DOI: 10.5061/dryad.6djh9w17p
title: Sucrose-preferring gut microbes prevent host obesity by producing exopolysaccharides

description: Commensal bacteria affect host health by producing various metabolites
 from dietary carbohydrates via bacterial glycometabolism; however, the
 underlying mechanism of action remains unclear. Here, we identified
 Streptococcus salivarius as a unique anti-obesity commensal bacterium. We
 found that S. salivarius may prevent host obesity caused by excess sucrose
 intake via the exopolysaccharide (EPS)-short-chain fatty acid
 (SCFA)-carbohydrate metabolic axis. Healthy human donor-derived S.
 salivarius produced high EPS levels from sucrose but not from other
 sugars. S. salivarius abundance was significantly decreased in human
 donors with obesity, and the EPS-SCFA bacterial carbohydrate metabolic
 process was attenuated. Our findings reveal an important mechanism by
 which host–commensal interactions in glycometabolism affect energy
 regulation, suggesting an approach for preventing lifestyle-related
 diseases via prebiotics and probiotics by targeting bacteria and EPS
 metabolites.
description: # Sucrose-preferring gut microbes prevent host obesity by producing
 exopolysaccharides
 [https://doi.org/10.5061/dryad.6djh9w17p](https://doi.org/10.5061/dryad.6djh9w17p) We have submitted a description of the data and file structure, sharing/access information, and computational scripts. ## Description of the data and file structure Figure 1: Isolation and characterisation of exopolysaccharide (EPS)-producing human commensal bacterium *Streptococcus salivarius*. (c) Correlation between high-EPS-producing bacterial abundance in human faeces and donor body mass index (BMI). (n = 132,48 independent experiments). (e) Growth curves of EPS biosynthesis and optical density at 600 nm (OD600) (n = 3 independent experiments). (g) Expression of putative levansucrase and glycosyltransferases mRNAs in MRS medium containing sucrose or glucose during bacterial culture for 10 h determined using RNA-seq (n = 4 independent experiments). (h) Expression of putative levansucrase and glycosyltransferases mRNAs in MRS medium containing sucrose or glucose during bacterial culture for 10 h was measured using RT-qPCR (n = 4 independent experiments). Figure 2: Gut microbial analysis in human faeces. (a) Spearman’s rank correlation between faecal levels of total short-chain fatty acids (SCFAs) such as acetate, propionate, and butyrate, exopolysaccharides (EPS) and donor body mass index (BMI) in human faeces (n = 132, 48 independent experiments). (c) Comparison of polysaccharide synthesis, polysaccharide decomposition, glycolytic pathway enrichment, and SCFA synthesis between lean donors and donors with obesity in shotgun metagenomic sequencing analysis (n = 26 independent experiments). Figure 3: Metabolic improvement effect of SsEPS intake on high-fat diet (HFD)-induced obesity. (a) Bacterial SCFA levels in the culture supernatants of each gut bacterium (n = 6 independent experiments). (b–e) C57BL/6J and *Gpr41Gpr43* double-deficient mice were fed an HFD supplemented with 10% cellulose or SsEPS for 12 weeks. (b, c) Faecal and plasma SCFA levels were measured using GC-MS (b), changes in body and tissue weight (c) (n = 9, 10 independent experiments). (d–f) Blood glucose, plasma non-esterified fatty acid (NEFAs) (d), GLP-1 (e), and PYY levels (f) were measured at the end of the experimental period (n = 9, 10 independent experiments). (g) Daily food intake at 12 weeks of age (n = 5 independent experiments). (h) Expression of *Ucp 1* mRNA (n = 10 independent experiments) was measured by RT-qPCR in subcutaneous WAT. (i, j) Following 24 h of fasting, the mice were fed 0.2 g AIN-93G, containing 50% cellulose or 50% SsEPS, and an intraperitoneal glucose tolerance test was performed 1 h after feeding. Wild-type (n = 10 independent experiments), *Gpr41Gpr43* double-deficient (n = 7, 8 independent experiments), ICR (n = 8, 9 independent experiments), and GF-ICR (n = 8 independent experiments) mice were used. Plasma insulin levels were measured 15 min after intraperitoneal glucose administration. Wild-type (n = 8, 9 independent experiments), *Gpr41Gpr43* double-deficient (n = 7, 8 independent experiments), ICR and GF-ICR (n = 8 independent experiments) mice were used. Figure 4: Identification of gut bacterial SCFAs production pathway by SsEPS intake. (a, c) Gut microbial composition was evaluated to perform principal coordinate analysis and determine the relative abundance at the phylum level (a), and genus level (c) (n = 10 independent experiments). (e) SsEPS-utilizing *Bacteroides* and *Bacteroidales* S24-7 group species were detected by qPCR (n = 10 independent experiments). (f) EPS degradation, SCFA synthesis, and glycolysis pathways were compared between cellulose- and SsEPS-fed mice in shotgun metagenomic sequencing analysis (n = 5 independent experiments). Figure 5: Improvement of sucrose-induced metabolic function in *S. salivarius* colonised mice. Germ-free (GF) and colonised mice were generated and consumed sterilised water containing 20% sucrose. (a) Faecal EPS was measured by HPLC (n = 10, 10, 8, 10, 10, 8 independent experiments). (b) Faecal SCFA levels were measured by GC/MS (n = 10, 9, 8, 10, 10, 8 independent experiments). (c) After colonisation, an intraperitoneal GTT was performed (n = 8, 7, 10, 9, 6, 6 independent experiments). (d) Plasma insulin (left; n = 8, 9, 10, 7, 9, 7, 7 independent experiments) and GLP-1 (right; n = 7, 8, 8, 7, 9, 7, 7 independent experiments) levels were measured 15 min after intraperitoneal glucose administration. (f–k) After colonisation, the mice were fed an AIN-93G diet or high-fat diet (HFD) for 9 weeks. (f, g) Changes in body and tissue weights (f) and blood glucose (g) (n = 10, 10, 8, 8, 8, 8 independent experiments) under an AIN-93G diet feeding with 20% sucrose drinking water. (h) Plasma GLP-1 (n = 7, 7, 8, 8, 8, 8 independent experiments) levels were measured at the end of the experimental period. (i–k) Changes in body and tissue weights under HFD feeding (i), blood glucose (j) plasma GLP-1 and insulin (k) levels were measured at the end of the experimental period (n = 5, 8, 8, 9, 9 independent experiments). Figure 6: Improvement of sucrose-induced metabolic function in *S. salivarius* dominant human gut microbiota in mice. (b–h) *S. salivarius*-dominant [Ss (+)] or -nondominant human gut microbiota culture colonized [Ss (-)] mice were generated from GF mice through transplantation with human faecal culture solution and fed a high-fat diet supplemented with sugars (sucrose, glucose, or fructose). (b, c) Faecal total SCFAs (b) and EPS (c) were measured by GC/MS and HPLC. (n=7, 7, 5, 7, 5, 7 independent experiments). (d) Faecal *S. salivarius*, *B. ovatus*,* and *B. thetaiotaomicron* were detected by qPCR (n= 7, 7, 5, 7, 5, 7 independent experiments). (e, f) Changes in body and tissue weight (n= 7, 7, 5, 7, 5, 7 independent experiments). Blood glucose (g) and plasma GLP-1 (h) levels were measured at the end of the experimental period (n = 7, 7, 5, 7, 5, 7 independent experiments). Supplementary Figure 3: RNA-sequencing data analysis of *S. salivarius* in MRS medium containing sucrose or glucose. KEGG pathway enrichment of the EPS synthesis pathway in MRS medium containing sucrose or glucose during bacterial culture for 10 h (n = 4 independent experiments). Supplementary Figure 4: Shotgun metagenomic sequencing data analysis in human faeces. The gut microbial composition between lean donors and donors with obesity at the genus level (n = 26 independent experiments). Supplementary Figure 5: Beneficial effect of SsEPS intake on high-fat diet (HFD)-induced obesity. C57BL/6J and *Gpr41Gpr43* double-deficient mice were fed an HFD supplemented with 10% cellulose or SsEPS for 12 weeks. (a) Plasma triglycerides (TGs) and total cholesterol levels were measured at the end of the experimental period (n = 9, 10 independent experiments). (b) Oral glucose (left; C57BL/6J: n = 8, 10, *Gpr41Gpr43 *double-deficient: n = 9 independent experiments) and insulin tolerance tests (right; C57BL/6J: n = 10, 10, *Gpr41Gpr43* double-deficient: n = 9 independent experiments) were performed at 13–14 weeks of age. (c) Plasma insulin levels were measured at the end of the experimental period (n = 9, 10, 9, 9 independent experiments). (d) Plasma GLP-1 levels were measured 15 min after intraperitoneal glucose administration. Wild-type (n = 8 independent experiments), *Gpr41Gpr43* double-deficient (n = 7, 7, 8 independent experiments), conventional ICR (n = 9, 8, 9 independent experiments), and GF-ICR (n = 7 independent experiments) mice were used. Supplementary Figure 6: Shotgun metagenomic sequencing data analysis in mouse faeces. The top 20 Gene Ontology (GO) terms are related to biological processes and molecular function. Significant pathways increased in SsEPS as determined by HUMAnN3 and MaAsLin2. Supplementary Figure 7: Generation of levansucrase deficiency in *S. salivarius* strain. (d) Growth curves of optical density at 600 nm (OD600) (n = 3 independent experiments). (e) EPS biosynthesis of *S. salivarius* in MRS medium containing 15% sucrose after 24 h of cultivation (n = 6 independent experiments). (f) Expression of putative levansucrase and glycosyltransferase mRNAs in MRS medium containing sucrose or glucose during bacterial culture for 10 h were measured using RT-qPCR (n = 4 independent experiments). Supplementary Figure 8: Beneficial effects of sucrose-induced metabolic function by *S. salivarius*. (b, c) *S. salivarius*, *B. ovatus*, and *B. thetaiotaomicro*n were detected by qPCR in gnotobiotic mice (n = 10, 9, 8, 10, 10, 8 independent experiments). (d) EPS analysis of the jejunum, ileum, and caecum in colonised *S. salivarius* mice were performed using HPLC (n = 6, 5 independent experiments). (e, f) Plasma triglyceride (TGs), non-esterified fatty acid (NEFA), total cholesterol, and insulin levels were measured at the end of the experimental period (n = 7, 7, 8, 8, 8, 8 independent experiments). (g) Faecal *S. salivarius, B. ovatus* and *B. thetaiotaomicron* were detected by qPCR (n = 5, 8, 8, 9, 9 independent experiments). (h) Faecal SCFA levels were measured using GC-MS (n = 5, 8, 8, 9, 9 independent experiments). (i) Faecal EPS in *S. salivarius* gnotobiotic mice after drinking sterilised water containing 20% sucrose, glucose, or fructose for 2 weeks (n = 6, 5, 6, 5, 6, 5 independent experiments). Supplementary Figure 9: Gut microbial composition in human-flora-transplanted mice. (a) Relative abundance at the genus level and quantification of *S. salivarius, B. ovatus*, and *B. thetaiotaomicron* at the species level in *S. salivarius*-dominant [Ss (+)] or -nondominant [Ss (-)] human gut microbiome culture-collection (n = 3 independent experiments). (c) The gut microbial composition in [Ss (+)] or [Ss (-)]-colonised mice was evaluated using relative abundance at the genus level. (c) (n = 7, 7, 7, 5, 7, 5 independent experiments). ## Sharing/Access information The 16S rRNA sequencing data generated in this study have been deposited in the DNA Data Bank of Japan (DDBJ) under accession Nos. [DRA017528](https://jpn01.safelinks.protection.outlook.com/?url=https%3A%2F%2Fddbj.nig.ac.jp%2Fsearch%2Fentry%2Fsra-submission%2FDRA017528&data=05%7C02%7Chidenori.shimizu%40shiseido.com%7C7ad98a1683a24aaf189708dd3145db3a%7C602fb21270b34ef8938e9ba98c5ab37a%7C1%7C0%7C638720898298808924%7CUnknown%7CTWFpbGZsb3d8eyJFbXB0eU1hcGkiOnRydWUsIlYiOiIwLjAuMDAwMCIsIlAiOiJXaW4zMiIsIkFOIjoiTWFpbCIsIldUIjoyfQ%3D%3D%7C0%7C%7C%7C&sdata=ULY%2B8%2F7pB1TCWNPK7K9AnA9CE83gLvSLbxI%2Bu4qZf1g%3D&reserved=0 "https://ddbj.nig.ac.jp/search/entry/sra-submission/DRA017528"), [DRA017529](https://jpn01.safelinks.protection.outlook.com/?url=https%3A%2F%2Fddbj.nig.ac.jp%2Fsearch%2Fentry%2Fsra-submission%2FDRA017529&data=05%7C02%7Chidenori.shimizu%40shiseido.com%7C7ad98a1683a24aaf189708dd3145db3a%7C602fb21270b34ef8938e9ba98c5ab37a%7C1%7C0%7C638720898298822752%7CUnknown%7CTWFpbGZsb3d8eyJFbXB0eU1hcGkiOnRydWUsIlYiOiIwLjAuMDAwMCIsIlAiOiJXaW4zMiIsIkFOIjoiTWFpbCIsIldUIjoyfQ%3D%3D%7C0%7C%7C%7C&sdata=r2UK9TFuNkG4l3jLNBJ8ynGgCEFEVUPFAurhb1lJBrY%3D&reserved=0 "https://ddbj.nig.ac.jp/search/entry/sra-submission/DRA017529"), and [DRA017628](https://jpn01.safelinks.protection.outlook.com/?url=https%3A%2F%2Fddbj.nig.ac.jp%2Fsearch%2Fentry%2Fsra-submission%2FDRA017628&data=05%7C02%7Chidenori.shimizu%40shiseido.com%7C7ad98a1683a24aaf189708dd3145db3a%7C602fb21270b34ef8938e9ba98c5ab37a%7C1%7C0%7C638720898298836047%7CUnknown%7CTWFpbGZsb3d8eyJFbXB0eU1hcGkiOnRydWUsIlYiOiIwLjAuMDAwMCIsIlAiOiJXaW4zMiIsIkFOIjoiTWFpbCIsIldUIjoyfQ%3D%3D%7C0%7C%7C%7C&sdata=X6XiYvAGXEunhmcxK9s0XeJ1THLttuKUH%2B1hQAi7HoM%3D&reserved=0 "https://ddbj.nig.ac.jp/search/entry/sra-submission/DRA017628"). The shotgun metagenomic sequencing data are available under restricted accession Nos. [DRA017626](https://jpn01.safelinks.protection.outlook.com/?url=https%3A%2F%2Fddbj.nig.ac.jp%2Fsearch%2Fentry%2Fsra-submission%2FDRA017626&data=05%7C02%7Chidenori.shimizu%40shiseido.com%7C7ad98a1683a24aaf189708dd3145db3a%7C602fb21270b34ef8938e9ba98c5ab37a%7C1%7C0%7C638720898298850809%7CUnknown%7CTWFpbGZsb3d8eyJFbXB0eU1hcGkiOnRydWUsIlYiOiIwLjAuMDAwMCIsIlAiOiJXaW4zMiIsIkFOIjoiTWFpbCIsIldUIjoyfQ%3D%3D%7C0%7C%7C%7C&sdata=L74NMBxqP%2BA44cszHXmKmSwUMcVsRqTsfaa4t33UxzI%3D&reserved=0 "https://ddbj.nig.ac.jp/search/entry/sra-submission/DRA017626") and [DRA017627](https://jpn01.safelinks.protection.outlook.com/?url=https%3A%2F%2Fddbj.nig.ac.jp%2Fsearch%2Fentry%2Fsra-submission%2FDRA017627&data=05%7C02%7Chidenori.shimizu%40shiseido.com%7C7ad98a1683a24aaf189708dd3145db3a%7C602fb21270b34ef8938e9ba98c5ab37a%7C1%7C0%7C638720898298865677%7CUnknown%7CTWFpbGZsb3d8eyJFbXB0eU1hcGkiOnRydWUsIlYiOiIwLjAuMDAwMCIsIlAiOiJXaW4zMiIsIkFOIjoiTWFpbCIsIldUIjoyfQ%3D%3D%7C0%7C%7C%7C&sdata=CiyRk7dAdg%2BFhL2XVKPcMR4TAHYXDFKag9eqjC5Plgg%3D&reserved=0 "https://ddbj.nig.ac.jp/search/entry/sra-submission/DRA017627"). RNA sequencing data are accessible via accession Nos. [DRA017530](https://jpn01.safelinks.protection.outlook.com/?url=https%3A%2F%2Fddbj.nig.ac.jp%2Fsearch%2Fentry%2Fsra-submission%2FDRA017530&data=05%7C02%7Chidenori.shimizu%40shiseido.com%7C7ad98a1683a24aaf189708dd3145db3a%7C602fb21270b34ef8938e9ba98c5ab37a%7C1%7C0%7C638720898298879314%7CUnknown%7CTWFpbGZsb3d8eyJFbXB0eU1hcGkiOnRydWUsIlYiOiIwLjAuMDAwMCIsIlAiOiJXaW4zMiIsIkFOIjoiTWFpbCIsIldUIjoyfQ%3D%3D%7C0%7C%7C%7C&sdata=dfODXUqiqZ0MXsnGKYd%2FtbC1M9YHPyHu9O7ldkPcwFM%3D&reserved=0 "https://ddbj.nig.ac.jp/search/entry/sra-submission/DRA017530") and [E-GEAD-664](https://jpn01.safelinks.protection.outlook.com/?url=https%3A%2F%2Fddbj.nig.ac.jp%2Fpublic%2Fddbj_database%2Fgea%2Fexperiment%2FE-GEAD-000%2FE-GEAD-664%2F&data=05%7C02%7Chidenori.shimizu%40shiseido.com%7C7ad98a1683a24aaf189708dd3145db3a%7C602fb21270b34ef8938e9ba98c5ab37a%7C1%7C0%7C638720898298893106%7CUnknown%7CTWFpbGZsb3d8eyJFbXB0eU1hcGkiOnRydWUsIlYiOiIwLjAuMDAwMCIsIlAiOiJXaW4zMiIsIkFOIjoiTWFpbCIsIldUIjoyfQ%3D%3D%7C0%7C%7C%7C&sdata=HyORfgB2GCzVoM0YmZs8iN%2Fw9F9VHcorLo17q8U4Mkw%3D&reserved=0 "https://ddbj.nig.ac.jp/public/ddbj_database/gea/experiment/E-GEAD-000/E-GEAD-664/"). The draft genome sequencing data of *S. salivarius* are available under restricted accession Nos. [DRA018992](https://jpn01.safelinks.protection.outlook.com/?url=https%3A%2F%2Fddbj.nig.ac.jp%2Fsearch%2Fentry%2Fsra-submission%2FDRA018992&data=05%7C02%7Chidenori.shimizu%40shiseido.com%7C7ad98a1683a24aaf189708dd3145db3a%7C602fb21270b34ef8938e9ba98c5ab37a%7C1%7C0%7C638720898298907171%7CUnknown%7CTWFpbGZsb3d8eyJFbXB0eU1hcGkiOnRydWUsIlYiOiIwLjAuMDAwMCIsIlAiOiJXaW4zMiIsIkFOIjoiTWFpbCIsIldUIjoyfQ%3D%3D%7C0%7C%7C%7C&sdata=scrou%2BsiLiwlx6OtWN%2Fzs4FLR%2FWY%2FxJiXv2F2jiNFgA%3D&reserved=0 "https://ddbj.nig.ac.jp/search/entry/sra-submission/DRA018992") and [BAAFPP010000000](https://jpn01.safelinks.protection.outlook.com/?url=https%3A%2F%2Fgetentry.ddbj.nig.ac.jp%2Fgetentry%2Fna%2FBAAFPP010000000&data=05%7C02%7Chidenori.shimizu%40shiseido.com%7Cc8a32293020b4aff765c08dd314c4542%7C602fb21270b34ef8938e9ba98c5ab37a%7C1%7C0%7C638720925845732178%7CUnknown%7CTWFpbGZsb3d8eyJFbXB0eU1hcGkiOnRydWUsIlYiOiIwLjAuMDAwMCIsIlAiOiJXaW4zMiIsIkFOIjoiTWFpbCIsIldUIjoyfQ%3D%3D%7C0%7C%7C%7C&sdata=2d9tiTNpNec2oq0HNsHVd98NNOO8%2FGhy0F1g%2FJK2THA%3D&reserved=0 "https://getentry.ddbj.nig.ac.jp/getentry/na/BAAFPP010000000"). The Figures 1–6, Supplementary Figures 1–9 data generated in this study are provided in the Supplementary Information/Source Data file. The Figures 1–6, Supplementary Figures 1–9, 16S rRNA sequencing, shotgun metagenomic sequencing, and RNA sequencing data used in this study are available in the Dryad repository under accession code ([https://doi.org/10.5061/dryad.6djh9w17p](https://doi.org/10.5061/dryad.6djh9w17p)). Source data are provided with this paper. ## Computational scripts All computational scripts are available on GitHub [[https://github.com/petadimensionlab/EPS](https://github.com/petadimensionlab/EPS)] and Zenodo ([https://doi.org/10.5281/zenodo.14550033](https://doi.org/10.5281/zenodo.14550033)).