The pathogenic fungus Aspergillus fumigatus utilizes a cyclic ferrioxamine E (FOXE) siderophore to acquire iron from the host. Biomimetic FOXE analogues were labeled with gallium-68 for molecular imaging with PET. [68Ga]Ga(III)-FOXE analogues were internalized in A. fumigatus cells via Sit1. Uptake of [68Ga]Ga(III)-FOX 2-5, the most structurally alike analogue to FOXE, was high by both A. fumigatus and bacterial Staphylococcus aureus. However, altering the ring size provoked species-specific uptake between these two microbes: ring size shortening by one methylene unit (FOX 2-4) increased uptake by A. fumigatus compared to that by S. aureus, whereas lengthening the ring (FOX 2-6 and 3-5) had the opposite effect. These results were consistent both in vitro and in vivo, including PET imaging in infection models. Overall, this study provided valuable structural insights into the specificity of siderophore uptake and, for the first time, opened up ways for selective targeting and imaging of microbial pathogens by siderophore derivatization.

• MeSH
• Aspergillus fumigatus * metabolism   chemistry   MeSH
• Aspergillosis * diagnostic imaging   microbiology   MeSH
• Biomimetic Materials chemistry   metabolism   MeSH
• Peptides, Cyclic MeSH
• Deferoxamine chemistry   MeSH
• Species Specificity MeSH
• Mice MeSH
• Positron-Emission Tomography * methods   MeSH
• Gallium Radioisotopes * chemistry   MeSH
• Siderophores * chemistry   metabolism   MeSH
• Staphylococcus aureus * metabolism   MeSH
• Ferric Compounds chemistry   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Peptides, Cyclic MeSH
• Deferoxamine MeSH
• ferrioxamine E MeSH Browser
• Gallium-68 MeSH Browser
• Gallium Radioisotopes * MeSH
• Siderophores * MeSH
• Ferric Compounds MeSH

Invasive fungal infections have become a major challenge for public health, mainly due to the growing numbers of immunocompromised patients, with high morbidity and mortality. Currently, conventional imaging modalities such as computed tomography and magnetic resonance imaging contribute largely to the noninvasive diagnosis and treatment evaluation of those infections. These techniques, however, often fall short when a fast, noninvasive and specific diagnosis of fungal infection is necessary. Molecular imaging, especially using nuclear medicine-based techniques, aims to develop fungal-specific radiotracers that can be tested in preclinical models and eventually translated to human applications. In the last few decades, multiple radioligands have been developed and tested as potential fungal-specific tracers. These include radiolabeled peptides, antifungal drugs, siderophores, fungal-specific antibodies, and sugars. In this review, we provide an overview of the pros and cons of the available radiotracers. We also address the future prospects of fungal-specific imaging.

• Keywords
• PET, immunoPET, invasive fungal infection, radionuclide imaging, siderophores,
• MeSH
• Antifungal Agents therapeutic use   MeSH
• Invasive Fungal Infections * MeSH
• Humans MeSH
• Mycoses * diagnostic imaging   MeSH
• Tomography, X-Ray Computed MeSH
• Positron-Emission Tomography methods   MeSH
• Antibodies, Fungal MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Research Support, N.I.H., Intramural MeSH
• Names of Substances
• Antifungal Agents MeSH
• Antibodies, Fungal MeSH

Fungal infections are a serious threat, especially for immunocompromised patients. Early and reliable diagnosis is crucial to treat such infections. The bacterially produced siderophore desferrioxamine B (DFO-B) is utilized by a variety of microorganisms for iron acquisition, while mammalian cells lack the uptake of DFO-B chelates. DFO-B is clinically approved for a variety of long-term chelation therapies. Recently, DFO-B-complexed gallium-68 ([68Ga]Ga-DFO-B) was shown to enable molecular imaging of bacterial infections by positron emission tomography (PET). Here, we demonstrate that [68Ga]Ga-DFO-B can also be used for the preclinical molecular imaging of pulmonary infection caused by the fungal pathogen Aspergillus fumigatus in a rat aspergillosis model. Moreover, by combining in vitro uptake studies and the chemical modification of DFO-B, we show that the cellular transport efficacy of ferrioxamine-type siderophores is impacted by the charge of the molecule and, consequently, the environmental pH. The chemical derivatization has potential implications for its diagnostic use and characterizes transport features of ferrioxamine-type siderophores.

• Keywords
• Aspergillus fumigatus, PET, desferrioxamine B, gallium-68, imaging, infection, invasive pulmonary aspergillosis, pH, positron emission tomography, siderophore,
• Publication type
• Journal Article MeSH

Invasive pulmonary aspergillosis (IPA) is a life-threatening form of fungal infection, primarily in immunocompromised patients and associated with significant mortality. Diagnostic procedures are often invasive and/or time consuming and existing antifungals can be constrained by dose-limiting toxicity and drug interaction. In this study, we modified triacetylfusarinine C (TAFC), the main siderophore produced by the opportunistic pathogen Aspergillus fumigatus (A. fumigatus), with antifungal molecules to perform antifungal susceptibility tests and molecular imaging. A variation of small organic molecules (eflornithine, fludioxonil, thiomersal, fluoroorotic acid (FOA), cyanine 5 (Cy5) with antifungal activity were coupled to diacetylfusarinine C (DAFC), resulting in a "Trojan horse" to deliver antifungal compounds specifically into A. fumigatus hyphae by the major facilitator transporter MirB. Radioactive labeling with gallium-68 allowed us to perform in vitro characterization (distribution coefficient, stability, uptake assay) as well as biodistribution experiments and PET/CT imaging in an IPA rat infection model. Compounds chelated with stable gallium were used for antifungal susceptibility tests. [Ga]DAFC-fludioxonil, -FOA, and -Cy5 revealed a MirB-dependent active uptake with fungal growth inhibition at 16 µg/mL after 24 h. Visualization of an A. fumigatus infection in lungs of a rat was possible with gallium-68-labeled compounds using PET/CT. Heterogeneous biodistribution patterns revealed the immense influence of the antifungal moiety conjugated to DAFC. Overall, novel antifungal siderophore conjugates with promising fungal growth inhibition and the possibility to perform PET imaging combine both therapeutic and diagnostic potential in a theranostic compound for IPA caused by A. fumigatus.

• Keywords
• Aspergillus fumigatus, PET/CT, TAFC, antifungal, antifungal susceptibility testing, invasive pulmonary aspergillosis, siderophore, theranostics,
• Publication type
• Journal Article MeSH

Invasive fungal infections such as aspergillosis are life-threatening diseases mainly affecting immuno-compromised patients. The diagnosis of fungal infections is difficult, lacking specificity and sensitivity. This review covers findings on the preclinical use of siderophores for the molecular imaging of infections. Siderophores are low molecular mass chelators produced by bacteria and fungi to scavenge the essential metal iron. Replacing iron in siderophores by radionuclides such as gallium-68 allowed the targeted imaging of infection by positron emission tomography (PET). The proof of principle was the imaging of pulmonary Aspergillus fumigatus infection using [68Ga]Ga-triacetylfusarinine C. Recently, this approach was expanded to imaging of bacterial infections, i.e., with Pseudomonas aeruginosa. Moreover, the conjugation of siderophores and fluorescent dyes enabled the generation of hybrid imaging compounds, allowing the combination of PET and optical imaging. Nevertheless, the high potential of these imaging probes still awaits translation into clinics.

• Keywords
• bacterial, fluorescence, fungal, imaging, infection, positron emission tomography, siderophore,
• Publication type
• Journal Article MeSH
• Review MeSH

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.

• Keywords
• PET, fluorescence microscopy, gallium-68, invasive pulmonary aspergillosis, near infrared, siderophores,
• MeSH
• Aspergillus fumigatus MeSH
• Fluorescent Dyes MeSH
• Microscopy, Fluorescence MeSH
• Invasive Pulmonary Aspergillosis diagnostic imaging   microbiology   MeSH
• Binding, Competitive MeSH
• Hydrogen-Ion Concentration MeSH
• Rats MeSH
• Disease Models, Animal MeSH
• Positron Emission Tomography Computed Tomography MeSH
• Rats, Inbred Lew MeSH
• Gallium Radioisotopes chemistry   MeSH
• Siderophores metabolism   MeSH
• Protein Binding MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Fluorescent Dyes MeSH
• Gallium-68 MeSH Browser
• Gallium Radioisotopes MeSH
• Siderophores MeSH