cells undergo programmed cell death when they are cultured in media containing either 15% or 22% ethanol [33
]. To determine if S. boulardii
also undergoes PCD, we began by comparing the viabilities of both these strains in ethanol. While the W303α strain shows almost 50% viability after three hours suspended in 22% ethanol, S. boulardii
shows less than 10% viability after growth in the same media (Figure ). Our data suggests that S. boulardii
is less viable in ethanol than this common laboratory strain of S. cerevisiae
, which is not surprising given the adaptations of brewing yeast, S. cerevisiae,
that allow it to undergo fermentation efficiently. (Note that after 3 hr, cells cultured in rich media without any cell death inducing agents were able to grow and to divide, hence the relative viability levels that are greater than 100%).
Figure 1 S. boulardii has decreased viability in ethanol, similar to S. cerevisiae.S. boulardii (Florastor) and S. cerevisiae (W303α) were cultured in rich YPD media overnight and resuspended in fresh media and allowed to reach exponential phase. They (more ...)
Next, we examined the S. boulardii
cells dying either in 15% or in 22% ethanol for markers indicative of PCD in yeast, including mitochondrial fragmentation, ROS accumulation, and caspase-like enzyme activation. As shown in Figure A, S. boulardii
cells cultured in 15% ethanol for 1.5 hr had fragmented mitochondria – punctate fluorescence rather than the tubular fluorescence normally seen in wildtype yeast cells – as revealed by MitoTracker Green staining. Cells cultured in ethanol also accumulated ROS (Figure B) and manifested a caspase-like activity as measured by a FLICA assay (Figure C). Similar findings were obtained with S. boulardii
cells cultured in 160 mM acetic acid (data not shown), another known inducer of PCD in S. cerevisiae
]. Together, these results suggest that Saccharomyces boulardii,
like Saccharomyces cerevisiae
, undergoes programmed cell death.
Figure 2 Like S. cerevisiae, S. boulardii cells undergo programmed cell death in ethanol.S. Boulardii cells were cultured in rich YPD media overnight and resuspended in fresh media and allowed to reach exponential phase. They were then resuspended either in water (more ...)
Studies have reported that only between 1-3% of live S. boulardii
yeast is recovered in human feces after oral administration [27
] as the acidic conditions disrupt cell wall function and cause morphological alterations that lead to cell death [27
]. However, the nature of this cell death in acidic environments remains unclear.
To determine the type of cell death experienced by S. boulardii
cells in an acidic environment, we began by determining the viability of S. boulardii
in low pH conditions. Our results show that S. boulardii
cells have an increased viability in acidic conditions as compared to their S. cerevisiae
counterpart. After six hours in 50 mM HCl media, W303α cells showed almost no viability, while S. boulardii
cells were more than 70% viable (Figure ). This confirms the findings of others who have shown that S. boulardii
cells are more resistant to acidic conditions than their S. cerevisiae
Figure 3 S. boulardii cells are more viable in 50 mM HCl than their S. cerevisiae counterparts. S. boulardii (Florastor) and S. cerevisiae (W303α) were cultured in rich YPD media overnight and resuspended in fresh media and allowed to reach exponential (more ...)
To determine if the S. boulardii
cells were undergoing PCD in the acidic environment, we repeated our cell death assays with cells cultured in 75 mM HCl (pH 1.5), a scenario that mimics the conditions in the stomach [48
]. DHR staining revealed that 92% of the S. boulardii
cells cultured in an acidic environment contained ROS as compared to cells grown in rich YPD media (Figure A). FLICA staining also showed that 90% of the S. boulardii
cells in the HCl solution, but only 1% of the control cell population had activated caspase-like activity (Figure B).
Figure 4 S. boulardii undergoes programmed cell death in an acidic environment. S. boulardii (Florastor) was cultured in rich YPD media overnight and resuspended in fresh media and allowed to reach exponential phase. They were then resuspended in water or water (more ...)
Finally, to better understand the mechanism of cell death at the molecular level, we generated microarray gene expression profiles of S. boulardii
cells cultured in an acidic environment. We found that a total of 947 genes were differentially expressed (log2 values greater than 2 or less that −2) of which 470 were up-regulated and 457 down-regulated (Additional file 1
). Significantly, functional annotation revealed that the up-regulated genes were significantly (p<0.0005) over-represented in cell death pathways (Figure ; Table ). One of these up-regulated cell death genes, RNY1
, encodes a RNase T2 family member that is released from the vacuole into the cytosol during oxidative stress to promote yeast cell death [49
]. Since the vacuole is the organelle most responsible for pH homeostasis in yeast [50
], this may suggest that a similar mechanism of cell death may be occurring in S. boulardii
cells cultured in an acidic environment. Finally, a significant majority of the other up-regulated cell death genes (80%) were ORFs involved in mitochondrial function, including numerous genes encoding proteins involved in the electron transport chain (Table ). These microarray results together with our characterization of the cell death phenotype described above suggest that S. boulardii
cells undergo PCD when they are cultured in acidic conditions similar to those found in the stomach.
Figure 5 Functional classification/GO analysis of differentially transcribed genes in S. boulardii cells cultured in 50 mM HCl. Genes showing 2-fold or greater increase (up-regulated) or decrease (down-regulated) in response to an acidic environment were grouped (more ...)
S. boulardii cell death genes differentially expressed in an acidic environment
Previously published work has shown that S-adenosyl-L-methionine (AdoMet) protects S. cerevisiae
from programmed cell death [34
]. To determine if AdoMet is also capable of rescuing S. boulardii
from inducers that induce PCD, we first suspended S. boulardii
cells either in 22% ethanol or in 22% ethanol containing 1 mM AdoMet, for 3 hours. We discovered that both S. cerevisiae
and S. boulardii
cells cultured in ethanol containing AdoMet had higher viabilities than cells cultured in ethanol alone (Figure A). These results suggest that AdoMet is also capable of rescuing S. boulardii
from programmed cell death.
Figure 6 AdoMet protects S. boulardii from ethanol and HCl-Induced cell death. S. boulardii cells (Florastor) were cultured in rich YPD media overnight and resuspended in fresh media and allowed to reach exponential phase. (A) They were then resuspended in fresh (more ...)
Next, we wanted to determine if AdoMet could also rescue S. boulardii cells undergoing HCl-induced programmed cell death. As shown in Figure B, the viability of Florastor cells cultured in an acidic environment was significantly enhanced in the presence of 2 mM AdoMet. Next, we showed that 2 mM AdoMet decreased both ROS generation (Figure C) and caspase activation (Figure D) in S. boulardii cells cultured in 50 mM HCl suggesting that this supplement may enhance cell viability by preventing programmed cell death.