FIG 1.

EVs from C. albicans inhibit biofilm formation and yeast-to-hyphae differentiation. Both protocols described in Materials and Methods for EV isolation were used with identical results. The results presented here were obtained from C. albicans 90028 EVs (yeast cells) isolated from liquid culture medium. (A) C. albicans (90028) (10⁵ yeasts) were inoculated into RPMI-MOPS in the presence or absence of C. albicans 90028 EVs. Different concentrations of EVs (0.5, 1, and 5 μg/mL, based on sterol content) were added to wells containing C. albicans (90028) yeasts, and cells were incubated to develop biofilm for 24 and 48 h in 96-well microplates. (B) C. albicans (90028) (10⁵ yeasts) were inoculated into RPMI-MOPS and incubated for 90 min. The nonadherent cells were washed out, and fresh medium containing C. albicans 90028 EVs (0.5, 1, and 5 μg/mL, based on sterol content) was added. PBS was used as control. Biofilm formation was quantified by crystal violet method. Group comparisons were submitted to one-way analysis of variance (ANOVA) with Dunnett’s correction (**, P < 0.002; ***, P < 0.001; ****, P < 0.0001). (C and D) For SEM, C. albicans (strain 90028) yeast cells, untreated (C) or treated with C. albicans EVs (5 μg/mL) (D), were inoculated onto coverslips previously coated with poly-l-lysine in RPMI-MOPS and incubated for 24 h. SEM images show the effect of EVs after 24 h of growth. Insets in panels C and D depict higher magnification of each condition. Bar, 30 μm. (E) C. albicans (90028) yeasts were inoculated into M199 medium to induce filamentation in the presence or absence of C. albicans 90028 EVs (5 μg/mL, based on sterol content). Cellular density was monitored, and the pictures represent the minutes of incubation (from 100 to 800 min). Bar, 30 μm. Growth under control conditions (absence of C. albicans 90028 EVs) and in the presence of C. albicans 90028 EVs. The numbers indicate the time in minutes. Bar, 30 μm. (F) C. albicans (90028) yeasts were inoculated into M199 medium to induce filamentation in the presence or absence of EVs (5 μg/mL, based on sterol content). SEM of cells after 24 h showing the hyphae and yeasts under control conditions (a and a′) in contrast with C. albicans 90028 EVs that led to the presence of pseudohyphae with multiple budding sites and yeasts (b and b′).