利用荧光蛋白可视化和定量<em>衣原体</em>液泡生长动态的活细胞
Summary
A live cell fluorescent protein based method for illuminating cellular vacuoles (inclusions) containing Chlamydia is described. This strategy enables rapid, automated determination of Chlamydia infectivity in samples and can be used to quantitatively investigate inclusion growth dynamics.
Abstract
The obligate intracellular bacterium Chlamydia elicits a great burden on global public health. C. trachomatis is the leading bacterial cause of sexually transmitted infection and also the primary cause of preventable blindness in the world. An essential determinant for successful infection of host cells by Chlamydia is the bacterium’s ability to manipulate host cell signaling from within a novel, vacuolar compartment called the inclusion. From within the inclusion, Chlamydia acquire nutrients required for their 2-3 day developmental growth, and they additionally secrete a panel of effector proteins onto the cytosolic face of the vacuole membrane and into the host cytosol. Gaps in our understanding of Chlamydia biology, however, present significant challenges for visualizing and analyzing this intracellular compartment. Recently, a reverse-imaging strategy for visualizing the inclusion using GFP expressing host cells was described. This approach rationally exploits the intrinsic impermeability of the inclusion membrane to large molecules such as GFP. In this work, we describe how GFP- or mCherry-expressing host cells are generated for subsequent visualization of chlamydial inclusions. Furthermore, this method is shown to effectively substitute for costly antibody-based enumeration methods, can be used in tandem with other fluorescent labels, such as GFP-expressing Chlamydia, and can be exploited to derive key quantitative data about inclusion membrane growth from a range of Chlamydia species and strains.
Introduction
引起细胞内细菌衣原体种传染病引起全球健康的一大负担，包括性传播疾病，盆腔炎，失明，肺炎和动脉粥样硬化有可能1-4。 衣原体到与宿主细胞相互作用，从一个液泡内的能力（称为包含），是一个重要的决定因素的成功细胞和宿主的感染。列入是一种新型的致病隔室，使衣原体生长和在整个衣原体 5的整个 2-3天的发育循环被动态地修改。衣原体的专性细胞内的性质提出了直接学习列入独特的生物学无数挑战，研究界，尤其如此。一个主要的障碍已经无法有效地可视化或者细胞内沙眼或将其列入荧光方法ES在活细胞。最近发现终于露出来产生绿色荧光蛋白表达C.手段衣原体 6;然而，这一发现还没有导致列入特异性标记。一些技术已经被描述用于细菌和夹杂物7,8的标记，但是它们从缺点，例如非特异性，顷刻和易感性漂白受损。一个重要的发现我们集团成立了一个新的战略，利用绿色荧光蛋白表达的宿主细胞9照明列入。这一战略合理利用纳入膜的固有抗渗性的分子大于520大10。当细胞改造成稳定表达特定的细胞内荧光蛋白（ 如 GFP或mCherry）， 衣原体夹杂物具有显着的清晰度荧光他们完全排除可见。这种倒车影像战略使夹杂物的所有叶绿素即时可视化amydia物种，它可以容易地适用于大多数感兴趣的宿主细胞。由于其效用的演示，这种方法以前用来揭示和定义细胞退出途径衣原体属9。
在这里，我们进一步说明此方法的执行，并且可以被利用来导出关于夹杂物的生长动力学键的定量数据。此外，它可以有效地替代昂贵的基于抗体的枚举的方法和可以串联与其他荧光标记，如mKate2表达衣原体 11被使用。这个强大的工具结合，使生活在宿主细胞内的衣原体包涵体膜的物理性质的探索。
Protocol
Representative Results
Discussion
在这里，我们描述实验的策略产生荧光宿主细胞进行实时可视化和衣原体夹杂物的分析。这种液泡可视化方法赋予强大能力来照亮，跟踪和定量测定衣原体夹杂物的动态属性跨细胞群或单细胞中随着时间的推移， 衣原体夹杂在荧光蛋白标记的细胞惊人地明确定义，使得它们很容易识别而不需要附加的免疫处理。此外，这种方法是足够敏感的解决签名形态差异，不同的衣原体物…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
The authors thank Ian Clarke and P. Scott Hefty for the pGFP-SW2 and pASK-GFP-mKate2-L2 plasmids, respectively. We thank Paul Miller for technical assistance and Richard Stephens for other resources. This work was funded by NIH grant AI095603 (KH).
Materials
| Lipofectamine 2000 | Invitrogen | 11668 | |
| Opti-Mem | Invitrogen | 31985 | |
| Polybrene | Sigma | H9268 | |
| 0.45 µm filters | Fisher | 09-719D | |
| G418 | Invitrogen | 10131 | |
| HBSS | Invitrogen | 14025 | |
| DMEM | Invitrogen | 11995 | |
| RPMI 1640 | Invitrogen | 11875 | |
| RPMI 1640 w/o phenol red | Cellgro | 17-105-CV | |
| Penicillin G | Sigma | 13752 | |
| Cycloheximide | Sigma | C7698 | |
| Glass bottom dishes | MatTek | P35G-1.5-14-C | |
| Chamber slides, Lab-Tek II | Nunc | 154526, 154534 |
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