Abstract
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-β-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.
Highlights
Research Park Doctoral Training Grant BB/ M011216/
We showed that the mucin-foraging strategy of R. gnavus is strain specific [26] and associated with the expression of specific glycoside hydrolases (GHs) active against terminal isothermal titration calorimetry; IT-sialidase, epitopes, including GH33 intramolecular trans-sialidase (IT-sialidase) [27,28,29] and GH29 or intramolecular trans-sialidase; LC-FD-MS/MS, liquid chromatography with fluorescence detection and mass spectrometric detection; N-term GBLD, N-terminal galactose-binding-like domain; purified pig gastric mucin (pPGM), GH95 fucosidases [30]
RgGH98 modular structure consists of an N-terminal galactose-binding-like domain (N-term GBLD) (52–260 aa), a central/catalytic domain (Cd) covering 274–589 aa, a C-terminal (C-term) domain (592–876 aa), a C-term GBLD (894–986 aa), and a C-term fibronectin type 3 domain (1,099–1,366 aa) (Fig 1B)
Summary
Research Park Doctoral Training Grant BB/ M011216/. WB was funded the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grantThe gut microbiota plays a major role in human health and an alteration in its structure and function has been implicated in several diseases (for a review, see [1]). Research Park Doctoral Training Grant BB/ M011216/. WB was funded the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant. Mucus covering the epithelium is critical to maintain a homeostatic relationship with the gut microagreement 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, biota by harbouring a microbial community at safe distance from the epithelium surface [2]. The mucin glycans composing the mucus layer provide binding sites and a sustainable source of nutrients to the bacteria inhabiting the mucus niche [3,4,5]. Mucin-type O-glycans consist of N-acetylgalactosamine (GalNAc), Gal and N-acetylglucosamine (GlcNAc) containing glycan chains usually
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