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Abstract

The human gut symbiont Ruminococcus gnavus displays a strain-specific repertoire of glycoside hydrolases (GHs) contributing to its spatial location in the gut. Sequence similarity networks showed that R. gnavus GH98 (RgGH98) sequence fell in a cluster different from sequences of GH98 enzymes functionally characterised to date. We heterologously expressed and purified RgGH98, and determined its substrate and linkage specificity. We showed that RgGH98 is specific for blood group A antigen (BgA), as also confirmed by isothermal titration calorimetry (ITC) and saturation transfer difference (STD) NMR, revealing affinity for blood group A over blood group B and H antigens. The molecular basis of RgGH98 specificity was further investigated using a combination of site-directed mutagenesis and X-ray crystallography. The crystal structure of the complex between RgGH98 and BgA trisaccharide and RgGH98 inactive mutant with BgA tetrasaccharide identified residues involved in RgGH98 unique specificity. RNAseq and qPCR analysis showed that the gene encoding RgGH98 is part of an operon that is overexpresssed in vitro when R. gnavusis grown on mucin as sole carbon source. We showed that RgGH98 releases BgA trisaccharide from mucin and that pretreatment of mucin with RgGH98 conferred other R. gnavusstrains lacking this enzyme the ability to grow through BgA metabolism and access to the underlying mucin glycan chain. These data further support that the GH repertoire of R. gnavus strains enables them to colonise different nutritional niches in the gut and provide a source of enzymes with unique specificities for potential applications in diagnostic or therapeutics.

  • This is an open-access article distributed under the terms of the Creative Commons Attribution License.
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/content/journal/acmi/10.1099/acmi.ac2021.po0311
2022-05-27
2022-07-06
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http://instance.metastore.ingenta.com/content/journal/acmi/10.1099/acmi.ac2021.po0311
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