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Elucidation of a sialic acid metabolism pathway in mucus-foraging Ruminococcus gnavus unravels mechanisms of bacterial adaptation to the gut


Sialic acid (N-acetylneuraminic acid (Neu5Ac)) is commonly found in the terminal location of colonic mucin glycans where it is a much-coveted nutrient for gut bacteria, including Ruminococcus gnavus. R. gnavus is part of the healthy gut microbiota in humans, but it is disproportionately represented in diseases. There is therefore a need to understand the molecular mechanisms that underpin the adaptation of R. gnavus to the gut. Previous in vitro research has demonstrated that the mucin-glycan-foraging strategy of R. gnavus is strain dependent and is associated with the expression of an intramolecular trans-sialidase, which releases 2,7-anhydro-Neu5Ac, rather than Neu5Ac, from mucins. Here, we unravelled the metabolism pathway of 2,7-anhydro-Neu5Ac in R. gnavus that is underpinned by the exquisite specificity of the sialic transporter for 2,7-anhydro-Neu5Ac and by the action of an oxidoreductase that converts 2,7-anhydro-Neu5Ac into Neu5Ac, which then becomes a substrate of a Neu5Ac-specific aldolase. Having generated an R. gnavus nan-cluster deletion mutant that lost the ability to grow on sialylated substrates, we showed that—in gnotobiotic mice colonized with R. gnavus wild-type (WT) and mutant strains—the fitness of the nan mutant was significantly impaired, with a reduced ability to colonize the mucus layer. Overall, we revealed a unique sialic acid pathway in bacteria that has important implications for the spatial adaptation of mucin-foraging gut symbionts in health and disease.

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Fig. 1: The nan operon of R. gnavus ATCC 29149.
Fig. 2: Steady-state fluorescence analysis of ligand binding to RgSBP.
Fig. 3: Biophysical analysis of ligand binding to RgSBP.
Fig. 4: Enzymic reaction of R. gnavus sialic acid aldolase.
Fig. 5: RgNanOx catalyses the conversion of 2,7-anhydro-Neu5Ac to Neu5Ac.
Fig. 6: Colonization of germ-free C57BL/6J mice with R. gnavus ATCC 29149 WT or nan mutant strains.

Data availability

Genome and protein sequences are available from NCBI and referenced within the text or the Supplementary Information. Accession numbers for all of the genomes used for multigene alignments are provided in Supplementary Table 5. Raw FASTQ files for the RNA-seq libraries were deposited to the NCBI Sequence Read Archive (SRA) under BioProject accession number PRJNA559470. The crystal structures described in this paper have been deposited in the protein data bank (PDB) under the following identifiers: 6RAB (WT), 6RB7 (K167A) and 6RD1 (RgNanA K167A–Neu5Ac complex). All other data are available from the corresponding author on reasonable request.

Code availability

All code used for statistical analysis is available from the corresponding author on reasonable request.


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We thank M. Rejzek (JIC) for help with the purification of 2,7-anhydro-Neu5Ac; N. Minton (University of Nottingham) for access to ClosTron technology; H. Wu, M. Philo and G. Savva at QIB for their help with the SSN, mass spectrometry analysis and statistical analyses, respectively; A. Brion and A. Goldson for their technical help with gnotobiotic mouse experiments; staff at Diamond Light Source beamlines VMXi, I03, I04 and I24 for beamtime and assistance; and R. Keegan of CCP4 and STFC for assistance with phasing the RgNanA crystal structure. We acknowledge the support of the Biotechnology and Biological Sciences Research Council (BBSRC); this research was funded by the BBSRC 42854000B responsive mode grant, the BBSRC Institute Strategic Programme Gut Microbes and Health BB/R012490/1 and its constituent project BBS/E/F/000PR10353 (Theme 1, Determinants of microbe-host responses in the gut across life). A.B. was supported by the BBSRC Norwich Research Park Biosciences Doctoral Training Partnership grant number BB/M011216/1.

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N.J. conceived the study and wrote the manuscript with contribution from the co-authors. A.B. performed bioinformatics analyses, transcriptomics, heterologous expression, site-directed mutagenesis, enzymatic assays, analytical product characterization (HPLC and mass spectrometry), protein–ligand interaction experiments (ITC and fluorescence spectroscopy) and R. gnavus mutagenesis. J.B. supervised the ClosTron mutagenesis. G.H.T. supervised the fluorescence spectroscopy experiments. D.L. developed the HPLC and mass spectrometry analysis protocols. C.D.O. performed the X-ray crystallography experiments under the supervision of M.A.W. L.V. performed the immunohistochemistry experiments. E.C. contributed to the mouse study and RNA-seq analyses. A.B., E.C., L.V. and D.L. worked under the supervision of N.J. R.N. performed the NMR experiments under the supervision of J.A. A.X. and W.L. synthesized the 2,7-anydro-Neu5Ac used in this study under the supervision of X.C. J.C. performed the cluster bioinformatics analyses. All of the authors reviewed and corrected the final manuscript.

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Correspondence to Nathalie Juge.

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Bell, A., Brunt, J., Crost, E. et al. Elucidation of a sialic acid metabolism pathway in mucus-foraging Ruminococcus gnavus unravels mechanisms of bacterial adaptation to the gut. Nat Microbiol 4, 2393–2404 (2019).

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