Abstract
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.
Highlights
Originally believed to be restricted to eukaryotes, protein glycosylation, i.e., the covalent attachment of a carbohydrate moiety to specific protein targets, is emerging as an important feature in bacteria and archaea, revealing an important diversity of glycan structures and pathways within and between microbial species (Schäffer and Messner 2017)
Using a combination of bioinformatics analysis, lectin screening, LC–MS-based sugar nucleotide profiling, MALDI-ToF and gas chromatography (GC)–MS analyses, we showed that the L. reuteri ATCC 53608 and 100-23C strains are capable of performing protein glycosylation and that SRRP100-23 and SRRP53608 are glycosylated with hexose (Hex)2-N-acetylhexosamine (HexNAc) and di-HexNAc moieties, respectively
To determine whether L. reuteri strains 100-23C and ATCC 53608 are capable of performing protein glycosylation of L. reuteri SRRPs (LrSRRPs), the proteins from the spent media (SM) were separated by SDS-PAGE and analyzed by western blot using a range of fluorescein (f)-labeled lectins
Summary
Originally believed to be restricted to eukaryotes, protein glycosylation, i.e., the covalent attachment of a carbohydrate moiety to specific protein targets, is emerging as an important feature in bacteria and archaea, revealing an important diversity of glycan structures and pathways within and between microbial species (Schäffer and Messner 2017). The nature and function of protein glycosylation in gut commensal bacteria remains largely unexplored (Latousakis and Juge 2018). One of the mechanisms mediating specific interaction of L. reuteri strains with the host is provided by cell surface proteins that facilitate adherence to epithelial or mucosal surface along the GI tract, depending on the niche colonized by the bacteria (Mackenzie et al 2010; Etzold et al 2014; Sequeira et al 2018). Inactivation of SRRP100-23 completely abrogated epithelial association, indicating that initial adhesion represented the most significant step in biofilm formation, likely conferring host specificity (Frese et al 2013)
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