2Aa, b, and c) ( 10?9, Fig

2Aa, b, and c) ( 10?9, Fig. roles as pioneer colonizers in the development of dental plaque (35). In addition, these streptococci are also well known for their ability to colonize damaged heart valves and are the most frequently identified bacteria as primary etiological agents of infective endocarditis (2, 3, 14). adhere to saliva-coated hydroxyapatite, an experimental model of the tooth surface, and attach to host cells such as erythrocytes, platelets, and polymorphonuclear leukocytes (PMNs) (18, 19, 21, 39, 46). A common mechanism involved in these interactions is the recognition of surface-associated host sialoglycoconjugates. Recently, such interactions have been found to involve the binding of streptococcal adhesins identified as large serine-rich glycoproteins (6, 33, 47) to membrane sialoglycoproteins of host cells (6, 33, 49, 52). We previously reported that the DL1 gene encodes a large serine-rich repeat protein (Hsa) composed of 2,178 amino acid residues. Hsa consists of an N-terminal nonrepetitive region (NR1), a serine-rich repeat region (SR1), another nonrepetitive region (NR2), an additional serine-rich repeat region (SR2), and a C-terminal cell wall anchoring domain (47). NR2 of Hsa is considered to be a binding site for 2-3-linked sialic acid (46, 47, 49). SR2, which accounts for over 75% of the length of Hsa, is a glycosylated region containing GlcNAc (46, 49). This glycosylation may confer an extended rod-shaped conformation on the serine-rich region, enabling this region to function as a molecular stalk for cell surface presentation of the putative amino-terminal receptor-binding JAK-3 domain (49). Hsa binds to the 2-3-linked sialic acid termini OPC21268 of O-glycosylated mucin-type glycoproteins, including salivary mucin MG2, platelet glycoprotein Ib (GPIb), and leukosialin, the major surface glycoprotein of human PMNs (7, 39, 40, 47, 48, 49, 52). Moreover, fibronectin and GPIIb, another platelet sialoglycoprotein, have been identified as receptors for Hsa (24, 52). Hsa of DL1 and SraP, a Hsa homologue of strains in the rat model of infective endocarditis do not appear to be correlated with the adhesion of these bacteria to isolated platelets or the fibrin-platelet matrix but instead are correlated with the biological consequence of bacterial binding to PMNs (54). The latter finding suggests that the ability of to survive in PMNs following adhesin-mediated phagocytosis may be an important virulence determinant of infective endocarditis. The mechanism by which streptococci escape from the immune response, including phagocytosis, during the progression of infective endocarditis is not well understood. In the present study, we showed that DL1 interacts with phagocytes such OPC21268 as monocytes, granulocytes, and macrophages. Furthermore, we identified the receptors bound to DL1 Hsa. Our data strongly suggest that CD11b, CD43, and CD50 are the host receptors for DL1 Hsa. MATERIALS AND METHODS Cell culture. The human promyelocytic leukemia HL-60 cell line was maintained in RPMI 1640 medium with 10% fetal calf serum. For the differentiation assay, 2 105 HL-60 cells/ml were treated with either 100 nM 1,25-dihydroxyvitamin D3 (VD3) (Calbiochem, Darmstadt, Germany) for 24 h, 1 M all-retinoic acid (RA; Sigma-Aldrich, St. Louis, MO) for 4 days after the addition of 1 1.25% OPC21268 dimethyl sulfoxide (DMSO) for 16 h, or 32 nM 12-strains used in the present study were DL1 (wild type) and its mutant EM230 (DL1 test. For the binding inhibition assay, cells were pretreated with various concentrations of GST or GST-HsaNR2 for 30 min at 37C. Cell extracts and SDS-PAGE. Cells were lysed in TMN buffer containing 20 mM Tris-HCl (pH 7.8)-150 mM NaCl (TBS) containing 5 mM MgCl2 and 0.1% Nonidet P-40. Aliquots of 20 g of cell lysate were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on a 4 to 12% gradient gel (Invitrogen, Carlsbad, CA). The proteins were electrophoretically transferred to a nitrocellulose membrane (Millipore, Billerica, MA). Bacterial overlay. DL1 at 2 109 cells/ml in 1 PBS were biotin labeled by incubation with sulfosuccinimidyl-6-(biotinamido) hexanoate (NHS-LC)-biotin (Pierce, Rockford, IL) at 100 g/ml for 1 h at room temperature. Untreated nitrocellulose transfers, or transfers incubated with 1 U of neuraminidase/ml (Sigma-Aldrich) in 1 PBS for 1 h at 37C, were blocked in TBS containing 5% BSA,.