Objetives: We aimed to characterize the extracellular vesicles (EVs) produced by Candida albicans stimulated by farnesol, tyrosol and oxidative stress. EVs are closed structures, surrounded by a lipid bilayer membrane, which transport cellular components to the outside environment in all kinds of prokaryotic and eukaryotic organisms. The molecules already described in fungal EVs vary from protein, lipid, polysaccharide, melanin, mRNA, and s(small)RNA. EVs are involved in cell signaling and immune modulation.
Methods: We used concentration and differential centrifugation steps to isolate EVs from culture supernatants of C. albicans cultivated for one day under shaking at 37oC in Ham’s F12 defined medium (pH 6.3) + 1,5% glucose (controls), in the presence of farnesol (50 µM), tyrosol (20 µM) or 25 mM H2O2. The stimuli conditions were optimized to evoke a stressful environment without viability loss or change in cell morphology. EVs were analyzed by nanoparticle-tracking for number and size, by microscopy for interaction with C. albicans and macrophages, and by liquid chromatography coupled with in-tandem mass spectrometry for quantitative proteomics. We used Bioanalyzer and RNA-seq (Proton platform, Ion Torrent) for characterization of the mRNA and sRNA content in both EVs and C. albicans cells for each condition in biological triplicates. 
Results: Farnesol evoked about 8-fold increase in the C. albicans EV yields and a decrease in the mean EV size when compared with controls. Tyrosol and oxidative stress had the opposite effect. We observed a total of 202 proteins in control EVs versus 203 (oxidative stress), 252 (tyrosol), and 248 (farnesol), with 61% found in all samples. Most sequences were associated with catalytic activities. The most abundant proteins were associated with cell surface and morphology. In general, the proteins were comparatively more abundant in tyrosol-derived EVs. We observed that our stimulation conditions also modulated the EV mRNA population. We identified 842 differentially represented mRNA sequences among EV and C. albicans samples. The EV sequences, although in lower number than C. albicans sequences, showed higher proportion of modulation and exclusive mRNAs. Most importantly, comparison between EV and total C. albicans transcriptome suggested that the EV population does not reflect the cell mRNA population. Differential sRNA analysis is ongoing. Uptake assays with PKH26-labelled fungal EVs showed that they were either bound to or internalized by 10% of cultured C. albicans cells. On the other hand, when C. albicans was previously incubated with fungal EVs from both control and stressed cultures, the association of fungal cells with RAW 264.7 was inhibited by 15-20% in 2-to-4-h co-cultures. EVs isolated from tyrosol-stimulated cultures accelerated C. albicans filamentation under specific co-culture conditions. 
Conclusions: Extracellular vesicles from C. albicans can apparently signal both fungal cells and macrophages. Quorum sense molecules and oxidative stress can modulate the EV size, number, proteome, and transcriptome, thus suggesting that EVs might have a fundamental role in the fungal biology and virulence.
Financial support: FAPESP, CNPq, CAPES.

1 / Farnesol, tyrosol, and oxidative stress modulate the extracellular vesicle proteome and transcriptome in Candida albicans