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

Joachim Pfister,Clemens Decristoforo,Dominik Summer,Piriya Kaeopookum,Thomas Orasch,Laurin Kochinke,Marta Khoylou,Hubertus Haas,Alexander Lichius,Milos Petrik

doi:10.3390/biom10020168

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.

Highlights

Fungal infections of humans are widespread and can appear in many different forms
One of the most prominent examples is invasive pulmonary aspergillosis (IPA), an opportunistic infection of the lung mainly caused by Aspergillus fumigatus (A. fumigatus)
Doyle et al modified [Fe]fusarinine C (FsC) with the fluorescent dye NBD (6-(N-(7-nitrobenz-2-oxa,1,3-diazol-4-yl)amino)Hexanoate) for fluorescent microscopy of A. fumigatus showing the feasibility of modifying siderophores retaining recognition by pathogenic fungi [30]

Summary

Introduction

Fungal infections of humans are widespread and can appear in many different forms. Infections of the skin and nails are very common, like onychomycosis or oral thrush [1]. Most people will suffer from those at least once in their lifetime, but these infections are diagnosed and respectively curable. One of the most prominent examples is invasive pulmonary aspergillosis (IPA), an opportunistic infection of the lung mainly caused by Aspergillus fumigatus (A. fumigatus). The vegetatively produced spores (conidia), A. fumigatus uses for its own distribution, are the prominent infectious agent in air samples, including air from hospitals. In case the immune system is suppressed including genetic reasons, medication (e.g., chemotherapy, after organ transplantation and concomitant immunosuppressive drug therapy) or deficient lung functions due to comorbidity (e.g., COPD or mechanical ventilation in intensive care units), the conidia can germinate, invade the lung tissue and develop IPA. One reason for the high lethality rates of IPA originate from insufficient diagnostic methods. New non-invasive methods to diagnose the early onset of IPA are urgently needed

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