1. Academic Validation
  2. Live-cell imaging with Aspergillus fumigatus-specific fluorescent siderophore conjugates

Live-cell imaging with Aspergillus fumigatus-specific fluorescent siderophore conjugates

  • Sci Rep. 2020 Sep 23;10(1):15519. doi: 10.1038/s41598-020-72452-2.
Joachim Pfister  # 1 Alexander Lichius  # 2 Dominik Summer 1 Hubertus Haas 3 Thines Kanagasundaram 4 Klaus Kopka 4 5 6 Clemens Decristoforo 7
Affiliations

Affiliations

  • 1 Department of Nuclear Medicine, Medical University Innsbruck, Innsbruck, Austria.
  • 2 Department of Microbiology, University Innsbruck, Innsbruck, Austria.
  • 3 Division of Molecular Biology, Medical University Innsbruck, Innsbruck, Austria.
  • 4 Division of Radiopharmaceutical Chemistry, German Cancer Research Center, (DKFZ), Im Neuenheimer Feld 280, 69120, Heidelberg, Germany.
  • 5 Institute of Radiopharmaceutical Cancer Research, Helmholtz-Zentrum Dresden-Rossendorf, Dresden, Germany.
  • 6 Faculty of Chemistry and Food Chemistry, Technische Universität Dresden, Dresden, Germany.
  • 7 Department of Nuclear Medicine, Medical University Innsbruck, Innsbruck, Austria. Clemens.decristoforo@i-med.ac.at.
  • # Contributed equally.
Abstract

Live-cell imaging allows the in vivo analysis of subcellular localisation dynamics of physiological processes with high spatial-temporal resolution. However, only few fluorescent dyes have been custom-designed to facilitate species-specific live-cell imaging approaches in filamentous fungi to date. Therefore, we developed Fluorescent Dye conjugates based on the sophisticated iron acquisition system of Aspergillus fumigatus by chemical modification of the siderophore triacetylfusarinine C (TAFC). Various fluorophores (FITC, NBD, Ocean Blue, BODIPY 630/650, SiR, TAMRA and Cy5) were conjugated to diacetylfusarinine C (DAFC). Gallium-68 labelling enabled in vitro and in vivo characterisations. LogD, uptake assays and growth assays were performed and complemented by live-cell imaging in different Aspergillus species. Siderophore conjugates were specifically recognised by the TAFC transporter MirB and utilized as an iron source in growth assays. Fluorescence microscopy revealed uptake dynamics and differential subcellular accumulation patterns of all compounds inside Fungal hyphae.[Fe]DAFC-NBD and -Ocean Blue accumulated in vacuoles, whereas [Fe]DAFC-BODIPY, -SiR and -Cy5 localised to mitochondria. [Fe]DAFC -FITC showed a uniform cytoplasmic distribution, whereas [Fe]DAFC-TAMRA was not internalised at all. Co-staining experiments with commercially available fluorescent dyes confirmed these findings. Overall, we developed a new class of fluorescent dyes that vary in intracellular Fungal targeting , thereby providing novel tools for live-cell imaging applications for Aspergillus fumigatus.

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