Characterization of the Accessory Sec System of Staphylococcus aureus

The binding of bacteria to platelets is a postulated central event in the pathogenesis of infective endocarditis (23). Initial colonization of damaged endothelium on the valve surface may be mediated by the attachment of blood-borne bacteria to platelets bound at the site of injury (9, 24, 31). Platelets may subsequently be recruited to the site of infection through direct adhesion to immobilized bacteria (6). These events, in combination with bacterial proliferation, are thought to produce the hallmark lesion of infective endocarditis, the macroscopic vegetation. Staphylococcus aureus has been shown to bind to human platelets through a variety of adhesins. Many of these surface components bind platelets through their interaction with bridging molecules, such as fibrinogen or fibronectin (7, 13, 15, 17, 22). In addition, our laboratory has identified a large surface glycoprotein of S. aureus, the serine-rich adhesin for platelets (SraP) (Fig. ​(Fig.1)1) that also mediates binding to human platelets (21). Although the receptor for SraP binding has not been identified, it appears that SraP can bind directly to the platelet surface (21). Loss of SraP expression is associated with reduced virulence in an animal model of endocarditis, indicating that this interaction is important for the pathogenesis of endovascular infection (21). FIG. 1. Schematic diagram of the accessory Sec loci of S.aureus and S. gordonii. (A) The export pathways include two sets of common genes: (a) the export-related genes secY2, asp1, asp2, asp3, and secA2 and (b) the glycosyltransferase genes gtfA and gtfB that ... SraP shares similarity with a group of cell wall-associated glycoproteins found in a number of organisms including, Streptococcus sanguinis, Streptococcus pneumoniae, and Streptococcus agalactiae (4, 12, 16, 19, 20, 25, 30). Among the best-characterized members of the family is GspB of Streptococcus gordonii. This large, extensively glycosylated surface protein has a multidomain structure that includes two serine-rich domains, an N-terminal receptor binding domain, and an atypically long signal peptide (Fig. ​(Fig.1).1). Export of GspB occurs exclusively through an accessory Sec system, whose sole function appears to be the transport of this substrate to the bacterial surface. This pathway is comprised of SecY2 and SecA2 (homologs of SecY and SecA, respectively, of the general Sec system) and five accessory Sec proteins (Asp1 to Asp5) that lack homology to any proteins of known function (4, 28). The components for GspB glycosylation and export are encoded in a locus located immediately downstream of the gene encoding the adhesin (Fig. ​(Fig.1).1). Although the organization of this locus is well conserved across species, the SraP accessory Sec locus of S. aureus has a number of distinctive features. First, it contains only two glycosylation-related genes (gtfA and gtfB) compared to four such genes in S. gordonii (gly, nss, gtfA, and gtfB) (Fig. ​(Fig.1)1) (26), suggesting that SraP may be less extensively glycosylated compared to GspB. Since glycosylation is a major structural feature of GspB that precludes its export by the canonical Sec system, these findings suggest that SraP may be less stringent in its export requirements. In addition, the signal peptide of SraP differs from that of GspB in that its signal peptide contains fewer glycine residues in the hydrophobic region, which block the entry of the protein into the canonical Sec pathway (3). Third, two essential components of the accessory Sec system in S. gordonii (asp4 and asp5) (28) are absent in S. aureus (Fig. ​(Fig.1).1). The lack of Asp4 and Asp5 in S. aureus indicates that the accessory Sec system of S. aureus may function differently from its ortholog in S. gordonii. Taken together, these differences in the export substrate and sec locus indicate that the accessory Sec system in S. aureus may have altered substrate specificity compared to S. gordonii. To address these issues, we examined the effects of disrupting the accessory Sec system on SraP-specific and general protein transport by S. aureus.