Characterization of the accessory Sec system of Staphylococcus aureus.

The SraP adhesin of Staphylococcus aureus is a member of a highly conserved family of serine-rich surface glycoproteins of gram-positive bacteria. For streptococci, export of the SraP homologs requires a specialized transport pathway (the accessory Sec system). Compared to streptococci, however, SraP is predicted to differ in its signal peptide and glycosylation, which may affect its dependence on a specialized system for transport. In addition, two genes (asp4 and asp5) essential for export in Streptococcus gordonii are missing in S. aureus. Thus, the selectivity of the accessory Sec system in S. aureus may also differ compared to streptococci. To address these issues, the five genes encoding the putative accessory Sec system (secY2, secA2, and asp1-3) were disrupted individually in S. aureus ISP479C, and the resultant mutants were examined for SraP export. Disruption of secA2 resulted in the near complete loss of SraP surface expression. Similar results were seen with disruption of secY2 and asp1, asp2, or asp3. To assess whether the accessory Sec system transported other substrates, we compared secreted proteomes of ISP479C and a secA2 isogenic mutant, by two-dimensional fluorescence difference gel electrophoresis. Although two consistent differences in proteome content were noted between the strains, neither protein appeared to be a likely substrate for accessory Sec export. Thus, the accessory Sec system of S. aureus is required for the export of SraP, and it appears to be dedicated to the transport of this substrate exclusively.

Mechanism of cell surface expression of the Streptococcus mitis platelet binding proteins PblA and PblB.

PblA and PblB are prophage-encoded proteins of Streptococcus mitis strain SF100 that mediate binding to human platelets. The mechanism for surface expression of these proteins has been unknown, as they do not contain signal sequences or cell wall sorting motifs. We therefore assessed whether expression of these proteins was linked the lytic cycle of the prophage. Deletion of either the holin or lysin gene resulted in retention of PblA and PblB in the cytoplasm, and loss of these proteins from the cell wall. Flow cytometric analysis revealed that induction of phage replication in SF100 produced a subpopulation of cells with increased permeability. This effect was abrogated by disruption of the holin and lysin genes. Treatment of these mutants with exogenous PblA and PblB restored surface expression, apparently via binding of the proteins to cell wall choline. Loss of PblA and PblB expression was associated with decreased platelet binding in vitro, and reduced virulence in an animal model of endocarditis. Thus, expression of PblA and PblB occurs via a novel mechanism, whereby phage induction increases bacterial permeability and release of the proteins, followed by their binding to surface of viable cells. This mechanism may be important for endovascular infection.

Glycine residues in the hydrophobic core of the GspB signal sequence route export toward the accessory Sec pathway.

The Streptococcus gordonii cell surface glycoprotein GspB mediates high-affinity binding to distinct sialylated carbohydrate structures on human platelets and salivary proteins. GspB is glycosylated in the cytoplasm of S. gordonii and is then transported to the cell surface via a dedicated transport system that includes the accessory Sec components SecA2 and SecY2. The means by which the GspB preprotein is selectively recognized by the accessory Sec system have not been characterized fully. GspB has a 90-residue amino-terminal signal sequence that displays a traditional tripartite structure, with an atypically long amino-terminal (N) region followed by hydrophobic (H) and cleavage regions. In this report, we investigate the relative importance of the N and H regions of the GspB signal peptide for trafficking of the preprotein. The results show that the extended N region does not prevent export by the canonical Sec system. Instead, three glycine residues in the H region not only are necessary for export via the accessory Sec pathway but also interfere with export via the canonical Sec route. Replacement of the H-region glycine residues with helix-promoting residues led to a decrease in the efficiency of SecA2-dependent transport of the preprotein and a simultaneous increase in SecA2-independent translocation. Thus, the hydrophobic core of the GspB signal sequence is responsible primarily for routing towards the accessory Sec system.

Binding of the Streptococcus gordonii surface glycoproteins GspB and Hsa to specific carbohydrate structures on platelet membrane glycoprotein Ibalpha.

GspB and Hsa are homologous serine-rich surface glycoproteins of Streptococcus gordonii strains M99 and Challis, respectively, that mediate the binding of these organisms to platelet membrane glycoprotein (GP) Ibalpha. Both GspB and Hsa consist of an N-terminal putative signal peptide, a short serine-rich region, a region (BR) that is rich in basic amino acids, a longer serine-rich region and a C-terminal cell wall anchoring domain. To further assess the mechanisms for GspB and Hsa binding, we investigated the binding of the BRs of GspB and Hsa (expressed as glutathione S-tranferase fusion proteins) to sialylated glycoproteins in vitro. Both fusion proteins showed significant levels of binding to sialylated moieties on fetuin and GPIbalpha. In contrast, the corresponding region of a GspB homologue of Streptococcus agalactiae, which is acidic rather than basic, showed no binding to either fetuin or GPIbalpha. As measured by surface plasmon resonance kinetic analysis, GspB- and Hsa-derived fusion proteins had high affinity for GPIbalpha, but with somewhat different dissociation constants. Dot blot analysis using a panel of synthesized oligosaccharides revealed that the BR of Hsa can bind both alpha(2-3) sialyllactosamine [NeuAcalpha(2-3)Galbeta(1-4)GlcNAc] and sialyl-T antigen [NeuAcalpha(2-3)Galbeta(1-3)GalNAc], whereas the BR of GspB only bound sialyl-T antigen. Moreover, far Western blotting using platelet membrane proteins revealed that GPIbalpha is the principal receptor for GspB and Hsa on human platelets. The combined results indicate that the BRs of GspB and Hsa are the binding domains of these adhesins. However, the subsets of carbohydrate structures on GPIbalpha recognized by the binding domains appear to be different between the two proteins.

Role of SraP, a Serine-Rich Surface Protein of Staphylococcus aureus, in binding to human platelets.

The binding of bacteria to platelets is a postulated central event in the pathogenesis of infective endocarditis. Platelet binding by Streptococcus gordonii is mediated in large part by GspB, a high-molecular-mass cell wall glycoprotein. Although Staphylococcus aureus has a GspB homolog (SraP), little is known about its function. SraP has a calculated molecular mass of 227 kDa and, like GspB, is predicted to contain an atypical N-terminal signal sequence, two serine-rich repeat regions (srr1 and srr2) separated by a nonrepeat region, and a C-terminal cell wall anchoring motif (LPDTG). To assess whether SraP contributes to platelet binding, we compared the binding to human platelets of S. aureus strain ISP479C and of an isogenic variant (strain PS767) in which sraP had been disrupted by allelic replacement. Platelet binding in vitro by PS767 was 47% +/- 17% (mean +/- standard deviation) lower than that of ISP479C (P < 0.001). In addition, a recombinant fragment of SraP containing srr1 and the nonrepeat region was found to bind platelets directly. Binding was saturable, suggesting a receptor-ligand interaction. When tested in a rabbit model of endocarditis, in which each animal was simultaneously infected with ISP479C and PS767 at a ratio of approximately 1:1, the titers of the mutant strain within vegetations were significantly lower than those of the parent strain at 1 and 24 h postinfection. These results indicate that SraP can mediate the direct binding of S. aureus to platelets and that the platelet-binding domain of this glycoprotein is located within its N-terminal region. Moreover, the expression of SraP appears to be a virulence determinant in endovascular infection.

Genomic organization and molecular characterization of SM1, a temperate bacteriophage of Streptococcus mitis.

The direct binding of Streptococcus mitis to human platelets is mediated in part by two proteins (PblA and PblB) encoded by a lysogenic bacteriophage (SM1). Since SM1 is the first prophage of S. mitis that has been identified and because of the possible role of these phage-encoded proteins in virulence, we sought to characterize SM1 in greater detail. Sequencing of the SM1 genome revealed that it consisted of 34,692 bp, with an overall G+C content of 39 mol%. Fifty-six genes encoding proteins of 40 or more amino acids were identified. The genes of SM1 appear to be arranged in a modular, life cycle-specific organization. BLAST analysis also revealed that the proteins of SM1 have homologies to proteins from a wide variety of lambdoid phages. Bioinformatic analyses, in addition to N-terminal sequencing of the proteins, led to the assignment of possible functions to a number of proteins, including the integrase, the terminase, and two major structural proteins. Examination of the phage structural components indicates that the phage head may assemble using stable multimers of the major capsid protein, in a process similar to that of phage r1t. These findings indicate that SM1 may be part of a discrete subfamily of the Siphoviridae that includes at least phages r1t of Lactococcus lactis and SF370.3 of Streptococcus pyogenes.