Publications by authors named "Hubertus Haas"

144 Publications

Generation and characterisation of a semi-synthetic siderophore-immunogen conjugate and a derivative recombinant triacetylfusarinine C-specific monoclonal antibody with fungal diagnostic application.

Anal Biochem 2021 Sep 17:114384. Epub 2021 Sep 17.

Department of Biology, Maynooth University, Maynooth, Co. Kildare, W23 F2H6, Ireland. Electronic address:

Invasive pulmonary aspergillosis (IPA) is a severe life-threatening condition. Diagnosis of fungal disease in general, and especially that caused by Aspergillus fumigatus is problematic. A. fumigatus secretes siderophores to acquire iron during infection, which are also essential for virulence. We describe the chemoacetylation of ferrated fusarinine C to diacetylated fusarinine C (DAFC), followed by protein conjugation, which facilitated triacetylfusarinine C (TAFC)-specific monoclonal antibody production with specific recognition of the ferrated form of TAFC. A single monoclonal antibody sequence was ultimately elucidated by a combinational strategy involving protein LC-MS/MS, cDNA sequencing and RNAseq. The resultant murine IgG2 monoclonal antibody was secreted in, and purified from, mammalian cell culture (5 mg) and demonstrated to be highly specific for TAFC detection by competitive ELISA (detection limit: 15 nM) and in a lateral flow test system (detection limit: 3 ng), using gold nanoparticle conjugated- DAFC-bovine serum albumin for competition. Overall, this work reveals for the first time a recombinant TAFC-specific monoclonal antibody with diagnostic potential for IPA diagnosis in traditional and emerging patient groups (e.g., COVID-19) and presents a useful strategy for murine Ig sequence determination, and expression in HEK293 cells, to overcome unexpected limitations associated with aberrant or deficient murine monoclonal antibody production.
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http://dx.doi.org/10.1016/j.ab.2021.114384DOI Listing
September 2021

Transcriptional response of to copper and the role of the Cu chaperones.

Virulence 2021 12;12(1):2186-2200

Department of Clinical Microbiology and Immunology, Sackler School of Medicine Ramat-Aviv, Tel-Aviv, Israel.

is the leading cause of life-threatening invasive mold infections in immunocompromised individuals. This ubiquitous saprophyte possesses several natural attributes allowing it to evade the immune system, including the ability to withstand high toxic Cu concentrations within the phagosomes of macrophages and neutrophils. We previously established that at high levels, Cu binds and activates the transcription factor AceA, which upregulates the expression of the Cu exporter CrpA to expel excess Cu. Deletion of or result in extreme Cu sensitivity and attenuated virulence.To identify other elements participating in resistance to Cu, we performed a genome-wide analysis of the transcriptome by RNAseq to analyze the AceA-dependent response of to excess Cu. We deleted key genes whose transcription was strongly upregulated by high Cu, including those encoding homologs of the three Cu chaperones and . Detailed analysis of these genes indicates that in is an essential gene with a possible role in respiration, the gene product participates in reductive iron uptake and encodes the Cu chaperone activating Sod1. Interestingly, although the -null strain was extremely sensitive to high Cu and oxidative stress, it was not attenuated in virulence in a mouse model of invasive pulmonary aspergillosis.Our work provides (i) a detailed view of the genome-wide transcriptional response of to excess Cu, (ii) identification of the AceA-dependent transcriptome and (iii) analysis of the roles of the three Cu chaperones and
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http://dx.doi.org/10.1080/21505594.2021.1958057DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8425704PMC
December 2021

Peroxiredoxin Asp f3 Is Essential for Aspergillus fumigatus To Overcome Iron Limitation during Infection.

mBio 2021 Aug 17;12(4):e0097621. Epub 2021 Aug 17.

Max von Pettenkofer-Institut für Hygiene und Medizinische Mikrobiologie, Medizinische Fakultät, LMU München, Munich, Germany.

Aspergillus fumigatus is an important fungal pathogen that causes allergic reactions but also life-threatening infections. One of the most abundant A. fumigatus proteins is Asp f3. This peroxiredoxin is a major fungal allergen and known for its role as a virulence factor, vaccine candidate, and scavenger of reactive oxygen species. Based on the hypothesis that Asp f3 protects A. fumigatus against killing by immune cells, we investigated the susceptibility of a conditional mutant by employing a novel assay. Surprisingly, Asp f3-depleted hyphae were killed as efficiently as the wild type by human granulocytes. However, we identified an unexpected growth defect of mutants that lack Asp f3 under low-iron conditions, which explains the avirulence of the Δ deletion mutant in a murine infection model. A. fumigatus encodes two Asp f3 homologues which we named Af3l (Asp f3-like) 1 and Af3l2. Inactivation of Af3l1, but not of Af3l2, exacerbated the growth defect of the conditional mutant under iron limitation, which ultimately led to death of the double mutant. Inactivation of the iron acquisition repressor SreA partially compensated for loss of Asp f3 and Af3l1. However, Asp f3 was not required for maintaining iron homeostasis or siderophore biosynthesis. Instead, we show that it compensates for a loss of iron-dependent antioxidant enzymes. Iron supplementation restored the virulence of the Δ deletion mutant in a murine infection model. Our results unveil the crucial importance of Asp f3 to overcome nutritional immunity and reveal a new biological role of peroxiredoxins in adaptation to iron limitation. Asp f3 is one of the most abundant proteins in the pathogenic mold Aspergillus fumigatus. It has an enigmatic multifaceted role as a fungal allergen, virulence factor, reactive oxygen species (ROS) scavenger, and vaccine candidate. Our study provides new insights into the cellular role of this conserved peroxiredoxin. We show that the avirulence of a Δ mutant in a murine infection model is linked to a low-iron growth defect of this mutant, which we describe for the first time. Our analyses indicated that Asp f3 is not required for maintaining iron homeostasis. Instead, we found that Asp f3 compensates for a loss of iron-dependent antioxidant enzymes. Furthermore, we identified an Asp f3-like protein which is partially functionally redundant with Asp f3. We highlight an unexpected key role of Asp f3 and its partially redundant homologue Af3l1 in overcoming the host's nutritional immunity. In addition, we uncovered a new biological role of peroxiredoxins.
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http://dx.doi.org/10.1128/mBio.00976-21DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8406167PMC
August 2021

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

J Fungi (Basel) 2021 Jul 14;7(7). Epub 2021 Jul 14.

Department of Nuclear Medicine, Medical University Innsbruck, A-6020 Innsbruck, Austria.

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 (), 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 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 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 .
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http://dx.doi.org/10.3390/jof7070558DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8304796PMC
July 2021

The bZIP Transcription Factor HapX Is Post-Translationally Regulated to Control Iron Homeostasis in .

Int J Mol Sci 2021 Jul 20;22(14). Epub 2021 Jul 20.

Institute of Molecular Biology, Biocenter, Medical University of Innsbruck, 6020 Innsbruck, Austria.

The airborne fungus causes opportunistic infections in humans with high mortality rates in immunocompromised patients. Previous work established that the bZIP transcription factor HapX is essential for virulence via adaptation to iron limitation by repressing iron-consuming pathways and activating iron acquisition mechanisms. Moreover, HapX was shown to be essential for transcriptional activation of vacuolar iron storage and iron-dependent pathways in response to iron availability. Here, we demonstrate that HapX has a very short half-life during iron starvation, which is further decreased in response to iron, while siderophore biosynthetic enzymes are very stable. We identified Fbx22 and SumO as HapX interactors and, in agreement, HapX post-translational modifications including ubiquitination of lysine, sumoylation of lysine and phosphorylation of threonine. All three modifications were enriched in the immediate adaptation from iron-limiting to iron-replete conditions. Interfering with these post-translational modifications, either by point mutations or by inactivation, of Fbx22 or SumO, altered HapX degradation, heme biosynthesis and iron resistance to different extents. Consistent with the need to precisely regulate HapX protein levels, overexpression of caused significant growth defects under iron sufficiency. Taken together, our results indicate that post-translational regulation of HapX is important to control iron homeostasis in . .
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http://dx.doi.org/10.3390/ijms22147739DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8307855PMC
July 2021

Inducible Selectable Marker Genes to Improve Genetic Manipulation.

J Fungi (Basel) 2021 Jun 24;7(7). Epub 2021 Jun 24.

Institute of Molecular Biology, Biocenter, Medical University of Innsbruck, 6020 Innsbruck, Austria.

The hygromycin B phosphotransferase gene from and the pyrithiamine resistance gene from are two dominant selectable marker genes widely used to genetically manipulate several fungal species. Despite the recent development of CRISPR/Cas9 and marker-free systems, in vitro molecular tools to study  , which is a saprophytic fungus causing life-threatening diseases in immunocompromised hosts, still rely extensively on the use of dominant selectable markers. The limited number of drug selectable markers is already a critical aspect, but the possibility that their introduction into a microorganism could induce enhanced virulence or undesired effects on metabolic behavior constitutes another problem. In this context, here, we demonstrate that the use of in leads to the secretion of a compound that allows the recovery of thiamine auxotrophy. In this study, we developed a simple modification of the two commonly used dominant markers in which the development of resistance can be controlled by the xylose-inducible promoter from . This strategy provides an easy solution to avoid undesired side effects, since the marker expression can be readily silenced when not required.
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http://dx.doi.org/10.3390/jof7070506DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8305790PMC
June 2021

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

J Fungi (Basel) 2020 Dec 15;6(4). Epub 2020 Dec 15.

Department of Nuclear Medicine, Medical University Innsbruck, 6020 Innsbruck, Austria.

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 , 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 . 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.
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http://dx.doi.org/10.3390/jof6040367DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7765500PMC
December 2020

Fungal iron homeostasis with a focus on Aspergillus fumigatus.

Biochim Biophys Acta Mol Cell Res 2021 01 10;1868(1):118885. Epub 2020 Oct 10.

Institute of Molecular Biology - Biocenter, Medical University of Innsbruck, Innsbruck, Austria. Electronic address:

To maintain iron homeostasis, fungi have to balance iron acquisition, storage, and utilization to ensure sufficient supply and to avoid toxic excess of this essential trace element. As pathogens usually encounter iron limitation in the host niche, this metal plays a particular role during virulence. Siderophores are iron-chelators synthesized by most, but not all fungal species to sequester iron extra- and intracellularly. In recent years, the facultative human pathogen Aspergillus fumigatus has become a model for fungal iron homeostasis of siderophore-producing fungal species. This article summarizes the knowledge on fungal iron homeostasis and its links to virulence with a focus on A. fumigatus. It covers mechanisms for iron acquisition, storage, and detoxification, as well as the modes of transcriptional iron regulation and iron sensing in A. fumigatus in comparison to other fungal species. Moreover, potential translational applications of the peculiarities of fungal iron metabolism for treatment and diagnosis of fungal infections is addressed.
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http://dx.doi.org/10.1016/j.bbamcr.2020.118885DOI Listing
January 2021

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

Sci Rep 2020 09 23;10(1):15519. Epub 2020 Sep 23.

Department of Nuclear Medicine, Medical University Innsbruck, Innsbruck, Austria.

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.
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http://dx.doi.org/10.1038/s41598-020-72452-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7511942PMC
September 2020

Absent regulation of iron acquisition by the copper regulator Mac1 in A. fumigatus.

Biochem J 2020 08;477(16):2967-2970

Institute of Molecular Biology, Medical University of Innsbruck, A6020 Innsbruck, Austria.

Aspergillus fumigatus is the most common cause of invasive aspergillosis, a life-threatening infection mainly affecting immunocompromised patients. The essential metals copper and iron play crucial roles in virulence of this mold. Recently, the copper-regulatory transcription factor Mac1 was reported to additionally be involved in the control of iron acquisition. However, in the current study, neither growth assays on solid and in liquid media, analysis of siderophore production nor expression analysis of genes involved in iron acquisition indicated the involvement of Mac1 in the regulation of iron uptake in A. fumigatus.
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http://dx.doi.org/10.1042/BCJ20200286DOI Listing
August 2020

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

J Fungi (Basel) 2020 May 29;6(2). Epub 2020 May 29.

Institute of Molecular Biology, Medical University Innsbruck, 6020 Innsbruck, Austria.

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 infection using [Ga]Ga-triacetylfusarinine C. Recently, this approach was expanded to imaging of bacterial infections, i.e., with 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.
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http://dx.doi.org/10.3390/jof6020073DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7345832PMC
May 2020

Structural basis of HapE-linked antifungal triazole resistance in .

Life Sci Alliance 2020 07 28;3(7). Epub 2020 May 28.

Center for Integrated Protein Science Munich at the Department Chemistry, Technical University of Munich, Garching, Germany

Azoles are first-line therapeutics for human and plant fungal infections, but their broad use has promoted the development of resistances. Recently, a pan-azole-resistant clinical isolate was identified to carry the mutation P88L in subunit HapE of the CCAAT-binding complex (CBC), a conserved eukaryotic transcription factor. Here, we define the mechanistic basis for resistance in this isolate by showing that the HapE mutation interferes with the CBC's ability to bend and sense CCAAT motifs. This failure leads to transcriptional derepression of the gene, which encodes the target of azoles, the 14-α sterol demethylase Cyp51A, and ultimately causes drug resistance. In addition, we demonstrate that the CBC-associated transcriptional regulator HapX assists repression in low-iron environments and that this iron-dependent effect is lost in the HapE mutant. Altogether, these results indicate that the mutation HapE confers increased resistance to azoles compared with wt , particularly in low-iron clinical niches such as the lung.
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http://dx.doi.org/10.26508/lsa.202000729DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7266990PMC
July 2020

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

Molecules 2020 May 1;25(9). Epub 2020 May 1.

Department of Nuclear Medicine, Medical University Innsbruck, A-6020 Innsbruck, Austria.

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 -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. Ga-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.
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http://dx.doi.org/10.3390/molecules25092123DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7249120PMC
May 2020

Siderophores in plant root tissue: Tagetes patula nana colonized by the arbuscular mycorrhizal fungus Gigaspora margarita.

Biometals 2020 06 3;33(2-3):137-146. Epub 2020 May 3.

Department of Microbiology & Biotechnology, University of Tübingen, Auf der Morgenstelle 28, 72076, Tübingen, Germany.

More than 70% of vascular plant species live in symbiosis with arbuscular mycorrhizal (AM) fungi. In addition to other effects this symbiosis is known for its significance for plant nutrition including iron. Fungal iron mobilization from soil is commonly dependent on siderophores. This study reports on a search for such iron-chelators in root tissue of Tagetes patula nana var. plena colonized by Gigaspora margarita. The AM colonized plants and uninoculated controls were grown under strictly axenic conditions. HPLC analyses of aqueous extracts from plant roots have provided clear evidence for the presence of a rhizoferrin type siderophore, named glomuferrin, in root tissue of mycorrhizal seedlings. Results from HPLC analytical work are seconded by molecular biological data: A BLASTp search revealed that the AM fungal species Gigaspora rosea, Rhizophagus irregularis (formerly Glomus intraradices), Glomus cerebriformis and Diversispora epigea encode a non-ribosomal peptide synthetase (NRPS)-independent siderophore synthase (NIS), which is homologous to the rhizoferrin synthetase of Rhizopus delemar. Thus this study indicates that the biosynthesis of rhizoferrin type siderophores such as glomuferrin (= bis-imidorhizoferrin) may be widespread in the AM symbiosis.
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http://dx.doi.org/10.1007/s10534-020-00238-0DOI Listing
June 2020

Arginine Auxotrophy Affects Siderophore Biosynthesis and Attenuates Virulence of .

Genes (Basel) 2020 04 15;11(4). Epub 2020 Apr 15.

Institute of Molecular Biology, Biocenter, Medical University of Innsbruck, 6020 Innsbruck, Austria.

is an opportunistic human pathogen mainly infecting immunocompromised patients. The aim of this study was to characterize the role of arginine biosynthesis in virulence of via genetic inactivation of two key arginine biosynthetic enzymes, the bifunctional acetylglutamate synthase/ornithine acetyltransferase (/AFUA_5G08120) and the ornithine carbamoyltransferase (/AFUA_4G07190). Arginine biosynthesis is intimately linked to the biosynthesis of ornithine, a precursor for siderophore production that has previously been shown to be essential for virulence in . ArgJ is of particular interest as it is the only arginine biosynthetic enzyme lacking mammalian homologs. Inactivation of either ArgJ or ArgB resulted in arginine auxotrophy. Lack of ArgJ, which is essential for mitochondrial ornithine biosynthesis, significantly decreased siderophore production during limited arginine supply with glutamine as nitrogen source, but not with arginine as sole nitrogen source. In contrast, siderophore production reached wild-type levels under both growth conditions in ArgB null strains. These data indicate that siderophore biosynthesis is mainly fueled by mitochondrial ornithine production during limited arginine availability, but by cytosolic ornithine production during high arginine availability via cytosolic arginine hydrolysis. Lack of ArgJ or ArgB attenuated virulence of in the insect model and in murine models for invasive aspergillosis, indicating limited arginine availability in the investigated host niches.
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http://dx.doi.org/10.3390/genes11040423DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7231135PMC
April 2020

Multiplex Genetic Engineering Exploiting Pyrimidine Salvage Pathway-Based Endogenous Counterselectable Markers.

mBio 2020 04 7;11(2). Epub 2020 Apr 7.

Institute of Molecular Biology/Biocenter, Innsbruck Medical University, Innsbruck, Austria

Selectable markers are indispensable for genetic engineering, yet their number and variety are limited. Most selection procedures for prototrophic cells rely on the introduction of antibiotic resistance genes. New minimally invasive tools are needed to facilitate sophisticated genetic manipulations. Here, we characterized three endogenous genes in the human fungal pathogen for their potential as markers for targeted genomic insertions of DNAs of interest (DOIs). Since these genes are involved in uptake and metabolization of pyrimidines, resistance to the toxic effects of prodrugs 5-fluorocytosine and 5-fluorouracil can be used to select successfully integrated DOIs. We show that DOI integration, resulting in the inactivation of these genes, caused no adverse effects with respect to nutrient requirements, stress resistance, or virulence. Beside the individual use of markers for site-directed integration of reporter cassettes, including the 17-kb penicillin biosynthetic cluster, we demonstrate their sequential use by inserting three genes encoding fluorescent proteins into a single strain for simultaneous multicolor localization microscopy. In addition to , we validated the applicability of this novel toolbox in and Enabling multiple targeted insertions of DOIs without the necessity for exogenous markers, this technology has the potential to significantly advance genetic engineering. This work reports the discovery of a novel genetic toolbox comprising multiple, endogenous selectable markers for targeted genomic insertions of DNAs of interest (DOIs). Marker genes encode proteins involved in 5-fluorocytosine uptake and pyrimidine salvage activities mediating 5-fluorocytosine deamination as well as 5-fluorouracil phosphoribosylation. The requirement for their genomic replacement by DOIs to confer 5-fluorocytosine or 5-fluorouracil resistance for transformation selection enforces site-specific integrations. Due to the fact that the described markers are endogenously encoded, there is no necessity for the exogenous introduction of commonly employed markers such as auxotrophy-complementing genes or antibiotic resistance cassettes. Importantly, inactivation of the described marker genes had no adverse effects on nutrient requirements, growth, or virulence of the human pathogen Given the limited number and distinct types of selectable markers available for the genetic manipulation of prototrophic strains such as wild-type strains, we anticipate that the proposed methodology will significantly advance genetic as well as metabolic engineering of fungal species.
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http://dx.doi.org/10.1128/mBio.00230-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7157766PMC
April 2020

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

Anal Bioanal Chem 2020 Sep 14;412(24):6351-6360. Epub 2020 Mar 14.

Institute of Physical Chemistry and Abbe Center of Photonics, Friedrich Schiller University Jena, Helmholtzweg 4, 07743, Jena, Germany.

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.
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http://dx.doi.org/10.1007/s00216-020-02571-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7442771PMC
September 2020

The fungal CCAAT-binding complex and HapX display highly variable but evolutionary conserved synergetic promoter-specific DNA recognition.

Nucleic Acids Res 2020 04;48(7):3567-3590

Department of Molecular and Applied Microbiology, Leibniz Institute for Natural Product Research and Infection Biology (HKI), Jena D-07745, Germany.

To sustain iron homeostasis, microorganisms have evolved fine-tuned mechanisms for uptake, storage and detoxification of the essential metal iron. In the human pathogen Aspergillus fumigatus, the fungal-specific bZIP-type transcription factor HapX coordinates adaption to both iron starvation and iron excess and is thereby crucial for virulence. Previous studies indicated that a HapX homodimer interacts with the CCAAT-binding complex (CBC) to cooperatively bind bipartite DNA motifs; however, the mode of HapX-DNA recognition had not been resolved. Here, combination of in vivo (genetics and ChIP-seq), in vitro (surface plasmon resonance) and phylogenetic analyses identified an astonishing plasticity of CBC:HapX:DNA interaction. DNA motifs recognized by the CBC:HapX protein complex comprise a bipartite DNA binding site 5'-CSAATN12RWT-3' and an additional 5'-TKAN-3' motif positioned 11-23 bp downstream of the CCAAT motif, i.e. occasionally overlapping the 3'-end of the bipartite binding site. Phylogenetic comparison taking advantage of 20 resolved Aspergillus species genomes revealed that DNA recognition by the CBC:HapX complex shows promoter-specific cross-species conservation rather than regulon-specific conservation. Moreover, we show that CBC:HapX interaction is absolutely required for all known functions of HapX. The plasticity of the CBC:HapX:DNA interaction permits fine tuning of CBC:HapX binding specificities that could support adaptation of pathogens to their host niches.
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http://dx.doi.org/10.1093/nar/gkaa109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7144946PMC
April 2020

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

Biomolecules 2020 01 22;10(2). Epub 2020 Jan 22.

Department of Nuclear Medicine, Medical University Innsbruck, A-6020 Innsbruck, Austria.

() is a human pathogen causing severe invasive fungal infections, lacking sensitive and selective diagnostic tools. 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. [Ga]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.
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http://dx.doi.org/10.3390/biom10020168DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7072563PMC
January 2020

The Lysine Deacetylase RpdA Is Essential for Virulence in .

Front Microbiol 2019 4;10:2773. Epub 2019 Dec 4.

Institute of Molecular Biology, Biocenter, Medical University of Innsbruck, Innsbruck, Austria.

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. is the most common airborne opportunistic mold pathogen causing invasive and often fatal disease. Establishing a novel conditional gene expression system, we demonstrate that downregulation of the class 1 lysine deacetylase (KDAC) RpdA leads to avirulence of in a murine model for pulmonary aspergillosis. The 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 tuning of gene activity by supplying xylose in the drinking water of mice. In the absence of xylose, an strain expressing under control of the promoter, , was avirulent and lung histology showed significantly less fungal growth. With xylose, however, displayed full virulence demonstrating that xylose was taken up by the mouse, transported to the site of fungal infection and caused induction . These results demonstrate that (i) RpdA is a promising target for novel antifungal therapies and (ii) the expression system is a powerful new tool for gene silencing in .
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http://dx.doi.org/10.3389/fmicb.2019.02773DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6905131PMC
December 2019

Novel intermicrobial molecular interaction: Quinolone Signal (PQS) modulates response to iron.

Microbiology (Reading) 2020 01 15;166(1):44-55. Epub 2019 Nov 15.

California Institute for Medical Research, San Jose, CA, USA.

(Pa) and (Af), the commonest bacterium and fungus in compromised host airways, compete for iron (Fe). The Pseudomonas quinolone signal (PQS), a Pa quorum sensing molecule, also chelates Fe, and delivers Fe to the Pa cell membrane using Pa siderophores. In models of Af biofilm formation or preformed biofilms, PQS inhibited Af in a low Fe environment. AfΔ (mutant unable to produce siderophores) biofilm was more sensitive to PQS inhibition than wild-type (WT), as was planktonic AfΔ growth. PQS decreased WT Af growth on agar. All these inhibitory actions were reversed by Fe. The Pa siderophore pyoverdin, or Af siderophore inhibitor celastrol, act cooperatively with PQS in Af inhibition. These findings all indicate PQS inhibition is owing to Fe chelation. , in high Fe environments PQS Af biofilm at 1/100 to 1/2000 Fe concentration required for Fe alone to enhance. Planktonic Af growth, and on agar, Af conidiation, were also enhanced by PQS+Fe compared to Fe alone. In contrast, neither AfΔ biofilm, nor planktonic AfΔ, were enhanced by PQS-Fe compared to Fe. When Af siderophore ferricrocin (FC),+PQS, were added to AfΔ Af was then boosted more than by FC alone. Moreover, FC+PQS+Fe boosted AfΔ more than Fe, FC, FC+Fe, PQS+FC or PQS+Fe. Thus PQS-Fe maximal stimulation requires Af siderophores. PQS inhibits Af via chelation under low Fe conditions. In a high Fe environment, PQS paradoxically stimulates Af efficiently, and this involves Af siderophores. PQS production by Pa could stimulate Af in cystic fibrosis airways, where Fe homeostasis is altered and Fe levels increase, supporting fungal growth.
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http://dx.doi.org/10.1099/mic.0.000858DOI Listing
January 2020

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

Virulence 2019 12;10(1):925-934

Institute of Molecular Biology, Medical University of Innsbruck, Innsbruck, Austria.

In contrast to mammalia, fungi are able to synthesize the branched-chain amino acid leucine . Recently, the transcription factor LeuB has been shown to cross-regulate leucine biosynthesis, nitrogen metabolism and iron homeostasis in , 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 and . Here, we demonstrate that in 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 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 and that the leucine biosynthetic pathway is an attractive target for the development of antifungal drugs.
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http://dx.doi.org/10.1080/21505594.2019.1682760DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6844326PMC
December 2019

Comparison of Ga-labeled RGD mono- and multimers based on a clickable siderophore-based scaffold.

Nucl Med Biol 2019 Nov - Dec;78-79:1-10. Epub 2019 Oct 25.

Department of Nuclear Medicine, Medical University Innsbruck, Innsbruck, Austria. Electronic address:

Cyclic pentapeptides containing the amino acid sequence arginine-glycine-aspartic (RGD) have been widely applied to target αβ integrin, which is upregulated in various tumors during tumor-induced angiogenesis. Multimeric cyclic RGD peptides have been reported to be advantageous over monomeric counterparts for angiogenesis imaging. Here, we prepared mono-, di-, and trimeric cyclic arginine-glycine-aspartic-D-phenylalanine-lysine (c (RGDfK)) derivatives by conjugation with the natural chelator fusarinine C (FSC) using click chemistry based on copper (I)-catalyzed azide-alkyne cycloaddition (CuAAC). The αβ binding properties of Ga-labeled mono-, di-, and trimeric c(RGDfK) peptides were evaluated in vitro as well as in vivo and compared with the references monomeric [Ga]GaNODAGA-c(RGDfK) and trimeric [Ga]GaFSC(suc-c(RGDfK)). All Ga-labeled c(RGDfK) peptides displayed hydrophilicity (logD = -2.96 to -3.80), low protein binding and were stable in phosphate buffered-saline (PBS) and serum up to 2 h. In vitro internalization assays with human melanoma M21 (αβ-positive) and M21-L (αβ-negative) cell lines showed specific uptake of all derivatives and increased in the series: mono- < di- < trimeric peptide. The highest tumor uptake, tumor-to-background ratios, and image contrast were found for the dimeric [Ga]GaMAFC(c(RGDfK)aza). In conclusion, we developed a novel strategy for direct, straight forward preparation of mono-, di-, and trimeric c(RGDfK) conjugates based on the FSC scaffold. Interestingly, the best αβ imaging properties were found for the dimeric [Ga]GaMAFC(c(RGDfK)aza).
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http://dx.doi.org/10.1016/j.nucmedbio.2019.09.002DOI Listing
July 2020

The monothiol glutaredoxin GrxD is essential for sensing iron starvation in Aspergillus fumigatus.

PLoS Genet 2019 09 16;15(9):e1008379. Epub 2019 Sep 16.

Institute of Molecular Biology, Biocenter, Medical University of Innsbruck, Innsbruck, Austria.

Efficient adaptation to iron starvation is an essential virulence determinant of the most common human mold pathogen, Aspergillus fumigatus. Here, we demonstrate that the cytosolic monothiol glutaredoxin GrxD plays an essential role in iron sensing in this fungus. Our studies revealed that (i) GrxD is essential for growth; (ii) expression of the encoding gene, grxD, is repressed by the transcription factor SreA in iron replete conditions and upregulated during iron starvation; (iii) during iron starvation but not iron sufficiency, GrxD displays predominant nuclear localization; (iv) downregulation of grxD expression results in de-repression of genes involved in iron-dependent pathways and repression of genes involved in iron acquisition during iron starvation, but did not significantly affect these genes during iron sufficiency; (v) GrxD displays protein-protein interaction with components of the cytosolic iron-sulfur cluster biosynthetic machinery, indicating a role in this process, and with the transcription factors SreA and HapX, which mediate iron regulation of iron acquisition and iron-dependent pathways; (vi) UV-Vis spectra of recombinant HapX or the complex of HapX and GrxD indicate coordination of iron-sulfur clusters; (vii) the cysteine required for iron-sulfur cluster coordination in GrxD is in vitro dispensable for interaction with HapX; and (viii) there is a GrxD-independent mechanism for sensing iron sufficiency by HapX; (ix) inactivation of SreA suppresses the lethal effect caused by GrxD inactivation. Taken together, this study demonstrates that GrxD is crucial for iron homeostasis in A. fumigatus.
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http://dx.doi.org/10.1371/journal.pgen.1008379DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6762210PMC
September 2019

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

Antimicrob Agents Chemother 2019 10 23;63(10). Epub 2019 Sep 23.

Division of Molecular Biology, Biocenter, Medical University of Innsbruck, Innsbruck, Austria

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 resistant to VL-2397. Moreover, expression of the endogenous gene under the control of a xylose-inducible promoter (to uncouple 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 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 expression indicated that the reduced antifungal activity of the iron-chelated VL-2397 is caused by downregulation of expression by the imported iron. Furthermore, we demonstrate that defects in iron homeostatic mechanisms modulate the activity of VL-2397. In contrast to and , displays intrinsic resistance to VL-2397 antifungal activity. However, expression of from , or its homologue from , resulted in susceptibility to VL-2397, which suggests that the intrinsic resistance of is based on lack of uptake and that , , and share an intracellular target for VL-2397.
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http://dx.doi.org/10.1128/AAC.00807-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6761561PMC
October 2019

Intermicrobial interaction: Aspergillus fumigatus siderophores protect against competition by Pseudomonas aeruginosa.

PLoS One 2019 8;14(5):e0216085. Epub 2019 May 8.

California Institute for Medical Research, San Jose, California, United States of America.

Pseudomonas aeruginosa and Aspergillus fumigatus are pathogens frequently co-inhabiting immunocompromised patient airways, particularly in people with cystic fibrosis. Both microbes depend on the availability of iron, and compete for iron in their microenvironment. We showed previously that the P. aeruginosa siderophore pyoverdine is the main instrument in battling A. fumigatus biofilms, by iron chelation and denial of iron to the fungus. Here we show that A. fumigatus siderophores defend against anti-fungal P. aeruginosa effects. P. aeruginosa supernatants produced in the presence of wildtype A. fumigatus planktonic supernatants (Afsup) showed less activity against A. fumigatus biofilms than P. aeruginosa supernatants without Afsup, despite higher production of pyoverdine by P. aeruginosa. Supernatants of A. fumigatus cultures lacking the sidA gene (AfΔsidA), unable to produce hydroxamate siderophores, were less capable of protecting A. fumigatus biofilms from P. aeruginosa supernatants and pyoverdine. AfΔsidA biofilm was more sensitive towards inhibitory effects of pyoverdine, the iron chelator deferiprone (DFP), or amphothericin B than wildtype A. fumigatus biofilm. Supplementation of sidA-deficient A. fumigatus biofilm with A. fumigatus siderophores restored resistance to pyoverdine. The A. fumigatus siderophore production inhibitor celastrol sensitized wildtype A. fumigatus biofilms towards the anti-fungal activity of DFP. In conclusion, A. fumigatus hydroxamate siderophores play a pivotal role in A. fumigatus competition for iron against P. aeruginosa.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0216085PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6505954PMC
January 2020

Rational Design, Synthesis and Preliminary Evaluation of Novel Fusarinine C-Based Chelators for Radiolabeling with Zirconium-89.

Biomolecules 2019 03 6;9(3). Epub 2019 Mar 6.

Department of Nuclear Medicine, Medical University Innsbruck, 6020 Innsbruck, Austria.

Fusarinine C (FSC) has recently been shown to be a promising and novel chelator for Zr. Here, FSC has been further derivatized to optimize the complexation properties of FSC-based chelators for Zr-labeling by introducing additional carboxylic groups. These were expected to improve the stability of Zr-complexes by saturating the 8-coordination sphere of [Zr] Zr, and also to introduce functionalities suitable for conjugation to targeting vectors such as monoclonal antibodies. For proof of concept, succinic acid derivatization at the amine groups of FSC was carried out, resulting in FSC(succ)₂ and FSC(succ)₃. FSC(succ)₂ was further derivatized to FSC(succ)₂ AA by reacting with acetic anhydride (AA). The Zr complexation properties of these chelators were studied by reacting with ZrCl₄. Partition coefficient, protein binding, serum stability, acid dissociation, and transchelation studies of Zr-complexes were carried out in vitro and the results were compared with those for Zr-desferrioxamine B ([Zr]Zr-DFO) and Zr-triacetylfusarinine C ([Zr]Zr-TAFC). The in vivo properties of [Zr]Zr-FSC(succ)₃ were further compared with [Zr]Zr-TAFC in BALB/c mice using micro-positron emission tomography/computer tomography (microPET/CT) imaging. Fusarinine C (succ)₂AA and FSC(succ)₃ were synthesized with satisfactory yields. Complexation with ZrCl₄ was achieved using a simple strategy resulting in high-purity Zr-FSC(succ)₂AA and Zr-FSC(succ)₃ with 1:1 stoichiometry. Distribution coefficients of Zr-complexes revealed increased hydrophilic character compared to [Zr]Zr-TAFC. All radioligands showed high stability in phosphate buffered saline (PBS) and human serum and low protein-bound activity over a period of seven days. Acid dissociation and transchelation studies exhibited a range of in vitro stabilities following the order: [Zr]Zr-FSC(succ)₃ > [Zr]Zr-TAFC > [Zr]Zr-FSC(succ)₂AA >> [Zr]Zr-DFO. Biodistribution studies of [Zr]Zr-FSC(succ)₃ revealed a slower excretion pattern compared to [Zr]Zr-TAFC. In conclusion, [Zr]Zr-FSC(succ)₃ showed the best stability and inertness. The promising results obtained with [Zr]Zr-FSC(succ)₂AA highlight the potential of FSC(succ)₂ as a monovalent chelator for conjugation to targeted biomolecules, in particular, monoclonal antibodies.
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http://dx.doi.org/10.3390/biom9030091DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6468543PMC
March 2019

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

Mol Imaging Biol 2019 12;21(6):1097-1106

Department of Nuclear Medicine, Medical University Innsbruck, Innsbruck, Austria.

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 [Ga]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, [Ga]tripropanoyl(FSC) ([Ga]TPFC), [Ga]diacetylbutanoyl(FSC) ([Ga]DABuFC), and [Ga]trisuccinyl(FSC) ([Ga]FSC(suc)), 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 [Ga]TPFC and [Ga]DABuFC were comparable to [Ga]TAFC, whereas no uptake in the infected region was observed with [Ga]FSC(suc).

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.
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http://dx.doi.org/10.1007/s11307-019-01325-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6877352PMC
December 2019

Aspergillus-Pseudomonas interaction, relevant to competition in airways.

Med Mycol 2019 Apr;57(Supplement_2):S228-S232

California Institute for Medical Research, San Jose, California, USA.

In airways of immunocompromised patients and individuals with cystic fibrosis, Pseudomonas aeruginosa and Aspergillus fumigatus are the most common opportunistic bacterial and fungal pathogens. Both pathogens form biofilms and cause acute and chronic illnesses. Previous studies revealed that P. aeruginosa is able to inhibit A. fumigatus biofilms in vitro. While numerous P. aeruginosa molecules have been shown to affect A. fumigatus, there never has been a systematic approach to define the principal causative agent. We studied 24 P. aeruginosa mutants, with deletions in genes important for virulence, iron acquisition, or quorum sensing, for their ability to interfere with A. fumigatus biofilms. Cells, planktonic or biofilm culture filtrates of four P. aeruginosa mutants, pvdD-pchE-, pvdD-, lasR-rhlR-, and lasR-, inhibited A. fumigatus biofilm metabolism or planktonic A. fumigatus growth significantly less than P. aeruginosa wild type. The common defect of these four mutants was a lack in the production of the P. aeruginosa siderophore pyoverdine. Pure pyoverdine affected A. fumigatus biofilm metabolism, and restored inhibition by the above mutants. In lungs from cystic fibrosis patients, pyoverdine production and antifungal activity correlated. The key inhibitory mechanism for pyoverdine was iron-chelation and denial of iron to A. fumigatus. Further experiments revealed a counteracting, self-protective mechanism by A. fumigatus, based on A. fumigatus siderophore production.
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http://dx.doi.org/10.1093/mmy/myy087DOI Listing
April 2019
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