Hybrid Imaging of Aspergillus fumigatus Pulmonary Infection with Fluorescent, 68Ga-Labelled Siderophores

Abstract

Aspergillus fumigatus (A. fumigatus) is a human pathogen causing severe invasive fungal infections, lacking sensitive and selective diagnostic tools. A. fumigatus secretes the siderophore desferri-triacetylfusarinine C (TAFC) to acquire iron from the human host. TAFC can be labelled with gallium-68 to perform positron emission tomography (PET/CT) scans. Here, we aimed to chemically modify TAFC with fluorescent dyes to combine PET/CT with optical imaging for hybrid imaging applications. Starting from ferric diacetylfusarinine C ([Fe]DAFC), different fluorescent dyes were conjugated (Cy5, SulfoCy5, SulfoCy7, IRDye 800CW, ATTO700) and labelled with gallium-68 for in vitro and in vivo characterisation. Uptake assays, growth assays and live-cell imaging as well as biodistribution, PET/CT and ex vivo optical imaging in an infection model was performed. Novel fluorophore conjugates were recognized by the fungal TAFC transporter MirB and could be utilized as iron source. Fluorescence microscopy showed partial accumulation into hyphae. µPET/CT scans of an invasive pulmonary aspergillosis (IPA) rat model revealed diverse biodistribution patterns for each fluorophore. [68Ga]Ga-DAFC-Cy5/SufloCy7 and -IRDye 800CW lead to a visualization of the infected region of the lung. Optical imaging of ex vivo lungs corresponded to PET images with high contrast of infection versus non-infected areas. Although fluorophores had a decisive influence on targeting and pharmacokinetics, these siderophores have potential as a hybrid imaging compounds combining PET/CT with optical imaging applications.

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

“…To obtain the iron free siderophores, the product was treated with a 1,000-fold excess of ethylenediaminetetraacetic acid (EDTA) at pH 4 and purified by preparative RP-HPLC, as described earlier 18 . 68 Ga-compounds were prepared as previously described 21 using gallium-68 from a commercial 68 Ge/ 68 Ga-generator (IGG100, Eckert & Ziegler Isotope products, Berlin, Germany) and incubating with iron free siderophore in acetate buffer at pH 4.5.…”

“…Spores were point inoculated (104 conidia) in 24-well plates, containing 0.5 mL of Aspergillus minimal medium agar and an increasing concentration of iron containing siderophore ranging from 0.1-50 μM. Plates were incubated for 48 h at 37 °C in a humidity chamber and visually assessed 18 . Without siderophore supplementation, no growth of this mutant strain was observed.…”

“…Z-stack acquisition is indicated in the image description where applicable. Apart from brightness and contrast adjustments and cropping using the ImageJ 1.52a open source software platform (Wayne Rasband, NIH, Bethesda, MD, USA), images were not subjected to further manipulation 18 .…”

“…Uptake assays demonstrated that various modifications are possible without losing recognition of TAFC by MirB 17 . During the development of novel hybrid imaging applications for fungal infection diagnostics, combining positron emission tomography (PET) with optical imaging by introduction of a near-infrared (NIR) dye 18 , we successfully visualized A. fumigatus infections. However, we also observed loss of uptake depending on the fluorescent label used.…”

“…Starting from the diacetylated form (diacetylfusarinine C, DAFC) 18 we coupled a series of different fluorescent dyes and characterized their recognition by MirB.…”