Publications by authors named "Binder U"

67 Publications

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

Molecular recognition of structurally disordered Pro/Ala-rich sequences (PAS) by antibodies involves an Ala residue at the hot spot of the epitope.

J Mol Biol 2021 09 20;433(18):167113. Epub 2021 Jun 20.

Lehrstuhl für Biologische Chemie, Technische Universität München, Emil-Erlenmeyer-Forum 5, 85354 Freising, Germany; XL-protein GmbH, Lise-Meitner-Strasse 30, 85354 Freising, Germany. Electronic address:

Pro/Ala-rich sequences (PAS) are polypeptides that were developed as a biological alternative to poly-ethylene glycol (PEG) to generate biopharmaceuticals with extended plasma half-life. Like PEG, PAS polypeptides are conformationally disordered and show high solubility in water. Devoid of any charged or prominent hydrophobic side chains, these biosynthetic polymers represent an extreme case of intrinsically disordered proteins. Despite lack of immunogenicity of PAS tags in numerous animal studies we now succeeded in generating monoclonal antibodies (MAbs) against three different PAS versions. To this end, mice were immunized with a PAS#1, P/A#1 or APSA 40mer peptide conjugated to keyhole limpet hemocyanin as highly immunogenic carrier protein. In each case, one MAb with high binding activity and specificity towards a particular PAS motif was obtained. The apparent affinity was strongly dependent on the avidity effect and most pronounced for the bivalent MAb when interacting with a long PAS repeat. X-ray structural analysis of four representative anti-PAS Fab fragments in complex with their cognate PAS epitope peptides revealed interactions dominated by hydrogen bond networks involving the peptide backbone as well as multiple Van der Waals contacts arising from intimate shape complementarity. Surprisingly, Ala, the L-amino acid with the smallest side chain, emerged as a crucial feature for epitope recognition, contributing specific contacts at the center of the paratope in several anti-PAS complexes. Apart from these insights into how antibodies can recognize feature-less peptides without secondary structure, the MAbs characterized in this study offer valuable reagents for the preclinical and clinical development of PASylated biologics.
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http://dx.doi.org/10.1016/j.jmb.2021.167113DOI Listing
September 2021

The impact of episporic modification of on virulence and interaction with phagocytes.

Comput Struct Biotechnol J 2021 20;19:880-896. Epub 2021 Jan 20.

Leibniz Institute for Natural Product Research and Infection Biology - Hans Knöll Institute (HKI), Jena, Germany.

Fungal infections caused by the ancient lineage Mucorales are emerging and increasingly reported in humans. Comprehensive surveys on promising attributes from a multitude of possible virulence factors are limited and so far, focused on and . This study addresses a systematic approach to monitor phagocytosis after physical and enzymatic modification of the outer spore wall of , one of the major causative agents of mucormycosis. Episporic modifications were performed and their consequences on phagocytosis, intracellular survival and virulence by murine alveolar macrophages and in an invertebrate infection model were elucidated. While depletion of lipids did not affect the phagocytosis of both strains, delipidation led to attenuation of LCA strain but appears to be dispensable for infection with LCV strain in the settings used in this study. Combined glucano-proteolytic treatment was necessary to achieve a significant decrease of virulence of the LCV strain in during maintenance of the full potential for spore germination as shown by a novel automated germination assay. Proteolytic and glucanolytic treatments largely increased phagocytosis compared to alive resting and swollen spores. Whilst resting spores barely (1-2%) fuse to lysosomes after invagination in to phagosomes, spore trypsinization led to a 10-fold increase of phagolysosomal fusion as measured by intracellular acidification. This is the first report of a polyphasic measurement of the consequences of episporic modification of a mucormycotic pathogen in spore germination, spore surface ultrastructure, phagocytosis, stimulation of Toll-like receptors (TLRs), phagolysosomal fusion and intracellular acidification, apoptosis, generation of reactive oxygen species (ROS) and virulence.
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http://dx.doi.org/10.1016/j.csbj.2021.01.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7851798PMC
January 2021

PASylated Thymosin α1: A Long-Acting Immunostimulatory Peptide for Applications in Oncology and Virology.

Int J Mol Sci 2020 Dec 24;22(1). Epub 2020 Dec 24.

Lehrstuhl für Biologische Chemie, Technische Universität München, Emil-Erlenmeyer-Forum 5, 85354 Freising, Germany.

Thymosin α1 (Tα1) is an immunostimulatory peptide for the treatment of hepatitis B virus (HBV) and hepatitis C virus (HCV) infections and used as an immune enhancer, which also offers prospects in the context of COVID-19 infections and cancer. Manufacturing of this N-terminally acetylated 28-residue peptide is demanding, and its short plasma half-life limits in vivo efficacy and requires frequent dosing. Here, we combined the PASylation technology with enzymatic in situ N-acetylation by RimJ to produce a long-acting version of Tα1 in at high yield. ESI-MS analysis of the purified fusion protein indicated the expected composition without any signs of proteolysis. SEC analysis revealed a 10-fold expanded hydrodynamic volume resulting from the fusion with a conformationally disordered Pro/Ala/Ser (PAS) polypeptide of 600 residues. This size effect led to a plasma half-life in rats extended by more than a factor 8 compared to the original synthetic peptide due to retarded kidney filtration. Our study provides the basis for therapeutic development of a next generation thymosin α1 with prolonged circulation. Generally, the strategy of producing an N-terminally protected PASylated peptide solves three major problems of peptide drugs: (i) instability in the expression host, (ii) rapid degradation by serum exopeptidases, and (iii) low bioactivity because of fast renal clearance.
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http://dx.doi.org/10.3390/ijms22010124DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7795856PMC
December 2020

Efficacy of LAMB against Emerging Azole- and Multidrug-Resistant Isolates in the Model.

J Fungi (Basel) 2020 Dec 18;6(4). Epub 2020 Dec 18.

Institute of Hygiene and Medical Microbiology, Medical University Innsbruck, Schöpfstrasse 41, 6020 Innsbruck, Austria.

While being the third leading cause of candidemia worldwide, numerous studies have shown severe clonal outbreaks due to fluconazole-resistant (FLCR) isolates associated with fluconazole therapeutic failure (FTF) with enhanced mortality. More recently, multidrug resistant (MDR) blood isolates have also been identified that are resistant to both azole and echinocandin drugs. Amphotericin B (AMB) resistance is rarely reported among isolates and proper management of bloodstream infections due to FLZR and MDR isolates requires prompt action at the time of outbreak. Therefore, using a well-established model, we assessed whether (a) laboratory-based findings on azole or echinocandin (micafungin) resistance in lead to therapeutic failure, (b) LAMB could serve as an efficient salvage treatment option, and (c) distinct mutations in impact mortality. Our in vivo data confirm fluconazole inefficacy against FLCR isolates carrying Y132F, Y132F + K143R, Y132F + G307A, and G307A + G458S in Erg11p, while LAMB proved to be an efficacious accessible option against both FLCR and MDR isolates. Moreover, positive correlation of in vitro and in vivo data further highlights the utility of as a reliable model to investigate azole and polyene drug efficacy.
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http://dx.doi.org/10.3390/jof6040377DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7767002PMC
December 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

RcLS2F - A Novel Fungal Class 1 KDAC Co-repressor Complex in .

Front Microbiol 2020 4;11:43. Epub 2020 Feb 4.

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

The fungal class 1 lysine deacetylase (KDAC) RpdA is a promising target for prevention and treatment of invasive fungal infection. RpdA is essential for survival of the most common air-borne mold pathogen and the model organism . In , RpdA depletion induced production of previously unknown small bioactive substances. As known from yeasts and mammals, class 1 KDACs act as components of multimeric protein complexes, which previously was indicated also for . Composition of these complexes, however, remained obscure. In this study, we used tandem affinity purification to characterize different RpdA complexes and their composition in . In addition to known class 1 KDAC interactors, we identified a novel RpdA complex, which was termed RcLS2F. It contains ScrC, previously described as suppressor of the transcription factor CrzA, as well as the uncharacterized protein FscA. We show that recruitment of FscA depends on ScrC and we provide clear evidence that Δ suppression by ScrC depletion is due to a lack of transcriptional repression caused by loss of the novel RcLS2F complex. Moreover, RcLS2F is essential for sexual development and engaged in an autoregulatory feed-back loop.
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http://dx.doi.org/10.3389/fmicb.2020.00043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7010864PMC
February 2020

PASylation of IL-1 receptor antagonist (IL-1Ra) retains IL-1 blockade and extends its duration in mouse urate crystal-induced peritonitis.

J Biol Chem 2020 01 9;295(3):868-882. Epub 2019 Dec 9.

Department of Medicine, University of Colorado, Aurora, Colorado 80045.

Interleukin-1 (IL-1) is a key mediator of inflammation and immunity. Naturally-occurring IL-1 receptor antagonist (IL-1Ra) binds and blocks the IL-1 receptor-1 (IL-1R1), preventing signaling. Anakinra, a recombinant form of IL-1Ra, is used to treat a spectrum of inflammatory diseases. However, anakinra is rapidly cleared from the body and requires daily administration. To create a longer-lasting alternative, PASylated IL-1Ra (PAS-IL-1Ra) has been generated by in-frame fusion of a long, defined-length, N-terminal Pro/Ala/Ser (PAS) random-coil polypeptide with IL-1Ra. Here, we compared the efficacy of two PAS-IL-1Ra molecules, PAS600-IL-1Ra and PAS800-IL-1Ra (carrying 600 and 800 PAS residues, respectively), with that of anakinra in mice. PAS600-IL-1Ra displayed markedly extended blood plasma levels 3 days post-administration, whereas anakinra was undetectable after 24 h. We also studied PAS600-IL-1Ra and PAS800-IL-1Ra for efficacy in monosodium urate (MSU) crystal-induced peritonitis. 5 days post-administration, PAS800-IL-1Ra significantly reduced leukocyte influx and inflammatory markers in MSU-induced peritonitis, whereas equimolar anakinra administered 24 h before MSU challenge was ineffective. The 6-h pretreatment with equimolar anakinra or PAS800-IL-1Ra before MSU challenge similarly reduced inflammatory markers. In cultured A549 lung carcinoma cells, anakinra, PAS600-IL-1Ra, and PAS800-IL-Ra reduced IL-1α-induced IL-6 and IL-8 levels with comparable potency. In human peripheral blood mononuclear cells, these molecules suppressed -induced production of the cancer-promoting cytokine IL-22. Surface plasmon resonance analyses revealed significant binding between PAS-IL-1Ra and IL-1R1, although with a slightly lower affinity than anakinra. These results validate PAS-IL-1Ra as an active IL-1 antagonist with marked potency and a significantly extended half-life compared with anakinra.
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http://dx.doi.org/10.1074/jbc.RA119.010340DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6970921PMC
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

Minimal Inhibitory Concentration (MIC)-Phenomena in and Their Impact on the Diagnosis of Antifungal Resistance.

J Fungi (Basel) 2019 Sep 4;5(3). Epub 2019 Sep 4.

Institute of Hygiene and Medical Microbiology, Medical University Innsbruck, 6020 Innsbruck, Austria.

Antifungal susceptibility testing (AFST) of clinical isolates is a tool in routine diagnostics to facilitate decision making on optimal antifungal therapy. The minimal inhibitory concentration (MIC)-phenomena (trailing and paradoxical effects (PXE)) observed in AFST complicate the unambiguous and reproducible determination of MICs and the impact of these phenomena on in vivo outcome are not fully understood. We aimed to link the MIC-phenomena with in vivo treatment response using the alternative infection model . We found that strains exhibiting PXE for caspofungin (CAS) had variable treatment outcomes in the model. In contrast, strains showing trailing for voriconazole failed to respond in vivo. Caspofungin- and voriconazole-susceptible strains responded to the respective antifungal therapy in vivo. In conclusion, MIC data and subsequent susceptibility interpretation of strains exhibiting PXE and/or trailing should be carried out with caution, as both effects are linked to drug adaptation and treatment response is uncertain to predict.
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http://dx.doi.org/10.3390/jof5030083DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6787722PMC
September 2019

Hypoxia Decreases Diagnostic Biomarkers for Aspergillosis In Vitro.

J Fungi (Basel) 2019 Jul 11;5(3). Epub 2019 Jul 11.

Department of Hygiene, Medical Microbiology and Public Health, Institute of Hygiene and Medical Microbiology, Medical University of Innsbruck, Schöpfstrasse 41, 6020 Innsbruck, Austria.

The aim of the study was to evaluate the influence of hypoxia on galactomannan and (1,3)-β-d-glucan release of clinically relevant in vitro. Hypoxia decreased biomass and consequently led to lower biomarker release. However, when normalized to biomass, hypoxia led to increased levels of biomarkers at early growth stages (24 h). Antifungals (amphotericin B and voriconazole) decreased the galactomannan amount of , even more prominently in hypoxia.
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http://dx.doi.org/10.3390/jof5030061DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6787614PMC
July 2019

Cryptic species of section display essential physiological features to cause infection and are similar in their virulence potential in .

Virulence 2019 12;10(1):542-554

a Division of Hygiene and Medical Microbiology , Medical University Innsbruck , Austria.

species account for the majority of invasive mold infections in immunocompromised patients. Most commonly, members of the section are isolated from clinical material, followed by isolates belonging to section . The section contains 16 accepted species. Six species were found to be of clinical relevance and studied for differences in growth adaptability and virulence potential. Therefore, a set of 73 isolates (22 s.s., 8 , 27 , 2 , 13 , and 1 ) was studied to determine differences in (a) germination kinetics, (b) temperature tolerance, (c) oxygen stress tolerance (1% O), and (d) a combination of the latter two. Virulence potential of phialidic (PC) and accessory conidia (AC) was studied in larvae, using survival as read out. Further, the formation of AC was evaluated in larval tissue. All isolates were able to grow at elevated temperature and hypoxia, with highest growth and germination rates at 37°C. , and exhibited highest growth rates. Virulence potential in larvae was inoculum and temperature dependent. All species except formed AC and germination kinetics of AC was variable. Significantly higher virulence potential of AC was found for one isolate. AC could be detected in larval tissue 96 h post infection. Based on these findings, cryptic species of section are well adapted to the host environment and have similar potential to cause infections.
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http://dx.doi.org/10.1080/21505594.2019.1614382DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6592363PMC
December 2019

Half-life extension and non-human primate pharmacokinetic safety studies of i-body AD-114 targeting human CXCR4.

MAbs 2019 10 23;11(7):1331-1340. Epub 2019 Aug 23.

The Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University , Bundoora , Melbourne , Australia.

Single domain antibodies that combine antigen specificity with high tissue penetration are an attractive alternative to conventional antibodies. However, rapid clearance from the bloodstream owing to their small size can be a limitation of therapeutic single domain antibodies. Here, we describe and evaluate the conjugation of a single domain i-body, AD-114, which targets CXCR4, to a panel of half-life extension technologies including a human serum albumin-binding peptide, linear and branched PEG, and PASylation (PA600). The conjugates were assessed in murine, rat and cynomolgus monkey pharmacokinetic studies and showed that the branched PEG was most effective at extending circulating half-life in mice; however, manufacturing limitations of PEGylated test material precluded scale-up and assessment in larger animals. PA600, by comparison, was amenable to scale-up and afforded considerable half-life improvements in mice, rats and cynomolgus monkeys. In mice, the circulating half-life of AD-114 was extended from 0.18 h to 7.77 h following conjugation to PA600, and in cynomolgus monkeys, the circulating half-life of AD-114-PA600 was 24.27 h. AD-114-PA600 was well tolerated in cynomolgus monkeys at dose rates up to 100 mg/kg with no mortalities or drug-related clinical signs.
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http://dx.doi.org/10.1080/19420862.2019.1626652DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6748587PMC
October 2019

Precise genome editing using a CRISPR-Cas9 method highlights the role of CoERG11 amino acid substitutions in azole resistance in Candida orthopsilosis.

J Antimicrob Chemother 2019 08;74(8):2230-2238

Laboratoire de Parasitologie-Mycologie, CHU de Nantes, Nantes, France.

Background: Azoles are one of the main antifungal classes for the treatment of candidiasis. In the current context of emerging drug resistance, most studies have focused on Candida albicans, Candida glabrata or Candida auris but, so far, less is known about the underlying mechanisms of resistance in other species, including Candida orthopsilosis.

Objectives: We investigated azole resistance in a C. orthopsilosis clinical isolate recovered from a patient with haematological malignancy receiving fluconazole prophylaxis.

Methods: Antifungal susceptibility to fluconazole was determined in vitro (CLSI M27-A3) and in vivo (in a Galleria mellonella model of invasive candidiasis). The CoERG11 gene was then sequenced and amino acid substitutions identified were mapped on the predicted 3D structure of CoErg11p. A clustered regularly interspaced short palindromic repeat-Cas9 (CRISPR-Cas9) genome-editing strategy was used to introduce relevant mutations into a fluconazole-susceptible C. orthopsilosis isolate.

Results: Compared with unrelated C. orthopsilosis isolates, the clinical isolate exhibited both in vitro and in vivo fluconazole resistance. Sequencing of the CoERG11 gene identified several amino acid substitutions, including two possibly involved in fluconazole resistance (L376I and G458S). Both mutations mapped close to the active site of CoErg11p. Engineering these mutations in a different genetic background using CRISPR-Cas9 demonstrated that G458S, but not L376I, confers resistance to fluconazole and voriconazole.

Conclusions: Our data show that the G458S amino acid substitution in CoERG11p, but not L376I, contributes to azole resistance in C. orthopsilosis. In addition to highlighting the potential of CRISPR-Cas9 technology for precise genome editing in the field of antifungal resistance, we discuss some points that are critical to improving its efficiency.
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http://dx.doi.org/10.1093/jac/dkz204DOI Listing
August 2019

Author Correction: Consistent success in life-supporting porcine cardiac xenotransplantation.

Nature 2019 Apr;568(7752):E7

Institute of Molecular Animal Breeding and Biotechnology, Gene Center, LMU Munich, Munich, Germany.

In this Letter, Mayuko Kurome and Valeri Zakhartchenko have been added to the author list (affiliated with Institute of Molecular Animal Breeding and Biotechnology, Gene Center, LMU Munich, Munich, Germany). The author list and 'Author contributions' section have been corrected online; see accompanying Amendment.
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http://dx.doi.org/10.1038/s41586-019-1108-4DOI Listing
April 2019

Factor H Binding Molecule Hgt1p - A Low Glucose-Induced Transmembrane Protein Is Trafficked to the Cell Wall and Impairs Phagocytosis and Killing by Human Neutrophils.

Front Microbiol 2018 15;9:3319. Epub 2019 Jan 15.

Division of Hygiene and Medical Microbiology, Medical University of Innsbruck, Innsbruck, Austria.

Complement is a tightly controlled arm of the innate immune system, facilitating phagocytosis and killing of invading pathogens. Factor H (FH) is the main fluid-phase inhibitor of the alternative pathway. Many pathogens can hijack FH from the host and protect themselves from complement-dependent killing. is a clinically important opportunistic yeast, expressing different FH binding molecules on its cell surface, which allow complement evasion. One such FH binding molecule is the transmembrane protein "High affinity glucose transporter 1" (Hgt1p), involved in glucose metabolism. This study demonstrated that Hgt1p transcription and expression is induced and highest at the low, but physiological glucose concentration of 0.1%. Thus, this concentration was used throughout the study. We also demonstrated the transport of Hgt1p to the fungal cell wall surface by vesicle trafficking and its release by exosomes containing Hgt1p integrated in the vesicular membrane. We corroborated Hgt1p as FH binding molecule. A polyclonal anti-Hgt1p antibody was created which interfered with the binding of FH, present in normal human serum to the fungal cell wall. A chimeric molecule consisting of FH domains 6 and 7 fused to human IgG1 Fc (FH6.7/Fc) even more comprehensively blocked FH binding, likely because FH6.7/Fc diverted FH away from fungal FH ligands other than Hgt1p. Reduced FH binding to the yeast was associated with a concomitant increase in C3b/iC3b deposition and resulted in significantly increased phagocytosis and killing by human neutrophils. In conclusion, Hgt1p also exhibits non-canonical functions such as binding FH after its export to the cell wall. Blocking Hgt1p-FH interactions may represent a tool to enhance complement activation on the fungal surface to promote phagocytosis and killing of
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http://dx.doi.org/10.3389/fmicb.2018.03319DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6340940PMC
January 2019

Generation of A Mucor circinelloides Reporter Strain-A Promising New Tool to Study Antifungal Drug Efficacy and Mucormycosis.

Genes (Basel) 2018 Dec 7;9(12). Epub 2018 Dec 7.

Departamento de Genética y Microbiología, Facultad de Biología, Universidad de Murcia, 30100 Murcia, Spain.

Invasive fungal infections caused by Mucorales (mucormycosis) have increased worldwide. These life-threatening infections affect mainly, but not exclusively, immunocompromised patients, and are characterized by rapid progression, severe tissue damage and an unacceptably high rate of mortality. Still, little is known about this disease and its successful therapy. New tools to understand mucormycosis and a screening method for novel antimycotics are required. Bioluminescent imaging is a powerful tool for in vitro and in vivo approaches. Hence, the objective of this work was to generate and functionally analyze bioluminescent reporter strains of , one mucormycosis-causing pathogen. Reporter strains were constructed by targeted integration of the firefly luciferase gene under control of the promoter P. The luciferase gene was sufficiently expressed, and light emission was detected under several conditions. Phenotypic characteristics, virulence potential and antifungal susceptibility were indifferent to the wild-type strains. Light intensity was dependent on growth conditions and biomass, being suitable to determine antifungal efficacy in vitro. This work describes for the first time the generation of reporter strains in a basal fungus that will allow real-time, non-invasive infection monitoring in insect and murine models, and the testing of antifungal efficacy by means other than survival.
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http://dx.doi.org/10.3390/genes9120613DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6315630PMC
December 2018

Consistent success in life-supporting porcine cardiac xenotransplantation.

Nature 2018 12 5;564(7736):430-433. Epub 2018 Dec 5.

Institute of Molecular Animal Breeding and Biotechnology, Gene Center, LMU Munich, Munich, Germany.

Heart transplantation is the only cure for patients with terminal cardiac failure, but the supply of allogeneic donor organs falls far short of the clinical need. Xenotransplantation of genetically modified pig hearts has been discussed as a potential alternative. Genetically multi-modified pig hearts that lack galactose-α1,3-galactose epitopes (α1,3-galactosyltransferase knockout) and express a human membrane cofactor protein (CD46) and human thrombomodulin have survived for up to 945 days after heterotopic abdominal transplantation in baboons. This model demonstrated long-term acceptance of discordant xenografts with safe immunosuppression but did not predict their life-supporting function. Despite 25 years of extensive research, the maximum survival of a baboon after heart replacement with a porcine xenograft was only 57 days and this was achieved, to our knowledge, only once. Here we show that α1,3-galactosyltransferase-knockout pig hearts that express human CD46 and thrombomodulin require non-ischaemic preservation with continuous perfusion and control of post-transplantation growth to ensure long-term orthotopic function of the xenograft in baboons, the most stringent preclinical xenotransplantation model. Consistent life-supporting function of xenografted hearts for up to 195 days is a milestone on the way to clinical cardiac xenotransplantation.
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http://dx.doi.org/10.1038/s41586-018-0765-zDOI Listing
December 2018

Siroheme Is Essential for Assimilation of Nitrate and Sulfate as Well as Detoxification of Nitric Oxide but Dispensable for Murine Virulence of .

Front Microbiol 2018 12;9:2615. Epub 2018 Nov 12.

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

The saprophytic mold is the most common airborne fungal pathogen causing severe invasive fungal infections in immunocompromised patients. Siroheme is a heme-like prosthetic group used by plants and microorganisms for sulfate and nitrate assimilation but is absent in higher eukaryotes. Here, we investigated the role of siroheme in by deletion of the gene encoding the bifunctional dehydrogenase/ferrochelatase enzyme Met8. Met8-deficiency resulted in the inability to utilize sulfate and nitrate as sulfur and nitrogen sources, respectively. These results match previous data demonstrating that siroheme is an essential cofactor for nitrite and sulfite reductases. Moreover, Met8-deficiency caused significantly decreased resistance against nitric oxide (NO) underlining the importance of nitrite reductase in NO detoxification. Met8-deficiency did not affect virulence in murine models for invasive aspergillosis indicating that neither NO-detoxification nor assimilation of sulfate and nitrate play major roles in virulence in this host. Interestingly, Met8-deficiency resulted in mild virulence attenuation in the infection model revealing differences in interaction of with and mouse.
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http://dx.doi.org/10.3389/fmicb.2018.02615DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6240589PMC
November 2018

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

PLoS Genet 2018 10 26;14(10):e1007762. Epub 2018 Oct 26.

Jiangsu Key Laboratory for Microbes and Functional Genomics, College of Life Sciences, Nanjing Normal University, Nanjing, China.

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.
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http://dx.doi.org/10.1371/journal.pgen.1007762DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6221358PMC
October 2018

Dihydroorotate dehydrogenase inhibitor olorofim exhibits promising activity against all clinically relevant species within Aspergillus section Terrei.

J Antimicrob Chemother 2018 11;73(11):3068-3073

Department of Hygiene, Microbiology and Public Health, Division of Hygiene and Medical Microbiology, Medical University of Innsbruck, Innsbruck, Austria.

Objectives: In vitro and in vivo activity of the dihydroorotate dehydrogenase inhibitor olorofim (formerly F901318) (F2G Limited, UK) against clinically relevant species of the Aspergillus section Terrei was evaluated.

Methods: A total of 92 clinical Aspergillus section Terrei isolates [42 Aspergillus terreus sensu stricto and 50 cryptic species: Aspergillus alabamensis (n = 8), Aspergillus citrinoterreus (n = 27), Aspergillus floccosus (n = 1), Aspergillus hortai (n = 13) and Aspergillus neoafricanus (n = 1)] were evaluated. MICs were determined using the CLSI M38-A2 method. MICs of olorofim were compared with those of posaconazole, voriconazole, itraconazole and amphotericin B. The in vivo efficacy of olorofim was determined in an immunosuppressed murine model of disseminated aspergillosis.

Results: Olorofim was highly active against all tested Aspergillus section Terrei isolates, exhibiting an MIC range of 0.002-0.063 mg/L. Slightly higher MICs were observed for A. terreus cryptic species. Olorofim MICs were lower than those observed for the azoles. Selected strains with elevated MICs of azoles were highly susceptible to olorofim. Olorofim administered by oral and intravenous routes produced survival rates of 90%-100% in A. terreus-infected mice.

Conclusions: Olorofim showed potent and consistent in vitro activity against all A. terreus strains tested, including those with elevated MICs of other antifungal substances. Overall, growth inhibition by olorofim was superior to that of azoles. In vivo data showed that olorofim was highly efficacious in prolonging survival of mice with disseminated aspergillosis due to A. terreus sensu stricto.
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http://dx.doi.org/10.1093/jac/dky329DOI Listing
November 2018

Galleria mellonella as a model system to study virulence potential of mucormycetes and evaluation of antifungal treatment.

Med Mycol 2019 Apr;57(3):351-362

Department of Hygiene, Microbiology and Public Health, Division of Hygiene and Medical Microbiology, Medical University Innsbruck, Austria.

Mucorales can cause cutaneous to deep-seated infections, mainly in the immunocompromised host, resulting in high mortality rates due to late and inefficient treatment. In this study, Galleria mellonella larvae were evaluated as a heterologous invertebrate host to study pathogenicity of clinically relevant mucormycetes (Rhizopus spp., Rhizomucor spp., Lichtheimia spp., Mucor spp.). All tested species were able to infect G. mellonella larvae. Virulence potential was species-specific and correlated to clinical relevance. Survival of infected larvae was dependent on (a) the species (growth speed and spore size), (b) the infection dose, (c) the incubation temperature, (d) oxidative stress tolerance, and (e) iron availability in the growth medium. Moreover, we exploited the G. mellonella system to determine antifungal efficacy of liposomal amphotericin B, posaconazole, isavuconazole, and nystatin-intralipid. Outcome of in vivo treatment was strongly dependent upon the drug applied and the species tested. Nystatin-intralipid exhibited best activity against Mucorales, followed by posaconazole, while limited efficacy was seen for liposomal amphotericin B and isavuconazole. Pharmacokinetic properties of the tested antifungals within this alternative host system partly explain the limited treatment efficacy. In conclusion, G. mellonella represents a useful invertebrate infection model for studying virulence of mucormycetes, while evaluation of treatment response was limited.
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http://dx.doi.org/10.1093/mmy/myy042DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6398984PMC
April 2019

Sterol Composition of Clinically Relevant Mucorales and Changes Resulting from Posaconazole Treatment.

Molecules 2018 May 19;23(5). Epub 2018 May 19.

Department of Hygiene, Microbiology and Public Health, Division of Hygiene and Medical Microbiology, Medical University Innsbruck, Schöpfstr. 41, 6020 Innsbruck, Austria.

Mucorales are fungi with increasing importance in the clinics. Infections take a rapidly progressive course resulting in high mortality rates. The ergosterol biosynthesis pathway and sterol composition are of interest, since they are targeted by currently applied antifungal drugs. Nevertheless, Mucorales often exhibit resistance to these drugs, resulting in therapeutic failure. Here, sterol patterns of six clinically relevant Mucorales (, , , , , and ) were analysed in a targeted metabolomics fashion after derivatization by gas chromatography-mass spectrometry. Additionally, the effect of posaconazole (POS) treatment on the sterol pattern of was evaluated. Overall, fifteen different sterols were detected with species dependent variations in the total and relative sterol amount. Sterol analysis from hyphae confronted with sublethal concentrations of posaconazole revealed the accumulation of 14-methylergosta-8,24-diene-3,6-diol, which is a toxic sterol that was previously only detected in yeasts. Sterol content and composition were further compared to the well-characterized pathogenic mold . This work contributes to a better understanding of the ergosterol biosynthesis pathway of Mucorales, which is essential to improve antifungal efficacy, the identification of targets for novel drug design, and to investigate the combinatorial effects of drugs targeting this pathway.
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http://dx.doi.org/10.3390/molecules23051218DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6100088PMC
May 2018

Impact of spectral features of common LED lighting systems on TM-30 color indices.

Opt Express 2017 Feb;25(3):1824-1830

We examine the difference between the newly developed IES TM-30 color indices and some of the most common previously established color indices for LED systems which are used for lighting purposes, focusing on the influence of realistic spectral variations among different system designs and manufacturing runs. We find a significantly stronger influence of the employed blue InGaN wavelength on TM-30 Rf and Rg than on CRI Ra and FCI. In addition, for the established combination of green converted InGaN chips with red InGaAlP chips, we observe large differences in the effect of the wavelength of the red emission of InGaAlP chips on Rf and Ra.
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http://dx.doi.org/10.1364/OE.25.001824DOI Listing
February 2017

Anti-fibrotic Effects of CXCR4-Targeting i-body AD-114 in Preclinical Models of Pulmonary Fibrosis.

Sci Rep 2018 02 16;8(1):3212. Epub 2018 Feb 16.

AdAlta Limited, La Trobe University, 15/2 Park Drive, Bundoora, 3083, Australia.

Idiopathic pulmonary fibrosis (IPF) is a chronic fibrotic lung disease that is prevalent in individuals >50 years of age, with a median survival of 3-5 years and limited therapeutic options. The disease is characterized by collagen deposition and remodeling of the lung parenchyma in a process that is thought to be driven by collagen-expressing immune and structural cells. The G-protein coupled C-X-C chemokine receptor 4, CXCR4, is a candidate therapeutic target for IPF owing to its role in the recruitment of CXCR4 fibrocytes from the bone marrow to fibrotic lung tissue and its increased expression levels by structural cells in fibrotic lung tissue. We have engineered a novel fully human single domain antibody "i-body" called AD-114 that binds with high affinity to human CXCR4. We demonstrate here that AD-114 inhibits invasive wound healing and collagen 1 secretion by human IPF fibroblasts but not non-diseased control lung fibroblasts. Furthermore, in a murine bleomycin model of pulmonary fibrosis, AD-114 reduced the accumulation of fibrocytes (CXCR4/Col1/CD45) in fibrotic murine lungs and ameliorated the degree of lung injury. Collectively, these studies demonstrate that AD-114 holds promise as a new biological therapeutic for the treatment of IPF.
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http://dx.doi.org/10.1038/s41598-018-20811-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5816662PMC
February 2018

Impact of Morphological Sectors on Antifungal Susceptibility Testing and Virulence Studies.

Antimicrob Agents Chemother 2017 12 22;61(12). Epub 2017 Nov 22.

Division of Hygiene and Medical Microbiology, Medical University of Innsbruck, Innsbruck, Austria

Morphological heterogeneity of cultures was observed during continued cultivation of amphotericin B (AMB)-resistant isolates on drug-free medium. Outgrowth leads to the emergence of multiple sectors that might result from increased growth rates at drug-free conditions. We evaluated the differences in AMB susceptibility and virulence between sector subcultures (ATSec), AMB-resistant (ATR) strains, and AMB-susceptible (ATS) strains. By comparing AMB-resistant (ATR) strains and sector (ATSec) cultures we observed a highly significant reduction of AMB MICs in ATSec (ATR MIC, 2 to 32 μg/ml; ATSec MIC, 0.12 to 5 μg/ml). Furthermore, survival studies revealed an enhanced virulence of ATSec, which was comparable with that of AMB-sensitive strains (median survival rates for ATS isolates, 72 h; for ATSec isolate ATSec, 84 h; for ATR isolates, 144 h). Our findings clearly demonstrate that spontaneous culture degeneration occurs in and, most importantly, crucially impacts drug efficacy and virulence.
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http://dx.doi.org/10.1128/AAC.00755-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5700351PMC
December 2017

Fast-track development of an in vitro 3D lung/immune cell model to study Aspergillus infections.

Sci Rep 2017 09 14;7(1):11644. Epub 2017 Sep 14.

Division of Hygiene and Medical Microbiology, Medical University of Innsbruck, Innsbruck, Austria.

To study interactions of airborne pathogens, e.g. Aspergillus (A.) fumigatus with upper and lower respiratory tract epithelial and immune cells, we set up a perfused 3D human bronchial and small airway epithelial cell system. Culturing of normal human bronchial or small airway epithelial (NHBE, SAE) cells under air liquid interphase (ALI) and perfusion resulted in a significantly accelerated development of the lung epithelia associated with higher ciliogenesis, cilia movement, mucus-production and improved barrier function compared to growth under static conditions. Following the accelerated differentiation under perfusion, epithelial cells were transferred into static conditions and antigen-presenting cells (APCs) added to study their functionality upon infection with A. fumigatus. Fungi were efficiently sensed by apically applied macrophages or basolaterally adhered dendritic cells (DCs), as illustrated by phagocytosis, maturation and migration characteristics. We illustrate here that perfusion greatly improves differentiation of primary epithelial cells in vitro, which enables fast-track addition of primary immune cells and significant shortening of experimental procedures. Additionally, co-cultured primary DCs and macrophages were fully functional and fulfilled their tasks of sensing and sampling fungal pathogens present at the apical surface of epithelial cells, thereby promoting novel possibilities to study airborne infections under conditions mimicking the in vivo situation.
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http://dx.doi.org/10.1038/s41598-017-11271-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5599647PMC
September 2017

Oxidative Stress Response Tips the Balance in Aspergillus terreus Amphotericin B Resistance.

Antimicrob Agents Chemother 2017 10 22;61(10). Epub 2017 Sep 22.

Division of Hygiene and Medical Microbiology, Medical University of Innsbruck, Innsbruck, Austria

In this study, we characterize the impact of antioxidative enzymes in amphotericin B (AmB)-resistant (ATR) and rare AmB-susceptible (ATS) clinical isolates. We elucidate expression profiles of superoxide dismutase (SOD)- and catalase (CAT)-encoding genes, enzymatic activities of SODs, and superoxide anion production and signaling pathways involved in the oxidative stress response (OSR) in ATS and ATR strains under AmB treatment conditions. We show that ATR strains possess almost doubled basal SOD activity compared to that of ATS strains and that ATR strains exhibit an enhanced OSR, with significantly higher mRNA levels and significantly increased transcripts in ATR strains upon AmB treatment. In particular, inhibition of SOD and CAT proteins renders resistant isolates considerably susceptible to the drug In conclusion, this study shows that SODs and CATs are crucial for AmB resistance in and that targeting the OSR might offer new treatment perspectives for resistant species.
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http://dx.doi.org/10.1128/AAC.00670-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5610508PMC
October 2017

Antifungal drug testing by combining minimal inhibitory concentration testing with target identification by gas chromatography-mass spectrometry.

Nat Protoc 2017 05 6;12(5):947-963. Epub 2017 Apr 6.

Department of Pharmacy, Ludwig-Maximilians-University Munich, Munich, Germany.

Fungal infections and their increasing resistance to antibiotics are an emerging threat to public health. Novel antifungal drugs, as well technologies that can help us bolster the antimicrobial pipeline and understand resistance mechanisms, are needed. The ergosterol biosynthetic pathway is one potential target for antifungal drugs. Here we describe how antifungal susceptibility testing can be combined with target identification in distal ergosterol biosynthesis by means of gas chromatography-mass spectrometry. The fungi are treated with sublethal doses of active components that block ergosterol biosynthesis, and the ergosterol biosynthesis intermediates are analyzed in a targeted metabolomics manner after derivatization (trimethylsilylation). Drug treatment results in distinct sterol patterns that are characteristic of the affected enzyme. Sterol identification based on relative retention times and electron ionization (EI) mass spectra, as well as semiquantitative assessment of ergosterol intermediates, is described. The protocol is applicable to yeasts and molds. The overall analysis time from incubation to test result is not more than 3 d. The assay can be used to determine whether an antifungal compound of interest targets sterol biosynthesis, and, if so, to determine which enzyme in the pathway it targets.
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http://dx.doi.org/10.1038/nprot.2017.005DOI Listing
May 2017

Histidine biosynthesis plays a crucial role in metal homeostasis and virulence of Aspergillus fumigatus.

Virulence 2016 05 6;7(4):465-76. Epub 2016 Feb 6.

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

Aspergillus fumigatus is the most prevalent airborne fungal pathogen causing invasive fungal infections in immunosuppressed individuals. The histidine biosynthetic pathway is found in bacteria, archaebacteria, lower eukaryotes, and plants, but is absent in mammals. Here we demonstrate that deletion of the gene encoding imidazoleglycerol-phosphate dehydratase (HisB) in A. fumigatus causes (i) histidine auxotrophy, (ii) decreased resistance to both starvation and excess of various heavy metals, including iron, copper and zinc, which play a pivotal role in antimicrobial host defense, (iii) attenuation of pathogenicity in 4 virulence models: murine pulmonary infection, murine systemic infection, murine corneal infection, and wax moth larvae. In agreement with the in vivo importance of histidine biosynthesis, the HisB inhibitor 3-amino-1,2,4-triazole reduced the virulence of the A. fumigatus wild type and histidine supplementation partially rescued virulence of the histidine-auxotrophic mutant in the wax moth model. Taken together, this study reveals limited histidine availability in diverse A. fumigatus host niches, a crucial role for histidine in metal homeostasis, and the histidine biosynthetic pathway as being an attractive target for development of novel antifungal therapy approaches.
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http://dx.doi.org/10.1080/21505594.2016.1146848DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4871644PMC
May 2016
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