Targeting of phagolysosomes containing conidia of the fungus Aspergillus fumigatus with polymeric particles.

Conidia of the airborne human-pathogenic fungus Aspergillus fumigatus are inhaled by humans. In the lung, they are phagocytosed by alveolar macrophages and intracellularly processed. In macrophages, however, conidia can interfere with the maturation of phagolysosomes to avoid their elimination. To investigate whether polymeric particles (PPs) can reach this intracellular pathogen in macrophages, we formulated dye-labeled PPs with a size allowing for their phagocytosis. PPs were efficiently taken up by RAW 264.7 macrophages and were found in phagolysosomes. When macrophages were infected with conidia prior to the addition of PPs, we found that they co-localized in the same phagolysosomes. Mechanistically, the fusion of phagolysosomes containing PPs with phagolysosomes containing conidia was observed. Increasing concentrations of PPs increased fusion events, resulting in 14% of phagolysosomes containing both conidia and PPs. We demonstrate that PPs can reach conidia-containing phagolysosomes, making these particles a promising carrier system for antimicrobial drugs to target intracellular pathogens. KEY POINTS: • Polymer particles of a size larger than 500 nm are internalized by macrophages and localized in phagolysosomes. • These particles can be delivered to Aspergillus fumigatus conidia-containing phagolysosomes of macrophages. • Enhanced phagolysosome fusion by the use of vacuolin1 can increase particle delivery.

The Zn2Cys6-type transcription factor LeuB cross-links regulation of leucine biosynthesis and iron acquisition in Aspergillus fumigatus.

Both branched-chain amino acids (BCAA) and iron are essential nutrients for eukaryotic cells. Previously, the Zn2Cys6-type transcription factor Leu3/LeuB was shown to play a crucial role in regulation of BCAA biosynthesis and nitrogen metabolism in Saccharomyces cerevisiae and Aspergillus nidulans. In this study, we found that the A. fumigatus homolog LeuB is involved in regulation of not only BCAA biosynthesis and nitrogen metabolism but also iron acquisition including siderophore metabolism. Lack of LeuB caused a growth defect, which was cured by supplementation with leucine or iron. Moreover, simultaneous inactivation of LeuB and HapX, a bZIP transcription factor required for adaptation to iron starvation, significantly aggravated the growth defect caused by inactivation of one of these regulators during iron starvation. In agreement with a direct role in regulation of both BCAA and iron metabolism, LeuB was found to bind to phylogenetically conserved motifs in promoters of genes involved in BCAA biosynthesis, nitrogen metabolism, and iron acquisition in vitro and in vivo, and was required for full activation of their expression. Lack of LeuB also caused activation of protease activity and autophagy via leucine depletion. Moreover, LeuB inactivation resulted in virulence attenuation of A. fumigatus in Galleria mellonella. Taken together, this study identified a previously uncharacterized direct cross-regulation of BCCA biosynthesis, nitrogen metabolism and iron homeostasis as well as proteolysis.

Caspofungin Functionalized Polymethacrylates with Antifungal Properties.

We describe the synthesis of hydrophilic poly(poly(ethylene glycol) methyl ether methacrylate) (PmPEGMA) and hydrophobic poly(methyl methacrylate) (PMMA) caspofungin conjugates by a post-polymerization modification of copolymers containing 10 mol % pentafluorophenyl methacrylate (PFPMA), which were obtained via reversible addition-fragmentation chain transfer copolymerization. The coupling of the clinically used antifungal caspofungin was confirmed and quantified in detail by a combination of 1H-, 19F- and diffusion-ordered NMR spectroscopy, UV-vis spectroscopy, and size exclusion chromatography. The trifunctional amine-containing antifungal was attached via several amide bonds to the hydrophobic PMMA, but sterical hindrance induced by the mPEGMA side chains prohibited intramolecular double functionalization. Both polymer-drug conjugates revealed activity against important human-pathogenic fungi, that is, two strains of Aspergillus fumigatus and one strain of Candida albicans (2.5 mg L-1 < MEC < 8 mg L-1, MIC50 = 4 mg L-1), whereas RAW 264.7 macrophages as well as HeLa cells remained unaffected at these concentrations.

Rapid detection of the aspergillosis biomarker triacetylfusarinine C using interference-enhanced Raman spectroscopy.

Triacetylfusarinine C (TAFC) is a siderophore produced by certain fungal species and might serve as a highly useful biomarker for the fast diagnosis of invasive aspergillosis. Due to its renal elimination, the biomarker is found in urine samples of patients suffering from Aspergillus infections. Accordingly, non-invasive diagnosis from this easily obtainable body fluid is possible. Within our contribution, we demonstrate how Raman microspectroscopy enables a sensitive and specific detection of TAFC. We characterized the TAFC iron complex and its iron-free form using conventional and interference-enhanced Raman spectroscopy (IERS) and compared the spectra with the related compound ferrioxamine B, which is produced by bacterial species. Even though IERS only offers a moderate enhancement of the Raman signal, the employment of respective substrates allowed lowering the detection limit to reach the clinically relevant range. The achieved limit of detection using IERS was 0.5 ng of TAFC, which is already well within the clinically relevant range. By using an extraction protocol, we were able to detect 1.4 µg/mL TAFC via IERS from urine within less than 3 h including sample preparation and data analysis. We could further show that TAFC and ferrioxamine B can be clearly distinguished by means of their Raman spectra even in very low concentrations.

The Lipid Raft-Associated Protein Stomatin Is Required for Accumulation of Dectin-1 in the Phagosomal Membrane and for Full Activity of Macrophages against Aspergillus fumigatus.

Alveolar macrophages belong to the first line of defense against inhaled conidia of the human-pathogenic fungus Aspergillus fumigatus. In lung alveoli, they contribute to phagocytosis and elimination of conidia. As a counterdefense, conidia have a gray-green pigment that enables them to survive in phagosomes of macrophages for some time. Previously, we showed that this conidial pigment interferes with the formation of flotillin-dependent lipid raft microdomains in the phagosomal membrane, thereby preventing the formation of functional phagolysosomes. Besides flotillins, stomatin is a major component of lipid rafts and can be targeted to the membrane. However, only limited information on stomatin is available, in particular on its role in defense against pathogens. To determine the function of this integral membrane protein, a stomatin-deficient macrophage line was generated by CRISPR/Cas9 gene editing. Immunofluorescence microscopy and flow cytometry revealed that stomatin contributes to the phagocytosis of conidia and is important for recruitment of the ß-glucan receptor dectin-1 to both the cytoplasmic membrane and phagosomal membrane. In stomatin knockout cells, fusion of phagosomes and lysosomes, recruitment of the vATPase to phagosomes, and tumor necrosis factor alpha (TNF-α) levels were reduced when cells were infected with pigmentless conidia. Thus, our data suggest that stomatin is involved in maturation of phagosomes via fostering fusion of phagosomes with lysosomes. IMPORTANCE Stomatin is an integral membrane protein that contributes to the uptake of microbes, e.g., spores of the human-pathogenic fungus Aspergillus fumigatus. By generation of a stomatin-deficient macrophage line by advanced genetic engineering, we found that stomatin is involved in the recruitment of the ß-glucan receptor dectin-1 to the phagosomal membrane of macrophages. Furthermore, stomatin is involved in maturation of phagosomes via fostering fusion of phagosomes with lysosomes. The data provide new insights on the important role of stomatin in the immune response against human-pathogenic fungi.

Polymer-based particles against pathogenic fungi: A non-uptake delivery of compounds.

The therapy of life-threatening fungal infections is limited and needs urgent improvement. This is in part due to toxic side effects of clinically used antifungal compounds or their limited delivery to fungal structures. Until today, it is a matter of debate how drugs or drug-delivery systems can efficiently reach the intracellular lumen of fungal cells and how this can be improved. Here, we addressed both questions by applying two different polymeric particles for delivery of compounds. Their formulation was based on two biocompatible polymers, i.e., poly(lactic-co-glycolic acid)50:50 and poly(methyl methacrylate-stat-methacrylic acid)90:10 yielding particles with hydrodynamic diameters ranging from 100 to 300 nm. The polymers were covalently labeled with the fluorescent dye DY-550 to monitor the interaction between particles and fungi by confocal laser scanning microscopy. Furthermore, the fluorescent dye coumarin-6 and the antifungal drug itraconazole were successfully encapsulated in particles to study the fate of both the cargo and the particle when interacting with the clinically most important human-pathogenic fungi Aspergillus fumigatus, A. terreus, Candida albicans, and Cryptococcus neoformans. While the polymers were exclusively located on the fungal surface, the encapsulated cargo was efficiently transported into fungal hyphae, indicated by increased intracellular fluorescence signals due to coumarin-6. In accordance with this finding, compared to the pristine drug a reduced minimal inhibitory concentration for itraconazole was determined, when it was encapsulated. Together, the herein used polymeric particles were not internalized by pathogenic fungi but were able to efficiently deliver hydrophobic cargos into fungal cells.

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.

The Siderophore Transporters Sit1 and Sit2 Are Essential for Utilization of Ferrichrome-, Ferrioxamine- and Coprogen-Type Siderophores in Aspergillus fumigatus.

Siderophore-mediated acquisition of iron has been shown to be indispensable for the virulence of several fungal pathogens, the siderophore transporter Sit1 was found to mediate uptake of the novel antifungal drug VL-2397, and siderophores were shown to be useful as biomarkers as well as for imaging of fungal infections. However, siderophore uptake in filamentous fungi is poorly characterized. The opportunistic human pathogen Aspergillus fumigatus possesses five putative siderophore transporters. Here, we demonstrate that the siderophore transporters Sit1 and Sit2 have overlapping, as well as unique, substrate specificities. With respect to ferrichrome-type siderophores, the utilization of ferrirhodin and ferrirubin depended exclusively on Sit2, use of ferrichrome A depended mainly on Sit1, and utilization of ferrichrome, ferricrocin, and ferrichrysin was mediated by both transporters. Moreover, both Sit1 and Sit2 mediated use of the coprogen-type siderophores coprogen and coprogen B, while only Sit1 transported the bacterial ferrioxamine-type xenosiderophores ferrioxamines B, G, and E. Neither Sit1 nor Sit2 were important for the utilization of the endogenous siderophores fusarinine C and triacetylfusarinine C. Furthermore, A. fumigatus was found to lack utilization of the xenosiderophores schizokinen, basidiochrome, rhizoferrin, ornibactin, rhodotorulic acid, and enterobactin. Taken together, this study characterized siderophore use by A. fumigatus and substrate characteristics of Sit1 and Sit2.

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 leucine biosynthetic pathway is crucial for adaptation to iron starvation and virulence in Aspergillus fumigatus.

In contrast to mammalia, fungi are able to synthesize the branched-chain amino acid leucine de novo. Recently, the transcription factor LeuB has been shown to cross-regulate leucine biosynthesis, nitrogen metabolism and iron homeostasis in Aspergillus fumigatus, the most common human mold pathogen. Moreover, the leucine biosynthetic pathway intermediate α-isopropylmalate (α-IPM) has previously been shown to posttranslationally activate LeuB homologs in S. cerevisiae and A. nidulans. Here, we demonstrate that in A. fumigatus inactivation of both leucine biosynthetic enzymes α-IPM synthase (LeuC), which disrupts α-IPM synthesis, and α-IPM isomerase (LeuA), which causes cellular α-IPM accumulation, results in leucine auxotrophy. However, compared to lack of LeuA, lack of LeuC resulted in increased leucine dependence, a growth defect during iron starvation and decreased expression of LeuB-regulated genes including genes involved in iron acquisition. Lack of either LeuA or LeuC decreased virulence in an insect infection model, and inactivation of LeuC rendered A. fumigatus avirulent in a pulmonary aspergillosis mouse model. Taken together, we demonstrate that the lack of two leucine biosynthetic enzymes, LeuA and LeuC, results in significant phenotypic consequences indicating that the regulator LeuB is activated by α-IPM in A. fumigatus and that the leucine biosynthetic pathway is an attractive target for the development of antifungal drugs.

The Lysine Deacetylase RpdA Is Essential for Virulence in Aspergillus fumigatus.

Current suboptimal treatment options of invasive fungal infections and emerging resistance of the corresponding pathogens urge the need for alternative therapy strategies and require the identification of novel antifungal targets. Aspergillus fumigatus is the most common airborne opportunistic mold pathogen causing invasive and often fatal disease. Establishing a novel in vivo conditional gene expression system, we demonstrate that downregulation of the class 1 lysine deacetylase (KDAC) RpdA leads to avirulence of A. fumigatus in a murine model for pulmonary aspergillosis. The xylP promoter used has previously been shown to allow xylose-induced gene expression in different molds. Here, we demonstrate for the first time that this promoter also allows in vivo tuning of A. fumigatus gene activity by supplying xylose in the drinking water of mice. In the absence of xylose, an A. fumigatus strain expressing rpdA under control of the xylP promoter, rpdA xylP , was avirulent and lung histology showed significantly less fungal growth. With xylose, however, rpdA xylP displayed full virulence demonstrating that xylose was taken up by the mouse, transported to the site of fungal infection and caused rpdA induction in vivo. These results demonstrate that (i) RpdA is a promising target for novel antifungal therapies and (ii) the xylP expression system is a powerful new tool for in vivo gene silencing in A. fumigatus.

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.

Triacetylfusarinine C: A urine biomarker for diagnosis of invasive aspergillosis.

OBJECTIVES: Early diagnosis of invasive aspergillosis (IA) remains challenging, with available diagnostics being limited by inadequate sensitivities and specificities. Triacetylfusarinine C, a fungal siderophore that has been shown to accumulate in urine in animal models, is a potential new biomarker for diagnosis of IA. METHODS: We developed a method allowing absolute and matrix-independent mass spectrometric quantification of TAFC. Urine TAFC, normalized to creatinine, was determined in 44 samples from 24 patients with underlying hematologic malignancies and probable, possible or no IA according to current EORTC/MSG criteria and compared to other established biomarkers measured in urine and same-day blood samples. RESULTS: TAFC/creatinine sensitivity, specificity, positive and negative likelihood ratio for probable versus no IA (cut-off ≥ 3) were 0.86, 0.88, 6.86, 0.16 per patient. CONCLUSION: For the first time, we provide proof for the occurrence of TAFC in human urine. TAFC/creatinine index determination in urine showed promising results for diagnosis of IA offering the advantages of non-invasive sampling. Sensitivity and specificity were similar as reported for GM determination in serum and bronchoalveolar lavage, the gold standard mycological criterion for IA diagnosis.

Exploiting the Concept of Multivalency with 68Ga- and 89Zr-Labelled Fusarinine C-Minigastrin Bioconjugates for Targeting CCK2R Expression.

Cholecystokinin-2 receptors (CCK2R) are overexpressed in a variety of malignant diseases and therefore have gained certain attention for peptide receptor radionuclide imaging. Among extensive approaches to improve pharmacokinetics and metabolic stability of minigastrin (MG) based radioligands, the concept of multivalency for enhanced tumour targeting has not been investigated extensively. We therefore utilized fusarinine C (FSC) as chelating scaffold for novel mono-, di-, and trimeric bioconjugates for targeting CCK2R expression. FSC-based imaging probes were radiolabelled with positron emitting radionuclides (gallium-68 and zirconium-89) and characterized in vitro (log⁡D, IC50, and cell uptake) and in vivo (metabolic stability in BALB/c mice, biodistribution profile, and microPET/CT imaging in A431-CCK2R/A431-mock tumour xenografted BALB/c nude mice). Improved targeting did not fully correlate with the grade of multimerization. The divalent probe showed higher receptor affinity and increased CCK2R mediated cell uptake while the trimer remained comparable to the monomer. In vivo biodistribution studies 1 h after administration of the 68Ga-labelled radioligands confirmed this trend, but imaging at late time point (24 h) with 89Zr-labelled counterparts showed a clearly enhanced imaging contrast of the trimeric probe compared to the mono- and dimer. Furthermore, in vivo stability studies showed a higher metabolic stability for multimeric probes compared to the monomeric bioconjugate. In summary, we could show that FSC can be utilized as suitable scaffold for novel mono- and multivalent imaging probes for CCK2R-related malignancies with partly improved targeting properties for multivalent conjugates. The increased tumour accumulation of the trimer 24 h postinjection (p.i.) can be explained by slower clearance and increased metabolic stability of multimeric conjugates.