Biology subjects

Brakhage, A. A.

Publications and source records attributed to Brakhage, A. A..

26 records · Page 2Linked to original sources

bioRxiv2021

Dynamic optimization reveals alveolar epithelial cells as key mediators of host defense in invasive aspergillosis

Aspergillus fumigatus is an important human fungal pathogen and its conidia are constantly inhaled by humans. In immunocompromised individuals, conidia can grow out as hyphae that damage lung epithelium. The resulting invasive aspergillosis is associated with devastating mortality rates. Since infection is a race between the innate immune system and the outgrowth of A. fumigatus conidia, we use dynamic optimization to obtain insight into the recruitment and depletion of alveolar macrophages and neutrophils. Using this model, we obtain key insights into major determinants of infection outcome on host and pathogen side. On the pathogen side, we predict in silico and confirm in vitro that germination speed is a key virulence trait of fungal pathogens due to the vulnerability of conidia against host defense. On the host side, we find that epithelial cells play a so far underappreciated role in fungal clearance and are potent mediators of cytokine release which we confirm ex vivo. Further, our model affirms the importance of neutrophils in invasive aspergillosis and underlines that the role of macrophages remains elusive. We expect that our model will contribute to improvement of treatment protocols by focusing on the critical components of immune response to fungi but also fungal virulence.

systems biology↗

bioRxiv2021

The termite fungal cultivar Termitomyces combines diverse enzymes and oxidative reactions for plant biomass conversion

Macrotermitine termites have domesticated fungi in the genus Termitomyces as their primary food source using pre-digested plant biomass. To access the full nutritional value of lignin-enriched plant biomass, the termite-fungus symbiosis requires the depolymerization of this complex phenolic polymer. While most previous work suggests that lignocellulose degradation is accomplished predominantly by the fungal cultivar, our current understanding of the underlying biomolecular mechanisms remains rudimentary. Here, we provide conclusive OMICs and activity-based evidence that Termitomyces partially depolymerizes lignocellulose through the combined actions of high-redox potential oxidizing enzymes (laccases, aryl-alcohol oxidases and a manganese peroxidase), the production of extracellular H2O2 and Fenton-based oxidative degradation, which is catalyzed by a newly described 2-methoxybenzoquinone/hydroquinone redox shuttle system and mediated by secreted chelating dicarboxylic acids. In combination, our approaches reveal a comprehensive depiction of how the efficient biomass degradation mechanism in this ancient insect agricultural symbiosis is accomplished through a combination of white- and brown-rot mechanisms. ImportanceFungus-growing termites have perfected the decomposition of recalcitrant plant biomass to access valuable nutrients by engaging in a tripartite symbiosis with complementary contributions from a fungal mutualist and a co-diversified gut microbiome. This complex symbiotic interplay makes them one of the most successful and important decomposers for carbon cycling in Old World ecosystems. To date, most research has focused on the enzymatic contributions of microbial partners to carbohydrate decomposition. Here we provide genomic, transcriptomic and enzymatic evidence that Termitomyces also employs redox mechanisms, including diverse ligninolytic enzymes and a Fenton-based hydroquinone-catalyzed lignin-degradation mechanism, to break down lignin-rich plant material. Insights into these efficient decomposition mechanisms open new sources of efficient ligninolytic agents applicable for energy generation from renewable sources.

microbiology↗

bioRxiv2020

Aspergillus fumigatus versus Genus Aspergillus: Conservation, adaptive evolution and specific virulence genes

Aspergillus is an important fungal genus containing economically important species, as well as pathogenic species of animals and plants. Using eighteen fungal species of the genus Aspergillus, we conducted a comprehensive investigation of conserved genes and their evolution. This also allows to investigate the selection pressure driving the adaptive evolution in the pathogenic species A. fumigatus. Among single-copy orthologs (SCOs) for A. fumigatus and the closely related species A. fischeri, we identified 122 versus 50 positively selected genes (PSGs), respectively. Moreover, twenty conserved genes of unknown function were established to be clearly positively selected and thus important for adaption. A. fumigatus PSGs interacting with human host proteins show over-representation of adaptive, symbiosis-related, immunomodulatory, and virulence related pathways such as the TGF-{beta} pathway, insulin receptor signaling, IL1 pathway and interfering with phagosomal of GTPase signaling. Additionally, among the virulence factor coding genes, secretory and membrane protein coding genes in multi-copy gene families, 212 genes underwent positive selection and also suggest increased adaptation such as fungal immune evasion mechanisms (aspf2), siderophore biosynthesis (sidD), fumarylalanine production (sidE), stress tolerance (atfA) and thermotolerance (sodA). These genes presumably contribute to host adaptation strategies. Genes for the biosynthesis of gliotoxin are shared among all the close relatives of A. fumigatus as ancient defense mechanism. Positive selection plays a crucial role in the adaptive evolution of A. fumigatus. The genome-wide profile of PSGs provides valuable targets for further research on the mechanisms of immune evasion, for antimycotic targeting and understanding fundamental processes of virulence.

bioinformatics↗

bioRxiv2020

Biotinylated surfome profiling identifies potential biomarkers for diagnosis and therapy of Aspergillus fumigatus infection

Aspergillus fumigatus is one of the most common airborne fungi capable of causing invasive mycoses in immunocompromised patients and allergic diseases in susceptible individuals. In both cases, 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 to study the surface proteome (surfome) of A. fumigatus, like trypsin shaving, glucanase treatment, or formic acid extraction. Biotinylation coupled with 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 314 different surface proteins, including several well-known proteins like RodA, CcpA, and DppV, as well as several allergens, heat shock proteins (HSPs), and previously undescribed surface proteins. Using immunofluorescence microscopy, we confirmed the surface localization of three HSPs, which may have moonlighting functions. Collectively, our study generated a comprehensive data set of the A. fumigatus surfome, which complements already existing A. fumigatus surface proteome data and allows us to propose a common core set of A. fumigatus surface proteins. In addition, our study uncovers the surface-exposed regions of many proteins on the surface of spores 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, the comprehensive datasets provided and compiled here represent reasonable immunotherapy and diagnostic targets for future investigations. HIGHLIGHTSO_LISurface protein biotinylation coupled with LC-MS/MS analysis provides a comprehensive dataset of the A. fumigatus surface proteome. C_LIO_LI314 different A. fumigatus proteins (including immunoreactive proteins, and virulence factors) with surface exposed regions were detected. C_LIO_LISurface localization of three Hsp70 chaperones was confirmed by protein tagging coupled with immunofluorescence. C_LIO_LIBy comparison with other surfome datasets, a core surfome of A. fumigatus was defined, which provides possible biomarkers for diagnosis or therapy. C_LI SIGNIFICANCEAspergillus fumigatus is the most important airborne human pathogenic mold, capable of causing both life-threatening invasive pulmonary aspergillosis in immunocompromised patients and allergic infections in atopic individuals. Despite its obvious medical relevance, timely diagnosis and efficient antifungal treatment of A. fumigatus infection remains a major challenge. 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 A. fumigatus and other organisms, and sometimes even 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 A. fumigatus surface proteins with exposed regions, further defining putative targets for possible diagnostic and immunotherapeutic design.

microbiology↗

bioRxiv2020

The dynamic surface proteomes of allergenic fungal conidia

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 Aspergillus fumigatus conidia, respectively. We investigated the surface proteome under different conditions, including temperature variation and germination. We found that the surface proteome of resting A. fumigatus conidia is not static, but instead unexpectedly dynamic, as evidenced by drastically different surface proteomes under different growth conditions. Knockouts of two abundant A. fumigatus 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 A. fumigatus to other allergy-inducing molds, including Alternaria alternata, Penicillium rubens, and Cladosporium herbarum, 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 A. fumigatus. Ultimately, this study highlights the dynamic nature of the A. fumigatus conidial surface and provides targets for future diagnostics and immunotherapy.

microbiology↗

bioRxiv2019

Human neutrophils produce antifungal extracellular vesicles against Aspergillus fumigatus

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 Aspergillus fumigatus triggers PMNs to release a distinct set of antifungal EVs (afEVs). Proteome analyses indicated that afEVs are enriched in antimicrobial proteins. The cargo and 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. Two human proteins enriched in afEVs, cathepsin G and azurocidin, were heterologously expressed in fungal hyphae, which led to reduced fungal growth relative to a control retinol binding protein 7 producing strain. In conclusion, the production of afEVs by PMNs offers an intriguing, previously overlooked mechanism of antifungal defense against A. fumigatus. ImportanceInvasive fungal infections caused by the mold Aspergillus fumigatus 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 mortality as treatment and diagnostic options remain limited. In healthy individuals, neutrophilic granulocytes are critical for elimination of A. fumigatus 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 A. fumigatus not previously described. We find that extracellular vesicles produced by neutrophils in response to A. fumigatus 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 A. fumigatus host pathogenesis and opens up novel diagnostic and therapeutic possibilities.

microbiology↗

bioRxiv2019

Flotillin-dependent lipid-raft microdomains are required for functional phagolysosomes against fungal infections

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 the phagolysosomal membrane of macrophages. Genetic deletion of the lipidraft chaperons flotillin-1 and flotillin-2 demonstrate that the assembly of both major defense complexes vATPase and NADPH oxidase on the phagolysosomal membrane requires lipid rafts. Furthermore, we discovered a new virulence mechanism leading to the dysregulation of lipid-raft formation by melanized wild-type conidia of the important human-pathogenic fungus Aspergillus fumigatus. This results in reduced phagolysosomal acidification. Phagolysosomes with ingested melanized conidia contain a reduced amount of free Ca2+ ions as compared to phagolysosomes with melanin-free conidia. In agreement with a role of Ca2+ for generation of functional lipid rafts, we show that Ca2+-dependent calmodulin activity is required for lipid-raft formation on the phagolysosome. We identified a single nucleotide polymorphism in the human FLOT1 gene that results in heightened susceptibility for invasive aspergillosis in hematopoietic stem-cell transplant recipients. Collectively, flotillin-dependent lipid rafts on the phagolysosomal membrane play an essential role in protective antifungal immunity in humans.

microbiology↗

bioRxiv2017

Bacterium-triggered remodeling of chromatin identifies BasR, a novel regulator of fungal natural product biosynthesis

The eukaryotic epigenetic machinery is targeted by bacteria to reprogram the response of eukaryotes during their interaction with microorganisms. In line, we discovered that the bacterium Streptomyces rapamycinicus triggered increased chromatin acetylation and thus activation of the silent secondary metabolism ors gene cluster leading to the production of orsellinic acid in the fungus Aspergillus nidulans. Using this model we aim at understanding molecular mechanisms of communication between bacteria and eukaryotic microorganisms based on bacteria-triggered chromatin modification. By genome-wide ChIP-seq analysis of acetylated histone H3 (H3K9ac, H3K14ac) we uncovered the unique chromatin landscape in A. nidulans upon co-cultivation with S. rapamycinicus. Genome-wide acetylation of H3K9 correlated with increased gene expression, whereas H3K14 appears to function in transcriptional initiation by providing a docking side for regulatory proteins. In total, histones belonging to six secondary metabolism gene clusters showed higher acetylation during co-cultivation including the ors, aspercryptin, cichorine, sterigmatocystin, anthrone and 2,4-dihydroxy-3-methyl-6-(2-oxopropyl)benzaldehyde gene cluster with the emericellamide cluster being the only one with reduced acetylation and expression. Differentially acetylated histones were also detected in genes involved in amino acid and nitrogen metabolism, signaling, and genes encoding transcription factors. In conjunction with LC-MS/MS and MALDI-MS imaging, molecular analyses revealed the cross-pathway control and Myb-like transcription factor BasR as regulatory nodes for transduction of the bacterial signal in the fungus. The presence of basR in other fungal species allowed forecasting the inducibility of ors-like gene clusters by S. rapamycinicus in these fungi, and thus their effective interaction with activation of otherwise silent gene clusters.

microbiology↗