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Macrotermitine termites have domesticated fungi in the genus Termitomyces as their primary food source using predigested 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 employs not only a broad array of carbohydrate-active enzymes (CAZymes) but also a restricted set of oxidizing enzymes (manganese peroxidase, dye decolorization peroxidase, an unspecific peroxygenase, laccases, and aryl-alcohol oxidases) and Fenton chemistry for biomass degradation. We propose for the first time that Termitomyces induces hydroquinone-mediated Fenton chemistry (Fe2+ + H2O2 + H+ → Fe3+ + •OH + H2O) using a herein newly described 2-methoxy-1,4-dihydroxybenzene (2-MH2Q, compound 19)-based electron shuttle system to complement the enzymatic degradation pathways. This study provides a comprehensive depiction of how efficient biomass degradation by means of this ancient insects agricultural symbiosis is accomplished. IMPORTANCE Fungus-growing termites have optimized 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 codiversified 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 chemistry-based hydroquinone-catalyzed lignin degradation mechanism, to break down lignin-rich plant material. Insights into these efficient decomposition mechanisms reveal new sources of efficient ligninolytic agents applicable for energy generation from renewable sources.

Gliotoxin (GT), a secondary metabolite and virulence factor of the fungal pathogen Aspergillus fumigatus, suppresses innate immunity and supports the suppression of host immune responses. Recently, we revealed that GT blocks the formation of the chemotactic lipid mediator leukotriene (LT)B4 in activated human neutrophils and monocytes, and in rodents in vivo, by directly inhibiting LTA4 hydrolase. Here, we elucidated the impact of GT on LTB4 biosynthesis and the entire lipid mediator networks in human M1‐ and M2‐like monocyte‐derived macrophages (MDMs) and in human tissue‐resident alveolar macrophages. In activated M1‐MDMs with high capacities to generate LTs, the formation of LTB4 was effectively suppressed by GT, connected to attenuated macrophage phagocytic activity as well as human neutrophil movement and migration. In resting macrophages, especially in M1‐MDMs, GT elicited strong formation of prostaglandins, while bacterial exotoxins from Staphylococcus aureus evoked a broad spectrum of lipid mediator biosynthesis in both MDM phenotypes. We conclude that GT impairs functions of activated innate immune cells through selective suppression of LTB4 biosynthesis, while GT may also prime the immune system by provoking prostaglandin formation in macrophages. [ABSTRACT FROM AUTHOR]

Aspergillus fumigatus can cause invasive pulmonary aspergillosis (IPA). Fungicidal azoles and fungistatic caspofungin (CAS) are the first- and second-line therapies, respectively, used to treat IPA. Treatment of A. fumigatus with CAS or micafungin induces the production of the oxylipin 5,8-diHODE by the fungal oxygenase PpoA. For this article, we investigated the influence of ppo genes, which encode the fatty acid oxygenases responsible for oxylipin biosynthesis, on CAS tolerance. The influence of PpoA and PpoC on CAS tolerance is mediated by MpkA phosphorylation and protein kinase A (PKA) activity. RNAseq transcriptional profiling and the label-free quantitative proteomics of the ppoA and ppoC mutants showed that differentially expressed genes and proteins are related to secondary metabolites and carbohydrate metabolism. We also characterized two clinical isolates, CM7555 and IFM61407, which decrease and increase susceptibility to CAS, respectively. CM7555 does not exhibit increased oxylipin production in the presence of CAS but oxylipin induction upon CAS exposure is increased in IFM61407, suggesting that oxylipins are not the only mechanism involved in CAS tolerance in these isolates. Upon CAS exposure, CM7555 has higher MpkA phosphorylation and PKA activity than IFM61407. Our results reveal the different aspects and genetic determinants involved in A. fumigatus CAS tolerance. [ABSTRACT FROM AUTHOR]

Aspergillus fumigatus is a common airborne fungal pathogen of humans and a significant source of mortality in immunocompromised individuals. Here, we provide the most extensive cell wall proteome profiling to date of A. fumigatus resting conidia, the fungal morphotype pertinent to first contact with the host. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), we identified proteins within the conidial cell wall by hydrogen-fluoride (HF)-pyridine extraction and proteins exposed on the surface using a trypsin-shaving approach. One protein, designated conidial cell wall protein A (CcpA), was identified by both methods and was found to be nearly as abundant as hydrophobic rodlet layer-forming protein RodA. CcpA, an amphiphilic protein, like RodA, peaks in expression during sporulation on resting conidia. Despite high cell wall abundance, the cell surface structure of ΔccpA resting conidia appeared normal. However, trypsin shaving of ΔccpA conidia revealed novel surface-exposed proteins not detected on conidia of the wild-type strain. Interestingly, the presence of swollen ΔccpA conidia led to higher activation of neutrophils and dendritic cells than was seen with wild-type conidia and caused significantly less damage to epithelial cells in vitro In addition, virulence was highly attenuated when cortisone-treated, immunosuppressed mice were infected with ΔccpA conidia. CcpA-specific memory T cell responses were detectable in healthy human donors naturally exposed to A. fumigatus conidia, suggesting a role for CcpA as a structural protein impacting conidial immunogenicity rather than possessing a protein-intrinsic immunosuppressive effect. Together, these data suggest that CcpA serves as a conidial stealth protein by altering the conidial surface structure to minimize innate immune recognition.IMPORTANCE The mammalian immune system relies on recognition of pathogen surface antigens for targeting and clearance. In the absence of immune evasion strategies, pathogen clearance is rapid. In the case of Aspergillus fumigatus, the successful fungus must avoid phagocytosis in the lung to establish invasive infection. In healthy individuals, fungal spores are cleared by immune cells; however, in immunocompromised patients, clearance mechanisms are impaired. Here, using proteome analyses, we identified CcpA as an important fungal spore protein involved in pathogenesis. A. fumigatus lacking CcpA was more susceptible to immune recognition and prompt eradication and, consequently, exhibited drastically attenuated virulence. In infection studies, CcpA was required for virulence in infected immunocompromised mice, suggesting that it could be used as a possible immunotherapeutic or diagnostic target in the future. In summary, our report adds a protein to the list of those known to be critical to the complex fungal spore surface environment and, more importantly, identifies a protein important for conidial immunogenicity during infection.

BACKGROUND. The fungus Aspergillus fumigatus causes a variety of clinical phenotypes in patients with cystic fibrosis (pwCF). Th cells orchestrate immune responses against fungi, but the types of A. fumigatus-specific Th cells in pwCF and their contribution to protective immunity or inflammation remain poorly characterized. METHODS. We used antigen-reactive T cell enrichment (ARTE) to investigate fungus-reactive Th cells in peripheral blood of pwCF and healthy controls. RESULTS. We show that clonally expanded, high-avidity A. fumigatus-specific effector Th cells, which were absent in healthy donors, developed in pwCF. Individual patients were characterized by distinct Th1-, Th2-, or Th17- dominated responses that remained stable over several years. These different Th subsets target different A. fumigatus proteins, indicating that differential antigen uptake and presentation directs Th cell subset development. Patients with allergic bronchopulmonary aspergillosis (ABPA) are characterized by high frequencies of Th2 cells that cross- recognize various filamentous fungi. CONCLUSION. Our data highlight the development of heterogenous Th responses targeting different protein fractions of a single fungal pathogen and identify the development of multispecies cross- reactive Th2 cells as a potential risk factor for ABPA. FUNDING. German Research Foundation (DFG), under Germany's Excellence Strategy (EXC 2167-390884018 'Precision Medicine in Chronic Inflammation' and EXC 2051-390713860 'Balance of the Microverse'); Oskar Helene Heim Stiftung; Christiane Herzog Stiftung; Mukoviszidose Institut gGmb; German Cystic Fibrosis Association Mukoviszidose e.V; German Federal Ministry of Education and Science (BMBF) InfectControl 2020 Projects AnDiPath (BMBF 03ZZ0838A+B)., Introduction Humans are constantly exposed to ubiquitous airborne fungi such as Aspergillus fumigatus. Despite this continuous challenge, healthy individuals rarely develop Aspergillus-associated diseases. This is likely due to rapid clearing [...]

Extracellular vesicles (EVs) have gained attention as facilitators of intercellular as well as interkingdom communication during host–microbe interactions. Recently we showed that upon infection, host polymorphonuclear leukocytes produce antifungal EVs targeting the clinically important fungal pathogen Aspergillus fumigatus ; however, the small size of EVs (<1 µm) complicates their functional analysis. Here, we employed a more tractable, reporter-based system to label host alveolar epithelial cell-derived EVs and enable their visualization during in vitro A. fumigatus interaction. Fusion of EV marker proteins (CD63, CD9, and CD81) with a Nanoluciferase (NLuc) and a green fluorescent protein (GFP) facilitated their relative quantification by luminescence and visualization by a fluorescence signal. The use of an NLuc fused with a GFP is advantageous as it allows for quantification and visualization of EVs simultaneously without additional external manipulation and to distinguish subpopulations of EVs. Using this system, visualization and tracking of EVs was possible using confocal laser scanning microscopy and advanced imaging analysis. These experiments revealed the propensity of host cell-derived EVs to associate with the fungal cell wall and ultimately colocalize with the cell membrane of A. fumigatus hyphae in large numbers. In conclusion, we have created a series of tools to better define the complex interplay of host-derived EVs with microbial pathogens. [ABSTRACT FROM AUTHOR]

Cytolytic proteins and peptide toxins are classical virulence factors of several bacterial pathogens which disrupt epithelial barrier function, damage cells and activate or modulate host immune responses. Such toxins have not been identified previously in human pathogenic fungi. Here we identify the first, to our knowledge, fungal cytolytic peptide toxin in the opportunistic pathogen Candida albicans. This secreted toxin directly damages epithelial membranes, triggers a danger response signalling pathway and activates epithelial immunity. Membrane permeabilization is enhanced by a positive charge at the carboxy terminus of the peptide, which triggers an inward current concomitant with calcium influx. C. albicans strains lacking this toxin do not activate or damage epithelial cells and are avirulent in animal models of mucosal infection. We propose the name 'Candidalysin' for this cytolytic peptide toxin; a newly identified, critical molecular determinant of epithelial damage and host recognition of the clinically important fungus, C. albicans., The ability of mucosal surfaces to discriminate between commensal and pathogenic microbes is essential to human health. The fungus Candida albicans is normally a benign member of the human microbiota, [...]