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Lactobacillus reuteri is a gut symbiont inhabiting the gastrointestinal tract of numerous vertebrates. The surface-exposed serine-rich repeat protein (SRRP) is a major adhesin in Gram-positive bacteria. Using lectin and sugar nucleotide profiling of wild-type or L. reuteri isogenic mutants, MALDI-ToF-MS, LC-MS and GC-MS analyses of SRRPs, we showed that L. reuteri strains 100-23C (from rodent) and ATCC 53608 (from pig) can perform protein O-glycosylation and modify SRRP100-23 and SRRP53608 with Hex-Glc-GlcNAc and di-GlcNAc moieties, respectively. Furthermore, in vivo glycoengineering in E. coli led to glycosylation of SRRP53608 variants with α-GlcNAc and GlcNAcβ(1→6)GlcNAcα moieties. The glycosyltransferases involved in the modification of these adhesins were identified within the SecA2/Y2 accessory secretion system and their sugar nucleotide preference determined by saturation transfer difference NMR spectroscopy and differential scanning fluorimetry. Together, these findings provide novel insights into the cellular O-protein glycosylation pathways of gut commensal bacteria and potential routes for glycoengineering applications.

Lactobacillus reuteri , a Gram-positive bacterial species inhabiting the gastrointestinal tract of vertebrates, displays remarkable host adaptation. Previous mutational analyses of rodent strain L. reuteri 100-23C identified a gene encoding a predicted surface-exposed serine-rich repeat protein (SRRP 100-23 ) that was vital for L. reuteri biofilm formation in mice. SRRPs have emerged as an important group of surface proteins on many pathogens, but no structural information is available in commensal bacteria. Here we report the 2.00-Å and 1.92-Å crystal structures of the binding regions (BRs) of SRRP 100-23 and SRRP 53608 from L. reuteri ATCC 53608, revealing a unique β-solenoid fold in this important adhesin family. SRRP 53608 -BR bound to host epithelial cells and DNA at neutral pH and recognized polygalacturonic acid (PGA), rhamnogalacturonan I, or chondroitin sulfate A at acidic pH. Mutagenesis confirmed the role of the BR putative binding site in the interaction of SRRP 53608 -BR with PGA. Long molecular dynamics simulations showed that SRRP 53608 -BR undergoes a pH-dependent conformational change. Together, these findings provide mechanistic insights into the role of SRRPs in host-microbe interactions and open avenues of research into the use of biofilm-forming probiotics against clinically important pathogens., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)