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

Siderophores play an important role in fungal virulence, serving as trackers for in vivo imaging and as biomarkers of fungal infections. However, siderophore uptake is only partially characterized. As the major cause of aspergillosis, Aspergillus fumigatus is one of the most common airborne fungal pathogens of humans. Here, we demonstrate that this mold species mediates the uptake of iron chelated by the secreted siderophores triacetylfusarinine C (TAFC) and fusarinine C by the major facilitator-type transporters MirB and MirD, respectively. In a murine aspergillosis model, MirB but not MirD was found to be crucial for virulence, indicating that TAFC-mediated uptake plays a dominant role during infection. In the absence of MirB, TAFC becomes inhibitory by decreasing iron availability because the mutant is not able to recognize iron that is chelated by TAFC. MirB-mediated transport was found to tolerate the conjugation of fluorescein isothiocyanate to triacetylfusarinine C, which might aid in the development of siderophore-based antifungals in a Trojan horse approach, particularly as the role of MirB in pathogenicity restrains its mutational inactivation. Taken together, this study identified the first eukaryotic siderophore transporter that is crucial for virulence and elucidated its translational potential as well as its evolutionary conservation. IMPORTANCE Aspergillus fumigatus is responsible for thousands of cases of invasive fungal disease annually. For iron uptake, A. fumigatus secretes so-called siderophores, which are taken up after the binding of environmental iron. Moreover, A. fumigatus can utilize siderophore types that are produced by other fungi or bacteria. Fungal siderophores raised considerable interest due to their role in virulence and their potential for the diagnosis and treatment of fungal infections. Here, we demonstrate that the siderophore transporter MirB is crucial for the virulence of A. fumigatus, which reveals that its substrate, triacetylfusarinine C, is the most important siderophore during infection. We found that in the absence of MirB, TAFC becomes inhibitory by decreasing the availability of environmental iron and that MirB-mediated transport tolerates the derivatization of its substrate, which might aid in the development of siderophore-based antifungals. This study significantly improved the understanding of fungal iron homeostasis and the role of siderophores in interactions with the host.

Automated Synthesis of 68Ga-Labeled DOTA-MGS8 and Preclinical Characterization of Cholecystokinin-2 Receptor Targeting.

The new minigastrin analog DOTA-MGS8 targeting the cholecystokinin-2 receptor (CCK2R) used in this study displays the combination of two site-specific modifications within the C-terminal receptor binding sequence together with an additional N-terminal amino acid substitution preventing fast metabolic degradation. Within this study, the preparation of 68Ga-labeled DOTA-MGS8 was validated using an automated synthesis module, describing the specifications and analytical methods for quality control for possible clinical use. In addition, preclinical studies were carried out to characterize the targeting potential. [68Ga]Ga-DOTA-MGS8 showed a high receptor-specific cell internalization into AR42J rat pancreatic cells (~40%) with physiological expression of rat CCK2R as well as A431-CCK2R cells transfected to stably express human CCK2R (~47%). A favorable biodistribution profile was observed in BALB/c nude mice xenografted with A431-CCK2R cells and mock-transfected A431 cells as control. The high tumor uptake of ~27% IA/g together with low background activity and limited uptake in non-target tissue confirms the potential for high-sensitivity positron emission tomography of stabilized MG analogs in patients with MTC and other CCK2R-related malignancies.

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

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.

Antifungal Siderophore Conjugates for Theranostic Applications in Invasive Pulmonary Aspergillosis Using Low-Molecular TAFC Scaffolds.

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.

68Ga-labelled desferrioxamine-B for bacterial infection imaging.

PURPOSE: With the increase of especially hospital-acquired infections, timely and accurate diagnosis of bacterial infections is crucial for effective patient care. Molecular imaging has the potential for specific and sensitive detection of infections. Siderophores are iron-specific chelators recognized by specific bacterial transporters, representing one of few fundamental differences between bacterial and mammalian cells. Replacing iron by gallium-68 without loss of bioactivity is possible allowing molecular imaging by positron emission tomography (PET). Here, we report on the preclinical evaluation of the clinically used siderophore, desferrioxamine-B (Desferal®, DFO-B), radiolabelled with 68Ga for imaging of bacterial infections. METHODS: In vitro characterization of [68Ga]Ga-DFO-B included partition coefficient, protein binding and stability determination. Specific uptake of [68Ga]Ga-DFO-B was tested in vitro in different microbial cultures. In vivo biodistribution was studied in healthy mice and dosimetric estimation for human setting performed. PET/CT imaging was carried out in animal infection models, representing the most common pathogens. RESULTS: DFO-B was labelled with 68Ga with high radiochemical purity and displayed hydrophilic properties, low protein binding and high stability in human serum and PBS. The high in vitro uptake of [68Ga]Ga-DFO-B in selected strains of Pseudomonas aeruginosa, Staphylococcus aureus and Streptococcus agalactiae could be blocked with an excess of iron-DFO-B. [68Ga]Ga-DFO-B showed rapid renal excretion and minimal retention in blood and other organs in healthy mice. Estimated human absorbed dose was 0.02 mSv/MBq. PET/CT images of animal infection models displayed high and specific accumulation of [68Ga]Ga-DFO-B in both P. aeruginosa and S. aureus infections with excellent image contrast. No uptake was found in sterile inflammation, heat-inactivated P. aeruginosa or S. aureus and Escherichia coli lacking DFO-B transporters. CONCLUSION: DFO-B can be easily radiolabelled with 68Ga and displayed suitable in vitro characteristics and excellent pharmacokinetics in mice. The high and specific uptake of [68Ga]Ga-DFO-B by P. aeruginosa and S. aureus was confirmed both in vitro and in vivo, proving the potential of [68Ga]Ga-DFO-B for specific imaging of bacterial infections. As DFO-B is used in clinic for many years and the estimated radiation dose is lower than for other 68Ga-labelled radiopharmaceuticals, we believe that [68Ga]Ga-DFO-B has a great potential for clinical translation.

Siderophore Scaffold as Carrier for Antifungal Peptides in Therapy of Aspergillus fumigatus Infections.

Antifungal resistance of human fungal pathogens represents an increasing challenge in modern medicine. Short antimicrobial peptides (AMP) display a promising class of antifungals with a different mode of action, but lack target specificity and metabolic stability. In this study the hexapeptide PAF26 (Ac-dArg-dLys-dLys-dTrp-dPhe-dTrp-NH2) and the three amino acid long peptide NLF (H2N-Asn-Leu-dPhe-COOH) were coupled to diacetylfusarinine C (DAFC), a derivative of the siderophore triacetylfusarinine C (TAFC) of Aspergillus fumigatus, to achieve targeted delivery for treatment of invasive aspergillosis. Conjugated compounds in various modifications were labelled with radioactive gallium-68 to perform in vitro and in vivo characterizations. LogD, serum stability, uptake- growth promotion- and minimal inhibitory concentration assays were performed, as well as in vivo stability tests and biodistribution in BALB/c mice. Uptake and growth assays revealed specific internalization of the siderophore conjugates by A. fumigatus. They showed a high stability in human serum and also in the blood of BALB/c mice but metabolites in urine, probably due to degradation in the kidneys. Only PAF26 showed growth inhibition at 8 µg/ml which was lost after conjugation to DAFC. Despite their lacking antifungal activity conjugates based on a siderophore scaffold have a potential to provide the basis for a new class of antifungals, which allow the combination of imaging by using PET/CT with targeted treatment, thereby opening a theranostic approach for personalized therapy.

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

Live-cell imaging allows the in vivo analysis of subcellular localisation dynamics of physiological processes with high spatial-temporal resolution. However, only few fluorescent dyes have been custom-designed to facilitate species-specific live-cell imaging approaches in filamentous fungi to date. Therefore, we developed fluorescent dye conjugates based on the sophisticated iron acquisition system of Aspergillus fumigatus by chemical modification of the siderophore triacetylfusarinine C (TAFC). Various fluorophores (FITC, NBD, Ocean Blue, BODIPY 630/650, SiR, TAMRA and Cy5) were conjugated to diacetylfusarinine C (DAFC). Gallium-68 labelling enabled in vitro and in vivo characterisations. LogD, uptake assays and growth assays were performed and complemented by live-cell imaging in different Aspergillus species. Siderophore conjugates were specifically recognised by the TAFC transporter MirB and utilized as an iron source in growth assays. Fluorescence microscopy revealed uptake dynamics and differential subcellular accumulation patterns of all compounds inside fungal hyphae.[Fe]DAFC-NBD and -Ocean Blue accumulated in vacuoles, whereas [Fe]DAFC-BODIPY, -SiR and -Cy5 localised to mitochondria. [Fe]DAFC -FITC showed a uniform cytoplasmic distribution, whereas [Fe]DAFC-TAMRA was not internalised at all. Co-staining experiments with commercially available fluorescent dyes confirmed these findings. Overall, we developed a new class of fluorescent dyes that vary in intracellular fungal targeting , thereby providing novel tools for live-cell imaging applications for Aspergillus fumigatus.

Siderophore-Based Molecular Imaging of Fungal and Bacterial Infections-Current Status and Future Perspectives.

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.

Hybrid Imaging Agents for Pretargeting Applications Based on Fusarinine C-Proof of Concept.

Hybrid imaging combining the beneficial properties of radioactivity and optical imaging within one imaging probe has gained increasing interest in radiopharmaceutical research. In this study, we modified the macrocyclic gallium-68 chelator fusarinine C (FSC) by conjugating a fluorescent moiety and tetrazine (Tz) moieties. The resulting hybrid imaging agents were used for pretargeting applications utilizing click reactions with a trans-cyclooctene (TCO) tagged targeting vector for a proof of principle both in vitro and in vivo. Starting from FSC, the fluorophores Sulfocyanine-5, Sulfocyanine-7, or IRDye800CW were conjugated, followed by introduction of one or two Tz motifs, resulting in mono and dimeric Tz conjugates. Evaluation included fluorescence microscopy, binding studies, logD, protein binding, in vivo biodistribution, µPET (micro-positron emission tomography), and optical imaging (OI) studies. 68Ga-labeled conjugates showed suitable hydrophilicity, high stability, and specific targeting properties towards Rituximab-TCO pre-treated CD20 expressing Raji cells. Biodistribution studies showed fast clearance and low accumulation in non-targeted organs for both SulfoCy5- and IRDye800CW-conjugates. In an alendronate-TCO based bone targeting model the dimeric IRDye800CW-conjugate resulted in specific targeting using PET and OI, superior to the monomer. This proof of concept study showed that the preparation of FSC-Tz hybrid imaging agents for pretargeting applications is feasible, making such compounds suitable for hybrid imaging applications.

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

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.

The Siderophore Transporter Sit1 Determines Susceptibility to the Antifungal VL-2397.

VL-2397 (previously termed ASP2397) is an antifungal, aluminum-chelating cyclic hexapeptide with a structure analogous to that of ferrichrome-type siderophores, whereby replacement of aluminum by iron was shown to decrease the antifungal activity of this compound. Here, we found that inactivation of an importer for ferrichrome-type siderophores, termed Sit1, renders Aspergillus fumigatus resistant to VL-2397. Moreover, expression of the endogenous sit1 gene under the control of a xylose-inducible promoter (to uncouple sit1 expression from iron repression) combined with C-terminal tagging with a fluorescent protein demonstrated localization of Sit1 in the plasma membrane and xylose-dependent VL-2397 susceptibility. This underlines that Sit1-mediated uptake is essential for VL-2397 susceptibility. Under xylose-induced sit1 expression, VL-2397 also retained antifungal activity after replacing aluminum with iron, which demonstrates that VL-2397 bears antifungal activity independent of cellular aluminum importation. Analysis of sit1 expression indicated that the reduced antifungal activity of the iron-chelated VL-2397 is caused by downregulation of sit1 expression by the imported iron. Furthermore, we demonstrate that defects in iron homeostatic mechanisms modulate the activity of VL-2397. In contrast to A. fumigatus and Candida glabrata, Saccharomyces cerevisiae displays intrinsic resistance to VL-2397 antifungal activity. However, expression of sit1 from A. fumigatus, or its homologue from C. glabrata, resulted in susceptibility to VL-2397, which suggests that the intrinsic resistance of S. cerevisiae is based on lack of uptake and that A. fumigatus, C. glabrata, and S. cerevisiae share an intracellular target for VL-2397.

Modifying the Siderophore Triacetylfusarinine C for Molecular Imaging of Fungal Infection.

PURPOSE: Aspergillus fumigatus produces the siderophore triacetylfusarinine C (TAFC) for iron acquisition which is essential for its virulence. Therefore, TAFC is a specific marker for invasive aspergillosis. We have shown previously that positron emission tomography (PET) imaging with [68Ga]TAFC exhibited excellent targeting properties in an A. fumigatus rat infection model. In this study, we aimed to prepare TAFC analogs modifying fusarinine C (FSC) by acylation with different carbon chain lengths as well as with charged substituents and investigated the influence of introduced substituents on preservation of TAFC characteristics in vitro and in vivo. PROCEDURES: Fifteen TAFC derivatives were prepared and labeled with gallium-68. In vitro uptake assays were carried out in A. fumigatus under iron-replete as well as iron-depleted conditions and distribution coefficient was determined. Based on these assays, three compounds, [68Ga]tripropanoyl(FSC) ([68Ga]TPFC), [68Ga]diacetylbutanoyl(FSC) ([68Ga]DABuFC), and [68Ga]trisuccinyl(FSC) ([68Ga]FSC(suc)3), with high, medium, and low in vitro uptake in fungal cultures, were selected for further evaluation. Stability and protein binding were evaluated and in vivo imaging performed in the A. fumigatus rat infection model. RESULTS: In vitro uptake studies using A. fumigatus revealed specific uptake of mono- and trisubstituted TAFC derivatives at RT. Lipophilicities as expressed by logD were 0.34 to - 3.80. The selected compounds displayed low protein binding and were stable in PBS and serum. Biodistribution and image contrast in PET/X-ray computed tomography of [68Ga]TPFC and [68Ga]DABuFC were comparable to [68Ga]TAFC, whereas no uptake in the infected region was observed with [68Ga]FSC(suc)3. CONCLUSIONS: Our studies show the possibility to modify TAFC without losing its properties and specific recognition by A. fumigatus. This opens also new ways for multimodality imaging or theranostics of fungal infection by introducing functionalities such as fluorescent dyes or antifungal moieties.

Influence of a novel, versatile bifunctional chelator on theranostic properties of a minigastrin analogue.

BACKGROUND: 6-[Bis(carboxymethyl)amino]-1,4-bis(carboxymethyl)-6-methyl-1,4-diazepane (AAZTA ) is a promising chelator with potential advantages over 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) for radiopharmaceutical applications. Its mesocyclic structure enables fast radiolabelling under mild conditions with trivalent metals including not only (68)Ga for positron emission tomography (PET) but also (177)Lu and (111)In for single-photon emission computed tomography (SPECT) and radionuclide therapy. Here, we describe the evaluation of a bifunctional AAZTA derivative conjugated to a model minigastrin derivative as a potential theranostic agent. METHODS: An AAZTA derivative with an aliphatic C9 chain as linker was coupled to a minigastrin, namely [AAZTA(0), D-Glu(1), desGlu(2-6)]-minigastrin (AAZTA-MG), and labelled with (68)Ga, (177)Lu and (111)In. The characterisation in vitro included stability studies in different media and determination of logD (octanol/PBS). Affinity determination (IC50) and cell uptake studies were performed in A431-CCK2R cells expressing the human CCK2 receptor. µPET/CT and ex vivo biodistribution studies were performed in CCK2 tumour xenograft-bearing nude mice and normal mice. RESULTS: AAZTA-MG showed high radiochemical yields for (68)Ga (>95 %), (177)Lu (>98 %) and (111)In (>98 %). The logD value of -3.7 for both [(68)Ga]- and [(177)Lu]-AAZTA-MG indicates a highly hydrophilic character. Stability tests showed overall high stability in solution with some degradation in human plasma for [(68)Ga]- and transchelation towards DTPA for and [(177)Lu]-AAZTA-MG. An IC50 value of 10.0 nM was determined, which indicates a high affinity for the CCK2 receptor. Specific cell uptake after 60 min was >7.5 % for [(68)Ga]-AAZTA-MG and >9.5 % for [(177)Lu]-AAZTA-MG, comparable to other DOTA-MG-analogues. µPET/CT studies in CCK2 receptor tumour xenografted mice not only revealed high selective accumulation in A431-CCK2R positive tumours of (68)Ga-labelled AAZTA-MG (1.5 % ID/g in 1 h post injection) but also higher blood levels as corresponding DOTA-analogues. The (111)In-labelled peptide had a tumour uptake of 1.7 % ID/g. Biodistribution in normal mice with the [(177)Lu]-AAZTA-MG showed a considerable uptake in intestine (7.3 % ID/g) and liver (1.5 % ID/g). CONCLUSION: Overall, AAZTA showed interesting properties as bifunctional chelator for peptides providing mild radiolabelling conditions for both (68)Ga and trivalent metals having advantages over the currently used chelator DOTA. Studies are ongoing to further investigate in vivo targeting properties and stability issues and the influence of spacer length on biodistribution of AAZTA.