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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]

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]

The choanoflagellate Salpingoeca rosetta is an important model system to study the evolution of multicellularity. In this study we developed a new, modular, and scalable synthesis of sulfonolipid IOR‐1A (six steps, 27 % overall yield), which acts as bacterial inhibitor of rosette formation in S. rosetta. The synthesis features a decarboxylative cross‐coupling reaction of a sulfonic acid‐containing tartaric acid derivative with alkyl zinc reagents. Synthesis of 15 modified IOR‐1A derivatives, including fluorescent and photoaffinity‐based probes, allowed quantification of IOR‐1A, localization studies within S. rosetta cells, and evaluation of structure‐activity relations. In a proof of concept study, an inhibitory bifunctional probe was employed in proteomic profiling studies, which allowed to deduce binding partners in bacteria and S. rosetta. These results showcase the power of synthetic chemistry to decipher the biochemical basis of cell differentiation processes within S. rosetta. [ABSTRACT FROM AUTHOR]

Patients suffering from coronavirus disease-2019 (COVID-19) are susceptible to deadly secondary fungal infections such as COVID-19-associated pulmonary aspergillosis and COVID-19-associated mucormycosis. Despite this clinical observation, direct experimental evidence for severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2)-driven alterations of antifungal immunity is scarce. Using an ex-vivo whole blood stimulation assay, we challenged blood from twelve COVID-19 patients with Aspergillus fumigatus and Rhizopus arrhizus antigens and studied the expression of activation, maturation, and exhaustion markers, as well as cytokine secretion. Compared to healthy controls, T-helper cells from COVID-19 patients displayed increased expression levels of the exhaustion marker PD-1 and weakened A. fumigatus- and R. arrhizus-induced activation. While baseline secretion of proinflammatory cytokines was massively elevated, whole blood from COVID-19 patients elicited diminished release of T-cellular (e.g., IFN-g, IL-2) and innate immune cell-derived (e.g., CXCL9, CXCL10) cytokines in response to A. fumigatus and R. arrhizus antigens. Additionally, samples from COVID-19 patients showed deficient granulocyte activation by mold antigens and reduced fungal killing capacity of neutrophils. These features of weakened anti-mold immune responses were largely decoupled from COVID-19 severity, the time elapsed since diagnosis of COVID-19, and recent corticosteroid uptake, suggesting that impaired anti-mold defense is a common denominator of the underlying SARS-CoV-2 infection. Taken together, these results expand our understanding of the immune predisposition to post-viral mold infections and could inform future studies of immunotherapeutic strategies to prevent and treat fungal superinfections in COVID-19 patients. [ABSTRACT FROM AUTHOR]