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

Background:   Saccharomyces cerevisiae is generally considered safe, and is involved in the production of many types of foods and dietary supplements. However, some isolates, which are genetically related to strains used in brewing and baking, have shown virulent traits, being able to produce infections in humans, mainly in immunodeficient patients. This can lead to systemic infections in humans.
Methods: In this work, we studied S. cerevisiae isolates in an in vitro human endothelial barrier model, comparing their behaviour with that of several strains of the related pathogens Candida glabrata and Candida albicans.
Results: The results showed that this food related yeast is able to cross the endothelial barrier in vitro. However, in contrast to C. glabrata and C. albicans, S. cerevisiae showed very low levels of traversal. Conclusions: We conclude that using an in vitro human endothelial barrier model with S. cerevisiae can be useful to evaluate the safety of S. cerevisiae strains isolated from foods.

Introduction

Saccharomyces cerevisiae is generally considered safe, and is involved in the production of a variety of foods and dietary supplements. Several types of food and beverage still contain viable yeast cells [1– 5]. However, in the last years human infections with Saccharomyces cerevisiae have increased [6– 8]. Consequently, S. cerevisiae is considered an emerging pathogen [9– 11]. Different parts of the body can be affected in immunocompromised [12– 15] and healthy patients [16– 18]. The potential virulence of this yeast has been analysed with different methods in vitro [19– 22] and in vivo [23– 27], for example by measuring epithelial barrier traversal [28]. These reports have suggested that certain strains can cause disease and death in murine models. However, the bio-therapeutic agent Ultralevure ( S. cerevisiae var. boulardii) and other supplements are consumed in high doses, ranging from 10 7 to 10 10 live yeast cells per day and for long periods.

The study of yeast virulence includes studying their behaviour when they encounter endothelial barriers. Opportunistic pathogenic yeasts such as C. glabrata and C. albicans are able to pass the intestinal barrier [29, 30] and generate systemic infections [31– 33]. Also, C. albicans can cross the blood-brain barrier (BBB) to reach the brain [34, 35]. Regarding S. cerevisiae, infections after oral ingestion [16] or digestive translocation [12, 14, 36] show that it can reach brain in murine models [25]. However, few studies have investigated the behaviour of S. cerevisiae when they reach endothelial barriers [28].

Methods

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.

Table 1.

Yeast strains used in this study.

Strain	Species	Source
W303	S. cerevisiae	From our collection
102	S. cerevisiae	Vall d’Hebron Hospital (Barcelona, Spain) [19]
60	S. cerevisiae	Vall d’Hebron Hospital (Barcelona, Spain) [19]
Cb	S. cerevisiae	Vall d’Hebron Hospital (Barcelona, Spain) [19]
Co	C. glabrata	Vall d’Hebron Hospital (Barcelona, Spain)
C2	C. glabrata	Provided by B. Hube (Friedrich Schiller University; Jena, Germany)
C4	C. glabrata	Provided by B. Hube (Friedrich Schiller University; Jena, Germany)
C5	C. glabrata	Provided by B. Hube (Friedrich Schiller University; Jena, Germany)
CA-1	C. albicans	Statens Serum Institute (Copenhagen, Denmark)
SC5314	C. albicans	Provided by A. Yañez [22] (Universitat de Valencia, Spain)
ATCC26555	C. albicans	Provided by A. Yañez [22] (Universitat de Valencia, Spain)
CBS562	C. albicans	From our collection

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 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.

Trans-epithelial electrical resistance (TEER) assay

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.

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 = (Δ A /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.

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 (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.

Results

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 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].

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 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.

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.

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.

Figure 2.

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

Click here for additional data file.

Discussion

A model for traversal across the e in vitro has been used to study behaviour and pathogenicity mechanisms of yeast strains such as C. albicans [34, 35]. Here, we have shown that S. cerevisiae strains are able to cross the endothelial barrier. This data is in accordance with previous studies, where S. cerevisiae cells were observed in the brain after systemic infections in murine models [25]. When comparing to other well-known yeast pathogens such as C. glabrata and C. albicans, none of the S. cerevisiae strains were able to cross the endothelial barrier at high levels. Despite S. cerevisiae pathogenicity levels being lower than other opportunistic yeasts, we recommend the potential risk of new S. cerevisiae strains to be evaluated before using them in food production.

Data availability

The data referenced by this article are under copyright with the following copyright statement: Copyright: © 2017 Pérez-Torrado R and Querol A

Data associated with the article are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).

Dataset 1: Raw data of permeability measurements and cell counts for endothelium traversal. DOI, 10.5256/f1000research.11782.d177554 [41]

By stating that their model is an endothelial barrier in vitro model rather than a blood-brain barrier model, the authors have addressed my main concern. Considering that they submitted this work as a 'Note', defined as a small, often preliminary study, I believe it is suitable for indexing at F1000. I, nevertheless, encourage them to consider my previous minor comments on their follow up studies.

I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

Although the article is interesting and the data clear, I believe the authors are overstating the findings. First, HUVECs are not considered a good model for blood-brain barrier anymore. They used to be a favorite one because they are a human cell line, however, they are not of cerebral origin, and deviate considerably from the behavior of cerebral endothelial cells. They could fix this by calling their model an "endothelial" monolayer instead, or repeat the experiment using "real" BBB cell lines (i.e. hCMEC/D3, which is commercially available). Also, they report the TEER values (which by the way the correct units should be resistance (ohms) times area (cm 2) rather than dividing by it) before the start of the experiment, but they should also measure the integrity of the monolayer at the end of the experiment, to rule out that the amount of S. cerevisiae crossing is due to rupture of the monolayer. This assay is also hard to interpret in the absence of a negative control - in fact, S. cerevisiae has been traditionally used as a negative control on this type of assays! Would inert beads also cross? Would any other organisms cross at the same rate? Maybe they can check this by using fluorescent beads and measuring fluorescence on the bottom. Or if easier to do by CFUs, they could add another organism known to not been able to cross and count CFUs. Overall, it is a nice preliminary report, one worth the time pursuing. Considering this was submitted as a Research Note, I believe is appropriate for indexing once they address my comments above.

I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

Introduction:

I would consider to change the sentence “Consequently, S, cerevisiae is considered an emerging pathogen” with “Consequently, S, cerevisiae is considered an emerging pathogen of low virulence”

Origin of isolation of the yeast strains could be included in Table 1.

Methods:

Abbreviation of BBB should be added in the title Ability to cross the blood-brain barrier.

Results:

In Figure 1 there are different colours but not information about the meaning of it has been included. In Figure 2, there is a mistake for C. glabrata and C. albicans, they are named as S.glabrata and S.albicans.

I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.