Results and Discussion Saccharomyces GS-1101 purchase cerevisiae 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 RG7112 clinical trial in the same media (Figure 1). 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 were then resuspended in fresh media or in fresh media containing 22% ethanol, allowed to grow at 30°C for the indicated times, serially diluted onto YPD plates, and cultured at 30°C for 2 days. Viability was measured as percentage colony forming units. At least three independent selleck kinase inhibitor cultures were tested and compared. 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%. The differences in viabilities were deemed

statistically significant by the Student’s t-test (p<0.05) 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 2A, 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 Aspartate ROS (Figure 2B) and manifested a caspase-like activity as measured by a FLICA assay (Figure 2C). 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 [46, 47]. 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.