牙龈卟啉单作为模式生物的评价厌氧细菌的相互作用与宿主细胞
Summary
This article presents two protocols: one to measure anaerobic bacteria that can successfully invade and survive within the host, and the other to visualize anaerobic bacteria interacting with host cells. This study can be applied to any cultivable anaerobe and any eukaryotic cell type.
Abstract
厌氧菌的数量远远超过许多人的壁龛，如肠道，口腔和阴道需氧菌。此外，厌氧菌感染是常见的，经常土著血统的。的一些厌氧病原体侵入人体细胞的能力使它们适应措施逃避先天免疫以及调节宿主细胞中的行为。然而，确保厌氧细菌是试验研究的事件可能会带来的挑战过程中活。 牙龈卟啉 ，革兰氏阴性厌氧菌，能够侵入的各种真核非吞噬细胞的。本文概述了如何成功培养和评估P的能力牙龈侵入人脐静脉内皮细胞（HUVEC）。两个协议被开发：一个用于测量细菌，可以成功地侵入和主机内生存，而其他的可视化的细菌与宿主细胞相互作用。这些技术需要使用一个ANAE的罗比克室提供P.牙龈与最佳生长厌氧环境。
所述第一协议是基于抗生素保护分析，这在很大程度上是用于研究的宿主细胞的入侵的细菌。然而，抗生素保护测定是有限的;仅胞内细菌是培养的下列抗生素治疗和宿主细胞裂解计量。为了评估与宿主细胞，无论是活的和死的相互作用所有的细菌，我们开发了使用荧光显微镜检查宿主 – 病原体相互作用的协议。细菌荧光标记与2'，7'-二（2-羧乙基）-5-（和-6） – 羧基乙酰甲酯（BCECF-AM）和用于感染的真核细胞在厌氧条件下。以下定影用多聚甲醛和透用0.2％的Triton X-100，宿主细胞被标以TRITC鬼笔环肽和DAPI分别以标记细胞骨架和细胞核。多IMA在不同的联络点（Z堆栈）采取GES是为细菌的时空可视化获得。在这项研究中使用的方法可以适用于任何可耕厌氧菌和任何真核细胞类型。
Introduction
厌氧细菌定殖于人的身体的几乎所有表面。虽然主要在肠道和泌尿生殖道，其中氧气浓度是低的植物，它们也存在着在高水平上的皮肤，口，鼻，喉和1。厌氧细菌是内源性感染的常见原因，并且经常从患病部位分离。由于它们的性质挑剔然而，厌氧菌可能很难分离和培养。研究涉及厌氧菌一定限制条件下进行。现代厌氧培养技术使研究人员模仿学习许多厌氧菌实验室菌株，甚至临床分离2,3所需的厌氧设置。
致病性厌氧菌已经开发出一种动态关系和共同进化与它们所在的宿主细胞。最厌氧菌易受到达infecti之前杀死被宿主的免疫反应OU的水平。然而，一些病原菌已经开发机制从逃脱或破坏宿主的免疫反应。他们实现这一目标，通过机制，如免疫识别，免疫介导的中和，改变细胞介导的免疫，侵入宿主细胞，并改变免疫信号4的逃避。 牙龈卟啉 ，革兰氏阴性厌氧菌参与了口头和口外疾病， 是一个高度适于细菌病原体能够引起在宿主5-7致病变化的一个例子。
生物膜斑块口袋累积在牙齿和牙龈粘膜组织间形成能够携带受保护的大气中的氧气8厌氧菌深的裂缝。这些牙周袋作为利基关于各种厌氧病原体， 诸如铜绿牙龈 9 P。牙龈是一个梯形的病原体，它能够改造的荷兰国际集团的口腔微生物群落的促进发展的牙周病10和发展方式。它产生了大量的有效对抗宿主蛋白广谱并提供机制宿主防御11的逃避毒力因子。它也能够侵入上皮细胞，内皮细胞，成纤维细胞，和牙周膜细胞体外 12-14中 和体内15。通过有效地侵入宿主细胞中，P。牙龈可以逃避宿主免疫。有效侵入宿主细胞不仅允许细菌逃离宿主防御但也作为贮存以备将来再感染以及改变宿主细胞。需要由宿主细胞参与粘附和细菌的内化的分子机制的研究进展。研究几个实验室的重点是了解与体育的内在关联的分子事件牙龈由宿主细胞以及用于抑制并劫持免疫反应和生存的敌对宿主防御机制的机制。
有能够识别和表征的病原体，其能够侵入宿主细胞的许多测定法。 但是 ，在体外研究与厌氧病原体提出了许多实验问题，为研究者主要是因为它是难以执行依赖于在不存在氧的笨重仪器研究。这是由真核细胞所需要的氧气，以成长，因此必须在组织培养孵化器分别制备的事实复杂。避免这种障碍之一的方法是将大气中的氧气下进行研究，但是这将使厌氧细菌的生长是不可能的。另一种方法是使用热灭活的细菌感染和研究宿主 – 细胞相互作用。然而，不同的削弱宿主 – 病原体interacti的相关性热灭活和可行的细菌之间存在16。这是中央对学习活菌与不变的表达与宿主细胞相互作用的;因此，方法培养P.在厌氧环境牙龈给出。此外，两个简单的成本效益的协议被证明为评估P的能力牙龈由人脐静脉内皮细胞（HUVECs）被内化。所述第一协议是基于流行的抗生素保护测定。虽然实验非常简单，利用厌氧微生物时的注意事项给出。第二协议要求使用荧光扫描显微镜的可视化和交互内在P.牙龈 。每个测定有其局限性和将要讨论，以提供研究者的轮廓为研究厌氧菌的侵袭优点。虽然目前的手稿研究P.牙龈和内皮细胞，这些协议可以用于许多其它厌氧细菌以及作为其他类型的宿主细胞。
Protocol
Representative Results
Discussion
所有上述方法可用于设计特异性试验，以评估厌氧菌与真核细胞的相互作用。然而，有几个方面的考虑，以成功地执行该实验。第一是在一个研究中所用的微生物菌株。
它是在两个菌株均存活测定法的比较以及由显微镜分析至关重要的是，它们是在类似的生长阶段和达到相似的细胞浓度如在上述的任何差异可以影响入侵效率13。当生长曲线两株细菌对之间的不同，调?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
We would like to thank Dr. Hiroshi Miyazaki, Dr. Scott Henderson, Dr. Todd Kitten, Dr. Justin Hutcherson, Dr. Catherine Jauregui, and Collin R. Berry. This work was supported by NIH NIDCR grants R01DE016124, R01DE018039, and R01DE023304 to J.P. Lewis.
Microscopy was performed at the VCU Department of Anatomy and Neurobiology Microscopy Facility, supported, in part, with funding from NIH-NINDS Center core grant (5P30NS047463).
Materials
| Vinyl Anaerobic Chamber-Type B | Coy Laboratory Products | Model 2000 incubator | |
| TSA II Trypticase Soy Agar w/5% Sheep Blood | BBL | 221261 | |
| Human Umbilical Vein Endothelial Cells 10-donor Pool | LifeLine Technology | FC-0044 | |
| VascuLife VEGF Medium Complete Kit | LifeLine Technology | LL-0003 | |
| TrypKit | LifeLine | LL-0013 | |
| Saponin | Riedel-de Haen | 16109 | |
| Gentamicin Sulfate Salt | Sigma-Aldrich | G-1264 | |
| Metronidazole | Sigma-Aldrich | M-3761 | |
| BCECF-AM | LifeTechnologies | B1150 | |
| TRITC Phalloidin | Sigma-Aldrich | P1951 | |
| 18 mm Circular Coverslips | Electron Microscopy Sciences | 72222-01 | |
| VectaShield Mounting Medium with DAPI | Vector Laboratories | H-1200 |
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