A tracer-based method enables tracking of Plasmodium falciparum malaria parasites during human skin infection.,Theranostics

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A tracer-based method enables tracking of Plasmodium falciparum malaria parasites during human skin infection.
Theranostics ( IF 12.4 ) Pub Date : 2019-01-01 , DOI: 10.7150/thno.33467
Béatrice M F Winkel _{1,

2} , Clarize M de Korne _{1,

2} , Matthias N van Oosterom ₂ , Diego Staphorst ₂ , Anton Bunschoten _{2,

3} , Marijke C C Langenberg ₁ , Séverine C Chevalley-Maurel ₁ , Chris J Janse ₁ , Blandine Franke-Fayard ₁ , Fijs W B van Leeuwen ₂ , Meta Roestenberg _{1,

4}

Affiliation

Introduction: The skin stage of malaria is a vital and vulnerable migratory life stage of the parasite. It has been characterised in rodent models, but remains wholly uninvestigated for human malaria parasites. To enable in depth analysis of not genetically modified (non-GMO) Plasmodium falciparum (Pf) sporozoite behaviour in human skin, we devised a labelling technology (Cy5M2, targeting the sporozoite mitochondrion) that supports tracking of individual non-GMO sporozoites in human skin. Methods: Sporozoite labelling with Cy5M2 was performed in vitro as well as via the feed of infected Anopheles mosquitos. Labelling was validated using confocal microscopy and flow cytometry and the fitness of labelled sporozoites was determined by analysis of infectivity to human hepatocytes in vitro, and in vivo in a rodent infection model. Using confocal video microscopy and custom software, single-sporozoite tracking studies in human skin-explants were performed. Results: Both in vitro and in mosquito labelling strategies yielded brightly fluorescent sporozoites of three different Plasmodium species. Cy5M2 uptake colocalized with MitoTracker® green and could be blocked using the known Translocator protein (TSPO)-inhibitor PK11195. This method supported the visualization and subsequent quantitative analysis of the migration patterns of individual non-GMO Pf sporozoites in human skin and did not affect the fitness of sporozoites. Conclusions: The ability to label and image non-GMO Plasmodium sporozoites provides the basis for detailed studies on the human skin stage of malaria with potential for in vivo translation. As such, it is an important tool for development of vaccines based on attenuated sporozoites and their route of administration.

中文翻译：

基于示踪剂的方法能够在人类皮肤感染期间追踪恶性疟原虫疟原虫。

简介：疟疾的皮肤阶段是该寄生虫至关重要且脆弱的迁徙生命阶段。在啮齿动物模型中已对其进行了表征，但对于人类疟疾寄生虫仍未进行调查。为了能够深入分析人类皮肤中非转基因（非GMO）的恶性疟原虫（Pf）子孢子的行为，我们设计了一种标记技术（针对子孢子线粒体的Cy5M2），支持跟踪人类皮肤中的各个非GMO子孢子。。方法：在体外以及通过感染的蚊子的饲料对Cy5M2进行亚孢子标记。使用共聚焦显微镜和流式细胞术验证标记，并通过在体外和在啮齿动物感染模型中体内对人肝细胞的感染性分析，确定标记的子孢子的适应性。使用共聚焦显微镜和定制软件，在人类皮肤外植体中进行了单子孢子跟踪研究。结果：体外和蚊子标记策略均产生了三种不同疟原虫物种的明亮的子孢子体。Cy5M2的摄取与MitoTracker®green共存，可以使用已知的Translocator蛋白（TSPO）抑制剂PK11195阻止。此方法支持对人类皮肤中各个非GMO Pf子孢子的迁移模式进行可视化和后续定量分析，并且不影响子孢子的适应性。结论：标记和成像非转基因疟原虫子孢子的能力为详细研究人类疟疾的皮肤阶段提供了基础，并具有体内翻译的潜力。因此，

更新日期：2019-01-01

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