Engineered spermidine-secreting Saccharomyces boulardii ameliorates colitis and colon cancer in mice

Abstract

Experimental studies suggest that the probiotic yeast Saccharomyces boulardii can mitigate the symptoms of inflammatory bowel disease. However, these results are equivocal and S. boulardii probiotic therapy has not gained widespread acceptance in clinical practice. To assess whether the therapeutic properties of S. boulardii might be improved upon, we engineered S. boulardii to overproduce and secrete spermidine, a pro-regenerative natural metabolite. We employed CRISPR gene deletion and integration of gene cassettes at the Ty2 locus to achieve high level polyamine synthesis and transport. We tested whether spermidine secreting S. boulardii could reduce disease symptoms in dextran sulfate sodium (DSS) and azoxymethane induced models of intestinal inflammation and cancer. We demonstrate that oral delivery of spermidine-secreting S. boulardii in mice populates the gastrointestinal tract with viable spermidine-secreting S. boulardii cells and raises free spermidine levels in the gastrointestinal tract. Strikingly, spermidine-secreting S. boulardii strains were significantly more effective than wild-type S. boulardii in reducing colitis symptoms as well as colitis-associated carcinogenesis in mice. These results suggest that in situ spermidine secretion by engineered synthetic biotic yeast strains may be an effective and low-cost therapy to mitigate inflammatory bowel disease and associated colon cancer.

Keywords: S. boulardii; Colitis; Colon cancer; Inflammatory bowel disease; Spermidine.

Fig. 1

Engineering S. boulardii to overproduce and secrete spermidine. A. Polyamine synthetic pathway in S cerevisiae. In eukaryotic cells, ornithine decarboxylase (ODC; Spe1 in yeast) is the first and rate-limiting enzyme in the polyamine synthetic pathway, converting L-ornithine to putrescine. Two additional enzymes are involved in spermidine synthesis. First, S-adenosylmethionine decarboxylase (SAM-DC; Spe2 in yeast) converts SAM to decarboxylated SAM (dcSAM). Spermidine synthase (Spe3 in yeast) then transfers an amino prolyl group from dcSAM to putrescine to produce spermidine, and 5’-methylthioadenosine (MTA). Similarly, spermine synthase (Spe4 in yeast) transfers an amino propyl group to spermidine to produce spermine and MTA. ODC is subjected to feedback inhibition by ODC antizyme (OAZ; Oaz1 in yeast), a polyamine-induced protein that inhibits ODC activity and targets ODC for ubiquitin-independent degradation. Tpo1 (pH dependent) and Tpo5 (pH-independent) are two possible polyamine exporters in S cerevisiae and S boulardii. B. Integration cassette for overexpression of TPO5, SPE1 and SPE2. The Ty2 element is an LTR-containing retrotransposon present in several copies in the genomes of S. cerevisiae and S. boulardii. The TPO5, SPE1 and/or SPE2 genes were expressed from the TDH3 promoter. The kanMX marker allows growth selection on G418 medium. 5’ and 3’ Ty2 recombination arms flank the entire cassette. The graphs show growth rates at 30 and 37 °C respectively. C. Intracellular (left) and culture medium (right) polyamine concentrations in saturated yeast cultures grown in synthetic complete (SC) medium. Means ± SEM for three independent experiments are shown. PUT: putrescine; SPD: spermidine; SPM: spermine. Significance was calculated by one-way ANOVA and Tukey’s multiple comparisons test.

Fig. 2

Sb576 increases colonic spermidine. A. Schematic of AOM-DSS protocol (see text for details). B. Yeast CFU after five days of oral gavage with SbWT or Sb576 (both at 1 × 10⁸ cells per mouse per day). Fecal samples from Sb576-treated mice were plated on both ampicillin (Amp)- and G418-containing plates. Shown are means ± SEM (CFU per g fresh fecal sample). Each dot represents one mouse. C. Female C57BL (five-month old, n = 5) were administered a single dose of streptomycin followed by a single dose of Sb576 (1 × 10⁸ yeast cells per mouse) a day later. Fecal samples were collected 16, 40, and 64 h after Sb576 administration for CFU determination on G418-containing yeast culture plates. Shown are means with SEM. D. Free spermidine levels in fresh fecal samples following five days of oral gavage of vehicle, SbWT or Sb576. Shown are means ± SEM. One-way ANOVA and Tukey’s multiple comparisons test.

Fig. 3

Sb576 reduces DSS-induced colitis symptoms. A. Daily body weight (means ± SEM) expressed as % of the weight of individual animals on day 5 (prior to the first DSS cycle) of mice receiving vehicle (n = 7), SbWT (n = 10) or Sb576 (n = 10), 10 consecutive days for each DSS cycle starting one day after the beginning of DSS treatment. Body weight of control mice (n = 3) from the same cohort is shown on intermittent days. Shown on the right are mean body weights (means ± SEM) of individual mice over the 30-day course. Significance was assessed by one-way ANOVA and Tukey’s multiple comparisons test. Arrows indicate cessation of DSS water (details in Fig. 2A). B. Daily DAI (means ± SEM) of mice receiving vehicle (n = 7), SbWT (n = 10) or Sb576 (n = 10) following each of the three DSS cycles, as above. Shown on the right are mean DAIs (means ± SEM) of individual mice over the 30-day course. Significance was assessed by oOne-way ANOVA and Tukey’s multiple comparisons test. Arrows indicate cessation of DSS water (details in Fig. 2A).

Fig. 4

Sb576 restores colon length in AOM/DSS-treated mice. A. Colon length of the four groups of mice. Shown are means ± SEM. Significance was assessed by one-way ANOVA and Tukey’s multiple comparisons test. B. Spleen weight of the four groups of mice. Shown are means ± SEM. Significance was assessed by one-way ANOVA and Tukey’s multiple comparisons test. Colon and spleen images of the entire cohort of mice are shown below. A dashed line is drawn across the 8 cm length marks.

Fig. 5

Sb576 reduces macroscopic colon tumors. A. Representative colon images (taken by cellphone camera during tumor counting/measuring) of untreated control, AOM-DSS-treated mice receiving vehicle, SbWT or Sb576. Distal ends on the right. White line marks tumors in the colon of AOM-DSS mice treated with vehicle. Tumor counting and measuring were done under a dissecting microscope. B. Tumor numbers. C. Tumor burden (sum of tumor area). Shown are means ± SEM. Significance was assessed by one-way ANOVA and Tukey’s multiple comparisons test.

Fig. 6

Sb576 reduces microscopic neoplastic lesions. Hematoxylin and Eosin staining images of colonic neoplasia. Control: a section through the middle of the colon Swiss roll (distal end at center) of a control mouse showing normal colonic crypts. AOM-DSS: A colon Swiss roll section of a mouse in the AOM/DSS-vehicle group depicting low-grade and high-grade adenomas, and adenocarcinoma. The graph summarizes the numbers of low-grade or high-grade adenomas, adenocarcinomas and total neoplastic lesions in AOM-DSS treated mice receiving vehicle, SbWT or Sb576 treatment. Shown are means ± SEM (n = 7 for vehicle; n = 10 for SbWT or Sb576). Significance was assessed by one-way ANOVA and Tukey’s multiple comparisons test.