破译和成像发病机制与盘带<em>脓肿分枝杆菌</em>斑马鱼胚胎
Summary
Optically transparent zebrafish embryos are widely used to study and visualize in real time the interactions between pathogenic microorganisms and the innate immune cells. Micro-injection of Mycobacterium abscessus, combined with fluorescence imaging, is used to scrutinize essential pathogenic features such as cord formation in zebrafish embryos.
Abstract
Zebrafish (Danio rerio) embryos are increasingly used as an infection model to study the function of the vertebrate innate immune system in host-pathogen interactions. The ease of obtaining large numbers of embryos, their accessibility due to external development, their optical transparency as well as the availability of a wide panoply of genetic/immunological tools and transgenic reporter line collections, contribute to the versatility of this model. In this respect, the present manuscript describes the use of zebrafish as an in vivo model system to investigate the chronology of Mycobacterium abscessus infection. This human pathogen can exist either as smooth (S) or rough (R) variants, depending on cell wall composition, and their respective virulence can be imaged and compared in zebrafish embryos and larvae. Micro-injection of either S or R fluorescent variants directly in the blood circulation via the caudal vein, leads to chronic or acute/lethal infections, respectively. This biological system allows high resolution visualization and analysis of the role of mycobacterial cording in promoting abscess formation. In addition, the use of fluorescent bacteria along with transgenic zebrafish lines harbouring fluorescent macrophages produces a unique opportunity for multi-color imaging of the host-pathogen interactions. This article describes detailed protocols for the preparation of homogenous M. abscessus inoculum and for intravenous injection of zebrafish embryos for subsequent fluorescence imaging of the interaction with macrophages. These techniques open the avenue to future investigations involving mutants defective in cord formation and are dedicated to understand how this impacts on M. abscessus pathogenicity in a whole vertebrate.
Introduction
脓肿分枝杆菌是一种新兴的病原体，导致广泛的人类临床综合征谱。这些包括皮肤感染以及严重的慢性肺部感染，免疫功能低下和囊性纤维化患者1,2,3,4大多遇到。M.脓肿也算是一大快速增长的分枝杆菌负责人和医院和医源性感染的物种。此外，最近的几份报告强调了可能性，即M.脓肿可以穿过血-脑屏障并诱导在中枢神经系统（CNS）5,6-重要病变。尽管是一个快速的种植者，M。脓肿展品还与相关的那些结核分枝杆菌的致病性的几个功能，包括肉芽肿结构中保持沉默多年，产生干酪样病变的肺部7的能力。更令人震惊的是低仙M的敏度脓肿抗生素，使这些感染极难治疗导致显著治疗失败率8,9。这一物种的重要威胁，主要是其对抗生素的内在阻力，这是公共卫生机构10重大关切和禁忌肺移植11。
脓肿分支杆菌显示平滑（S）或粗（R）的菌落形态，导致不同临床结果。与此相反的S品系，R细菌有增加的趋势端到端，导致绳索或条索状结构12,13。基于两种细胞或动物模型有几个独立的研究揭示了R的超毒力型形态型14,15。从流行病学研究，M的最严重的情况下， 脓肿肺部感染似乎被R相关联的变体16，它是唯一的变体一直被视为持续数年被感染主机 3。该形态类型的差异依赖于表面相关glycopeptidolipids（GPL）12的存在（在S）或损失（在读）。然而，由于目前可用的蜂窝/动物模型的固有局限性用于研究M.脓肿感染，我们对R或S变体的病理生理事件的知识仍然是模糊的。 通过静脉注射或气溶胶途径免疫能力的小鼠感染导致短暂的殖民统治，阻碍利用老鼠来研究持续感染和体内药物敏感试验17。因此，开发的动物模型适合于宿主反应的操作是一项重大的挑战。在这种背景下，近来已经开发感染的非哺乳动物模型，包括果蝇 18，提供了几个优点，例如成本，速度和伦理可接受ö版本1.00的小鼠模型。斑马鱼（ 斑马鱼 ）感染模型也已经探索了可视化，通过无创成像，M的进展和年表脓肿感染的活的动物19。重要的是，概念证明也成立了证明其适合在体内对M.抗生素评估脓肿 17,20。
斑马鱼已经在过去二十年期间已广泛用于研究各种病原体和宿主的免疫系统 21之间的交互。这种替代脊椎动物模式的日益成功，依赖于激励和验证其用于更好地了解众多的病毒和细菌感染19,22,23,24,25,26,27,28,29重大而独特的机会。相对于大多数其他的动物模型，斑马鱼的胚胎是光学透明的，从而允许非侵入性荧光成像30。此HA为首的研究M.脓肿感染斑马鱼的胚胎以前所未有的细节，与细胞外录制的说明中，表示细菌形态可塑性的一例最终。盘带代表免疫系统颠覆的新机制和一个关键机制促进急性M的发病机制脓肿感染19。
这份报告描述了使用斑马鱼胚胎破译M的病理生理特点新的工具和方法脓肿感染并研究杆菌和先天免疫系统之间的亲密互动。首先，将详细显微注射协议，它包括的细菌接种物，胚胎制备，和本身感染的处理，提出。方法特别适用于评估M.脓肿毒力通过测量各种参数，如主机存活和细菌负荷，现介绍。特别重点就如何进行监控，在时空水平，感染的命运和进展以及为 M.宿主的免疫反应脓肿利用视频显微镜。此外，为了调查M.期间的贡献和巨噬细胞的作用脓肿感染，方法生成的巨噬细胞的贫胚胎（使用genetically-或化学为基础的方法）进行说明。最后，协议的可视化使用固定的或活的胚胎的巨噬细胞和中性粒细胞记录的特定交互。
本报告的目的是刺激进一步的研究阐明新的光转化为M.脓肿毒力机制和盘带在建立急性和不受控制的感染过程的特别的作用。
Protocol
Representative Results
Discussion
斑马鱼最近出现作为优良的脊椎动物模型系统用于使用实时36广阔的领域，共焦成像研究细菌感染的动态。分散分枝杆菌悬浮液（协议2.2）与显微注射方法（协议4）相结合，可重现的全身性感染，以及随后的监测和感染，特别注重与主机巨噬细胞细菌相互作用的进展可视化。 M的毒力脓肿体内可以调查由于使用的野生型金黄斑马鱼37。为了研究M之间的相?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
作者感谢K. Kissa有益的讨论，并提供脂，氯膦酸二钠和L.莱玛克里斯南的pTEC27和pTEC15，让tdTomato和芥末的表现，分别的一份厚礼。这项工作形成了法国国家研究署的项目（ZebraFlam ANR-10-MIDI-009和DIMYVIR ANR-13-BSV3-007-01）和欧盟第七框架计划的一部分（FP7-人2011年ITN）根据赠款协议没有。 PITN-GA-2011-289209的玛丽 – 居里的初始培训网络FishForPharma。我们还要感谢该协会格雷戈里Lemarchal和Vaincre拉Mucoviscidose（RF20130500835），用于资助CM杜邦。
Materials
| BBL MGIT PANTA | BD Biosciences | 245114 | |
| Bovine Serum Albumin | Euromedex | 04-100-811-E | |
| Catalase from Bovine Liver | Sigma-Aldrich | C40 | |
| Difco Middlebrook 7H10 Agar | BD Biosciences | 262710 | |
| Difco Middlebrook 7H9 Broth | BD Biosciences | 271310 | |
| Ethyl 3-aminobenzoate methanesulfonate salt (Tricaine) | Sigma-Aldrich | A5040 | |
| Oleic Acid | Sigma-Aldrich | O1008 | |
| Paraformaldehyde | Delta Microscopie | 15710 | |
| Phenol Red | Sigma-Aldrich | 319244 | |
| Tween 80 | Sigma-Aldrich | P4780 | |
| Agar | Gibco Life Technologie | 30391-023 | |
| Low melting agarose | Sigma-Aldrich | ||
| Instant Ocean Sea Salts | Aquarium Systems Inc | ||
| Borosilicate glass capillaries | Sutter instrument Inc | BF100-78-10 | 1mm O.D. X 0.78 mm I.D. |
| Micropipette puller device | Sutter Instrument Inc | Flamming/Brown Micropipette Puller p-87 | |
| Microinjector | Tritech Research | Digital microINJECTOR, MINJ-D | |
| Tweezers | Sciences Tools inc | Dumont # M5S | |
| Microloader Tips | Eppendorf |
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