Nutritional Provision of Iron Complexes by the Major Allergen Alt a 1 to Human Immune Cells Decreases Its Presentation.

Alternaria alternata is a common fungus strongly related with severe allergic asthma, with 80% of affected individuals being sensitized solely to its major allergen Alt a 1. Here, we assessed the function of Alt a 1 as an innate defense protein binding to micronutrients, such as iron-quercetin complexes (FeQ2), and its impact on antigen presentation in vitro. Binding of Alt a 1 to FeQ2 was determined in docking calculations. Recombinant Alt a 1 was generated, and binding ability, as well as secondary and quaternary structure, assessed by UV-VIS, CD, and DLS spectroscopy. Proteolytic functions were determined by casein and gelatine zymography. Uptake of empty apo- or ligand-filled holoAlt a 1 were assessed in human monocytic THP1 cells under the presence of dynamin and clathrin-inhibitors, activation of the Arylhydrocarbon receptor (AhR) using the human reporter cellline AZ-AHR. Human PBMCs were stimulated and assessed for phenotypic changes in monocytes by flow cytometry. Alt a 1 bound strongly to FeQ2 as a tetramer with calculated Kd values reaching pico-molar levels and surpassing affinities to quercetin alone by a factor of 5000 for the tetramer. apoAlt a 1 but not holoAlta 1 showed low enzymatic activity against casein as a hexamer and gelatin as a trimer. Uptake of apo- and holo-Alt a 1 occurred partly clathrin-dependent, with apoAlt a 1 decreasing labile iron in THP1 cells and holoAlt a 1 facilitating quercetin-dependent AhR activation. In human PBMCs uptake of holoAlt a 1 but not apoAlt a 1 significantly decreased the surface expression of the costimulatory CD86, but also of HLADR, thereby reducing effective antigen presentation. We show here for the first time that the presence of nutritional iron complexes, such as FeQ2, significantly alters the function of Alt a 1 and dampens the human immune response, thereby supporting the notion that Alt a 1 only becomes immunogenic under nutritional deprivation.

Genome analysis of Cephalotrichum gorgonifer and identification of the biosynthetic pathway for rasfonin, an inhibitor of KRAS dependent cancer.

BACKGROUND: Fungi are important sources for bioactive compounds that find their applications in many important sectors like in the pharma-, food- or agricultural industries. In an environmental monitoring project for fungi involved in soil nitrogen cycling we also isolated Cephalotrichum gorgonifer (strain NG_p51). In the course of strain characterisation work we found that this strain is able to naturally produce high amounts of rasfonin, a polyketide inducing autophagy, apoptosis, necroptosis in human cell lines and showing anti-tumor activity in KRAS-dependent cancer cells. RESULTS: In order to elucidate the biosynthetic pathway of rasfonin, the strain was genome sequenced, annotated, submitted to transcriptome analysis and genetic transformation was established. Biosynthetic gene cluster (BGC) prediction revealed the existence of 22 BGCs of which the majority was not expressed under our experimental conditions. In silico prediction revealed two BGCs with a suite of enzymes possibly involved in rasfonin biosynthesis. Experimental verification by gene-knock out of the key enzyme genes showed that one of the predicted BGCs is indeed responsible for rasfonin biosynthesis. CONCLUSIONS: This study identified a biosynthetic gene cluster containing a key-gene responsible for rasfonin production. Additionally, molecular tools were established for the non-model fungus Cephalotrichum gorgonifer which allows strain engineering and heterologous expression of the BGC for high rasfonin producing strains and the biosynthesis of rasfonin derivates for diverse applications.

Agromyces aureus sp. nov., isolated from the rhizosphere of Salix caprea L. grown in a heavy-metal-contaminated soil.

A Gram-reaction-positive, motile, yellow-pigmented and rod-shaped bacterial strain, designated AR33T, was isolated from the rhizosphere of Salix caprea L. growing in a former zinc/lead mining and processing site in Austria. A polyphasic approach was applied to determine its taxonomic position. 16S rRNA gene sequence analysis, and morphological and chemotaxonomic properties showed that strain AR33T belongs to the genus Agromyces. Strain AR33T had peptidoglycan type B2γ and the major menaquinones were MK-11, MK-10 and MK-12. The main branched-chain fatty acids were anteiso-C15 : 0, anteiso-C17 : 0 and iso-C16 : 0. Strain AR33T showed catalase and oxidase activity and multiple heavy metal resistances to zinc, lead and cadmium. The DNA G+C content was 70.1 mol%. Levels of 16S rRNA gene sequence similarity with closely related recognized species of the genus Agromyces ranged between 98 and 99 %. However, DNA-DNA hybridization between strain AR33T and the type strains of three Agromyces species showed values lower than 42 % relatedness. Therefore, differential phenotypic characteristics together with DNA-DNA relatedness suggested that strain AR33T can be recognized as representing a distinct Agromyces species, for which the name Agromyces aureus sp. nov. is proposed. The type strain is AR33T (=DSM 101731T=LMG 29235T).

Fungi treated with small chemicals exhibit increased antimicrobial activity against facultative bacterial and yeast pathogens.

For decades, fungi have been the main source for the discovery of novel antimicrobial drugs. Recent sequencing efforts revealed a still high number of so far unknown "cryptic" secondary metabolites. The production of these metabolites is presumably epigenetically silenced under standard laboratory conditions. In this study, we investigated the effect of six small mass chemicals, of which some are known to act as epigenetic modulators, on the production of antimicrobial compounds in 54 spore forming fungi. The antimicrobial effect of fungal samples was tested against clinically facultative pathogens and multiresistant clinical isolates. In total, 30 samples of treated fungi belonging to six different genera reduced significantly growth of different test organisms compared to the untreated fungal sample (growth log reduction 0.3-4.3). For instance, the pellet of Penicillium restrictum grown in the presence of butyrate revealed significant higher antimicrobial activity against Staphylococcus (S.) aureus and multiresistant S. aureus strains and displayed no cytotoxicity against human cells, thus making it an ideal candidate for antimicrobial compound discovery. Our study shows that every presumable fungus, even well described fungi, has the potential to produce novel antimicrobial compounds and that our approach is capable of rapidly filling the pipeline for yet undiscovered antimicrobial substances.

N-glycosylation engineering of plants for the biosynthesis of glycoproteins with bisected and branched complex N-glycans.

Glycoengineering is increasingly being recognized as a powerful tool to generate recombinant glycoproteins with a customized N-glycosylation pattern. Here, we demonstrate the modulation of the plant glycosylation pathway toward the formation of human-type bisected and branched complex N-glycans. Glycoengineered Nicotiana benthamiana lacking plant-specific N-glycosylation (i.e. ß1,2-xylose and core α1,3-fucose) was used to transiently express human erythropoietin (hEPO) and human transferrin (hTF) together with modified versions of human ß1,4-mannosyl-ß1,4-N-acetylglucosaminyltransferase (GnTIII), α1,3-mannosyl-ß1,4-N-acetylglucosaminyltransferase (GnTIV) and α1,6-mannosyl-ß1,6-N-acetylglucosaminyltransferase (GnTV). hEPO was expressed as a fusion to the IgG-Fc domain (EPO-Fc) and purified via protein A affinity chromatography. Recombinant hTF was isolated from the intracellular fluid of infiltrated plant leaves. Mass spectrometry-based N-glycan analysis of hEPO and hTF revealed the quantitative formation of bisected (GnGnbi) and tri- as well as tetraantennary complex N-glycans (Gn[GnGn], [GnGn]Gn and [GnGn][GnGn]). Co-expression of GnTIII together with GnTIV and GnTV resulted in the efficient generation of bisected tetraantennary complex N-glycans. Our results show the generation of recombinant proteins with human-type N-glycosylation at great uniformity. The strategy described here provides a robust and straightforward method for producing mammalian-type N-linked glycans of defined structures on recombinant glycoproteins, which can advance glycoprotein research and accelerate the development of protein-based therapeutics.

Identification of heavy metal regulated genes from the root associated ascomycete Cadophora finlandica using a genomic microarray.

The ascomycete Cadophora finlandica, which can form mycorrhizas with ectomycorrhizal and ericoid hosts, is commonly found in heavy metal polluted soils. To understand the selective advantage of this organism at contaminated sites heavy metal regulated genes from C. finlandica were investigated. For gene identification a strategy based on a genomic microarray was chosen, which allows a rapid, genome-wide screening in genetically poorly characterized organisms. In a preliminary screen eleven plasmids covering eight distinct genomic regions and encoding a total of ten Cd-regulated genes were identified. Northern analyses with RNA from C. finlandica grown in the presence of either Cd, Pb or Zn revealed different transcription patterns in response to the heavy metals present in the growth medium. The Cd-regulated genes are predicted to encode several extracellular proteins with unknown functions, transporters, a centaurin-type regulator of intracellular membrane trafficking, a GNAT-family acetyltransferase and a B-type cyclin.