在研究隐球菌-阿米巴相互作用中,显微技术和荧光读数的补充应用
Summary
本文详细介绍了利用静止光图像和高分辨率透射电子显微镜图像进行制备隐球细胞和阿米巴共同培养的协议。此处说明定量数据如何补充此类定性信息。
Abstract
为了模拟隐球菌感染,阿米巴是环境中隐球细胞的天然捕食者,可以作为巨噬细胞的模型。这种食肉生物,类似于巨噬细胞,利用噬菌体杀死内化细胞。借助共聚焦激光扫描显微镜,可以捕获描述隐球细胞和阿米巴之间交互时刻的图像。电子显微镜的分辨率也有助于揭示隐球细胞的超结构细节,当被困在阿米巴食物真空中时。由于噬菌体是一个连续的过程,因此定量数据随后被集成到分析中,以解释捕获图像的时间点时发生的情况。具体来说,读取相对荧光单位是为了量化阿米巴在隐球细胞内化中的效率。为此,隐球细胞被染色的染料,使他们荧光一旦被困在食物的酸性环境中。当一起使用时,通过此类技术收集的信息可以提供关键信息,以帮助得出细胞在阿米巴内部化时以及可能由其他噬菌体细胞内化时的行为和命运的结论。
Introduction
微生物经过一段时间的进化,在不同的生态区中占据并茁壮成长,如土壤和水的开放物理边界,等等。在这些利基中,微生物往往直接争夺有限的资源;重要的是,对于营养,他们用来支持他们的生长或空间,他们需要容纳不断扩大的人口2,3。在某些情况下,一些霍洛索生物,如阿米巴甚至可能早于隐球细胞,作为从生物量4,5中提取营养物质的一种方式。反过来,这允许这些生物通过控制猎物的数量来确立领土优势。由于这种掠夺性压力,一些猎物可能被选择来产生微生物因子,如隐球胶囊6,以调和压力的负面影响。然而,作为这种压力的意外后果,一些微生物获得一些因素,使它们能够跨越物种屏障,寻找新的利基来殖民7,如人体的封闭空间,富含营养,具有理想条件。后者可以解释陆地微生物如何像隐球菌(C.新福曼人可以转化为致病性。
为此,必须研究隐球细胞与阿米巴的初始接触,以及如何选择它们成为致病菌。更具体地说,这可能为隐球细胞在感染期间受到巨噬细胞作用时的行为提供线索。正是出于这个原因,阿米巴被选为这里巨噬细胞的模型,因为它是相对便宜和容易保持阿米巴文化在实验室8。感兴趣的也是研究隐球菌二次代谢物(即3-羟基脂肪酸9,10)如何影响阿米巴和隐球细胞之间的相互作用。
用肉眼感知阿米巴和猎物之间的相互作用的一种简单方法是在琼脂盘表面使用猎物创建草坪,并发现阿米巴。琼脂板上的斑块或透明区域的可视化描绘了阿米巴可能以猎物为食的区域。然而,在这个宏观层面上,只注意到这一进程的结果,并且不能观察到噬菌体过程是机械化的。因此,为了在细胞到细胞的基础上欣赏这个过程,有几种微观方法可以使用11,12。例如,带孵化室的倒置显微镜可用于视频记录噬菌细胞与其目标13之间的事件延时。不幸的是,由于具有延时功能的显微镜的成本,实验室并不总是能够购买这种显微镜,尤其是在资源贫乏的环境中。
为了规避上述限制,本研究提出了一个顺序探索设计,评估C.新福曼斯(C.新福曼斯UOFS Y-1378)和C.新福曼斯LMPE 046与阿坎塔莫埃巴·卡斯特拉尼的相互作用.首先,使用在定量方法之前的定性方法。静止图像使用倒置荧光显微镜以及透射电子显微镜拍摄,以描绘阿米巴-隐球菌相互作用。随后,使用板读取器对荧光进行量化,以估计阿米巴将隐球细胞内化的效率。在数据解释阶段协调这些方法的发现时,这可能与浏览方位体延时视频一样多的关键信息。
Protocol
隐球新毒杆菌和一些阿坎塔莫埃巴卡斯特兰尼菌株被视为生物安全-2级(BSL-2)病原体;因此,研究人员在使用这些生物时必须采取适当的预防措施。例如,实验室人员应接受专门的培训和个人防护设备 (PPE),如实验室外套、手套和眼睛防护。生物安全柜(2级)应用于可能导致感染的程序14。 1. 真菌细胞的培养和标准化(从Madu等人改性15) …
Representative Results
微生物是肉眼无法感知的微生物。然而,它们的影响可能导致可观察到的临床明显疾病,如皮肤感染。在研究微生物的某些方面时,从微生物的形态、副产品和相互作用,能够提供图片和视频证据至关重要。 我们首先试图可视化隐球细胞和阿米巴之间的相互作用。为此,首先研究了显示2小时共育细胞的明亮场图像。一张图片显示一?…
Discussion
在本文中,成功地使用了不同的技术来揭示当阿米巴与隐球菌细胞相互作用时可能出现的结果。此外,我们有兴趣展示3-羟基脂肪酸对隐球菌-阿米巴相互作用结果的影响。
第一种技术是共聚焦显微镜,它渲染静止图像。此处此技术的主要缺点是,它只为我们提供了仅限于特定时间点的信息。任何可以根据结果得出的结论都有助于归纳推理,其中人们可以根据一组观察<sup class="…
Divulgations
The authors have nothing to disclose.
Acknowledgements
这项工作得到了南非国家研究基金会(赠款号:87903年)和自由州大学的赠款的支持。我们也感谢彼得·范·怀克和汉莉·格罗布勒在我们的显微镜研究中提供的服务和协助。
Materials
| 1,4-Diazabicyclo-[2.2.2]-octane | Sigma-Aldrich | D27802 | – |
| 1.5-mL plastic tube | Thermo Fisher Scientific | 69715 | – |
| 15-mL Centrifuge tube | Thermo Fisher Scientific | 7252018 | – |
| 50-mL Centrifuge tube | Thermo Fisher Scientific | 1132017 | – |
| 8-Well chamber slide | Thermo Fisher Scientific | 1109650 | – |
| Acetone | Merck | SAAR1022040LC | – |
| Amoeba strain | ATCCÒ | 30234TM | – |
| ATCC medium 712 | ATCCÒ | 712TM | Amoeba medium |
| Black 96-well microtiter plate | Thermo Fisher Scientific | 152089 | – |
| Centrifuge | Hermle | – | – |
| Chloroform | Sigma-Aldrich | C2432 | – |
| Confocal microscope | Nikon | Nikon TE 2000 | – |
| Epoxy resin: | |||
| [1] NSA | [1] ALS | [1] R1054 | – |
| [2] DER 736 | [2] ALS | [2] R1073 | – |
| [3] ERL Y221 resin | [3] ALS | [3] R1047R | – |
| [4] S1 (2-dimethylaminoethanol) | [4] ALS | [4] R1067 | – |
| Fluorescein isothiocyanate | Sigma-Aldrich | F4274 | – |
| Formic Acid | Sigma-Aldrich | 489441 | – |
| Fluoroskan Ascent FL | Thermo Fisher Scientific | 374-91038C | Microplate reader |
| Glucose | Sigma-Aldrich | G8270 | – |
| Glutaraldehyde | ALS | R1009 | – |
| Hemocytometer | Boeco | – | – |
| Lead citrate | ALS | R1209 | – |
| Liquid Chromatography Mass Spectrometer | Thermo Fisher Scientific | – | |
| Methanol | Sigma-Aldrich | R 34,860 | – |
| Orbital shaker | Lasec | – | – |
| Osmium tetroxide | ALS | R1015 | – |
| pHrodo Green Zymosan A BioParticles | Life Technologies | P35365 | This is the pH-sensitive dye |
| Physiological buffer solution | Sigma-Aldrich | P4417-50TAB | – |
| Rotary shaker | Labcon | – | – |
| Sodium phosphate buffer: | |||
| [1] di-sodium hydrogen orthophosphate dihydrate | [1] Merck | [1] 106580 | – |
| [2] sodium di-hydrogen orthophosphate dihydrate | [2] Merck | [2] 106345 | |
| Transmission electron microscope | Philips | Philips EM 100 | – |
| Trypan blue | Sigma-Aldrich | T8154 | – |
| Ultramicrotome | Leica | EM UC7 | – |
| Uranyl acetate | ALS | R1260A | – |
| Vacuum dessicator | Lasec | – | – |
| Vial | Sigma-Aldrich | 29651-U | – |
| YNB | Lasec | 239210 | – |
| YPD agar | Sigma-Aldrich | Y-1500 | – |
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Tags
Microscopic TechniquesFluorescence ReadingCryptococcus-Amoeba InteractionsPhagocytosisPathogenic FungusCryptococcus NeoformansEnvironmental PredatorAmoebaConfocal Laser-scanning MicroscopyTransmission Electron MicroscopyYPD AgarYNB BrothGlucoseAmoeba CellsPeptone Yeast Extract Glucose BrothTrypan BlueFluorescein IsothiocyanatePBSGlutaraldehydeCo-culture
Citer Cet Article
Madu, U. L., Sebolai, O. M. Complementary Use of Microscopic Techniques and Fluorescence Reading in Studying Cryptococcus-Amoeba Interactions. J. Vis. Exp. (148), e58698, doi:10.3791/58698 (2019).