Publications by authors named "Ulrike Binder"

43 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

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

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

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

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

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

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 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

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

Galleria mellonella: An invertebrate model to study pathogenicity in correctly defined fungal species.

Fungal Biol 2016 Feb 16;120(2):288-95. Epub 2015 Jun 16.

Department of Hygiene, Microbiology and Social Medicine, Division of Hygiene and Medical Microbiology, Medical University Innsbruck, Schöpfstrasse 41, A-6020 Innsbruck, Tirol, Austria.

The high mortality rates and economic burden associated with fungal infections, plus the emergence of fungal strains resistant to antifungal drugs, make it necessary to get a deeper understanding of fungal pathogenesis, as well as to identify new target structures for antifungal drug development. Still, murine models are considered as the gold standard for studying pathogenesis, quantifying virulence, and analysing the efficacy of antifungal drugs. However, invertebrates, such as the larvae of the greater wax moth Galleria mellonella, are promising alternative hosts to address some of these questions, especially when a large number of fungal strains need to be evaluated. The purpose of this review is to summarize the benefits and drawbacks, explain the utilization of the invertebrate model host G. mellonella, and compare the virulence potential of the most important human fungal pathogens, with the focus on different virulence potential of closely related species.
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http://dx.doi.org/10.1016/j.funbio.2015.06.002DOI Listing
February 2016

N-Chlorotaurine Exhibits Fungicidal Activity against Therapy-Refractory Scedosporium Species and Lomentospora prolificans.

Antimicrob Agents Chemother 2015 Oct 3;59(10):6454-62. Epub 2015 Aug 3.

Department of Hygiene, Microbiology and Social Medicine, Division of Hygiene and Medical Microbiology, Medical University of Innsbruck, Innsbruck, Austria

N-Chlorotaurine (NCT), a well-tolerated endogenous long-lived oxidant that can be applied topically as an antiseptic, was tested on its fungicidal activity against Scedosporium and Lomentospora, opportunistic fungi that cause severe infections with limited treatment options, mainly in immunocompromised patients. In quantitative killing assays, both hyphae and conidia of Scedosporium apiospermum, Scedosporium boydii, and Lomentospora prolificans (formerly Scedosporium prolificans) were killed by 55 mM (1.0%) NCT at pH 7.1 and 37°C, with a 1- to 4-log10 reduction in CFU after 4 h and a 4- to >6-log10 reduction after 24 h. The addition of ammonium chloride to NCT markedly increased this activity. LIVE/DEAD staining of conidia treated with 1.0% NCT for 0.5 to 3 h increased the permeability of the cell wall and membrane. Preincubation of the test fungi in 1.0% NCT for 10 to 60 min delayed the time to germination of conidia by 2 h to >12 h and reduced their germination rate by 10.0 to 100.0%. Larvae of Galleria mellonella infected with 1.0 × 10(7) conidia of S. apiospermum and S. boydii died at a rate of 90.0 to 100% after 8 to 12 days. The mortality rate was reduced to 20 to 50.0% if conidia were preincubated in 1.0% NCT for 0.5 h or if heat-inactivated conidia were used. Our study demonstrates the fungicidal activity of NCT against different Scedosporium and Lomentospora species. A postantifungal effect connected with a loss of virulence occurs after sublethal incubation times. The augmenting effect of ammonium chloride can be explained by the formation of monochloramine.
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http://dx.doi.org/10.1128/AAC.00957-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4576043PMC
October 2015

Galleria mellonella as a host model to study Aspergillus terreus virulence and amphotericin B resistance.

Virulence 2015 24;6(6):591-8. Epub 2015 Jun 24.

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

The aim of this study was to investigate if the alternative in vivo model Galleria mellonella can be used (i) to determine differences in pathogenicity of amphotericin B (AMB) resistant and susceptible A. terreus isolates, (ii) to evaluate AMB efficacy in vivo (iii) and to correlate outcome to in vitro susceptibility data. Larvae were infected with 2 A. terreus AMB resistant (ATR) and 3 AMB susceptible (ATS) isolates and survival rates were correlated to physiological attributes and killing ability of larval haemocytes. Additionally, infected larvae were treated with different concentrations of L-AMB. Haemocyte density were ascertained to evaluate the influence of L-AMB on the larval immune cells. Larvae were sensitive to A. terreus infection in an inoculum-size and temperature dependent manner. In vitro susceptibility to L-AMB correlated with in vivo outcome of antifungal treatment, defining an AMB susceptible strain cluster of A. terreus. Susceptibility to L-AMB increased virulence potential in the larval model, but this increase was also in accordance with faster growth and less damage caused by larval haemocytes. L-AMB treatment primed the larval immune response by increasing haemocyte density. G. mellonella provides a convenient model for the in vivo screening of A. terreus virulence and treatment options, contributing to the generation of a hypothesis that can be further tested in refined experiments in mammalian models.
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http://dx.doi.org/10.1080/21505594.2015.1045183DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4720272PMC
June 2016

Adaptation to thermotolerance in Rhizopus coincides with virulence as revealed by avian and invertebrate infection models, phylogeny, physiological and metabolic flexibility.

Virulence 2015 ;6(4):395-403

a Jena Microbial Resource Collection ; Jena , Germany.

Mucormycoses are fungal infections caused by the ancient Mucorales. They are rare, but increasingly reported. Predisposing conditions supporting and favoring mucormycoses in humans and animals include diabetic ketoacidosis, immunosuppression and haematological malignancies. However, comprehensive surveys to elucidate fungal virulence in ancient fungi are limited and so far focused on Lichtheimia and Mucor. The presented study focused on one of the most important causative agent of mucormycoses, the genus Rhizopus (Rhizopodaceae). All known clinically-relevant species are thermotolerant and are monophyletic. They are more virulent compared to non-clinically, mesophilic species. Although adaptation to elevated temperatures correlated with the virulence of the species, mesophilic strains showed also lower virulence in Galleria mellonella incubated at permissive temperatures indicating the existence of additional factors involved in the pathogenesis of clinical Rhizopus species. However, neither specific adaptation to nutritional requirements nor stress resistance correlated with virulence, supporting the idea that Mucorales are predominantly saprotrophs without a specific adaptation to warm blooded hosts.
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http://dx.doi.org/10.1080/21505594.2015.1029219DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4604701PMC
March 2016

Amphotericin B Resistance in Aspergillus terreus Is Overpowered by Coapplication of Pro-oxidants.

Antioxid Redox Signal 2015 Dec 8;23(18):1424-38. Epub 2015 Oct 8.

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

Aims: Invasive fungal infections have significantly increased over the past decades in immunocompromised individuals and high-risk patients. Amphotericin B (AmB) exerts a powerful and broad activity against a vast array of fungi and has a remarkably low rate of microbial resistance. However, most isolates of Aspergillus terreus developed an intrinsic resistance against AmB, and during this study, we characterized the mode of action of this polyene antifungal drug in more detail in resistant (ATR) and rare susceptible (ATS) clinical isolates of A. terreus.

Results: We illustrate that AmB treatment changes cellular redox status and promotes the generation of high levels of reactive oxygen species (ROS) in ATS. In contrast, ATR isolates were able to cope better with AmB-induced oxidative stress.

Innovation: Most importantly, we demonstrate in this study that coapplication of anti- and pro-oxidants significantly affects AmB efficacy in an antithetic manner--antioxidants and ROS-scavenging agents increase AmB tolerance in susceptible strains, while pro-oxidants render formerly resistant isolates considerably susceptible to the antifungal drug also in vivo in a Galleria animal model.

Conclusion: Thereby, our study provides novel therapeutic options to treat formerly resistant fungal strains by a combination of AmB and pro-oxidant compounds.
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http://dx.doi.org/10.1089/ars.2014.6220DOI Listing
December 2015

Fungal sterol C22-desaturase is not an antimycotic target as shown by selective inhibitors and testing on clinical isolates.

Steroids 2015 Sep 27;101:1-6. Epub 2015 May 27.

Department of Pharmacy - Center for Drug Research, Ludwig-Maximilians University of Munich, Butenandtstr. 5-13, 81377 Munich, Germany.

Inhibition of concise enzymes in ergosterol biosynthesis is one of the most prominent strategies for antifungal chemotherapy. Nevertheless, the enzymes sterol C5-desaturase and sterol C22-desaturase, which introduce double bonds into the sterol core and side chain, have not been fully investigated yet for their potential as antifungal drug targets. Lathosterol side chain amides bearing N-alkyl groups of proper length are known as potent inhibitors of the enzymes sterol C5-desaturase and sterol Δ(24)-reductase in mammalian cholesterol biosynthesis. Here we present the results of our evaluation of these amides for their ability to inhibit enzymes in fungal ergosterol biosynthesis. In the presence of inhibitor(s) an accumulation of sterols lacking a double bond at C22/23 (mainly ergosta-5,7-dien-3β-ol) was observed in Candida glabrata, Saccharomyces cerevisiae, and Yarrowia lipolytica. Hence, the lathosterol side chain amides were identified as selective inhibitors of the fungal sterol C22-desaturase, which was discussed as a specific target for novel antifungals. One representative inhibitor, (3S,20S)-20-N-butylcarbamoylpregn-7-en-3β-ol was subjected to antifungal susceptibility testing on patient isolates according to modified EUCAST guidelines. But, the test organisms showed no significant reduction of cell growth and/or viability up to an inhibitor concentration of 100μg/mL. This leads to the conclusion that sterol C22-desaturase is not an attractive target for the development of antifungals.
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http://dx.doi.org/10.1016/j.steroids.2015.05.004DOI Listing
September 2015

AmcA-a putative mitochondrial ornithine transporter supporting fungal siderophore biosynthesis.

Front Microbiol 2015 7;6:252. Epub 2015 Apr 7.

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

Iron is an essential nutrient required for a wide range of cellular processes. The opportunistic fungal pathogen Aspergillus fumigatus employs low-molecular mass iron-specific chelators, termed siderophores, for uptake, storage and intracellular iron distribution, which play a crucial role in the pathogenicity of this fungus. Siderophore biosynthesis (SB) depends on coordination with the supply of its precursor ornithine, produced mitochondrially from glutamate or cytosolically via hydrolysis of arginine. In this study, we demonstrate a role of the putative mitochondrial transporter AmcA (AFUA_8G02760) in SB of A. fumigatus. Consistent with a role in cellular ornithine handling, AmcA-deficiency resulted in decreased cellular ornithine and arginine contents as well as decreased siderophore production on medium containing glutamine as the sole nitrogen source. In support, arginine and ornithine as nitrogen sources did not impact SB due to cytosolic ornithine availability. As revealed by Northern blot analysis, transcript levels of siderophore biosynthetic genes were unresponsive to the cellular ornithine level. In contrast to siderophore production, AmcA deficiency did only mildly decrease the cellular polyamine content, demonstrating cellular prioritization of ornithine use. Nevertheless, AmcA-deficiency increased the susceptibility of A. fumigatus to the polyamine biosynthesis inhibitor eflornithine, most likely due to the decreased ornithine pool. AmcA-deficiency decreased the growth rate particularly on ornithine as the sole nitrogen source during iron starvation and sufficiency, indicating an additional role in the metabolism and fitness of A. fumigatus, possibly in mitochondrial ornithine import. In the Galleria mellonella infection model, AmcA-deficiency did not affect virulence of A. fumigatus, most likely due to the residual siderophore production and arginine availability in this host niche.
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http://dx.doi.org/10.3389/fmicb.2015.00252DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4387927PMC
April 2015

Protein kinase A signaling and calcium ions are major players in PAF mediated toxicity against Aspergillus niger.

FEBS Lett 2015 May 14;589(11):1266-71. Epub 2015 Apr 14.

Biocenter, Division of Molecular Biology, Medical University of Innsbruck, Innrain 80, A-6020 Innsbruck, Austria. Electronic address:

The Penicillium chrysogenum antifungal protein PAF is toxic against potentially pathogenic Ascomycetes. We used the highly sensitive aequorin-expressing model Aspergillus niger to identify a defined change in cytoplasmic free Ca(2+) dynamics in response to PAF. This Ca(2+) signature depended on an intact positively charged lysine-rich PAF motif. By combining Ca(2+) measurements in A. niger mutants with deregulated cAMP/protein kinase A (PKA) signaling, we proved the interconnection of Ca(2+) perturbation and cAMP/PKA signaling in the mechanistic function of PAF. A deep understanding of the mode of action of PAF is an invaluable prerequisite for its future application as new antifungal drug.
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http://dx.doi.org/10.1016/j.febslet.2015.03.037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4424949PMC
May 2015

Blocking Hsp70 enhances the efficiency of amphotericin B treatment against resistant Aspergillus terreus strains.

Antimicrob Agents Chemother 2015 Jul 13;59(7):3778-88. Epub 2015 Apr 13.

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

The polyene antifungal amphotericin B (AmB) is widely used to treat life-threatening fungal infections. Even though AmB resistance is exceptionally rare in fungi, most Aspergillus terreus isolates exhibit an intrinsic resistance against the drug in vivo and in vitro. Heat shock proteins perform a fundamental protective role against a multitude of stress responses, thereby maintaining protein homeostasis in the organism. In this study, we elucidated the role of heat shock protein 70 (Hsp70) family members and compared resistant and susceptible A. terreus clinical isolates. The upregulation of cytoplasmic Hsp70 members at the transcriptional as well as translational levels was significantly higher with AmB treatment than without AmB treatment, particularly in resistant A. terreus isolates, thereby indicating a role of Hsp70 proteins in the AmB response. We found that Hsp70 inhibitors considerably increased the susceptibility of resistant A. terreus isolates to AmB but exerted little impact on susceptible isolates. Also, in in vivo experiments, using the Galleria mellonella infection model, cotreatment of resistant A. terreus strains with AmB and the Hsp70 inhibitor pifithrin-μ resulted in significantly improved survival compared with that achieved with AmB alone. Our results point to an important mechanism of regulation of AmB resistance by Hsp70 family members in A. terreus and suggest novel drug targets for the treatment of infections caused by resistant fungal isolates.
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http://dx.doi.org/10.1128/AAC.05164-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4468683PMC
July 2015
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