Name: identification of virulence markers of mycobacterium abscessus for intracellular replication in phagocytes
Uploaded: 2018-09-27T13:00:00
Description: Watch this Scientific Journal Video about Identification of Virulence Markers of Mycobacterium abscessus for Intracellular Replication in Phagocytes at JoVE.com

我们非常感谢公关. E.J. 鲁宾 (哈佛医学院, 美国波士顿) 为变种人图书馆的珍贵礼物, 并且马修博士 (医学学院, 英国南安普敦大学) 为原稿的更正。我们非常感谢法国病人协会的囊性纤维化 "Vaincre la Mucoviscidose" 和 "L ' 协会格雷戈里 Lemarchal" 为他们的财政支持 (RF20150501377)。我们还感谢国家研究机构 (DIMIVYR 项目 (ANR-13-BSV3-0007-01)) 和 Région 法国 (酒庄 Intérêt 不可抗力疾病 Infectieuses et Emergentes) 为博士后奖学金提供给 v. m。李涅是 "部 Enseignement 高级研究所" 的博士研究员。

1. 图书馆筛选
<ol><li>Tn突变体库的构建
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<li>获取座子库。 注: 本实验中, 美国波士顿大学公共卫生学院 E.J. 鲁宾 (座子) 提供了一个变种人图书馆。图书馆是由m. 脓肿综合症 (m 脓肿亚种. massiliense) 的平滑临床应变 (43S) 构建的, phagemid 引入m 脓肿允许随机插入单个Tn成 ta 二核苷酸 (91240 TA 序列在m 脓肿基因组)。有关详细信息, 请参阅鲁?...

<ol>
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m. 脓肿的行为与 RGM2的其他分枝杆菌相比, 与结核菌病等致病性通用的行为更为相似。该病致病性的关键因素是它们在抗原呈现细胞中存活甚至繁殖的能力, 如巨噬细胞和树突状体细胞。
m. 脓肿已经获得了某些基因组优势, 如其基因组14的总序列所示, 以便在真核吞噬细胞中生存。m 脓肿的基因组已经被证明是迅速发...

分枝杆菌属包括从无害的腐生有机体到主要人类病原体的种类。众所周知的致病物种, 如结核分枝杆菌, marinum 分枝杆菌和杆菌分枝杆菌属于慢生长的分枝杆菌 (上海市) 亚组。与此相反, 快速生长的分枝杆菌 (RGM) 亚群的特征是它们在琼脂培养基上在不到7天内形成可见菌落的能力。RGM 组包括180多种, 主要为非致病性腐生分枝杆菌。RGM 与寄主的相互作用的研究主要集中在耻垢分支杆菌上, 并表明这些分枝杆菌被巨噬细胞的杀菌作用迅速消除。
脓肿分枝杆菌是一种罕见的 RGM, 是人类的致病性, 并负责广泛的感染范围从皮肤和软组织感染到肺部和传播感染。m. 脓肿与杆菌分枝杆菌一起被认为是囊性纤维化患者1的主要结核杆菌病原体。
在m. 脓肿上进行的各种研究表明, 这种分枝杆菌的行为类似于胞内病原体, 能够在肺部和皮肤中存活的巨噬细胞和成纤维细胞的杀菌反应, 而这通常不是在 RGM2,3,4. m. 脓肿基因组分析确定了在与土壤、植物和水生环境接触的环境微生物中通常发现的代谢通路, 那里的自由阿米巴经常存在5。他们还表明, m. 脓肿被赋予在腐生和非致病性 RGM 中未发现的几种毒力基因, 可能是在有利基因交换的利基水平基因转移中获得的, 可能会聚集各种抗虫病菌。
实验中, 第一个显著的结果是观察脓肿 m . 型巨噬细胞细胞内生长和结核6。m. 脓肿也抵抗吞噬体的酸化, 细胞凋亡和自噬, 三细胞抵抗的基本的机制对传染2。它甚至表明, m.脓肿能够建立一个即时沟通之间的吞噬体和细胞质, 一个更富营养的环境, 可能有利于细菌增殖2。很少有人知道m. 脓肿拥有或已经获得的基因组优势, 以允许在细胞内环境中生存。变形虫共培养是一种有效的方法, 允许分离许多新的抗虫菌massiliense7,8分枝杆菌。在小鼠脓肿的 aerosolization 模型中观察到在阿米巴内繁殖的能力, 这会使m 脓肿4的毒力增加。一个假说是, m. 脓肿在这个环境中发现了遗传特征, 在吞噬细胞中生存, 这与其他非致病性 RGM 不同。这些收购可能有利于传播能力和它在人类宿主的毒力。
本报告介绍了一些工具和方法, 以突出基因组优势授予m. 脓肿生存在阿米巴环境中。为此, 首先描述了 m.脓肿座子突变体的筛选,棘阿米巴 castellanii型菌株, 它允许鉴定突变体细胞内生长缺陷。另外还报告了巨噬细胞的第二次筛查, 以确认这种缺陷是否在人类宿主中持续存在。其次, 为了了解在脓肿中利用哪些机制来适应吞噬细胞的生命, 增加其在动物宿主中的毒力, 在共培养后, 开发了一种特别适合m 脓肿的方法。在阿米巴的存在, 允许从 amoebal 内细菌中提取总 RNA。因此, 对细胞内生命所需的m 脓肿基因的综合看法得到了发展。

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	<tbody>
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			<td height="21" style="height:21px;width:341px;">Name of Material/ Equipment</td>
			<td style="width:128px;"></td>
			<td style="width:135px;"></td>
			<td style="width:211px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">24-well plates</td>
			<td style="width:128px;">Thermofisher</td>
			<td>11874235</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">96-well plates</td>
			<td style="width:128px;">Thermofisher</td>
			<td style="width:135px;">10687551</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;">Beadbeater&#160;</td>
			<td style="width:128px;">Bertin</td>
			<td style="width:135px;">Precellys 24</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:341px;">Bioanalyzer</td>
			<td style="width:128px;">Agilent</td>
			<td style="width:135px;"></td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:341px;">Genepulser Xcell</td>
			<td style="width:128px;">Biorad</td>
			<td style="width:135px;"></td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;">Nanodrop spectrophotometer 2000</td>
			<td style="width:128px;">Thermofisher</td>
			<td style="width:135px;"></td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;">QuBit fluorometer</td>
			<td style="width:128px;">Thermofisher</td>
			<td style="width:135px;">Q33226</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">zirconium beads/silica beads</td>
			<td style="width:128px;">Biospec products</td>
			<td style="width:135px;">11079101Z</td>
			<td>Beads</td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:341px;">Name of reagent/cells</td>
			<td style="width:128px;"></td>
			<td style="width:135px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acanthamoeba&#160;castellanii&#160;</td>
			<td style="width:128px;">ATCC</td>
			<td style="width:135px;">30010</td>
			<td>strain</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Amikacin&#160;</td>
			<td style="width:128px;">Mylan</td>
			<td style="width:135px;">150927-A</td>
			<td>powder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">B-mercaptoethanol&#160;</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td style="width:135px;">M6250</td>
			<td>solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CaCl2</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td style="width:135px;">C1016</td>
			<td>&#62;93% granular anhydrous</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Chloroform&#160;</td>
			<td style="width:128px;">Fluka</td>
			<td style="width:135px;">25666</td>
			<td>solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ClaI enzyme</td>
			<td style="width:128px;">New England Biolabs</td>
			<td style="width:135px;">R0197S</td>
			<td>enzyme</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Columbia agar&#160;</td>
			<td style="width:128px;">Biomerieux</td>
			<td style="width:135px;">43041</td>
			<td>90 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">D-Glucose</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td style="width:135px;">G8270&#160;</td>
			<td>powder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">DMEM&#160;</td>
			<td style="width:128px;">Thermofisher</td>
			<td style="width:135px;">11500596</td>
			<td>medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DNase and RNase free water&#160;</td>
			<td style="width:128px;">Invitrogen</td>
			<td style="width:135px;">10977-035</td>
			<td>solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">E.&#160;coli electrocompetent&#160;</td>
			<td style="width:128px;">Thermofisher</td>
			<td style="width:135px;">18265017</td>
			<td>bacteria</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EDTA</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td style="width:135px;">E4884</td>
			<td>powder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Escherichia coli&#160;</td>
			<td style="width:128px;">Clinical isolate</td>
			<td style="width:135px;">personal stock</td>
			<td>bacteria</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fe(NH4)2(SO4)-6H20&#160;</td>
			<td>EMS</td>
			<td style="width:135px;">15505-40</td>
			<td>sulfate solution 4% aqueous</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal Calf Serum</td>
			<td style="width:128px;">Gibco</td>
			<td style="width:135px;">10270</td>
			<td>serum</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">Glycerol</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td style="width:135px;">G5516</td>
			<td>solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Guanidium thiocyanate&#160;</td>
			<td style="width:128px;">Euromedex</td>
			<td style="width:135px;">EU0046-D</td>
			<td>powder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isopropanol&#160;</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td style="width:135px;">I9516</td>
			<td>solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">J774.2 macrophages</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td style="width:135px;">J774.2</td>
			<td>Strain</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">kanamycin&#160;</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td>60615</td>
			<td>powder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">KH2PO4</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td style="width:135px;">P0662</td>
			<td>Monobasic, anhydrous</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LB liquid medium&#160;</td>
			<td style="width:128px;">Invitrogen</td>
			<td style="width:135px;">12795-027</td>
			<td>powder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lysozyme</td>
			<td style="width:128px;">Roche</td>
			<td style="width:135px;">10837059001</td>
			<td>powder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MgSO4</td>
			<td>Labosi</td>
			<td style="width:135px;">M275</td>
			<td>pur</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">Microbank TM (cryotubes with beads)</td>
			<td style="width:128px;">Pro-Lab Diagnostic</td>
			<td style="width:135px;">PL.170/M</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">Middlebrook 7H11 medium</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td>M0428</td>
			<td>powder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">Middlebrook 7H9 medium</td>
			<td style="width:128px;">Thermofisher</td>
			<td style="width:135px;">11753473</td>
			<td>powder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">M&#252;ller-Hinton agar</td>
			<td style="width:128px;">Biorad</td>
			<td>3563901</td>
			<td>powder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N-Lauryl-sarcosine</td>
			<td style="width:128px;">Merck</td>
			<td style="width:135px;">S37700 416</td>
			<td>powder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Na2HPO4-7H2O</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td style="width:135px;">S9390</td>
			<td>98-102%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phenol/chloroforme&#160;</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td style="width:135px;">77617</td>
			<td>solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Proteinase K</td>
			<td style="width:128px;">Thermofisher</td>
			<td style="width:135px;">EO0491</td>
			<td>powder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">proteose peptone</td>
			<td>BD</td>
			<td style="width:135px;">211684</td>
			<td>enzymatic digest of animal tissue</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pUC19 plasmid</td>
			<td style="width:128px;">New England Biolabs</td>
			<td style="width:135px;">54357</td>
			<td>plasmid</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SDS&#160; 20%</td>
			<td style="width:128px;">Biorad</td>
			<td style="width:135px;">1610418</td>
			<td>solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium citrate</td>
			<td>Calbiochem</td>
			<td style="width:135px;">567446</td>
			<td>powder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thiourea</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td style="width:135px;">88810</td>
			<td>powder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">Tris</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td style="width:135px;">154563</td>
			<td>powder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trizol&#160;</td>
			<td style="width:128px;">Thermofisher</td>
			<td>12044977</td>
			<td>solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tween 80</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td style="width:135px;">P1754&#160;</td>
			<td>solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Yeast extract&#160;</td>
			<td>BD</td>
			<td style="width:135px;">212750</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">Kit</td>
			<td style="width:128px;"></td>
			<td style="width:135px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AMBION DNase kit&#160;</td>
			<td style="width:128px;">Thermofisher</td>
			<td>10792877</td>
			<td>kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DNA Agilent Chip</td>
			<td style="width:128px;">Agilent</td>
			<td>5067-1504</td>
			<td>kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GeneJET Plasmid Miniprep kit&#160;</td>
			<td style="width:128px;">Thermofisher</td>
			<td style="width:135px;">K0503</td>
			<td>kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PureLink PCR Purification kit</td>
			<td style="width:128px;">Invitrogen</td>
			<td style="width:135px;">K310001</td>
			<td>kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Quant-It&#34; assays kit</td>
			<td style="width:128px;">Thermofisher</td>
			<td style="width:135px;">Q33140/Q32884</td>
			<td>kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">T4 DNA ligase&#160;</td>
			<td style="width:128px;">Invitrogen</td>
			<td style="width:135px;">Y90001</td>
			<td>kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TruSeq Stranded RNA LT prep kit</td>
			<td style="width:128px;">Illumina</td>
			<td>15032611</td>
			<td>kit</td>
		</tr>
	</tbody>
</table>

identification of virulence markers of mycobacterium abscessus for intracellular replication in phagocytes

区分脓肿分枝杆菌与其他腐生分支杆菌的区别是能够抵抗人类巨噬细胞吞噬功能, 以及在这种细胞内繁殖的能力。这些毒力特征使m. 脓肿致病性, 特别是在易受感染的宿主与潜在的结构性肺病, 如囊性纤维化, 支气管扩张症或肺结核。患者如何感染 m.脓肿仍然不清楚。与许多分枝杆菌不同, m. 脓肿在环境中找不到, 但可能居住在阿米巴, 环境吞噬, 代表m 脓肿的潜在水库。事实上, m. 脓肿对 amoebal 吞噬功能有抵抗力, 而在实验性感染模型中, 变形虫的生命似乎增加了m 脓肿的毒力。然而, 对m. 脓肿的毒力本身知之甚少。为了破译对m 脓肿细胞内生命具有优势的基因,脓肿座子突变体文库进行了筛选。同时, 建立了阿米巴共培养后细胞内分枝杆菌的 RNA 提取方法。验证了该方法, 并允许细胞内全m 脓肿transcriptomes 的测序;第一次提供了关于m. 脓肿适应细胞内生活的全球观点。这两种方法都让我们对m. 脓肿致病因子的洞察力, 使m 脓肿能够在人类中殖民呼吸道。

m. 脓肿具有抵御和逃避巨噬细胞和环境原生动物如阿米巴的杀菌反应的能力。m. 脓肿在接触阿米巴时表达毒力因子, 使其在小鼠中更具毒性4。这些方法的第一个目的是识别m 脓肿中存在的基因, 使其在阿米巴中存活和增殖。
为此, 由Tn交付9获得的m . 脓肿</e...

Watch this Scientific Journal Video about Identification of Virulence Markers of Mycobacterium abscessus for Intracellular Replication in Phagocytes at JoVE.com

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

在这里, 我们提出两个协议来研究吞噬-分枝杆菌脓肿的相互作用: 座子突变文库的筛选和 RNA 中细菌细胞内转录的测定测 序。这两种方法都提供了对基因组优势和 transcriptomic 适应增强细胞内细菌适应性的洞察力。

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 ...

细胞内病原体的突变库筛选和转录研究可以帮助回答有关在宿主体内生存的病原体的基因组和调控适应的关键问题。这些技术的主要优点是，它们是大规模调查技术，并且提供了对细胞内寿命适应的客观洞察。这些技术的影响延伸到与这种疾病关联的分枝杆菌脓肿的治疗，通过深入了解疫苗治疗的潜在目标。首先在室温下75平方厘米组织培养瓶中培养30毫升Pepton酵母葡萄糖培养基中的阿米巴阿坎塔莫巴卡斯特拉尼或Ac。一小时后，使用25毫升移液器去除上流液，用10毫升的交流缓冲液洗三次，每次洗涤时不带碳或氮气。上次洗涤后，用一次剧烈的摇动将阿米巴从烧瓶底部分离，并调整猎鹰管中的细胞，在新鲜 Ac 缓冲液中每密耳集中五次 10 到五个阿米巴浓度。种子五倍 10 到四阿米巴每井在 96 井板。将板在32摄氏度下放置一小时，不摇晃，让漂浮的阿米巴龙虫粘附在井上。同时，在冰上加热细菌，并在孵化结束时将解冻的细菌的5微升加入到每一个井中。在 32 摄氏度下 1.5 小时后，将上经剂倒置在烟罩下消毒金属托盘中的无菌纱布上，将上经下一杯丢弃。每井用200微升的新鲜交流介质清洗每井三次。上次洗涤后，用20微升的新鲜培养剂孵育，每井辅以阿米卡辛，以消除任何剩余的细胞外分枝杆菌两小时。接着是三次洗在新鲜的Ac介质，刚刚证明。然后，添加200微升的介质，并辅以新鲜的阿米卡辛到每一个井，然后添加5倍10到7热灭活大肠杆菌。48小时后，将每井10微升10%多西基硫酸钠的细胞在32摄氏度下，在32摄氏度下30分钟，并在含有5%羊血的哥伦比亚阿加盘子里传播5微升的碱酸盐。通过在每滴上加入25微升无菌水，进一步稀释酸盐。当所有的落酸都镀上时，用塑料石蜡膜密封板，并在37摄氏度下孵育培养物三到四天。当菌落出现在板块上时，拍摄培养物，并识别细菌菌落数量最低的沉积物中受损的突变体，促进细胞内生长。对于细胞内分枝杆菌RNA提取，首先在7H9介质中生长M.脓肿，并在120RPM下在50毫升管中补充甘油和葡萄糖至中指数阶段。在培养结束时，每次洗涤用10毫升的Ac缓冲液洗两次细菌，并测量O.D.600以估计细菌浓度。接下来，在8个分枝杆菌中加入8.5倍10，将6倍10添加到6个阿米巴的新鲜Ac介质中，在30摄氏度和80RPM下孵育一小时。在孵化结束时，通过离心收获细胞，并在相同的离心机条件下，每次洗涤10毫升新鲜Ac缓冲液中洗涤三次。然后将细胞重新悬浮在10毫升新鲜Ac缓冲液中，辅以阿米卡辛，以消除任何细胞外细菌，并在30摄氏度和80RPM下孵育细胞4小时，然后在新鲜Ac缓冲液中洗两次。上次洗涤后，将受感染的阿米巴重新悬浮在四毫升的冷溶液三中，辅以 280 微升的β甲醇，以在冰上破坏阿米巴 10 分钟。在孵育结束时，将细胞离心分离，对释放的细胞内细菌进行颗粒化，并在一毫升的冷溶液三中重新悬浮细菌颗粒。清除所有上一代，以避免被阿米巴RNA或可能制造样品的蛋白酶污染，这一点很重要。用第二个离心收集释放的细菌，并在一毫升的萃取缓冲液中重新悬浮颗粒。然后在负80摄氏度下孵育24小时。将分枝杆溶液转移到两个毫升螺杆中，其中含有500微升梯度标记的硅珠，并在负80摄氏度下将管再储存24小时，以方便RNA合成活化和细胞溶解。分析当天，在冰上解冻样品，在6000 RPM下用两轮均质化细胞，25秒，随后在6000 RPM下进行一轮均质化20秒。在每轮均质之间，在冰上冷却样品一分钟，以避免RNA降解。第三轮均质化后，在冰上冷却样品20分钟，并在每管中加入200微升在异丙醇中稀释的氯仿。涡流一分钟，然后离心样品，并在水相中加入0.8毫升的冷异丙醇，在负20摄氏度下孵育2至3小时。在孵化结束时，再次离心样品，并在500微升的冷70%乙醇中清洗颗粒，无需混合。然后立即通过离心去除乙醇。吸制上一液，用于在离心浓缩器中干燥颗粒，并在 100 微升脱氧核糖核酸/无RNA水中重新悬浮颗粒。分枝杆菌 MRNA 提取如所证明的，允许通过根据基因在响应其营养和矿物质来源有限的环境或低氧或酸性环境方面的角色对诱导和抑制基因家族进行分组，对诱导和抑制基因家族进行评估。在抗氧化和亚硝化应激，或在表达毒性因素的反应环境阿米巴。在尝试这些程序时，重要的是要记住同时对受控和受感染样品进行培养和RNA分离，以避免批量效应。按照这个程序，其他方法，如双RNA测序可以预先形成，以回答有关宿主病原体相互作用的其他问题。开发后，该技术为微生物学领域的研究人员在阿米巴宿主模型中实现分枝杆菌毒性铺平了道路。不要忘记，使用阿米巴可能非常危险，因为如果您不按照程序中描述的步骤操作，阿米巴可能会变成囊肿。

Research

JoVE Journal

Immunology and Infection

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

Dissecting Host-virus Interaction in Lytic Replication of a Model Herpesvirus

解剖宿主 - 病毒相互作用模型疱疹病毒裂解性复制

In response to viral infection, a host develops various defensive responses, such as activating innate immune signaling pathways that lead to antiviral ...

科研

免疫与感染

Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies

Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies

样品制备<em&gt;结核分枝杆菌</em提取物对核磁共振的代谢组学研究

Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) ...

Growth of Mycobacterium tuberculosis Biofilms

Growth of Mycobacterium tuberculosis Biofilms

增长结核分枝杆菌生物膜

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including ...

Purification of Pathogen Vacuoles from Legionella-infected Phagocytes

Purification of Pathogen Vacuoles from Legionella-infected Phagocytes

病原液泡的分离纯化<em&gt;军团</em&gt;感染吞噬细胞

The opportunistic pathogen Legionella pneumophila is an amoeba-resistant bacterium, which also replicates in alveolar macrophages thus causing the severe ...

Following in Real Time the Impact of Pneumococcal Virulence Factors in an Acute Mouse Pneumonia Model Using Bioluminescent Bacteria

在实时以下的肺炎球菌毒力因子在小鼠急性肺炎模型的影响采用发光细菌

Pneumonia is one of the major health care problems in developing and industrialized countries and is associated with considerable morbidity and mortality. ...

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 ...

Deciphering and Imaging Pathogenesis and Cording of Mycobacterium abscessus in Zebrafish Embryos

Deciphering and Imaging Pathogenesis and Cording of Mycobacterium abscessus in Zebrafish Embryos

破译和成像发病机制与盘带<em&gt;脓肿分枝杆菌</em&gt;斑马鱼胚胎

Zebrafish (Danio rerio) embryos are increasingly used as an infection model to study the function of the vertebrate innate immune system in host-pathogen ...

Development and Identification of a Novel Subpopulation of Human Neutrophil-derived Giant Phagocytes In Vitro

Development and Identification of a Novel Subpopulation of Human Neutrophil-derived Giant Phagocytes In Vitro

开发和鉴定人的中性粒细胞源性巨噬细胞新型亚群<em&gt;体外</em

Neutrophils (PMN) are best known for their phagocytic functions against invading pathogens and microorganisms. They have the shortest half-life amongst ...

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

系统抑制剂靶向细胞内的疗效和毒性筛选<em&gt;结核分枝杆菌</em

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread ...

Quantification of Intracellular Growth Inside Macrophages is a Fast and Reliable Method for Assessing the Virulence of Leishmania Parasites

Quantification of Intracellular Growth Inside Macrophages is a Fast and Reliable Method for Assessing the Virulence of Leishmania Parasites

巨噬细胞内细胞内生长的定量化是评价利什曼原虫寄生虫毒力的快速可靠方法

The lifecycle of Leishmania, the causative agent of leishmaniasis, alternates between promastigote and amastigote stages inside the insect and vertebrate ...