Extracellular vesicles released by Candida albicans yeast cells impact candidiasis lethality in insect and murine models

ABSTRACT Extracellular vesicles (EVs) are nano-sized lipid bilayer structures released by diverse cell types. For pathogenic fungi, EVs play critical roles by modulating immune responses and influencing disease progression. Previously, we demonstrated that vaccination with EVs from Candida albicans promoted protection in immunosuppressed mice subsequently infected with lethal doses of this organism. However, the impact of EVs in the presence of fungi is poorly investigated in vivo . Here, we demonstrated that co-injection of C. albicans yeasts and their EVs into Galleria mellonella larvae led to a rapid larval death compared to yeast cells alone. This effect seems to be associated with thermosensitive compounds, such as secreted aspartic proteases (Saps), virulence factors carried by C. albicans EVs. EVs from SAP knockout (KO) strains ( sap1 / 2 / 3 Δ and sap9 Δ) impaired this harmful effect, confirming the crucial role of Saps. Deletion of SAP1–3 prevented the EV-associated decrease in hemocyte density, whereas SAP9 deletion impaired the increase in oxidative response and melanin production. In a murine model of disseminated candidiasis with intraperitoneal infection, the deleterious effect of C. albicans EVs was observed only when co-injected intraperitoneally with yeasts, but not when EVs were administered intramuscularly. Therefore, both fungal cells and EVs are required for the pathogenic effects. Our findings confirm the aggressive role of fungal EVs in the presence of C. albicans and reinforce their contribution to candidiasis, elucidating EV–pathogen interactions. Although these results do not preclude the use of C. albicans EVs in vaccine formulations, they emphasize the need to consider delivery route and presence of virulence factors. IMPORTANCE Fungal infections caused by Candida albicans represent a significant threat, particularly to immunocompromised individuals. Extracellular vesicles (EVs) from fungi have been explored as promising vaccine formulations, with demonstrated protective effects. However, previous studies have also suggested that EVs may contribute to disease progression. Here, we demonstrate that EVs enhance fungal pathogenicity in vivo and identify key factors driving this effect. We show that EV-associated aspartyl proteases (secreted aspartic proteases [Saps]), along with other virulence components, directly contribute to accelerated host mortality when EVs are present alongside fungal cells, and that this outcome depends on both their composition and route of delivery. By demonstrating the involvement of EV-associated Saps in disease progression, this work advances our understanding of host–pathogen interactions and reveals critical considerations for the safe development of EV-based therapeutic and vaccine strategies.