Biomedical subjects

Paul M Sullam

Publications and source records attributed to Paul M Sullam.

12 recordsLinked to original sources

PubMed2006

Binding of the streptococcal surface glycoproteins GspB and Hsa to human salivary proteins.

GspB and Hsa are homologous surface glycoproteins of Streptococcus gordonii that bind sialic acid moieties on platelet membrane glycoprotein Ibalpha. Since this species is an important member of the oral flora, we examined the direct binding of these adhesins to human salivary proteins. Both GspB and Hsa bound low-molecular-weight salivary mucin MG2 and salivary agglutinin. Hsa also bound several other salivary proteins, including secretory immunoglobulin A. Screening of six oral streptococcal isolates revealed that at least two of the strains expressed GspB homologues. These results indicate that GspB-like adhesins may be important for oral bacterial colonization.

Adhesins, Bacterial↗

PubMed2005

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.

Adhesins, Bacterial↗

PubMed2005

Determinants of the streptococcal surface glycoprotein GspB that facilitate export by the accessory Sec system.

GspB is a large cell-surface glycoprotein expressed by Streptococcus gordonii M99 that mediates binding of this organism to human platelets. This adhesin is glycosylated in the cytoplasm, and is then transported to the cell surface via an accessory Sec system. To assess the structural features of GspB that are needed for export, we examined the effects of altering the carbohydrate moieties or the polypeptide backbone of GspB. Truncated, glycosylated variants of GspB were exported exclusively via the accessory Sec pathway. When glycosylation was abolished, the GspB variants were still exported by this pathway, but minor amounts could also be transported by the canonical Sec system. GspB variants with in-frame insertions or deletions in the N-terminus were not secreted, indicating that this domain is necessary for export. However, the N-terminus is not sufficient for the transport of heterologous proteins, because C-terminal fusion of passenger proteins to this domain hindered export. In contrast, fusion of GspB to a canonical signal peptide resulted in the efficient export of non-glycosylated forms of the fusion protein via the canonical Sec pathway, whereas glycosylated forms could not be exported. Thus, the carbohydrate moieties and the atypical signal sequence of GspB interfere with export via the canonical pathway, and direct GspB towards the accessory Sec system.

Adenosine Triphosphatases↗

PubMed2005

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.

Adhesins, Bacterial↗

PubMed2005

Two additional components of the accessory sec system mediating export of the Streptococcus gordonii platelet-binding protein GspB.

The gspB-secY2A2 locus of Streptococcus gordonii strain M99 encodes the platelet-binding glycoprotein GspB, along with proteins that mediate its glycosylation and export. We have identified two additional components of the accessory Sec system (Asp4 and Asp5) encoded just downstream of gtfB in the gspB-secY2A2 locus. These proteins are required for GspB export and for normal levels of platelet binding by M99. Asp4 and Asp5 may be functional homologues of SecE and SecG, respectively.

Amino Acid Sequence↗

PubMed2004

Genes in the accessory sec locus of Streptococcus gordonii have three functionally distinct effects on the expression of the platelet-binding protein GspB.

Platelet binding by Streptococcus gordonii strain M99 is strongly correlated with the expression of the large surface glycoprotein GspB. A 14 kb chromosomal region downstream of gspB was previously shown to be required for the expression of this protein. The region encodes SecA2 and SecY2, which are components of an accessory secretion system dedicated specifically to the export of GspB. The region also includes three genes (gly, nss and gtf) that encode proteins likely to function in carbohydrate metabolism, and four genes (orf1-4) that encode proteins of unknown function. In this report, we have investigated the role of these genes in GspB expression. We found that disruption of orf1, orf2 or orf3 resulted in a loss of GspB export and the intracellular accumulation of GspB. As they are apparently essential components of the accessory secretion system, these genes were renamed asp1-3 (for accessory secretory protein). In gtf and orf4 mutants, gspB was transcribed, but no GspB was detected. These results suggest that Gtf and Orf4 are required for the translation or for the stability of GspB. In contrast, gly and nss mutants were able to express and export GspB. However, disruption of these genes appeared to affect the carbohydrate composition of this glycoprotein. As asp1-3, gtf and orf4, but not gly and nss, are conserved in the accessory sec loci of several staphylococcal and streptococcal species, these genes may also have crucial roles in the expression and export of GspB homologues in the other Gram-positive bacteria.

Adenosine Triphosphatases↗

PubMed2004

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

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.

Adhesins, Bacterial↗

PubMed2004

Four proteins encoded in the gspB-secY2A2 operon of Streptococcus gordonii mediate the intracellular glycosylation of the platelet-binding protein GspB.

Platelet binding by Streptococcus gordonii strain M99 is mediated predominantly by the cell surface glycoprotein GspB. This adhesin consists of a putative N-terminal signal peptide, two serine-rich regions (SRR1 and SRR2), a basic region between SRR1 and SRR2, and a C-terminal cell wall anchoring domain. The glycosylation of GspB is mediated at least in part by Gly and Nss, which are encoded in the secY2A2 locus immediately downstream of gspB. This region also encodes two proteins (Gtf and Orf4) that are required for the expression of GspB but whose functions have not been delineated. In this study, we further characterized the roles of Gly, Nss, Gtf, and Orf4 by investigating the expression and glycosylation of a series of glutathione S-transferase-GspB fusion proteins in M99 and in gly, nss, gtf, and orf4 mutants. Compared with fusion proteins expressed in the wild-type background, fusion proteins expressed in the mutant strain backgrounds showed altered electrophoretic mobility. In addition, the fusion proteins formed insoluble aggregates in protoplasts of the gtf and orf4 mutants. Glycan detection and lectin blot analysis revealed that SRR1 and SRR2 were glycosylated but that the basic region was unmodified. When the fusion protein was expressed in Escherichia coli, glycosylation of this protein was observed only in the presence of both gtf and orf4. These results demonstrate that Gly, Nss, Gtf, and Orf4 are all involved in the intracellular glycosylation of SRRs. Moreover, Gtf and Orf4 are essential for glycosylation, which in turn is important for the solubility of GspB.

Adenosine Triphosphatases↗

PubMed2004

The Streptococcus gordonii platelet binding protein GspB undergoes glycosylation independently of export.

The binding of bacteria and platelets may play a central role in the pathogenesis of infective endocarditis. Platelet binding by Streptococcus gordonii strain M99 is predominantly mediated by the 286-kDa cell wall-anchored protein GspB. This unusually large protein lacks a typical amino-terminal signal peptide and is translocated from the cytoplasm via a dedicated transport system. A 14-kb segment just downstream of gspB encodes SecA2 and SecY2, two components of the GspB-specific transport system. The downstream segment also encodes several putative glycosyl transferases that may be responsible for the posttranslational modification of GspB. In this study, we compared the abilities of M99 and two GspB(-) mutant strains to bind various lectins. GspB was found to have affinity for lectins that bind N-acetylglucosamine. We also examined variant forms of GspB that lack a carboxy-terminal cell wall-anchoring domain and thus are free of covalent linkage to cell wall peptidoglycan. Like native GspB, these truncated proteins appear to be heavily glycosylated, as evidenced by migration during sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular mass >100 kDa in excess of the predicted mass, negligible staining with conventional protein stains, and reactivity with hydrazide following periodate oxidation. Furthermore, analysis of the carbohydrate associated with the GspB variants by high-pH anion-exchange chromatography revealed the presence of approximately 70 to 100 monosaccharide residues per GspB polypeptide (primarily N-acetylglucosamine and glucose). Analysis of GspB in protoplasts of secA2 or secY2 mutant strains, which do not export GspB, indicates that GspB is glycosylated in the cytoplasm of these strains. The combined data suggest that the native GspB is a glycoprotein and that it may be glycosylated prior to export.

Bacterial Adhesion↗

PubMed2003

Pattern recognition by TREM-2: binding of anionic ligands.

We recently described the cloning of murine triggering receptor expressed by myeloid cells (TREM) 2, a single Ig domain DNAX adaptor protein 12-associated receptor expressed by cells of the myeloid lineage. In this study, we describe the identification of ligands for TREM-2 on both bacteria and mammalian cells. First, by using a TREM-2A/IgG1-Fc fusion protein, we demonstrate specific binding to a number of Gram-negative and Gram-positive bacteria and to yeast. Furthermore, we show that fluorescently labeled Escherichia coli and Staphylococcus aureus bind specifically to TREM-2-transfected cells. The binding of TREM-2A/Ig fusion protein to E. coli can be inhibited by the bacterial products LPS, lipoteichoic acid, and peptidoglycan. Additionally, binding can be inhibited by a number of other anionic carbohydrate molecules, including dextran sulfate, suggesting that ligand recognition is based partly on charge. Using a sensitive reporter assay, we demonstrate activation of a TREM-2A/CD3zeta chimeric receptor by both bacteria and dextran sulfate. Finally, we demonstrate binding of TREM-2A/Ig fusion to a series of human astrocytoma lines but not to a variety of other cell lines. The binding to astrocytomas, like binding to bacteria, is inhibited by anionic bacterial products, suggesting either a similar charge-based ligand recognition method or overlapping binding sites for recognition of self- and pathogen-expressed ligands.

Animals↗

PubMed2003

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.

Base Composition↗

PubMed2002

An accessory sec locus of Streptococcus gordonii is required for export of the surface protein GspB and for normal levels of binding to human platelets.

The translocation of proteins across the bacterial cell membrane is carried out by highly conserved components of the Sec system. Most bacterial species have a single copy of the genes encoding SecA and SecY, which are essential for viability. However, Streptococcus gordonii strain M99 encodes SecA and SecY homologues that are not required for viability or for the translocation of most exported proteins. The genes (secA2 and secY2) reside in a region of the chromosome required for the export of GspB, a 286 kDa cell wall-anchored protein. Loss of GspB surface expression is associated with a significant reduction in the binding of M99 to human platelets, suggesting that it may be an adhesin. Genetic analyses indicate that M99 has a second, canonical SecA homologue that is essential for viability. At least two other Gram-positive species, Streptococcus pneumoniae and Staphylococcus aureus, encode two sets of SecA and SecY homologues. One set is more similar to SecA and SecY of Escherichia coli, whereas the other set is more similar to SecA2 and SecY2 of strain M99. The conserved organization of genes in the secY2-secA2 loci suggests that, in each of these Gram-positive species, SecA2 and SecY2 may constitute a specialized system for the transport of a very large serine-rich repeat protein.

Adenosine Triphosphatases↗