The yeast strains are described in Table 1. Strains were propagated in YPD media (1% glucose, 1% BactoPeptone, 0.5% yeast extract) for 24 h at 30°C.

Human umbilical endothelial cells (HUVECs) (Clonetics®) were grown in minimum essential medium (Earle’s salt, 25 mM HEPES and GlutaMAX™, Invitrogen) supplemented with 10% foetal bovine serum (FBS, Cambrex Bio Science), 1% nonessential amino acids (Invitrogen) and 50 μg mL ^–1 gentamicin (Invitrogen). The cells were grown in 150 cm ² culture flasks (TPP) at 37°C in a humidified atmosphere of 5% CO ₂ and 95% air until a confluence. Culture medium was changed every second day.

HUVEC cells (1×10 ⁵ cells/cm ²) were seeded on Transwell® filter inserts (8 μm, Corning Incorporated) in 24-well plates (Corning Incorporated). A volume of 200 μL cell growth medium was added to the apical compartment and 1250 μL to the basolateral compartment. The TEER was measured using the Millicell-ERS Electrical Resistance System (Millipore). The net value of the TEER (Ωcm ²) was corrected for background resistance by subtracting the contribution of the cell-free filter and the medium (110 Ωcm ²). The TEER was measured before the addition of yeasts.

1 μg/mL of fluorescein (Sigma) was added to the media in the apical compartment of the transwell, with or without established HUVEC monolayers, and fluorescence was measured over time in the media of the apical and basolateral compartment. The apparent permeability, Papp, was defined as (Hilgers et al., 1990):

                                                                                                 Papp = (Δ A _R /Δt))/ C _D,0                                                                                                (1)

(ΔA _R/Δt) is the rate of drug appearance in the receiver side, S is the surface area of the Transwell (0.33 cm ² for Transwell® inserts (8 μm pore size, Corning) of 6.5-mm insert diameter), and C _D,0 is the initial drug concentration in the donor side at time = 0. Values are expressed in cm/s.

HUVEC cells were seeded on Transwell® filter as described above. Yeasts grown overnight at 30°C in YPD were resuspended (10 ⁶ cells mL ^–1) in the apical compartment and incubated at 37°C in a humidified atmosphere of 5% CO ₂ and 95% air. After 12 h, the basolateral compartment medium was replaced. Colony forming units were counted in YPD plate triplicates after two days. Control wells used to evaluate yeast growth showed no significant growth after 12 h. Negative control HUVEC Transwells without adding cells were performed to control TEER stability across the experiment.

To establish an in vitro human endothelial barrier, we used HUVEC monolayers, a methodology that has been widely used ^{37, 38} . Monolayers were formed in transwells and two different methods were used to determine the robustness, consistency and integrity of the barrier. First, we studied the TEER, indicative of physical separation. After seeding the HUVECs, TEER was measured and we observed increased values over time that were overcoming 450 Ωcm ², which correlates with the establishment of a monolayer barrier. Second, we studied the monolayer permeability. The value obtained was 1.82±0.13 (10 ^-6 cm/s) on average, which indicates an integral barrier with low permeability ³⁹ .

To determine whether S. cerevisiae is able to cross the human endothelial barrier, we used an in vitro model of the endothelium with HUVECs ⁴⁰ . The number of cells in the basolateral compartment was measured 12 hours after addition of S. cerevisiae, C. albicans and C. glabrata strains to the apical compartment ( Figure 1). The results showed that all yeast strains were able to cross the endothelial barrier. While elevated number of cells from C. glabrata and C. albicans strains were able to cross the endothelial barrier, S. cerevisiae values were low. Furthermore, while the S. cerevisiae control strain W303 showed the lowest levels of yeast transcytosis, the other opportunistic pathogenic strains presented higher levels.

To compare the different species, the average level of cell transcytosis for all strains of each species was calculated ( Figure 2). After 12 h, Candida species showed a high number of cells in the basolateral chamber (4.9–5.7 Log ₁₀ units). On the contrary, we observed that S. cerevisiae showed significantly lower levels (1.0–3.3 Log ₁₀ units) than the Candida species.