Live-cell Imaging with Aspergillus Fumigatus-specific fluorescent Siderophore Conjugates

Live-cell imaging allows to analyse the subcellular localisation dynamics of physiological processes with high spatial-temporal resolution in vivo. So far, only few fluorescent dyes have been custom-designed to facilitate species-specific live-cell imaging approaches in filamentous fungi. 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 in growth assays as an iron source. 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 varying in intracellular fungal targeting providing novel tools for live-cell imaging applications for Aspergillus fumigatus.