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Biologia

使用单蠕虫数据量化 秀丽隐杆线虫的异质性 - 细菌相互作用

Published: July 22, 2022
doi:
10.3791/64027
, ,
1Department of Biology,Emory University, 2Department of Physics,Emory University

Summary

该协议描述了在冷麻痹和表面漂白以去除外部细菌后，对单个细菌定植 的秀丽隐杆线虫炎的 96孔破坏。将得到的悬浮液接种在琼脂平板上，以便对大量单个蠕虫中的细菌负荷进行准确的中等通量定量。

Abstract

线虫 秀丽隐杆线虫 是宿主-微生物和宿主-微生物组相互作用的模型系统。迄今为止，许多研究使用间歇消化物而不是单个蠕虫样本来量化该生物体中的细菌负荷。这里认为，在 秀丽隐杆线虫 肠道的细菌定植中看到的较大的个体间变异性是信息性的，并且批量消化方法丢弃了对跨条件准确比较很重要的信息。由于描述这些样品固有的变异需要大量的个体，因此建立了一种方便的96孔板方案，用于单个蠕虫的破坏和菌落电镀。

Introduction

宿主 – 微生物关联的异质性无处不在，个体之间的变异越来越被认为是从竞争和共存1到疾病传播2，3，4的人群水平过程的一个促成因素。在秀丽隐杆线虫中，反复观察到等基因群体内的“隐藏的异质性”，个体亚群在热休克反应5，6，衰老和寿命7，8，9，10，11以及生理学和发育的许多其他方面显示出不同的表型12.大多数试图确定亚种群结构的分析为等基因同步蠕虫实验种群中的两个亚种群提供了证据5，7，8，尽管其他数据表明性状的种群内分布的可能性，而不是不同的群体7，12，13.与此相关的是，即使在从共享的微生物来源定植的蠕虫的等基因群体中，也观察到肠道群体中的大量异质性13，14，15，16，并且这种异质性可以通过批量消化测量来掩盖，该测量广泛用于蠕虫中的细菌定量17，18，19，20。

这项工作提供的数据表明，在宿主 – 微生物关联中需要更多地依赖单蠕虫测量，以及提高单蠕虫破坏准确性和通量的方案。这些方案旨在促进对大量单个 秀丽隐杆线虫 的机械破坏，以定量活的细菌负荷，同时提供比基于杵的单个蠕虫更好的可重复性和更低的每个样品的努力。包括推荐的肠道清除步骤，其中允许蠕虫在准备破坏之前以热杀死 的大肠杆菌 为食，以尽量减少最近摄入的细菌和其他瞬时（非粘附）细菌的贡献。这些方案包括用低浓度表面漂白处理清洁角质层的冷麻痹方法;表面漂白可用作单一蠕虫破坏的准备步骤，或作为制备活的、外部无菌蠕虫的方法。这种表面漂白方法足以去除广泛的外部微生物，冷处理提供了常规左旋咪唑类麻痹的替代方案;虽然左旋咪唑将首选用于冷敏感实验，但冷麻痹可最大限度地减少对危险废物流的贡献，并允许迅速恢复正常活动。虽然完整的方案描述了一个实验室实验，其中蠕虫被已知细菌定植，但清洁蠕虫和单蠕虫破坏的程序可以很容易地应用于从野生样本中分离出或在微观实验中定植的蠕虫。这里描述的方案产生从蠕虫肠中提取的活细菌，适用于单个蠕虫中菌落形成单元（CFU）的电镀和定量;对于基于测序的肠道群落分析，应在这些方案中添加随后的细胞裂解和核酸提取步骤。

Protocol

这些实验中使用的蠕虫是从 Caenorhabditis 遗传中心获得的，该中心由NIH研究基础设施计划办公室（P40 OD010440）资助。布里斯托尔N2是野生型。DAF-2/IGF 突变体 daf-16（mu86） I （CGC CF1038） 和 DAF-2（e1370） III （CGC CB1370） 用于说明肠道细菌负荷的差异。 携带 pos-1 RNAi 载体的大 肠杆菌 HT115 （DE3） 来自阿林格文库21。 秀丽隐杆线?…

Representative Results

活虫的漂白灭菌表面漂白的蠕虫实际上没有外部细菌，直到运动恢复并恢复排泄。在这里使用的条件下，观察到缓冲液中细菌的快速灭绝（图1A-C，补充图2，视频1），而不会干扰冷麻痹蠕虫中的肠道相关细菌（图1D-F，视频2）。这些数据表明，表面漂白可以有效地用于外部消毒蠕虫，而…

Discussion

这里提供了关于 秀丽隐杆线虫中细菌负荷的单蠕虫定量的优势的数据，以及96孔破坏方案，以允许快速和一致地获取这种类型的大型数据集。与现有方法33相比，这些方案允许对蠕虫中的肠道微生物群落进行更高通量的测量。

这种方法将电镀作为速率限制步骤，并不是真正的“高吞吐量”。大物体流式细胞术（图3C，D</stron…

Declarações

The authors have nothing to disclose.

Acknowledgements

作者要感谢H.舒伦伯格和C.拉洛克在这些实验中使用的细菌菌株的慷慨分享。这项工作得到了埃默里大学和NSF（PHY2014173）的资助。

Materials

96-well flat-bottom polypropylene plates, 300 uL Evergreen Labware 290-8350-03F
96-well plate sealing mat, silicon, square wells (AxyMat) Axygen AM-2ML-SQ
96-well plates, 2 mL, square wells Axygen P-2ML-SQ-C-S
96-well polypropylene plate lids Evergreen Labware 290-8020-03L
Agar Fisher Scientific 443570050
Bead mill adapter set for 96-well plates QIAGEN 119900 Adapter plates for use with two 96-well plates on the TissueLyser II
Bead mill tissue homogenizer (TissueLyser II) QIAGEN 85300 Mechanical homogenizer for medium to high-throughput sample disruption
BioSorter Union Biometrica By quotation Large object sorter equipped with a 250 micron focus for C. elegans
Bleach, commercial, 8.25% sodium hypochlorite Clorox
Breathe-Easy 96-well gas permeable sealing membrane Diversified Biotech BEM-1 Multiwell plate gas permeable polyurethane membranes. Thin sealing film is permeable to O2, CO2, and water vapors and is UV transparent down to 300 nm. Sterile, 100/box.
Calcium chloride dihydrate Fisher Scientific AC423525000
Cholesterol VWR AAA11470-30
Citric acid monohydrate Fisher Scientific AC124910010
Copper (II) sulfate pentahydrate Fisher Scientific AC197722500
Corning 6765 LSE Mini Microcentrifuge Corning  COR-6765
Disodium EDTA Fisher Scientific 409971000
DL 1,4 Dithiothreitol, 99+%, for mol biology, DNAse, RNAse and Protease free, ACROS Organics Fisher Scientific 327190010
Eppendorf 1.5 mL microcentrifuge tubes, natural Eppendorf
Eppendorf 5424R microcentrifuge Eppendorf 5406000640 24-place refrigerated benchtop microcentrifuge
Eppendorf 5810R centrifuge with rotor S-4-104 Eppendorf 22627040 3L benchtop centrifuge with adaptors for 15-50 mL tubes and plates
Eppendorf plate bucket (x2), for Rotor S-4-104 Eppendorf 22638930
Ethanol 100% Fisher Scientific BP2818500
Glass beads, 2.7 mm Life Science Products LS-79127
Glass beads, acid-washed, 425-600 µm Sigma G877-500G
Glass plating beads VWR 76005-124
Hydrochloric acid VWR BDH7204-1
Iron (II) sulfate heptahydrate Fisher Scientific 423731000
Kimble Kontes pellet pestle motor DWK Life Sciences 749540-0000
Kimble Kontes polypropylene pellet pestles and microtubes, 0.5 mL DWK Life Sciences 749520-0590
Leica DMi8 motorized inverted microscope with motorized stage Leica 11889113
Leica LAS X Premium software Leica 11640687
Magnesium sulfate heptahydrate Fisher Scientific AC124900010
Manganese(II) chloride tetrahydrate VWR 470301-706
PARAFILM M flexible laboratory sealing film Amcor PM996
Peptone Fisher Scientific BP1420-500
Petri dishes, round, 10 cm VWR 25384-094
Petri dishes, round, 6 cm VWR 25384-092
Petri dishes, square, 10 x 10 cm VWR 10799-140
Phospho-buffered saline (1X PBS) Gold Bio P-271-200
Polypropylene autoclave tray, shallow Fisher Scientific 13-361-10
Potassium hydroxide Fisher Scientific AC134062500
Potassium phosphate dibasic Fisher Scientific BP363-1
Potassium phosphate monobasic Fisher Scientific BP362-1
R 4.1.3/RStudio 2022.02.0 build 443 R Foundation n/a
Scoop-type laboratory spatula, metal VWR 470149-438
Silicon carbide 36 grit MJR Tumblers n/a Black extra coarse silicon carbide grit. Available in 0.5-5 lb sizes from this vendor.
Sodium dodecyl sulfate Fisher Scientific BP166-100
Sodium hydroxide VWR BDH7247-1
Sodium phosphate dibasic anhydrous Fisher Scientific BP332-500
Sodum chloride Fisher Scientific BP358-1
Sucrose Fisher Scientific AC419760010
Tri-potassium citrate monohydrate Fisher Scientific AC611755000
Triton X-100 Fisher Scientific BP151-100
Zinc sulfate heptahydrate Fisher Scientific AC205982500
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Tags

Keywords: Caenorhabditis ElegansBacterial InteractionsSingle-worm DataMechanical Disruption96-well FormatMedium ThroughputQuantificationWorm MeasurementsM9TX01OP50SDS/DTT SolutionChemical PermeabilizationWorm Cuticle
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Taylor, M. N., Spandana Boddu, S., Vega, N. M. Using Single-Worm Data to Quantify Heterogeneity in Caenorhabditis elegans-Bacterial Interactions. J. Vis. Exp. (185), e64027, doi:10.3791/64027 (2022).
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