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

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.

Siderophore Scaffold as Carrier for Antifungal Peptides in Therapy of Aspergillus fumigatus Infections

“…Briefly, peptides were produced by solid-phase peptide synthesis (SPPS) using rink amide resin and 2-Chlortrityl resin [ 24 ]. For conjugation of the individual peptides a O-(7-Azabenzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium-hexafluorphosophat (HATU) coupling strategy was selected to bind the peptides to the free amine group of [Fe]DAFC, or using the carboxylic group of [Fe]DAFC succ [ 8 ]. Conjugates were purified using preparative RP-HPLC and the identity was confirmed by MALDI-TOF MS.…”

“…Assays were performed as previously described [ 8 , 25 ]. Briefly, uptake and competition assays were performed with A. fumigatus in iron-depleted and iron-replete Aspergillus minimal media (AMM) [ 26 ], using a 96-well MultiScreen Filter Plates HTS (1 μm glass fiber filter, Merck Millipore, Darmstadt, Germany).…”

“…After chelating iron, TAFC is reabsorbed by a specific siderophore transporter called MirB [ 5 ], while the transporter for [Fe]FsC has not been identified yet. Modifying TAFC using FsC as scaffold allows the creation of molecules that act as specific and target-oriented carriers to the fungal cell [ 6 , 7 , 8 ]. For this purpose, the diacetylated form of TAFC, diacetylfusarinine C (DAFC) was used as starting material.…”

Antifungal siderophore conjugates for theranostic applications in invasive pulmonary aspergillosis using low molecular TAFC scaffolds

“…Furthermore, by using diacetylfusarinine C ([Fe]DAFC), which possesses a free amine group, chemical modifications are possible [17]. In previous experiments by our group, we used fluorescent dyes coupled to DAFC to perform fluorescence-microscopy of A. fumigatus hyphae [18] and labelled with gallium-68, hybrid imaging of an infection in a rat model [19]. This proof of principle using TAFC as a "Trojan horse" to channel a variety of molecules specifically into A. fumigatus via the MirB transporter, led us to the idea to adopt this system for antifungal molecules.…”

“…A similar concept could be applied for thiomersal which was commonly used as a preservative substance in vaccines and ophthalmic products [29,30] but adverse health effects of ethylmercury (active metabolite) are still controversially discussed [31]. The last compound Cy5 was originally designed as an agent for hybrid imaging and microscopy, whereby an antifungal activity was observed during growth experiments by our group [18,19].…”