Structural and glycosylation characterization of extracelular vesicles released by clinical isolates of Candida albicans

Candida albicans is an opportunistic fungal pathogen commonly present in the human microbiota. It is responsible for causing candidiasis, which can be lethal in immunocompromised patients. The cell wall of C. albicans consists of an outer layer that is made up of highly mannosylated proteins, which are associated with fungal adherence, drug resistance, and virulence. Together with other fungal pathogens, C. albicans release extracellular vesicles (EVs), lipid nanoparticles enclosing a diverse molecular cargo and reported to play an important role in cell-to-cell communication. C. albicans EVs bear a fibrillar decoration known to be mainly composed by mannoproteins. These EVs can activate phagocytic cells, such as macrophages and dendritic cells, and can serve as vaccine platforms in mice models. The search for alternative treatments for candidiasis is crucial due to toxicity related to the current used antifungal agents and the absence of vaccines for fungal infections. This study aims to analyze the EV morphological and compositional properties, mainly concerning the mannoproteins present on EVs of different C. albicans clinical isolates (granted from the Instituto de Controle de Qualidade em Saúde-INCQS) and their potential to stimulate immune cells. EVs were obtained by inoculating the solid media with 300 µL of a cell suspension containing 3.5 x 107 cells/mL. After 24 h of incubation at 30°C, the cells were gently harvested in a sterile tube containing 20 mL of 0.22 µm-filtered PBS. The cell suspensions were centrifuged at 5,000 x g for 15 min at 4°C, and the supernatant was transferred to another sterile tube and centrifuged at 15,000 x g for the same time and temperature. Subsequently, the supernatant was filtered through a 0.45 µm filter and centrifuged at 100,000 x g for 1 h at 4°C. Once the supernatant was discarded, the resulting EV pellet was suspended in filtered PBS and stored at -80°C. The main analyses included protein and lipid measurements, transmission electron microscopy, Nanoparticle Tracking Analysis (NTA), and Western Blot. Our data indicate that EV obtained from clinical isolates exhibit peculiar characteristics concerning size and glycosylation patterns. Future assays, including EV proteomic analysis and immunoassays with dendritic cells, will be crucial in elucidating the potential biological implications of this EV heterogeneity in C. albicans infections.