Identification of Polymorphonuclear Leukocyte and HL-60 Cell Receptors for Adhesins ofStreptococcus gordoniiandActinomyces naeslundii

Abstract

Interactions of oral streptococci and actinomyces with polymorphonuclear leukocytes (PMNs), mediated by sialic acid-and Gal/GalNAc-reactive adhesins, respectively, result in activation of the PMNs and thereby may contribute to the initiation of oral inflammation. Sialidase treatment of PMNs or HL-60 cells abolished adhesion of Streptococcus gordonii but was required for adhesion of Actinomyces naeslundii. The same effects of sialidase were noted for adhesion of these bacteria to a major 150-kDa surface glycoprotein of either PMNs or undifferentiated HL-60 cells and to a 130-kDa surface glycoprotein of differentiated HL-60 cells. These glycoproteins were both identified as leukosialin (CD43) by immunoprecipitation with a specific monoclonal antibody (MAb). Adhesion of streptococci and actinomyces to a 200-kDa minor PMN surface glycoprotein was also detected by bacterial overlay of untreated and sialidase-treated nitrocellulose transfers, respectively. This glycoprotein was identified as leukocyte common antigen (CD45) by immunoprecipitation with a specific MAb. CD43 and CD45 both possess extracellular mucinlike domains in addition to intracellular domains that are implicated in signal transduction. Consequently, the interactions of streptococci and actinomyces with the mucinlike domains of these mammalian cell surface glycoproteins result not only in adhesion but, in addition, may represent the initial step in PMN activation by these bacteria.

Identification of Platelet Receptors for the Streptococcus gordonii DL1 Sialic Acid‐Binding Adhesin

“…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 (1, 2, 7).Strains of S. gordonii, Streptococcus sanguinis, and Streptococcus oralis adhere to saliva-coated hydroxyapatite, an experimental model of the tooth surface, and also attach to host cells including erythrocytes and polymorphonuclear leukocytes (8,9,12,19,20). A common mechanism involved in these interactions is recognition of surface-associated host sialoglycoconjugates.…”

“…We have previously identified and functionally characterized the gene (hsa) encoding the sialic acidbinding adhesin (Hsa) of S. gordonii DL1, a glycoprotein containing GlcNAc (24,25,27). Hsa binds α2-3-linked sialic acid termini of O-glycosylated mucin-type glycoproteins, including salivary mucin MG2 and leukosialin, the major surface glycoprotein of human polymorphonuclear leukocytes (20,21,(24)(25)(26).…”

“…We have previously identified and functionally characterized the gene (hsa) encoding the sialic acidbinding adhesin (Hsa) of S. gordonii DL1, a glycoprotein containing GlcNAc (24,25,27). Hsa binds α2-3-linked sialic acid termini of O-glycosylated mucin-type glycoproteins, including salivary mucin MG2 and leukosialin, the major surface glycoprotein of human polymorphonuclear leukocytes (20,21,(24)(25)(26).We have also shown that the aggregation of human platelets by S. gordonii DL1, an interaction implicated in the pathogenesis of infective endocarditis (11), required the expression of hsa (27). In addition, neuraminidase-treatment of the platelets abolished this interaction, further supporting the essential role of Hsa in the bacteria-platelet interaction (27).…”

“…Labeled surface glycoconjugates were extracted with 0.5% Triton X-100 (Sigma-Aldrich) in 0.5 ml of TBS containing 2 mM phenylmethylsulfonyl fluoride for 30 min at 4 C, boiled for 10 min in reducing buffer, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using 3-8% or 10% polyacrylamide gels (16), and electrophoretically transferred to nitrocellulose membranes (28). Transfers were blocked with TBS containing 3% bovine serum albumin (BSA, Sigma-Aldrich) for 1 hr and subsequently incubated with 0.2 units/ml of avidin-D-horse radish peroxidase (Vector Laboratories, Inc., Burlingame, Calif., U.S.A.) in the same buffer for 30 min, washed, and developed with 4-chloro-1-naphthol and hydrogen peroxide as previously described (20). As shown in Fig.…”

“…Surface proteins were extracted from unlabeled washed platelets with 0.5% Triton X-100, boiled for 10 min in reducing buffer, separated by SDS-PAGE (10% polyacrylamide gels), and transferred to nitrocellulose membrane as described above. The transfers were untreated or treated with 1 unit/ml type X neuraminidase in TBS for 1 hr at 37 C, and subjected to bacterial overlay as described previously (20,24). Briefly, bacteria at 2ϫ10 9 /ml in PBS were biotin-labeled by incubation with 100 µg/ml of sulfosuccinimidyl 6-(biotinamido) hexanoate (NHS-LC)-biotin (Pierce) for 1 hr at room temperature.…”