Human Neutrophils Produce Antifungal Extracellular Vesicles against Aspergillus fumigatus

Abstract

Polymorphonuclear granulocytes (PMNs) are indispensable for controlling life-threatening fungal infections. In addition to various effector mechanisms, PMNs also produce extracellular vesicles (EVs). Their contribution to antifungal defense has remained unexplored. We reveal that the clinically important human-pathogenic fungus Aspergillus fumigatus triggers PMNs to release a distinct set of antifungal EVs (afEVs). Proteome analyses indicated that afEVs are enriched in antimicrobial proteins. The cargo and the release kinetics of EVs are modulated by the fungal strain confronted. Tracking of afEVs indicated that they associated with fungal cells and even entered fungal hyphae, resulting in alterations in the morphology of the fungal cell wall and dose-dependent antifungal effects. To assess as a proof of concept whether the antimicrobial proteins found in afEVs might contribute to growth inhibition of hyphae when present in the fungal cytoplasm, two human proteins enriched in afEVs, cathepsin G and azurocidin, were heterologously expressed in fungal hyphae. This led to reduced fungal growth relative to that of a control strain producing the human retinol binding protein 7. In conclusion, extracellular vesicles produced by neutrophils in response to A. fumigatus infection are able to associate with the fungus, limit growth, and elicit cell damage by delivering antifungal cargo. This finding offers an intriguing, previously overlooked mechanism of antifungal defense against A. fumigatus. IMPORTANCE Invasive fungal infections caused by the mold Aspergillus fumigatus are a growing concern in the clinic due to the increasing use of immunosuppressive therapies and increasing antifungal drug resistance. These infections result in high rates of mortality, as treatment and diagnostic options remain limited. In healthy individuals, neutrophilic granulocytes are critical for elimination of A. fumigatus from the host; however, the exact extracellular mechanism of neutrophil-mediated antifungal activity remains unresolved. Here, we present a mode of antifungal defense employed by human neutrophils against A. fumigatus not previously described. We found that extracellular vesicles produced by neutrophils in response to A. fumigatus infection are able to associate with the fungus, limit growth, and elicit cell damage by delivering antifungal cargo. In the end, antifungal extracellular vesicle biology provides a significant step forward in our understanding of A. fumigatus host pathogenesis and opens up novel diagnostic and therapeutic possibilities.

Extracellular Vesicles in Fungi: Past, Present, and Future Perspectives

“…EVs released by neutrophils exposed to conidia had antifungal properties and inhibited the growth of A. fumigatus hyphae. The same outcome was not observed for EVs released by uninfected neutrophils (Shopova et al, 2020). These data suggest that EV release by mammalian cells represent a still unexplored mechanism of antifungal defense during host-pathogen interactions.…”

“…EVs produced by germinating conidial protoplasts increased in number and differed in cargo when the cells were incubated under cell wall regeneration conditions (Rizzo et al, 2020). Finally, another recent study demonstrated that polymorphonuclear granulocytes produce EVs that associate with the cell wall of A. fumigatus and even enter fungal hyphae, resulting in alterations in the morphology of the fungal cell wall (Shopova et al, 2020). These studies highlight the cell wall as a dynamic structure with flexible viscoelastic properties and provide new insights into how vesicles can cross the cell wall to reach the extracellular space.…”

RevisitingCryptococcusextracellular vesicles properties and their use as vaccine platforms

“…In mammalian cells, two major classes of EVs have been defined, microvesicles and exosomes, according to their size and cellular origin (Meldolesi 2018, van Niel et al 2018. In these organisms, a large body of literature describes how EVs participate in intercellular signaling within an organism but also in organism-toorganism communication, including carcinogenesis and host-pathogen interactions (Xu et al 2018, Shopova et al 2020. In fungi, the first report of fungal EVs was published in 2007 (Rodrigues et al 2007), and, since then, their existence has been reported in many species of pathogenic and nonpathogenic fungi .…”

Immune modulation by complement receptor 3-dependent human monocyte TGF-β1-transporting vesicles

“…Finally, like monocytes, the vesicles also expressed CD11b and CD14. EV formation in response to C. albicans 32 and Aspergillus fumigatus 33 was recently also reported. However, experimental conditions were substantially different in these studies regarding cell type and incubation time.…”