Interspecies Competition Impacts Targeted Manipulation of Human Gut Bacteria by Fiber-Derived Glycans

Abstract

Development of microbiota-directed foods (MDFs) that selectively increase the abundance of beneficial human gut microbes, and their expressed functions, requires knowledge of both the bioactive components of MDFs and the mechanisms underlying microbe-microbe interactions. Here, gnotobiotic mice were colonized with a defined consortium of human-gut-derived bacterial strains and fed different combinations of 34 food-grade fibers added to a representative low-fiber diet consumed in the United States. Bioactive carbohydrates in fiber preparations targeting particular Bacteroides species were identified using community-wide quantitative proteomic analyses of bacterial gene expression coupled with forward genetic screens. Deliberate manipulation of community membership combined with administration of retrievable artificial food particles, consisting of paramagnetic microscopic beads coated with dietary polysaccharides, disclosed the contributions of targeted species to fiber degradation. Our approach, including the use of bead-based biosensors, defines nutrient-harvesting strategies that underlie, as well as alleviate, competition between Bacteroides and control the selectivity of MDF components.

Keywords: biosensors; community ecology; interspecies competition; microbiota-directed foods; polysaccharide utilization.

Conflict of interest statement

Declaration of interests:

J.I.G. is a co-founder of Matatu, Inc., a company characterizing the role of diet-by-microbiota interactions in animal health. Elements of this report are the subject of patent applications that are currently being submitted.

Copyright © 2019 Elsevier Inc. All rights reserved.

Figures

Figure 1.. In vivo screen of the effects of food-grade fiber preparations on members of a defined human gut microbiota.

(A) Schematic design of screen (one of three similar screens). Gnotobiotic mice harboring a consortium of bacterial strains obtained from a single human donor. Animals received a series of supplemented HiSF-LoFV diets, each containing one fiber type at 8% (w/w) and another at 2% (w/w) (colored boxes). Control animals received the unsupplemented HiSF-LoFV or LoSF-HiFV diet monotonously for four weeks. (B,C) Average relative abundance values for B. thetaiotaomicron and B. caccae during administration of the indicated fiber. Bars show mean values. Circles denote individual mice. Arrowheads mark mice that received pea fiber (B) or high molecular weight (MW) inulin (C) as the minor fiber type (2% w/w). (D) Estimated coefficients from linear models (with at least one estimated coefficient > 0.4) for bacterial strains across the three screening experiments. Statistically significant coefficients (P < 0.01; ANOVA) are shaded according to the color bar. (See also Table S1 and S2).

Figure 2.. Proteomics and forward genetics identify arabinan in pea fiber as a nutrient source for multiple species

(A) Schematic representation of polysaccharide structures detected in pea fiber based on monosaccharide and linkage analyses (with stereochemistry of anomeric carbon inferred). (B-E) Gnotobiotic mice harboring a 15-member community consisting of INSeq libraries representing four Bacteroides species together with 10 additional bacterial strains used in screening experiments (Figure 1). Relative abundance is shown for each bacterial strain at each of the indicated time points in mice monotonously fed the HiSF-LoFV diet with (green) or without (grey) addition of 10% (w/w) pea fiber. Key: circles, individual mice; lines, mean values; shading, 95%CI (n=15 individually-caged mice per group; Tables S3A-S3C). *, P < 0.05; (pea fiber diet versus control HiSF-LoFV diet; ANOVA). (F-I) Proteomic and INSeq analyses of fecal samples collected on experimental day 6. On the x-axis, the position of each dot denotes the mean value for the abundance of a single bacterial protein in samples obtained from mice fed a HiSF-LoFV + pea fiber diet (relative to control diet). The y-axis indicates the mean value of the differential enrichment of mutant strains with Tn disruptions in the gene encoding each protein in the pea fiber versus HiSF-LoFV diet groups. All genes represented in both the protein dataset and INSeq mutant pool are plotted as grey dots. Green dots highlight genes that are significantly affected by pea fiber (P < 0.05, ∣fold change∣ > log2(1.2); limma or limma-voom) as judged by levels of their protein products or their contribution to fitness; open circles mark the subset of these genes that are encoded by PULs. Genes that are present in the three homologous arabinan-processing PULs (see below) are labeled with their PUL number (Table S7). Genes in an arabinose-processing operon in B. vulgatus are labeled with an ‘A’. Genes in the B. ovatus RGI-processing PUL97 are also labeled. (J) Alignment of B. thetaiotaomicron PUL7, B. cellulosilyticus PUL5, B. vulgatus PUL27, and the B. vulgatus arabinose operon. The direction of transcription is left to right (unless marked by a leftward pointing arrowhead). The first and last genes are labeled above with their locus tag number. Genes are color-coded according to their functional annotation (see key). GH families for enzymes in the CAZy database are shown as numbers inside the gene boxes. Shaded regions connecting genes denote significant BLAST homology (E-value <10−9); the percent amino acid identity of their protein products is shown. (See also Figure S2, Figure S1, Table S3-S5, and Table S7).

Figure 3.. Deliberate manipulation of community composition demonstrates interspecies competition for pea fiber arabinan.

(A,B) Relative abundance of each bacterial strain in fecal samples from gnotobiotic mice harboring the 15-member community. Mice were fed the control HiSF-LoFV diet in the presence (light grey, closed circles), or absence (dark grey, open circles) of B. cellulosilyticus (B.c.), or fed the HiSF-LoFV diet + 10% (w/w) pea fiber in the presence (green, closed circles) or absence (magenta, open circles) of B.c. Key: circles, individual mice; lines, mean values; shading, 95%CI (n=4-10 mice per group). *, P < 0.05; (diet-by-community interaction; ANOVA). (C,D) Proteomics analysis of fecal communities sampled on experimental days 6, 12, 19, and 25. Genes in PULs of interest are shown along the x-axis (as locus tag number only; BT_XXXX or BVU_XXXX). Mean values ± SD (vertical shading) are indicated (n=5 animals/treatment group). Genes are color-coded according to their functional annotation (see key). GH families for enzymes in the CAZy database are shown as numbers inside the gene boxes. Key for circles is identical to that used in panels A and B. *, P < 0.05, ∣fold change∣ > log2(1.2) (HiSF-LoFV + pea fiber versus HiSF-LoFV diet; limma). (See also Figure S3 and Table S3-S4).

Figure 4.. Characterizing glycan processing as a function of community membership with artificial food particles.

(A) Schematic depiction of the bead-based in vivo glycan degradation assay. (B) Fluorescence intensities of pooled bead types before and after transit though the guts of mice representing two colonization conditions. Axes are labeled with the fluorophore detected in each channel. (C) Gnotobiotic mice, mono-colonized with either B. cellulosilyticus (B.c.) or B. vulgatus, were gavaged with a pool of glycan-coated and ‘empty’ uncoated beads. Beads were purified from cecal and colonic contents four hours after gavage. The mass of arabinose associated with each bead type is plotted before (black) and after (green) passage through the intestine. Circles denote individual animals. Bars show mean values and 95%CI. (D,E) Glycan degradation in mice colonized with derivatives of the 15-member community (with or without B.c.) fed the HiSF-LoFV diet (Table S3B). The mass of bead-associated arabinose (panel D) or glucose (panel E) is plotted before (black) and after collection from cecal and colonic contents on experimental day 12 (grey, 15-member community group; magenta, minus B.c. group). Circles denote individual mice. Mean values + 95%CI are shown (n=3-6 animals/group). *, P < 0.05 (Mann-Whitney U test). (See also Figure S4 and Table S6).

Figure 5.. Detecting acclimation to the presence of a potential competitor using proteomics and forward genetics.

(A,B) Relative abundance of each bacterial strain in fecal samples from gnotobiotic mice harboring derivatives of the 15-member community. Mice received the control HiSF-LoFV diet in the presence (grey closed circles) or absence of B. cellulosilyticus (B.c.) or B. vulgatus (open circles; magenta and brown respectively). Key: circles, individual mice; lines, mean values; shading, 95%CI. (C,D) Protein abundance and INSeq data for genes in arabinoxylan PULs shown along the x-axis (as locus tag number only; Bovatus_0XXXX). Mean values ± SD (vertical shading) are indicated (n=5 animals/treatment group). Genes are color-coded according to functional annotation. Key for circles: grey, 15-member community; magenta or brown, mice harboring communities without B.c. or B. vulgatus, respectively. *, P < 0.05, ∣fold change∣ > log2(1.2) [plus B.c. versus minus B.c.); limma or limma-voom]. (E) Mean protein abundance in fecal samples from day 6 in the minus B.c. group relative to the plus B.c. group; proteins whose levels are significantly different between the groups and encoded by genes in PULs are highlighted with open circles, while those encoded by genes in arabinoxylan processing PULs are labeled with their PUL number. (F) INSeq analysis showing the mean change in abundance of mutant strains from experimental day 2 to day 6 in the minus B. c. group relative to the plus B. c. group. Genes in PULs that have a significant effect on fitness are highlighted with open circles; those located in arabinoxylan processing PULs are labeled with their PUL number. (See also Figure S5 and Table S3-S5).

Figure 6.. Alleviation of competition between arabinoxylan-consuming Bacteroides.

(A-C) Relative abundance of each bacterial strain in fecal samples from gnotobiotic mice harboring derivatives of the 15-member community. Animals fed the control HiSF-LoFV diet in the presence (closed circles) or absence of B. cellulosilyticus (B.c.) or B. ovatus (B.o.) or both species (open circles; magenta, orange, or cyan respectively). Key: circles, individual mice; lines, mean values; shading, 95%CI. (D,E) Mean protein abundances in fecal samples obtained on experimental day 6. Genes in B.o. and B. c. arabinoxylan-processing PULs are shown along the x-axis (as locus tag number only; Bovatus_0XXXX or BcellWH2_0XXXX) relative to full 15-member community condition. Mean values ± SD (vertical shading) are indicated (n=5-7 animals/treatment group). Genes are color-coded according to functional annotation (see key). Key for circles: grey, 15-member community; magenta, orange, or cyan, mice harboring communities without B.c., B.o., or both species, respectively. *, P < 0.05 [15-member community versus 14-member (minus B. cellulosilyticus); limma]. (F,G) Bead-based assay of polysaccharide degradation in mice fed the HiSF-LoFV diet and colonized with the complete 15-member community, with or without B.c., B.o., or both species. (Table S3E). The mass of bead-associated arabinose (panel F) or mannose (panel G) is plotted before (black) and after exposure to the indicated communities (grey, complete 15-member community; magenta, minus B.c. group; orange, minus B.o. group; cyan, minus B.c. and B.o. group). Circles denote individual mice. Mean values + 95%CI are shown (n=5-7 animals/group). *, P < 0.05 (Mann-Whitney U test). (See also Figure S5, Table S3, Table S4, and Table S6).