Publications by authors named "Axel A Brakhage"

232 Publications

Discovery of fungal surface NADases predominantly present in pathogenic species.

Nat Commun 2021 03 12;12(1):1631. Epub 2021 Mar 12.

Department of Biomedicine, University of Bergen, Bergen, Norway.

Nicotinamide adenine dinucleotide (NAD) is a key molecule in cellular bioenergetics and signalling. Various bacterial pathogens release NADase enzymes into the host cell that deplete the host's NAD pool, thereby causing rapid cell death. Here, we report the identification of NADases on the surface of fungi such as the pathogen Aspergillus fumigatus and the saprophyte Neurospora crassa. The enzymes harbour a tuberculosis necrotizing toxin (TNT) domain and are predominately present in pathogenic species. The 1.6 Å X-ray structure of the homodimeric A. fumigatus protein reveals unique properties including N-linked glycosylation and a Ca-binding site whose occupancy regulates activity. The structure in complex with a substrate analogue suggests a catalytic mechanism that is distinct from those of known NADases, ADP-ribosyl cyclases and transferases. We propose that fungal NADases may convey advantages during interaction with the host or competing microorganisms.
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http://dx.doi.org/10.1038/s41467-021-21307-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7955114PMC
March 2021

Direct Visualization of Fungal Burden in Filamentous Fungus-Infected Silkworms.

J Fungi (Basel) 2021 Feb 13;7(2). Epub 2021 Feb 13.

Interdisciplinary Center for Clinical Research Laboratory, Department of Internal Medicine II, Würzburg University Hospital, 97080 Würzburg, Germany.

Invasive fungal infections (IFIs) are difficult to diagnose and to treat and, despite several available antifungal drugs, cause high mortality rates. In the past decades, the incidence of IFIs has continuously increased. More recently, SARS-CoV-2-associated lethal IFIs have been reported worldwide in critically ill patients. Combating IFIs requires a more profound understanding of fungal pathogenicity to facilitate the development of novel antifungal strategies. Animal models are indispensable for studying fungal infections and to develop new antifungals. However, using mammalian animal models faces various hurdles including ethical issues and high costs, which makes large-scale infection experiments extremely challenging. To overcome these limitations, we optimized an invertebrate model and introduced a simple calcofluor white (CW) staining protocol to macroscopically and microscopically monitor disease progression in silkworms () infected with the human pathogenic filamentous fungi and . This advanced silkworm infection model could validate knockout mutants with either attenuated, strongly attenuated or unchanged virulence. Finally, CW staining allowed us to efficiently visualize antifungal treatment outcomes in infected silkworms. Conclusively, we here present a powerful animal model combined with a straightforward staining protocol to expedite large-scale in vivo research of fungal pathogenicity and to investigate novel antifungal candidates.
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http://dx.doi.org/10.3390/jof7020136DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7918154PMC
February 2021

Isolation and immunofluorescence staining of conidia-containing phagolysosomes.

STAR Protoc 2021 Mar 5;2(1):100328. Epub 2021 Feb 5.

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

The analysis of phagolysosomes within professional phagocytic cells is facilitated by their isolation. Here, we optimized a protocol for the isolation of intact phagolysosomes from macrophages infected with the spores of . Purified phagolysosomes allow improved immunostaining, e.g., of phagolysosomal membrane proteins, or proteome analysis. For complete details on the use and execution of this protocol, please refer to Schmidt et al. (2020).
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http://dx.doi.org/10.1016/j.xpro.2021.100328DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7868627PMC
March 2021

Carbon Catabolite Repression in Filamentous Fungi Is Regulated by Phosphorylation of the Transcription Factor CreA.

mBio 2021 01 5;12(1). Epub 2021 Jan 5.

Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Ribeirao Preto, Brazil

Filamentous fungi of the genus are of particular interest for biotechnological applications due to their natural capacity to secrete carbohydrate-active enzymes (CAZy) that target plant biomass. The presence of easily metabolizable sugars such as glucose, whose concentrations increase during plant biomass hydrolysis, results in the repression of CAZy-encoding genes in a process known as carbon catabolite repression (CCR), which is undesired for the purpose of large-scale enzyme production. To date, the CH transcription factor CreA has been described as the major CC repressor in spp., although little is known about the role of posttranslational modifications in this process. In this work, phosphorylation sites were identified by mass spectrometry on CreA, and subsequently, the previously identified but uncharacterized site S262, the characterized site S319, and the newly identified sites S268 and T308 were chosen to be mutated to nonphosphorylatable residues before their effect on CCR was investigated. Sites S262, S268, and T308 are important for CreA protein accumulation and cellular localization, DNA binding, and repression of enzyme activities. In agreement with a previous study, site S319 was not important for several here-tested phenotypes but is key for CreA degradation and induction of enzyme activities. All sites were shown to be important for glycogen and trehalose metabolism. This study highlights the importance of CreA phosphorylation sites for the regulation of CCR. These sites are interesting targets for biotechnological strain engineering without the need to delete essential genes, which could result in undesired side effects. In filamentous fungi, the transcription factor CreA controls carbohydrate metabolism through the regulation of genes encoding enzymes required for the use of alternative carbon sources. In this work, phosphorylation sites were identified on CreA, and subsequently, the two newly identified sites S268 and T308, the previously identified but uncharacterized site S262, and the previously characterized site S319 were chosen to be mutated to nonphosphorylatable residues before their effect on CCR was characterized. Sites S262, S268, and T308 are important for CreA protein accumulation and cellular localization, DNA binding, and repression of enzyme activities. In agreement with a previous study, site S319 is not important for several here-tested phenotypes but is key for CreA degradation and induction of enzyme activities. This work characterized novel CreA phosphorylation sites under carbon catabolite-repressing conditions and showed that they are crucial for CreA protein turnover, control of carbohydrate utilization, and biotechnologically relevant enzyme production.
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http://dx.doi.org/10.1128/mBio.03146-20DOI Listing
January 2021

Fungal iron homeostasis with a focus on Aspergillus fumigatus.

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

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

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

The Role of RodA-Conserved Cysteine Residues in the Conidial Surface Organization.

J Fungi (Basel) 2020 Aug 26;6(3). Epub 2020 Aug 26.

Molecular Mycology Unit, Institut Pasteur, CNRS-UMR-2000, 10098 Paris, France.

Immune inertness of conidia is attributed to its surface rodlet-layer made up of RodAp, characterized by eight conserved cysteine residues forming four disulfide bonds. Earlier, we showed that the conserved cysteine residue point (ccrp mutations result in conidia devoid of the rodlet layer. Here, we extended our study comparing the surface organization and immunoreactivity of conidia carrying ccrp-mutations with the deletion mutant (∆). Western blot analysis using anti-RodAp antibodies indicated the absence of RodAp in the cytoplasm of ccrpmutant conidia. Immunolabeling revealed differential reactivity to conidial surface glucans, the ccrp-mutant conidia preferentially binding to α-(1,3)-glucan, ∆ conidia selectively bound to β-(1,3)-glucan; the parental strain conidia showed negative labeling. However, permeability of ccrp-mutants and ∆ was similar to the parental strain conidia. Proteomic analyses of the conidial surface exposed proteins of the ccrp-mutants showed more similarities with the parental strain, but were significantly different from the ∆. Ccrp-mutant conidia were less immunostimulatory compared to ∆ conidia. Our data suggest that (i) the conserved cysteine residues are essential for the trafficking of RodAp and the organization of the rodlet layer on the conidial surface, and (ii) targeted point mutation could be an alternative approach to study the role of fungal cell-wall genes in host-fungal interaction.
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http://dx.doi.org/10.3390/jof6030151DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7558875PMC
August 2020

N-Heterocyclization in Gliotoxin Biosynthesis is Catalyzed by a Distinct Cytochrome P450 Monooxygenase.

Chembiochem 2021 Jan 5;22(2):336-339. Epub 2020 Oct 5.

Department of Biomolecular Chemistry, Leibniz Institute for Natural Product Research and Infection Biology (HKI), Beutenbergstrasse 11a, 07745, Jena, Germany.

Gliotoxin and related epidithiodiketopiperazines (ETP) from diverse fungi feature highly functionalized hydroindole scaffolds with an array of medicinally and ecologically relevant activities. Mutation analysis, heterologous reconstitution, and biotransformation experiments revealed that a cytochrome P450 monooxygenase (GliF) from the human-pathogenic fungus Aspergillus fumigatus plays a key role in the formation of the complex heterocycle. In vitro assays using a biosynthetic precursor from a blocked mutant showed that GliF is specific to ETPs and catalyzes an unprecedented heterocyclization reaction that cannot be emulated with current synthetic methods. In silico analyses indicate that this rare biotransformation takes place in related ETP biosynthetic pathways.
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http://dx.doi.org/10.1002/cbic.202000550DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7891397PMC
January 2021

Biotinylated Surfome Profiling Identifies Potential Biomarkers for Diagnosis and Therapy of Aspergillus fumigatus Infection.

mSphere 2020 08 12;5(4). Epub 2020 Aug 12.

Department of Molecular and Applied Microbiology, Leibniz Institute for Natural Product Research and Infection Biology-Hans Knöll Institute, Jena, Germany

is one of the most common airborne molds capable of causing mycoses and allergies in humans. During infection, fungal surface proteins mediate the first contact with the human immune system to evade immune responses or to induce hypersensitivity. Several methods have been established for surface proteomics (surfomics). Biotinylation coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) identification of peptides is a particularly efficient method to identify the surface-exposed regions of proteins that potentially mediate interaction with the host. After biotinylation of surface proteins during spore germination, we detected 231 different biotinylated surface proteins (including several well-known proteins such as RodA, CcpA, and DppV; allergens; and heat shock proteins [HSPs]), as well as some previously undescribed surface proteins. The dynamic change of the surface proteome was illustrated by detection of a relatively high number of proteins exclusively at one developmental stage. Using immunofluorescence microscopy, we confirmed the surface localization of several HSPs of the HSP70 family, which may have moonlighting functions. Collectively, by comparing our data with data representative of previously published surface proteomes, our study generated a comprehensive data set corresponding to the surfome and uncovered the surface-exposed regions of many proteins on the surface of conidia or hyphae. These surface-exposed regions are candidates for direct interaction with host cells and may represent antigenic epitopes that either induce protective immune responses or mediate immune evasion. Thus, our data sets provided and compiled here represent reasonable immunotherapy and diagnostic targets for future investigations. is the most important airborne human-pathogenic mold, capable of causing both life-threatening invasive pulmonary aspergillosis in immunocompromised patients and allergy-inducing infections in individuals with atopic allergy. Despite its obvious medical relevance, timely diagnosis and efficient antifungal treatment of infection remain major challenges. Proteins on the surface of conidia (asexually produced spores) and mycelium directly mediate host-pathogen interaction and also may serve as targets for diagnosis and immunotherapy. However, the similarity of protein sequences between and other organisms, sometimes even including the human host, makes selection of targets for immunological-based studies difficult. Here, using surface protein biotinylation coupled with LC-MS/MS analysis, we identified hundreds of surface proteins with exposed regions, further defining putative targets for possible diagnostic and immunotherapeutic design.
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http://dx.doi.org/10.1128/mSphere.00535-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7426169PMC
August 2020

Flotillin-Dependent Membrane Microdomains Are Required for Functional Phagolysosomes against Fungal Infections.

Cell Rep 2020 08;32(7):108017

Department of Molecular and Applied Microbiology, Leibniz Institute for Natural Product Research and Infection Biology - Hans Knöll Institute (HKI), 07745 Jena, Germany; Department of Microbiology and Molecular Biology, Institute of Microbiology, Friedrich Schiller University Jena, 07745 Jena, Germany. Electronic address:

Lipid rafts form signaling platforms on biological membranes with incompletely characterized role in immune response to infection. Here we report that lipid-raft microdomains are essential components of phagolysosomal membranes of macrophages and depend on flotillins. Genetic deletion of flotillins demonstrates that the assembly of both major defense complexes vATPase and NADPH oxidase requires membrane microdomains. Furthermore, we describe a virulence mechanism leading to dysregulation of membrane microdomains by melanized wild-type conidia of the important human-pathogenic fungus Aspergillus fumigatus resulting in reduced phagolysosomal acidification. We show that phagolysosomes with ingested melanized conidia contain a reduced amount of free Ca ions and that inhibition of Ca-dependent calmodulin activity led to reduced lipid-raft formation. We identify a single-nucleotide polymorphism in the human FLOT1 gene resulting in heightened susceptibility for invasive aspergillosis in hematopoietic stem cell transplant recipients. Collectively, flotillin-dependent microdomains on the phagolysosomal membrane play an essential role in protective antifungal immunity.
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http://dx.doi.org/10.1016/j.celrep.2020.108017DOI Listing
August 2020

Lichen-like association of Chlamydomonas reinhardtii and Aspergillus nidulans protects algal cells from bacteria.

ISME J 2020 11 4;14(11):2794-2805. Epub 2020 Aug 4.

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

Organismal interactions within microbial consortia and their responses to harmful intruders remain largely understudied. An important step toward the goal of understanding functional ecological interactions and their evolutionary selection is the study of increasingly complex microbial interaction systems. Here, we discovered a tripartite biosystem consisting of the fungus Aspergillus nidulans, the unicellular green alga Chlamydomonas reinhardtii, and the algicidal bacterium Streptomyces iranensis. Genetic analyses and MALDI-IMS demonstrate that the bacterium secretes the algicidal compound azalomycin F upon contact with C. reinhardtii. In co-culture, A. nidulans attracts the motile alga C. reinhardtii, which becomes embedded and surrounded by fungal mycelium and is shielded from the algicide. The filamentous fungus Sordaria macrospora was susceptible to azalomycin F and failed to protect C. reinhardtii despite chemotactically attracting the alga. Because S. macrospora was susceptible to azalomycin F, this data imply that for protection the fungus needs to be resistant. Formation of the lichen-like association between C. reinhardtii and A. nidulans increased algal growth. The protection depends on the increased amounts of membrane lipids provided by resistant fungi, thereby generating a protective shelter against the bacterial toxin. Our findings reveal a strategy whereby algae survive lethal environmental algicides through cooperation with fungi.
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http://dx.doi.org/10.1038/s41396-020-0731-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7784976PMC
November 2020

Functional surface proteomic profiling reveals the host heat-shock protein A8 as a mediator of Lichtheimia corymbifera recognition by murine alveolar macrophages.

Environ Microbiol 2020 09 21;22(9):3722-3740. Epub 2020 Jul 21.

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

Mucormycosis is an emergent, fatal fungal infection of humans and warm-blooded animals caused by species of the order Mucorales. Immune cells of the innate immune system serve as the first line of defence against inhaled spores. Alveolar macrophages were challenged with the mucoralean fungus Lichtheimia corymbifera and subjected to biotinylation and streptavidin enrichment procedures followed by LC-MS/MS analyses. A total of 28 host proteins enriched for binding to macrophage-L. corymbifera interaction. Among those, the HSP70-family protein Hspa8 was found to be predominantly responsive to living and heat-killed spores of a virulent and an attenuated strain of L. corymbifera. Confocal scanning laser microscopy of infected macrophages revealed colocalization of Hspa8 with phagocytosed spores of L. corymbifera. The amount of detectable Hspa8 was dependent on the multiplicity of infection. Incubation of alveolar macrophages with an anti-Hspa8 antibody prior to infection reduced their capability to phagocytose spores of L. corymbifera. In contrast, anti-Hspa8 antibodies did not abrogate the phagocytosis of Aspergillus fumigatus conidia by macrophages. These results suggest an important contribution of the heat-shock family protein Hspa8 in the recognition of spores of the mucoralean fungus L. corymbifera by host alveolar macrophages and define a potential immunomodulatory therapeutic target.
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http://dx.doi.org/10.1111/1462-2920.15140DOI Listing
September 2020

The domestic pig as human-relevant large animal model to study adaptive antifungal immune responses against airborne Aspergillus fumigatus.

Eur J Immunol 2020 11 13;50(11):1712-1728. Epub 2020 Jul 13.

Institute of Immunology, Centre for Infection Medicine, Department of Veterinary Medicine, Freie Universität Berlin, Berlin, Germany.

Pulmonary mucosal immune response is critical for preventing opportunistic Aspergillus fumigatus infections. Although fungus-specific CD4 T cells in blood are described to reflect the actual host-pathogen interaction status, little is known about Aspergillus-specific pulmonary T-cell responses. Here, we exploit the domestic pig as human-relevant large animal model and introduce antigen-specific T-cell enrichment in pigs to address Aspergillus-specific T cells in the lung compared to peripheral blood. In healthy, environmentally Aspergillus-exposed pigs, the fungus-specific T cells are detectable in blood in similar frequencies as observed in healthy humans and exhibit a Th1 phenotype. Exposing pigs to 10 cfu/m conidia induces a long-lasting accumulation of Aspergillus-specific Th1 cells locally in the lung and also systemically. Temporary immunosuppression during Aspergillus-exposure showed a drastic reduction in the lung-infiltrating antifungal T-cell responses more than 2 weeks after abrogation of the suppressive treatment. This was reflected in blood, but to a much lesser extent. In conclusion, by using the human-relevant large animal model the pig, this study highlights that the blood clearly reflects the mucosal fungal-specific T-cell reactivity in environmentally exposed as well as experimentally exposed healthy pigs. But, immunosuppression significantly impacts the mucosal site in contrast to the initial systemic immune response.
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http://dx.doi.org/10.1002/eji.201948524DOI Listing
November 2020

Structural basis of HapE-linked antifungal triazole resistance in .

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

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

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

The Pheromone Module SteC-MkkB-MpkB-SteD-HamE Regulates Development, Stress Responses and Secondary Metabolism in .

Front Microbiol 2020 7;11:811. Epub 2020 May 7.

Department of Biology, Fungal Genetics and Secondary Metabolism Laboratory, Maynooth University, Maynooth, Ireland.

In order for eukaryotes to efficiently detect and respond to environmental stimuli, a myriad of protein signaling pathways are utilized. An example of highly conserved signaling pathways in eukaryotes are the mitogen-activated protein kinase (MAPK) pathways. In fungi, MAPK pathways have been shown to regulate a diverse array of biological processes, such as asexual and sexual development, stress responses and the production of secondary metabolites (SMs). In the model fungus , a MAPK pathway known as the pheromone module is utilized to regulate both development and SM production. This signaling cascade consists of the three kinases SteC, MkkB, and MpkB, as well as the SteD adaptor protein and the HamE scaffold. In this study, homologs of each of these proteins have been identified in the opportunistic human pathogen By performing epitope tagging and mass spectrometry experiments, we have shown that these proteins form a pentameric complex, similar to what is observed in This complex has been shown to assemble in the cytoplasm and MpkB enters the nucleus, where it would presumably interact with various transcription factors. Pheromone module mutant strains exhibit drastic reductions in asexual sporulation, vegetative growth rate and production of SMs, such as gliotoxin. Mutants also display increased sensitivity to cell wall and oxidative stress agents. Overall, these data provide evidence of the existence of a conserved MAP kinase signaling pathway in species and suggest that this pathway is critical for the regulation of fungal development and secondary metabolism.
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http://dx.doi.org/10.3389/fmicb.2020.00811DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7223695PMC
May 2020

Phagosomal removal of fungal melanin reprograms macrophage metabolism to promote antifungal immunity.

Nat Commun 2020 05 8;11(1):2282. Epub 2020 May 8.

Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, 4710-057, Braga, Portugal.

In response to infection, macrophages adapt their metabolism rapidly to enhance glycolysis and fuel specialized antimicrobial effector functions. Here we show that fungal melanin is an essential molecule required for the metabolic rewiring of macrophages during infection with the fungal pathogen Aspergillus fumigatus. Using pharmacological and genetic tools, we reveal a molecular link between calcium sequestration by melanin inside the phagosome and induction of glycolysis required for efficient innate immune responses. By remodeling the intracellular calcium machinery and impairing signaling via calmodulin, melanin drives an immunometabolic signaling axis towards glycolysis with activation of hypoxia-inducible factor 1 subunit alpha (HIF-1α) and phagosomal recruitment of mammalian target of rapamycin (mTOR). These data demonstrate a pivotal mechanism in the immunometabolic regulation of macrophages during fungal infection and highlight the metabolic repurposing of immune cells as a potential therapeutic strategy.
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http://dx.doi.org/10.1038/s41467-020-16120-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7210971PMC
May 2020

Ahr1 and Tup1 Contribute to the Transcriptional Control of Virulence-Associated Genes in Candida albicans.

mBio 2020 04 28;11(2). Epub 2020 Apr 28.

Research Group Fungal Septomics, Leibniz Institute for Natural Product Research and Infection Biology-Hans Knoell Institute, Jena, Germany

The capacity of to reversibly change its morphology between yeast and filamentous stages is crucial for its virulence. Formation of hyphae correlates with the upregulation of genes and , which are involved in pathogenicity processes such as invasion, iron acquisition, and host cell damage. The global repressor Tup1 and its cofactor Nrg1 are considered to be the main antagonists of hyphal development in However, our experiments revealed that Tup1, but not Nrg1, was required for full expression of and In contrast to , overexpression of was found to inhibit neither filamentous growth nor transcription of and In addition, we identified the transcription factor Ahr1 as being required for full expression of both genes. A hyperactive version of Ahr1 bound directly to the promoters of and and induced their transcription even in the absence of environmental stimuli. This regulation worked even in the absence of the crucial hyphal growth regulators Cph1 and Efg1 but was dependent on the presence of Tup1. Overall, our results show that Ahr1 and Tup1 are key contributors in the complex regulation of virulence-associated genes in the different morphologies. is a major human fungal pathogen and the leading cause of systemic infections. In recent years, Als3 and Ece1 were identified as important factors for fungal virulence. Transcription of both corresponding genes is closely associated with hyphal growth. Here, we describe how Tup1, normally a global repressor of gene expression as well as of filamentation, and the transcription factor Ahr1 contribute to full expression of and in hyphae. Both regulators are required for high mRNA amounts of the two genes to ensure functional relevant protein synthesis and localization. These observations identified a new aspect of regulation in the complex transcriptional control of virulence-associated genes in .
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http://dx.doi.org/10.1128/mBio.00206-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7188989PMC
April 2020

Human Neutrophils Produce Antifungal Extracellular Vesicles against Aspergillus fumigatus.

mBio 2020 04 14;11(2). Epub 2020 Apr 14.

Institute of Microbiology, Friedrich Schiller University, Jena, Germany

Polymorphonuclear granulocytes (PMNs) are indispensable for controlling life-threatening fungal infections. In addition to various effector mechanisms, PMNs also produce extracellular vesicles (EVs). Their contribution to antifungal defense has remained unexplored. We reveal that the clinically important human-pathogenic fungus triggers PMNs to release a distinct set of antifungal EVs (afEVs). Proteome analyses indicated that afEVs are enriched in antimicrobial proteins. The cargo and the release kinetics of EVs are modulated by the fungal strain confronted. Tracking of afEVs indicated that they associated with fungal cells and even entered fungal hyphae, resulting in alterations in the morphology of the fungal cell wall and dose-dependent antifungal effects. To assess as a proof of concept whether the antimicrobial proteins found in afEVs might contribute to growth inhibition of hyphae when present in the fungal cytoplasm, two human proteins enriched in afEVs, cathepsin G and azurocidin, were heterologously expressed in fungal hyphae. This led to reduced fungal growth relative to that of a control strain producing the human retinol binding protein 7. In conclusion, extracellular vesicles produced by neutrophils in response to infection are able to associate with the fungus, limit growth, and elicit cell damage by delivering antifungal cargo. This finding offers an intriguing, previously overlooked mechanism of antifungal defense against Invasive fungal infections caused by the mold are a growing concern in the clinic due to the increasing use of immunosuppressive therapies and increasing antifungal drug resistance. These infections result in high rates of mortality, as treatment and diagnostic options remain limited. In healthy individuals, neutrophilic granulocytes are critical for elimination of from the host; however, the exact extracellular mechanism of neutrophil-mediated antifungal activity remains unresolved. Here, we present a mode of antifungal defense employed by human neutrophils against not previously described. We found that extracellular vesicles produced by neutrophils in response to infection are able to associate with the fungus, limit growth, and elicit cell damage by delivering antifungal cargo. In the end, antifungal extracellular vesicle biology provides a significant step forward in our understanding of host pathogenesis and opens up novel diagnostic and therapeutic possibilities.
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http://dx.doi.org/10.1128/mBio.00596-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7157820PMC
April 2020

Caspofungin Functionalized Polymethacrylates with Antifungal Properties.

Biomacromolecules 2020 06 13;21(6):2104-2115. Epub 2020 May 13.

Jena Center for Soft Matter (JCSM), Friedrich Schiller University Jena, Philosophenweg 7, 07743 Jena, Germany.

We describe the synthesis of hydrophilic poly(poly(ethylene glycol) methyl ether methacrylate) (PmPEGMA) and hydrophobic poly(methyl methacrylate) (PMMA) caspofungin conjugates by a post-polymerization modification of copolymers containing 10 mol % pentafluorophenyl methacrylate (PFPMA), which were obtained via reversible addition-fragmentation chain transfer copolymerization. The coupling of the clinically used antifungal caspofungin was confirmed and quantified in detail by a combination of H-, F- and diffusion-ordered NMR spectroscopy, UV-vis spectroscopy, and size exclusion chromatography. The trifunctional amine-containing antifungal was attached via several amide bonds to the hydrophobic PMMA, but sterical hindrance induced by the mPEGMA side chains prohibited intramolecular double functionalization. Both polymer-drug conjugates revealed activity against important human-pathogenic fungi, that is, two strains of and one strain of (2.5 mg L < MEC < 8 mg L, MIC = 4 mg L), whereas RAW 264.7 macrophages as well as HeLa cells remained unaffected at these concentrations.
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http://dx.doi.org/10.1021/acs.biomac.0c00096DOI Listing
June 2020

Dynamic Surface Proteomes of Allergenic Fungal Conidia.

J Proteome Res 2020 May 14;19(5):2092-2104. Epub 2020 Apr 14.

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

Fungal spores and hyphal fragments play an important role as allergens in respiratory diseases. In this study, we performed trypsin shaving and secretome analyses to identify the surface-exposed proteins and secreted/shed proteins of conidia, respectively. We investigated the surface proteome under different conditions, including temperature variation and germination. We found that the surface proteome of resting conidia is not static but instead unexpectedly dynamic, as evidenced by drastically different surface proteomes under different growth conditions. Knockouts of two abundant surface proteins, ScwA and CweA, were found to function only in fine-tuning the cell wall stress response, implying that the conidial surface is very robust against perturbations. We then compared the surface proteome of to other allergy-inducing molds, including , , and , and performed comparative proteomics on resting and swollen conidia, as well as secreted proteins from germinating conidia. We detected 125 protein ortholog groups, including 80 with putative catalytic activity, in the extracellular region of all four molds, and 42 nonorthologous proteins produced solely by . Ultimately, this study highlights the dynamic nature of the conidial surface and provides targets for future diagnostics and immunotherapy.
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http://dx.doi.org/10.1021/acs.jproteome.0c00013DOI Listing
May 2020

Genome Sequence of Escherichia coli KI683, Isolated from a Urosepsis Patient.

Microbiol Resour Announc 2020 Feb 27;9(9). Epub 2020 Feb 27.

Leibniz Institute for Natural Product Research and Infection Biology-Hans Knöll Institute, Molecular and Applied Microbiology, Jena, Germany

KI683 was isolated from blood of a patient who developed septicemia as a complication of a urinary tract infection. Genome sequencing resulted in three contigs with a total genome size of 5,243,173 bp encoding 5,143 genes.
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http://dx.doi.org/10.1128/MRA.01297-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7046818PMC
February 2020

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

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

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

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

Targeted induction of a silent fungal gene cluster encoding the bacteria-specific germination inhibitor fumigermin.

Elife 2020 02 21;9. Epub 2020 Feb 21.

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

Microorganisms produce numerous secondary metabolites (SMs) with various biological activities. Many of their encoding gene clusters are silent under standard laboratory conditions because for their activation they need the ecological context, such as the presence of other microorganisms. The true ecological function of most SMs remains obscure, but understanding of both the activation of silent gene clusters and the ecological function of the produced compounds is of importance to reveal functional interactions in microbiomes. Here, we report the identification of an as-yet uncharacterized silent gene cluster of the fungus , which is activated by the bacterium during the bacterial-fungal interaction. The resulting natural product is the novel fungal metabolite fumigermin, the biosynthesis of which requires the polyketide synthase FgnA. Fumigermin inhibits germination of spores of the inducing and thus helps the fungus to defend resources in the shared habitat against a bacterial competitor.
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http://dx.doi.org/10.7554/eLife.52541DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7034978PMC
February 2020

Enolase From Is a Moonlighting Protein That Binds the Human Plasma Complement Proteins Factor H, FHL-1, C4BP, and Plasminogen.

Front Immunol 2019 22;10:2573. Epub 2019 Nov 22.

Department of Infection Biology, Leibniz Institute for Natural Product Research and Infection Biology, Hans Knöll Institute, Jena, Germany.

The opportunistic fungal pathogen can cause severe infections, particularly in immunocompromised individuals. Upon infection, faces the powerful and directly acting immune defense of the human host. The mechanisms on how evades innate immune attack and complement are still poorly understood. Here, we identify enolase, AfEno1, which was also characterized as fungal allergen, as a surface ligand for human plasma complement regulators. AfEno1 binds factor H, factor-H-like protein 1 (FHL-1), C4b binding protein (C4BP), and plasminogen. Factor H attaches to AfEno1 via two regions, via short conserved repeats (SCRs) 6-7 and 19-20, and FHL-1 contacts AfEno1 via SCRs 6-7. Both regulators when bound to AfEno1 retain cofactor activity and assist in C3b inactivation. Similarly, the classical pathway regulator C4BP binds to AfEno1 and bound to AfEno1; C4BP assists in C4b inactivation. Plasminogen which binds to AfEno1 via lysine residues is accessible for the tissue-type plasminogen activator (tPA), and active plasmin cleaves the chromogenic substrate S2251, degrades fibrinogen, and inactivates C3 and C3b. Plasmin attached to swollen conidia damages human A549 lung epithelial cells, reduces the cellular metabolic activity, and induces cell retraction, which results in exposure of the extracellular matrix. Thus, AfEno1 is a moonlighting protein and virulence factor which recruits several human regulators. The attached human regulators allow the fungal pathogen to control complement at the level of C3 and to damage endothelial cell layers and tissue components.
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http://dx.doi.org/10.3389/fimmu.2019.02573DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6883375PMC
November 2020

Towards Raman spectroscopy of urine as screening tool.

J Biophotonics 2020 01 19;13(1):e201900143. Epub 2019 Nov 19.

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

For the screening purposes urine is an especially attractive biofluid, since it offers easy and noninvasive sample collection and provides a snapshot of the whole metabolic status of the organism, which may change under different pathological conditions. Raman spectroscopy (RS) has the potential to monitor these changes and utilize them for disease diagnostics. The current study utilizes mouse models aiming to compare the feasibility of the urine based RS combined with chemometrics for diagnosing kidney diseases directly influencing urine composition and respiratory tract diseases having no direct connection to urine formation. The diagnostic models for included diseases were built using principal component analysis with linear discriminant analysis and validated with a leave-one-mouse-out cross-validation approach. Considering kidney disorders, the accuracy of 100% was obtained in discrimination between sick and healthy mice, as well as between two different kidney diseases. For asthma and invasive pulmonary aspergillosis achieved accuracies were noticeably lower, being, respectively, 77.27% and 78.57%. In conclusion, our results suggest that RS of urine samples not only provides a solution for a rapid, sensitive and noninvasive diagnosis of kidney disorders, but also holds some promises for the screening of nonurinary tract diseases.
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http://dx.doi.org/10.1002/jbio.201900143DOI Listing
January 2020

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

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

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

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

High-Throughput Gene Replacement in Aspergillus fumigatus.

Curr Protoc Microbiol 2019 09;54(1):e88

Manchester Fungal Infection Group, Division of Infection, Immunity and Respiratory Medicine, School of Biological Sciences, Faculty of Biology, Medicine and Health, Core Technology Facility, University of Manchester, Manchester, United Kingdom.

Aspergillus fumigatus is a human pathogen and the principal etiologic agent of invasive and chronic aspergillosis leading to several hundreds of thousands of deaths every year. Very few antifungals are available to treat infections caused by A. fumigatus, and resistance is developing to those we have. Our understanding of the molecular mechanisms that drive pathogenicity and drug resistance have been hampered by the lack of large mutant collections, which limits our ability to perform functional genomics analysis. Here we present a high-throughput gene knockout method that combines a highly reproducible fusion PCR method to enable generation of gene replacement cassettes with a multiwell format transformation procedure. This process can be used to generate 96 null mutants within 5 days by a single person at a cost of less than £18 ($24) per mutant and is being employed in our laboratory to generate a barcoded genome-wide knockout library in A. fumigatus. © 2019 The Authors.
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http://dx.doi.org/10.1002/cpmc.88DOI Listing
September 2019

Conidial surface proteins at the interface of fungal infections.

PLoS Pathog 2019 09 12;15(9):e1007939. Epub 2019 Sep 12.

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

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http://dx.doi.org/10.1371/journal.ppat.1007939DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6742213PMC
September 2019

Stress-Induced Changes in the Lipid Microenvironment of β-(1,3)-d-Glucan Synthase Cause Clinically Important Echinocandin Resistance in Aspergillus fumigatus.

mBio 2019 06 4;10(3). Epub 2019 Jun 4.

Center for Discovery and Innovation, Hackensack Meridian Health, Nutley, New Jersey, USA

is a leading cause of invasive fungal infections. Resistance to first-line triazole antifungals has led to therapy with echinocandin drugs. Recently, we identified several high-minimum-effective-concentration (MEC) clinical isolates from patients failing echinocandin therapy. Echinocandin resistance is known to arise from amino acid substitutions in β-(1,3)-d-glucan synthase encoded by the gene. Yet these clinical isolates did not contain mutations in , indicating an undefined resistance mechanism. To explore this new mechanism, we used a laboratory-derived strain, RG101, with a nearly identical caspofungin (CAS) susceptibility phenotype that also does not contain mutations. Glucan synthase isolated from RG101 was fully sensitive to echinocandins. Yet exposure of RG101 to CAS during growth yielded a modified enzyme that was drug insensitive (4 log orders) in kinetic inhibition assays, and this insensitivity was also observed for enzymes isolated from clinical isolates. To understand this alteration, we analyzed whole-enzyme posttranslational modifications (PTMs) but found none linked to resistance. However, analysis of the lipid microenvironment of the enzyme with resistance induced by CAS revealed a prominent increase in the abundances of dihydrosphingosine (DhSph) and phytosphingosine (PhSph). Exogenous addition of DhSph and PhSph to the sensitive enzyme recapitulated the drug insensitivity of the CAS-derived enzyme. Further analysis demonstrated that CAS induces mitochondrion-derived reactive oxygen species (ROS) and that dampening ROS formation by antimycin A or thiourea eliminated drug-induced resistance. We conclude that CAS induces cellular stress, promoting formation of ROS and triggering an alteration in the composition of plasma membrane lipids surrounding glucan synthase, rendering it insensitive to echinocandins. Resistance to first-line triazole antifungal agents among species has prompted the use of second-line therapy with echinocandins. As the number of -infected patients treated with echinocandins is rising, clinical observations of drug resistance are also increasing, indicating an emerging global health threat. Our knowledge regarding the development of clinical echinocandin resistance is largely derived from spp., while little is known about resistance in Therefore, it is important to understand the specific cellular responses raised by against echinocandins. We discovered a new mechanism of resistance in that is independent of the well-characterized mutation mechanism observed in This study identified an off-target effect of CAS, i.e., ROS production, and integrated oxidative stress and sphingolipid alterations into a novel mechanism of resistance. This stress-induced response has implications for drug resistance and/or tolerance mechanisms in other fungal pathogens.
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http://dx.doi.org/10.1128/mBio.00779-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6550521PMC
June 2019

Recreation of in-host acquired single nucleotide polymorphisms by CRISPR-Cas9 reveals an uncharacterised gene playing a role in Aspergillus fumigatus azole resistance via a non-cyp51A mediated resistance mechanism.

Fungal Genet Biol 2019 09 23;130:98-106. Epub 2019 May 23.

MRC Centre for Medical Mycology at the University of Aberdeen, Aberdeen Fungal Group, Institute of Medical Sciences, University of Aberdeen, UK. Electronic address:

The human host comprises a range of specific niche environments. In order to successfully persist, pathogens such as Aspergillus fumigatus must adapt to these environments. One key example of in-host adaptation is the development of resistance to azole antifungals. Azole resistance in A. fumigatus is increasingly reported worldwide and the most commonly reported mechanisms are cyp51A mediated. Using a unique series of A. fumigatus isolates, obtained from a patient suffering from persistent and recurrent invasive aspergillosis over 2 years, this study aimed to gain insight into the genetic basis of in-host adaptation. Single nucleotide polymorphisms (SNPs) unique to a single isolate in this series, which had developed multi-azole resistance in-host, were identified. Two nonsense SNPs were recreated using CRISPR-Cas9; these were 213 in svf1 and 167 in uncharacterised gene AFUA_7G01960. Phenotypic analyses including antifungal susceptibility testing, mycelial growth rate assessment, lipidomics analysis and statin susceptibility testing were performed to associate genotypes to phenotypes. This revealed a role for svf1 in A. fumigatus oxidative stress sensitivity. In contrast, recapitulation of 167 in AFUA_7G01960 resulted in increased itraconazole resistance. Comprehensive lipidomics analysis revealed decreased ergosterol levels in strains containing this SNP, providing insight to the observed itraconazole resistance. Decreases in ergosterol levels were reflected in increased resistance to lovastatin and nystatin. Importantly, this study has identified a SNP in an uncharacterised gene playing a role in azole resistance via a non-cyp51A mediated resistance mechanism. This mechanism is of clinical importance, as this SNP was identified in a clinical isolate, which acquired azole resistance in-host.
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http://dx.doi.org/10.1016/j.fgb.2019.05.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6876285PMC
September 2019