Yeasts, which have been a component of the human diet for at least 7,000 years, possess an elaborate cell wall α-mannan. The influence of yeast mannan on the ecology of the human microbiota is unknown. Here we show that yeast α-mannan is a viable food source for the Gram-negative bacterium Bacteroides thetaiotaomicron, a dominant member of the microbiota. Detailed biochemical analysis and targeted gene disruption studies support a model whereby limited cleavage of α-mannan on the surface generates large oligosaccharides that are subsequently depolymerized to mannose by the action of periplasmic enzymes. Co-culturing studies showed that metabolism of yeast mannan by B. thetaiotaomicron presents a ‘selfish’ model for the catabolism of this difficult to breakdown polysaccharide. Genomic comparison with B. thetaiotaomicron in conjunction with cell culture studies show that a cohort of highly successful members of the microbiota has evolved to consume sterically-restricted yeast glycans, an adaptation that may reflect the incorporation of eukaryotic microorganisms into the human diet.
At a glance
- Enterotypes of the human gut microbiome. Nature 473, 174–180 (2011) et al.
- Host-bacterial mutualism in the human intestine. Science 307, 1915–1920 (2005) , , , &
- Metabolites produced by commensal bacteria promote peripheral regulatory T-cell generation. Nature 504, 451–455 (2013) et al.
- Polysaccharide utilization by gut bacteria: potential for new insights from genomic analysis. Nature Rev. Microbiol. 6, 121–131 (2008) , , , &
- Human nutrition, the gut microbiome and the immune system. Nature 474, 327–336 (2011) , , , &
- The gut microbiota shapes intestinal immune responses during health and disease. Nature Rev. Immunol. 9, 313–323 (2009) &
- Complex glycan catabolism by the human gut microbiota: the Bacteroidetes Sus-like paradigm. J. Biol. Chem. 284, 24673–24677 (2009) , , &
- The abundance and variety of carbohydrate-active enzymes in the human gut microbiota. Nature Rev. Microbiol. 11, 497–504 (2013) , , , &
- The carbohydrate-active enzymes database (CAZy) in 2013. Nucleic Acids Res. 42, D490–D495 (2014) , , , &
- Immune sensitization to yeast antigens in ASCA-positive patients with Crohn’s disease. Inflamm. Bowel Dis. 10, 97–105 (2004) et al.
- Microbial mannan inhibits bacterial killing by macrophages: a possible pathogenic mechanism for Crohn’s disease. Gastroenterology 133, 1487–1498 (2007) et al.
- A genomic view of the human–Bacteroides thetaiotaomicron symbiosis. Science 299, 2074–2076 (2003) et al.
- Mucosal glycan foraging enhances fitness and transmission of a saccharolytic human gut bacterial symbiont. Cell Host Microbe 4, 447–457 (2008) , &
- Schizosaccharomyces pombe glycosylation mutant with altered cell surface properties. Proc. Natl Acad. Sci. USA 91, 9327–9331 (1994) , &
- Genetic control of yeast mannan structure. Isolation and characterization of mannan mutants. J. Biol. Chem. 248, 4660–4666 (1973) , , &
- Analysis of a new family of widely distributed metal-independent alpha-mannosidases provides unique insight into the processing of N-linked glycans. J. Biol. Chem. 286, 15586–15596 (2011) et al.
- Mechanistic insights into a Ca2+-dependent family of α-mannosidases in a human gut symbiont. Nature Chem. Biol. 6, 125–132 (2010) et al.
- An ecological network of polysaccharide utilization among human intestinal symbionts. Curr. Biol. 24, 40–49 (2014) , &
- Ruminococcus bromii is a keystone species for the degradation of resistant starch in the human colon. ISME J. 6, 1535–1543 (2012) , , &
- Transfer of carbohydrate-active enzymes from marine bacteria to Japanese gut microbiota. Nature 464, 908–912 (2010) et al.
- A discrete genetic locus confers xyloglucan metabolism in select human gut Bacteroidetes. Nature 506, 498–502 (2014) et al.
- The devil lies in the details: how variations in polysaccharide fine-structure impact the physiology and evolution of gut microbes. J. Mol. Biol. 426, 3851–3865 (2014) , , &
- Recognition and degradation of plant cell wall polysaccharides by two human gut symbionts. PLoS Biol. 9, e1001221 (2011) et al.
- Saccharomyces boulardii administration changes gut microbiota and reduces hepatic steatosis, low-grade inflammation, and fat mass in obese and type 2 diabetic db/db mice. MBio. 5, e01011–e01014 (2014) , , , &
- Key residues in subsite F play a critical role in the activity of Pseudomonas fluorescens subspecies cellulosa xylanase A against xylooligosaccharides but not against highly polymeric substrates such as xylan. J. Biol. Chem. 272, 2942–2951 (1997) et al.
- Protein production by auto-induction in high density shaking cultures. Protein Expr. Purif. 41, 207–234 (2005)
- Structure of a family 15 carbohydrate-binding module in complex with xylopentaose. Evidence that xylan binds in an approximate 3-fold helical conformation. J. Biol. Chem. 276, 49061–49065 (2001) et al.
- Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem. 31, 426–428 (1959)
- The topology of the substrate binding clefts of glycosyl hydrolase family 10 xylanases are not conserved. J. Biol. Chem. 273, 32187–32199 (1998) et al.
- Structural and mechanistic insight into N-glycan processing by endo-α-mannosidase. Proc. Natl Acad. Sci. USA 109, 781–786 (2012) et al.
- Preparation of a mannopentaose, mannohexaose, and mannoheptaose from Saccharomyces cerevisiae mannan. Biochemistry 7, 1843–1854 (1968) , &
- Glycome mapping on DNA sequencing equipment. Nature Protocols 1, 397–405 (2006) , &
- Per-O-methylation reaction for structural analysis of carbohydrates by mass spectrometry. Anal. Chim. Acta 576, 147–155 (2006)
- A comprehensive procedure for preparation of partially methylated alditol acetates from glycoprotein carbohydrates. Anal. Biochem. 203, 101–108 (1992) &
- Starch catabolism by a prominent human gut symbiont is directed by the recognition of amylose helices. Structure 16, 1105–1115 (2008) , , &
- A discrete genetic locus confers xyloglucan metabolism in select human gut Bacteroidetes. Nature 506, 498–502 (2014) et al.
- 41–51 (Springer, 2007) & in Evolving Methods for Macromolecular Crystallography (eds & ) Ch. 4,
- Overview of the CCP4 suite and current developments. Acta Crystallogr. D 67, 235–242 (2011) et al.
- Xds. Acta Crystallogr. D 66, 125–132 (2010)
- Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007) et al.
- Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010) , , &
- MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D 66, 12–21 (2010) et al.
- How good are my data and what is the resolution? Acta Crystallogr. D 69, 1204–1214 (2013) &
Extended data figures and tables
Extended Data Figures
- Extended Data Figure 1: The role of specific B. thetaiotaomicron PULs and enzymes in utilization of mannan from S. cerevisiae and other yeast species. (288 KB)
a, Growth of wild-type B. thetaiotaomicron on Candida albicans mannan and glucose. b, Growth of wild-type B. thetaiotaomicron and the mutant lacking MAN-PUL1 and MAN-PUL3 (ΔMAN-PUL1/3) on Schizosaccharomyces pombe α-mannan. c, Growth of wild-type B. thetaiotaomicron, and the B. thetaiotaomicron mutants lacking MAN-PUL2 (ΔMAN-PUL2), or all three mannan PULs (ΔMAN-PUL1/2/3) on S. cerevisiae α-mannan. d, The growth profile of wild-type B. thetaiotaomicron and the B. thetaiotaomicron mutant lacking bt3774 (Δbt3774) on S. cerevisiae mannan. In panels a, b, c and d, each point on the growth curve represents the mean of three biological replicates. e, Enzymes at 1 μM at 37 °C were incubated with either undecorated α-1,6-mannan (derived from mnn2 mutant of S. cerevisiae) (lanes 1–3) or mannan from S. pombe (lanes 4–9). Lanes 1 and 4, the mannans incubated in the absence of the enzymes; lanes 2 and 6, mannans incubated with the periplasmic mannanase BT3782; lanes 3 and 7, mannans incubated with the surface mannanase BT3792; lane 5, S. pombe mannan incubated with the GH97 α-galactosidase BT2620; lanes 8 and 9, S. pombe mannan incubated with BT2620/BT3782 and BT2620/BT3792, respectively. Lane 10 galactose standard; lane 11 α-1,6-mannooligosaccharides: mannose (M1), mannobiose (M2), mannotriose (M3) and mannotetraose (M4).
- Extended Data Figure 2: Product profiling of GT32 glycosyltransferases encoded by MAN-PUL2. (305 KB)
a, HPAEC of biosynthetic reactions using mannose as an acceptor and GDP-α-Man as the donor. Mannobiose is formed in the presence of BT3775 (black) and mannotriose with BT3775 and BT3776 (red). The blue trace is a mannose standard. b, HPAEC of biosynthetic reactions with α-1,3-mannobiose as the acceptor and GDP-α-Man as the donor. BT3775 is not capable of extending mannobiose (black). In the presence of BT3775 and BT3776 mannotriose is produced (red). The blue trace is an α-1,3-mannobiose standard. c, d, MALDI-TOF analysis of the reaction products of BT3775 + BT3776 using mannose (c) and α-1,3-mannobiose (d) as an acceptor. e, NMR analysis of the α-1,3-mannobiose substrate. Peaks 1 and 3 correspond to the α-anomer and β-anomer of the mannose at the reducing end, respectively; peak 2 corresponds to the terminal α-mannosyl residue linked to O3 of the mannose at the reducing end. f, NMR analysis of the α-1,3,( α-1,6)-mannotriose BT3776 product. The numbering of the peaks are the same as in e. Peak 4 corresponds to the terminal α-mannosyl residue linked to O6 of the 3,6-linked mannose at the reducing end. g, Alditol-acetate linkage analysis of mannobiose produced by BT3775 from mannose. h, Alditol-acetate linkage analysis of branched (α-1,3),(α-1,6)-mannotriose produced by BT3776 from α-1,3-mannobiose. The green circles indicate the mannose residues present in carbohydrates identified by HPAEC, MALDI-TOF and NMR.
- Extended Data Figure 3: The structures of enzymes that play a key role in yeast mannan degradation. (753 KB)
a, Overlay of the hydrophobic conserved residues in the predicted substrate-binding cleft of BT3792 (yellow), BT2949 (cyan) and the Listeria protein Lin0763 (green; PDB code 3K7X), and the predicted catalytic aspartates. b, Solvent representation of BT3792 in which the predicted catalytic residues, Asp258 and Asp259, are coloured green. c, Overlay of BT3862 (cyan) with a homologue of the enzyme from B. xylanisolvens, BxGH99 (green; PDB code 4UTF) in complex with Man-α-1,3-isofagomine and α-1,2-mannobiose (Man residues coloured yellow and isofagomine pink). d, Solvent-exposed surface of the substrate binding cleft of the BxGH99 (teal) ligand complex overlaid with BT3862 (grey). The subsites are numbered with the catalytic residues, Glu 333 and Glu 336, coloured red and the solvent exposed O2 of Man bound at the −2 subsite and O1 and O6 of the Man located at the +2 subsite are coloured bright green. e, Overlay of BT3781 (green; PDB code 2P0V) with the substrate and catalytic residues of the Clostridium perfringens GH125 α-mannosidase CpGH125 (cyan; PDB code 3QT9), in which the ligand 6-S-α-d-mannopyranosyl-6-thio-a-d-mannopyranose (Man-S-Man) is shown in yellow. f, Solvent-exposed surface of BT3781 in the vicinity of the active site in which the catalytic residues (Glu 174 and Glu 439) are depicted in green. The position of Man-S-Man is based on the overlay shown in e. g, Overlay of BT3783 with the catalytic and substrate binding residues of a tyrosyl-DNA phosphodiesterase (PDB code 4GYZ) in complex with Mg2+ (slate-blue sphere) and phosphate (coloured orange). h, A region of the solvent-accessible surface of BT3783 in which the catalytic residues are coloured green. The figure was prepared using PyMOL. A detailed description of the structures of these proteins is provided in Supplementary Information section 5.0.
- Extended Data Figure 4: The degradation of yeast mannan by B. thetaiotaomicron in culture and the selected enzymes expressed by the bacterium, and the stereochemistry of the reaction catalysed by GH76 endo-α-1,6-mannanases. (392 KB)
a, GH92 α-mannosidases at high concentrations (50 μM) were incubated with yeast mannan for 5 h in the absence (labelled GH92) or in the presence (GH92/GH76) of the endo-α-1,6-mannanase BT3782. The GH92 α-mannosidases in this example were BT2199 (1), BT2130 (2) and BT3773 (3). The GH76 endo-α-1,6-mannanase only releases mannooligosaccharides in the presence of BT2199; see also Supplementary Information section 4.1. b, B. thetaiotaomicron was grown on yeast mannan or glucose. Yeast mannan was incubated with no bacterium (1), B. thetaiotaomicron previously cultured on yeast mannan (2) and B. thetaiotaomicron grown on glucose (3). The cells were incubated for 5 h at 37 °C with the polysaccharide without a nitrogen source and thus were not growing. The products released by the B. thetaiotaomicron cells, analysed by TLC, were mediated by the activity of enzymes presented on the surface of B. thetaiotaomicron, and not through the action of periplasmic mannanases and mannosidases. The black box highlights very low levels of high molecular weight mannooligosaccharides generated by the cells incubated in yeast mannan. c, B. thetaiotaomicron was cultured for up to 48 h (stationary phase) on yeast mannan. The supernatant of the culture at the time points indicated were analysed by TLC. In all panels the samples were chromatographed with the following α-1,6-mannooligosaccharides: mannose, M1; mannobiose, M2; mannotriose, M3; mannotetraose, M4. d, The absorbance of the culture used in c. e, BT3792 (GH76) endo-α-1,6-mannosidase is a retaining glycoside hydrolase. Enzymatic hydrolysis of 4-nitrophenyl α-d-mannopyranosyl-1,6-α-d-mannopyranoside (S) was monitored by 1H-NMR spectroscopy (500 MHz). The stacked spectra show the reaction progress over time. SH1α is the anomeric proton of the reducing end mannopyranoside of the substrate, and SH1′α is the anomeric proton of the non-reducing end mannopyranoside. The reaction proceeds with the initial formation of the product, the α-anomer of α-1,6-mannobiose (P-α, peaks PH1α and PH1′α), which slowly mutarotates to the β-anomer (P-β, peaks PH1β and PH1′β). f, TLC analysis of S. cerevisiae mannan incubated without enzyme (lane 1), BT3774 (lane 2), BT3792 (lane 3), and BT3774 and BT3792 (lane 4). M1–M4 are α-mannnooligosaccharide standards numbered according to their d.p. GH76 mannanase BT3792 does not attack the backbone of S. cerevisiae mannan unless the side chains are first removed by the GH38 α-mannosidase BT3774, confirming that this enzyme cleaves the mannose α-1,2-linked to the mannan backbone. The data in a, b and c are representative of two biological replicates, while the data in f are representative of two technical replicates.
- Extended Data Figure 5: The activity of periplasmic α-mannosidases and the growth of different species of Bacteroides against yeast mannan. (360 KB)
Structures of the mannans derived from wild-type and mutants of S. cerevisiae. The tables adjacent to the different yeast structures depict the initial rate of mannan hydrolysis by the four enzymes. The growth curves adjacent to the different mannan structures show the growth profile of B. thetaiotaomicron (black), B. ovatus (red) and B. xylanisolvens (blue) on the glycans (each point represents the mean growth of 3 separate cultures ± s.d.). The porcine-derived B. xylanisolvens strain shown here acquired MAN-PUL1 by lateral gene transfer (Extended Data Fig. 9), explaining its capacity to degrade processed mannans. Vertical error bars represent standard deviation of three separate replicates in each condition.
- Extended Data Figure 6: The activity of GH76 α-mannanases and GH125 α-mannosidases. (413 KB)
a, b, BT3792 and BT3782, respectively, were incubated with α-1,6-mannotetraose at a concentration KM. Substrate depletion was measured using HPAEC and the rate (right of a and b) enabled kcat/KM to be determined. c, BT3792 and BT3782 were incubated with unbranched yeast mannan (derived from the S. cerevisiae mutant MNN2). The yeast mannan at 0.1% was incubated with the two GH76 α-1,6-mannanases for 1 h at 37 °C in 50 mM sodium phosphate buffer, pH 7.0. The limit products were analysed by TLC. α-1,6-Mannooligosaccharides are identified by their degree of polymerization (M1, mannose; M2, mannobiose; M3, mannotriose; M4, mannotetraose); IP, injection peak. d, e, BT2632 and BT3781 at 100 nM were incubated with 1 mg ml−1 of the debranched mannan for 1 h in the buffer described above. TLC analysis (d) and HPAEC traces (e) of the reactions are shown. The data in c and d are representative of two technical replicates.
- Extended Data Figure 7: HMNG deconstruction by B. thetaiotaomicron. (286 KB)
a, Structure of the HMNG PUL. Genes drawn to scale with their orientation indicated. Genes encoding known or predicted functionalities are colour-coded and, where appropriate, are also annotated according to their CAZy glycoside hydrolase (GH) family number. SGBP represents a surface glycan binding protein. b, BT3994 was incubated with α-1,6-mannotetraose (Man4) or the high mannose N-glycan Man5GlcNAc2, with both oligosaccharides labelled with 2-aminobenzamide (AB). At the indicated time points aliquots were removed and analysed by HPAEC using a fluorescence detection system. While Man5GlcNac2-AB was hydrolysed by BT3994, the enzyme was not active against Man4-AB. c, Chicken ovalbumin was incubated with buffer (1) or 1 μM of BT3987 (2) in 20 mM Na-HEPES buffer, pH 7.5, for 5 h at 37 °C, and the soluble material was permethylated and analysed by MALDI-TOF mass spectrometry. The high mannose N-glycans released are labelled. d, Western blot of B. thetaiotaomicron cells cultured on yeast mannan that were untreated with proteinase K (0 h) or incubated with 2 mg ml−1 proteinase K for 16 h (16 h). The lane labelled RP contained a purified recombinant form of BT3990. The blots were probed with antibodies against BT3990. The data in d are representative of two biological replicates. e, f, Representative isothermal calorimetry titrations for BT3984 titrated with Gal-β1,4-GlcNAc (LacNAc; 25 mM) (e), and for BT3986 titrated with mannose (50 mM) (f). The top half of each panel shows the raw isothermal calorimetry titration heats; the bottom half, the integrated peak areas fitted using a single binding model by MicroCal Origin software. ITC was carried out in 50 mM Na-HEPES, pH 7.5 at 25 °C. The affinities and thermodynamic parameters of binding are shown in Supplementary Table 5. g, Growth profile of wild-type B. thetaiotaomicron (WT Bt; black) and the mutant Δbt3993 (red), which lacks the extra-cellular factor σ regulator gene of HMNG-PUL, cultured on Man8GlcNAc2 (each curve shows the mean ± s.d. of 3 separate cultures).
- Extended Data Figure 8: Metagenomic analysis of the occurrence of the yeast mannan PULs in humans. (520 KB)
Abundance of Bacteroides mannan PULs in humans from a survey of metagenomic sequencing data from a total of 250 adult human samples (211 healthy, 27 ulcerative colitis, 12 Crohn’s disease; see Methods for references). Data sets were individually queried by BLAST using either each entire mannan PUL (PULs 2,3) or a sub-fragment that was trimmed to eliminate cross-detection of other species genomes beyond B. thetaiotaomicron and porcine B. xylanisolvens (PUL1; see Methods for additional search details). Each horizontal line represents the presence of a hit in a single individual. The leftmost column summarizes the total mannan PUL content in each person (annotated according to the colour key in the upper right corner). The mannan PUL frequency across all 250 samples is shown at the bottom for each condition and is compared to the frequency of several other PULs implicated in xyloglucan and porphyran utilization. Graph at far right illustrates the variation in sequencing depth for each sample/study; black lines show the average depth in megabase pairs (Mbp) for each study; the light grey line shows the depth for each individual sample.
- Extended Data Figure 9: Presence of a conjugative transposon (cTn) that contains MAN-PUL1 in the genomes of porcine B. xylanisolvens strains and the mannan presented to these organisms. (207 KB)
a, Shown across the top is a schematic of a cTn that has been integrated into the 3′ end of a tRNAphe gene in B. thetaiotaomicron strain VPI-5482. Integration is mediated by a 22 bp direct repeat sequence that is contained in tRNAphe and repeated again at the other side of the cTn (right insertion site). The location of B. thetaiotaomicron MAN-PUL1 is denoted within the larger cTn element using a colour scheme identical to Fig. 1a. The lower panel shows an expanded view of the MAN-PUL1 locus in five sequenced strains of B. xylanisolvens from the faeces of pigs fed a diet enriched with distillers grains that were fermented with yeast. A nearly identical copy (both by amino acid homology and syntenic organization) of this genomic region is present in B. thetaiotaomicron and the porcine B. xylanisolvens strains. Although the draft genomes of the B. xylanisolvens strains contains gaps in all five assemblies at the left side of the MAN-PUL1, the right side insertion site was resolved in all genomes, suggesting that the B. xylanisolvens loci were also transferred by lateral transfer at some point in the history of these strains. b, Forty-three different strains from five Bacteroidetes isolated from animal guts (each indicated with a solid circle) were inoculated into minimal media containing S. cerevisiae mannan as the sole carbon source. The growth of the cultures was measured over 48 h by recording the optical density at 600 nm. c, TLC analysis of the products generated by incubating BT3774 and BT3792 with α-mannan extracted from the distillers grain fed to the pigs from which the B. xylanisolvens were isolated. The data are representative of two technical replicates.
- Extended Data Figure 10: In vivo and in vitro expression of the mannan PULs. (227 KB)
a, Level of susC-like transcripts derived from MAN-PUL1 (BT2626), MAN-PUL2 (BT3788) and MAN-PUL3 (BT3854) from B. thetaiotaomicron in monocolonized gnotobiotic mice fed a glycan-free diet deficient in B. thetaiotaomicron-digestible glycans (red), the same diet with added yeast mannan (1% w/v in drinking water) as the only usable polysaccharide (green), and a diet containing 50% bread (blue). The levels of the susC transcripts were quantified relative to the same mRNA species in B. thetaiotaomicron cultured in vitro on glucose minimal medium (MM-G). Note that in all cases, expression of MAN-PUL genes is equally high in vivo. b, Transcription of the same mannan susC-like genes in response to increasing concentrations of yeast mannan in the media after 30 min exposure. The prototypic susC (BT3701) involved in starch metabolism is shown for comparison. c, An identical exposure experiment to that shown in b, except that glucose-grown B. thetaiotaomicron cells were exposed to aqueous extracts of the cereal grain diet (natural diet) fed to mice before the experiment, or the digestible glycan-free control diet (glycan-free diet) used as a base for all feeding treatments. Exposure was conducted for 30 min to determine if any diet extract contained contaminating levels of mannan that could be detected by B. thetaiotaomicron cells; inclusion of purified mannan (5 mg ml−1) in addition to the glycan-free diet served as positive controls. In all panels, the results represent the mean of 3 biological replicates and error bars represent s.d.
- Supplementary Information (406 KB)
This file contains Supplementary Text, Supplementary References and Supplementary Tables 1-6.