Serine-rich repeat protein adhesins from Lactobacillus reuteri display strain specific glycosylation profiles

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.

Fig. 1.

Lectin screening of L. reuteri SM proteins. (A) Western blot analysis of L. reuteri 100-23C and ATCC 53608 SM proteins, using f-WGA, f-RCA and f-SNA. The arrow indicates SRRP in L. reuteri 100-23C. (B) Western blot analysis of L. reuteri ATCC 53608 SM proteins with f-WGA and anti-SRRP-BR₅₃₆₀₈ antibody. (C) Western blot analysis of L. reuteri 100-23C WT, Δasp2, ΔgtfB and Δsrr mutant SM proteins with f-WGA. (D) Purification of LrSRRPs by affinity chromatography, using agWGA. LrSRRPs were eluted with 0.5 M GlcNAc.

Fig. 2.

LC–MS sugar nucleotide profiling of L. reuteri 100-23C and ATCC 53608 strains. The bars represent the standard error of three biological replicates. See also Table S1 for MRM transitions, retention times and quantity of the sugar nucleotides.

Fig. 3.

Structural analysis of SRRP_100-23 glycosylation. (A) MALDI-ToF analysis of SRRP_100-23 released glycans found in the 35% ACN elution fraction. (B) Fragmentation of the 738 Da peak. (C) Western blot analysis of enzymatically deglycosylated SRRP_100-23. 1. SRRP_100-23 (1), treated with α- and β-glucosidase (2), or α- and β- galactosidase (3). (D) Monosaccharide composition analysis of SRRP_100-23 glycans. Extracted ion chromatogram for ions at 204 and 173 Da, characteristic for monosaccharides. See also Figure S1 for comparison of MALDI-ToF spectra of the fraction containing the released glycans of L. reuteri 100-23 WT and ΔgtfB mutant.

Fig. 4.

Structural analysis of SRRP₅₃₆₀₈ glycosylation (A) MALDI-ToF analysis of SRRP₅₃₆₀₈ released glycans. (B) Fragmentation of the 575 Da peak. (C) Monosaccharide composition analysis of SRRP₅₃₆₀₈ glycans. Extracted ion chromatogram for ions at 204 and 173 Da, characteristic for monosaccharides.

Fig. 5.

Schematic representation of the accessory SecA2/Y2 clusters from L. reuteri 100-23C and ATCC 53608.

Fig. 6.

Analysis of GtfC_100-23 and GtfC₅₃₆₀₈ ligand specificity. (A–F) Differential scanning fluorimetry (DSF) analysis. (A) Melt curve of GtfC₅₃₆₀₈ in the presence of increasing concentrations of UDP-GlcNAc. (B) Tm of GtfC₅₃₆₀₈ in the presence of increasing concentrations of UDP, UDP-Gal, UDP-Glc and UDP-GlcNAc. Error bars represent the standard error of the mean of four technical replicates. (C) Melt curve of GtfC₅₃₆₀₈ in the presence of 4 mM UDP-GlcNAc, UDP-Glc, UDP-Gal and UDP. (D) Melt curve of GtfC_100-23C in the presence of 4 mM UDP-GlcNAc, UDP-Glc, UDP-Gal and UDP. (E) Melt curves of GtfC₅₃₆₀₈ in the presence of 5 mM Mn²⁺ (left), or 5 mM Mn²⁺ and 4 mM UDP-GlcNAc. (F) Melt curves of GtfC_100-23C in the presence of 5 mM Mn²⁺ (left), or 5 mM Mn²⁺ and 4 mM UDP-Glc. Since no significant difference was observed between the different divalent ions, only Mn²⁺ is shown. (G–L) Saturation Transfer Difference (STD) NMR analysis. (G), (H), (I) Binding epitope maps for the complexes of GtfC_100-23 with UDP-GlcNAc, UDP-Glc and UDP-Gal, respectively. Bottom row, (J), (K), (L) binding epitope maps for the complexes of GtfC₅₃₆₀₈ with UDP-GlcNAc, UDP-Glc and UDP-Gal, respectively. See also Table I and Figure S2 for the competition assays of the sugar nucleotides against GtfC_100-23 and GtfC₅₃₆₀₈.