I S R Storer, L E Sastré-Velásquez, T Easter, B Mertens, A Dallemulle, M Bottery, R Tank, M Offterdinger, M J Bromley, N van Rhijn, F Gsaller
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引用次数: 0
摘要
荧光蛋白(FPs)是分子成像、单细胞动力学、感染模型成像等领域不可或缺的工具。然而,下一代荧光蛋白尚未在曲霉菌中得到表征。在这里,我们描述了致病性丝状真菌曲霉(Aspergillus fumigatus)中跨越可见光光谱的 18 种 FPs。我们报告了在菌丝和孢子形态中 FP 的活体亮度,并展示了如何利用基于氟嘧啶的选择系统迭代引入四种不同的 FP,从而实现细胞膜、线粒体、过氧化物酶体和液泡的同时可视化。利用这种菌株,我们描述并比较了细胞器对伏立康唑、两性霉素 B 以及新型抗真菌药物奥罗菲姆和马诺吉匹克诱导的压力的动态反应。荧光基因工具箱的扩展将突破丝状真菌荧光成像研究应用的界限。
Shining a light on the impact of antifungals on Aspergillus fumigatus subcellular dynamics through fluorescence imaging.
Fluorescent proteins (FPs) are indispensable tools used for molecular imaging, single-cell dynamics, imaging in infection models, and more. However, next-generation FPs have yet to be characterized in Aspergillus. Here, we characterize 18 FPs in the pathogenic filamentous fungus Aspergillus fumigatus spanning the visible light spectrum. We report on in vivo FP brightness in hyphal and spore morphotypes and show how a fluoropyrimidine-based selection system can be used to iteratively introduce four distinct FPs enabling the simultaneous visualization of the cell membrane, mitochondria, peroxisomes, and vacuoles. Using this strain, we describe and compare the dynamic responses of organelles to stresses induced by voriconazole, amphotericin B, and the novel antifungal drugs olorofim and manogepix. The expansion to the fluorescent genetic toolbox will overcome boundaries in research applications that involve fluorescence imaging in filamentous fungi.
期刊介绍:
Antimicrobial Agents and Chemotherapy (AAC) features interdisciplinary studies that build our understanding of the underlying mechanisms and therapeutic applications of antimicrobial and antiparasitic agents and chemotherapy.