获得单一菌落。</li>
	<li>将大肠杆菌的单个菌落接种成2毫升的 LB 培养基, 在滚筒滚筒或37摄氏度的振动筛中孵化一夜。</li>
	<li>把隔夜大肠杆菌培养到室温。使用木棍, 接种阿米巴从冻结的库存保持在-80 °c 到750µL 的隔夜文化。 注: 使用D. 柄菌株 AX3, 无菌生长能力为18。这一步不需要无菌增长, 但随后的步骤将有他的要求。</li>
	<li>立即将阿米巴大肠杆菌混合物的700µL 添加到葡萄糖酵母蛋白胨 (别扭) 板上。将文化均匀地放在盘子的表面上, 使其在需要的时候向后倾斜和侧向一侧。避免使用摊铺机。</li>
	<li>将别扭板与琼脂正面朝上, 保持高湿度。使用两个一次性塑料容器 (183 厘米 x 183 厘米 x 91 厘米) 堆叠在彼此的顶部。用大约25毫升的水填充底部容器, 并将顶部容器与0.5 厘米直径的孔一起钻入容器的地板上, 允许湿气从蓄水池移动到顶容器。</li>
	<li>孵化的别扭板和盒在22°c 4-5 天, 直到变形虫孢子被观察到生长在板块表面附近 (图 2)。使用冷藏孵化器孵化板块, 而不是在室温下孵化。 注: 在孢子生长后, 它们在盘子中的存活时间在4摄氏度时保持不变。将印有琼脂面的盘子朝上。经过 3-4 天的孵化, 可以观察到细菌草坪内的清除区, 这表明孢子虫的发病。<ol>
<li>观察 4-5 天的孵化后, 在板表面上方的小阿米巴孢子 (图 2)。 注意: 不要长超过一周的阿米巴, 因为孢子的质量在这个时间段内会减少。</li>
	</ol>
</li>
	<li>收集阿米巴孢子, 以准备无菌生长的阿米巴通过扫一个不育的1毫升吸管尖端平行的板表面。收集10厘米培养皿的一半孢子。 注意: 避免接触到琼脂盘表面的尖端, 因为这项运动将收集大量大肠杆菌。</li>
	<li>并用重悬将尖端浸入1毫升的 PS 培养基中, 轻轻吹打上下, 将阿米巴放在吸管尖端。</li>
	<li>将接种后的混合物转化为含有25毫升 PS 培养基的250毫升烧瓶, 辅以0.25x 浓度的抗生素防霉溶液。 注: 将抗生素防霉溶液贮存为250µL 整除数-20 摄氏度。避免重复循环的解冻和冻结这个解决方案, 因为这可能会降低其功效。</li>
	<li>生长阿米巴 axenically 在22°c 在旋转振动筛在100转每分钟。对于步骤5中的毒力检测, 将细胞生长成光学密度, 测量 600 nm (OD600) 0.2 到0.5。 注: 这一步骤需要 3-4 天的增长从接种时间 (步骤 2.7)。如果细胞生长到更高的密度, 将培养基稀释到600的0.05 或更低, 然后长到600到0.2 到0.5 的 od。</li>
</ol>3. 铜绿假单胞菌的生长
<ol><li>从 LB 盘中挑选一只单胞菌, 将其接种成2毫升 PS: DB 培养, 并在37摄氏度一夜之间生长到饱和度。 注意: 不要将抗生素防霉溶液添加到 PS: DB 介质中。</li>
	<li>稀释隔夜文化1:100 或 1:1, 000 成6厘米直径培养皿使用 PS: DB。如果浮游细胞的毒力仅被测定, 那么就用传统的试管来培养它们。摇动的文化在一个台式旋转设置在 100 rpm 在37摄氏度。</li>
	<li>收获文化在特定的时间点, 以确保重现性。在评估毒力前30分钟到1小时, 准备一个琼脂垫, 如第4节所述。 注意: 铜绿假单胞菌的毒力是由表面大约8小时的生长引起的。</li>
	<li>要隔离表面附着的细胞, 从培养皿中取出液体培养基, 立即用1毫升的 DB 缓冲器清洗, 以去除浮游细胞, 然后立即进入步骤5.1。 注意: 不要洗涤长于三十年代, 因为这将扰乱和分离表面附着细胞, 可能会影响毒力测量。</li>
	<li>要隔离浮游细胞, 将10µL 的培养物从培养皿转移到干净的培养皿中, 然后立即进行步骤5.1。</li>
</ol>4. 显微术. 琼脂垫的制备
<ol><li>在阿米巴准备与铜绿假单胞菌混合之前, 准备琼脂垫约30分钟至1小时。</li>
	<li>微波50毫升 1% (瓦特/v) 琼脂在 DB 缓冲器在250毫升瓶。每二十年代混合, 直到琼脂丛消失。</li>
	<li>将熔融琼脂放在预热的55°c 水浴中冷却15分钟。</li>
	<li>添加15µL 的 calcein 的股票溶液到15毫升的熔融琼脂在锥形管。混合通过轻轻地反转管 4-5 次, 并立即进行下一步。 注: 在聚丙烯15毫升离心管中执行此步骤。不要使用厚玻璃容器, 因为这会使琼脂凝固得太快。</li>
	<li>将熔融琼脂混合物倒入12厘米 x 10 厘米的玻璃板上, 使琼脂在室温下凝固10分钟。通过将玻璃板放在印刷网格上, 并用金属尺沿网格切割, 将凝固琼脂垫切成较小的1.5 厘米 x 1.5 厘米的部分。</li>
	<li>保持凝胶在潮湿的盒子里水合, 直到它可以使用。</li>
</ol>5. 显微术. 细菌-变形虫样的制备. 成像
<ol><li>为了检测表面附着细菌细胞的毒力, 从步骤3.4 中添加10µL 的变形细胞, 从2.10 步到表面附着细菌。 注: 本步骤描述了阿米巴与细菌的混合。</li>
	<li>为了测定浮游细胞的毒力, 从步骤2.10 中将10µL 的变形细胞与10µL 的浮游细菌混合在步骤3.5 中。 注: 在与细菌混合之前, 不要清洗 axenically 生长的变形虫细胞 (在600到0.2 到0.5 的 OD)。由于使用了抗生素防霉溶液, 在变形虫的培养中不会发现大肠杆菌的生长。</li>
	<li>立即把1.5 厘米 x 1.5 厘米 calcein 琼脂垫从步骤4.5 上的细菌-变形虫混合物上的培养皿表面上。 注: 使用快速平滑运动将琼脂垫放在混合物的顶部。不要缓慢地降低垫在混合物, 因为这项运动倾向于推动细胞向外围和远离垫的底部。这一步骤将确保细菌和阿米巴均匀混合, 固定化, 并且两种细胞类型被限制在同一平面上。</li>
	<li>通过轻轻吹打在琼脂垫周围残留的液体, 去除多余的液体。允许培养皿在室温下干燥20分钟。</li>
	<li>盖上带盖子的培养皿, 在室温下孵化固定化的变形虫-细菌混合物, 再加40分钟. 继续步骤6.1。 注意: 在相同的时间内孵化所有样本是很重要的, 因为背景荧光随着时间的推移而增加。推荐1小时的孵化时间。孵化超过1小时的重复性较差, 因为阿米巴细胞可能开始爆裂。</li>
</ol>6. 显微图像采集
<ol><li>图像阿米巴使用荧光显微镜, 能够获得 brightfield 和绿色荧光图像。对于绿色荧光, 分别使用 474/27 nm 和 525/45 nm 的绿色荧光蛋白 (GFP) 过滤器进行激发和发射。使用 10X (≥0.3 数字光圈) 的目标来图像的阿米巴。 注: calcein 在琼脂中会导致阿米巴在绿色荧光通道中的荧光。健康的阿米巴否则不是荧光的。</li>
	<li>调整相衬的采集时间, GFP 通道限制毒性, 优化图像强度的动态范围。如有必要, 替换差动干涉对比 (DIC) 相衬。 注: 如果可能, 使用 100-200 ms 曝光的相位对比和 GFP 荧光。在整个实验中保持曝光率和仪器设置不变。这对于计算主机杀死索引非常重要。</li>
	<li>获取至少100个变形虫细胞的图像, 以获得计算宿主死亡指数的良好统计数据。</li>
</ol>7. 图像分析和主机杀伤指数
<ol><li>使用 ImageJ 进行图像分析。</li>
	<li>在 ImageJ 中打开相位对比度图像文件, 点击文件 |打开并选择图像。点击图像调整阈值|分析 |门槛。调整下下限和上阈值, 只选择强度峰值 (图 3A)。</li>
	<li>单击 "处理" | "转换为二进制图像" 二进制 |做二进制。通过选择过程填充孔|二进制 |填充孔。 注意: 使用图 1中的图像作为源, 步骤 7.1-7.2 将产生一个图像, 其中阿米巴和表面附着的细菌显示为深色区域 (图 3B)。</li>
	<li>确保在分析中检查区域和平均值的灰色值框|设置测量。通过在主工具栏上选择椭圆工具来测量阿米巴的近似大小。点击分析 |测量和注意代表阿米巴的面积。</li>
	<li>通过单击 "分析" 来创建阿米巴的掩码|分析粒子。在 "大小" 框中, 输入将包括阿米巴但排除细菌的区域。在 "放映" 选项的下拉菜单栏中选择 "掩码"。确保选中 "添加到管理器" 框。</li>
	<li>单击"确定" (OK ), 将显示仅显示阿米巴和 ROI 管理器对话框的图像 (图 3C)。</li>
	<li>使用文件打开荧光图像|打开并选择适当的文件。通过按住 Shift 键并单击列表中的每个区域, 选择ROI 管理器中的所有区域 (图 3D)。</li>
	<li>单击ROI 管理器中的 "度量" 按钮。这将打开一个结果窗口, 显示每个变形虫的平均强度值。</li>
	<li>将每个变形虫的荧光值复制到电子表格程序中, 并通过取所有荧光值的平均值来计算主机杀死指数。</li>
</ol>

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作者没有什么可透露的。

本协议描述一种快速、定量的方法来测定铜绿假单胞菌的毒力。此协议可与其他细菌进行测试。然而, 重要的是要记住, 生长培养基应与变形虫生长条件兼容。特别是, 我们已经优化的协议使用 PS: DB 作为细菌生长培养基。如果使用其他介质, 则可能需要执行仅生长介质控制, 在该控件中没有细菌细胞来验证介质是否与阿米巴兼容。
这一方法可以扩展, 以测定毒力的生长阶段依赖性。特别是, 细菌培养可以在很大范围内生长, 而不是固定的时间点。根据我们的经验, 在特定时间点收获细菌培养很重要, 因为铜绿假单胞菌的毒力似乎依赖于生长阶段。我们观察到, 在培养皿中, 铜绿假单胞菌的毒力在 6-8 小时之间被诱导。
与生长环境相关的许多变量影响宿主健康和细菌毒性。因此, 重要的是要使用适当的积极和消极的控制, 每个实验。我们用野生型表面附着的铜绿假单胞菌作为阳性对照, ΔlasR为阴性对照。我们建议每次执行毒力试验时都要执行这些控制。这些控制对于验证宿主细胞死亡不是由于任何外部因素, 如阿米巴的年龄、温度的变化等, 都是很重要的.此外, 我们建议执行所有生物复制实验, 以建立毒力表型的重现性。
在稀释至少1:10 后, 我们用阿米巴在0.2 到0.5 的光密度下进行实验, 并将阿米巴的温度精确地调节到22摄氏度。在这些生长条件之外, 我们发现阿米巴对剧毒细菌的易感性和毒力测定的重现性发生了变化。变形虫细胞的颗粒相对较快。确保在进行光学密度测量之前, 吹打上下悬浮的区域性。不要将变形虫的培养量提高到600密度的 1, 因为高密度的生长会影响实验的重现性。传播无菌文化不超过1周。此外, 重要的是要验证阿米巴是增长 axenically (从步骤2.7 开始) 通过20X 或高放大显微镜, 使其他微生物不存在的文化。如果在最初接种后的2天内, 在步骤2.10 中的文化混浊, 这可能表明存在微生物污染。如果怀疑细菌污染, 我们建议1毫升的阿米巴培养37摄氏度, 4-8 小时。在这种情况下, 观察混浊的文化会提示细菌污染。如果观察到重复的细菌污染, 应更换抗生素防霉溶液。
宿主杀死指数是一个可靠的指标, 说明细菌的数量是致命的还是无毒的。对于不同的中毒性 (即低毒力与中低毒力) 的比较和对结果的过度解释, 该方法尚未得到优化。解决这个问题的潜在方法是在多天内重复使用不同批次的宿主细胞进行毒力检测, 以建立对结果的信心, 执行额外的复制实验, 并执行适当的统计分析。
通过对细菌培养的光密度进行规范化, 可以控制浮游细胞的感染多样性。然而, 这种检测并不能控制表面附着细菌细胞的语言。我们观察到细菌的表面密度随着时间的推移而增加。因此, 调整培养皿中的生长时间可能会导致表面密度的相应变化。另外, 表面密度取决于表面的材料。在这里所描述的化验中,铜绿假单胞细胞附着在聚苯乙烯表面。然而, 其他表面, 包括玻璃, 琼脂, 和聚丙烯酰胺也可以测定13。单层细菌种群的表面密度可以用100X 放大相显微镜测量。如果表面密度是多层次的, 共焦显微镜可能是适当的。
在这里, 我们描述了一种快速和稳健的方法来测量细菌的毒力。通过修改个别参数, 如潜伏期, 荧光染料, 细菌菌株, 宿主细胞类型, 或生长培养基, 这种方法可以扩大, 以量化的毒力, 在广泛的有机体和生长条件。

细菌感染是人类和动物死亡的主要原因之一,1,2。检测细菌在培养或生物膜中的毒力的能力在医疗保健和研究环境中很重要。在这里, 我们描述了一种通用的, 快速的, 相对简单的方法来量化细菌的毒性。以真核生物盘基柄柄(变形虫) 为模型宿主有机体。柄已被用作鉴定铜绿假单胞菌致病因子的宿主 (绿脓杆菌)3,4,5和其他细菌6,7 ,8 , 并且很容易受到同样的毒力因素的影响, 杀死哺乳动物细胞, 包括 III. 型分泌物9,10。以前使用柄的毒力测定, 在3、4、5小时的过程中, 会使细菌与d. 柄细胞长期接触。在这里, 该协议提出了一种快速的方法来确定毒力使用这种变形虫。本协议 (图 1) 描述了如何: (1) 生长阿米巴 axenically (在没有细菌的情况下), (2) 为化验培养细菌, (3) 为显微镜准备细菌和宿主细胞, (4) 执行荧光显微术, (5) 分析变形虫荧光。
阿米巴最初是从冰冻的储存和生长在大肠杆菌 (大肠杆菌) 的草坪上, 阿米巴产生孢子。这些孢子被采摘并接种成富含培养基无菌生长。阿米巴是通过无菌生长营养丰富的条件来维持的, 直到它们准备与细菌混合以评估细菌的毒力。阿米巴的生存或死亡通过测量 calcein-乙酰甲基 (calcein AM) 的荧光量量化, 这是由细胞内酯, 从而激活荧光11,12。活阿米巴表现很少或没有荧光, 而压力和垂死的细胞荧光强烈。这一结果是由于很少或没有纳入 calcein 的健康阿米巴和成立和分裂的基质在强调阿米巴13。这种行为明显有别于哺乳动物细胞11、14、15、16的 calcein 荧光。
将被评估为毒力的细菌分别生长。在这里, 我们描述如何测量机会病原菌的毒力, 并详细说明如何量化的毒性的浮游 (游泳) 和表面附着的亚人口。本议定书可用于测试其他细菌的毒力。在有代表性的结果部分, 我们表明, 毒力是激活的表面附着细胞和低的浮游细胞, 这是以前报告13。毒力活化的表面附着的铜绿假单胞菌杀死阿米巴, 而非毒性浮游细胞消耗的阿米巴。如果浮游细菌的毒力仅被测定, 细菌可以在普通的试管中培养, 而不是按照议定书中所描述的那样使用培养皿。
阿米巴和铜绿假单胞菌培养的生长必须协调, 以使铜绿假单胞菌培养达到预期的生长阶段, 而阿米巴在营养丰富的条件下生长在稳定的状态。这种情况通常要求阿米巴文化在与细菌混合之前至少1天被稀释。阿米巴和细菌是固定使用琼脂垫, 共孵化1小时, 并成像使用低分辨率 (10X, 数字光圈 0.3) 目标, 绿色荧光蛋白 (GFP) 过滤器, 和成像相机。可以使用自由可用的 ImageJ 软件或自定义图像分析软件进行分析。我们的分析是使用我们自己的软件编写的, 使用一个科学的分析包13。该软件应创建一个面具使用相对比图像和提取荧光值从蒙面区域的荧光图像。荧光值平均在至少100个细胞, 导致一个数字主机杀死指数。

<table><tbody><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Bacto agar, dehydrated</td><td>BD Difco</td><td>214010</td><td></td></tr><tr><td>Antibiotic-Antimycotic (100X)</td><td>Life Technologies</td><td>15240062</td><td>Aliquot &amp;lt; 1 mL and store at -20 &amp;deg;C</td></tr><tr><td>Calcein-acetoxymethyl ester (calcein-AM)</td><td>Life Technologies</td><td>C34852</td><td>Calcein Acetoxymethyl (AM)</td></tr><tr><td>Calcium chloride, anhydrous</td><td>Sigma-Aldrich</td><td>C1016</td><td></td></tr><tr><td>D-Glucose</td><td>Fisher Chemical</td><td>D16500</td><td>Dextrose</td></tr><tr><td>Dimethyl sulfoxide</td><td>Sigma-Aldrich</td><td>D5879</td><td></td></tr><tr><td>Folic acid</td><td>Sigma-Aldrich</td><td>F8758</td><td></td></tr><tr><td>LB-Miller</td><td>BD Difco</td><td>244620</td><td></td></tr><tr><td>Magnesium chloride</td><td>Sigma-Aldrich</td><td>M8266</td><td></td></tr><tr><td>Yeast extract</td><td>Oxoid</td><td>LP0021</td><td></td></tr><tr><td>Special peptone</td><td>Oxoid</td><td>LP0072</td><td></td></tr><tr><td>Potassium hyroxide</td><td>Fisher Chemical</td><td>P250</td><td></td></tr><tr><td>Potassium phosphate monobasic&amp;nbsp;</td><td>Sigma-Aldrich</td><td>P0662</td><td></td></tr><tr><td>Sodium phosphate dibasic heptahydrate</td><td>Fisher Chemical</td><td>S373</td><td></td></tr><tr><td>Vitamin B12</td><td>Sigma-Aldrich</td><td>V2876</td><td></td></tr><tr><td>&lt;strong&gt;Strains&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Dictyostelium discoideum</td><td>Siryaporn lab</td><td>Strain AX3&lt;sup&gt;18&lt;/sup&gt;</td><td></td></tr><tr><td>Escherichia coli</td><td>Siryaporn lab</td><td>Strain B/r&lt;sup&gt;17&lt;/sup&gt;</td><td></td></tr><tr><td>Pseudomonas aeruginosa</td><td>Siryaporn lab</td><td>PA14</td><td>PA14 strain&lt;sup&gt;19&lt;/sup&gt;</td></tr><tr><td>Pseudomonas aeruginosa &amp;Delta;lasR</td><td>Siryaporn lab</td><td>AFS20.1</td><td>PA14-derived strain&lt;sup&gt;20&lt;/sup&gt;</td></tr><tr><td>&lt;strong&gt;Supplies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>0.22 &amp;micro;m filter&amp;nbsp;</td><td>Millipore</td><td>SCGPT01RE</td><td>For filter sterilization</td></tr><tr><td>Conical tube, 15 mL</td><td>Corning</td><td>352097</td><td></td></tr><tr><td>Glass storage bottles</td><td>Pyrex</td><td>13951L</td><td>250 mL, 500 mL, 1000 mL</td></tr><tr><td>Petri dish, 6 cm diameter</td><td>Corning</td><td>351007</td><td>60 x 15 mm polystyrene plates</td></tr><tr><td>Petri dish, 10 cm diameter</td><td>Fisher</td><td>FB0875712</td><td>100 x 15 mm polystyrene plates</td></tr><tr><td>Plastic containers with lid</td><td>Ziploc</td><td>2.57E+09</td><td>Square 3-cup containers</td></tr><tr><td>Glass plates</td><td>Bio-Rad</td><td>1653308</td><td>For preparing agar pads. Other glass plates may be used with similar dimensions.</td></tr><tr><td>Wooden sticks</td><td>Fisher</td><td>23-400-102&amp;nbsp;</td><td></td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Eclipse Ti-E microscope&amp;nbsp;</td><td>Nikon</td><td>MEA53100</td><td>Microscope setup</td></tr><tr><td>10X Plan Fluor Ph1 objective&amp;nbsp; 0.3 NA</td><td>Nikon</td><td>MRH20101</td><td>Microscope setup</td></tr><tr><td>Fluorescence excitation source</td><td>Lumencor</td><td>Sola light engine</td><td>Microscope setup</td></tr><tr><td>Fluorescence filter set</td><td>Semrock</td><td>LED-DA/FI/TX-3X3M-A-000&amp;nbsp;</td><td>Microscope setup</td></tr><tr><td>Orca Flash 4.0 V2 Camera</td><td>Hamamatsu</td><td>77054098</td><td>Microscope setup</td></tr><tr><td>ImageJ</td><td>NIH</td><td>v. 1.49</td><td>Software for image analysis</td></tr><tr><td>MATLAB</td><td>Mathworks</td><td>R2013</td><td>Software for image analysis</td></tr><tr><td>Orbital shaker incubator</td><td>VWR</td><td>89032-092</td><td>For growth of bacteria at 37 &amp;deg;C</td></tr><tr><td>Platform shaker</td><td>Fisher</td><td>13-687-700</td><td>For growth of amoebae at 22 &amp;deg;C</td></tr><tr><td>Spectrophotometer</td><td>Biochrom</td><td>Ultrospec 10</td><td></td></tr><tr><td>Undercounter refrigerated incubator</td><td>Fisher</td><td>97990E</td><td>For growth of amoebae at 22 &amp;deg;C</td></tr><tr><td>Isotemp waterbath&amp;nbsp;</td><td>Fisher</td><td>15-462-21Q</td><td>For cooling media to 55 &amp;deg;C</td></tr></tbody></table>

a rapid image-based bacterial virulence assay using amoeba

传统的细菌毒力检测包括在几个小时内长期暴露细菌, 以宿主细胞。在这段时间内, 由于接触宿主生长环境和宿主细胞的存在, 细菌可以经受生理变化。我们开发了一种快速测量细菌的毒力状态的检测方法, 以最大限度地减少细菌在宿主细胞存在时的生长程度。细菌和阿米巴混合在一起, 并固定在一个单一的成像平面上使用琼脂垫。该程序使用单细胞荧光成像与 calcein-乙酰甲基酯 (calcein) 作为一个指标的宿主细胞健康。利用荧光显微术对宿主细胞的1小时暴露后的宿主细胞进行荧光分析。图像分析软件用于计算主机的杀戮指数。该方法用于测定生物膜形成初期浮游和表面附着铜绿假单胞菌亚群中的毒力, 并可适应其他细菌和生物膜生长的其他阶段。该协议提供了一种快速和稳健的方法来测量毒力, 并避免了许多与生长和维护哺乳动物细胞系相关的复杂性。使用阿米巴测定的毒力表型也得到了使用小鼠巨噬细胞的验证。特别是, 这一检测被用来建立的表面附着上调毒力在铜绿假单胞菌。

我们在6厘米直径的培养皿中生长野生型铜绿假单胞菌 PA1419或ΔlasR菌株20 , 并测定浮游和表面附着细胞的毒力。文化从单一殖民地接种了入 PS: DB 文化, 在养殖管子隔夜生长在37°c 到饱和, 稀释1:100 入 ps: DB, 增长 8 h 在 6 cm 直径培养皿颤抖在100转, 并且浮游物和表面附上铜绿假单胞菌亚群被隔离, 如3节所述。
表面附着野生型铜绿假单胞杆菌杀死阿米巴, 这是由一个圆形的变形细胞形状和观察 calcein 荧光 (图 4a, 左上板)。这导致了一个高主机杀死索引 (图 4B)。浮游细胞被阿米巴消耗, 这是由无定形的变形细胞形状和观察很少或没有 calcein 荧光在背景之上 (图 4A, 右上板)。这导致了一个相对较低的主机杀戮指数 (图 4B)。ΔlasR菌株的表面附着和浮游细胞都被阿米巴所消耗, 这是由低宿主杀死指数 (图 4B, 底部面板) 所指示的。结果表明, 在表面附着的种群中, 毒力是上调的, LasR 是表面活化的毒力所必需的, 前文描述了13。这些实验为未来的实验建立了强有力的正负控制。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57844/57844fig1.jpg"> 图 1: 描述毒力测定概述的示意图.(1) 变形虫宿主细胞生长, (2) 细菌培养, (3) 浮游或表面附着的细菌细胞与阿米巴混合, 并使用琼脂垫固定在同一成像平面上。宿主细胞使用 calcein、荧光显微镜和图像分析来量化健康。刻度条代表50µm.<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/57844/57844fig1large.jpg" target="_blank">请点击这里查看这个数字的大版本</a>。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57844/57844fig2.jpg"> 图 2: 变形虫孢子在一个别扭10厘米直径的培养皿板经过5天的生长.孢子形成于培养皿表面之上。该嵌入图显示了盘面的单个截面的放大视图。表面可能含有细菌, 这项决议不能看出。主要和嵌入图像中的刻度条分别代表1厘米和2毫米。<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/57844/57844fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57844/57844fig3.jpg"> 图 3: 用 ImageJ 对显微数据进行图像分析。(A)使用阈值工具选择峰值强度。(B) 图 1中的相位对比度源图像转换为二进制图像后产生的图像。(C)图像随后转换为掩码后产生的图像。(D)使用 ROI 管理器选择感兴趣区域的截图。刻度条代表50µm.<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/57844/57844fig3large.jpg" target="_blank">请点击这里查看这个数字的大版本</a>。
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57844/57844fig4.jpg"> 图 4: 用铜绿假单胞细胞杀死变形虫的典型结果。(A) Calcein 荧光覆盖在相显微图像上, (B)对表面附着或浮游野生型或ΔlasR 铜绿假单胞杆菌的寄主杀伤指标。野生型表面附着的铜绿假单胞菌杀死阿米巴, 这表现出显著的 calcein 荧光, 因此是一个重要的宿主杀死指数。浮游细胞被阿米巴所消耗, 它表现出很少的 calcein 荧光, 并产生低宿主杀死指数。表面附着和浮游ΔlasR都是由阿米巴消耗的, 导致了低宿主杀死指数。刻度条代表50µm. 条形指示三独立实验的平均值和误差条表示标准偏差。<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/57844/57844fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about A Rapid Image-based Bacterial Virulence Assay Using Amoeba at JoVE.com

A Rapid Image-based Bacterial Virulence Assay Using Amoeba

一种基于变形虫的快速图像细菌毒力检测方法

在这里, 我们提出了一个协议, 以测量浮游或表面附着细菌的毒力, 使用柄(变形虫) 作为宿主。在1小时内对毒力进行测量, 并使用荧光显微镜和图像分析对宿主杀死进行量化。我们用铜绿假单胞菌证明了这个协议。

Traditional bacterial virulence assays involve prolonged exposure of bacteria over the course of several hours to host cells. During this time, bacteria ...

a-rapid-image-based-bacterial-virulence-assay-using-amoeba

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

8.1K 次观看.  University of California. 在这里, 我们提出了一个协议, 以测量浮游或表面附着细菌的毒力, 使用柄(变形虫) 作为宿主。在1小时内对毒力进行测量, 并使用荧光显微镜和图像分析对宿主杀死进行量化。我们用铜绿假单胞菌证明了这个协议。

视频: A Rapid Image-based Bacterial Virulence Assay Using Amoeba

A Visual Assay to Monitor T6SS-mediated Bacterial Competition

Type VI secretion systems (T6SSs) are molecular nanomachines allowing Gram-negative bacteria to transport and inject proteins into a wide variety of target cells1,2. The T6SS is composed of 13 core components and displays structural similarities with the tail-tube of bacteriophages3. The phage uses a tube and a puncturing device to penetrate the cell envelope of target bacteria and inject DNA. It is proposed that the T6SS is an inverted bacteriophage device creating a specific path in the bacterial cell envelope to drive effectors and toxins to the surface. The process could be taken further and the T6SS device could perforate other cells with which the bacterium is in contact, thus injecting the effectors into these targets. The tail tube and puncturing device parts of the T6SS are made with Hcp and VgrG proteins, respectively4,5.
The versatility of the T6SS has been demonstrated through studies using various bacterial pathogens. The Vibrio cholerae T6SS can remodel the cytoskeleton of eukaryotic host cells by injecting an "evolved" VgrG carrying a C-terminal actin cross-linking domain6,7. Another striking example was recently documented using Pseudomonas aeruginosa which is able to target and kill bacteria in a T6SS-dependent manner, therefore promoting the establishment of bacteria in specific microbial niches and competitive environment8,9,10.
In the latter case, three T6SS-secreted proteins, namely Tse1, Tse2 and Tse3 have been identified as the toxins injected in the target bacteria (Figure 1). The donor cell is protected from the deleterious effect of these effectors via an anti-toxin mechanism, mediated by the Tsi1, Tsi2 and Tsi3 immunity proteins8,9,10. This antimicrobial activity can be monitored when T6SS-proficient bacteria are co-cultivated on solid surfaces in competition with other bacterial species or with T6SS-inactive bacteria of the same species8,11,12,13.
The data available emphasized a numerical approach to the bacterial competition assay, including time-consuming CFU counting that depends greatly on antibiotic makers. In the case of antibiotic resistant strains like P. aeruginosa, these methods can be inappropriate. Moreover, with the identification of about 200 different T6SS loci in more than 100 bacterial genomes14, a convenient screening tool is highly desirable. We developed an assay that is easy to use and requires standard laboratory material and reagents. The method offers a rapid and qualitative technique to monitor the T6SS-dependent bactericidal/bacteriostasis activity by using a reporter strain as a prey (in this case Escherichia coli DH5&alpha;) allowing a-complementation of the lacZ gene. Overall, this method is graphic and allows rapid identification of T6SS-related phenotypes on agar plates. This experimental protocol may be adapted to other strains or bacterial species taking into account specific conditions such as growth media, temperature or time of contact.

We describe a qualitative assay to monitor bacterial competition mediated by the Pseudomonas aeruginosa type VI secretion system (T6SS). The assay relies on the survival/killing of Escherichia coli target cells carrying a lacZ-reporter. This technique is adjustable to assess the bactericidal/bacteriostasis activity of T6SS-proficient microorganisms.

的视觉检测，以监测T6SS介导的细菌竞争

Type VI secretion systems (T6SSs) are molecular  nanomachines allowing Gram-negative bacteria to transport and inject proteins  into a wide variety of ...

科研

免疫与感染

Single Cell Measurements of Vacuolar Rupture Caused by Intracellular Pathogens

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed compartment allows bacteria to move within the cytosol, proliferate and further invade neighboring cells. Mycobacterium tuberculosis is phagocytosed by immune cells, and has recently been shown to rupture phagosomal membrane in macrophages. We developed a robust assay for tracking phagosomal membrane disruption after host cell entry of Shigella flexneri or Mycobacterium tuberculosis. The approach makes use of CCF4, a FRET reporter sensitive to &beta;-lactamase that equilibrates in the cytosol of host cells. Upon invasion of host cells by bacterial pathogens, the probe remains intact as long as the bacteria reside in membrane-enclosed compartments. After disruption of the vacuole, &beta;-lactamase activity on the surface of the intracellular pathogen cleaves CCF4 instantly leading to a loss of FRET signal and switching its emission spectrum. This robust ratiometric assay yields accurate information about the timing of vacuolar rupture induced by the invading bacteria, and it can be coupled to automated microscopy and image processing by specialized algorithms for the detection of the emission signals of the FRET donor and acceptor. Further, it allows investigating the dynamics of vacuolar disruption elicited by intracellular bacteria in real time in single cells. Finally, it is perfectly suited for high-throughput analysis with a spatio-temporal resolution exceeding previous methods. Here, we provide the experimental details of exemplary protocols for the CCF4 vacuolar rupture assay on HeLa cells and THP-1 macrophages for time-lapse experiments or end points experiments using Shigella flexneri as well as multiple mycobacterial strains such as Mycobacterium marinum, Mycobacterium bovis, and Mycobacterium tuberculosis.

We describe a method for tracking the endomembrane rupture elicited by the intracellular bacteria Shigella flexneri and Mycobacterium tuberculosis upon host cell invasion. Our assay makes use of CCF4, a host cytoplasmic FRET probe in live or fixed cells. This reporter is degraded by an enzyme activity present on the bacterial surface.

单细胞测量液泡破裂引起细胞内病原体

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed ...

Tractable Mammalian Cell Infections with Protozoan-primed Bacteria

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

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

听话的哺乳动物细胞原生动物引发的细菌感染

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

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 ...

Methods for Detecting Cytotoxic Amyloids Following Infection of Pulmonary Endothelial Cells by Pseudomonas aeruginosa

Patients who survive pneumonia have elevated death rates in the months following hospital discharge. It has been hypothesized that infection of pulmonary tissue during pneumonia results in the production of long-lived cytotoxins that can lead to subsequent end organ failure. We have developed in vitro assays to test the hypothesis that cytotoxins are produced during pulmonary infection. Isolated rat pulmonary endothelial cells and the bacterium Pseudomonas aeruginosa are used as model systems, and the production of cytoxins following infection of the endothelial cells by the bacteria is demonstrated using cell culture followed by direct quantitation using lactate dehydrogenase assays and a novel microscopic method utilizing ImageJ technology. The amyloid nature of these cytotoxins was demonstrated by thioflavin T binding assays and by immunoblotting and immunodepletion using A11 anti-amyloid antibody. Further analyses using immunoblotting demonstrated that oligomeric tau and A&#946; were produced and released by endothelial cells following infection by P. aeruginosa. These methods should be readily adaptable to analyses of human clinical samples.

Methods for Detecting Cytotoxic Amyloids Following Infection of Pulmonary Endothelial Cells by Pseudomonas aeruginosa

Simple methods are described for demonstrating the production of cytotoxic amyloids following infection of pulmonary endothelium by Pseudomonas aeruginosa.

铜绿假单胞菌感染肺内皮细胞后细胞毒性 Amyloids 的检测方法

Patients who survive pneumonia have elevated death rates in the months following hospital discharge. It has been hypothesized that infection of pulmonary ...

Identification of Virulence Markers of Mycobacterium abscessus for Intracellular Replication in Phagocytes

What differentiates Mycobacterium abscessus from other saprophytic mycobacteria is the ability to resist phagocytosis by human macrophages and the ability to multiply inside such cells. These virulence traits render M. abscessus pathogenic, especially in vulnerable hosts with underlying structural lung disease, such as cystic fibrosis, bronchiectasis or tuberculosis. How patients become infected with M. abscessus remains unclear. Unlike many mycobacteria, M. abscessus is not found in the environment but might reside inside amoebae, environmental phagocytes that represent a potential reservoir for M. abscessus. Indeed, M. abscessus is resistant to amoebal phagocytosis and the intra-amoeba life seems to increase M. abscessus virulence in an experimental model of infection. However, little is known about M. abscessus virulence in itself. To decipher the genes conferring an advantage to M. abscessus intracellular life, a screening of a M. abscessus transposon mutant library was developed. In parallel, a method of RNA extraction from intracellular Mycobacteria after co-culture with amoebae was developed. This method was validated and allowed the sequencing of whole M. abscessus transcriptomes inside the cells; providing, for the first time, a global view on M. abscessus adaptation to intracellular life. Both approaches give us an insight into M. abscessus virulence factors that enable M. abscessus to colonize the airways in humans.

Identification of Virulence Markers of Mycobacterium abscessus for Intracellular Replication in Phagocytes

Here, we present two protocols to study Phagocyte-Mycobacterium abscessus interactions: the screening of a transposon mutant library for bacterial intracellular deficiency and the determination of bacterial intracellular transcriptome from RNA sequencing. Both approaches provide insight into the genomic advantages and transcriptomic adaptations enhancing intracellular bacteria fitness.

吞噬细胞内复制脓肿分枝杆菌毒力标志物的鉴定

What differentiates Mycobacterium abscessus from other saprophytic mycobacteria is the ability to resist phagocytosis by human macrophages and the ability ...

A High-throughput, High-content, Liquid-based C. elegans Pathosystem

The number of new drugs identified by traditional, in vitro screens has waned, reducing the success of this approach in the search for new weapons to combat multiple drug resistance. This has led to the conclusion that researchers do not only need to find new drugs, but also need to develop new ways of finding them. Amongst the most promising candidate methods are whole-organism, in vivo assays that use high-throughput, phenotypic readouts and hosts that range from Caenorhabditis elegans to Danio rerio. These hosts have several powerful advantages, including dramatic reductions in false positive hits, as compounds that are toxic to the host and/or biounavailable are typically dropped in the initial screen, prior to costly follow up.
Here we show how our assay has been used to interrogate host variation in the well-documented C. elegans&#8212;Pseudomonas aeruginosa liquid killing pathosystem. We also demonstrate several extensions of this well-worked out technique. For example, we are able to carry out high-throughput genetic screens using RNAi in 24- or 96-well plate formats to query host factors in this host-pathogen interaction. Using this assay, whole genome screens can be completed in only a few months, which can dramatically simplify the task of identifying drug targets, potentially without the need for laborious biochemical purification approaches.
We also report here a variation of our method that substitutes the gram-positive bacterium Enterococcus faecalis for the gram-negative pathogen P. aeruginosa. Much as is the case for P. aeruginosa, killing by E. faecalis is time-dependent. Unlike previous C. elegans&#8212;E. faecalis assays, our assay for E. faecalis does not require preinfection, improving its safety profile and reducing the chances of contaminating liquid-handling equipment. The assay is highly robust, showing ~95% death rates 96 h post infection.

A High-throughput, High-content, Liquid-based C. elegans Pathosystem

Here we describe a protocol that is an adaptable, whole host, high-content screening tool that can be utilized to study host-pathogen interactions and be used for drug discovery.

一种高通量、高含量、液态的 Pathosystem线虫

The number of new drugs identified by traditional, in vitro screens has waned, reducing the success of this approach in the search for new weapons to ...

<meta charset="utf8"></head><body>棘 阿米巴 物种运动的长期跟踪和定量

Quantification of Acanthamoeba spp. Motility

Acanthamoeba keratitis is a severe ocular infection that poses treatment challenges and can lead to blindness. Despite its ubiquity and potential contamination of contact lenses after water exposure, the natural behavior of this pathogen remains elusive. Understanding Acanthamoeba's movement patterns can inform us about how it colonizes contact lenses and contaminates the patient's cornea. It is fortunate that Acanthamoeba spp. are visible via brightfield microscopy starting at 4x magnification. Previous techniques have been developed to quantify Acanthamoeba motility in regard to cytopathic effects or under-exposure to the electric field. Here, we describe a method to track and quantify Acanthamoeba spp. motility long-term (hours to days), which is a protocol applicable to multiple amoeba strains, surfaces, and nutritional status of amoeba. This procedure is germane to determining many core motility quantifications, such as distance, speed, confinement, and directionality, which are necessary to monitor different stages of infection, proliferation, or behavioral change.

<meta charset="utf8"></head><body>作者聚焦：研究 棘阿米巴 的行为以制定预防 棘阿米巴 角膜炎的针对性策略

This procedure describes how to visualize, track, and quantify Acanthamoeba spp. motility.

一种基于变形虫的快速图像细菌毒力检测方法

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棘阿米巴属的定量运动性

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棘阿米巴属的定量运动性