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A surface endogalactanase in Bacteroides thetaiotaomicron confers keystone status for arabinogalactan degradation

Nature Microbiology volume 3, pages 1314–1326 (2018)Cite this article

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Abstract

Glycans are major nutrients for the human gut microbiota (HGM). Arabinogalactan proteins (AGPs) comprise a heterogenous group of plant glycans in which a β1,3-galactan backbone and β1,6-galactan side chains are conserved. Diversity is provided by the variable nature of the sugars that decorate the galactans. The mechanisms by which nutritionally relevant AGPs are degraded in the HGM are poorly understood. Here we explore how the HGM organism Bacteroides thetaiotaomicron metabolizes AGPs. We propose a sequential degradative model in which exo-acting glycoside hydrolase (GH) family 43 β1,3-galactanases release the side chains. These oligosaccharide side chains are depolymerized by the synergistic action of exo-acting enzymes in which catalytic interactions are dependent on whether degradation is initiated by a lyase or GH. We identified two GHs that establish two previously undiscovered GH families. The crystal structures of the exo-β1,3-galactanases identified a key specificity determinant and departure from the canonical catalytic apparatus of GH43 enzymes. Growth studies of Bacteroidetes spp. on complex AGP revealed 3 keystone organisms that facilitated utilization of the glycan by 17 recipient bacteria, which included B. thetaiotaomicron. A surface endo-β1,3-galactanase, when engineered into B. thetaiotaomicron, enabled the bacterium to utilize complex AGPs and act as a keystone organism.

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Fig. 1: The structure of arabinogalactans, PULs upregulated by the glycans and enzymes that attack these glycans.
Fig. 2: The crystal structure of GH43_24 β1,3-d-galactosidases in complex with ligands.
Fig. 3: HPAEC analysis of the activity of GH43_24 β1,3-d-galactanases.
Fig. 4: Degradation of GA-AGP side chains.
Fig. 5: Cell localization and growth of Bacteroides on complex AGPs.
Fig. 6: Growth profile of keystone and recipient Bacteroides species on complex AGPs.

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Data availability

The authors declare that the data supporting the findings of this study are available within the paper and the Supplementary Information. The crystal structure data sets generated (coordinate files and structure factors) have been deposited in the Protein Data Bank (PDB) and are listed in Supplementary Table 6 together with the PDB accession codes.

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  • 20 September 2019

    An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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Acknowledgements

This work was supported in part by an Advanced Grant from the European Research Council (grant no. 322820) awarded to H.J.G. and B.H. supporting D.A.N., A.C., J.M.-M., J.B. and N.T., and a Wellcome Trust Senior Investigator Award to H.J.G. (grant no. WT097907MA) that supported E.C.L. The Biotechnology and Biological Research Council project ‘Ricefuel’ (grant number BB/K020358/1) awarded to H.J.G. supported A.L. We thank Diamond Light Source for access to beamline I02, I04-1 and I24 (mx1960, mx7854 and mx9948) that contributed to the results presented here.

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Author notes

  1. These authors contributed equally: Alan Cartmell, Jose Muñoz-Muñoz, Jonathon Briggs, Didier A. Ndeh.

Authors and Affiliations

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

    Alan Cartmell, Jose Muñoz-Muñoz, Jonathon A. Briggs, Didier A. Ndeh, Elisabeth C. Lowe, Arnaud Baslé, Tiaan Heunis, Joe Gray, Aurore Labourel, Matthias Trost & Harry J. Gilbert

  2. Department of Applied Sciences, Faculty of Health and Life Sciences, Northumbria University, Newcastle upon Tyne, UK

    Jose Muñoz-Muñoz

  3. Architecture et Fonction des Macromolécules Biologiques, Centre National de la Recherche Scientifique (CNRS), Aix-Marseille University, Marseille, France

    Nicolas Terrapon & Bernard Henrissat

  4. INRA, USC 1408 AFMB, Marseille, France

    Nicolas Terrapon & Bernard Henrissat

  5. Department of Biochemistry, University of Cambridge, Cambridge, UK

    Katherine Stott, Li Yu & Paul Dupree

  6. School of Chemistry and Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Victoria, Australia

    Pearl Z. Fernandes, Sayali Shah & Spencer J. Williams

  7. Department of Biological Sciences, King Abdulaziz University, Jeddah, Saudi Arabia

    Bernard Henrissat

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Contributions

Enzyme characterization and oligosaccharide purification were performed by A.C., D.A.N. and J.M.-M. Gene deletion strains were constructed by D.N. and A.L. Co-culturing experiments were carried out by A.C, J.B. and D.A.N. Western blots were carried out by D.A.N. Phylogenetic reconstruction and metagenomic analysis were performed by N.T. and B.H. Bacterial growth and transcriptomic experiments: E.C.L. and D.A.N. X-ray protein crystallography was carried out by A.C., A.B. J.M.-M. NMR experiments were performed by A.C. and K.S. Mass spectrometry was carried out by J.G., L.Y. and P.D. Chemical synthesis was performed by P.Z.F., S.S. and S.J.W. T.H., M.T. and E.C.L. performed the whole-cell proteomics. Experiments were designed by H.J.G. A.C. J.M.-M. and D.A.N. The manuscript was written by H.J.G. with substantial contributions from N.T., B.H. and S.J.W. Figures were prepared by J.M.-M. and E.C.L.

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Correspondence to Harry J. Gilbert.

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Cartmell, A., Muñoz-Muñoz, J., Briggs, J.A. et al. A surface endogalactanase in Bacteroides thetaiotaomicron confers keystone status for arabinogalactan degradation. Nat Microbiol 3, 1314–1326 (2018). https://doi.org/10.1038/s41564-018-0258-8

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