Saccharomyces cerevisiae show low levels of traversal across human endothelial barrier in vitro

Yeast strains and growth media

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.

Growth of mammalian cells

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 cm² culture flasks (TPP) at 37°C in a humidified atmosphere of 5% CO₂ and 95% air until a confluence. Culture medium was changed every second day.

Trans-epithelial electrical resistance (TEER) assay

HUVEC cells (1×10⁵ cells/cm²) 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 (Ωcm²) was corrected for background resistance by subtracting the contribution of the cell-free filter and the medium (110 Ωcm²). The TEER was measured before the addition of yeasts.

Determination of permeability coefficient

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 = (ΔA_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 cm² 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.

Ability to cross the endothelial barrier

HUVEC cells were seeded on Transwell® filter as described above. Yeasts grown overnight at 30°C in YPD were resuspended (10⁶ cells mL^–1) in the apical compartment and incubated at 37°C in a humidified atmosphere of 5% CO₂ 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.

Evaluation of the endothelial barrier integrity

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 Ωcm², 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 permeability³⁹.

Study of the ability of yeast species to cross the human endothelial barrier in vitro

To determine whether S. cerevisiae is able to cross the human endothelial barrier, we used an in vitro model of the endothelium with HUVECs⁴⁰. 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 Log₁₀ units). On the contrary, we observed that S. cerevisiae showed significantly lower levels (1.0–3.3 Log₁₀ units) than the Candida species.

Figure 1. Number of yeast cells that were able to cross the endothelial barrier.

To perform this assay we established HUVEC monolayers in Transwell® filter inserts in 24 well plates. 24 hours after apical addition of various strains of S. cerevisiae, C. albicans and C. glabrata, yeast cells from the basolateral compartment were incubated on YPD plates and colonies were counted after one day of growth. Values were obtained after plating several dilutions of the basolateral compartment media. Average of three experiments and standard deviation is shown. To determine statistically significant data, Student t-tests were performed in Excel with 0.05 as the p-value.

Figure 2. Box graph comparing the number of cells able to cross the endothelial barrier in the three yeast species.