The Streptococcus gordonii surface proteins GspB and Hsa mediate binding to sialylated carbohydrate epitopes on the platelet membrane glycoprotein Ibalpha

Abstract

Platelet binding by Streptococcus gordonii strain M99 is dependent on expression of the cell wall-anchored glycoprotein GspB. This large cell surface protein is exported from the M99 cytoplasm via a dedicated transport system that includes SecA2 and SecY2. GspB is highly similar to Hsa, a protein expressed by S. gordonii Challis that has been characterized as a sialic acid binding hemagglutinin. In this study, we compared the contribution of GspB and Hsa to the adherence of S. gordonii to selected glycoproteins. Our results indicate that GspB can mediate binding to a variety of sialylated glycoproteins. GspB facilitates binding to carbohydrates bearing sialic acid in either alpha(2-3) or alpha(2-6) linkages, with a slight preference for alpha(2-3) linkages. Furthermore, GspB readily mediates binding to sialic acid residues on immobilized glycocalicin, the extracellular portion of the platelet membrane glycoprotein (GP) Ibalpha (the ligand binding subunit of the platelet von Willebrand factor receptor complex GPIb-IX-V). Although Hsa is required for the binding of S. gordonii Challis to sialic acid, most of the Hsa expressed by Challis is retained in the cytoplasm. The deficiency in export is due, at least in part, to a nonsense mutation in secA2. Hsa export can be enhanced by complementation with secA2 from M99, which also results in significantly greater binding to sialylated glycoproteins, including glycocalicin. The combined results indicate that GspB and Hsa contribute similar binding capabilities to M99 and Challis, respectively, but there may be subtle differences in the preferred epitopes to which these adhesins bind.

FIG. 1.

Domain structure of the GspB and Hsa polypeptides. SP, putative signal peptide; srr1, first serine-rich region; BR, basic region; srr2, second serine-rich region; CWA, cell wall anchoring domain (an LPXTG motif, hydrophobic region, and charged tail, characterized as a signal for covalent linkage to the cell wall peptidoglycan [9, 20, 21]). The predicted molecular mass of each protein is indicated.

FIG. 2.

Comparison of the gspB-secY2/A2 locus of M99 with the hsa-secY2/A2 locus of Challis. Gly, Nss, and Gtf are likely to function in carbohydrate metabolism: Gly is predicted to be a cytoplasmic glycosyl transferase (family 8); Nss is similar to nucleotide sugar synthetases; Gtf is a likely glucosyl transferase; SecA2 and SecY2 are similar to the SecA ATPase and the SecY transmembrane translocase of various organisms, respectively (components of the general secretory pathway), and are required specifically for the export of GspB. Asp1, Asp2, and Asp3 are additional accessory secretory proteins. Orf4 is not similar to any protein of known function. The asterisk denotes a frameshift mutation at codon 361 of 682 in gly; the double asterisk indicates a nonsense mutation at codon 770 of 793 in secA2.

FIG. 3.

Binding of M99 and the gspB mutant strain PS436 to immobilized glycoproteins. Microtiter wells were coated with the indicated amounts of each protein. Binding is expressed as the mean ± standard deviation of the results of three independent experiments. Binding of these strains to plastic alone was approximately 0.2% of the applied inoculum. BSM, bovine submaxillary mucin; SL, sialyllactose; glycocalicin, the extracellular portion of the platelet membrane GPIbα.

FIG. 4.

Features of the platelet membrane glycoprotein GPIb. (A) Domains of the GPIb heterodimer (adapted from reference 13). GPIb consists of two covalently linked transmembrane proteins. The amino-terminal globular domain contains leucine-rich (leu-rich) repeats and binding sites for von Willebrand factor (vWF) and thrombin. The mucin-like core has approximately 60 O-linked oligosaccharide chains. (B) Diagram of the O-linked carbohydrates (12). (C) Diagram of the N-linked carbohydrates (11). NeuAc, N-acetyl neuraminic acid; Gal, galactose; GalNAc, N-acetyl galactosamine; GlcNAc, N-acetyl glucosamine.

FIG. 5.

Inhibition of M99 binding to glycocalicin by carbohydrates. Wells were coated with 1.25 μg of glycocalicin. Binding is expressed as the mean ± standard deviation of the results of two independent experiments. NANA, N-acetyl neuraminic acid.

FIG. 6.

Expression of Hsa by S. gordonii Challis and derivative strains. Blots were probed with a polyclonal anti-GspB serum. (A) Proteins were extracted from the cell wall by using mutanolysin. Each lane was loaded with proteins extracted from bacteria in 730 μl of a broth culture. (B) Proteins associated with protoplasts. Lanes were loaded with protoplasts from 120 μl of the culture. Lane 1, S. gordonii Challis (parental strain); lane 2, PS779 (Δhsa); lane 3, PS796 (Δnss-gtf); lane 4, PS787 (srr2::his6).

FIG. 7.

Repair of the secA2 mutation in Challis by replacement of the 3′ region of secA2 with the corresponding region from M99. The Challis secA2 sequence has a nonsense mutation (asterisk) at codon 770 of 793. Recombination upstream of the nonsense mutation results in a chimeric secA2 sequence and leaves an intact gtf downstream of the integrated vector.

FIG. 8.

Export of Hsa by Challis and derivative strains complemented with M99 secA2 sequences. (A) Cell wall proteins and protoplasts of secA2-complemented Challis (lanes 1 to 4). Lanes were loaded with cell wall proteins extracted from 730 μl of culture (upper panel) or with protoplasts from 120 μl of culture (lower panel). GspB expressed by M99 was used for comparison (lane 5; the predicted molecular mass of GspB is 286 kDa, and the apparent mass is approximately 450 kDa). Lane 5 was loaded with protoplasts from 120 μl of culture (lower panel) or with cell wall proteins extracted from 150 μl of the M99 culture (upper panel). Membrane was probed with a polyclonal anti-GspB serum. Lane 1, PS779 (Δhsa); lane 2, Challis; lane 3, PS795 (pM99secA2); lane 4, PS798 (pM993′A2); lane 5, M99. (B) Secreted proteins and protoplasts of secA2-complemented PS787.Lanes were loaded with proteins from 500 μl of the spent culture medium or with protoplasts from 120 μl of the culture. Lane 1, PS779 (Δhsa); lane 2, PS787 (srr2::his6); lane 3, PS797 (srr2::his6 pM99secA2); lane 4, PS799 (srr2::his6 pM993′A2). Upper panels: proteins were detected using the polyclonal anti-GspB serum. Lower panels: proteins were detected using an anti-His₆ monoclonal antibody.

FIG. 9.

Lectin binding to S. gordonii strain Challis and various derivative strains. Biotinylated forms of the indicated lectins were assessed for binding to bacteria that were immobilized in 96-well plates. WGA, wheat germ agglutinin (affinity for sialic acid and GlcNAc); sWGA, succinylated wheat germ agglutinin (affinity for GlcNAc); GSL-II, Griffonia simplicifolia lectin II (affinity for terminal GlcNAc); ConA, concanavalin A (affinity for glucose and mannose).

FIG. 10.

Binding of Challis and derivative strains to immobilized fetuin and asialofetuin. Microtiter wells were coated with 50 μg of either protein. Binding is expressed as the mean ± standard deviation of the results of three independent experiments. For each of the strains, approximately 0.2% of the inoculum bound to wells that had been treated only with the blocking reagent.

FIG. 11.

Effect of Hsa expression on binding to sialyllactose and glycocalicin. Binding is expressed as the mean ± standard deviation of the results of two independent experiments. (A) Microtiter wells were coated with 5 μg of either NeuAcα(2-3)Galβ(1-4)-Glc or NeuAcα(2-6)Galβ(1-4)Glc multivalently conjugated to human serum albumin. (B) Microtiter wells were coated with 1.25 μg of either glycocalicin or desialylated glycocalicin.

FIG. 12.

Inhibition of PS798 binding to glycocalicin by carbohydrates. Wells were coated with 1.25 μg of glycocalicin. Binding is expressed as the mean ± standard deviation of the results of two independent experiments. NANA, N-acetyl neuraminic acid.