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 66Histidine-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. © 2013 Nishiyama et al.
Nishiyama, K., Ochiai, A., Tsubokawa, D., Ishihara, K., Yamamoto, Y., & Mukai, T. (2013). Identification and characterization of sulfated carbohydrate-binding protein from Lactobacillus reuteri. PLoS ONE, 8(12). https://doi.org/10.1371/journal.pone.0083703