Visualizing the interaction of Acanthamoeba castellanii with human retinal epithelial cells by spontaneous Raman and CARS imaging
Visualizing the interaction of Acanthamoeba castellanii with human retinal epithelial cells by spontaneous Raman and CARS imaging
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DOI：
10.1002/jrs.5296
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发表时间：
2018-03
影响因子：
2.5
通讯作者：
Abida Naemat;F. Sinjab;A. McDonald;A. Downes;A. Elfick;H. Elsheikha;I. Notingher
中科院分区：
文献类型：
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作者：
Abida Naemat;F. Sinjab;A. McDonald;A. Downes;A. Elfick;H. Elsheikha;I. Notingher
对营养吸收机制的更好理解可以使对具有重要医学意义的病原体的营养感知途径的针对性操作达到比目前可能的程度更大的程度。在这种背景下，我们提出使用自发拉曼光谱和相干反斯托克斯拉曼光谱来可视化原生动物卡氏棘阿米巴和宿主人类细胞之间的依赖于时间的分子相互作用。人视网膜色素上皮(ARPE-19)细胞预先用氚代苯丙氨酸(L-Phe[D8])标记，用拉曼光谱检测宿主来源的L-苯丙氨酸(D8)在感染后48小时内被锥虫滋养体摄取。这一方法揭示了在最初的24 HPI中，卡氏疟原虫滋养体对这种必需氨基酸的摄取与时间相关的模式，并在48 HPI时在滋养体中检测到L-Phe(D8)的完全浓缩。相比之下，在3、24和48小时的HPI后，无细胞的滋养体仅从添加L-Phe(D8)的培养液中适度摄取16-18%的L-Phe(D8)。相干反斯托克斯拉曼光谱显微镜被成功地用来监测滋养体内与宿主细胞接触时脂类的重新编程。本文介绍的方法学提供了新的进展，能够分析从宿主细胞和细胞外环境获取氨基酸的动力学，并可视化滋养体内的脂重新编程。
Improved understanding of the mechanism of nutrient's uptake can enable targeted manipulation of nutrient sensing pathways in medically important pathogens to a greater degree than is currently possible. In this context, we present the use of spontaneous Raman microspectroscopy and coherent anti-Stokes Raman spectroscopy to visualize the time-dependent molecular interactions between the protozoan Acanthamoeba castellanii and host human cells. Human retinal pigment epithelial (ARPE-19) cells were pre-labelled with deuterated Phe (L-Phe[D8]) and the uptake of the host derived L-Phe(D8) by A. castellanii trophozoites was measured by Raman microspectroscopy for up to 48 hr post infection (hpi). This approach revealed a time-dependent uptake pattern of this essential amino acid by A. castellanii trophozoites during the first 24 hpi with complete enrichment with L-Phe(D8) detected in trophozoites at 48 hpi. In contrast, cell free A. castellanii trophozoites showed a modest uptake of only 16–18% L-Phe(D8) from L-Phe(D8)–supplemented culture medium after 3, 24, and 48 hr hpi. Coherent anti-Stokes Raman spectroscopy microscopy was successfully used to monitor the reprogramming of lipids within the trophozoites as they engaged with host cells. The methodology presented here provides new advances in the ability to analyze the kinetic of amino acid acquisition by A. castellanii from host cell and extracellular environment, and to visualize lipid reprogramming within the trophozoite.