Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

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

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.

This is a preview of subscription content

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

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.

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.

References

  1. 1.

    Johansson, M. E. et al. The inner of the two Muc2 mucin-dependent mucus layers in colon is devoid of bacteria. Proc. Natl Acad. Sci. USA 105, 15064–15069 (2008).

    ADS  CAS  PubMed  PubMed Central  Article  Google Scholar 

  2. 2.

    Benjdia, A., Martens, E. C., Gordon, J. I. & Berteau, O. Sulfatases and a radical S-adenosyl-l-methionine (AdoMet) enzyme are key for mucosal foraging and fitness of the prominent human gut symbiont, Bacteroides thetaiotaomicron. J. Biol. Chem. 286, 25973–25982 (2011).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  3. 3.

    Hickey, C. A. et al. Colitogenic Bacteroides thetaiotaomicron antigens access host immune cells in a sulfatase-dependent manner via outer membrane vesicles. Cell Host Microbe 17, 672–680 (2015).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  4. 4.

    Packey, C. D. & Sartor, R. B. Commensal bacteria, traditional and opportunistic pathogens, dysbiosis and bacterial killing in inflammatory bowel diseases. Curr. Opin. Infect. Dis. 22, 292–301 (2009).

    PubMed  PubMed Central  Article  Google Scholar 

  5. 5.

    Sears, C. L. & Garrett, W. S. Microbes, microbiota, and colon cancer. Cell Host Microbe 15, 317–328 (2014).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  6. 6.

    Van der Sluis, M. et al. Muc2-deficient mice spontaneously develop colitis, indicating that Muc2 is critical for colonic protection. Gastroenterology 131, 117–129 (2006).

    PubMed  Article  CAS  Google Scholar 

  7. 7.

    Velcich, A. et al. Colorectal cancer in mice genetically deficient in the mucin Muc2. Science 295, 1726–1729 (2002).

    ADS  CAS  PubMed  Article  Google Scholar 

  8. 8.

    Bergstrom, K. et al. Core 1- and 3-derived O-glycans collectively maintain the colonic mucus barrier and protect against spontaneous colitis in mice. Mucosal Immunol. 10, 91–103 (2016).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  9. 9.

    Larsson, J. M. et al. Altered O-glycosylation profile of MUC2 mucin occurs in active ulcerative colitis and is associated with increased inflammation. Inflamm. Bowel Dis. 17, 2299–2307 (2011).

    PubMed  Article  Google Scholar 

  10. 10.

    Fu, J. et al. Loss of intestinal core 1-derived O-glycans causes spontaneous colitis in mice. J. Clin. Invest. 121, 1657–1666 (2011).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  11. 11.

    Kudelka, M. R. et al. Cosmc is an X-linked inflammatory bowel disease risk gene that spatially regulates gut microbiota and contributes to sex-specific risk. Proc. Natl. Acad. Sci. USA 113, 14787–14792 (2016).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  12. 12.

    Larsson, J. M., Karlsson, H., Sjovall, H. & Hansson, G. C. A complex, but uniform O-glycosylation of the human MUC2 mucin from colonic biopsies analyzed by nanoLC/MSn. Glycobiology 19, 756–766 (2009).

    PubMed  Article  CAS  Google Scholar 

  13. 13.

    Holmen Larsson, J. M., Thomsson, K. A., Rodriguez-Pineiro, A. M., Karlsson, H. & Hansson, G. C. Studies of mucus in mouse stomach, small intestine, and colon. III. Gastrointestinal Muc5ac and Muc2 mucin O-glycan patterns reveal a regiospecific distribution. Am. J. Physiol. Gastrointest. Liver Physiol. 305, G357–G363 (2013).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  14. 14.

    Thomsson, K. A. et al. Detailed O-glycomics of the Muc2 mucin from colon of wild-type, core 1- and core 3-transferase-deficient mice highlights differences compared with human MUC2. Glycobiology 22, 1128–1139 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  15. 15.

    Robbe, C. et al. Evidence of regio-specific glycosylation in human intestinal mucins—presence of an acidic gradient along the intestinal tract. J. Biol. Chem. 278, 46337–46348 (2003).

    CAS  PubMed  Article  Google Scholar 

  16. 16.

    Katoh, T. et al. Identification and characterization of a sulfoglycosidase from Bifidobacterium bifidum implicated in mucin glycan utilization. Biosci. Biotechnol. Biochem. 81, 2018–2027 (2017).

    CAS  PubMed  Article  Google Scholar 

  17. 17.

    Martens, E. C., Chiang, H. C. & Gordon, J. I. Mucosal glycan foraging enhances fitness and transmission of a saccharolytic human gut bacterial symbiont. Cell Host Microbe 4, 447–457 (2008).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  18. 18.

    Diez-Roux, G. & Ballabio, A. Sulfatases and human disease. Annu. Rev. Genomics Hum. Genet. 6, 355–379 (2005).

    CAS  PubMed  Article  Google Scholar 

  19. 19.

    Wlodarska, M. et al. Indoleacrylic acid produced by commensal Peptostreptococcus species suppresses inflammation. Cell Host Microbe 22, 25–37 (2017).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  20. 20.

    Tramontano, M. et al. Nutritional preferences of human gut bacteria reveal their metabolic idiosyncrasies. Nat. Microbiol. 3, 514–522 (2018).

    CAS  Article  Google Scholar 

  21. 21.

    Derrien, M., Vaughan, E. E., Plugge, C. M. & de Vos, W. M. Akkermansia muciniphila gen. nov., sp. nov., a human intestinal mucin-degrading bacterium. Int. J. Syst. Evol. Microbiol. 54, 1469–1476 (2004).

    CAS  PubMed  Article  Google Scholar 

  22. 22.

    Pudlo, N. A. et al. Symbiotic human gut bacteria with variable metabolic priorities for host mucosal glycans. mBio 6, e01282 (2015).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  23. 23.

    Barbeyron, T. et al. Matching the diversity of sulfated biomolecules: creation of a classification database for sulfatases reflecting their substrate specificity. PLoS ONE 11, e0164846 (2016).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  24. 24.

    Benjdia, A. et al. Anaerobic sulfatase-maturating enzymes, first dual substrate radical S-adenosylmethionine enzymes. J. Biol. Chem. 283, 17815–17826 (2008).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  25. 25.

    Cartmell, A. et al. How members of the human gut microbiota overcome the sulfation problem posed by glycosaminoglycans. Proc. Natl Acad. Sci. USA 114, 7037–7042 (2017).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  26. 26.

    Ndeh, D. et al. Metabolism of multiple glycosaminoglycans by Bacteroides thetaiotaomicron is orchestrated by a versatile core genetic locus. Nat. Commun. 11, 646 (2020).

    ADS  CAS  PubMed  PubMed Central  Article  Google Scholar 

  27. 27.

    Ulmer, J. E. et al. Characterization of glycosaminoglycan (GAG) sulfatases from the human gut symbiont Bacteroides thetaiotaomicron reveals the first GAG-specific bacterial endosulfatase. J. Biol. Chem. 289, 24289–24303 (2014).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  28. 28.

    Tobisawa, Y., Imai, Y., Fukuda, M. & Kawashima, H. Sulfation of colonic mucins by N-acetylglucosamine 6-O-sulfotransferase-2 and its protective function in experimental colitis in mice. J. Biol. Chem. 285, 6750–6760 (2010).

    CAS  PubMed  Article  Google Scholar 

  29. 29.

    Neelamegham, S. et al. Updates to the Symbol Nomenclature for Glycans guidelines. Glycobiology 29, 620–624 (2019).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  30. 30.

    Egan, M., Jiang, H., O’Connell Motherway, M., Oscarson, S. & van Sinderen, D. Glycosulfatase-encoding gene cluster in Bifidobacterium breve UCC2003. Appl. Environ. Microbiol. 82, 6611–6623 (2016).

    ADS  CAS  PubMed  PubMed Central  Article  Google Scholar 

  31. 31.

    Briliute, J. et al. Complex N-glycan breakdown by gut Bacteroides involves an extensive enzymatic apparatus encoded by multiple co-regulated genetic loci. Nat. Microbiol. 4, 1571–1581 (2019).

    CAS  PubMed  Article  Google Scholar 

  32. 32.

    Packer, N. H., Lawson, M. A., Jardine, D. R. & Redmond, J. W. A general approach to desalting oligosaccharides released from glycoproteins. Glycoconjugate J. 15, 737–747 (1998).

    CAS  Article  Google Scholar 

  33. 33.

    Hayes, C. A. et al. UniCarb-DB: a database resource for glycomic discovery. Bioinformatics 27, 1343–1344 (2011).

    CAS  PubMed  Article  Google Scholar 

  34. 34.

    Everest-Dass, A. V., Abrahams, J. L., Kolarich, D., Packer, N. H. & Campbell, M. P. Structural feature ions for distinguishing N- and O-linked glycan isomers by LC–ESI–IT MS/MS. J. Am. Soc. Mass Spectrom. 24, 895–906 (2013).

    ADS  CAS  PubMed  Article  Google Scholar 

  35. 35.

    Domon, B. & Costello, C. E. Structure elucidation of glycosphingolipids and gangliosides using high-performance tandem mass spectrometry. Biochemistry 27, 1534–1543 (1988).

    CAS  PubMed  Article  Google Scholar 

  36. 36.

    Byrne, D. P. et al. New tools for carbohydrate sulfation analysis: heparan sulfate 2-O-sulfotransferase (HS2ST) is a target for small-molecule protein kinase inhibitors. Biochem. J. 475, 2417–2433 (2018).

    CAS  PubMed  Article  Google Scholar 

  37. 37.

    Byrne, D. P. et al. cAMP-dependent protein kinase (PKA) complexes probed by complementary differential scanning fluorimetry and ion mobility–mass spectrometry. Biochem. J. 473, 3159–3175 (2016).

    CAS  PubMed  Article  Google Scholar 

  38. 38.

    Das, T. M., Rao, C. P. & Kolehmainen, E. Synthesis and characterisation of N-glycosyl amines from the reaction between 4,6-O-benzylidene-d-glucopyranose and substituted aromatic amines and also between 2-(O-aminophenyl) benzimidazole and pentoses or hexoses. Carbohydr. Res. 334, 261–269 (2001).

    CAS  PubMed  Article  Google Scholar 

  39. 39.

    Kabsch, W. Xds. Acta Crystallogr. D Biol. Crystallogr. 66, 125–132 (2010).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  40. 40.

    Winter, S. E., Lopez, C. A. & Baumler, A. J. The dynamics of gut-associated microbial communities during inflammation. EMBO Rep. 14, 319–327 (2013).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  41. 41.

    Evans, P. R. & Murshudov, G. N. How good are my data and what is the resolution? Acta Crystallogr. D Biol. Crystallogr. 69, 1204–1214 (2013).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  42. 42.

    Long, F., Vagin, A. A., Young, P. & Murshudov, G. N. BALBES: a molecular-replacement pipeline. Acta Crystallogr. D Biol. Crystallogr. 64, 125–132 (2008).

    CAS  PubMed  Article  Google Scholar 

  43. 43.

    Vagin, A. & Teplyakov, A. Molecular replacement with MOLREP. Acta Crystallogr. D Biol. Crystallogr. 66, 22–25 (2010).

    CAS  PubMed  Article  Google Scholar 

  44. 44.

    Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  45. 45.

    Murshudov, G. N. et al. REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr. D Biol. Crystallogr. 67, 355–367 (2011).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  46. 46.

    Lebedev, A. A. et al. JLigand: a graphical tool for the CCP4 template-restraint library. Acta Crystallogr. D Biol. Crystallogr. 68, 431–440 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  47. 47.

    Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D Biol. Crystallogr. 66, 12–21 (2010).

    CAS  PubMed  Article  Google Scholar 

  48. 48.

    Agirre, J. et al. Privateer: software for the conformational validation of carbohydrate structures. Nat. Struct. Mol. Biol. 22, 833–834 (2015).

    CAS  PubMed  Article  Google Scholar 

  49. 49.

    Terwilliger, T. C. et al. Iterative-build OMIT maps: map improvement by iterative model building and refinement without model bias. Acta Crystallogr. D Biol. Crystallogr. 64, 515–524 (2008).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  50. 50.

    Collaborative Computational Project, Number 4. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D Biol. Crystallogr. 50, 760–763 (1994).

    Article  Google Scholar 

  51. 51.

    Potterton, L. et al. CCP4i2: the new graphical user interface to the CCP4 program suite. Acta Crystallogr. D Struct. Biol. 74, 68–84 (2018).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  52. 52.

    Micsonai, A. et al. BeStSel: a web server for accurate protein secondary structure prediction and fold recognition from the circular dichroism spectra. Nucleic Acids Res. 46, W315–W322 (2018).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  53. 53.

    Desai, M. S. et al. A dietary fiber-deprived gut microbiota degrades the colonic mucus barrier and enhances pathogen susceptibility. Cell 167, 1339–1353 (2016).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  54. 54.

    Koropatkin, N. M., Martens, E. C., Gordon, J. I. & Smith, T. J. Starch catabolism by a prominent human gut symbiont is directed by the recognition of amylose helices. Structure 16, 1105–1115 (2008).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  55. 55.

    Degnan, P. H., Barry, N. A., Mok, K. C., Taga, M. E. & Goodman, A. L. Human gut microbes use multiple transporters to distinguish vitamin B12 analogs and compete in the gut. Cell Host Microbe 15, 47–57 (2014).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  56. 56.

    Katoh, K., Misawa, K., Kuma, K. & Miyata, T. MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic Acids Res. 30, 3059–3066 (2002).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  57. 57.

    Clamp, M., Cuff, J., Searle, S. M. & Barton, G. J. The Jalview Java alignment editor. Bioinformatics 20, 426–427 (2004).

    CAS  PubMed  Article  Google Scholar 

  58. 58.

    Stamatakis, A. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 30, 1312–1313 (2014).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  59. 59.

    Felsenstein, J. Evolutionary trees from DNA sequences: a maximum likelihood approach. J. Mol. Evol. 17, 368–376 (1981).

    ADS  CAS  PubMed  Article  Google Scholar 

  60. 60.

    Le, S. Q. & Gascuel, O. An improved general amino acid replacement matrix. Mol. Biol. Evol. 25, 1307–1320 (2008).

    CAS  PubMed  Article  Google Scholar 

  61. 61.

    Felsenstein, J. Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39, 783–791 (1985).

    PubMed  PubMed Central  Article  Google Scholar 

  62. 62.

    Kumar, S., Stecher, G. & Tamura, K. MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets. Mol. Biol. Evol. 33, 1870–1874 (2016).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

Download references

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

Affiliations

Authors

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.

Ethics declarations

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-GlcNAc6S-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.

Source data

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.

Source data

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing