The Streptococcus gordonii Platelet Binding Protein GspB Undergoes Glycosylation Independently of Export

The binding of bacteria and platelets is thought to play a central role in the pathogenesis of infective endocarditis. Platelets on the surfaces of damaged cardiac valves may provide an attachment site for bacteria circulating in the bloodstream, thereby initiating infection (6, 8, 9, 13). The subsequent deposition of platelets onto the infected valve surface may also be facilitated by bacterium-platelet binding (5, 21), leading to the formation of the hallmark lesion of this disease, the macroscopic endovascular vegetation. Numerous mechanisms for the attachment of bacteria to platelets have been proposed. Our own studies indicate that platelet binding by Streptococcus gordonii strain M99 is predominantly mediated by the cell surface protein GspB (2). This 280-kDa protein has a cell wall-anchoring domain characteristic of many gram-positive bacterial surface proteins (an LPXTG motif, a hydrophobic domain, and a charged tail [10, 18, 19]). However, several features of GspB are unusual. First, it does not have a typical amino-terminal signal for export. Instead, GspB is predicted to have a 90-amino-acid signal peptide, which is approximately three times longer than signals for export mediated by the general protein secretion (Sec) system in gram-positive bacteria (25, 27). Second, GspB migrates during sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with an apparent molecular mass that is much greater than predicted. Third, a majority of the protein is comprised of ∼190 semiconserved repeats of the peptide motif SASESASTSASV. This feature of an extended region of serine-rich repeats has recently been noted for several other streptococcal surface proteins, including the S. gordonii DL1 sialic acid-binding adhesin Hsa (23) and the Streptococcus parasanguis fimbria-associated protein Fap1 (28). GspB is also unusual because a 13.6-kb region downstream of the 9.2-kb gspB structural gene (Fig. ​(Fig.1)1) is required for its expression. This region encodes SecA2 and SecY2, which are homologues of two highly conserved components of the general Sec system. Mutation of either secA2 or secY2 results in the accumulation of GspB in the cytoplasm, indicating that they are required for the export of GspB. However, mutation of these genes has no apparent effect on the transport of other proteins, which suggests that SecA2 and SecY2 selectively mediate the export of GspB (2). Although the export of GspB by a dedicated export pathway is consistent with the fact that GspB does not have a typical N-terminal signal peptide, the specific features of the GspB sequence or structure that are recognized by SecA2 and SecY2 for export are unknown. FIG. 1. gspB-sec locus of M99. GspB is a cell surface-anchored platelet binding adhesin. 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 ... The segment downstream of gspB also encodes several proteins that are likely to function in carbohydrate metabolism, including two that have conserved domains characteristic of glycosyl transferases. Mutation of at least one of the putative glycosyl transferase genes (gtf) abolished GspB expression (2). We proposed that Gtf, along with the other transferases encoded within the gspB-sec operon, might be responsible for the glycosylation of GspB, which would account for the aberrant electrophoretic mobility of the protein. We now report that GspB is indeed a glycoprotein and that its glycosylation occurs independently of its export.