Biomimetic Analogues of the Desferrioxamine E Siderophore for PET Imaging of Invasive Aspergillosis: Targeting Properties and Species Specificity

Abstract

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.

Figure 1

FOXE and its FOX analogues were investigated in this work. Iron-chelating hydroxamic acid groups marked in red.

Figure 2

Growth promotion analysis of FOX analogues. 10⁴ conidia of A. fumigatus mutant strains ΔsidAΔftrA (A) and ΔsidAΔftrAΔsit1 (B) were point-inoculated for every condition. All natural and artificial siderophores were tested in the ferri-form in the concentrations 0.1, 1.0, 10, and 50 μM. As controls, TAFC, which is taken up in a Sit1-independent manner, and FOXE were included. Moreover, controls without siderophore supplementation with (+) or without (−) inoculation of conidia were included. Greenish coloration of the fungal colonies indicates asexual sporulation. Pictures were taken after 48 h of incubation at 37 °C.

Figure 3

Uptake of ⁶⁸Ga-labeled FOXE analogues by Fe(III)-FOXE-blocked and -unblocked iron-starved (Fe(−)) A. fumigatus cultures after 45 min of incubation. * indicates significant difference of blocking compared with nonblocking (mean of three independent experiments, paired t test, p < 0.01).

Figure 4

Uptake of [⁶⁸Ga]Ga(III)-FOXE and [⁶⁸Ga]Ga(III)-FOX 2–5 by A. fumigatus WT, Δsit1, and sit1^xyl prom. Uptake of [⁶⁸Ga]Ga(III)-FOXE was normalized to WT. The results indicate relevant uptake only by the WT under iron deplete conditions (Fe(−)) and in the sit1^xyl prom mutant with xylose (Xyl(+)), which promotes artificial Sit1 upregulation, in contrast to the sit1^xyl prom mutant without xylose (Xyl(−)), a mutant lacking Sit1 and WT grown under iron-sufficient conditions (Fe(+)); (mean of four replicates from one experiment).

Figure 5

Ex vivo biodistribution data of (A) [⁶⁸Ga]Ga-FOX 2–5, (B) [⁶⁸Ga]Ga-FOX 2–4, and (C) [⁶⁸Ga]Ga-FOX 2–6 in healthy Balb/c mice 30 and 90 min postinjection (n = 3 per time interval).

Figure 6

(A) Dynamic imaging (PET/CT MIP images) of [⁶⁸Ga]Ga(III)-FOX 2–5 in a rat (n = 1) with A. fumigatus lung infection (48 h after infection) showing rapid uptake in the infected area and renal clearance of the unbound tracer up to 60 min postinjection. The yellow arrow indicates the site of infection. (B) Time–activity curves of [⁶⁸Ga]Ga(III)-FOX 2–5 in healthy and infected rat lung tissue from (A).

Figure 7

Static PET/MRI MIP images of [⁶⁸Ga]Ga(III)-FOX 2–5 in a rat model of A. fumigatus lung infection. Images of different rats imaged (A) 48 h and (B) 72 h after infection and 45 min after injection of [⁶⁸Ga]Ga(III)-FOX 2–5. Yellow arrows indicate the site of the infection.

Figure 8

Comparison of ⁶⁸Ga-labeled FOX derivatives uptake in A. fumigatus and S. aureus cultures. (mean of three independent experiments).

Figure 9

(A) Static PET/CT imaging data: MIP images of [⁶⁸Ga]Ga-FOX 2–4 and [⁶⁸Ga]Ga-FOX 2–6 in A. fumigatus-infected rats 45 min after injection. The red arrow indicates A. fumigatus infection. (B) Comparison of radioactive signal uptake in the lungs of A. fumigatus-infected rats (n = 4). Results are expressed as the mean of percentage of injected dose per gram of organ (%ID/g); *P < 0.05.

Figure 10

(A) Static PET/CT imaging data: MIP images of [⁶⁸Ga]Ga(III)-FOX 2–4 and [⁶⁸Ga]Ga(III)-FOX 2–6 in S. aureus-infected Balb/c mice 45 min after injection. Yellow arrows indicate S. aureus infection. (B) Comparison of radioactive signal uptake in the muscles of S. aureus-infected mice (n = 4). Results are expressed as the mean of percentage of injected dose per gram of organ (%ID/g); **P < 0.01.