Functional properties of Candida albicans extracellular vesicles released in the presence of the antifungal drugs amphotericin B, fluconazole and caspofungin

The prevalence of diseases caused by pathogenic fungi of the Candida genus is currently a significant problem, particularly due to the emerging antifungal drug resistance and increasing number of immunocompromised individuals susceptible to opportunistic infections. Recently, it has been shown that fungal extracellular vesicles (EVs) – nanometre-sized structures of cellular origin, equipped with varied cargo enclosed by lipid bilayer – may play a vital role in the response of pathogen cells to antifungal treatment. In this work, we demonstrated that Candida albicans yeast cells grown at the subinhibitory concentrations of three commonly used antifungal drugs – amphotericin B, fluconazole and caspofungin – released a greater number of EVs than fungal cells grown under drug-free conditions. Moreover, these EVs exhibited some variability in size and protein composition, yet they consistently induced the production of the pro-inflammatory cytokine IL-8 by THP-1 macrophage-like cells at levels comparable to control EVs. In studies using the invertebrate model organism Galleria mellonella, EVs released by cells exposed to antifungals did not cause a significant increase in larval mortality, similar to control EVs, although they triggered a remarkably lower activation of phenol oxidase in larval haemolymph. In addition, EVs produced in the presence of caspofungin interacted more noticeably with the membrane of U-937 pro-monocytic cells as indicated by measurements of zeta potential changes. Furthermore, tested EVs contributed to increased tolerance of C. albicans cells to the antifungal drugs. These observations underscore the unmissable role of EVs in the response of pathogen cells to antifungal treatment and highlight the importance of understanding EV functionalities in antifungal resistance.