Characterization and functional exploration of extracellular vesicles secreted by Aspergillus fumigatus

Abstract

Aspergillus fumigatus is a human-pathogenic fungus that causes a range of diseases from superficial to invasive infections, particularly in immunocompromised individuals. However, effectiveness of antifungal treatment is limited and no preventive vaccines are available. Fungal extracellular vesicles (EVs) contain various bioactive components including proteins, nucleic acids, polysaccharides and lipids, which play roles in different physiological and pathological processes. There is growing interest in investigating their applications in host-pathogen interactions, theragnostic biomarkers, drug delivery systems, and vaccine platforms. This raises the hypothesis that A. fumigatus may release EVs with similar properties, which could have potential biological implications. To confirm the prevalence of and characterize EVs from Aspergillus spp., the first critical step is to optimize EVs isolation methods using suitable culture medium without contaminating EVs. In this study, EVs were found to be pre-existing in many commonly used microbial culture media, likely originating from the medium components. These medium-associated EVs exhibited similar morphology and size distribution to microbial EVs as observed through Transmission Electron Microscopy (TEM) and analyzed using nanoparticle counter-ZetaView. Importantly, these medium-associated EVs stimulated the expression of immune genes in host macrophages in vitro. This suggests that liquid culture medium is not suitable for the current study. Besides, classical ultracentrifugation methods for isolating EVs from culture supernatant failed to yield sufficient EVs from Aspergillus spp. To address this issue, a new solid medium-based sequential centrifugation procedure for scaling up and streamlining the isolation of Aspergillus spp. EVs was therefore established. This newly developed method enables efficient and faster isolation of Aspergillus spp. EVs with high yield. Using this method, Aspergillus spp. EVs were successfully isolated and characterized, as confirmed by TEM and ZetaView analysis. EVs were mainly secreted during the log phase rather than the stationary phase of the fungal culture. Scanning Electron Microscopy (SEM) suggested that the conidia in the log phase have rougher surfaces than the stationary phase. A. fumigatus EVs (AFEV) were purified using density gradient centrifugation and used for all downstream functional studies. Through proteomics analysis, 1620 proteins were identified in AFEV, and these proteins were classified into functional subtypes according to UniProt and GENE ONTOLOGY(GO). Both macrophage-like J774 and alveolar epithelial-like A549 cell lines internalized AFEV. AFEV was non-cytotoxic to tested cell lines (including J774, A549, THP-1, and HL60) and induced a pro-inflammatory M1-like response in immune cell lines (e.g., J774, HL60, PMA-stimulated macrophage-like THP-1), as well as alveolar epithelial cells (e.g., A549). AFEV was also found to react with sera from patients with aspergillosis. The galactomannoprotein of A. fumigatus, AFMP1P, was associated with AFEV. Furthermore, conidia were unable to germinate once being internalized by macrophages. AFEV was shown to enhance the ability of conidia to adhere to cell surrounding and promote cell death, potentially contributing to fungal virulence. These findings provide important insights for future studies in pathogenic fungal EVs and highlight the potential of AFEV as a therapeutic target for aspergillosis intervention.