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NJ, HW, DN, AMG received funding from InnovateUK Biocatalyst grant Glycoenzymes for Bioindustries (grant BB/M029042/). NJ, EHC,DL received funding from Biotechnology and Biological Sciences Research Council (BBSRC) Institute Strategic Programme Gut Microbes and Health (grant BB/R012490/1). JA and SM acknowledge support of BBSRC (grant BB/ P010660/1). JA was also supported by the Spanish Ministry of Science, Innovation and Universities through the grant PID2019-109395GB-I00. TH, RSR, SW were funded by BBSRC Norwich Research Park Doctoral Training Grant BB/ M011216/. WB was funded the European Unions Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No 814102. We acknowledge the participation of the Protein-Glycan Interaction Resource of the Consortium for Functional Glycomics (supporting grant R24 GM098791) and the National Center for Functional Glycomics (NCFG) at Beth Israel Deaconess Medical Center, Harvard Medical School (supporting grant P41 GM10369). We acknowledge George Savva (Quadram Institute Bioscience) supported by the BBSRC Core Capability Grant BB/CCG1860/1 for help with statistical analyses of growth assays. We would like to thank Diamond Light Source beamlines VMXi, I24, I03 and I04 for beamtime and assistance, as well as the crystallisation facility at Harwell for access and support. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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The human gut symbiont Ruminococcus gnavus shows specificity to blood group A antigen during mucin glycan foraging: Implication for niche colonisation in the gastrointestinal tract

Publicado en:Plos Biology. 19 (12): e3001498- - 2021-12-01 19(12), DOI: 10.1371/journal.pbio.3001498

Autores: Wu, Haiyang; Crost, Emmanuelle H; Owen, C David; van Bakel, Wouter; Martinez Gascuena, Ana; Latousakis, Dimitrios; Hicks, Thomas; Walpole, Samuel; Urbanowicz, Paulina A; Ndeh, Didier; Monaco, Serena; Sanchez Salom, Laura; Griffiths, Ryan; Reynolds, Raven S; Colvile, Anna; Spencer, Daniel I R; Walsh, Martin; Angulo, Jesus; Juge, Nathalie

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The human gut symbiont Ruminococcus gnavus displays strain-specific repertoires of glycoside hydrolases (GHs) contributing to its spatial location in the gut. Sequence similarity network analysis identified strain-specific differences in blood-group endo-beta-1,4-galactosidase belonging to the GH98 family. We determined the substrate and linkage specificities of GH98 from R. gnavus ATCC 29149, RgGH98, against a range of defined oligosaccharides and glycoconjugates including mucin. We showed by HPAEC-PAD and LC-FD-MS/MS that RgGH98 is specific for blood group A tetrasaccharide type II (BgA II). Isothermal titration calorimetry (ITC) and saturation transfer difference (STD) NMR confirmed RgGH98 affinity for blood group A over blood group B and H antigens. The molecular basis of RgGH98 strict specificity was further investigated using a combination of glycan microarrays, site-directed mutagenesis, and X-ray crystallography. The crystal structures of RgGH98 in complex with BgA trisaccharide (BgAtri) and of RgGH98 E411A with BgA II revealed a dedicated hydrogen network of residues, which were shown by site-directed mutagenesis to be critical to the recognition of the BgA epitope. We demonstrated experimentally that RgGH98 is part of an operon of 10 genes that is overexpresssed in vitro when R. gnavus ATCC 29149 is grown on mucin as sole carbon source as shown by RNAseq analysis and RT-qPCR confirmed RgGH98 expression on BgA II growth. Using MALDI-ToF MS, we showed that RgGH98 releases BgAtri from mucin and that pretreatment of mucin with RgGH98 confered R. gnavus E1 the ability to grow, by enabling the E1 strain to metabolise BgAtri and access the underlying mucin glycan chain. These data further support that the GH repertoire of R. gnavus strains enable them to colonise different nutritional niches in the human gut and has potential applications in diagnostic and therapeutics against infection.

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