The specific role of neutrophil- and epithelial cell-derived extracellular vesicles in antifungal defence against Aspergillus fumigatus

oai:www.db-thueringen.de:dbt_mods_00055686In this work I contributed to the elucidation of the role and physiological meaning of extracellular vesicles (EVs) released from epithelial cells and neutrophils after interaction with the pathogenic fungus Aspergillus fumigatus. With my work I untangled more aspects of the interaction between neutrophils and this fungus and came to the finding that EVs generated from neutrophils infected with A. fumigatus are highly specific in the killing of A. fumigatus. Evaluation of the hyphal damage induced by EVs was performed with extensive imaging, based on confocal laser scanning microscopy, bioinformatic 3D reconstruction and quantification of signals, and a metabolic assay. In the course of my research on extracellular vesicles I also characterized EVs from epithelial cells. I evaluated the cell response after confrontation with different conidia morphotypes of A. fumigatus. Analysing the EVs after these different co-incubations revealed that their protein and cytokine contents are changed. This proves that the cells can sense different stimuli and modify their EV content

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.</p