Erythrocyte Membrane Receptors for Streptococcus gordonii Sialic Acid-Binding Adhesin

Bacterial recognition of host sialic acid-containing receptors plays an important role in microbial colonization of the human oral cavity. The aggregation of human platelets by Streptococcus gordonii DL1 is implicated in the pathogenesis of infective endocarditis. In addition, we consider hemagglutination of this organism may act as an additive factor to increase a severity of this disease. We previously reported this interaction requires the bacterial expression of a 203-kDa protein (Hsa), which has sialic acid-binding activity, because the hsa mutant, a derivative of S. gordonii DL1, does not hemagglutinate. Objectives: In the present study, we confirmed that erythrocyte surface sialoglycoproteins are the receptors for Hsa. Methods: 1) The effects of proteinase K, chymotrypsin, phospholipase C, and α(2-3) or α(2-3, 6, 8) neuraminidase on hemagglutination activity were examined. 2) Dot blot analysis of glycophorin A and band 3 with recombinant NR2, which is the putative binding domain of Hsa, fused with glutathione S-transferase (GST) in Escherichia coli BL21. 3) GST pull-down assays using GST-HsaNR2 were performed for erythrocyte membrane proteins. Results: 1) Proteinase K, chymotrypsin, and α(2-3) or α(2-3, 6, 8) neuraminidase were effective to reduce bacterial hemagglutination, but phospholipase C was not. 2) Dot blot analysis indicated the recombinant GST-HsaNR2 bound to both glycophorin A and band 3. 3) Glycophorin A and a small amount of band 3 were pulled down by GST-HsaNR2. Conclusion: These findings indicate that S. gordonii Hsa specifically binds to glycophorin A and band 3, α2-3-linked sialic acid-containing membrane glycoproteins.