Specificity of polysaccharide use in intestinal bacteroides species determines diet-induced microbiota alterations

Abstract

The intestinal microbiota impacts many facets of human health and is associated with human diseases. Diet impacts microbiota composition, yet mechanisms that link dietary changes to microbiota alterations remain ill-defined. Here we elucidate the basis of Bacteroides proliferation in response to fructans, a class of fructose-based dietary polysaccharides. Structural and genetic analysis disclosed a fructose-binding, hybrid two-component signaling sensor that controls the fructan utilization locus in Bacteroides thetaiotaomicron. Gene content of this locus differs among Bacteroides species and dictates the specificity and breadth of utilizable fructans. BT1760, an extracellular beta2-6 endo-fructanase, distinguishes B. thetaiotaomicron genetically and functionally, and enables the use of the beta2-6-linked fructan levan. The genetic and functional differences between Bacteroides species are predictive of in vivo competitiveness in the presence of dietary fructans. Gene sequences that distinguish species' metabolic capacity serve as potential biomarkers in microbiomic datasets to enable rational manipulation of the microbiota via diet.

Figure 1. Bt’s use of fructose-containing carbohydrates corresponds to induction of the polysaccharide utilization locus BT1757-1763 and BT1765

A. Genomic organization of Bt’s Sus locus (top) and putative fructan utilization locus (bottom). Genes of similar function are coded by color; intervening unrelated genes are white; genes without corresponding homologs are grey. B. Gene expression patterns of differentially regulated susC and susD homologs from Bt grown in rich medium (TYG) at five time points from early log (3.5h) to stationary phase (8.8h) in duplicate. C. Growth curves of Bt in minimal medium containing indicated carbon source at 0.5% w/v. FOS, fructo-oligosaccharide. D. RNA abundance for genes relevant to fructan use in cells grown in different carbon sources, relative to growth in minimal medium plus glucose. Standard errors of expression levels from three biological replicate cultures are shown.

Figure 2. BT1754 HTCS binds fructose and is required for growth on fructose-containing carbohydrates

A. Growth curves of Bt-ΔBT1754 compared to wild type Bt (WT) and the complemented mutant (ΔBT1754::BT1754) on fructose-based carbon sources. B. Domain organization of BT1754. C. Interaction of the N-terminal periplasmic domain of BT1754 with fructose or levanbiose assessed by isothermal calorimetry, showing the raw heats of binding (upper panel) and integrated data (lower panel) fit to a single site binding model (fructose only). Values areaverages and SDs of at least three independent titrations.

Figure 3. Structure of BT1754-PD in complex with fructose

A. Representation of the homodimer of BT1754-PD present in the asymmetric unit, with each monomer separated by a dotted line; molecule of fructose (pink); the flexible hinge between the two subdomains (circle). B. Overlay of BT1754-PD (green) with TtRBP (blue); the extended C-terminal helix in BT1754-PD (bracket) is unique to BT1754. C. Side view of the binding site illustrating hydrophobic interactions of BT1754-PD and fructose. Fo-Fc electron density prior to modeling the single molecule of fructose in the β-furanose form is shown (blue mesh contoured at 3σ). D. Top view of the binding site of BT1754-PD illustrating the numerous H-bonds (dotted black lines) with fructose.

Figure 4. BT1760 encodes an extracellular endo-levanase required for Bt growth in levan

A. Growth curves of Bt -ΔBT1760 compared to the complemented mutant (ΔBT1760::BT1760) in levan (top) or FOS (bottom panel). B. TLC analysis of the products of levan digestion by the Bt GH32 enzymes, BT1760, BT1759, BT1765 and BT3082. Frc, fructose; L2, levanbiose; L3, levantriose; L4, levantetraose. C. Degradation of levan by Bt cells grown in minimal medium plus fructose. Error bars show the SDs from three independent experiments.

Figure 5. The SusD-homolog encoded by BT1762 is required for efficient Bt utilizaton of levan and binds β2-6 but not β2-1 fructan

A. Growth curves of wild type Bt, Bt-ΔBT1762, and Bt-ΔBT1762::BT1762 in levan (left) or FOS (right). B. Interaction of BT1762 with fructans as assessed by isothermal calorimetry. Levan binding data integrated and fit to a single site binding model (bottom left). Values are averages and SDs of at least three independent titrations.

Figure 6. Comparative genomic and functional analysis of fructan utilization among Bacteroides species

Fructan-utilization loci from Bacteroides species (left). Common predicted functions are color coded, intervening unrelated genes are white. PL19, polysaccharide lyase family 19; GH32, glycoside hydrolase family 32. Growth curves (right) of each Bacteroides species in fructose-based carbohydrates.

Figure 7. Effect of dietary fructans on Bacteorides competition within the intestine

A. Experimental design for in vivo experiments. GF, germ-free. B. Average relative fecal proportion (% total bacteria) of Bt and B. caccae at 4, 6, 14, and 21 days after colonization; n=7 mice. C. Average relative fecal proportion (% total bacteria) of Bt and B. vulgatus at 4, 6, 14, and 21 days after colonization; n=3 mice. D. Increase in proportion (%) of B. caccae over Bt from day 6 (1 day prior to diet change) to day 21 (14 days after diet change). All groups received a standard diet on days 1-7; type of diet and whether the mice received inulin in their water on days 7-21 is indicated; n=3-7 individually housed mice. E. Average relative fecal proportion (% total bacteria) of inulin-utilizing Bt(In+) and B. caccae at 4, 6, 14, and 21 days after colonization; n=7 individually housed mice.