Species-Specific Differences in the Susceptibility of Fungi to the Antifungal Protein AFP Depend on C-3 Saturation of Glycosylceramides

AFP is an antimicrobial peptide (AMP) produced by the filamentous fungus Aspergillus giganteus and is a very potent inhibitor of fungal growth that does not affect the viability of bacteria, plant, or mammalian cells. It targets chitin synthesis and causes plasma membrane permeabilization in many human- and plant-pathogenic fungi, but its exact mode of action is not known. After adoption of the “damage-response framework of microbial pathogenesis” regarding the analysis of interactions between AMPs and microorganisms, we have recently proposed that the cytotoxic capacity of a given AMP depends not only on the presence/absence of its target(s) in the host and the AMP concentration applied but also on other variables, such as microbial survival strategies. We show here using the examples of three filamentous fungi (Aspergillus niger, Aspergillus fumigatus, and Fusarium graminearum) and two yeasts (Saccharomyces cerevisiae and Pichia pastoris) that the important parameters defining the AFP susceptibilities of these fungi are (i) the presence/absence of glycosylceramides, (ii) the presence/absence of Δ3(E) desaturation of the fatty acid chain therein, and (iii) the (dis)ability of these fungi to respond to AFP inhibitory effects with the fortification of their cell walls via increased chitin and β-(1,3)-glucan synthesis. These observations support the idea of the adoption of the damage-response framework to holistically understand the outcome of AFP inhibitory effects.TU Berlin, Open-Access-Mittel - 201

Purification and properties of bacterial pyrimidine nucleosidase

The metabolism of pyrimidine nucleosides was found to be similar to the dissimilation of purine nucleosides. Thus pyrimidine base is cleaved from the nucleoside and is accumulated. Ribose disappears from the reaction mixture; it is rapidly oxidised to carbon dioxide and water;Pyrimidine-bound ribose cannot be determined by the orcinol test of Mejbaum (1939). A modified orcinol reaction for this purpose was developed and used in the study of pyrimidine riboside metabolism;Pyrimidine nucleoside phosphorylase was isolated from extracts of E. coli and brought to a high degree of purity by fractional precipitation with ammonium sulfate and adsorption on and elution from alumina C-gamma. The purified enzyme shows a six-hundred-fold increase in activity over that of the cell-free extract from which it was prepared;Pyrimidine nucleoside phosphorylase is a phosphorolytic enzyme. Inorganic phosphate is necessary for its action. Ribose-1-phosphate is accumulated as a result of its reaction upon nucleosides. The presence of ribose-1-phosphate was determined by analytical methods and by isolation as barium salt;The enzyme is highly specific. It does not attack cytidine. It is inactive against purine nucleosides. It will not split thymine desoxyriboside. Cytosine, thymine, and orotic acid do not react with ribose-1-phosphate in the presence of the enzyme. The role of orotic acid in the anabolism of pyrimidine nucleosides is discussed;Uridine was synthesised. Uracil when incubated with ribose-1-phosphate in the presence of pyrimidine nucleoside phosphorylase was converted to uridine. The equilibrium between uridine synthesis and splitting was determined

Updating genome annotation for the microbial cell factory Aspergillus niger using gene co-expression networks

A significant challenge in our understanding of biological systems is the high number of genes with unknown function in many genomes. The fungal genus Aspergillus contains important pathogens of humans, model organisms, and microbial cell factories. Aspergillus niger is used to produce organic acids, proteins, and is a promising source of new bioactive secondary metabolites. Out of the 14,165 open reading frames predicted in the A. niger genome only 2% have been experimentally verified and over 6,000 are hypothetical. Here, we show that gene co-expression network analysis can be used to overcome this limitation. A meta-analysis of 155 transcriptomics experiments generated co-expression networks for 9,579 genes (∼65%) of the A. niger genome. By populating this dataset with over 1,200 gene functional experiments from the genus Aspergillus and performing gene ontology enrichment, we could infer biological processes for 9,263 of A. niger genes, including 2,970 hypothetical genes. Experimental validation of selected co-expression sub-networks uncovered four transcription factors involved in secondary metabolite synthesis, which were used to activate production of multiple natural products. This study constitutes a significant step towards systems-level understanding of A. niger, and the datasets can be used to fuel discoveries of model systems, fungal pathogens, and biotechnology.DFG, 325093850, Open Access Publizieren 2017 - 2018 / Technische Universität BerlinEC/FP7/607332/EU/Quantitative Biology for Fungal Secondary Metabolite Producers/QuantFun

A Computational Modeling Approach Predicts Interaction of the Antifungal Protein AFP from Aspergillus giganteus with Fungal Membranes via Its γ-Core Motif

This work is licensed under a Creative Commons Attribution 4.0 International License.Fungal pathogens kill more people per year globally than malaria or tuberculosis and threaten international food security through crop destruction. New sophisticated strategies to inhibit fungal growth are thus urgently needed. Among the potential candidate molecules that strongly inhibit fungal spore germination are small cationic, cysteine-stabilized proteins of the AFP family secreted by a group of filamentous Ascomycetes. Its founding member, AFP from Aspergillus giganteus, is of particular interest since it selectively inhibits the growth of filamentous fungi without affecting the viability of mammalian, plant, or bacterial cells. AFPs are also characterized by their high efficacy and stability. Thus, AFP can serve as a lead compound for the development of novel antifungals. Notably, all members of the AFP family comprise a γ-core motif which is conserved in all antimicrobial proteins from pro- and eukaryotes and known to interfere with the integrity of cytoplasmic plasma membranes. In this study, we used classical molecular dynamics simulations combined with wet laboratory experiments and nuclear magnetic resonance (NMR) spectroscopy to characterize the structure and dynamical behavior of AFP isomers in solution and their interaction with fungal model membranes. We demonstrate that the γ-core motif of structurally conserved AFP is the key for its membrane interaction, thus verifying for the first time that the conserved γ-core motif of antimicrobial proteins is directly involved in protein-membrane interactions. Furthermore, molecular dynamic simulations suggested that AFP does not destroy the fungal membrane by pore formation but covers its surface in a well-defined manner, using a multistep mechanism to destroy the membranes integrity.NIH GM31749NIH GM103426Deutsche Forschungsgemeinschaft (Cluster of Excellence ‘Unifying Concepts in Catalysis’ and SFB1078

Storytelling and design

Access to thesis permanently restricted to Ball State community only.Access to accompanying material permanently restricted to Ball State community onlyAccess to abstract permanently restricted to Ball State community only.Thesis (M.A.)Department of Telecommunication

Storytelling and design

Access to thesis permanently restricted to Ball State community only.Access to accompanying material permanently restricted to Ball State community onlyAccess to abstract permanently restricted to Ball State community only.Department of TelecommunicationsThesis (M.A.

Re-imagineering :a redesign of a project entry in the 2014 Disney Imaginations Competition

If you ask any Walt Disney Imagineer what they do, they will tell you that their primary role is a storyteller. They are also the creative machine behind all things Disney—the starting point for the ideas behind the magic. Disney Imagineering holds a prestigious design competition each year, called Disney Imaginations. When my three teammates and I poured ourselves into a submission for the 2014 Disney Imaginations competition, we were, needless to say, disheartened when we failed to finish any better than the “participant” level. As a first try, this competition was an adventure for all four of us. At the very least, it will remain a valuable advantage in experience for next year’s competition. In preparation for the 2015 Disney Imaginations Competition, I have analyzed our shortcomings and, as an exercise, have built upon our previous design.Honors CollegeThesis (B.?

Re-imagineering :a redesign of a project entry in the 2014 Disney Imaginations Competition

If you ask any Walt Disney Imagineer what they do, they will tell you that their primary role is a storyteller. They are also the creative machine behind all things Disney—the starting point for the ideas behind the magic. Disney Imagineering holds a prestigious design competition each year, called Disney Imaginations. When my three teammates and I poured ourselves into a submission for the 2014 Disney Imaginations competition, we were, needless to say, disheartened when we failed to finish any better than the “participant” level. As a first try, this competition was an adventure for all four of us. At the very least, it will remain a valuable advantage in experience for next year’s competition. In preparation for the 2015 Disney Imaginations Competition, I have analyzed our shortcomings and, as an exercise, have built upon our previous design.Thesis (B.?)Honors Colleg