从果蝇幼虫中提取包囊微生物感染及分析
Summary
该方法展示了如何用三维 (3D) 模型来可视化病原体侵入昆虫细胞。包囊从果蝇幼虫感染病毒或细菌病原体, 无论是体内或体内。感染的包囊然后固定和染色的成像与共聚焦显微镜和随后的3D 细胞重建。
Abstract
在果蝇的致病性感染中,包囊在整个感染的免疫应答中起着重要作用。因此, 本议定书的目的是开发一种方法, 以可视化病原体入侵在特定的免疫室的苍蝇, 即包囊。使用此处提供的方法, 可从 200果蝇3rd龄幼虫到30分钟 (前体感染) 中获得多达 3 x 106实时包囊。或者, 通过注射 3rd龄幼虫, 然后全血细胞提取多达24小时后感染, 包囊可以感染体内。这些受感染的主要细胞是固定的, 染色, 并使用共聚焦显微镜成像。然后, 从图像中生成3D 表示, 最终显示病原体侵袭。此外, 还可以获得高质量的 qRT PCR RNA, 用于检测感染后的病原 mRNA, 并从这些细胞中提取足够的蛋白质进行印迹分析。结合, 我们提出了一个确定的方法, 对病原体入侵和证实感染的细菌和病毒病原体类型和有效的全血细胞提取方法获得足够的活包囊从果蝇用于前体和体内感染实验的幼虫。
Introduction
果蝇是一种成熟的模型有机体, 用于研究先天免疫的1。在先天免疫反应中, 包囊在对病原体的挑战中起着重要的作用。包囊是关键的封装寄生虫, 以及有一个重要的功能, 通过吞噬行动在真菌, 病毒和细菌感染的病原体2,3。
为了更好地了解宿主对致病性微生物感染的先天免疫反应, 重要的是要想象病原体是如何侵入宿主细胞感染的。这种可视化有助于理解入侵的机制。这些数据连同病原体细胞内定位和细胞反应的细节, 可以提供关于宿主对感染的反应和微生物相互作用的细胞器的线索。因此, 采用显微成像技术进行3D 模型重建, 有助于确定宿主细胞病原体的准确定位。在本研究中, 我们可视化了贝柯克斯柯克斯 (柯克斯) 的入侵, “Q 发热” 的致病剂, 这是一种对人类和动物健康构成严重威胁的人畜共患病病毒, 成为主要的果蝇包囊. 最近, 据证明,果蝇易受生物安全级别2九英里第二阶段 (NMII) 克隆4菌株的C. 柯克斯的影响, 该菌株能够在果蝇4中复制, 表明果蝇可作为模型有机体研究C. 柯克斯发病机制。
以前的研究已经使用包囊检查宿主的先天免疫反应。包囊已用于形态学观察5,6,7, 形态分析2,8, 吞噬功能分析2,3, qRT PCR2, 9, 免疫沉淀 10, 11, 免疫荧光分析 10, 12, 染色 13, 免疫印迹法3,10, 11和免疫组化9,14。尽管果蝇S2 细胞也可用于各种体外实验, 但永生和潜在的病毒感染可能会改变其行为15,16。使用主细胞, 而不是永生化细胞系, 如 S2 细胞, 允许研究先天免疫功能的系统更具代表性的整个有机体。此外, 在提取之前, 包囊体内的感染允许细胞与其他宿主蛋白和组织进行交互, 这比在前体感染之前提取包囊更有利。许多不同的方法被用来在短时间内获得足够数量的包囊, 以保持包囊活着的8,17,18,19。
在本研究中, 我们提出了一种从果蝇3rd龄幼虫中提取包囊的方法, 用于致病性微生物感染C. 柯克斯,李斯特菌增生(李斯特菌), 或无脊椎动物彩虹病毒 6 (IIV6)。我们描述了体内和前体全血细胞感染的方法。体内-和前体感染的包囊通过共焦显微镜进行可视化, 用于构建C 柯克斯入侵的3D 模型。此外, 使用提取协议, 体外感染的包囊被用于基因和蛋白质表达测定. 具体地说, 为了检查感染的程度与 IIV6 和李斯特菌, 总 RNA 或蛋白质被分离从细胞为 qRT PCR 或西方印迹分析。结合起来, 该协议提供了从3个rd龄幼虫中快速收集大量包囊的方法, 并证明主要包囊感染了体内或前体内, 是一种适合微生物的平台。病原体感染研究及适用的下游分析, 如显微术、转录组学和蛋白质组学。
Protocol
1.体内感染 介质和设备 在无菌条件下, 准备新鲜的果蝇全血细胞隔离培养基 (DHIM), 含有75% 施耐德的果蝇培养基, 25% 胎牛血清 (血清) 和过滤消毒。 层2-3 片10厘米 x 10 厘米的石蜡膜下显微镜。 准备玻璃毛细管。将毛细管拉拔加热器设置为最大值的55%。将毛细管拉到大约10µm 的尖点。 用矿物油回填毛细管。 将填充的毛细管装入 nanoinj…
Representative Results
要收集前体感染的实时包囊, 可从 200果蝇3rd龄幼虫中提取多达 3×106包囊。为了发展我们的方法, 我们尝试了许多不同的技术。个体幼虫解剖可达1.5 小时, 使用此方法18获得平均8000个细胞, 其中大部分在采集结束时未存活。接下来, 我们试图提取血淋巴, 其中包含包囊, 从20幼虫一次使用玻璃毛细管管19, 但毛细?…
Discussion
为了更好地了解宿主细胞是如何感染的, 重要的是要澄清细胞中病原体的定位, 尤其是在试验以前未经测试的病原体和细胞类型组合4时。在对感染后细胞反应级联进行研究时, 可能表明有生产性致病菌侵袭, 而影像学和细胞反应数据的结合对于证明病原菌侵袭和感染是必不可少的。虽然有报告显示病原体侵入宿主细胞的2D 图像往往表明有生产性感染, 但仍有一些问题可能存在于宿?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢罗伯特 Heinzen 博士提供了 mCherry 表达贝柯克斯柯克斯的股票。我们感谢路易斯特谢拉博士提供的无脊椎动物彩虹病毒6和布卢明顿股票中心提供飞行股票。该项目部分由 NIH 赠款 R00 AI106963 (A.G.G.) 和华盛顿州立大学资助。
Materials
| Schneider's Drosophila Medium | Thermo Fisher Scientific (Gibco) | 21720024 | 1.1.1), 2.1.2) |
| Fetal Bovine Serum | GE Healthcare Life Sciences (HyClone) | SH30070.03HI | 1.1.1), 2.1.2) |
| Filter (0.22 µL) | RESTEK | 26158 | 1.1.1) |
| Strainer (100 µm) | Greiner bio-one | 542000 | 1.2.1), 2) |
| Stereo microscope | Amscope | SM-1BSZ-L6W | 1.2), 2) |
| Glass capillary | Fisher Scientific | 21-171-4 | 1.1), 1.2), 2) |
| Capillary puller | Narishige International USA, Inc. | PC-10 | 1.1.3) |
| Mineral oil | Snow River Products | 1.1.4) | |
| Nanoinjector | Drummond Scientific Company | 3-000-204 | 1.1), 1.2), 2.2) |
| Forceps | VWR | 82027-402 | 1.1.5), 1.2), 2), 3.1.7) |
| CO2 delivery apparatus | Genesee Scientific | 59-122BC | 1.2), 2) |
| Trypan Blue | Thermo Fisher Scientific (Gibco) | 15250061 | 1.3) |
| Hemocytometer | Hausser Scientific | 3100 | 1.3) |
| 24 well plate | Greiner bio-one | 662160 | 1.4), 2.2) |
| Coxiella burnetii – mCherry | Dr. Heinzen, R. | 1.4), 2.2) | |
| Drosophila fruit juice plates | Cold Spring Harbor Protocols | 2.1) http://cshprotocols.cshlp.org/content/2007/9/pdb.rec11113.full | |
| Agar | Fisher Bioreagents | BP1423-500 | 2.1.1.1) |
| Methyl paraben | Amresco | 0572-500G | 2.1.1.2) |
| Absolute ethanol | Fisher Bioreagents | BP2818-500 | 2.1.1.2) |
| Welch's 100% Grape juice frozen concentrate, 340 mL | Amazon | B0025UJVGM | 2.1.1.3) |
| Petri dishes, 10 x 35 mm | Fisher Scientific | 08-757-100A | 2.1.1.4) |
| Microscope cover glass | Fisher Scientific | 12-545-80 | 1.4.4), 2.2.2) |
| Yeast, Bakers Dried Active | MP Biomedicals | 0210140001 | 2.1) Add 2 parts of water to 1 part of yeast (v/v) |
| Tungsten needle | Fine Science Tools | 10130-20 | 2.1) |
| Holding forceps | VWR | HS8313 | 2.1) |
| Paraformaldehyde | Fisher Scientific | FLO4042-500 | 3.1.3) |
| Triton X-100 | Fisher Scientific | BP151-500 | 3.1.3) |
| Bovine Serum Albumin | Fisher Scientific | BP9706-100 | 3.1.3) |
| 4',6-diamidino-2-phenylindole | Thermo Fisher Scientific | 62247 | 3.1.4) |
| Antifade mounting medium | Thermo Fisher Scientific | P36930 | 3.1.6) |
| Confocal microsope | Leica | TCS SP8-X White Light Confocal Laser Scanning Microscope | 3.2) |
| 3D imaging reconstruction software | Leica | LASX with 3D visualization module | 3.3) |
| Microscope slides | Fisher Scientific | 12-552-3 | 3.1.6) |
| Invertebrate iridescent virus 6 (IIV6) | Dr. Teixeria, L. | 4) PLoS Biol, 6 (12), 2753-2763, doi: 10.1371/journal.pbio.1000002, (2008) | |
| Listeria monocytogenes | ATCC | strain: 10403S | 4) Listeria monocytogenes strain 10403S (Bishop and Hinrichs, 1987) was grown in Difco Brain-heart infusion (BHI) broth (BD Biosciences) containing 50 µg/ml streptomycin at 30 °C. |
| DNase I | Thermo Fisher Scientific(Invitrogen) | 18068015 | gDNA degradation |
| cDNA Synthesis Kit | Bio-Rad | 1708891 | cDNA synthesis |
| IIV6_193R_F | IDT | qRT-PCR, 5'- TCT TGT TTT CAG AAC CCC ATT -3' | |
| IIV6_193R_R | IDT | qRT-PCR, 5'- CAC GAA GAA TGA CCA CAA GG -3' | |
| RpII_qRTPCR_fwd | SIGMA-ALDRICH | qRT-PCR, 5'- GAA GCG TTT CTC CAA ACG -AG | |
| RpII_qRTPCR_rev | SIGMA-ALDRICH | qRT-PCR, 5'- TTG AGC GTA AGC ATC ACC -TG | |
| SYBR Green qRT-PCR reagent | Thermo Fisher Scientific | K0251, K0252, K0253 | qRT-PCR |
| Real-Time PCR System | Thermo Fisher Scientific | 4351107, 7500 Software v2.0 | qRT-PCR |
| Anti-Listeria monocytogenes antibody | abcam | ab35132 | Western blot |
| Anti-Actin antibody produced in rabbit | SIGMA-ALDRICH | A2066 | Western blot |
| Anti-Rabbit IgG (H+L), HRP Conjugate | Promega | W4011 | Western blot |
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Cite This Article
Hiroyasu, A., DeWitt, D. C., Goodman, A. G. Extraction of Hemocytes from Drosophila melanogaster Larvae for Microbial Infection and Analysis. J. Vis. Exp. (135), e57077, doi:10.3791/57077 (2018).