Abstract
We previously purified a putative sulfated-galactosylceramide (sulfatide)-binding protein with a molecular weight of 47 kDa from the cell surface of Lactobacillus reuteri JCM1081. The aim of this study was to identify the 47-kDa protein, examine its binding to sulfated glycolipids and mucins, and evaluate its role in bacterial adhesion to mucosal surfaces. By cloning and sequencing analysis, the 47-kDa protein was identified as elongation factor-Tu (EF-Tu). Adhesion properties were examined using 6×Histidine-fused EF-Tu (His6-EF-Tu). Surface plasmon resonance analysis demonstrated pH-dependent binding of His6-EF-Tu to sulfated glycolipids, but not to neutral or sialylated glycolipids, suggesting that a sulfated galactose residue was responsible for EF-Tu binding. Furthermore, His6-EF-Tu was found to bind to porcine gastric mucin (PGM) by enzyme-linked immunosorbent assay. Binding was markedly reduced by sulfatase treatment of PGM and in the presence of acidic and desialylated oligosaccharide fractions containing sulfated carbohydrate residues prepared from PGM, demonstrating that sulfated carbohydrate moieties mediated binding. Histochemical staining revealed similar localization of His6-EF-Tu and high iron diamine staining in porcine mucosa. These results indicated that EF-Tu bound PGM via sulfated carbohydrate moieties. To characterize the contribution of EF-Tu to the interaction between bacterial cells and PGM, we tested whether anti-EF-Tu antibodies could inhibit the interaction. Binding of L. reuteri JCM1081 to PGM was significantly blocked in a concentration-dependent matter, demonstrating the involvement of EF-Tu in bacterial adhesion. In conclusion, the present results demonstrated, for the first time, that EF-Tu bound sulfated carbohydrate moieties of sulfated glycolipids and sulfomucin, thereby promoting adhesion of L. reuteri to mucosal surfaces.
Highlights
Secreted extracellular mucins and cell surface glycocalyx prevent infection by the multitude of microorganisms that live in the healthy gastrointestinal (GI) tract
We demonstrated that the 47-kDa cell surface protein from L. reuteri JCM1081, purified in our previous paper as a putative sulfatide-binding protein, could be identified as elongation factor-Tu (EF-Tu), is a cytoplasmic protein that interacts with various partners during the elongation cycle of protein biosynthesis
The cell surface-associated EF-Tu from L. johnsonii NCC533 binds to human intestinal cells and mucins [12]
Summary
Secreted extracellular mucins and cell surface glycocalyx prevent infection by the multitude of microorganisms that live in the healthy gastrointestinal (GI) tract. Underneath the mucus layer, the cells present a dense array of highly diverse mucin glycoproteins and glycolipids forming the glycocalyx [2]. The carbohydrate chains of secreted or cell-surface mucin glycoproteins are highly diverse. Mucin oligosaccharides are joined to the protein core through an initial a-O-glycosidic linkage of acetylgalactosamine to the hydroxyl region of serine or threonine. These mucins can be broadly classified into neutral and acidic chemotypes, which are categorized further into sialomucins or sulfomucins on the basis of the presence of terminal sialic acid or sulfate groups, respectively, on the oligosaccharide chains [3,4]
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