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A single sulfatase is required to access colonic mucin by a gut bacterium

Nature volume 598, pages 332–337 (2021)Cite this article

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Abstract

Humans have co-evolved with a dense community of microbial symbionts that inhabit the lower intestine. In the colon, secreted mucus creates a barrier that separates these microorganisms from the intestinal epithelium1. Some gut bacteria are able to utilize mucin glycoproteins, the main mucus component, as a nutrient source. However, it remains unclear which bacterial enzymes initiate degradation of the complex O-glycans found in mucins. In the distal colon, these glycans are heavily sulfated, but specific sulfatases that are active on colonic mucins have not been identified. Here we show that sulfatases are essential to the utilization of distal colonic mucin O-glycans by the human gut symbiont Bacteroides thetaiotaomicron. We characterized the activity of 12 different sulfatases produced by this species, showing that they are collectively active on all known sulfate linkages in O-glycans. Crystal structures of three enzymes provide mechanistic insight into the molecular basis of substrate specificity. Unexpectedly, we found that a single sulfatase is essential for utilization of sulfated O-glycans in vitro and also has a major role in vivo. Our results provide insight into the mechanisms of mucin degradation by a prominent group of gut bacteria, an important process for both normal microbial gut colonization2 and diseases such as inflammatory bowel disease3.

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Fig 1: Bacterial growth on colonic mucin and B. thetaiotaomicron sulfatase activities.
Fig. 2: Activity of B. thetaiotaomicron sulfatases on colonic mucin O-glycans.
Fig. 3: Crystal structures of 3S-Gal/GalNAc sulfatases.
Fig. 4: BT16363S-Gal activity is required for the use of cMO and competitive fitness in vivo.

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Data availability

All data for the experiments, along with corresponding statistical test values, where appropriate, are provided within the paper and in its Supplementary Information. The crystal structure datasets generated have been deposited in the PDB under the following accession numbers: 7ANB, 7ANA, 7AN1, 7OQD and 7ALL. The MS raw files have been deposited in the GlycoPOST database under the following IDs: GPST000150 and GPST000196. Glycan structural annotations were deposited to the UniCarb database at https://unicarb-dr.glycosmos.org/references/462. There are no restrictions on data or biological resource availability. Data and biological resources can be obtained by contacting the corresponding authors. Source data are provided with this paper.

Code availability

No new codes were developed or compiled in this study.

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Acknowledgements

This project has received funding from the European Union’s Horizon 2020 research and innovation programme under Marie Skłodowska-Curie grant agreement no. 748336. This work was supported by National Institutes of Health grants (DK118024 and DK125445 awarded to E.C.M., U01AI095473 awarded to G.C.H.), the European Research Council (ERC; 694181), the Knut and Alice Wallenberg Foundation (2017.0028), the Swedish Research Council (2017-00958), Wilhelm och Martina Lundgrens Vetenskapsfond (2020.3597, awarded to A.S.L.) and the Academy of Medical Sciences/Wellcome Trust through Springboard grant SBF005\1065 163470 awarded to A.C. We acknowledge access to the SOLEIL and Diamond Light sources via both University of Liverpool and Newcastle University BAGs (proposal nos mx21970 and mx18598, respectively). We thank the staff of DIAMOND and SOLEIL and members of Liverpool’s molecular biophysics group for assistance with data collection. We thank members of the University of Michigan Mouse Facility and acknowledge the University of Michigan Center for Gastrointestinal Research (UMCGR; NIDDK 5P30DK034933) for support. MS analysis of glycans was performed in the Swedish Infrastructure for Biologic Mass Spectrometry (BioMS) supported by the Swedish Research Council. We are also grateful for E. Corre’s help regarding bioinformatics analyses (ABIMS platform, Station Biologique de Roscoff, France).

Author information

Authors and Affiliations

  1. Department of Microbiology and Immunology, University of Michigan, Ann Arbor, MI, USA

    Ana S. Luis, Gabriel Vasconcelos Pereira, Robert W. P. Glowacki, Sadie R. Gugel, Shaleni Singh, Nicholas A. Pudlo & Eric C. Martens

  2. Department of Medical Biochemistry, Institute for Biomedicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden

    Ana S. Luis, Chunsheng Jin, Gunnar C. Hansson & Niclas G. Karlsson

  3. Department of Biochemistry and Systems Biology, Institute of Systems, Molecular and Integrative Biology, University of Liverpool, Liverpool, UK

    Dominic P. Byrne, James A. London, Patrick A. Eyers, Edwin A. Yates & Alan Cartmell

  4. Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle upon Tyne, UK

    Arnaud Baslé

  5. Centre for Synthesis and Chemical Biology, University College Dublin, Belfield, Dublin, Ireland

    Mark Reihill & Stefan Oscarson

  6. Sorbonne Université, Univ Paris 06, CNRS, UMR 8227, Integrative Biology of Marine Models, Roscoff, Bretagne, France

    Mirjam Czjzek, Gurvan Michel & Tristan Barbeyron

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  1. Ana S. Luis
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Contributions

A.S.L., A.C. and E.C.M. designed experiments and wrote the manuscript. A.S.L. and A.C. cloned, expressed and purified sulfatases and performed the enzymatic assays. A.C., D.P.B., J.A.L. and P.A.E. carried out and analysed the data from kinetic and binding experiments. E.A.Y., M.R. and S.O. performed chemical syntheses. A.C. and A.B. performed structural biology experiments. C.J., A.S.L., G.C.H. and N.G.K. performed and interpreted data from analytical glycobiology experiments. A.S.L., G.V.P., R.W.P.G., S.G., S.S. and N.A.P. performed bacterial growth experiments and analysed in vivo competition data. M.C., G.M. and T.B. performed sulfatase phylogenetic analyses. All authors read and approved the manuscript.

Corresponding authors

Correspondence to Ana S. Luis, Alan Cartmell or Eric C. Martens.

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Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature thanks B. van den Berg and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 Growth of Bacteroides and Phocaeicola type strains and Akkermansia muciniphila in different mucin O-glycans.

a, Graphs showing the growth of strains that are able to utilize colonic or gastric O-glycans. Growths were performed in minimal media containing the indicated carbon source. b, Growth experiments performed identically to panel a, but with two species, P. massiliensis and A. muciniphila, that grow on gMO but not cMO. A control experiment was performed with A. muciniphila grown on cMO plus added GlcNAC to verify that cMO does not contain material that is inhibitory to this species (biological replicates n =3 for both panels, error bars denote the s.e.m. for each time point). Note that gMO were used at 10mg/ml final concentration, while cMO were used at 5mg/ml due to background turbidity. This reduced concentration and the higher amount of sulfate in cMO account for the lower growth on this substrate. cMO, colonic mucin O-glycans; gMO, gastric mucin O-glycans, GlcNAc, N-acetyl-D-glucosamine.

Source data

Extended Data Fig. 2 Schematic representation of polysaccharide utilization loci (PULs) encoding sulfatases (sulf).

Genes are colour coded according to the predicted function of the respective proteins. Glycoside hydrolases (GH) in known families are indicated by GHXX or GH*, where XX and * indicates the respective family number or non-classified, respectively.

Extended Data Fig. 3 Activity and affinity of sulfatases to targeted substrates.

a, Recombinant enzymes (1 μM) were incubated with 1 mM of substrate in 10 mM MES pH6.5 with 5 mM CaCl2 for 16h at 37 °C. Sulfated disaccharides were generated by adding 1 μM of a characterized α1,3/1,4-fucosidase (BT1625) in the enzymatic reaction. Control reactions without sulfatases were carried in the same conditions. Samples were analysed by mass spectrometry and the intensity of the substrate and reaction products was used for comparison of the relative abundance of these sugars after incubation with the respective enzymes. b, Affinity studies looking at the effect of ligand binding on the melting temperature of 3S and 6S-Gal sulfatases. All reactions were performed in 100 mM BTP, pH 7.0 with 150 mM NaCl. For sample melting temperatures see Supplementary Table 11. c, Activity of 3S-Gal/GalNAc sulfatases (10 μM) against 3S-GalNAc (10 mM). Reactions were performed in 10 mM Hepes, pH 7.0, with 150 mM NaCl and 5 mM CaCl2. The data shown are one representative from the biological replicates conducted (n = 3).

Source data

Extended Data Fig. 4 Enzymatic screen of Bt sulfatases using sulfated monosaccharides.

Recombinant enzymes (1 μM) were incubated with 1 mM of substrate in 10 mM MES pH6.5 with 5 mM CaCl2 for 16 h at 37 °C. Reactions were analyzed by thin layer chromatography (left side) or HPAEC with pulsed amperometric detection (right side). Control reactions without sulfatases were carried out in the same conditions. The standards in TLC and HPAEC-PAD are labelled on the left side and top, respectively. The different panel represent activities found for sulfatases targeting: (a) 4S-Gal/GalNAc; (b) 3S-GlcNAc (c) 6S-Gal/GalNAc; (d) 6S-GlcNAc. The data shown are representative from biological replicates (n = 3).

Source data

Extended Data Fig. 5 Activity of Bt sulfatases against colonic mucin O-glycans (cMO) analysed by mass spectrometry.

a, Relative abundance of structures detected in different samples organized by sulfate-linkage (top panel) or presence of one or several sugar substitutions such as sulfate, sialic acid and fucose (bottom panel). The colour-coded bars represent the relative abundance and the total number of the structures containing the specific linkage/substitution; b, Representation of O-glycans detected by mass spectrometry in cMO batch 2 (control) and after sulfatase treatment from the lower (top) to the higher (bottom) mass range; c, Relative abundance and putative structures for the specific m/z shown in panel b. The putative structure for the different mass is shown on the right side of the graphic. The reactions were performed with 1 μM of enzyme and 0.5% cMO in 10 mM MES pH 6.5 with 5 mM CaCl2 for 16 h at 37 °C. The complete dataset is provided in Supplementary Table 4 and 5 for cMO batch 1 and 2, respectively.

Source data

Extended Data Fig. 6 Schematic representation of 3S-Gal/GalNAc sulfatases.

a,(i) Cartoon representation colour ramped from blue (α/β/α N-terminal domain) to red (β-sheet C-terminal domain); (ii) the final 2mFobs-DFcalc maps contoured at 1σ for GalNAc in BT16223S-Gal/GalNAc (Top) LacNAc in BT16363S-Gal (middle) and BT46833S-Gal (bottom); (iii) represents the simulated annealed composite omit 2mFobs-DFcalc maps contoured at 1σ and (iv) represents the mFobs-DFcalc maps, prior to building of the ligand contoured at 3σ;. b,(i) Overlay of the active site S residues of BT16363S-Gal (green) BT16223S-Gal/GalNAc (blue) and BT46833S-Gal (pink). The putative catalytic residues are shown in bold. The calcium ion is represented as a grey sphere and its polar interactions indicated as dashed lines. The 3S-Gal substrate is from the BT16363S-Gal 3’S-Lewis-a complex, and BT16223S-Gal/GalNAc and BT46833S-Gal structures have been overlaid, (ii) the final 2mFobs-DFcalc maps of the observed 3’S-Lewis-a substrate contoured at 1σ, (iii) represents the simulated annealed composite omit 2mFobs-DFcalc maps contoured at 1σ, and (iv) represents the mFobs-DFcalc maps of the observed 3’S-Lewis-a substrate, prior to building of the ligand, contoured at 3σ; c, Docking of putative structures of O-glycans targeted by BT46833S-Gal using the LacNAc as reference point showing that this structure can accommodate a sialic acid in −1 subsite and additional sugars in positive subsites (left hand side). The docking sugars are shown as sticks (middle panel) and a schematic is represented inside the dashed box (right hand side). Using the LacNAc product as an ‘anchor’ additional sugars were built in manually with Coot 0.9 and regularized to low energy conformations.

Extended Data Fig. 7 Phylogenetic tree of S1_20 and S1_4 sulfatases.

The radial trees were constructed using the branched trees shown in Supplementary Figs. 3 and 4. For clarity, all labels and sequence accession codes have been omitted. Red filled circles designate sequences from B. thetaiotaomicron sulfatases. The residue is written in black without any attributes if present in the sequence, in grey and italics if the residue is mutated to any type in that sequence, or to a specific residue type if given in brackets. a, Radial representation of the phylogenetic tree constructed with representative sequences of the sulfatase S1_20 subfamily. The colour code is given as a pattern of presence or absence of the residues E100, Q173 H177, E334, R353, which are crucial in substrate recognition by BT1636 (acc-code Q8A789, coloured red). A grey X in italics specifically designates that the residue E100 is absent in that sequence, and no obvious orthologous residue can be found from the alignment. b, Radial representation of the phylogenetic tree constructed with representative sequences of the sulfatase S1_4 subfamily. The colour code is given as a pattern of presence or absence of the residues R72, E335 and W505, which are crucial in substrate recognition by BT4683 (acc-code Q89YP8, coloured red). A grey X in italics specifically designates that the residue W505 is absent in that sequence, and no obvious orthologous residue can be found from the alignment.

Extended Data Fig. 8 Sulfatase activity is required for growth in cMO and in vivo fitness.

a, Growth curves of Bt wild-type Δtdk (WT), different sulfatase mutants (ΔbtXXX) and complemented strains on glucose, colonic or gastric mucin O-glycans (cMO and gMO, respectively). The curves represent the average of biological replicates (n = 3) and the error bars denote s.e.m. b, Relative abundance of oligosaccharides detected by mass spectrometry in culture supernatant of WT and Δbt16363S-Gal after growth in cMO for 96h at anaerobic conditions. The control corresponds to cMOincubated in the same conditions without bacterium. The colours represent the relative abundance of structures grouped according to the presence of epitopes (sulfate, fucose and sialic acid) and the numbers represent the total number of structures that contain the respective substitution. c, Colonization of gnotobiotic mice fed a fiber-free diet by Bt WT and mutants lacking the full (ΔanSME, no S1 sulfatases active) or specific sulfatase activity (Δ6S-GlcNAc and Δ6S-GlcNAc+Δ6S-Gal/GalNAc). The fecal relative abundance of each strain was determined at regular intervals until day 42. The relative abundance of time 0 represents the abundance in the gavaged inoculum. At the experimental endpoint the relative abundance was also determined in small intestine and cecum. The graphs represent the average of n=3-7 and the error bars denote the s.e.m. The relative abundance in each individual animal is represented in a lighter colour in each of the respective graphics.

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Supplementary information

Supplementary Information

This file contains Supplementary Discussion and additional references.

Reporting Summary

Supplementary Figure 1

Characterization of negatively charged O-glycans from porcine colonic mucins using LC–MS/MS

Supplementary Figure 2

Phylogenetic tree of S1 sulfatases in the genomes of Bacteroides and Phocaeicola type strains and A. muciniphila

Supplementary Figure 3

Phylogenetic tree of representative sulfatases from subfamily S1_20

Supplementary Figure 4

Phylogenetic tree of representative sulfatases from subfamily S1_4

Supplementary Figure 5

Immobilized metal affinity chromatography purification of studied sulfatases

Supplementary Figure 6

Activity profiles of purified sulfatases showing pH optima

Supplementary Figure 7

Biophysical characteristics of inactive sulfatase mutants

Peer Review File

Supplementary Table 1

Family S1 sulfatase subfamiles encoded in the genomes of different Bacteroides and Phocaeicola type strains and Akkermansia.

Supplementary Table 2

List of sulfated saccharides used in the initial sulfatase activity screen.

Supplementary Table 3

Sulfatase kinetics for WT and mutants against different saccharides.

Supplementary Table 4

LC-MS analysis of colonic mucin oligosaccharides (cMO). Biological replicate 1.

Supplementary Table 5

LC-MS analysis of colonic mucin oligosaccharides (cMO). Biological replicate 2.

Supplementary Table 6

Sulfatase signal peptide and localization prediction.

Supplementary Table 7

LC-MS analysis of O-glycans in culture supernatant of bt16363S-Gal mutant by LC-MS/MS.

Supplementary Table 8

Conservation of S1_20 3S-Gal/GalNAc specificity residues in Bacteroides and Phocaeicola type strains, and Akkermansia muciniphila.

Supplementary Table 9

Primers designed to clone Bt sulfatases.

Supplementary Table 10

Primers designed to generate the site-directed mutants of Bt sulfatases.

Supplementary Table 11

Melting temperatures of galactose targeting sulfatases with and without ligands.

Supplementary Table 12

Analysis of carbohydrate structure ligands.

Supplementary Table 13

X-ray crystallographic and refinement statistics.

Supplementary Table 14

Primers designed to generate the in-frame gene deletions and complementations of Bt sulfatases.

Supplementary Table 15

S1_20 homologues of BT16363S-Gal.

Supplementary Table 16

S1_20 homologues of BT16223S-Gal/GalNAc.

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Luis, A.S., Jin, C., Pereira, G.V. et al. A single sulfatase is required to access colonic mucin by a gut bacterium. Nature 598, 332–337 (2021). https://doi.org/10.1038/s41586-021-03967-5

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