Genetic-based reporter systems for labelling and differentiation of host-derived extracellular vesicles reveal their association with a human-pathogenic fungus

ABSTRACT: Extracellular vesicles (EVs) have gained attention as a mechanism facilitating intercellular as well as interkingdom communication during host-microbe interactions. Recently we showed that upon infection, host polymorphonuclear leukocytes produce antifungal EVs targeting the clinically important fungal pathogen Aspergillus fumigatus; however, the small size of EVs complicates their tracking and functional analysis. We employed a more tractable, reporter-based system to label host alveolar epithelial cell-derived EVs and enable their visualization and tracking during in vitro A. fumigatus interaction. Proteome analysis of the cargo of these EVs confirmed the EV marker proteins, tetraspanins CD63, CD9, and CD81, and revealed limited antifungal components, which was corroborated by mito-GFP fungal killing assays. Fusion of the EV marker proteins with a nanoluciferase (NLuc) and a green fluorescent protein (GFP) facilitated their relative quantification by luminescence and visualization by fluorescence signal. The use of an NLuc fused with a GFP is advantageous as it allows quantification and visualisation of EVs simultaneously without additional external manipulation. Using this system, visualisation of EVs was possible using confocal laser scanning microscopy and revealed the ability of host cell-derived EVs to associate with A. fumigatus hyphae. In conclusion, we have created a series of tools to better define the complex interplay of host-derived EVs with microbial pathogens.

INSTRUMENT(S): Q Exactive Plus

ORGANISM(S): Homo Sapiens (human) Aspergillus Fumigatus Cea17

TISSUE(S): Extracellular Vesicle

SUBMITTER: Thomas Krüger  

LAB HEAD: Axel A. Brakhage

PROVIDER: PXD050398 | Pride | 2024-11-13

REPOSITORIES: Pride