高通量，实时，细胞内的抗菌活性的双读出测试和真核细胞的细胞毒性
Summary
A high throughput, real-time assay was developed to simultaneously identify (1) eukaryotic cell-penetrant antimicrobials targeting an intracellular bacterial pathogen, and (2) assess eukaryotic cell cytotoxicity. A variation on the same technology was thereafter combined with digital dispensing technology to enable facile, high-resolution, dose-response, and two- and three-dimensional synergy studies.
Abstract
细胞内的抗菌活性和真核细胞的细胞毒作用的传统措施，依靠端点检测。这样的端点检测要求，如细胞裂解，菌落形成单位的确定，或试剂除了读出之前一些额外的实验步骤。时，例如，在高通量筛选执行数以千计测定的，对于这些类型的测定所需要的下游努力是相当大的。因此，为了便于高通量抗菌发现，我们开发了实时测定以同时识别的细胞内细菌生长抑制剂和评估的真核细胞的细胞毒性。具体地讲，实时的细胞内细菌生长的检测用标记与任一细菌勒克斯操纵子（ 第一代测定法）或荧光蛋白记者（ 第二代，正交测定）细菌筛选菌株已启用。无毒的，细胞膜非透性，核酸结合染料巨噬细胞的最初感染过程中也被加入。这些染料被排除在活细胞。然而，非存活的宿主细胞失去膜完整性允许进入和核DNA的荧光标记（脱氧核糖核酸）。值得注意的是，DNA结合与荧光量子产率，提供了宿主细胞死亡的基于溶液的读出一个大的增加有关。我们已经使用该组合测定法来执行在微孔板格式的高通量筛选，并通过显微镜来评估细胞内的生长和细胞毒性。值得注意的是，抗微生物剂可显示协同作用，其中当一起施用两种或更多种抗微生物剂的组合效果是，当分别施加大于。测试体外抗细胞内病原体的协同作用，通常是一个很大的任务，因为在不同浓度的抗生素的组合排列必须评估。然而，我们发现，我们的实时检测与自动化，数字化配送技术相结合permitted轻便协同测试。使用这些方法，我们能够系统地调查了大量抗菌药物单独的行动，并结合对细胞内病原体， 嗜肺军团菌。
Introduction
在车厢内临时增加或居住病原体很难根除治疗。预留或相对专性细胞内的病原体，如嗜肺军团菌 ， 立克次体 ， 布鲁氏菌。 ，土拉弗朗西斯菌 ， 结核分枝杆菌。往往需要长时间抗菌治疗疗程治愈，可能范围从个月甚至数年。此外，细胞外的病原体可能暂时占据利基内并以这种方式通过抗生素治疗一般疗程逃脱通关，后来冒出来启动毒感染的新一轮。 金黄色葡萄球菌和1肠杆菌科细菌尿路感染2,3两日益认识到的例子。因此，从根本上药物发现的目标是，以确定渗透到细胞内的区室新颖抗微生物剂。最佳治疗迅速消灭胞内生物体和防止通过亚抑制抗微生物暴露性的发展是特别理想的。
为此，我们开发了一种高通量筛选技术来识别细胞内渗透剂抗菌药物靶向模型病原体， 嗜肺军团菌的细胞内生长。 4既往临床观察表明，标准的药敏试验并没有准确地对这种微生物体内的治疗功效预测。 5具体地，这是因为主要类别抗微生物剂，如β内酰胺类和氨基糖苷类的，虽然针对无菌生长军团菌非常有效，没有充分渗透到其中， 军团驻留的胞内区室。 5,6后来的证据表明，在技术上更复杂的细胞生长测定有效预测临床疗效。 7 </sup>不幸的是，这些测定是极其费力的端点测定法，要求感染巨噬细胞，用抗微生物剂处理过的，在不同的时间点被裂解为菌落形成单位计数。此类测定法是不实际的大规模做，不适合于高通量药物筛选。
因此，我们开发的技术为细胞内细菌生长的实时确定。 6这是通过使用或者通过一个细菌萤光素酶操纵子8的集成改性的细菌菌株的9记者（第二代，正交检测，这里描述的）来实现（第一代测定中，如前所述）4或荧光蛋白到细菌染色体中。以这种方式，发光或荧光信号提供细菌数的替代，实时读数。
然而，这些属性没有解决在细胞内infectio一个主要混杂因素Ñ测定法，对宿主细胞的脱靶效应。具体地，宿主细胞的死亡固有地限制细胞生长和导致的抗微生物效果的假阳性鉴定。在筛选库，许多化合物是真核细胞的毒性，例如假阳性将压倒真抗微生物剂，因此需要大量的后续，端点细胞毒性测定法进行解析。
因此，这是极大的兴趣的，以便能够同时评估真核细胞生存力和细胞内生长。值得注意的是，非存活的真核细胞的特征是细胞膜完整性的损失。该测试细胞膜的渗透性探针可以因此用于评估细胞生存力。我们以前的特点一系列推定细胞膜非透性，荧光，DNA结合染料的访问和染色死细胞的核DNA的能力。 4在结合核DNA，这些染料显示quantu大幅增加米荧光产量造成了后台解决方案增加了荧光信号。因此，这些染料提供真核细胞死亡的定量读数。 4值得注意的是，我们发现几个人正在无毒自己长期合作孵育J774巨噬细胞中。当最初的感染过程中加入的，它们提供了一种实时的，可以由一个微板荧光计来测量或观察到的微观真核细胞死亡的荧光读数。
因此，通过组合使用细菌记者和无毒，膜不通透性的，DNA结合染料，我们能够发展一种简单的，非破坏性的，实时的检测，以测量细菌负荷，同时真核细胞的细胞毒性。此法使我们能够在384孔板筛选〜万知的生物活性物质，包括〜250抗菌剂和> 24万的小分子与功能上未鉴定的活性，抑制细胞内生长的能力嗜肺军团菌 ，而在同一时间产生用于每个化合物的真核细胞的细胞毒性数据。 6我们对军团菌生长的细胞内已知的抗菌药物分析是这种类型迄今为止最全面的探索。 6
根据我们的测定形式的效率，在组合使用时，我们也随之探讨已知抗微生物剂的潜在的协同作用。其中最常见的协同作用试验中，所谓的棋盘测定中，被标准地通过评估两个或更多抗微生物剂的两倍连续稀释的组合效果进行。 10在这些试验中，当两个或多个抗微生物剂都大于每个单独施加的效果的总和施加一起协同作用是由更大的效果的观察中定义。值得注意的是，迄今为止，只有专注和反对细胞内嗜肺军团菌进行选择性的协同测试</eM>因为涉及传统的端点检测乘以所需的组合排列的巨大努力。
为了方便协同测试，我们在自动化数字胶技术相结合6利用我们的实时细胞生长/真核细胞毒性实验的。这种自动化允许我们以分配溶于DMSO或单独或在384孔格式组合水溶液的化合物的系列稀释。 11此外，这种强大的液体处理技术，使我们能够轻松地执行更高的分辨率，平方根的二（而不是标准的，较低的分辨率，加倍）稀释组合在我们的二维实现更高水平的特异性，棋盘的协同作用分析。用两倍稀释系列12时，该分辨率是在解决有关再现性的协同作用字段关注特别有价值。最后，我们的分析是定量的，也疗法安伏测量抑制等级。其结果，在测定捕获的抑制信息的全部，在等高线等效线图表达的，其中isocontours与生长抑制的类似水平连接的组合的浓度。 6该绘图策略允许组合剂量-反应曲线的可视化。为了说明我们的方法，我们描述我们的协议进行这些检测并显示代表性的结果。
Protocol
Representative Results
Discussion
我们描述了细胞内的细菌的生长和宿主细胞的细胞毒作用的同时检测的实时检测。有在协议中的几个关键步骤。首先，对于健壮测定性能，必须有细菌和细胞毒性读数之间有足够的光谱分离。这样的分离是本征为萤光素酶操纵子记者和荧光DNA结合染料的组合。然而，根据我们的经验（ 表1-3， 图2），使用双荧光读出要求的不重叠的荧光信号（ 例如 ，利用一个远红?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
研究这个手稿报告是由过敏和美国国立卫生研究院传染病研究所根据奖号R01AI099122到JEK的内容完全是作者的责任的支持，并不一定代表全国学院的官方意见健康。我们想从体外培育牛黄，朗伍德筛选基金和/或国家筛选实验室感谢詹尼弗·史密斯，大卫·罗贝尔，苏蒋道格洪水，肖恩·约翰斯顿，珍妮弗·纳莱，斯图尔特鲁德尼茨基，保仁，理查德兆，和Rachel监狱长卓越的生物防御和新兴他们在开发和高通量筛选试验的表现帮助传染病（由U54AI057159支持）新英格兰地区中心。我们还要感谢肯尼斯·P·史密斯对稿件有益的意见。
Materials
| J774A.1 cells | American Type Culture Collection | TIB-67 | Host cell |
| ACES | Sigma-Aldrich | A9758 | For making buffered charcoal yeast extract agar and buffered yeast extract medium |
| Yeast extract, ultrafiltered | Becton-Dickinson/Difco | 210929 | For making buffered charcoal yeast extract agar and buffered yeast extract medium; lower grades may cause impaired growth and/or alter sensitivity of Legionella to growth inhibitors |
| Alpha-ketoglutaric acid, monopotassium salt | Sigma-Aldrich | K2000 | For making buffered charcoal yeast extract agar and buffered yeast extract medium |
| Sodium pyruvate | Sigma-Aldrich | P5280 | For making buffered charcoal yeast extract agar and buffered yeast extract medium |
| Potassium phosphate, dibasic | Thermo Fisher Scientific | P288-500 | For making buffered charcoal yeast extract agar and buffered yeast extract medium |
| L-cysteine | Sigma-Aldrich | C-7755 | For making buffered charcoal yeast extract agar and buffered yeast extract medium |
| Ammonium iron(III) citrate | Sigma-Aldrich | F5879 | For making buffered charcoal yeast extract agar and buffered yeast extract medium; ferric pyrophosphate may be used instead but is more difficult to weigh accurately |
| Potassium hydroxide solution, concentrated | Thermo Fisher Scientific | SP236-500 | For making buffered charcoal yeast extract agar and buffered yeast extract medium |
| Deonized water | N/A | N/A | For making buffered charcoal yeast extract agar and buffered yeast extract medium |
| Thymidine (tissue culture grade) | Sigma-Aldrich | T1895 | For supplementing both RPMI 1640 and buffered yeast extract agar/medium — lower grade thymidine may be used for the latter, but may cause impaired cell growth and/or cell death in RPMI 1640 |
| RPMI 1640, standard formulation | Corning via Thermo Fisher Scientific | 10-040-CV | For growing J774A.1 cells prior to plating; includes 2 mM L-glutamine |
| RPMI 1640 lacking phenol red | Corning via Thermo Fisher Scientific | 17-105-CV | For plating J774A.1 cells in 384 well dishes (not suitable for growth prior to plating); also lacks L-glutamine — supplement to 2 mM before use |
| L-glutamine, 200 mM in 0.85% NaCl (tissue culture grade) | HyClone via Thermo Fisher Scientific | SH30034.02 | For supplementing RPMI 1640 lacking L-glutamine, to 2 mM final concentration |
| Iron-supplemented calf serum | Gemini Bioproducts | 100-510 | For supplementing RPMI 1640, to 9.1% final concentration |
| Trypan Blue solution | Sigma-Aldrich | T8154 | For staining for J774A.1 cell death determination while counting cell density |
| SYTOX Green, 5 mM solution in DMSO | Thermo Fisher Scientific | S7020 | For staining for J774A.1 cell death determination by fluorescence reading or epifluorescence microscopy (in conjunction with orange-red or far red fluorescent bacteria). Use at 125 nM final concentration. |
| GelRed, 10000X solution in water | Biotium | 41003 | For staining for J774A.1 cell death determination by epifluorescence microscopy (in conjunction with green fluorescent bacteria). Use Gel Red at 1X final concentration. |
| Cell culture incubator | Thermo Fisher Scientific | 13-255-26 | For incubation of J774A.1 cells (both before and after infection); can also be used for incubation of bacteria, or a standard atmosphere incubator can be used instead) |
| Orbital shaker | BellCo Glass | 7744-01010 | For shaking incubation of J774A.1 cells before infection; fits inside cell culture incubator; includes shaker base 7744-01000 and tray 7740-01010 (these are also available separately) |
| Shaker flasks (250 ml) | ChemGlass Life Sciences | CLS-2038-04 | For shaking incubation of J774A.1 cells before infection |
| Shaker clamps for flasks (250 ml) | BellCo Glass | 7744-16250 | For shaking incubation of J774A.1 cells before infection |
| Shaker flasks (1000 ml) | ChemGlass Life Sciences | CLS-2038-07 | For shaking incubation of J774A.1 cells before infection |
| Shaker clamps for flasks (1000 ml) | BellCo Glass | 7744-16100 | For shaking incubation of J774A.1 cells before infection |
| Sponge foam caps for flasks (250 ml – 1000 ml) | ChemGlass Life Sciences | CLS-1490-038 | For shaking incubation of J774A.1 cells before infection; reduces risk of contamination relative to standard metal caps |
| MultiDrop Combi programmable multichannel peristaltic pump | Thermo Fisher Scientific | 5840300 | For dispensing J774A.1 cells, medium, and bacterial suspension containing fluorophores to large numbers of 384 well dishes |
| Combi standard bore manifold | Thermo Fisher Scientific | 24072670 | Default predispense volume of 20 ml is insufficient to compensate for settling — increase to 80 ml |
| White 384 well dishes treated for tissue culture | Corning | 3570 | For reading luminescence and fluorescence; Greiner catalog # 781080 also tested successfully |
| DMSO (tissue culture grade, in sealed ampoules) | Sigma-Aldrich | D2650 | For dissolving positive control and test compounds |
| Azithromycin | Sigma-Aldrich | PHR1088 | Antibiotic positive control |
| Saponin (from Quillaja bark) | Sigma-Aldrich | S-4521 | Cytoxicity positive control |
| Multichannel pipettor | Thermo Fisher Scientific | Finnpipette | For transfer of fixed amounts of positive control compounds; pipettor must have digital dispensing with detents to enable repetitive fixed volume dispensing |
| Epson pin transfer robot | Epson/ICCB-L | (Custom equipment) | For transfer of fixed amounts of test compounds from library arrays |
| D300 digital dispensing system | Hewlett-Packard via Tecan | D300 | For transfer of variable amounts of test compounds ranging from 11 picoliters to 10 microliters |
| T8+ cartridges for D300 digital dispensing system | Hewlett-Packard via Tecan | T8+ | For dispensing test compounds |
| EnVision multi-mode plate reader | Perkin-Elmer | (Contact manufacturer) | For optimal detection of SYTOX Green fluorescence, use excitation filter 485/14, emission filter 535/25, and dichroic mirror 505 nm, with selection of minimum gain and transmittance, and “high concentration mode. For luminescence detection, use the "USLUM" protocol for high-sensitivity detection. For mNeptune2 detection, use excitation filter 600/8, emission filter 665/7.5, and dichroic mirror 658 nm, with selection of gain and transmittance to achieve the highest maximum signal possible without saturating the photomultiplier. |
| Epifluorescence microscope with computer-connected digital camera | Nikon | Ti | For live cell imaging; any standard fluorescent microscope can substitute, with phase contrast or DIC optics, capable of imaging green (fluorescein), orange-red to red (Texas Red), and far-red (Cy5) fluorescence, with 100X oil objective for highest resolution |
| Glass-bottom tissue culture dishes | MatTek Corporation | P35G-1.5-20-C | For live cell imaging. Dishes such as the MatTek allow microscopic visualization at 600X or 1000X magnification through use of an inverted epifluorescent or confocal microscope. These specific dishes are 3.5 cm nominal diameter, 3.3 cm inside diameter, with 20 mm diameter #1.5 thickness cover slips inserted into the bottoms. |
| Photoshop CS6 | Adobe | Adobe photoshop or similar programs can be used to pseudocolor and merge light microscopic and fluorescent images. | |
| Mathematica 10 | Wolfam | For generation of two-dimensioonal isocontour isobolograms and three-dimensional surface isobolograms. |
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