一种高通量、高含量、液态的 Pathosystem线虫
Summary
在这里, 我们描述了一个协议, 它是一个适应性强, 全宿主, 高内容筛选工具, 可用于研究宿主病原体相互作用, 并用于药物发现。
Abstract
传统的、体外筛查到的新药数量已经减退, 这就减少了这种方法在寻找新武器以对抗多种耐药性方面的成功。这就得出结论, 研究人员不仅需要找到新药, 还需要开发新的方法来找到它们。其中最有希望的候选方法是全有机体,在体内的检测, 使用高通量, 表型读数和寄主, 范围从秀丽线虫到斑马斑马。这些主机有几个强大的优势, 包括显着减少假阳性命中, 因为有毒的化合物, 对主机和/或 biounavailable 通常下降在最初的屏幕上, 在昂贵的后续行动之前。
在这里, 我们展示了我们的检测如何被用来审问寄主变异的良好记录的C. 线虫–铜绿假单胞菌液体杀死 pathosystem。我们还演示了这种精心编制的技术的几个扩展。例如, 我们能够使用24或96井板格式的 rna 干扰来进行高通量的基因筛, 以查询宿主-病原体相互作用中的宿主因素。使用这项化验, 整个基因组屏幕只能在几个月内完成, 这可以大大简化确定药物目标的任务, 可能不需要费力的生化纯化方法。
我们还报告了我们的方法的变化, 取代革兰氏阳性菌肠球菌的革兰氏阴性病原菌P.与铜绿假单胞菌的情况一样, 由大肠杆菌杀死是有时间依赖性的。与以前的 C.粪肠球菌化验不同, 我们对粪肠球菌的化验不需要 preinfection, 改善其安全状况, 减少污染液体处理设备的几率。该检测具有高度健壮性, 显示95% 死亡率 96 h 后感染。
Introduction
在近一个世纪以前, 确定和发展有效的广谱抗生素导致了公共卫生的一个分水岭时刻, 人们普遍认为, 传染病将是过去的祸害。在短短的几十年里, 这种乐观情绪开始消退, 因为病原体形成的抗病机制限制了这些曾经神奇的治疗。一段时间内, 药物发现努力与病原体之间的军备竞赛似乎是平衡的。然而, 滥用抗菌药物最近终于出现了耐药菌株的肺炎克雷伯杆菌, 不动杆菌鲍曼,沙雷质, 和铜绿假单胞菌1, 2,3,4。
铜绿假单胞菌是一种机会主义, 革兰阴性, 多宿主病原体, 严重威胁严重烧伤患者, 那些谁是免疫力低下, 或有囊性纤维化。它也日益被认定为严重医院感染的致病剂, 特别是由于其持续的耐药性的获得。为了应对这一威胁, 我们使用了记录良好的C.铜绿假单胞菌感染系统5。我们的实验室利用这个系统开发了一种基于液体的高通量高含量的筛选平台, 以确定新的化合物, 限制病原体杀死宿主6的能力。有趣的是, 这些化合物似乎属于至少三个一般类别, 包括抗菌素7和毒力抑制剂8。 tuberculosum 分枝杆菌、沙眼衣原体、鼠疫耶尔森菌、李斯特氏细胞增生、土拉弗 tularensis、金黄色葡萄球菌、白念珠病及粪肠球菌, 除其他9,10,11,12,13,14,15,16。这些类型的化验有几个公认的优势, 如限制假阳性命中可能对宿主和病原体, 增加生物有效性的可能性相比, 化学屏幕, 并有能力识别命中超出简单地限制微生物的生长, 例如抗 virulents, 免疫刺激分子, 或其他使宿主-病原体相互作用的平衡倾斜的化合物, 有利于前者。此外, 在这些屏幕上发现的化合物通常在哺乳动物宿主中有效。
值得注意的是, 至少有另外两种化验17,18可以在液体中的C. 线虫中进行高通量屏幕。然而, 每一个这些化验是一个修改, 允许原型肠道殖民化试验, 称为慢杀, 以液体进行, 增加的吞吐量, 并允许化合物更容易筛选。仔细的描述表明, 细菌毒力的机制是不同的, 这些化验和我们的液基筛7。由于两种类型的毒力都观察到哺乳动物系统, 重要的是要考虑哪些毒力决定因素是最相关的实验者的利益之前的化验选择。
在这里, 我们演示了一个优化版本的液态C 线虫-铜绿假单胞菌。我们还报告了我们的液基检测方法的适应性, 以适应革兰氏阳性菌粪肠球菌.与铜绿假单胞菌一样,粪肠球菌越来越多地被认定为严重的医院威胁, 并不断增加的武器耐药性途径1。尽管以前的高通量筛选方法存在14, 但它需要 preinfection 病原体, 这就使程序复杂化, 增加了诸如 COPAS FlowSort 等污染设备的可能性。我们的协议消除了感染前的需要, 改善了安全配置。最后, 我们报告一种方法, 其中任一检测可以结合喂养 rna 干扰, 允许用户搜索的宿主因素, 发挥作用, 建立或抵抗, 感染。
Protocol
Representative Results
Discussion
这种化验 (或类似的化验, 其他病原体被替代的铜绿假单胞菌或大肠杆菌) 是有用的各种目的, 包括药物发现。它还有助于解决基本的生物学问题, 如确定毒力因素, 阐明宿主防御通路, 并确定参与宿主-病原体相互作用的调控机制。
虽然铜绿假单胞菌液体杀灭试验具有较强的鲁棒性, 但有几点需要认真注意, 以尽量减少失败的几率。首先, 如前所述, 对铜绿假单…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项研究得到了德州癌症预防研究所 (CPRIT) 奖 RR150044、韦尔奇基金会研究补助金 C-1930 以及由国立卫生研究院 K22 AI110552 授予 NVK 的支持。资助者在研究设计、数据收集和分析、决定出版或编写手稿方面没有任何作用。
Materials
| COPAS FP BioSorter | Union Biometrica | Large object flow cytometer/worm sorter | |
| Cytation 5 | BioTek | ||
| EL406 Washer Dispenser | BioTek | ||
| Multitron Pro | Infors HT | ||
| 24 Deep-Well RB Block | Thermo Fisher Scientific | CS15124 | |
| 384-Well plate | Greiner Bio-One | MPG-781091 | |
| Nematode Growth Media (NGM) | Amount per liter: 18 grams agar, 3 grams NaCl, 2.5 grams Peptone, 1 mL CaCl2 (1 M), 1 mL MgSO4 (1 M), 25 mL Phospate buffer, and 973 mL of milli-Q water | ||
| Slow Killing (SK) plates | Amount per liter: 18 grams agar, 3 grams NaCl, 3.5 grams Peptone, 1 mL CaCl2 (1 M), 1 mL MgSO4 (1 M), 25 mL Phospate buffer, and 973 mL of milli-Q water | ||
| Slow Killing (SK) media | Amount per liter: 3 grams NaCl, 3.5 grams Peptone, 1 mL CaCl2 (1 M), 1 mL MgSO4 (1 M), 25 mL Phosphate buffer, and 973 mL of milli-Q water | ||
| Lysogeny Broth (LB) | USBiological Life Sciences | L1520 | |
| Brian Heart Infusion broth (BHI) | Research Products International Corp | 50-488-526 | |
| Worm Bleach Solution | Amount per 100 mL: 10 mL of 5 M NaOH solution, 20 mL of 5% Sodium Hypochlorite Solution, and 70 mL of sterile water | ||
| S Basal | Amount per liter: 5.85 grams NaCl, 6 grams KH2PO4, 1 gram K2HPO4, and 1 Liter of milli-Q water | ||
| Agar | USBiological Life Sciences | A0930 | |
| NaCl | USBiological Life Sciences | S5000 | |
| Peptone | USBiological Life Sciences | P3300 | |
| CaCl2 | USBiological Life Sciences | ||
| MgSO4 | Fisher Scientific | M63-500 | |
| Phospate buffer | amount per liter: 132 mL of K2HPO4 (1M) and 868 mL of KH2PO4 (1M) | ||
| KH2PO4 | Acros Organics | 7778-77-0 | |
| K2HPO4 | USBiological Life Sciences | P5100 | |
| 5% Sodium Hypochlorite Solution | BICCA | 7495.5-32 | |
| NaOH solution | Fisher Scientific | SS255-1 | |
| Breathe-easy | Diversified Biotech | BEM-1 | |
| SYTOX Orange Nucleic Acid Stain | Fisher Scientific | S11368 | |
| Bacterial Strains | |||
| P. aeruginosa (PA14) | |||
| E. faecalis(OG1RF) | |||
| E. coli superfood (OP50) | |||
| E. coli RNAi expressing bacteria (HT115) | |||
| Worm Strains | |||
| glp-4(bn2) (Beanan and Strome, 1992, PMID: 1289064) | |||
| PINK-1::GFP reporter (Kang et al., 2018, PMID: 29532717) |
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