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Updated: Dec 8, 2025
Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus
Published on: April 19, 2017
Live-cell imaging with Aspergillus fumigatus-specific fluorescent siderophore conjugates
Joachim Pfister1, Alexander Lichius2, Dominik Summer1
1Department of Nuclear Medicine, Medical University Innsbruck, Innsbruck, Austria.
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.
Insights
Researchers developed novel fluorescent dyes for live-cell imaging in Aspergillus fumigatus by modifying the triacetylfusarinine C (TAFC) siderophore. These TAFC-dye conjugates target specific cellular compartments, offering new tools for studying fungal biology.
Area of Science:
- Mycology
- Cell Biology
- Chemical Biology
Background:
- Live-cell imaging is crucial for analyzing subcellular dynamics in vivo.
- Few fluorescent dyes are optimized for filamentous fungi like Aspergillus fumigatus.
- The iron acquisition system of Aspergillus fumigatus presents a target for novel probe development.
Purpose of the Study:
- To develop and characterize novel fluorescent dye conjugates for live-cell imaging in Aspergillus species.
- To investigate the uptake, localization, and utilization of these conjugates by fungal cells.
- To establish new tools for studying fungal physiology and subcellular processes.
Main Methods:
- Chemical modification of the siderophore triacetylfusarinine C (TAFC) to create diacetylfusarinine C (DAFC) conjugates with various fluorophores (FITC, NBD, Ocean Blue, BODIPY 630/650, SiR, TAMRA, Cy5).
- Gallium-68 labeling for in vitro and in vivo characterization.
- LogD, uptake assays, growth assays, and live-cell fluorescence microscopy in Aspergillus species.
- Co-staining experiments with established fluorescent dyes.
Main Results:
- Siderophore conjugates were recognized by the TAFC transporter MirB and used as an iron source.
- Fluorescence microscopy revealed distinct intracellular accumulation patterns: [Fe]DAFC-NBD and -Ocean Blue localized to vacuoles, [Fe]DAFC-BODIPY, -SiR, and -Cy5 to mitochondria.
- [Fe]DAFC-FITC showed cytoplasmic distribution, while [Fe]DAFC-TAMRA was not internalized.
- Co-staining confirmed the observed localization patterns.
Conclusions:
- A new class of fluorescent dye conjugates targeting specific intracellular compartments in Aspergillus was successfully developed.
- These novel probes demonstrate differential subcellular localization (vacuoles, mitochondria, cytoplasm) within fungal hyphae.
- The developed fluorescent siderophores serve as valuable new tools for live-cell imaging and studying iron uptake mechanisms in filamentous fungi.
