Desferrioxamine B-Mediated Pre-Clinical In Vivo Imaging of Infection by the Mold Fungus Aspergillus fumigatus

Abstract

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.

Uptake of the Siderophore Triacetylfusarinine C, but Not Fusarinine C, Is Crucial for Virulence of Aspergillus fumigatus

“…Moreover, our data demonstrate that DAFC-FITC serves as a TAFC surrogate, allowing the analysis of TAFC uptake by epifluorescence microscopy. The elucidation of the substrate specificity of fungal siderophore transport is also helpful for the further development of siderophore-mediated in vivo imaging of fungal infections by 68 Ga positron emission tomography (PET), as shown previously with TAFC, desferrioxamine B, desferrioxamine E and fluorescent derivatives thereof ( 8 , 12 , 13 ). The elucidation of the substrate specificities of SITs will assist in the structural characterization of substrate discrimination.…”

“…Triacetylfusarinine C and fusarinine C were produced and isolated in-house from iron-starved liquid A. fumigatus cultures as described previously ( 30 , 66 ). The iron-free siderophores TAFC, FsC and FC were labeled for uptake assays as previously described ( 13 , 67 – 69 ), by using 200 μL of a 68 GaCl 3 eluate (~20 to 30 MBq) obtained by the fractionated elution of a 68 Ge/ 68 Ga generator (IGG100; Eckert and Ziegler Isotope Products, Berlin, Germany) with 0.1 M hydrochloric acid. Ten microliters of iron-free siderophores (5 to 8 nmol) was incubated with the 68 GaCl 3 eluate in 40 μL of sodium acetate (NaOAc) buffer (pH 4.0) for 15 min at room temperature.…”

“…The gold standard still relies on the detection of serum galactomannan, a fungal cell wall component ( 4 , 5 ), although many other diagnostic tools for aspergillosis are under development ( 6 – 9 ). Notably, siderophores (see below) were found to have great potential as biomarkers in blood, urine and bronchoalveolar lavage fluid for human A. fumigatus infections ( 6 , 7 , 10 , 11 ) and for the in vivo imaging of A. fumigatus infections by 68 Ga positron emission tomography (PET) ( 8 , 12 , 13 ). For the treatment of aspergillosis, only a few drug classes are currently used, which target either the fungal cell wall or the cell membrane, e.g., echinocandins, polyenes and azoles.…”

Siderophores: a potential role as a diagnostic for invasive fungal disease

“…Methods for the quantitative determination of siderophores such as TAFC in urine, serum and BALF have been established, although more rapid and cost-effective methods are needed for implementation into routine clinical practice. In animal models, siderophores have also been successfully utilized for functional imaging (PET), visualizing the fungal infection sites in the host [39,41 ▪ ,42,44,46–49,50 ▪ ]. 68 Ga proved to be useful, albeit not the only option in this regard.…”

“…However, invasive sampling is required for this diagnostic approach. This limitation is overcome by functional imaging techniques, particularly PET, as demonstrated in animal studies [39,41 ▪ ,42,44,46–49,50 ▪ ].…”