通过成像质谱分析 研究 感染期间琼脂和组织中生长的细菌菌落的微生物合作
Summary
展示了一种新型样品制备方法，用于通过基质辅助激光解吸/电离成像质谱 分析 琼脂基细菌大菌落。
Abstract
了解感染期间发生的微生物相互作用的代谢后果对生物医学成像领域提出了独特的挑战。基质辅助激光解吸/电离（MALDI）成像质谱代表了一种无标记的 原位 成像模式，能够生成各种代谢物的空间图。虽然现在通过该技术 常规 分析薄切片组织样品，但由于这些样品的高含水量和不均匀的地形，非传统底物（例如微生物学研究中通常在琼脂上生长的细菌菌落）的成像质谱分析仍然具有挑战性。本文演示了一种样品制备工作流程，以便对这些样品类型进行成像质谱分析。该过程使用两种胃肠道病原体的细菌共培养大菌落来举例说明： 艰难梭菌 和 粪肠球菌。研究这种明确定义的琼脂环境中的微生物相互作用也被证明可以补充组织研究，旨在了解小鼠感染模型中这两种致病生物之间的微生物代谢合作。氨基酸代谢物精氨酸和鸟氨酸的成像质谱分析作为代表性数据呈现。该方法广泛适用于其他分析物、微生物病原体或疾病以及需要细胞或组织生物化学空间测量的组织类型。
Introduction
人类微生物组是一个高度动态的生态系统，涉及细菌、病毒、古细菌和其他微生物真核生物的分子相互作用。虽然近年来对微生物关系进行了深入研究，但在化学水平上对微生物过程仍有许多了解1，2。这部分是由于没有能够准确测量复杂微生物环境的工具。过去十年来，成像质谱（IMS）领域的进展使生物底物中的许多代谢物，脂质和蛋白质的原位和无标记空间映射成为可能3，4。基质辅助激光解吸/电离（MALDI）已成为成像质谱中最常用的电离技术，涉及使用紫外激光从薄组织切片表面烧蚀材料，以通过质谱法4进行测量。通过将化学基质均匀地应用于样品表面，可以在整个样品表面上以光栅模式进行连续测量，从而促进了这一过程。然后在数据采集后生成分析物离子强度的热图。电离源和采样技术的最新进展使得能够分析非传统底物，例如在营养琼脂上生长的细菌5和哺乳动物6，7，8细胞标本。IMS提供的分子空间信息可以为感染过程中微生物-微生物和宿主-微生物相互作用的生化通讯提供独特的见解9，10，11，12，13，14。
艰难梭菌感染 （CDI） 后，艰难梭菌暴露于胃肠道中快速变化的微生物环境中，其中多种微生物相互作用可能会影响感染结果15，16。令人惊讶的是，人们对感染期间艰难梭菌和常驻微生物群之间相互作用的分子机制知之甚少。例如，肠球菌是肠道微生物组中的一类机会性共生病原体，与 CDI17、18、19、20 的易感性和严重程度增加有关。然而，对这些病原体之间相互作用的分子机制知之甚少。为了可视化肠道微生物组这些成员之间的小分子通讯，本文在琼脂上生长细菌大菌落以模拟受控环境中的微生物-微生物相互作用和细菌生物膜形成。然而，由于细菌培养标本的高含水量和不均匀的表面形貌，通过MALDI成像质谱分析获得具有代表性的代谢分布具有挑战性。这主要是由琼脂的高度亲水性和去除水分过程中琼脂表面反应不均匀引起的。
琼脂的高含水量也使得获得均匀的MALDI基质包衣变得具有挑战性，并可能干扰随后在真空中进行的MALDI分析21。例如，许多MALDI源在0.1-10托的压力下运行，这是一个足够的真空，可以从琼脂中去除水分，并可能导致样品变形。真空环境引起的琼脂中的这些形态变化导致干燥琼脂材料起泡和开裂。这些伪影会降低琼脂对载玻片的粘附，并可能导致样品拆卸或剥落到仪器真空系统中。琼脂样品的厚度可能与载玻片相距5 mm，这会导致仪器内部离子光学元件的间隙不足，从而导致仪器离子光学元件受到污染和/或损坏。这些累积效应可以导致反映表面形貌的离子信号减少，而不是潜在的微生物生化相互作用。琼脂样品必须均匀干燥，并在真空分析之前牢固地粘附在显微镜载玻片上。
本文展示了一种用于控制在琼脂培养基上生长的细菌培养物大菌落干燥的样品制备工作流程。这种多步骤、较慢的干燥过程（相对于先前报道的过程）可确保琼脂均匀脱水，同时最大限度地减少安装在显微镜载玻片上的琼脂样品冒泡或开裂的影响。通过使用这种逐渐干燥的方法，样品牢固地粘附在显微镜载玻片上，并适合随后的基质应用和MALDI分析。这使用在琼脂和鼠组织模型上生长的 艰难梭菌 模型来举例说明，这些模型具有和不存在共生和机会性病原体粪肠球菌。细菌和组织模型的MALDI成像质谱分析允许氨基酸代谢物谱的空间映射，为生物能量微生物代谢和通讯提供新的见解。
Protocol
注意：动物实验已获得费城儿童医院和宾夕法尼亚大学佩雷尔曼医学院动物护理和使用委员会的批准（协议IAC 18-001316和806279）。 注意：艰难梭菌（艰难梭菌）和粪肠球菌（ 粪肠球菌）是BSLII病原体，应格外小心处理。必要时使用适当的净化方案。 1. 细菌培养大菌落的生长和隔夜运输的准备 制备 艰难梭?…
Representative Results
我们对模型细菌菌落和与 粪肠杆菌 和 艰难梭菌 共定植的小鼠进行了代谢物MALDI成像质谱分析，以研究氨基酸在微生物 – 微生物相互作用中的作用。在琼脂上生长的细菌大菌落可作为分析细菌生物膜形成中不同生化变化的明确定义模型。重要的是要确保在琼脂培养基上生长的细菌培养大菌落的受控干燥过程，以最大限度地减少琼脂表面的变形和开裂。这是通过两步过程 实现 ?…
Discussion
在MALDI成像质谱分析过程中，重要的是具有平坦的样品表面，以使样品基板上入射MALDI激光的焦距一致。样品高度的偏差会导致 MALDI 激光束移出焦点，从而导致光束直径和强度的变化，从而影响 MALDI 电离效率。电离效率的这些变化可能导致整个组织表面的分析物强度差异，这些差异不反映底层组织生物化学，而是反映表面形貌。此外，大多数MALDI电离源在低于大气压下运行，水合琼脂样品在 真…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了美国国立卫生研究院（NIH）国家普通医学科学研究所（NIGMS）的支持，授予GM138660。J.T.S.得到了佛罗里达大学的查尔斯和莫妮卡·伯克特家庭暑期奖学金的支持。JPZ得到了NIH拨款K22AI7220（NIAID）和R35GM138369（NIGMS）的支持。A.B.S.得到了宾夕法尼亚大学细胞和分子生物学培训基金（T32GM07229）的支持。
Materials
| 0.2 μm Titan3 nylon syringe filters | Thermo Scientific | 42225-NN | |
| 1,5-diaminonaphthalene MALDI matrix | Sigma Aldrich | 2243-62-1 | |
| 20 mL Henke Ject luer lock syringes | Henke Sass Wolf | 4200.000V0 | |
| 275i series convection vacuum gauge | Kurt J. Lesker company | KJL275807LL | |
| 7T solariX FTICR mass spectrometer equipped with a Smartbeam II Nd:YAG MALDI laser system (2 kHz, 355 nm) | Bruker Daltonics | ||
| Acetic acid solution, suitable for HPLC | Sigma Aldrich | 64-19-7 | |
| Acetonitrile, suitable for HPLC, gradient grade, ≥99.9% | Sigma Aldrich | 75-05-8 | |
| Ammonium hydroxide solution, 28% NH3 in H2O, ≥99.99% trace metals basis | Sigma Aldrich | 1336-21-6 | |
| Autoclavable biohazard bags: 55 gal | Grainger | 45TV10 | |
| Biohazard specimen transport bags (8 x 8 in.) | Fisher Scientific | 01-800-07 | |
| Brain heart infusion broth | BD Biosciences | 90003-040 | |
| C57BL/6 male mice | Jackson Laboratories | ||
| CanoScan 9000F Mark II photo and document scanner | Canon | ||
| CM 3050S research cryomicrotome | Leica Biosystems | ||
| Desiccator cabinet | Sigma Aldrich | Z268135 | |
| Diamond tip scriber, Electron Microscopy Sciences | Fisher Scientific | 50-254-51 | |
| Drierite desiccant pellets | Drierite | 21005 | |
| Ethanol, 200 Proof | Decon Labs | 2701 | |
| flexImaging software | Bruker Daltonics | ||
| ftmsControl software | Bruker Daltonics | ||
| Glass vacuum trap | Sigma Aldrich | Z549460 | |
| HTX M5 TM robotic sprayer | HTX Technologies | ||
| Indium Tin Oxide (ITO)-coated microscope slides | Delta Technologies | CG-81IN-S115 | |
| In-line HEPA filter to vacuum pump | LABCONCO | 7386500 | |
| Methanol, HPLC Grade | Fisher Chemical | 67-56-1 | |
| MTP slide-adapter II | Bruker Daltonics | 235380 | |
| Optimal cutting temperature (OCT) compound | Fischer Scientific | 23-730-571 | |
| Peridox RTU Sporicide, Disinfectant and Cleaner | CONTEC | CR85335 | |
| PTFE (Teflon) printed slides, Electron Microscopy Sciences | VWR | 100488-874 | |
| Rotary vane vacuum pump RV8 | Edwards | A65401903 | |
| Tissue-Tek Accu-Edge Disposable High Profile Microtome Blades | Electron Microscopy Sciences | 63068-HP | |
| Transparent vacuum tubing | Cole Palmer | EW-06414-30 | |
| Ultragrade 19 vacuum pump oil | Edwards | H11025011 | |
| Variable voltage transformer | Powerstat | ||
| Water, suitable for HPLC | Sigma Aldrich | 7732-18-5 | |
| Wide-mouth dewar flask | Sigma Aldrich | Z120790 |
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Cite This Article
Specker, J. T., Smith, A. B., Keenan, O., Zackular, J. P., Prentice, B. M. Investigation of Microbial Cooperation via Imaging Mass Spectrometry Analysis of Bacterial Colonies Grown on Agar and in Tissue During Infection. J. Vis. Exp. (189), e64200, doi:10.3791/64200 (2022).