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

Joachim Pfister,Klaus Kopka,Hubertus Haas,Dominik Summer,Thines Kanagasundaram,Clemens Decristoforo,Alexander Lichius

doi:10.1038/s41598-020-72452-2

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.

Highlights

Live-cell imaging allows the in vivo analysis of subcellular localisation dynamics of physiological processes with high spatial–temporal resolution
A. fumigatus produces the hydroxamate-type siderophores desferri-fusarinine C (FsC) and desferri-N,N’,N’’-triacetylfusarinine C (TAFC) which are secreted in response to iron starvation, as well as desferri-ferricrocin (FC) and desferri-hydroxyferricrocin (HFC) which are used for internal iron handling in hyphae and conidia, respectively[9,10]
Fluorescent dyes were obtained in their carboxylic acid form, as N-hydroxysuccinimid-esters or isothiocyanates for coupling and were used without further purification: Fluorescein isothiocyanate (FITC) (Sigma Aldrich, Vienna, Austria)) 6-(7-Nitrobenzofurazan-4-ylamino)hexanoic acid (NBD) (Sigma Aldrich, Vienna, Austria), 5(6)-carboxy-2′,7′-dichlor-fluorescein-D (DFFDA) (Sigma Aldrich, Vienna, Austria), Calcofluor White stain (CFW) (Sigma Aldrich, Vienna, Austria), 4,4-difluoro-4-bora3a,4a-diaza-s-indacene (BODIPY 630/650), (Lumiprobe GmbH, Hannover, Germany), Cyanine 5-carboxylic acid (Cy5) (Lumiprobe GmbH, Hannover, Germany), 3-Carboxy-6,8-difluoro-7-hydroxycoumaryl succinimidyl ester (Ocean Blue); 5-Carboxytetramethylrhodamine-NHS (TAMRA) (Bio-Techne Ltd, Abingdon, UK), 10-(5-amino-2-methylphenyl) silicon-rhodamine (SiR), (N-(3-Triethylammoniumpropyl)4-(4-(Dibutylamino)Styryl)pyridinium dibromide) (FM1-43) (ThermoFischer Scientific, Watham, MA, USA)

Summary

Introduction

Live-cell imaging allows the in vivo analysis of subcellular localisation dynamics of physiological processes with high spatial–temporal resolution. We developed fluorescent dye conjugates based on the sophisticated iron acquisition system of Aspergillus fumigatus by chemical modification of the siderophore triacetylfusarinine C (TAFC). 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. Live-cell imaging is the key technology to analyse physiological processes inside filamentous fungi, including the uptake and distribution dynamics of fluorescent dyes. A considerable number of organelle-specific fluorescent probes are available for the visualisation of different cellular compartments in filamentous fungi[4,5], none are species-specific and they are not applicable for identification or targeted treatment in situ To overcome this limitation, we exploited the iron acquiring siderophore system of filamentous fungi. Modification of TAFC is a highly promising approach for the generation of novel and accurate diagnostic biomarkers for the detection of A. fumigatus infections

Methods

Results

Discussion

Conclusion