Sialic acid (Neu5Ac) is commonly found in terminal location of colonic mucins glycans where it is a much-coveted nutrient for gut bacteria including Ruminococcus gnavus. R. gnavus is part of Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

The human gut symbiont Ruminococcus gnavus scavenges host‐derived N‐acetylneuraminic acid (Neu5Ac) from mucins, by converting it to 2,7-anhydro-Neu5Ac. We previously showed that 2,7-anhydro-Neu5Ac is transported into R. gnavus ATCC 29149 before being converted back to Neu5Ac for further metabolic processing. However, the molecular mechanism leading to the conversion of 2,7-anhydro-Neu5Ac to Neu5Ac remained elusive. Using 1D and 2D nuclear magnetic resonance (NMR), we elucidated the multistep enzymatic mechanism of the oxidoreductase (RgNanOx) that leads to the reversible conversion of 2,7-anhydro-Neu5Ac to Neu5Ac through formation of a 4-keto-DANA intermediate and NAD+ regeneration. The crystal structure of RgNanOx in complex with the NAD+ cofactor showed a protein dimer with a Rossman fold. Guided by the RgNanOx structure, we identified catalytic residues by site-directed mutagenesis. Bioinformatics analyses revealed the presence of RgNanOx homologues across Gram negative and Gram positive bacterial species and co-occurrence with sialic acid transporters. We showed by electrospray ionisation spray mass spectrometry (ESI-MS) that the Escherichia coli homologue YjhC displayed activity against 2,7-anhydro-Neu5Ac and that E. coli could catabolise 2,7-anhydro-Neu5Ac. Differential scanning fluorimetry (DSF) analyses confirmed the binding of YjhC to the substrates 2,7-anhydro-Neu5Ac and Neu5Ac, as well as to co-factors NAD and NADH. Finally, using E. coli mutants and complementation growth assays, we demonstrated that 2,7-anhydro-Neu5Ac catabolism in E. coli was dependent on YjhC and on the predicted sialic acid transporter YjhB. These results revealed the molecular mechanisms of 2,7-anhydro-Neu5Ac catabolism across bacterial species and a novel sialic acid transport and catabolism pathway in E. coli.

SignificanceGut bacteria play a key role in health and disease, but the molecular mechanisms underpinning their interaction with the host remain elusive. The serine-rich repeat proteins (SRRPs) are a family of adhesins identified in many Gram-positive pathogenic bacteria. We previously showed that beneficial bacterial species found in the gut also express SRRPs and that SRRP was required for the ability of Lactobacillus reuteri strain to colonize mice. Here, our structural and biochemical data reveal that L. reuteri SRRP adopts a β-solenoid fold not observed in other structurally characterized SRRPs and functions as an adhesin via a pH-dependent mechanism, providing structural insights into the role of these adhesins in biofilm formation of gut symbionts.

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