Annual Conference 2025 of the Association for General and Applied Microbiology - Jahrestagung 2025 der Vereinigung für Allgemeine und Angewandte Mikrobiologie

Chair

Prof. Dr.

Flotillin-containing lipid rafts in Aspergillus nidulans are linked to calcium

Biology of yeast and fungi

Lipid rafts are tight assemblies of proteins and lipids in a biological membrane and are thought to be involved in many physiological processes such as immune signalling and host-pathogen interactions. However, due to their small sizes exceeding the resolution limit of conventional light microscopy, direct measurement and characterisation of lipid rafts in living membranes remains to be a challenge. While most studies on lipid rafts have been carried out on mammalian cells, here we use the genetic model fungus Aspergillus nidulans to allow a more versatile characterisation of lipid rafts at both molecular and organismic levels. In particular we investigate the role of flotillin (FloA), a lipid raft marker conserved across many organisms. Using a nanoluciferase reporter strain, we demonstrate that the A. nidulans flotillin FloA is highly expressed when the fungus is confronted with high calcium stress. Transcriptomic analysis further reveals that repression of FloA under these conditions leads to the upregulation of numerous mitochondrial genes, suggesting a functional connection between FloA and mitochondrial activity. In addition, in vivo protein-proximity labelling is performed to assess the physical interaction partner proteins of FloA. We also explore the role of FloA in microbial communication through co-cultivation experiments with the soil bacterium Streptomyces iranensis and show that the bacterium is able to induce the high expression of FloA, most likely by secreting a natural product. These findings contribute to a deeper understanding of lipid raft dynamics and the organisation of eukaryotic membranes.

Short lecture

Probing Aspergillus niger spore dynamics: A droplet – based microfluidic approach for single-spore analysis and stressor evaluation

Aspergillus niger spores demonstrate complex and heterogeneous responses to environmental stressors, which complicates efforts to control fungal growth and resistance. In this study, we used a droplet – based microfluidic system to encapsulate individual A. niger spores in hydrogel beads, creating a 3D microenvironment conducive to controlled, high-resolution monitoring. This platform facilitates single-cell time-lapse microscopy, enabling detailed observation of germination patterns and branching behaviors across up to 1500 spores in a single experiment.To evaluate the effects of copper ions (Cu²⁺, delivered as CuSO₄ solution) and the antifungal agent caspofungin, we measured parameters such as swelling events, swelling size, hyphal area and main hyphae length. Initial findings revealed a pronounced heterogeneity in germination and outgrowth behavior under copper stress, even in a species known for its robust copper homeostasis mechanisms. The treatment with caspofungin in comparison to led to a 175% increase in swelling size and elevated the fraction of swollen spores in wild-type strains, while exposure to a 10 mM CuSO₄ solution resulted in a reduction across all growth metrics. ICP-MS measurements of copper content before and after incubatio over 24h on chip further indicated potential uptake of Cu²⁺ ions by spores within the hydrogel matrix. Viability and cell wall integrity assessments via LIVE/DEAD fluorescence and calcofluor white staining confirmed the spores' responses to these treatments.This research highlights the nuanced, stress-induced heterogeneity in A. niger"s physiological responses and underscores the potential of microfluidic systems as powerful tools for real-time, high-throughput fungal analysis. This approach offers new insights for antifungal development and fungal behavior under metal-induced stress.

The lipid transfer protein PDR16 of the filamentous ascomycete Sordaria macrospora

Pulldown experiments with the Striatin interaction phosphatase and kinase (STRIPAK) component SCI1 identified an orthologue of the yeast PDR16 phosphatidylinositol (PtdIns) transfer protein as a putative target of the STRIPAK complex in S. macrospora.The S. cerevisiae homolog of the S. macrospora PDR16 protein is predominantly associated with lipid droplets or is localized either to the plasma membrane (PM) or the PM-associated ER. The role of the Pdr16 protein in yeast metabolism is not fully understood. However, the absence of Pdr16p leads to increased susceptibility of yeast cells to azole antifungals, indicating its role in sterol biosynthesis. By regulating the phospholipid/sterol composition of plasma- and endo-membranes, S. macrospora PDR16 may be involved in membrane recruiting of the STRIPAK signaling complex required for hyphal fusion.Deletion of Smpdr16 in S. macrospora resulted in no obvious changes in growth, fruiting-body morphology or sexual reproduction under regular conditions. Only when grown at decreased temperatures the ΔSmpdr16 mutant displayed a reduced growth rate in comparison to the wt. SmPDR16 is localized at septa and endo-membranes, but localization at the plasma membrane cannot be ruled out.To examine the functional conservation of PDR16 proteins, we heterologously expressed the S. macrospora pdr16 cDNA in a yeast Δpdr16 mutant and verified that the S. macrospora gene can substitute for the absence of the yeast protein. To address the lipid/sterol binding capacity of PDR16, we will overexpress SmPDR16 in E. coli followed by purification to perform a lipid binding assay in HL60 cells.

Sculpting fungal fate: the importance of RNA editing for developmental morphogenesis in filamentous fungi

RNA editing, the post-transcriptional selective insertion, substitution or deletion of nucleotides plays a crucial role in functional diversity of gene expression within all domains of life. In ascomycetes, a diverse group of filamentous fungi RNA editing primarily involves adenosine-to-inosine (A-to-I) conversions. In contrast to animals the A-to-I editing occur predominantly within coding regions of transcripts involved in sexual reproduction. Interestingly, not only amino acid codons but also stop codons tend to be affected by editing leading to a change of TAG or TGA codons to TGG tryptophan codons. The precise mechanisms regulating the conversion of the codons still need further investigation. However, A-to-I RNA editing is suggested to play a crucial role in the formation of sexual structures like ascospores and fruiting bodies and might also provide proteome diversity that benefits the progeny.To unravel the biological importance of A-to-I editing during development morphogenesis in filamentous fungi we analyzed so called efd (edited in fruiting body development) genes in the ascomycete Sordaria macrospora. Intriguingly, several deletion mutants of efd genes like efd4 and efd7 showed severe defects in their ascospore formation and/or discharge. Interestingly, complementation studies with constructs expressing either the edited version (TGG) or non-edited version (TAA) revealed putative functions for the editing sites during reproduction. Very recent results suggest a role for efd16 in cellular structure and physical properties of sexual reproduction structures. To investigate this further we are applying a novel method measuring structural integrity of ascospores. These data will provide better insight of the impact of A-to-I editing during developmental processes and its role for the adaptive capabilities and morphogenesis of fungi.

Structure-function analysis of Ubp3, Bre5 and the Ubp3/Bre5 complex

Ubiquitination is a post-translational protein modification in eukaryotes, with broad signaling functions. In this process, the small molecule ubiquitin is covalently attached to target proteins through a complex three-step enzymatic cascade, which can result in the formation of polyubiquitin chains. Ubiquitin-mediated signaling is involved in a multitude of cellular functions and quality control mechanisms, requiring precise regulation. These quality control pathways include different types of selective autophagy such as ribophagy or mitophagy. To maintain a balance between various quality control mechanisms, deubiquitinating enzymes can remove or modify ubiquitin signals by cleaving ubiquitin or polyubiquitin chains. In Saccharomyces cerevisiae, one such deubiquitinating enzyme is Ubp3, which relies on its cofactor Bre5 for specific activity. The Ubp3-Bre5 complex plays a vital role in various cellular processes, particularly in stress response. Interestingly, it exhibits a dual role in cellular functions. For instance, it protects mitochondria from mitophagy while simultaneously promoting other forms of general autophagy. This investigation aims to elucidate how these distinct functions are coordinated and to explore the nature of the interaction between Ubp3 and Bre5. We demonstrate the establishment of a heterologous expression and purification strategy, to obtain protein for detailed structure-function analyses. With this in vitro approach, isolation of individual full-size proteins and their complex is targeted. This is achieved through a series of chromatography steps utilizing different affinity tags. Isolated proteins are investigated regarding their activity, structure and interactions. With this approach, it should be possible to elucidate the molecular mechanisms that govern their natural function and the subsequent hypotheses can then be verified in vivo. This study should therefore help to entangle the complex regulatory network in which Ubp3 is involved and advance our understanding of deubiquitinases.

Prof. , Dr.

Basidiomycete model fungi and their communication in soil

For their life is soil, fungi are well-equipped. The filamentous lifestyle allows them to interact with different soil particles as well as plant roots. The hyphae spanning different niches in soil provide a route by which soil bacteria like Bacillus can access new environments. To delve into such interactions, different molecular signals are produced, among them volatiles. With respect to ectomycorrhizae, the involvement of fungal VOCs in modulating plant response is discussed.For soil attachment and growth in soil, a model using glass surfaces is presented that also allows to assess thigmotropic clues that lead hyphae to attach to surface veins and into crevices. This goes to show that basidiomycete fungi like Schizophyllum commune can sense surface structures and respond with specific gene expression profiles to different surfaces.Molecules involved in bacteria-fungi interaction are explored using Bacillus subtilis as a single celled bacterium as well as streptomycetes representing filamentous bacteria in interaction with fungi. Surface interactions of fungi enable bacterial movement, and the inhibition of swarming may indicate that the access of different niches is controlled by the fungus. In interaction with Schizophyllum commune, and in response to structured surfaces, bacterial signaling molecules are shown and the involvement of B pheromone receptor-like proteins, Brls, are discussed.The specific ectomycorrhizal symbiosis of Tricholoma vaccinum with its compatible host spruce and its low-compatibility host pine is used to show the involvement of volatile terpenoid molecules in host communication. The trees respond with different phytohormone profiles suggesting recognition of beneficial versus pathogenic soil microbes upon terpenes exuded by the fungus in a volatile-only chemical communication setting. Since ectomycorrhizal fungi determine the bacterial microbiome in forest soils, this specific volatile signaling prior to contact between the symbionts is seen as a first row of communication molecules in basidiomycete-host tree symbiosis.

Biology of yeasts and filamentous fungi

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