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线虫线虫线虫-一个多用途的体内模型研究宿主-微生物相互作用

Published: October 18, 2017
doi:
10.3791/56487
, ,
1Biology and Biotechnology,Worcester Polytechnic Institute

Summary

在这里, 我们提出线虫线虫线虫作为一个多用途的寄主模型来研究微生物的相互作用。

Abstract

我们演示了一种使用线虫线虫作为模型宿主来研究微生物相互作用的方法。通过饮食引入微生物, 使肠道成为疾病的主要部位。线虫肠道结构和功能模仿哺乳动物的肠道, 是透明的, 使其适于对殖民的微观研究。在这里, 我们表明病原体可以导致疾病和死亡。我们能够识别出具有改变毒性的微生物突变体。它对生物胁迫的固有反应使线虫成为一个极好的系统, 可以探测宿主先天免疫相互作用的方方面面。我们发现, 双氧化酶基因突变的寄主不能产生活性氧, 无法抵抗微生物的侮辱。我们进一步证明了所提出的生存试验的通用性, 表明它可以用于研究微生物生长抑制剂的影响。这种方法也可用于发现真菌致病因子作为发展新的抗真菌药物的目标, 以及提供一个机会, 进一步揭示宿主-微生物相互作用。这种检测方法的设计很好地适应了高通量全基因组的屏幕, 而低温保存蠕虫以备将来使用的能力使得它成为一个 cost-effective 和有吸引力的整体动物模型来研究。

Introduction

线虫已被用作一个强大的模型有机体超过50年。在二十世纪六十年代, 南非生物学家悉尼. 博纳率先使用了线虫来研究神经元的发育, 为长期的科学家们研究线虫的细胞和动物生物学的各个方面铺平了道路。这一血统包括诺贝尔奖得主克雷格. 梅洛和安德鲁火为他们的 rna 干扰工作1, 罗伯特 Horvitz 并且约翰 Sulston 为他们的工作器官发展和细胞凋亡2,3,4, 和马丁查尔菲为他的工作绿色荧光蛋白5。尽管这种模型生物体已经被传统用于研究分子和发育生物学, 但在过去15年中, 研究人员已经开始使用线虫来调查各种人类病原体的生物学, 包括假单胞菌铜绿假单胞菌,金黄色葡萄球菌,沙门氏菌 enterica, 和沙雷菌6,7,89,10。这些研究表明, 许多涉及人与病原体相互作用的机制都是在线虫中保存的, 但也有一些免疫机制是独特的, 这种模式生物体11,12。在自然界中,线虫遇到了来自土壤中的受感染病原体的各种威胁, 这为进化和维持一个复杂的先天免疫系统在其肠道腔内提供了强烈的选择性压力。许多涉及保护肠道腔的基因和机制都是由高度保守的元素组成的, 它们也存在于高哺乳动物中11,13。因此,线虫是研究胃肠病原体的一个很好的模型, 如沙门氏菌 enterica14,痢疾 boydii15, 或霍乱弧菌16。

在这里, 我们强调了显着的多功能性的c. 线虫作为一个模型的宿主, 以研究传染性药物, 如C. 白色念珠菌。C. 线虫作为模型宿主, 可以对毒性进行高通量筛选, 这比小鼠模型更便宜、更耗时, 通常用于研究念珠菌病42。

在这项研究中, 我们表明, 该模型和 assosiated 生存分析可以可靠地用于研究宿主先天免疫效应的重要性, 以抵消感染, 病原体的决定因素驱动毒力, 和药理化合物, 可以干预发病机制。与先前所描述的化验方法不同, 此法提供了一种研究动物生命周期中的病原体, 从幼虫阶段到成年, 而不是仅成年到死亡的手段43,44。总之, 我们的线虫—白色念珠菌模型是一种多功能、功能强大的工具, 不仅可以用于研究驱动感染和免疫的遗传基础, 还可用于识别治疗干预的新化合物。

Protocol

1. 制备线虫生长培养基 (NGM) 用于 1 l 介质, 结合 20 g 琼脂、2.5 g 有机氮源 ( 如 、bacto-蛋白胨) 和3克氯化钠在 2 l 烧瓶中。加入975毫升的无菌水. 在无菌搅拌栏中添加。如果使用自动介质倒, 高压釜管和介质为15分钟;如果有更高的音量, 介质应更长的时间进行蒸压. 在搅拌板上设置介质, 并允许冷却。 一旦媒体被热触碰 (大约60和 …

Representative Results

病机检测 (图 1) 使用c. 白色念珠菌和c. 线虫以前曾被我们的实验室描述17,18和其他实验室19,,。我们演示了使用可研究 白色念珠菌的毒力, 该病毒显示了c. 白念珠菌的细胞迅速被蠕虫吸收并积聚在肠道内, 导致运动迟缓, 肛门畸形?…

Discussion

用于检测线虫感染和生存期的方法对我们所描述的c. 白色念珠菌的影响可以通过修改来测试另一种病原体。另一种细菌或真菌的液体培养物可以用类似的方式制成并喂给C. 线虫。此外, 串行感染可以通过首先暴露幼虫到一个病原体, 然后转移到一个新的板块, 包含一个独立的病原体后, 成年。

在进行这种化验时, 注意时间是很重要的。首先, 当完成鸡蛋准备, …

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

这项工作是在伍斯特理工学院的支持下进行的。

Materials

Agar (granulated, bacterilogical grade) Apex BioResearch Products 20-248
Aluminum Wire (95% Pt, 32 Gauge) Genesee Scientific 59-1M32P
Axiovision Zeiss Inverted Microscope Axiovision Zeiss
Bacto-Peptone Fisher BioReagants BP1420-500
C. elegans strain Bli-3 Caenorhabditis Genetics Center Bli-3(e767) CB767
Calcium Chloride Fisher Scientific BP51-250
Cholesterol, Sigma Grade, minimum 99% Sigma C8667-25G
Disposable Culture Tubes (20 x 150 mm) FIsherBrand 14-961-33
Dissection Microscope (NI-150 High Intensity Illuminator) Nikon Instrument Inc.
E. coli Caenorhabditis Genetics Center OP50
GraphPad Prism (Survival Curve Analysis Software) GraphPad Software
LB Broth (Miller's) Apex BioResearch Products 11-120
Magnesium Sulfate Fisher Scientific 10034-99-8
Medium Petri Dishes (35 X 10 mm) Falcon 353001
Potassium Phosphate monobasic Sigma P0662-500G
Sodium Chloride Fisher Scientific BP358-1
Sodium Phosphate Fisher Scientific BP332-500
Wildtype C. albicans SC5314 ATCC SC5314
Wildtype C. elegans Caenorhabditis Genetics Center N2
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Tags

Keywords: Caenorhabditis ElegansIn Vivo ModelHost-microbe InteractionsFungal InfectionPathogen VirulenceHost Immune ResponseDrug DiscoveryInfectious DiseaseBacteriaMicrobial InteractionsWorm PickingAgar ChunkingM9 BufferOP50 E. Coli
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Issi, L., Rioux, M., Rao, R. The Nematode Caenorhabditis Elegans – A Versatile In Vivo Model to Study Host-microbe Interactions. J. Vis. Exp. (128), e56487, doi:10.3791/56487 (2017).
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