Differential Metabolism of Exopolysaccharides from Probiotic Lactobacilli by the Human Gut Symbiont Bacteroides thetaiotaomicron

Abstract

Probiotic microorganisms are ingested as food or supplements and impart positive health benefits to consumers. Previous studies have indicated that probiotics transiently reside in the gastrointestinal tract and, in addition to modulating commensal species diversity, increase the expression of genes for carbohydrate metabolism in resident commensal bacterial species. In this study, it is demonstrated that the human gut commensal species Bacteroides thetaiotaomicron efficiently metabolizes fructan exopolysaccharide (EPS) synthesized by probiotic Lactobacillus reuteri strain 121 while only partially degrading reuteran and isomalto/malto-polysaccharide (IMMP) α-glucan EPS polymers. B. thetaiotaomicron metabolized these EPS molecules via the activation of enzymes and transport systems encoded by dedicated polysaccharide utilization loci specific for β-fructans and α-glucans. Reduced metabolism of reuteran and IMMP α-glucan EPS molecules may be due to reduced substrate binding by components of the starch utilization system (sus). This study reveals that microbial EPS substrates activate genes for carbohydrate metabolism in B. thetaiotaomicron and suggests that microbially derived carbohydrates provide a carbohydrate-rich reservoir for B. thetaiotaomicron nutrient acquisition in the gastrointestinal tract.

FIG 1

(A) Graphical representation of β-fructan levan and α-glucans reuteran and IMMP exopolysaccharides produced by Lactobacillus reuteri 121 via transglycosylation reactions using glycoside hydrolase family 68 (GH68) or GH70 enzymes (www.cazy.org [39]). (B) NMR analysis of LrEPS products indicating the abundance of monosaccharides present as ratios of each peak representing the anomeric carbon present.

FIG 2

(A) Growth of B. thetaiotaomicron VPI-5482 and mutants derived on Lactobacillus reuteri EPS. Wild type, green; BtΔ3702 (sus mutant), yellow; BtΔ1763 (fructan-associated PUL mutant), red; and glucose control, blue. B. thetaiotaomicron and deletion mutants were grown under anaerobic conditions at 37°C in minimal growth media supplemented with wild-type LrEPS (levan plus reuteran) or glucose (control) at 5 mg/ml final concentration. Only one curve was carried out for each sample due to the limited availability of substrate, and results were verified by qPCR. (B) Graphical representation of B. thetaiotaomicron PULs mentioned in this study.

FIG 3

Growth of B. thetaiotaomicron VPI-5482 on individual α-glucan-based EPS components reuteran (red) and IMMP (purple) compared to LrEPS (green) and glucose (blue) controls. The lines indicate averages from six growth experiments, and error bars indicate the standard deviations for these experiments.

FIG 4

Thin-layer chromatography analysis and HPAEC-PAD profile of L. reuteri 121 β-fructan (levan) EPS degradation. The graph shows the levan (blue line) control and products from levan hydrolysis by B. thetaiotaomicron culture supernatants after growth on levan (red line). The inset is the corresponding TLC profile from enzymatic activity of CAZymes present in B. thetaiotaomicron culture supernatants from growth on wild-type LrEPS activity on LrEPS (lane 1) and reuteran (lane 2). The y axes show electric charges (in nanocoulombs [nC]).

FIG 5

Thin-layer chromatography analysis of the products of degradation on reuteran and IMMP incubated with culture supernatants (CS) of B. thetaiotaomicron grown in the presence of either substrate. Lane 1, reuteran and reuteran CS; lane 2, IMMP and IMMP CS; lane 3, reuteran and glucose CS; lane 4, IMMP and glucose CS. Culture supernatants were incubated with EPS substrate for 1 h before being spotted on a TLC plate.

FIG 6

Thin-layer chromatography of reuteran hydrolysis by SusG. All reaction mixtures contain 10 mg/ml polysaccharide and 22 μg SusG in 20 mM HEPES, pH 7.0, 150 mM NaCl, 5 mM CaCl₂. Reaction products were sampled after 4 h of incubation at 37°C. Lane 1, G1 to G7 maltooligosaccharide standards (Std); lane 2, reuteran (Reut); lane 3, Lr121EPS; lane 4, amylopectin (AP).

FIG 7

(A) Thin-layer chromatography analysis of the products of reuteran digestion by various α-glucan-degrading enzymes. Lane 1, Pseudomonas species isoamylase (EC 3.2.1.68); lane 2, B. licheniformis α-amylase (EC 3.2.1.1) (the dark spot is sucrose from enzyme storage solution); lane 3, Microbacterium aurum amylase A1 (EC3.2.1.1); lane 4, Aspergillus niger amyloglucosidase (EC 3.2.1.3); lane 5, Bacillus acidopullulyticus pullulanase (EC 3.2.1.41); lane 6, L. reuteri GTFA (EC 2.4.1.5); lane 7, GTF180; lane 8, Microbacterium aurum amylase B; lane 9, untreated reuteran control. (B) Activity of culture supernatants from B. thetaiotaomicron grown on reuteran with the addition of dextran- or pullulan-derived oligosaccharides isomaltose, maltose, and panose. Lane 1, reuteran plus isomaltose supernatant only; lane 2, reuteran plus isomaltose supernatant with reuteran; lane 3, reuteran plus maltose supernatant only; lane 4, reuteran plus maltose supernatant with reuteran; lane 5, reuteran plus panose culture supernatant only; lane 6, reuteran plus panose culture supernatant with reuteran. The addition of small oligosaccharides did not increase the ability of B. thetaiotaomicron to degrade reuteran. Results also indicate that isomaltose and panose are not transported by B. thetaiotaomicron, since they remain in the culture supernatant.

FIG 8

(A) Carbohydrate macroarray analysis of SusD binding to soluble α-glucans. A1, 1% pullulan; A2, 1% soluble potato starch; A3, 1% amylopectin; B1, 1% reuteran; B2, 1% IMMP; B3, 1% dextran. (B) Pulldown assay of SusD with insoluble wheat starch. TmCBM41 is a control for α-glucan binding activity and displays expected binding patterns (29). FITC-labeled SusD and TmCBM41 binding were carried out in 20 mM Tris, 150 mM NaCl, 5 mM CaCl₂, and 0.05% Tween 20 at room temperature for 1 h. Binding was visualized at 520 nm.