在梅瑞狄斯实验室的研究由埃伯利科学学院 (宾夕法尼亚州立大学) 提供的启动资金支持。我们感谢 Laremore 博士的专家技术咨询和获得设备在宾夕法尼亚州立大学的蛋白质组学和质谱的核心设施, 学院公园, PA, 在那里进行质谱分析。

1. 细胞生长和裂解
<ol><li>在15毫升胰蛋白酶大豆肉汤中生长细菌或类似的富媒体到晚期指数相 (OD600 1.0-1.5)。用离心法收割细胞, 用三缓冲盐水/EDTA (TBSE) 冲洗一次, 并继续使用协议或冷冻直至用途。 注: TBSE:20 毫米三盐酸盐 (HCl), pH 值 8.0, 130 毫米氯化钠 (氯化钠), 和5毫米乙二胺乙酸 (EDTA))。脂蛋白表达和修饰可能受生长条件 (例如酸度、盐度、生长介质) 和生长阶段15的影响。细胞生长可以按需要进行缩放, 但是建议用15毫升的细胞来单独制备脂蛋白, 因为过量的生物量可以降低脂蛋白的产量。一个15毫升的准备通常产生足够的样本, 以最佳负载2-4 车道上的标准 SDS 页迷你凝胶。</li>
	<li>并用重悬细胞在 800 ul TBSE 与1毫米苄磺酰氟 (PMSF) 和0.5 毫克/毫升溶菌酶。转移解决方案到一个2.0 毫升螺纹离心管与螺钉盖和 O 形环和孵化20分钟37摄氏度。 注意: 某些物种不容易溶菌酶溶解。应用适当的裂解酶代替以帮助裂解。</li>
	<li>将 800 ul 0.1 毫米氧化锆/二氧化硅微珠添加到管中, 在每一个周期之间的5个循环中, 以最大速度 (7000 rpm) 在均质机上摇晃, 以达到每一个循环之间的2分钟, 从而破坏细胞。</li>
	<li>离心样品在 3000 x g 5 分钟在4°c (对颗粒珠和未连续的细胞)。将上清液转移到新的2.0 毫升离心管上, 并保持冰。</li>
	<li>添加 200 ul TBSE 到剩余的颗粒, 并返回到均质机的一个额外的周期。离心机 (如上所述) 与上清液结合上清液 (应为 800-1000 ul 总容积)。</li>
</ol>2. TX-114 相分配法富集脂蛋白
<ol><li>用 TX-114 表面活性剂补充上清液到最终浓度为 2% (卷/量) 通过增加等量的 4% (音量/容量) 表面活性剂在冰冷 TBSE 和孵化在冰上1小时, 混合通过反转每 ~ 15 分钟。 注: 当冷却时, 上清和表面活性剂将被混溶。</li>
	<li>将管子转移到37摄氏度的水浴中, 孵化10分钟, 诱导相分离。在室温下, 将样品以 1万 x g 为10分钟离心, 以保持双相分离。</li>
	<li>轻轻地移开上水相并丢弃。在较低的表面活性剂阶段加入冰冷 TBSE, 将管子重新装入原体积, 并将其转化为混合。在冰上孵育10分钟。</li>
	<li>将管子转移到37摄氏度的水浴中, 孵化10分钟, 以诱导相分离, 然后离心机在 1万 x g 处室温下进行10分钟。</li>
	<li>重复步骤 2.3-2.4 次, 总共3个分色。移除上水相并丢弃。</li>
	<li>去除萃取过程中形成的沉淀蛋白颗粒 (可见于管底), 加入1的冰冷 TBSE 到表面活性剂相。离心机在4°c 在 1.6万 x g 为2分钟到颗粒不溶性蛋白质。 注: 样品应保持单一的阶段。如果相分离发生, 重新冷却样品和离心机。该颗粒一般由非脂蛋白污染物组成, 但可保留以供进一步分析。</li>
	<li>立即转移上清到一个新鲜的2.0 毫升离心管含有 1250 ul 100% 丙酮。吸管向上和向下彻底清洗样品从尖端, 因为它将是粘性的。混合通过反转和孵化隔夜在-20 °c 沉淀蛋白。</li>
	<li>离心样品在 1.6万 x g 在室温下为20分钟的颗粒脂蛋白, 注意管的方向。脂蛋白会沿着管子的外壁形成一层薄薄的白色薄膜。</li>
	<li>用100% 丙酮清洗颗粒两次。醒酒丙酮, 并允许样品风干。</li>
	<li>添加 20-40 ul 10 毫米三 HCl, pH 8.0 或标准 Laemmli SDS 页样本缓冲区16 , 并彻底并用重悬吹打上下对墙壁与沉淀脂蛋白。用吸管尖刮掉管子的一侧以去除脂蛋白。 注: 脂蛋白不会溶解在三个缓冲, 而是形成一个悬浮。<ol>
<li>储存脂蛋白在-20 °c, 直到使用。</li>
	</ol>
</li>
</ol>3. SDS 页、Electroblotting 和胭脂红的染色
<ol><li>使用标准方法16, 用 SDS 页分隔脂蛋白。 注: 适当的凝胶丙烯酰胺百分比将根据其预期用途和脂蛋白的大小而异。在这里,大肠杆菌脂蛋白被分离了10% 三甘氨酸凝胶, 而16.5% 三 tricine 凝胶用于小的大肠杆菌脂蛋白 Lpp17。</li>
	<li>使用标准的 electroblotting 程序将脂蛋白转移到硝化棉转移膜上。 注意: 使用 Bjerrum 谢弗-尼尔森缓冲器加 0.1% SDS 的半干转移是在 25 V, 1.3 mA 上执行15分钟18。交替地, 聚偏聚氟 (PVDF) 膜可以使用, 但由于它比硝化纤维素更疏水性, 它可能减少疏水性脂肽的有效洗脱从膜在以后的步骤。</li>
	<li>将硝化膜转移到容器中, 用胭脂红溶液 (0.2% (w/v) 胭脂红5% 乙酸) 覆盖。轻轻摇动5分钟, 或直到红色粉红色的条纹可见。</li>
	<li>倒入胭脂红溶液, 用 dH2O 小心冲洗硝化棉膜, 去除多余的污渍。 注: 胭脂红染色带将迅速 destain。如果乐队消失, 重复染色过程。</li>
	<li>用干净的刀片, 消费所需的带和转移到离心管。用1毫升 dH2O 洗涤三次, 完全 destain 带。 注意: 协议可以在这里暂停。将被切除的部分用水覆盖-20 摄氏度, 直到使用。</li>
	<li>将该部分转移到干净的表面, 用干净的剃刀刀片, 将硝化棉片切成大约1毫米 x 1 毫米的小块. 将这些碎片收集成一个0.5 毫升低蛋白结合离心管。</li>
	<li>用0.5 毫升新鲜制备的50毫米碳酸氢铵 (NH4HCO3), 用高效液相色谱 (HPLC) 级水中的 pH 值7.8 清洗两次工件。</li>
</ol>4. 胰蛋白酶消化, 从硝化棉膜中提取脂肽, 沉积到 MALDI 靶上, 并进行数据采集
<ol><li>并用重悬硝化棉片在 20 ul 20 微克/毫升溶液的胰蛋白酶在50毫米NH4HCO3, pH 7.8 在 HPLC 级水中。涡流混合, 然后简单地旋转, 以确保所有的部分完全覆盖的胰蛋白酶溶液。用石蜡膜覆盖管盖, 以防止蒸发和孵育消化过夜在37摄氏度。</li>
	<li>旋转样品在 1.6万 x g 三十年代和去除液体吹打。 注意: 这去除了 trypsinized 亲水性肽, 可以在以后被 MALDI 的 MS 用于蛋白质鉴定。</li>
	<li>在 HPLC 级水中加入 50 ul 0.5% 三氟乙酸酸 (TFA)。涡流混合, 然后简单地旋转样品, 以确保所有零件都被溶液覆盖。室温下孵育10分钟。用吹打除去液体。 警告:TFA 是有害的, 如果吸入。小心处理并在化学油烟机中使用。避免接触皮肤。</li>
	<li>重复步骤4.3 与 50 ul 10% 乙腈在 HPLC 级水。 警告:乙腈吸入有害。小心处理并在化学油烟机中使用。避免接触皮肤。</li>
	<li>重复步骤4.3 与 50 ul 20% 乙腈在 HPLC 级水。 注意: 这去除松散地束缚在硝化棉中的任何适度疏水肽。</li>
	<li>为洗脱紧密绑定的脂肽, 添加 15 ul 的新制成的10毫克/毫升α氰基-4-肉桂酸 (CHCA) 基质溶解在氯仿-甲醇 (2:1, v/v) 和孵化为10分钟在室温下, 间歇性涡流。<ol>
<li>交替地, 添加 15 ul 氯仿-甲醇洗脱脂肽, 并与基质混合在以后的时间。其他矩阵, 如 25-苯甲酸酸 (DHB), 应作为替代 CHCA, 如果获得不满意的结果, 为特定的脂肽组成。 警告:氯仿和甲醇都是有害的, 如果吸入。小心处理并在化学油烟机中使用。避免接触皮肤。</li>
		<li>
可选:为促进钠合物的形成, 用水碳酸氢钠 (NaHCO3) 补充氯仿-甲醇中 CHCA 的溶液, 最终浓度为1毫米。</li>
	</ol>
</li>
	<li>简单地旋转示例。用吸管, 小心地将液体转移到一个新的低蛋白结合离心管。此解决方案将包含 N 终端脂肽的大部分。 注: 硝化棉可部分或完全溶解在氯仿-甲醇溶液中。这不会对 MS 结果产生负面影响, 可以作为替代方法来增加脂肽的回报。PVDF 膜不会以同样的方式溶解。</li>
	<li>将洗脱脂肽的 1 ul 沉积在抛光钢 MALDI 靶上 CHCA。 注: 氯仿-甲醇将迅速蒸发, 留下结晶 CHCA 和脂肽。可选的第二 1 ul 整除可以放置到同一地点, 以增加样品浓度。氯仿-甲醇在沉积到靶上后可能会有很大的扩散, 因此应注意避免样品在靶上混合。建议将每个样品的多个斑点存入并拍摄。</li>
	<li>立即进行质谱分析。扫描现场的所有区域的脂肽信号, 特别是如果现场包含两层样本, 因为第二层可能推脂肽到现场的外缘。 注: 建议的启动激光强度为 25%, 但可能需要增加强度获取信号和总和多光谱 (20 或更多的扫描), 以达到足够的信噪比。在这里, 光谱是在一个 MALDI 的仪器上获得的 (参见材料表), 使用工厂配置的仪器方法对 700-3500 m/z范围的反射器进行正离子检测。该仪器用牛血清白蛋白 (BSA) 胰蛋白酶肽混合物进行校准。</li>
</ol>

<ol>
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作者没有什么可透露的。

本协议描述了两个不同阶段的脂蛋白表征: 富集 TX-114 相分配和结构确定的 MALDI MS。在 TX-114 萃取过程中, 额外的离心去除了在这个过程中沉淀的蛋白质, 其次是丙酮沉淀产生高浓度脂蛋白。通过将每个准备的规模限制为15毫升的细胞, 可以很容易地并行处理多个样本, 如果需要, 则在协议结束时将它们汇集在一起。
对于 MS 的结构分析, 将脂蛋白转移到硝化纤维素, 有利于胰蛋白酶...

细菌脂蛋白的特点是保守的 n-末端脂修饰半胱氨酸, 锚定球形蛋白领域的细胞膜表面。它们是普遍分布在细菌, 构成2到5% 的所有细胞基因在一个典型的基因组1。脂蛋白在多种细胞过程中起关键作用, 包括养分吸收、信号传导、蛋白质复合体的组装以及维持细胞包络结构完整性2。在致病性细菌中, 脂蛋白作为致病因子3,4。在感染过程中, 通过类似于收费受体 (TLR) 2 对 n-端脂肽的识别, 激发了先天免疫应答, 以清除入侵的病原体。根据 n-端酰化态, 脂蛋白通常由交替 TLR2 heterodimeric 配合物识别。TLR2-TLR1 承认 n-酰脂肽, 而 TLR2-TLR6 束缚自由脂肽α氨基终点。一旦绑定, 信号通路汇聚以诱导分泌促炎细胞因子3,4。
以前认为, 革兰氏阳性菌脂蛋白 diacylated, 革兰阴性菌 triacylated, 不同于不存在或存在酰胺相关脂肪酸的保守 n-端半胱氨酸残留。这种假设支持的是缺乏序列 orthologs 的革兰阳性基因组 Lnt, 革兰阴性 n-酰基转移酶, 形成 triacylated 脂蛋白5。然而, 最近的研究揭示了 Firmicutes 中缺乏lnt的脂蛋白 triacylation, 以及三种新的 n-端脂蛋白结构, 称为 peptidyl、溶血和n-乙酰形式67 ,8。这些发现引发了对可能尚未发现的脂蛋白形态的质疑, 还有关于这些新脂蛋白是如何制作的基本问题, 以及各种形式所传授的生理目的或优势。此外, 他们清楚地表明, 目前基因组无法预测脂蛋白结构。事实上, 我们最近发现了一种新的类脂蛋白 n-酰转移酶, 称为点燃, 从粪肠球菌和蜡样芽孢杆菌, 使溶血脂蛋白 9.这表明需要实验性地验证脂蛋白结构, 这可能是挑战, 由于其极其疏水性的性质和有限的方法, 以表征其分子结构。
为了促进对脂蛋白诱导宿主免疫应答以及 n 末端结构测定的研究, 我们已经修改了几种先前描述的协议, 以纯化细菌脂蛋白并制备 n-端胰蛋白酶。脂肽用于分析的 MALDI MS 6, 10, 11, 12.脂蛋白是丰富使用建立的海卫 X-114 (从今以后称为表面活性剂或 TX-114) 相分配方法, 优化去除污染的非脂蛋白和增加脂蛋白产量。这些脂蛋白适用于直接使用 TLR 化验或进一步纯化的 SDS 页。对于 MALDI MS, 脂蛋白转移到硝化棉膜提供了一个脚手架, 有效的原位胰蛋白酶消化, 洗涤, 并随后从膜表面洗脱, 导致高度纯化的 n-端脂肽。硝化棉已被证明可以促进样品处理和改善序列覆盖的高度疏水性肽从整体膜蛋白13,14, 以及脂蛋白9,10.该方法具有以极性为基础的分馏肽的额外优势, 在单实验中, 可以分析中间洗涤液与 n-末端结构测定同时进行高置信蛋白鉴定。.该协议独特的特点是有意的钠加成层, 以促进在 ms/毫秒内的父离子分裂为 dehydroalanyl 离子, 协助结构分配的N-酰化态。N 终点是与 TLR 识别脂蛋白有关的最大变量和关键特征。总之, 本议定书允许对脂蛋白进行深入和可重复性的研究, MALDI 的纯化和结构测定的各个阶段根据实验的总体目标容易适应。

<table>
	<tbody>
		<tr>
			<td>Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>0.01 mm Zirconia/silica beads</td>
			<td>BioSpec Products</td>
			<td>110791012</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic acid</td>
			<td>EMD</td>
			<td>AX0073-9</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>EMD</td>
			<td>AX0116-6</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetonitrile</td>
			<td>EMD</td>
			<td>AX0142-6</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium bicarbonate</td>
			<td>Fluka Analytical</td>
			<td>09830</td>
			<td></td>
		</tr>
		<tr>
			<td>BioTrace NT Nitrocellulose</td>
			<td>PALL Life Sciences</td>
			<td>66485</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA) digest standard</td>
			<td>Protea</td>
			<td>PS-204-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Acros Organics</td>
			<td>423550025</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid (EDTA)</td>
			<td>Fisher Scientific</td>
			<td>BP118</td>
			<td></td>
		</tr>
		<tr>
			<td>HPLC Grade water</td>
			<td>EMD</td>
			<td>WX0008-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Lysozyme</td>
			<td>Fisher Scientific</td>
			<td>BP535-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sigma-Aldrich</td>
			<td>34860</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenylmethyl sulfonyl fluoride (PMSF)</td>
			<td>Amresco</td>
			<td>0754</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce trypsin protease</td>
			<td>Thermo Scientific</td>
			<td>90057</td>
			<td></td>
		</tr>
		<tr>
			<td>Ponceau S</td>
			<td>Acros Organics</td>
			<td>161470100</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein LoBind Tube 0.5mL</td>
			<td>Eppendorf</td>
			<td>022431064</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Sigma-Aldrich</td>
			<td>792519</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Macron Fine Chemicals</td>
			<td>7581-06</td>
			<td></td>
		</tr>
		<tr>
			<td>Trifluoroacetic acid (TFA)</td>
			<td>Sigma-Aldrich</td>
			<td>299537</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-hydrochloride (HCl)</td>
			<td>Fisher Scientific</td>
			<td>BP152</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-114</td>
			<td>Sigma-Aldrich</td>
			<td>93422</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptic soy broth (TSB)</td>
			<td>BD</td>
			<td>211822</td>
			<td></td>
		</tr>
		<tr>
			<td>&#945;-cyano-4-hydroxycinnamic acid (MS Grade)</td>
			<td>Sigma-Aldrich</td>
			<td>C8982</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MagNALyser</td>
			<td>Roche</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trans-Blot Turbo Transfer System</td>
			<td>Bio-Rad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MALDI-TOF target</td>
			<td>Bruker Daltonics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ultraflextreme MALDI-TOF-TOF</td>
			<td>Bruker Daltonics</td>
			<td></td>
			<td>Equipped with a 355 nm frequency-tripled Nd:YAG smartbeam-II laser</td>
		</tr>
	</tbody>
</table>

enrichment of bacterial lipoproteins and preparation of n-terminal lipopeptides for structural determination by mass spectrometry

脂蛋白是哺乳动物先天免疫应答的细菌细胞包络和有效活化剂的重要成分。尽管它们对细胞生理学和免疫学都有重要意义, 但对于新的脂蛋白形态、合成方法以及各种形式对宿主免疫的影响, 仍有许多有待发现。为了对脂蛋白进行深入研究, 本协议描述了一种细菌脂蛋白的富集方法和 n-端胰蛋白酶脂肽的制备, 其结构由基质辅助激光解吸电离-飞行时间来确定。质谱 (MALDI)。在建立的海卫 X-114 相分治法中扩展了从细菌细胞膜中提取和富集的方法, 该协议包括去除非脂蛋白污染物的附加步骤, 增加脂蛋白产量和纯度。由于脂蛋白是常用的收费样受体 (TLR) 检测, 这是至关重要的第一个特点的 n-终端结构的 MALDI 的 MS. 本文提出了一种分离富集在 n-端的浓疏水性多肽的方法。脂肽适合直接分析的 MALDI 女士/ms. 经月桂酸钠分离的脂质体-丙烯酰胺凝胶电泳 (SDS 页) 被转移到硝化纤维膜上, 消化原位 胰蛋白酶, 依次洗涤去除极性胰蛋白酶肽, 最后洗脱与氯仿-甲醇。当与洗涤液中极性 trypsinized 肽的 MS 结合时, 这种方法提供了在单个实验中识别脂蛋白并表征其 n-终点的能力。故意的钠加成物也可以作为一种工具, 以促进更结构化的信息碎片谱。最终, 脂蛋白的丰富和对其 n-端结构的测定将允许对这种无处不在的细菌蛋白进行更广泛的研究。

协议的示意图在图 1中提供。图 2中显示了从粪肠球菌ATCC 19433 中提取的富含脂蛋白的分数 TX-114。为比较, 还显示了沉淀蛋白分数的带状模式。从这个分数的蛋白质证实了 MALDI-MS 是高度丰富的污染蛋白质以外的脂蛋白 (表 1)。在图 3中的质谱显示了 PnrA 的胰蛋白酶肽离子剖面, 该蛋白是在?...

Watch this Scientific Journal Video about Enrichment of Bacterial Lipoproteins and Preparation of N-terminal Lipopeptides for Structural Determination by Mass Spectrometry at JoVE.com

Enrichment of Bacterial Lipoproteins and Preparation of N-terminal Lipopeptides for Structural Determination by Mass Spectrometry

质谱法富集细菌脂蛋白及 n-端脂肽的制备结构测定

用非离子表面活性剂相分配法对细菌脂蛋白的富集进行了描述, 直接用于 TLR 化验或其他应用。进一步详细的步骤, 以准备 n-终端胰蛋白酶脂肽的结构表征的质谱。

Lipoproteins are important constituents of the bacterial cell envelope and potent activators of the mammalian innate immune response. Despite their ...

enrichment-bacterial-lipoproteins-preparation-n-terminal-lipopeptides

Research

JoVE Journal

Immunology and Infection

8.5K 次观看.  Pennsylvania State University. 用非离子表面活性剂相分配法对细菌脂蛋白的富集进行了描述, 直接用于 TLR 化验或其他应用。进一步详细的步骤, 以准备 n-终端胰蛋白酶脂肽的结构表征的质谱。

视频: Enrichment of Bacterial Lipoproteins and Preparation of N-terminal Lipopeptides for Structural Determination by Mass Spectrometry

Invasion of Human Cells by a Bacterial Pathogen

Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus . The general protocol can be applied to the study of cell invasion by virtually any culturable bacterium. The stages at which specific aspects of invasion can be studied, such as the role of actin rearrangement or caveolae, will be highlighted. Host cells are grown in flasks and when ready for use are seeded into 24-well plates containing Thermanox coverslips. Using coverslips allows subsequent removal of the cells from the wells to reduce interference from serum proteins deposited onto the sides of the wells (to which S. aureus would attach). Bacteria are grown to the required density and washed to remove any secreted proteins (e.g. toxins). Coverslips with confluent layers of endothelial cells are transferred to new 24-well plates containing fresh culture medium before the addition of bacteria. Bacteria and cells are then incubated together for the required amount of time in 5% CO2 at 37&deg;C. For S. aureus this is typically between 15-90 minutes. Thermanox coverslips are removed from each well and dip-washed in PBS to remove unattached bacteria. If total associated bacteria (adherent and internalised) are to be quantified, coverslips are then placed in a fresh well containing 0.5% Triton X-100 in PBS. Gentle pipetting leads to complete cell lysis and bacteria are enumerated by serial dilution and plating onto agar. If the number of bacteria that have invaded the cells is needed, coverslips are added to wells containing 500 &mu;l tissue culture medium supplemented with gentamicin and incubation continued for 1 h, which will kill all external bacteria. Coverslips can then be washed, cells lysed and bacteria enumerated by plating onto agar as described above. If the experiment requires direct visualisation, coverslips can be fixed and stained for light, fluorescence or confocal microscopy or prepared for electron microscopy.

A general protocol for the study of invasion of host cells by a bacterial pathogen, focusing on Staphylococcus aureus and human endothelial cells.

由细菌病原体入侵人体细胞

Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus . The general protocol can be ...

科研

免疫与感染

Structure of HIV-1 Capsid Assemblies by Cryo-electron Microscopy and Iterative Helical Real-space Reconstruction

Cryo-electron microscopy (cryo-EM), combined with image processing, is an increasingly powerful tool for structure determination of macromolecular protein complexes and assemblies. In fact, single particle electron microscopy1 and two-dimensional (2D) electron crystallography2 have become relatively routine methodologies and a large number of structures have been solved using these methods. At the same time, image processing and three-dimensional (3D) reconstruction of helical objects has rapidly developed, especially, the iterative helical real-space reconstruction (IHRSR) method3, which uses single particle analysis tools in conjunction with helical symmetry. Many biological entities function in filamentous or helical forms, including actin filaments4, microtubules5, amyloid fibers6, tobacco mosaic viruses7, and bacteria flagella8, and, because a 3D density map of a helical entity can be attained from a single projection image, compared to the many images required for 3D reconstruction of a non-helical object, with the IHRSR method, structural analysis of such flexible and disordered helical assemblies is now attainable.
In this video article, we provide detailed protocols for obtaining a 3D density map of a helical protein assembly (HIV-1 capsid9 is our example), including protocols for cryo-EM specimen preparation, low dose data collection by cryo-EM, indexing of helical diffraction patterns, and image processing and 3D reconstruction using IHRSR. Compared to other techniques, cryo-EM offers optimal specimen preservation under near native conditions. Samples are embedded in a thin layer of vitreous ice, by rapid freezing, and imaged in electron microscopes at liquid nitrogen temperature, under low dose conditions to minimize the radiation damage. Sample images are obtained under near native conditions at the expense of low signal and low contrast in the recorded micrographs. Fortunately, the process of helical reconstruction has largely been automated, with the exception of indexing the helical diffraction pattern. Here, we describe an approach to index helical structure and determine helical symmetries (helical parameters) from digitized micrographs, an essential step for 3D helical reconstruction. Briefly, we obtain an initial 3D density map by applying the IHRSR method. This initial map is then iteratively refined by introducing constraints for the alignment parameters of each segment, thus controlling their degrees of freedom. Further improvement is achieved by correcting for the contrast transfer function (CTF) of the electron microscope (amplitude and phase correction) and by optimizing the helical symmetry of the assembly.

This article describes a method to obtain a three-dimensional (3D) structure of helically assembled molecules using cryo-electron microscopy. In this protocol, we use HIV-1 capsid assemblies to illustrate the detailed 3D reconstruction procedure for achieving a density map by the iterative helical real-space reconstruction method.

HIV - 1衣壳大会通过低温电子显微镜的结构和迭代螺旋实时空间重构

Cryo-electron microscopy (cryo-EM), combined with image processing, is an increasingly powerful tool for structure determination of macromolecular protein ...

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne relapsing fever (Borrelia spp.), Rocky Mountain Spotted fever (Rickettsia rickettsii), ehrlichiosis (Ehrlichia chaffeensis and E. equi), anaplasmosis (Anaplasma phagocytophilum), encephalitis (tick-borne encephalitis virus), babesiosis (Babesia spp.), Colorado tick fever (Coltivirus), and tularemia (Francisella tularensis) 1-8. To be properly transmitted into the host these infectious agents differentially regulate gene expression, interact with tick proteins, and migrate through the tick 3,9-13. For example, the Lyme disease agent, Borrelia burgdorferi, adapts through differential gene expression to the feast and famine stages of the tick's enzootic cycle 14,15. Furthermore, as an Ixodes tick consumes a bloodmeal Borrelia replicate and migrate from the midgut into the hemocoel, where they travel to the salivary glands and are transmitted into the host with the expelled saliva 9,16-19.
As a tick feeds the host typically responds with a strong hemostatic and innate immune response 11,13,20-22. Despite these host responses, I. scapularis can feed for several days because tick saliva contains proteins that are immunomodulatory, lytic agents, anticoagulants, and fibrinolysins to aid the tick feeding 3,11,20,21,23. The immunomodulatory activities possessed by tick saliva or salivary gland extract (SGE) facilitate transmission, proliferation, and dissemination of numerous tick-borne pathogens 3,20,24-27. To further understand how tick-borne infectious agents cause disease it is essential to dissect actively feeding ticks and collect tick saliva. This video protocol demonstrates dissection techniques for the collection of hemolymph and the removal of salivary glands from actively feeding I. scapularis nymphs after 48 and 72 hours post mouse placement. We also demonstrate saliva collection from an adult female I. scapularis tick.

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

The collection of infected tick hemolymph, salivary glands, and saliva is important to study how tick-borne pathogens cause disease. In this protocol we demonstrate how to collect hemolymph and salivary glands from feeding Ixodes scapularis nymphs. We also demonstrate saliva collection from female I. scapularis adults.

唾液，唾液腺，淋巴收集肩突硬蜱蜱

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne ...

Discovery of New Intracellular Pathogens by Amoebal Coculture and Amoebal Enrichment Approaches

Intracellular pathogens such as legionella, mycobacteria and Chlamydia-like organisms are difficult to isolate because they often grow poorly or not at all on selective media that are usually used to cultivate bacteria. For this reason, many of these pathogens were discovered only recently or following important outbreaks. These pathogens are often associated with amoebae, which serve as host-cell and allow the survival and growth of the bacteria. We intend here to provide a demonstration of two techniques that allow isolation and characterization of intracellular pathogens present in clinical or environmental samples: the amoebal coculture and the amoebal enrichment. Amoebal coculture allows recovery of intracellular bacteria by inoculating the investigated sample onto an amoebal lawn that can be infected and lysed by the intracellular bacteria present in the sample. Amoebal enrichment allows recovery of amoebae present in a clinical or environmental sample. This can lead to discovery of new amoebal species but also of new intracellular bacteria growing specifically in these amoebae. Together, these two techniques help to discover new intracellular bacteria able to grow in amoebae. Because of their ability to infect amoebae and resist phagocytosis, these intracellular bacteria might also escape phagocytosis by macrophages and thus, be pathogenic for higher eukaryotes.

Amoebal coculture is a cell culture system using adherent amoebae to selectively grow intracellular pathogens able to resist phagocytic cells such as amoebae and macrophages. It thus represents a key tool to discover new infectious agents. Amoebal enrichment allows discovery of new amoebal species and of their specific intracellular bacteria.

新的细胞内病原体由Amoebal共培养和Amoebal富集途径探索

Intracellular pathogens such as legionella, mycobacteria and Chlamydia-like organisms are difficult to isolate because they often grow poorly or not at ...

Rapid Identification of Gram Negative Bacteria from Blood Culture Broth Using MALDI-TOF Mass Spectrometry

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional identification requires the sub-culture of signaled blood culture broth with identification available only after colonies on solid agar have matured. MALDI-TOF MS is a reliable, rapid method for identification of the majority of clinically relevant bacteria when applied to colonies on solid media. The application of MALDI-TOF MS directly to blood culture broth is an attractive approach as it has potential to accelerate species identification of bacteria and improve clinical management. However, an important problem to overcome is the pre-analysis removal of interfering resins, proteins and hemoglobin&#160;contained in blood culture specimens which, if not removed, interfere with the MS spectra and can result in insufficient or low discrimination identification scores. In addition it is necessary to concentrate bacteria to develop spectra of sufficient quality. The presented method describes the concentration, purification, and extraction of Gram negative bacteria allowing for the early identification of bacteria from a signaled blood culture broth.

The application of matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS) directly to blood culture broth expedites the identification of bacteria. The presented method is a rapid and reliable method for identification of Gram negative bacteria directly from blood culture broth.

革兰从血培养液使用MALDI-TOF质谱阴性细菌快速鉴定

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional ...

Preparation of a Blood Culture Pellet for Rapid Bacterial Identification and Antibiotic Susceptibility Testing

Bloodstream infections and sepsis are a major cause of morbidity and mortality. The successful outcome of patients suffering from bacteremia depends on a rapid identification of the infectious agent to guide optimal antibiotic treatment. The analysis of Gram stains from positive blood culture can be rapidly conducted and already significantly impact the antibiotic regimen. However, the accurate identification of the infectious agent is still required to establish the optimal targeted treatment. We present here a simple and fast bacterial pellet preparation from a positive blood culture that can be used as a sample for several essential downstream applications such as identification by MALDI-TOF MS, antibiotic susceptibility testing (AST) by disc diffusion assay or automated AST systems and by automated PCR-based diagnostic testing. The performance of these different identification and AST systems applied directly on the blood culture bacterial pellets is very similar to the performance normally obtained from isolated colonies grown on agar plates. Compared to conventional approaches, the rapid acquisition of a bacterial pellet significantly reduces the time to report both identification and AST. Thus, following blood culture positivity, identification by MALDI-TOF can be reported within less than 1 hr whereas results of AST by automated AST systems or disc diffusion assays within 8 to 18 hr, respectively. Similarly, the results of a rapid PCR-based assay can be communicated to the clinicians less than 2 hr following the report of a bacteremia. Together, these results demonstrate that the rapid preparation of a blood culture bacterial pellet has a significant impact on the identification and AST turnaround time and thus on the successful outcome of patients suffering from bloodstream infections.

A rapid bacterial pellet preparation from a positive blood culture can be used as a sample for applications such as identification by MALDI-TOF, Gram staining, antibiotic susceptibility testing and PCR-based test. The results can be rapidly communicated to clinicians to improve the outcome of patients suffering from bloodstream infections.

为快速细菌鉴定和药敏试验的准备血培养微丸

Bloodstream infections and sepsis are a major cause of morbidity and mortality. The successful outcome of patients suffering from bacteremia depends on a ...

Electroporation of Functional Bacterial Effectors into Mammalian Cells

The study of protein interactions in the context of living cells can generate critical information about localization, dynamics, and interacting partners. This information is particularly valuable in the context of host-pathogen interactions. Many pathogen proteins function within host cells in a variety of way such as, enabling evasion of the host immune system and survival within the intracellular environment. To study these pathogen-protein host-cell interactions, several approaches are commonly used, including: in vivo infection with a strain expressing a tagged or mutant protein, or introduction of pathogen genes via transfection or transduction. Each of these approaches has advantages and disadvantages. We sought a means to directly introduce exogenous proteins into cells. Electroporation is commonly used to introduce nucleic acids into cells, but has been more rarely applied to proteins although the biophysical basis is exactly the same. A standard electroporator was used to introduce affinity-tagged bacterial effectors into mammalian cells. Human epithelial and mouse macrophage cells were cultured by traditional methods, detached, and placed in 0.4 cm gap electroporation cuvettes with an exogenous bacterial pathogen protein of interest (e.g. Salmonella Typhimurium GtgE). After electroporation (0.3 kV) and a short (4 hr) recovery period, intracellular protein was verified by fluorescently labeling the protein via its affinity tag and examining spatial and temporal distribution by confocal microscopy. The electroporated protein was also shown to be functional inside the cell and capable of correct subcellular trafficking and protein-protein interaction. While the exogenous proteins tended to accumulate on the surface of the cells, the electroporated samples had large increases in intracellular effector concentration relative to incubation alone. The protocol is simple and fast enough to be done in a parallel fashion, allowing for high-throughput characterization of pathogen proteins in host cells including subcellular targeting and function of virulence proteins.

Electroporation was used to insert purified bacterial virulence effector proteins directly into living eukaryotic cells. Protein localization was monitored by confocal immunofluorescence microscopy. This method allows for studies on trafficking, function, and protein-protein interactions using active exogenous proteins, avoiding the need for heterologous expression in eukaryotic cells.

电功能性细菌效应器到哺乳动物细胞

The study of protein interactions in the context of living cells can generate critical information about localization, dynamics, and interacting partners. ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

生成和多表型高内涵筛选<em&gt;贝氏柯克斯体</em&gt;转座子突变体

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

Alternative In Vitro Methods for the Determination of Viral Capsid Structural Integrity

Human norovirus exacts considerable public health and economic losses worldwide. Emerging in vitro cultivation advances are not yet applicable for routine detection of the virus. The current detection and quantification techniques, which rely primarily on nucleic acid amplification, do not discriminate infectious from non-infectious viral particles. The purpose of this article is to present specific details on recent advances in techniques used together in order to acquire further information on the infectivity status of viral particles. One technique involves assessing binding of a norovirus ssDNA aptamer to capsids. Aptamers have the advantage of being easily synthesized and modified, and are inexpensive and stable. Another technique, dynamic light scattering (DLS), has the advantage of observing capsid behavior in solution. Electron microscopy allows for visualization of the structural integrity of the viral capsids. Although promising, there are some drawbacks to each technique, such as non-specific aptamer binding to positively-charged molecules from sample matrices, requirement of purified capsid for DLS, and poor sensitivity for electron microscopy. Nonetheless, when these techniques are used in combination, the body of data produced provides more comprehensive information on norovirus capsid integrity that can be used to infer infectivity, information which is essential for accurate evaluation of inactivation methods or interpretation of virus detection. This article provides protocols for using these methods to discriminate infectious human norovirus particles.

Alternative In Vitro Methods for the Determination of Viral Capsid Structural Integrity

Routine detection methods utilizing viral genome amplification are limited by their inability to discriminate infectious from non-infectious particles. The purpose of this article is to provide detailed protocols for alternative methods to aid in discrimination of infectious norovirus particles using aptamer binding, dynamic light scattering, and transmission electron microscopy.

替代体外测定病毒衣壳结构完整性的方法

Human norovirus exacts considerable public health and economic losses worldwide. Emerging in vitro cultivation advances are not yet applicable for routine ...

Identification of Nucleolar Factors During HIV-1 Replication Through Rev Immunoprecipitation and Mass Spectrometry

The HIV-1 infectious cycle requires viral protein interactions with host factors to facilitate viral replication, packaging, and release. The infectious cycle further requires the formation of viral/host protein complexes with HIV-1 RNA to regulate the splicing and enable nucleocytoplasmic transport. The HIV-1 Rev protein accomplishes the nuclear export of HIV-1 mRNAs through multimerization with intronic cis-acting targets - the Rev response element (RRE). A nucleolar localization signal (NoLS) exists within the COOH-terminus of the Rev arginine-rich motif (ARM), allowing the accumulation of Rev/RRE complexes in the nucleolus. Nucleolar factors are speculated to support the HIV-1 infectious cycle through various other functions in addition to mediating mRNA-independent nuclear export and splicing. We describe an immunoprecipitation method of wild-type (WT) Rev in comparison to Rev nucleolar mutations (deletion and single-point Rev-NoLS mutations) in the presence of HIV-1 replication for mass spectrometry. Nucleolar factors implicated in the nucleocytoplasmic transport (nucleophosmin B23 and nucleolin C23), as well as cellular splicing factors, lose interaction with Rev in the presence of Rev-NoLS mutations. Various other nucleolar factors, such as snoRNA C/D box 58, are identified to lose interaction with Rev mutations, yet their function in the HIV-1 replication cycle remain unknown. The results presented here demonstrate the use of this approach for the identification of viral/host nucleolar factors that maintain the HIV-1 infectious cycle. The concepts used in this approach are applicable to other viral and disease models requiring the characterization of understudied pathways.

Here we describe Rev immunoprecipitation in the presence of HIV-1 replication for mass spectrometry. The methods described can be used for the identification of nucleolar factors involved in the HIV-1 infectious cycle and are applicable to other disease models for the characterization of understudied pathways.

通过转版免疫沉淀和质谱测定,在HIV-1复制过程中核子因子的识别

The HIV-1 infectious cycle requires viral protein interactions with host factors to facilitate viral replication, packaging, and release. The infectious ...