Name: applying live cell imaging and cryo-electron tomography to resolve spatiotemporal features of the legionella pneumophila dot/icm secretion system
Uploaded: 2020-03-10T13:30:00
Description: Watch this Scientific Journal Video about Applying Live Cell Imaging and Cryo-Electron Tomography to Resolve Spatiotemporal Features of the Legionella pneumophila Dot/Icm Secretion System at JoVE.com

华盛顿特区和C.R.R.得到NIH的支持（R37AI041699和R21AI130671）。D.P.、B.H.和J.L得到国家卫生研究院（R01AI087946和R01GM107629）的支持。

注：所有涉及肺尘埃球菌生长、操作和成像的程序都应按照当地准则在生物安全2级实验室进行。
1. 使用全位交换和双选择策略将sfGFP插入L.肺炎嗜血杆菌染色体（图2，图3）
<ol><li>克隆成基因置换载体pSR47S11以下序列：1000bp上游感兴趣的站点，然后是sfGFP序列，然后是1000bp下游感兴趣的站点（<strong cl...

<ol>
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阐明细菌分泌系统的功能是全面了解宿主-病原体相互作用的关键。分泌系统是复杂的机器，可以将效应剂蛋白质注入宿主细胞，在某些情况下，它促进建立支持细菌复制的亚细胞利基。上述方法为研究呼吸系统病原体军团菌肺杆菌的Dot/Icm分泌系统提供了重要的新工具，为有效器易位机制提供了线索，对其致病性至关重要。这种方法可以通过利用活细菌细胞成像和结构研究来解剖其分子水?...

军团菌肺杆菌（L.肺炎杆菌），军团病的病因，栖息在淡水水库，细菌通过感染和复制水生自由游泳原生动物传播。L. 肺尘埃球菌在吸入饮用水源中的气溶胶细菌时，导致人类疾病爆发。在受感染的细胞中，宿主通路的颠覆使L.肺炎杆菌能够延缓其所在的真空细胞的内皮成熟，并促进支持细菌复制的细胞舱的生物生成。这个过程是由一个专门的细菌类型IVB分泌系统（T4BSS）称为Dot/Icm及其的300多个"效应器"蛋白质的品种，在感染期间被转移到宿主细胞群素，以促进细胞功能1，2，3，4，5的操纵。缺乏功能点/Icm仪器的突变体不能将效应器输送到宿主细胞索中，在细胞内复制方面存在缺陷，在疾病6、7的动物模型中具有avirule。
许多细菌物种已经开发出了感染过程所需的极其复杂和动态的多组分机。其他T4BSS，如Dot/Icm系统也是必不可少的细胞内复制细菌病原体，如科西拉布鲁内蒂和里基西拉格丽利。虽然T4BSS与原型型IVA系统具有进化关系，后者调解DNA转移，并能提供有限的有效蛋白，但Dot/Icm系统拥有几乎两倍的机器部件，并提供各种各样的效应器。据推测，这种组件数量的扩大使Dot/Icm设备能够轻松容纳和集成新的效应器8，9。
我们最近使用低温电子断层扫描（cryo-ET）就地解决了Dot/Icm仪器的结构，并表明它形成了一个细胞包络跨度通道，连接到细胞质复合体。进一步分析表明，通过与细胞体ATPase DotO的相互作用，圆细胞状ATPase DotB与L.肺尘埃球细胞极的点/Icm系统有关。我们发现，DotB在大多数细菌细胞中显示一个循环球状运动，这表明这种ATPase存在于动态的细胞群中，但也与极性点/Icm复合物有关联。此外，DotO 形成与内膜复合体相关的 DotO 二分器的 hexamic 组件，DotB hexamer 连接到此细胞质复合物的基座。DotB-DotO 能量复合体的组装创建了一个细胞质通道，通过 T4SS（图 1）10引导基材的易位。
尽管最近取得了这些进展，但关于Dot/Icm系统如何发挥作用以及每种蛋白质如何组装形成一个活性装置8知之甚少。发现Dot/Icm T4SS的调节回路对于理解宿主-病原体相互作用的分子机制至关重要。因此，我们讨论如何使用活细胞显微镜和低温-ET来检测和表征使用超级文件夹GFP（sfGFP）标记的基本L.肺活血杆菌/Icm系统组件。使用定量荧光显微镜，DotB 的极定位将在野生类型背景中定义，或者当 IV 型系统被删除时。延时显微镜将用于量化点/Icm细胞学的定位和动力学差异。
与其他体外系统相比，实时成像和低温ET等两种互补方法的组合应用提供了优势。这两种方法均以完整的细胞执行，并保护T4BSS的自然环境，从而最大限度地减少样品制备过程中原生结构的干扰。由于蛋白质的过度表达可能会损害分泌器的分泌器的分泌器，sfGFP融合通过异位交换返回到军团菌染色体，以便每个融合在单拷贝中编码，并且表达由内源促进者驱动。色谱编码聚变的可视化能够量化在规定的时间点表达的蛋白质的确切水平。Cryo-ET 在确定分泌系统结构方面也有许多优点。最显著的优点是低温-ET样品由冷冻完整细胞组成，在细菌细胞结构中保留原生复合物。因此，低温-ET可能比生化纯化方法更可取，后者提取膜复合物，并可能从核心设备中去除外周蛋白或修改整体结构。此外，用笨重的蛋白质（如 sfGFP）标记感兴趣的蛋白质，可添加低温-ET 可检测的质量，并有助于将 Dot/Icm 仪器的不同子复合物映射到低温-ET 获得的结构上。
这种方法是一个强大的工具，用于发现在细菌细胞膜中组装的多分子复合物的结构信息。解释使用这些技术阐明的结构将有助于现场了解 T4BSS 组件的工作原理、为什么函数需要这么多组件、组件如何在更复杂范围内交互以及这些组件的功能子组件执行。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10 nm colloidal gold particles</td>
			<td>Aurion</td>
			<td>25486</td>
			<td></td>
		</tr>
		<tr>
			<td>100x Plan Apo objective (1.4 NA)</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ACES</td>
			<td>Sigma-Aldrich</td>
			<td>A9758</td>
			<td></td>
		</tr>
		<tr>
			<td>Activated charcoal</td>
			<td>Sigma-Aldrich</td>
			<td>C5510</td>
			<td></td>
		</tr>
		<tr>
			<td>Agaroze GPG/LMP, low melt</td>
			<td>American bioanalytical</td>
			<td>AB00981</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto dehydrated agar</td>
			<td>BD</td>
			<td>214010</td>
			<td></td>
		</tr>
		<tr>
			<td>CoolSNAP EZ 20 MHz digital monochrome camera</td>
			<td>Photometrics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gene Frame, 1.7x2.8 cm, 125 &#181;L</td>
			<td>Fisher Scientific</td>
			<td>AB-0578</td>
			<td></td>
		</tr>
		<tr>
			<td>Holey Carbon grid R 2/1 Cu 200 mesh</td>
			<td>Quantifoil</td>
			<td>Q225-CR1</td>
			<td></td>
		</tr>
		<tr>
			<td>Iron(III) nitrate nonahydrate</td>
			<td>Sigma-Aldrich</td>
			<td>216828</td>
			<td></td>
		</tr>
		<tr>
			<td>K2 Summit camera for cryo-EM</td>
			<td>GATAN</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>L-Cysteine</td>
			<td>Sigma-Aldrich</td>
			<td>C7352</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope cover slides 22x22 mm</td>
			<td>Fisher Scientific</td>
			<td>12-542B</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope cover slides 24x50 mm</td>
			<td>Fisher Scientific</td>
			<td>12-545K</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope slides 25x75x1 mm</td>
			<td>Globe Scientific</td>
			<td>1380</td>
			<td></td>
		</tr>
		<tr>
			<td>SlideBook 6.0</td>
			<td>Intelligent Imaging Innovations</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Spectra X light engine</td>
			<td>Lumencor</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Taq 2X Master Mix</td>
			<td>New England BioLabs</td>
			<td>M0270</td>
			<td></td>
		</tr>
		<tr>
			<td>Titan Krios</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast Extract</td>
			<td>BD</td>
			<td>212750</td>
			<td></td>
		</tr>
	</tbody>
</table>

applying live cell imaging and cryo-electron tomography to resolve spatiotemporal features of the legionella pneumophila dot/icm secretion system

军团菌的点/Icm分泌系统是一种复杂的IV型分泌系统（T4SS）纳米机器，在细菌极处进行定位，并调解蛋白质和DNA基质向靶细胞的输送，这一过程通常需要细胞到细胞直接接触。我们最近通过低温电子断层扫描（cryo-ET）解决了Dot/Icm仪器的结构，并表明它形成了一个细胞包络跨度通道，连接到细胞质复合体。应用两种补充方法，保持标本的原生结构，荧光显微镜在活细胞和低温-ET中，允许蛋白质的原位可视化和同化每个机器部件相对于其他点/Icm子单位的生产时间。为了研究极性定位的要求，并描述与T4SS机器生物成因相关的动态特征，我们将编码超级文件夹绿色荧光蛋白的基因融合到Dot/Icm ATPase基因的原生位置。以下方法集成了活细胞和低温-ET的定量荧光显微镜，以量化这些蛋白质在完整细菌细胞中的极性定位、动力学和结构。应用这些方法研究军团菌肺炎T4SS有助于描述Dot/Icm系统的功能，并可用于研究利用T4SS或其他类型的细菌分泌复合物的各种细菌病原体。

在两个步骤中采用双选择进行同源重组，用于构造 sfGFP 的定义插入。在第一步中，进行了三亲交配，其中从大肠杆菌帮助器菌株MT616的pRK600偶联质质质体（IncP质粒）被调动到捐赠者大肠杆菌菌株与自杀载体pSR47S包含sfGFP基因的两个同源区域，转移奥里特的起源和杆菌子选择基因囊肿。接下来，从pRK600的合并系统协助将pSR47S衍生物动员到L.肺炎杆菌（Lp01，<str...

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Applying Live Cell Imaging and Cryo-Electron Tomography to Resolve Spatiotemporal Features of the Legionella pneumophila Dot/Icm Secretion System

细菌细胞成像是一种新兴的系统生物学方法，侧重于定义静态和动态过程，这些过程决定了大型大分子机器的功能。在这里，定量活细胞成像和低温电子断层扫描的集成用于研究军团菌肺瘤IV型分泌系统架构和功能。

应用活细胞成像和低温电子断层扫描解决军团菌肺瘤点/Icm分泌系统的时空特征

The Dot/Icm secretion system of Legionella pneumophila is a complex type IV secretion system (T4SS) nanomachine that localizes at the bacterial pole and ...

该协议可用于描述细菌分泌复合物的组装功能，并可对宿主病原体相互作用所需的分子机制有一个基本的了解。此技术集成了保持样本原生结构的补充方法，并允许和看到完整细菌细胞中蛋白质复合物的可视化。它增加了我们对细菌分泌系统的结构功能关系的理解。这种方法可以适应研究其他类型的分泌系统或其他细胞复合物在各种细菌物种。首先制作带活细胞成像的阿加罗斯垫。在水中准备约 30 mL 的 1% 低熔葡萄糖溶液，并在玻璃瓶中微波约 90 秒，偶尔旋转，直到红糖完全溶解。将两个 22 x 22 x 0.15mm 玻璃幻灯片放在 25 x 75 x 1.1mm 玻璃幻灯片的边缘，另一个放在顶部。然后，将两个较小的幻灯片堆叠到另一边。将约1 mL的熔融气液移入两个上部玻璃滑梯之间的中心滑轨中，并在熔融的气糖顶部放置另一个大滑轨，确保避免形成气泡。在4摄氏度下冷却滑梯15分钟，然后用手术刀或剃刀刀片轻轻地将垫子切成小方块。将双面胶架固定到 25 x 75 x 1.1mm 玻璃幻灯片上，并在幻灯片上放置多个垫片。在1 mL的双蒸馏水中溶解一块重的军团菌嗜血杆菌。然后涡流和移液器2至3微升稀释到垫上。轻轻将 58 x 24 x 0.15 mm 盖板滑过粘合框架。使用亮场照明选择用于成像的单元格。然后，在显微镜软件的捕获窗口中，将 ND 调整为 180，将装箱调整为 2 比 2。使用 488 纳米通道将样品暴露 500 到 1000 毫秒，并使用相同参数成像未标记的军团嗜血军团菌，验证荧光的特异性。要量化极性，请调整图像对比度，以便细菌清晰可见。然后放大感兴趣的区域，并使用区域工具将 0.25 到 1.3 微米的矩形从极点开始并延伸到细胞质，确保矩形保留在细菌边界内。标记至少 200 种细菌，并使用分析菜单中的区域来遮盖按钮，创建掩码。单击掩码统计信息并选择蒙版范围下的对象。然后在特征和强度下标记平均强度和方差。将数据导出为文本文件，并使用电子表格应用程序计算每个细菌的极性分数，作为方差和平均强度之间的比率。对于高吞吐量应用，请使用人脸对比度显微镜获取双通道图像。确保选择细菌完全分离的视场。接下来，将图像的面部通道对比度调整为细菌清晰可见的水平。打开双通道映像并启动创建段蒙版窗口。调整适当的阈值，然后通过单击定义对象按钮并调整最小大小到 100 像素来删除小对象。在"细化蒙版"下，选择移除边缘对象和彼此相邻的细菌分离掩膜。要量化融合蛋白的动态，请打开图像捕获窗口并标记时间推移。在时间点数框中输入两个。获得两个成功的军团性嗜血杆菌图像，表达感兴趣的荧光蛋白。调整图像对比度，直到单元格清晰可见。然后使用区域工具将 0.25 到 0.25 微米正方形放在至少 400 个单元格的中间。接下来，使用区域在分析菜单中蒙版按钮创建感兴趣的方块的蒙版。创建新的空蒙版，并使用像素工具标记至少 25 个随机单元格的整个单元格区域。创建另一个蒙版并使用大画笔工具标记单元格之间的区域。最后，选择蒙版统计信息，并确保在蒙版范围 1 下选择对象。在特征和强度下，选择平均强度。导出数据并重复掩码 2 的过程。对于蒙版 3，选择整个蒙版并导出平均强度。为了准备电子低温造影样本，在37摄氏度下在CYE猪链球菌板上种植一块重的军团性嗜血杆菌，48小时。将水中的细胞重新消耗到约0.7的OD 600。然后向细胞悬浮液的20微升中加入5个胶体金粒子微升。发光释放孔碳栅格和移液器五微升的细胞混合物。让它站立一分钟。用滤纸将网格混合，并使用重力驱动的柱塞装置将网格冻结在液体乙烷中。将超级折叠器GP插入军团性嗜血杆菌染色体后，进行活细胞荧光显微镜，将荧光信号与亲亲超级折叠器GP阴性应变的荧光信号进行比较。此外，还利用亮场显微镜检查具有特定荧光信号的细胞的比例。只有一小部分产生DotB超级折叠器GP的细胞显示融合蛋白的极性定位。在描述DotB荧光信号的分布时，发现双极点B超折叠器GP的强度比细胞糖高两倍。为了调查DotB超级折叠器GP极性突变是否依赖于完全组装的四型分泌系统，为野生型和四型分泌系统突变的单个细胞确定了DotB超级折叠器GP的极性分数。事实上，当Dot/ICM系统被删除时，DotB超级文件夹GP的极性定位消失了。动态模式表明，DotB ATPase存在于动态细胞体群体中，在后期组装反应时被招募到极性局部点/ICM四型分泌系统。高通量低温 ET 用于在表达 DotB 超级折叠器 GFP 的军团性嗜血菌株中可视化完整的四型分泌系统装置。细胞的重建揭示了细胞包络中嵌入的多个点/ICM机器。确定了DotB和超级折叠器GP相对于完整型四型分泌系统机器的整体定位。三维表面渲染表明，超级折叠器 GFP 位于 DotB 十六进制的下方，该六角形在细胞质 ATPase 复合物的基座上组装为圆盘。尝试此技术时，在进行图像采集之前，请务必评估融合蛋白的稳定性和标签分泌系统的功能。建模和拟合是分析动态模式和结构密度以及评估亚单元在结构中的构象变化或本地化的有效方法。此方法有助于了解不同类型的四个秘密系统组件如何发挥作用，它们如何在更大的复杂中交互，以及不同的子组件执行哪些功能。

Research

JoVE Journal

Immunology and Infection

Applying Live Cell Imaging and Cryo-Electron Tomography to Resolve Spatiotemporal Features of the Legionella pneumophila Dot/Icm Secretion System

Determination of Molecular Structures of HIV Envelope Glycoproteins using Cryo-Electron Tomography and Automated Sub-tomogram Averaging

使用低温电子断层扫描和自动子断层扫描平均HIV包膜糖蛋白分子结构的测定

Since its discovery nearly 30 years ago, more than 60 million people have been infected with the human immunodeficiency virus (HIV) (www.usaid.gov). The ...

科研

免疫与感染

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

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 ...

Real-time Live Imaging of T-cell Signaling Complex Formation

T细胞实时成像实时信号复合物的形成

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating ...

Live Cell Imaging of Alphaherpes Virus Anterograde Transport and Spread

顺行运输Alphaherpes病毒传播的活细胞成像

Advances in live cell fluorescence microscopy  techniques, as well as the construction of recombinant viral strains that  express fluorescent fusion ...

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

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

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 ...

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

成像InlC的分泌由细菌病原体调查感染蜂窝<em&gt;单核细胞增生李斯特氏菌</em

Bacterial intracellular  pathogens can be conceived as molecular tools to dissect cellular signaling  cascades due to their capacity to exquisitely ...

Applying an Inducible Expression System to Study Interference of Bacterial Virulence Factors with Intracellular Signaling

施加诱导表达系统研究与细胞内信号细菌毒力因子的干扰

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a ...

Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages

Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages

嗜肺军团菌外膜囊泡：隔离及对巨噬细胞促炎潜力分析

Bacteria are able to secrete a variety of molecules via various secretory systems. Besides the secretion of molecules into the extracellular space or ...

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

一种新型大鼠自然杀伤细胞无饲养系统的研究体外

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...

Isolation, Fixation, and Immunofluorescence Imaging of Mouse Adrenal Glands

小鼠肾上腺的分离、固定和免疫荧光成像

Immunofluorescence is a well-established technique for detection of antigens in tissues with the employment of fluorochrome-conjugated antibodies and has ...