尺寸事项:新生隐球菌中胶囊直径的测定
Summary
多糖胶囊是新生隐球菌的主要毒力因子, 其大小与菌株的毒力密切相关。胶囊直径测量用于表型检测和测量疗效。本文提出了一种标准的胶囊诱导方法, 并比较了两种染色法和测量直径法。
Abstract
新生隐球菌多糖胶囊是致病性酵母的主要毒力因子和最常见的研究领域之一。囊粒大小在菌株间有很大差异, 在引入压力或低养分条件时, 具有快速生长的能力, 与菌株的毒力呈正相关。由于这些原因, 胶囊的大小对C. 隐球菌研究人员非常感兴趣。在表型测试中诱导了C. 隐球菌胶囊的生长, 以帮助了解不同处理方法对酵母的影响以及菌株之间的大小差异。这里我们描述了胶囊诱导的标准方法之一, 并比较了两种被接受的染色和测量胶囊直径的方法: (i) 印度墨水, 一个负的污点, 使用与常规光镜和 (ii) 共染色与荧光染料的细胞壁和胶囊后, 共聚焦显微镜。最后, 我们展示了如何通过计算图像分析来自动测量来自印度墨水染色样品的胶囊直径。
Introduction
每年影响一季度百万人, 每年造成18万以上死亡,新生隐球菌是致病性, 胞内酵母和球菌1,2,的致病剂3. 受影响最严重的是贫穷国家的艾滋病毒阳性患者, 他们没有现成的抗逆转录病毒疗法, 使他们敏锐地易患疾病4,5,6。疾病控制中心的数据表明, 在撒哈拉以南非洲地区, 比起在记录中的 1上的任何埃博拉病毒爆发, 每年都有更多的人死于肺结核。最常见的接触途径是吸入在环境中常见的干燥孢子7。进入肺部后, 有几种致病因素有助于在受感染的个体中获得C. 隐球菌的成功。多糖胶囊被认为是微生物的主要毒力因子, 因为 acapsular 菌株不是毒性的8。
隐球菌胶囊由三个主要成分组成: glucuronoxylomannan (GXM)、galactoxylomannan (GalXM) 和 mannoproteins (MPs)9。虽然 MPs 是胶囊中相对较小的细胞壁相关组件, 但它们是免疫的, 可以促进一种主要支持炎症的响应9,10。相比之下, GXM 和 GalXM 构成了胶囊的大部分 (> 90% 的重量), 并有免疫抑制效应11。除了免疫调节作用外, 胶囊的快速扩增在体内形成了一个机械屏障, 可通过宿主吞噬细胞 (即、中性粒细胞和巨细胞)12摄取。C. 隐球菌胶囊及其合成是复杂的, 但总的来说, 增加的胶囊直径与增加的毒力6,13,14相关。考虑到这一点, 对于C. 隐球菌研究人员来说, 能够快速准确地量化胶囊测量是很重要的。
C. 隐球菌细胞及其多糖胶囊都是动态结构, 随着时间的推移15显示变化。该胶囊可以在密度、大小和组件上发生变化, 以响应主机环境中的更改16,17,18。低铁或营养水平, 接触血清, 人体生理 pH 值和增加 CO2是已知的启动胶囊生长16,18,19,20。此外, 研究人员还显示结构变化导致感染期间免疫反应有显著差异, 在其主机2122上为C. 隐球菌提供了优势。这是众所周知的, 因为C. 隐球菌胶囊的体系结构已经以多种方式进行了分析。例如, 电子显微镜显示, 该胶囊具有一个具有内部电子密层的异构矩阵, 在外层, 更透水层的下面是23。光散射和光镊的使用使研究人员得以进一步阐明其大分子特性24。通过对静态和动态光散射测量结果的分析, 我们知道多糖胶囊具有复杂的分支结构23。光学镊子用于测试结构的刚度, 并评估其抗体反应性24。然而, 到目前为止, 对C. 隐球菌胶囊最常用的分析是对其大小的测量。
为了量化胶囊的大小, 研究者使用了一个简单的测量方法: 胶囊的线性直径。数字显微镜用于捕获多个C. 隐球菌细胞 (一般上百个) 的图像, 它们都是印度墨水或荧光染料染色的。测量每个细胞体和周围胶囊的大小。通过对整个细胞直径 (细胞体 + 胶囊) 的减去细胞体直径, 计算出该胶囊的平均直径。直到这一点, 这些测量已经手动完成。虽然一般准确, 这种方法有缺点的研究人员。大型数据集可能需要几天甚至几周的时间来进行手工分析。由于这些测量是手工完成的, 所以主观和人为的错误可能会影响结果。
自动计算图像分析已成为许多分子细胞生物学领域的研究人员不可缺少的工具, 能够更快更可靠地分析生物图像25,26,27。精确的图像分析技术对于从通常复杂而庞大的数据集挖掘定量信息是必要的。然而, 一些测量, 特别是对C. 隐球菌胶囊的测量, 一直难以实现自动化。准确地识别细胞壁与胶囊之间的界面, 在相衬显微镜下成像时通常以暗环的形式出现, 用一个简单的阈值来解决会很麻烦。此外, 在文化中的C. 隐球菌细胞往往聚集在一起, 准确测量细胞的精确分割是必要的。
该项目的目的是 (i) 说明在C. 隐球菌中胶囊诱导的标准协议之一, (ii) 比较和对比印度墨水和荧光染色, 因为它们涉及胶囊直径测量, (iii) 发展简单,用印度墨水染色的图像测量胶囊直径的计算方法使用图像分析软件, (iv) 评估手动测量胶囊直径和使用软件自动化的好处和局限性。我们发现, 这两种染色方法, 荧光标记的细胞壁和胶囊, 而更费时, 提供了最一致的结果之间的实验。然而, 这两种方法都使我们能够成功地区分出不同胶囊大小的实验室和临床C. 隐球菌菌株。此外, 我们能够自动测量胶囊直径从印度墨水染色图像, 并发现这是一个可行的替代手工测量胶囊。
Protocol
Representative Results
Discussion
数十年来, 该胶囊一直是研究的主要焦点, 对 mycologists 和临床医生感兴趣的C. 隐球菌和球菌由于其作用的主要致病因素的病原体。用显微显微镜测量不同生长条件下的胶囊大小差异, 可以提供有关病原体及其对各种刺激的反应的重要信息 (即, 不同的环境条件,潜在的药物治疗,等) 在这里, 我们概述了一种方法, 以诱导生长的胶囊在C. 隐球菌, 并比较了两种不同的胶囊染色协?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢分子生物科学 (手机) 博士计划和中田纳西州立大学 (MTSU) 的生物学系为这项研究提供资金。该项目还部分由 MTSU 基金会授予 D.E.N. 的特别项目赠款提供经费。
Materials
| Capsule Induction | |||
| C. neoformans cells | The clinical lab strain, H99S, was a kind gift from Dr. John Perfect (Duke University). The clinical strains, B18 and B52, were kind gifts from Dr. Greg Bisson (University of Pennsylania). | ||
| Yeast Peptone Dextrose Broth (YPD) | Fisher Scientific | DF0428-17-5 | |
| Phosphate Buffered Saline (PBS) | This is made in the lab using standard recipe (137mM NaCl, 2.7 mM KCl, 10mM Na2HPO4O, 2 mM Kh2PO4O) | ||
| DMEM/high-glucose with L-glutamine, without sodium pyruvate | GE Life Sciences | SH30022.01 | |
| 6-well plates | Falcon | CL5335-5EA | |
| Shaking incubator | Thermo Scientific | MaxQ6000 | |
| CO2 incubator | Fisher Scientific | Isotemp | |
| Centrifuge | Thermo Scientific | Legend XTR | |
| Staining | |||
| Microcentrifuge | Thermo Scientific | Legend Micro 21R | |
| India ink | Fisher Scientific | 14-910-56 | |
| Calcofluor white | Sigma-Aldrich | 18909-100ML-F | |
| 18B7 mouse anti-GXM antibody conjugated to Alexafluor 488 | A kind gift from Dr. Arturo Casadevall (Johns Hopkins University) | ||
| PBS with 1% Bovine Serum Albumin (BSA) | PBS is the same recipe listed above (line 4) with 1% BSA added and filter sterilized. | ||
| Bovine Serum Albumin | Sigma-Aldrich | A9418 | |
| Superfrost microscope slides | Fisher Scientific | 12-550-143 | |
| Glass coverslips | Corning | 2855-18 | #1.5 thickness |
| Clear nail polish or other non-toxic sealant | |||
| Image Acquisition | |||
| Immersion oil | Cargille | 16484 | |
| Light microscope with immersion oil objective | Zeiss | Zeiss Axio A1 with a Plan – NEOFLUAR 100x oil immersion NA 1.30 objective | |
| Light microscope camera | Zeiss | Zeiss Axiocam ErCD camera | |
| Confocal microscope with oil immersion objective | Zeiss | LSM 700 laser scanning confocal equipped with a Plan-Apochromat 63X NA 1.4 oil immersion DIC M27 objective. | |
| Confocal microscope software | Zen 2009 | ||
| Confocal microscope camera | Nikon | Nikon Ti-Eclipse with a Intensilight epifluorescence illuminator (Nikon), CoolSNAP MYO microscope camera (Photometrics), Plan Apo 60x NA 1.40 oil immersion objective (Nikon) and 1.5x magnification changer. | |
| Widefield imaging software | Nikon Elements (Nikon) | ||
| Capsule Measurement | |||
| Image editing software | Photoshop (Adobe) | ||
| Microscope software for manual measurement | Axiovision (Carl Zeiss) | ||
| Image analysis software for automated meesurement | Aivia (DRVision Technologies) | ||
| Spreadsheet software | Excel (Microsoft) |
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