Human Anti-fungal Th17 Immunity and Pathology Rely on Cross-Reactivity against Candida albicans.

Th17 cells provide protection at barrier tissues but may also contribute to immune pathology. The relevance and induction mechanisms of pathologic Th17 responses in humans are poorly understood. Here, we identify the mucocutaneous pathobiont Candida albicans as the major direct inducer of human anti-fungal Th17 cells. Th17 cells directed against other fungi are induced by cross-reactivity to C. albicans. Intestinal inflammation expands total C. albicans and cross-reactive Th17 cells. Strikingly, Th17 cells cross-reactive to the airborne fungus Aspergillus fumigatus are selectively activated and expanded in patients with airway inflammation, especially during acute allergic bronchopulmonary aspergillosis. This indicates a direct link between protective intestinal Th17 responses against C. albicans and lung inflammation caused by airborne fungi. We identify heterologous immunity to a single, ubiquitous member of the microbiota as a central mechanism for systemic induction of human anti-fungal Th17 responses and as a potential risk factor for pulmonary inflammatory diseases.

Regulatory T Cell Specificity Directs Tolerance versus Allergy against Aeroantigens in Humans.

FOXP3+ regulatory T cells (Tregs) maintain tolerance against self-antigens and innocuous environmental antigens. However, it is still unknown whether Treg-mediated tolerance is antigen specific and how Treg specificity contributes to the selective loss of tolerance, as observed in human immunopathologies such as allergies. Here, we used antigen-reactive T cell enrichment to identify antigen-specific human Tregs. We demonstrate dominant Treg-mediated tolerance against particulate aeroallergens, such as pollen, house dust mites, and fungal spores. Surprisingly, we found no evidence of functional impairment of Treg responses in allergic donors. Rather, major allergenic proteins, known to rapidly dissociate from inhaled allergenic particles, have a generally reduced capability to generate Treg responses. Most strikingly, in individual allergic donors, Th2 cells and Tregs always target disparate proteins. Thus, our data highlight the importance of Treg antigen-specificity for tolerance in humans and identify antigen-specific escape from Treg control as an important mechanism enabling antigen-specific loss of tolerance in human allergy.

Functional modulation of chemical mediators in microbial communities.

Interactions between microorganisms are often mediated by specialized metabolites. Although the structures and biosynthesis of these compounds may have been elucidated, microbes exist within complex microbiomes and chemical signals can thus also be subject to community-dependent modifications. Increasingly powerful chemical and biological tools allow to shed light on this poorly understood aspect of chemical ecology. We provide an overview of loss-of-function and gain-of-function chemical mediator (CM) modifications within microbial multipartner relationships. Although loss-of-function modifications are abundant in the literature, few gain-of-function modifications have been described despite their important role in microbial interactions. Research in this field holds great potential for our understanding of microbial interactions and may also provide novel tools for targeted interference with microbial signaling.

Disruption of the Aspergillus fumigatus RNA interference machinery alters the conidial transcriptome.

The RNA interference (RNAi) pathway has evolved numerous functionalities in eukaryotes, with many on display in Kingdom Fungi. RNAi can regulate gene expression, facilitate drug resistance, or even be altogether lost to improve growth potential in some fungal pathogens. In the WHO fungal priority pathogen, Aspergillus fumigatus, the RNAi system is known to be intact and functional. To extend our limited understanding of A. fumigatus RNAi, we first investigated the genetic variation in RNAi-associated genes in a collection of 217 environmental and 83 clinical genomes, where we found that RNAi components are conserved even in clinical strains. Using endogenously expressed inverted-repeat transgenes complementary to a conditionally essential gene (pabA) or a nonessential gene (pksP), we determined that a subset of the RNAi componentry is active in inverted-repeat transgene silencing in conidia and mycelium. Analysis of mRNA-seq data from RNAi double-knockout strains linked the A. fumigatus dicer-like enzymes (DclA/B) and RNA-dependent RNA polymerases (RrpA/B) to regulation of conidial ribosome biogenesis genes; however, surprisingly few endogenous small RNAs were identified in conidia that could explain this broad change. Although RNAi was not clearly linked to growth or stress response defects in the RNAi knockouts, serial passaging of RNAi knockout strains for six generations resulted in lineages with diminished spore production over time, indicating that loss of RNAi can exert a fitness cost on the fungus. Cumulatively, A. fumigatus RNAi appears to play an active role in defense against double-stranded RNA species alongside a previously unappreciated housekeeping function in regulation of conidial ribosomal biogenesis genes.

The Toll pathway mediates Drosophila resilience to Aspergillus mycotoxins through specific Bomanins.

Host defense against infections encompasses both resistance, which targets microorganisms for neutralization or elimination, and resilience/disease tolerance, which allows the host to withstand/tolerate pathogens and repair damages. In Drosophila, the Toll signaling pathway is thought to mediate resistance against fungal infections by regulating the secretion of antimicrobial peptides, potentially including Bomanins. We find that Aspergillus fumigatus kills Drosophila Toll pathway mutants without invasion because its dissemination is blocked by melanization, suggesting a role for Toll in host defense distinct from resistance. We report that mutants affecting the Toll pathway or the 55C Bomanin locus are susceptible to the injection of two Aspergillus mycotoxins, restrictocin and verruculogen. The vulnerability of 55C deletion mutants to these mycotoxins is rescued by the overexpression of Bomanins specific to each challenge. Mechanistically, flies in which BomS6 is expressed in the nervous system exhibit an enhanced recovery from the tremors induced by injected verruculogen and display improved survival. Thus, innate immunity also protects the host against the action of microbial toxins through secreted peptides and thereby increases its resilience to infection.

Regulation and Role of Fungal Secondary Metabolites.

Fungi have the capability to produce a tremendous number of so-called secondary metabolites, which possess a multitude of functions, e.g., communication signals during coexistence with other microorganisms, virulence factors during pathogenic interactions with plants and animals, and in medical applications. Therefore, research on this topic has intensified significantly during the past 10 years and thus knowledge of regulatory mechanisms and the understanding of the role of secondary metabolites have drastically increased. This review aims to depict the complexity of all the regulatory elements involved in controlling the expression of secondary metabolite gene clusters, ranging from epigenetic control and signal transduction pathways to global and specific transcriptional regulators. Furthermore, we give a short overview on the role of secondary metabolites, focusing on the interaction with other microorganisms in the environment as well as on pathogenic relationships.

Recognition of DHN-melanin by a C-type lectin receptor is required for immunity to Aspergillus.

Resistance to infection is critically dependent on the ability of pattern recognition receptors to recognize microbial invasion and induce protective immune responses. One such family of receptors are the C-type lectins, which are central to antifungal immunity. These receptors activate key effector mechanisms upon recognition of conserved fungal cell-wall carbohydrates. However, several other immunologically active fungal ligands have been described; these include melanin, for which the mechanism of recognition is hitherto undefined. Here we identify a C-type lectin receptor, melanin-sensing C-type lectin receptor (MelLec), that has an essential role in antifungal immunity through recognition of the naphthalene-diol unit of 1,8-dihydroxynaphthalene (DHN)-melanin. MelLec recognizes melanin in conidial spores of Aspergillus fumigatus as well as in other DHN-melanized fungi. MelLec is ubiquitously expressed by CD31+ endothelial cells in mice, and is also expressed by a sub-population of these cells that co-express epithelial cell adhesion molecule and are detected only in the lung and the liver. In mouse models, MelLec was required for protection against disseminated infection with A. fumigatus. In humans, MelLec is also expressed by myeloid cells, and we identified a single nucleotide polymorphism of this receptor that negatively affected myeloid inflammatory responses and significantly increased the susceptibility of stem-cell transplant recipients to disseminated Aspergillus infections. MelLec therefore recognizes an immunologically active component commonly found on fungi and has an essential role in protective antifungal immunity in both mice and humans.

Bacterial marginolactones trigger formation of algal gloeocapsoids, protective aggregates on the verge of multicellularity.

Photosynthetic microorganisms including the green alga Chlamydomonas reinhardtii are essential to terrestrial habitats as they start the carbon cycle by conversion of CO2 to energy-rich organic carbohydrates. Terrestrial habitats are densely populated, and hence, microbial interactions mediated by natural products are inevitable. We previously discovered such an interaction between Streptomyces iranensis releasing the marginolactone azalomycin F in the presence of C. reinhardtii Whether the alga senses and reacts to azalomycin F remained unknown. Here, we report that sublethal concentrations of azalomycin F trigger the formation of a protective multicellular structure by C. reinhardtii, which we named gloeocapsoid. Gloeocapsoids contain several cells which share multiple cell membranes and cell walls and are surrounded by a spacious matrix consisting of acidic polysaccharides. After azalomycin F removal, gloeocapsoid aggregates readily disassemble, and single cells are released. The presence of marginolactone biosynthesis gene clusters in numerous streptomycetes, their ubiquity in soil, and our observation that other marginolactones such as desertomycin A and monazomycin also trigger the formation of gloeocapsoids suggests a cross-kingdom competition with ecological relevance. Furthermore, gloeocapsoids allow for the survival of C. reinhardtii at alkaline pH and otherwise lethal concentrations of azalomycin F. Their structure and polysaccharide matrix may be ancestral to the complex mucilage formed by multicellular members of the Chlamydomonadales such as Eudorina and Volvox Our finding suggests that multicellularity may have evolved to endure the presence of harmful competing bacteria. Additionally, it underlines the importance of natural products as microbial cues, which initiate interesting ecological scenarios of attack and counter defense.

Distinct transcriptional responses to fludioxonil in Aspergillus fumigatus and its ΔtcsC and Δskn7 mutants reveal a crucial role for Skn7 in the cell wall reorganizations triggered by this antifungal.

BACKGROUND: Aspergillus fumigatus is a major fungal pathogen that causes severe problems due to its increasing resistance to many therapeutic agents. Fludioxonil is a compound that triggers a lethal activation of the fungal-specific High Osmolarity Glycerol pathway. Its pronounced antifungal activity against A. fumigatus and other pathogenic molds renders this agent an attractive lead substance for the development of new therapeutics. The group III hydride histidine kinase TcsC and its downstream target Skn7 are key elements of the multistep phosphorelay that represents the initial section of the High Osmolarity Glycerol pathway. Loss of tcsC results in resistance to fludioxonil, whereas a Δskn7 mutant is partially, but not completely resistant. RESULTS: In this study, we compared the fludioxonil-induced transcriptional responses in the ΔtcsC and Δskn7 mutant and their parental A. fumigatus strain. The number of differentially expressed genes correlates well with the susceptibility level of the individual strains. The wild type and, to a lesser extend also the Δskn7 mutant, showed a multi-faceted stress response involving genes linked to ribosomal and peroxisomal function, iron homeostasis and oxidative stress. A marked difference between the sensitive wild type and the largely resistant Δskn7 mutant was evident for many cell wall-related genes and in particular those involved in the biosynthesis of chitin. Biochemical data corroborate this differential gene expression that does not occur in response to hyperosmotic stress. CONCLUSIONS: Our data reveal that fludioxonil induces a strong and TcsC-dependent stress that affects many aspects of the cellular machinery. The data also demonstrate a link between Skn7 and the cell wall reorganizations that foster the characteristic ballooning and the subsequent lysis of fludioxonil-treated cells.

Targeting of phagolysosomes containing conidia of the fungus Aspergillus fumigatus with polymeric particles.

Conidia of the airborne human-pathogenic fungus Aspergillus fumigatus are inhaled by humans. In the lung, they are phagocytosed by alveolar macrophages and intracellularly processed. In macrophages, however, conidia can interfere with the maturation of phagolysosomes to avoid their elimination. To investigate whether polymeric particles (PPs) can reach this intracellular pathogen in macrophages, we formulated dye-labeled PPs with a size allowing for their phagocytosis. PPs were efficiently taken up by RAW 264.7 macrophages and were found in phagolysosomes. When macrophages were infected with conidia prior to the addition of PPs, we found that they co-localized in the same phagolysosomes. Mechanistically, the fusion of phagolysosomes containing PPs with phagolysosomes containing conidia was observed. Increasing concentrations of PPs increased fusion events, resulting in 14% of phagolysosomes containing both conidia and PPs. We demonstrate that PPs can reach conidia-containing phagolysosomes, making these particles a promising carrier system for antimicrobial drugs to target intracellular pathogens. KEY POINTS: • Polymer particles of a size larger than 500 nm are internalized by macrophages and localized in phagolysosomes. • These particles can be delivered to Aspergillus fumigatus conidia-containing phagolysosomes of macrophages. • Enhanced phagolysosome fusion by the use of vacuolin1 can increase particle delivery.

Extremophile Metal Resistance: Plasmid-Encoded Functions in Streptomyces mirabilis.

The extreme metal tolerance of up to 130 mM NiSO4 in Streptomyces mirabilis P16B-1 was investigated. Genome sequencing revealed the presence of a large linear plasmid, pI. To identify plasmid-encoded determinants of metal resistance, a newly established transformation system was used to characterize the predicted plasmid-encoded loci nreB, hoxN, and copYZ. Reintroduction into the plasmid-cured S. mirabilis ΔpI confirmed that the predicted metal transporter gene nreB constitutes a nickel resistance factor, which was further supported by its heterologous expression in Escherichia coli. In contrast, the predicted nickel exporter gene hoxN decreased nickel tolerance, while copper tolerance was enhanced. The predicted copper-dependent transcriptional regulator gene copY did not induce tolerance toward either metal. Since genes for transfer were identified on the plasmid, its conjugational transfer to the metal-sensitive Streptomyces lividans TK24 was checked. This resulted in acquired tolerance toward 30 mM nickel and additionally increased the tolerance toward copper and cobalt, while oxidative stress tolerance remained unchanged. Intracellular nickel concentrations decreased in the transconjugant strain. The high extracellular nickel concentrations allowed for biomineralization. Plasmid transfer could also be confirmed into the co-occurring actinomycete Kribbella spp. in soil microcosms. IMPORTANCE Living in extremely metal-rich environments requires specific adaptations, and often, specific metal tolerance genes are encoded on a transferable plasmid. Here, Streptomyces mirabilis P16B-1, isolated from a former mining area and able to grow with up to 130 mM NiSO4, was investigated. The bacterial chromosome, as well as a giant plasmid, was sequenced. The plasmid-borne gene nreB was confirmed to confer metal resistance. A newly established transformation system allowed us to construct a plasmid-cured S. mirabilis as well as an nreB-rescued strain in addition to confirming nreB encoding nickel resistance if heterologously expressed in E. coli. The potential of intra- and interspecific plasmid transfer, together with the presence of metal resistance factors on that plasmid, underlines the importance of plasmids for transfer of resistance factors within a bacterial soil community.

The fungivorous amoeba Protostelium aurantium targets redox homeostasis and cell wall integrity during intracellular killing of Candida parapsilosis.

Predatory interactions among microbes are major evolutionary driving forces for biodiversity. The fungivorous amoeba Protostelium aurantium has a wide fungal food spectrum including foremost pathogenic members of the genus Candida. Here we show that upon phagocytic ingestion by the amoeba, Candida parapsilosis is confronted with an oxidative burst and undergoes lysis within minutes of processing in acidified phagolysosomes. On the fungal side, a functional genomic approach identified copper and redox homeostasis as primary targets of amoeba predation, with the highly expressed copper exporter gene CRP1 and the peroxiredoxin gene PRX1 contributing to survival when encountered with P. aurantium. The fungicidal activity was largely retained in intracellular vesicles of the amoebae. Following their isolation, the content of these vesicles induced immediate killing and lysis of C. parapsilosis in vitro. Proteomic analysis identified 56 vesicular proteins from P. aurantium. Although completely unknown proteins were dominant, many of them could be categorised as hydrolytic enzymes targeting the fungal cell wall, indicating that fungal cell wall structures are under selection pressure by predatory phagocytes in natural environments. TAKE AWAY: The amoeba Protostelium aurantium feeds on fungi, such as Candida parapsilosis. Ingested yeast cells are exposed to reactive oxygen species. A copper exporter and a peroxiredoxin contribute to fungal defence. Yeast cells undergo intracellular lysis. Lysis occurs via a cocktail of hydrolytic enzymes from intracellular vesicles.

Candidalysin delivery to the invasion pocket is critical for host epithelial damage induced by Candida albicans.

The human pathogenic fungus Candida albicans is a frequent cause of mucosal infections. Although the ability to transition from the yeast to the hypha morphology is essential for virulence, hypha formation and host cell invasion per se are not sufficient for the induction of epithelial damage. Rather, the hypha-associated peptide toxin, candidalysin, a product of the Ece1 polyprotein, is the critical damaging factor. While synthetic, exogenously added candidalysin is sufficient to damage epithelial cells, the level of damage does not reach the same level as invading C. albicans hyphae. Therefore, we hypothesized that a combination of fungal attributes is required to deliver candidalysin to the invasion pocket to enable the full damaging potential of C. albicans during infection. Utilising a panel of C. albicans mutants with known virulence defects, we demonstrate that the full damage potential of C. albicans requires the coordinated delivery of candidalysin to the invasion pocket. This process requires appropriate epithelial adhesion, hyphal extension and invasion, high levels of ECE1 transcription, proper Ece1 processing and secretion of candidalysin. To confirm candidalysin delivery, we generated camelid VH Hs (nanobodies) specific for candidalysin and demonstrate localization and accumulation of the toxin only in C. albicans-induced invasion pockets. In summary, a defined combination of virulence attributes and cellular processes is critical for delivering candidalysin to the invasion pocket to enable the full damage potential of C. albicans during mucosal infection. TAKE AWAYS: Candidalysin is a peptide toxin secreted by C. albicans causing epithelial damage. Candidalysin delivery to host cell membranes requires specific fungal attributes. Candidalysin accumulates in invasion pockets created by invasive hyphae. Camelid nanobodies enabled visualisation of candidalysin in the invasion pocket.

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 an important virulence trait of fungal pathogens due to the vulnerability of conidia against host defense. On the host side, we found that epithelial cells, which have been underappreciated, play a role in fungal clearance and are potent mediators of cytokine release. Both predictions were confirmed by in vitro experiments on established cell lines as well as primary lung cells. 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 traits.

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

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.

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

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.

Total Synthesis and Functional Evaluation of IORs, Sulfonolipid-based Inhibitors of Cell Differentiation in Salpingoeca rosetta.

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.

Bacterial cell wall-degrading enzymes induce basidiomycete natural product biosynthesis.

Natural products play a vital role for intermicrobial interactions. In the basidiomycete arena an important representative is variegatic acid, a lactone natural product pigment whose ecological relevance stems from both inhibiting bacterial swarming and from indirect participation in breakdown of organic matter by brown-rotting fungi. Previous work showed that the presence of bacteria stimulates variegatic acid production. However, the actual external molecular trigger that prompts its biosynthesis in the mushroom hyphae remained unknown. Here, we report on the identification of Bacillus subtilis subtilisin E (AprE) and chitosanase (Csn) as primary inducers of pulvinic acid pigment formation. Using the established co-culture system of B. subtilis and Serpula lacrymans, we used activity-guided FPLC-based fractionation of B. subtilis culture supernatants and subsequent peptide fingerprinting to identify candidates, and their role was corroborated by means of a pigment production assay using heterologously produced chitosanase and subtilisin. B. subtilis mutants defective in either the aprE or the csn gene still triggered pigmentation, yet to a lower degree, which points to a multicausal scenario and suggests the combined activity of these cell wall polymer-attacking enzymes as true stimulus.

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

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 106 cfu/m3 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.

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

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.