Functional Analysis of the Fusarielin Biosynthetic Gene Cluster.

Fusarielins are polyketides with a decalin core produced by various species of Aspergillus and Fusarium. Although the responsible gene cluster has been identified, the biosynthetic pathway remains to be elucidated. In the present study, members of the gene cluster were deleted individually in a Fusarium graminearum strain overexpressing the local transcription factor. The results suggest that a trans-acting enoyl reductase (FSL5) assists the polyketide synthase FSL1 in biosynthesis of a polyketide product, which is released by hydrolysis by a trans-acting thioesterase (FSL2). Deletion of the epimerase (FSL3) resulted in accumulation of an unstable compound, which could be the released product. A novel compound, named prefusarielin, accumulated in the deletion mutant of the cytochrome P450 monooxygenase FSL4. Unlike the known fusarielins from Fusarium, this compound does not contain oxygenized decalin rings, suggesting that FSL4 is responsible for the oxygenation.

Protein Binder Toolbox for Studies of Solute Carrier Transporters.

Transporters of the solute carrier superfamily (SLCs) are responsible for the transmembrane traffic of the majority of chemical substances in cells and tissues and are therefore of fundamental biological importance. As is often the case with membrane proteins that can be heavily glycosylated, a lack of reliable high-affinity binders hinders their functional analysis. Purifying and reconstituting transmembrane proteins in their lipidic environments remains challenging and standard approaches to generate binders for multi-transmembrane proteins, such as SLCs, channels or G protein-coupled receptors (GPCRs) are lacking. While generating protein binders to 27 SLCs, we produced full length protein or cell lines as input material for binder generation by selected binder generation platforms. As a result, we obtained 525 binders for 22 SLCs. We validated the binders with a cell-based validation workflow using immunofluorescent and immunoprecipitation methods to process all obtained binders. Finally, we demonstrated the potential applications of the binders that passed our validation pipeline in structural, biochemical, and biological applications using the exemplary protein SLC12A6, an ion transporter relevant in human disease. With this work, we were able to generate easily renewable and highly specific binders against SLCs, which will greatly facilitate the study of this neglected protein family. We hope that the process will serve as blueprint for the generation of binders against the entire superfamily of SLC transporters.

Real-time optical antimicrobial susceptibility testing.

Rapid antibiotic susceptibility testing is in high demand in health care fields as antimicrobial-resistant bacterial strains emerge and spread. Here, we describe an optical screening system (oCelloScope) which, based on time-lapse imaging of 96 bacteria-antibiotic combinations at a time, introduces real-time detection of bacterial growth and antimicrobial susceptibility with imaging material to support the automatically generated graphs. Automated antibiotic susceptibility tests of a monoculture showed statistically significant antibiotic effects within 6 min and within 30 min in complex samples from pigs suffering from catheter-associated urinary tract infections. The oCelloScope system provides a fast high-throughput screening method for detecting bacterial susceptibility that might entail an earlier diagnosis and introduction of appropriate targeted therapy and thus combat the threat from multidrug-resistant pathogenic bacteria. The oCelloScope system can be employed for a broad range of applications within bacteriology and might present new vistas as a point-of-care instrument in clinical and veterinary settings.

Loss of vps54 function leads to vesicle traffic impairment, protein mis-sorting and embryonic lethality.

The identification of the mutation causing the phenotype of the amyotrophic lateral sclerosis (ALS) model mouse, wobbler, has linked motor neuron degeneration with retrograde vesicle traffic. The wobbler mutation affects protein stability of Vps54, a ubiquitously expressed vesicle-tethering factor and leads to partial loss of Vps54 function. Moreover, the Vps54 null mutation causes embryonic lethality, which is associated with extensive membrane blebbing in the neural tube and is most likely a consequence of impaired vesicle transport. Investigation of cells derived from wobbler and Vps54 null mutant embryos demonstrates impaired retrograde transport of the Cholera-toxin B subunit to the trans-Golgi network and mis-sorting of mannose-6-phosphate receptors and cargo proteins dependent on retrograde vesicle transport. Endocytosis assays demonstrate no difference between wobbler and wild type cells, indicating that the retrograde vesicle traffic to the trans-Golgi network, but not endocytosis, is affected in Vps54 mutant cells. The results obtained on wobbler cells were extended to test the use of cultured skin fibroblasts from human ALS patients to investigate the retrograde vesicle traffic. Analysis of skin fibroblasts of ALS patients will support the investigation of the critical role of the retrograde vesicle transport in ALS pathogenesis and might yield a diagnostic prospect.

Resolving the Conflict between Strength and Toughness in Bioactive Silica-Polymer Hybrid Materials.

Simultaneously improving the strength and toughness of materials is a major challenge. Inorganic-polymer hybrids offer the potential to combine mechanical properties of a stiff inorganic glass with a flexible organic polymer. However, the toughening mechanism at the atomic scale remains largely unknown. Based on combined experimental and molecular dynamics simulation results, we find that the deformation and fracture behavior of hybrids are governed by noncovalent intermolecular interactions between polymer and silica networks rather than the breakage of covalent bonds. We then attempt three methods to improve the balance between strength and toughness of hybrids, namely the total inorganic/organic (I/O) weight ratio, the size of silica nanoparticles, and the ratio of -C-O vs -C-C bonds in the polymer chains. Specifically, for a hybrid with matched silica size and I/O ratio, we demonstrate optimized mechanical properties in terms of strength (1.75 MPa at breakage), degree of elongation at the fracture point (31%), toughness (219 kPa), hardness (1.08 MPa), as well as Young's modulus (3.0 MPa). We also demonstrate that this hybrid material shows excellent biocompatibility and ability to support cell attachment as well as proliferation. This supports the possible application of this material as a strong yet tough bone scaffold material.

PTR2 peptide transporters in Fusarium graminearum influence secondary metabolite production and sexual development.

Putative proton coupled di-peptide transporters, PTR2s, are found in filamentous fungi in different numbers and their function during fungal development and plant infection is unresolved. In Fusarium graminearum, the cause of head blight in cereals, we identified four putative PTR2 transporters (FgPTR2A-D). The genes did not cluster together in phylogenetic analyses and only FgPTR2A and FgPTR2C were able to complement a PTR2 deficient yeast mutant in uptake of di-peptides. All FgPTR2s are continuously expressed throughout the fungal lifecycle, although at different levels. In silico analyses of existing expression-data show that FgPTR2B is found at higher levels than the others in planta and during sexual development. Deletion mutants of FgPTR2A, FgPTR2C, and FgPTR2D had a higher production of deoxynivalenol (DON) and zearalenone and lower production of fusarielin H than the wild type. Perithecium development was reduced in these mutants but unaffected by deletion of FgPTR2B. Conidia production was reduced in the FgPTR2B mutant and unaffected by deletion of the other PTR2 transporters. Sexual development and secondary metabolite production are known to be linked at the regulatory level and the results suggest that PTR2s are active in nitrogen turnover and thereby influence signal processes.

Expression profiling and functional analyses of BghPTR2, a peptide transporter from Blumeria graminis f. sp. hordei.

The obligate ascomycete parasitic fungus Blumeria graminis f. sp. hordei (Bgh) has a unique lifestyle as it is completely dependent on living barley leaves as substrate for growth. Genes involved in inorganic nitrogen utilization are notably lacking, and the fungus relies on uptake of host-derived peptides and amino acids. The PTR2 transporter family takes up di- and tri- peptides in a proton coupled process and filamentous fungi typically have two or more di/tri peptide transporters. Here we show that Bgh appear to have one PTR2 that can restore dipeptide uptake in a Saccharomyces cerevisiae PTR2 deletion strain. The Bgh PTR2 gene is expressed in conidia and germinating conidia. During Bgh infection of barley the expression level of the BghPTR2 gene is high in the appressorial germ tube, low in the haustoria and high again during conidiation and secondary infection in the compatible and intermediate resistant interactions. BghPTR2 appears to be important for the initial establishment of fungal infection but not for uptake of di-tri-peptides at the haustorial interface. Based on the expression profile we suggest that BghPTR2 is active in internal transport of nutrient reserves and/or uptake of break down products from the plant surface during the early infection stages.

Glass bead cultivation of fungi: combining the best of liquid and agar media.

Production of bioactive compounds and enzymes from filamentous fungi is highly dependent on cultivation conditions. Here we present an easy way to cultivate filamentous fungi on glass beads that allow complete control of nutrient supply. Secondary metabolite production in Fusarium graminearum and Fusarium solani cultivated on agar plates, in shaking liquid culture or on glass beads was compared. Agar plate culture and glass bead cultivation yielded comparable results while liquid culture had lower production of secondary metabolites. RNA extraction from glass beads and liquid cultures was easier than from agar plates and the quality was superior. The system allows simple control of nutrient availability throughout fungal cultivation. This combined with the ease of extraction of nucleic acids and metabolites makes the system highly suitable for the study of gene regulation in response to specific nutrient factors.

Overexpression of NRPS4 leads to increased surface hydrophobicity in Fusarium graminearum.

The plant pathogen Fusarium graminearum is the infamous cause of Fusarium head blight worldwide resulting in significant losses of yield and reduced grain feed quality. It also has the potential to produce a range of small bioactive peptides produced by the non ribosomal peptide synthetases (NRPSs). Most of these are unknown as F. graminearum contains 19 NRPS encoding genes, but only three have been assigned products. For the first time, we use deletion and overexpression mutants to investigate the functions and product of NRPS4 in F. graminearum. Deletion of NRPS4 homologues in Alternaria brassicicola and Cochloibolus heterostrophus has been shown to result in mutants unable to repel water. In a time study of surface hydrophobicity we observed that water droplets could penetrate 7 d old colonies of the NRPS4 deletion mutants. Loss in ability to repel water was first observed on 13 d old cultures of the wild type strain, whereas the overexpression strain remained water repellant throughout the 38 d time study. The conidia of both mutants were examined and those of the overexpression mutant showed distinct morphological differences in form of collapsed cells. These observations might suggest that the peptide product of NRPS4 could be an architectural factor in the cell walls of Fusarium or an indirect regulator of hydrophobicity.

Autophagy provides nutrients for nonassimilating fungal structures and is necessary for plant colonization but not for infection in the necrotrophic plant pathogen Fusarium graminearum.

The role of autophagy in necrotrophic fungal physiology and infection biology is poorly understood. We have studied autophagy in the necrotrophic plant pathogen Fusarium graminearum in relation to development of nonassimilating structures and infection. We identified an ATG8 homolog F. graminearum ATG8 whose first 116 amino acids before the predicted ATG4 cleavage site are 100% identical to Podospora anserina ATG8. We generated a ΔFgatg8 mutant by gene replacement and showed that this cannot form autophagic compartments. The strain forms no perithecia, has reduced conidia production and the aerial mycelium collapses after a few days in culture. The collapsing aerial mycelium contains lipid droplets indicative of nitrogen starvation and/or an inability to use storage lipids. The capacity to use carbon/energy stored in lipid droplets after a shift from carbon rich conditions to carbon starvation is severely inhibited in the ΔFgatg8 strain demonstrating autophagy-dependent lipid utilization, lipophagy, in fungi. Radial growth rate of the ΔFgatg8 strain is reduced compared with the wild type and the mutant does not grow over inert plastic surfaces in contrast to the wild type. The ability to infect barley and wheat is normal but the mutant is unable to spread from spikelet to spikelet in wheat. Complementation by inserting the F. graminearum atg8 gene into a region adjacent to the actin gene in ΔFgatg8 fully restores the WT phenotype. The results showed that autophagy plays a pivotal role for supplying nutrients to nonassimilating structures necessary for growth and is important for plant colonization. This also indicates that autophagy is a central mechanism for fungal adaptation to nonoptimal C/N ratios.