Table 1.
Yeast strains used in the investigation.
Figure 1.
Comparison of the probiotic potential of S. cerevisiae var. boulardii and S. cerevisiae 1-1 strain.
(A) Experimental design. Low doses of DSS were used for 2 weeks followed by a 3-day restitution period. Either S. cerevisiae strains or β-glucan fractions were inoculated daily by oral gavage starting 3 days after C. albicans challenge. Three days post-C. albicans challenge was chosen to start S. cerevisiae treatment as both Candida colonization and colonic inflammation are well established at this time point. (B) Percentage survival of mice. The results are shown as percent survival from the time of C. albicans challenge and DSS treatment. The survival data were significantly different by the log-rank test. A total of 140 mice were divided into five control groups: water (n = 16 mice), Ca (n = 16 mice), Sb (n = 16 mice), Sc1-1 (n = 16 mice) and DSS alone (n = 16 mice), and three experimental groups: CaD (n = 20 mice), CaDSb (n = 20 mice), and CaDSc1-1 (n = 20 mice). (*P<0.05 for CaDSb mice vs. CaD mice; and ‡P<0.05 for CaDSc1-1 mice vs. CaD mice.) (C) Number of C. albicans colony forming units recovered from stools. Each data set represents the mean values of 10 mice/group. (*P<0.05 for CaDSb mice vs. CaD mice; and ‡P<0.05 for CaDSc1-1 mice vs. CaD mice.) (D) Clinical analysis of DSS-induced colitis in mice. Both S. cerevisiae var. boulardii and S. cerevisiae Sc1-1 significantly reduced the clinical score. (*P<0.05 for CaDSb mice vs. D mice; ‡P<0.05 for CaDSb mice vs. CaD mice; †P<0.05 for CaDSc1-1 mice vs. D mice; and **P<0.05 for CaDSc1-1 mice vs. CaD mice.) (E) Histological score. The histological score was determined by two independent, blinded examiners (degree of inflammation: 0, no changes, to 6, extensive cell infiltration and tissue damage). The histological score increased in both DSS and CaDSS groups. The histological score was significantly lower in both CaDSb and CaDSc1-1 groups when compared to CaDSS and DSS-treated groups. (*P<0.05 for CaDSb mice vs. D mice; ‡P<0.05 for CaDSb mice vs. CaD mice; †P<0.05 for CaDSc1-1 mice vs. D mice; and **P<0.05 for CaDSc1-1 mice vs. CaD mice.) (F and G) Relative expression levels, determined by real-time quantitative PCR, of TNF-α and IL-10 mRNA in the colon. Data are expressed as the mean ± SE of five mice in each group. (*P<0.05 for CaDSb mice vs. D mice; †P<0.05 for CaDSb mice vs. CaD mice; ‡P<0.05 for CaDSc1-1 mice vs. D mice; and **P<0.05 for CaDSc1-1 mice vs. CaD mice.)
Figure 2.
Histological analysis of DSS-induced colitis in mice.
Colon sections (4 µm thick) were stained with haematoxylin/eosin. Panel (a and b) show colon sections from a mouse colonized with C. albicans and treated with DSS; panel (c) shows colon sections from a C. albicans+Sc1-1 DSS-treated mouse; and panel (d), colon sections from a C. albicans+Sb DSS-treated mouse. Colon sections from the C albicans+DSS-treated mouse showed an inflammatory cell infiltrate in colonic wall structures (arrow, asterisk). Colon sections from the mouse with DSS-induced colitis colonized with C. albicans+either Sb or Sc1-1 showed an attenuated inflammatory infiltrate in the submucosa with the presence of occasional leukocytes in the lamina propria of the mucosa (panels c and d). The scale bars represent 50 µm (panels a, c, and d) and 10 µm (panel b).
Figure 3.
Assessment of biological effect of S. cerevisiae 1-1 versus other S. cerevisiae strains.
(A) Percentage survival of mice. Results are shown as percent survival from the time of C. albicans challenge and DSS treatment. Three days after C. albicans challenge, mice were given one of five S. cerevisiae strains: Sc1-1, Sc1-2, Sc2, Sc3 or Sc4 for 2 weeks. The survival data were significantly different by the log-rank test. A total of 60 mice were divided into six experimental groups: CaD (n = 10 mice), CaDSc1-1 (n = 10 mice), CaDSc1-2 (n = 10 mice), CaDSc2 (n = 10 mice), CaDSc3 (n = 10 mice) and CaDSc4 (n = 10 mice). (*P<0.05 for CaDSc3 mice vs. CaD mice; and ‡P<0.05 for CaDSc1-1 mice vs. CaD mice.) (B) Number of C. albicans colony forming units recovered from stools. Each data set represents the mean values of eight mice per group. (*P<0.05 for CaDSc3 mice vs. CaD mice; and ‡P<0.05 for CaDSc1-1 mice vs. CaD mice.) (C) Clinical analysis of DSS-induced colitis in mice. Clinical score was determined by assessing weight loss, change in stool consistency and the presence of gross bleeding. The clinical score ranged from 0 to 8. (*P<0.05 for CaDSc1-1 mice vs. CaD mice; ‡P<0.05 for CaDSc1-2 mice vs. CaD mice; †P<0.05 for CaDSc2 mice vs. CaD mice; ∂P<0.05 for CaDSc3 mice vs. CaD mice; and ¶P<0.05 for CaDSc3 mice vs. CaD mice.) (D) Histological score. Mice were exposed to 1.5% DSS in drinking water for 14 days. Degree of inflammation: 0, no changes, to 6, extensive cell infiltration and tissue damage. In CaDSc1-1 and CaDSc3 groups, the histological score was significantly lower than that of CaD mice. Sc4 administration dramatically increased the histological score in Candida DSS-treated mice. (*P<0.05 for CaDSc1-1 mice vs. CaD mice; ‡P<0.05 for CaDSc1-2 mice vs. CaD mice; †P<0.05 for CaDSc2 mice vs. CaD mice; ∂P<0.05 for CaDSc3 mice vs. CaD mice; and ¶P<0.05 for CaDSc3 mice vs. CaD mice.)
Figure 4.
Histological analysis of DSS-induced colitis in mice.
Panels (a), (c), (d) and (e) correspond to colon sections from CaDSc1-2, CaDSc2, CaDSc3 and CaDSc4 mice, respectively. The colon sections from CaDSc1-2, CaDSc2 and CaDSc4 mice showed an inflammatory cell infiltrate in colonic wall structures (asterisks, panels b and f) and tissue destruction with loss of both crypts and epithelial integrity (panels a, c and e). The colon sections from CaDSc3 revealed a lower inflammatory cell infiltrate in colonic wall structures and restoration of tissue destruction (panel D). The scale bars represent 50 µm (panels a, c, d and e) and 10 µm (panels b and f).
Figure 5.
Schematic diagram of glycan preparation from S. cerevisiae.
Figure 6.
Effect of glycan fractions derived from S. cerevisiae on Candida DSS-treated mice.
(A) Percentage survival of mice. The results are displayed as percent survival from the time of C. albicans challenge and DSS treatment. Three days after C. albicans challenge, mice were given either S. cerevisiae (Sc2), mannoproteins (Sc2MP) or β-glucan fractions (Sc2GP) for 2 weeks. The survival data were significantly different by the log-rank test. A total of 40 mice were divided into four experimental groups. (*P<0.05 for CaDSc2GP mice vs. CaD mice; and ‡P<0.05 for CaDSc2MP mice vs. CaD mice.) (B) Difference in body weight loss in mice. Each data set represents the mean value for each body weight. (*P<0.05 for CaDSc2GP mice vs. CaD mice, and †P<0.05 for CaDSc2MP mice vs. CaD mice.) (C) Number of C. albicans colony forming units recovered from stools. Each data set represents the mean value of 10 mice/group. (*P<0.05 for CaDSc2GP mice vs. CaD mice.) (D) Histological analysis of DSS-induced colitis in mice. Panel (a) shows a colon section from a C. albicans+mannoprotein DSS-treated mouse; panel (b) shows a colon section from a C. albicans+β-glucan DSS-treated mouse. Inflammatory cell infiltrates were insignificant in both CaDSc2MP and CaDSc2GP mice. (E) Clinical analysis of DSS-induced colitis in mice. Clinical score was determined by assessing weight loss, change in stool consistency and the presence of gross bleeding. The clinical score ranged from 0 to 8. (*P<0.05 for CaDSc2 mice vs. CaD mice; †P<0.05 for CaDSc2MP mice vs. CaD mice; and ‡P<0.05 for CaDSc2GP mice vs. CaD mice.) (F) Histological score. The histological score decreased significantly in CaDSc2GP mice. (*P<0.05 for CaDSc2 mice vs. CaD mice; †P<0.05 for CaDSc2MP mice vs. CaD mice; and ‡P<0.05 for CaDSc2GP mice vs. CaD mice.)
Figure 7.
Schematic diagram of β-glucan preparation from C. albicans.
Figure 8.
Immunofluorescence staining of C. albicans ghost cells and zymosan with various fluorescent probes specific for yeast cell wall glycans.
C. albicans ghost cells and zymosan were stained with an anti-chitin WGA monoclonal antibody (mAb) (a and b), anti-β-1,3 glucan (c and d), GNL (e and f), ConA (g and h) and DAPI (i and j), respectively. C. albicans ghost cells (b and d) and zymosan (a and c) were strongly stained with both anti-chitin WGA and anti-β-1,3 glucan mAb. Strong immunofluorescent staining was detected in zymosan using GNL and ConA (e and g), whereas no immunofluorescent signals were observed in C. albicans ghost cells (f and h). While zymosan was strongly stained with nuclear DAPI (i), only a weak signal was observed in C. albicans ghost cells (j).
Figure 9.
Structural analysis of glycan fraction.
(A) MALDI-MS analysis of the native fraction. Native glycan consisted of a large hexose polymer. (B) Zymolyase digestion of the polymer. This produced small hexose oligomers, 2–5 residues in size, which were identified by 1H-13C HSQC-NMR (C) as β-1,3-linked glucans partially substituted by single β-glucose residues linked to C-6 positions.
Figure 10.
Effect of β-oligoglucoside fractions derived from C. albicans on Candida DSS-treated mice.
(A) Percentage survival of mice. The results are shown as percent survival from the time of C. albicans challenge and DSS treatment. Three days after C. albicans challenge, mice were given C. albicans β-glucans (F2) for 2 weeks. The survival data were significantly different by the log-rank test. A total of 80 mice were divided into four experimental groups: D (n = 20 mice), CaD (n = 20 mice), DF2 (n = 20 mice), and CaDF2 (n = 20 mice). (*P<0.05 for DF2 mice vs. D mice; and ‡P<0.05 for CaDF2 mice vs. CaD mice.) (B) Clinical analysis of DSS-induced colitis in mice. Each data set represents the mean value for each body weight. (*P<0.05 for DF2 mice vs. D mice; and ‡P<0.05 for CaDF2 mice vs. CaD mice.) (C) Histological analysis of DSS-induced colitis in mice. Panels (a) and (b) correspond to colon sections from DF2 and CaDF2 mice, respectively. No histological signs of colonic inflammation were seen in either DF2 or CaDF2 mice. (D) Histological score. The histological score decreased significantly in both DF2 and CaDF2 mice. (†P<0.05 for DF2 mice vs. D mice; ‡P<0.05 for DF2 mice vs. CaD mice; *P<0.05 for CaDF2 mice vs. D mice; and ¶P<0.05 for CaDF2 mice vs. CaD mice.) (E) Number of C. albicans colony forming units recovered from stools. Each data set represents the mean values of eight mice/group. (*P<0.05 for CaDF2 mice vs. CaD mice.) (F) Number of C. albicans colony forming units (CFU) recovered from different gut compartments. Each data set represents the mean count of 16 mice/group. In mice receiving F2, numbers of C. albicans wild-type CFUs from the stomach, ileum and colon were significantly lower than those of CaD mice. (*, †, ‡ P<0.05 for CaDF2 mice vs. CaD mice.)
Figure 11.
Summary of the effects of S. cerevisiae strains or glycan fractions on Candida DSS-treated mice.
Radar blots showed the effect of each S. cerevisiae strain or glycan fraction on: (1) mortality; (2) body weight loss; (3) clinical score; (4) histological score; and (5) C. albicans colonization. Sb, Sc1-1 and Sc3 strains improved the intestinal damage due to DSS-induced colitis and C. albicans colonization. Sc1-2, Sc2 and Sc4 strains had a dramatic effect on all inflammation signs including Candida colonization. Regarding the glycan fractions, both β-glucans and F2 reduced all colitis parameters and eliminated C. albicans colonization, while MP was less effective at controlling these parameters.
Table 2.
Effect of different strains and cell wall extracts on C. albicans colonization and inflammation in the curative C. albicans DSS model.
Table 3.
Composition in dry matter of spray-dried Sc2 cell wall fractions.