吞噬细胞内复制脓肿分枝杆菌毒力标志物的鉴定
Summary
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。
Abstract
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。
Introduction
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。
Protocol
1. 图书馆筛选 Tn突变体库的构建 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?…
Representative Results
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…
Discussion
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…
Acknowledgements
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。
Materials
| Name of Material/ Equipment | |||
| 24-well plates | Thermofisher | 11874235 | |
| 96-well plates | Thermofisher | 10687551 | |
| Beadbeater | Bertin | Precellys 24 | |
| Bioanalyzer | Agilent | ||
| Genepulser Xcell | Biorad | ||
| Nanodrop spectrophotometer 2000 | Thermofisher | ||
| QuBit fluorometer | Thermofisher | Q33226 | |
| zirconium beads/silica beads | Biospec products | 11079101Z | Beads |
| Name of reagent/cells | |||
| Acanthamoeba castellanii | ATCC | 30010 | strain |
| Amikacin | Mylan | 150927-A | powder |
| B-mercaptoethanol | Sigma-Aldrich | M6250 | solution |
| CaCl2 | Sigma-Aldrich | C1016 | >93% granular anhydrous |
| Chloroform | Fluka | 25666 | solution |
| ClaI enzyme | New England Biolabs | R0197S | enzyme |
| Columbia agar | Biomerieux | 43041 | 90 mm |
| D-Glucose | Sigma-Aldrich | G8270 | powder |
| DMEM | Thermofisher | 11500596 | medium |
| DNase and RNase free water | Invitrogen | 10977-035 | solution |
| E. coli electrocompetent | Thermofisher | 18265017 | bacteria |
| EDTA | Sigma-Aldrich | E4884 | powder |
| Escherichia coli | Clinical isolate | personal stock | bacteria |
| Fe(NH4)2(SO4)-6H20 | EMS | 15505-40 | sulfate solution 4% aqueous |
| Fetal Calf Serum | Gibco | 10270 | serum |
| Glycerol | Sigma-Aldrich | G5516 | solution |
| Guanidium thiocyanate | Euromedex | EU0046-D | powder |
| Isopropanol | Sigma-Aldrich | I9516 | solution |
| J774.2 macrophages | Sigma-Aldrich | J774.2 | Strain |
| kanamycin | Sigma-Aldrich | 60615 | powder |
| KH2PO4 | Sigma-Aldrich | P0662 | Monobasic, anhydrous |
| LB liquid medium | Invitrogen | 12795-027 | powder |
| Lysozyme | Roche | 10837059001 | powder |
| MgSO4 | Labosi | M275 | pur |
| Microbank TM (cryotubes with beads) | Pro-Lab Diagnostic | PL.170/M | |
| Middlebrook 7H11 medium | Sigma-Aldrich | M0428 | powder |
| Middlebrook 7H9 medium | Thermofisher | 11753473 | powder |
| Müller-Hinton agar | Biorad | 3563901 | powder |
| N-Lauryl-sarcosine | Merck | S37700 416 | powder |
| Na2HPO4-7H2O | Sigma-Aldrich | S9390 | 98-102% |
| Phenol/chloroforme | Sigma-Aldrich | 77617 | solution |
| Proteinase K | Thermofisher | EO0491 | powder |
| proteose peptone | BD | 211684 | enzymatic digest of animal tissue |
| pUC19 plasmid | New England Biolabs | 54357 | plasmid |
| SDS 20% | Biorad | 1610418 | solution |
| Sodium citrate | Calbiochem | 567446 | powder |
| Thiourea | Sigma-Aldrich | 88810 | powder |
| Tris | Sigma-Aldrich | 154563 | powder |
| Trizol | Thermofisher | 12044977 | solution |
| Tween 80 | Sigma-Aldrich | P1754 | solution |
| Yeast extract | BD | 212750 | |
| Kit | |||
| AMBION DNase kit | Thermofisher | 10792877 | kit |
| DNA Agilent Chip | Agilent | 5067-1504 | kit |
| GeneJET Plasmid Miniprep kit | Thermofisher | K0503 | kit |
| PureLink PCR Purification kit | Invitrogen | K310001 | kit |
| Quant-It" assays kit | Thermofisher | Q33140/Q32884 | kit |
| T4 DNA ligase | Invitrogen | Y90001 | kit |
| TruSeq Stranded RNA LT prep kit | Illumina | 15032611 | kit |
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Cite This Article
Dubois, V., Laencina, L., Bories, A., Le Moigne, V., Pawlik, A., Herrmann, J., Girard-Misguich, F. Identification of Virulence Markers of Mycobacterium abscessus for Intracellular Replication in Phagocytes. J. Vis. Exp. (139), e57766, doi:10.3791/57766 (2018).