The carbohydrate binding region of the bacterial adhesin GspB from Streptococcus gordonii strain M99 (GspBBR) was expressed in Escherichia coli and purified using affinity and size exclusion chromatography. Separate sparse-matrix screening of GspBBR buffered in either 20 mM Tris pH 7.4 or 20 mM HEPES pH 7.5 resulted in different crystallographic behavior such that different precipitants, salts, and additives supported crystallization of GspBBR in each buffer. While both sets of conditions supported crystal growth in space group P212121, these had distinct unit cell dimensions of a=33.3 Å, b=86.6 Å, c=117.9 Å for crystal form one and a=34.6 Å, b=98.3 Å, c=99.0 Å for crystal form two. Additive screening improved the crystals grown in both conditions such that diffraction extended beyond 2 Å resolution. A complete data set has been collected to 1.3 Å resolution with an overall Rsym value of 0.04 and an Rsym value of 0.33 in the highest resolution shell.
GspB; glycoprotein; Streptococcus gordonii; sialic acid; adhesin; endocarditis; lectin
GspB is a serine-rich repeat (SRR) adhesin of Streptococcus gordonii that mediates binding of this organism to human platelets via its interaction with sialyl-T antigen on the receptor GPIbα. This interaction appears to be a major virulence determinant in the pathogenesis of infective endocarditis. To address the mechanism by which GspB recognizes its carbohydrate ligand, we determined the high-resolution x-ray crystal structure of the GspB binding region (GspBBR), both alone and in complex with a disaccharide precursor to sialyl-T antigen. Analysis of the GspBBR structure revealed that it is comprised of three independently folded subdomains or modules: 1) an Ig-fold resembling a CnaA domain from prokaryotic pathogens; 2) a second Ig-fold resembling the binding region of mammalian Siglecs; 3) a subdomain of unique fold. The disaccharide was found to bind in a pocket within the Siglec subdomain, but at a site distinct from that observed in mammalian Siglecs. Confirming the biological relevance of this binding pocket, we produced three isogenic variants of S. gordonii, each containing a single point mutation of a residue lining this binding pocket. These variants have reduced binding to carbohydrates of GPIbα. Further examination of purified GspBBR-R484E showed reduced binding to sialyl-T antigen while S. gordonii harboring this mutation did not efficiently bind platelets and showed a significant reduction in virulence, as measured by an animal model of endocarditis. Analysis of other SRR proteins revealed that the predicted binding regions of these adhesins also had a modular organization, with those known to bind carbohydrate receptors having modules homologous to the Siglec and Unique subdomains of GspBBR. This suggests that the binding specificity of the SRR family of adhesins is determined by the type and organization of discrete modules within the binding domains, which may affect the tropism of organisms for different tissues.
The binding of bacteria to human platelets is thought to be important for development of infective endocarditis, a life-threatening infection of the cardiovascular system. Streptococcus gordonii is a leading cause of endocarditis. This pathogen uses a protein called GspB to attach to carbohydrates on human platelets. While this binding interaction appears to be mediated by a specific, contiguous domain within GspB, little is known about the molecular details of the interaction between GspB and the carbohydrate receptors on its human host. We therefore determined the crystal structure of the region of GspB that binds to platelet carbohydrates, both alone and in complex with a synthetic carbohydrate receptor. Using this structure as a guide, we were able to produce three strains of S. gordonii that lacked the ability to bind to platelet carbohydrates. One of these isogenic variants was studied more in-depth and lacked the ability to bind to human platelets in vitro and was reduced in virulence when tested in vivo. These studies provide the first structural information detailing the molecular interactions between any serine-rich repeat adhesin and its host receptor, and identify how different, related adhesins may have evolved different specificities for host receptors.
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 ∼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.
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 α(2-3) or α(2-6) linkages, with a slight preference for α(2-3) linkages. Furthermore, GspB readily mediates binding to sialic acid residues on immobilized glycocalicin, the extracellular portion of the platelet membrane glycoprotein (GP) Ibα (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.
GspB is a serine-rich glycoprotein adhesin of Streptococcus gordonii that is exported to the bacterial surface by the accessory Sec system. This dedicated export pathway is comprised of seven components (SecA2, SecY2, and five accessory Sec proteins [Asp1 to Asp5]). The latter proteins have no known homologs beyond the Asps of other species. Asp1 to Asp3 are absolutely required for export of the substrate GspB, but their roles in this process are unknown. Using copurification analysis and far-Western blotting, we found that Asp2 and Asp3 could individually bind the serine-rich repeat (SRR) domains of GspB. Deletion of both SRR regions of GspB led to a decrease in its export, suggesting that binding of the Asps to the SRR regions is important for GspB transport by the accessory Sec system. The Asps also bound a heterologous substrate for the accessory Sec system containing a slow-folding MalE variant, but they did not bind wild-type MalE. The combined results indicate that the Asps may recognize the export substrate through preferential interactions with its unstructured or unfolded regions. Glycosylation of the SRR domains on GspB prevented Asp binding, suggesting that binding of the Asps to the preprotein occurs prior to its full glycosylation. Together, these findings suggest that Asp2 and Asp3 are likely to function in part as chaperones in the early phase of GspB transport.
Bacterial binding to human platelets is an important step in the pathogenesis of infective endocarditis. Streptococcus gordonii can mediate its platelet attachment through a cell wall glycoprotein termed GspB (“gordonii surface protein B”). GspB export is mediated by a seven component accessory Sec system, containing two homologues of the general secretory pathway (SecA2 & SecY2) and five accessory Sec proteins (Asps 1 – 5). Here we show that the Asps are required for optimal export of GspB independent of the glycosylation process. Furthermore, yeast two-hybrid screening of the accessory Sec system revealed interactions occurring between Asp3 and the other components of the system. Asp3 was shown to bind SecA2, Asp1, Asp2 and itself. Mutagenesis of Asp3 identified N and C-terminal regions that are essential for GspB transport, and conserved residues within the C-terminal domain mediated Asp3 binding to other accessory Sec components. The loss of binding by Asp3 also resulted in an impaired ability of S. gordonii to secrete GspB. These studies indicate that Asp3 is a central element mediating multiple interactions among accessory Sec components that are essential for GspB transport to the cell surface.
accessory Sec; secretion; glycoprotein; Streptococcus gordonii; Asp
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.
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.
The accessory Sec system is a specialized transport system that exports serine-rich repeat (SRR) glycoproteins of Gram-positive bacteria. This system contains two homologues of the general secretory (Sec) pathway (SecA2 and SecY2) and several other essential proteins (Asp1 to Asp5) that share no homology to proteins of known function. In Streptococcus gordonii, Asp2 is required for the transport of the SRR adhesin GspB, but its role in export is unknown. Tertiary structure predictions suggest that the carboxyl terminus of Asp2 resembles the catalytic region of numerous enzymes that function through a Ser-Asp-His catalytic triad. Sequence alignment of all Asp2 homologues identified a highly conserved pentapeptide motif (Gly-X-Ser362-X-Gly) typical of most Ser-Asp-His catalytic triads, where Ser forms the reactive residue. Site-directed mutagenesis of residues comprising the predicted catalytic triad of Asp2 of S. gordonii had no effect upon GspB transport but did result in a marked change in the electrophoretic mobility of the protein. Lectin-binding studies and monosaccharide content analysis of this altered glycoform revealed an increase in glucosamine deposition. Random mutagenesis of the Asp2 region containing this catalytic domain also disrupted GspB transport. Collectively, our findings suggest that Asp2 is a bifunctional protein that is essential for both GspB transport and correct glycosylation. The catalytic domain may be responsible for controlling the glycosylation of GspB, while other surrounding regions are functionally required for glycoprotein transport.
GspB and Hsa are homologous surface glycoproteins of Streptococcus gordonii that bind sialic acid moieties on platelet membrane glycoprotein Ibα. 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.
The direct binding of bacteria to platelets is a central interaction in the pathogenesis of infective endocarditis. GspB is a serine-rich, cell wall glycoprotein of Streptococcus gordonii that mediates the binding of this organism to human platelets in vitro. To assess the contribution of this adhesin to the pathogenesis of endocarditis, we compared the virulence of S. gordonii M99 (which expresses GspB) with an isogenic, gspB mutant (PS846) in two rat models of endovascular infection. In the first group of experiments, animals were infected intravenously with M99 or PS846, and sacrificed 72 h later, to assess levels of bacteria within cardiac vegetations, kidneys, and spleens. When inoculated with 105 CFU, rats infected with PS846 had significantly lower densities of organisms within vegetations (mean: 3.84 log10 CFU/g) as compared with M99-infected rats (6.67 log10 CFU/g; P < 0.001). Marked differences were also seen in rats co-infected with M99 and PS846, at a 1:1 ratio. While M99 was found at high levels within vegetations, kidneys and spleens (mean log10 CFU/g: 6.62, 5.07 and 4.18, respectively) PS846 was not detected within these tissues. Thus, platelet binding by GspB appears to be a major interaction in the pathogenesis of endocarditis due to S. gordonii.
Endocarditis; Platelets; Streptococci; Virulence; Adhesins; Bacterial
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.
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.
Damaged cardiac valves attract blood-borne bacteria, and infective endocarditis is often caused by viridans group streptococci. While such bacteria use multiple adhesins to maintain their normal oral commensal state, recognition of platelet sialoglycans provides an intermediary for binding to damaged valvular endocardium. We use a customized sialoglycan microarray to explore the varied binding properties of phylogenetically related serine-rich repeat adhesins, the GspB, Hsa, and SrpA homologs from Streptococcus gordonii and Streptococcus sanguinis species, which belong to a highly conserved family of glycoproteins that contribute to virulence for a broad range of Gram-positive pathogens. Binding profiles of recombinant soluble homologs containing novel sialic acid-recognizing Siglec-like domains correlate well with binding of corresponding whole bacteria to arrays. These bacteria show multiple modes of glycan, protein, or divalent cation-dependent binding to synthetic glycoconjugates and isolated glycoproteins in vitro. However, endogenous asialoglycan-recognizing clearance receptors are known to ensure that only fully sialylated glycans dominate in the endovascular system, wherein we find these particular streptococci become primarily dependent on their Siglec-like adhesins for glycan-mediated recognition events. Remarkably, despite an excess of alternate sialoglycan ligands in cellular and soluble blood components, these adhesins selectively target intact bacteria to sialylated ligands on platelets, within human whole blood. These preferred interactions are inhibited by corresponding recombinant soluble adhesins, which also preferentially recognize platelets. Our data indicate that circulating platelets may act as inadvertent Trojan horse carriers of oral streptococci to the site of damaged endocardium, and provide an explanation why it is that among innumerable microbes that gain occasional access to the bloodstream, certain viridans group streptococci have a selective advantage in colonizing damaged cardiac valves and cause infective endocarditis.
Bacterial infective endocarditis remains a disease with considerable morbidity and mortality. Of the numerous bacteria that can enter the bloodstream, certain oral commensal viridans group streptococci are among the major causative organisms of endocarditis. However, mechanisms underlying this selectivity are incompletely understood. Interactions between adhesins of such bacteria and human platelet sialoglycans are believed to play an important role in this selectivity, by facilitating bacterial adherence to damaged heart valves. Nevertheless, the molecular requirements for these interactions are not fully explored. Particularly, it is unclear whether selective targeting of platelets by these bacteria actually occurs in fluid human whole blood, an environment where numerous potential sialoglycan competitors exist. In the present work, we have addressed these important issues. We characterize in detail the glycan-binding spectra of a series of serine-rich repeat adhesins of oral streptococci. For the first time, we demonstrate that oral streptococci can indeed selectively target platelets in whole human blood. As a proof of concept, we also show that soluble recombinant bacterial adhesin binding region proteins can block the preferred platelet-bacterial interactions in whole blood. The knowledge gained from this study may help the development of novel preventive or therapeutic approaches against infective endocarditis.
Streptococcus gordonii colonizes multiple sites within the human oral cavity. This colonization depends upon the initial interactions of streptococcal adhesins with host receptors. The adhesins that bind salivary agglutinin glycoprotein (gp340) and human cell surface receptors include the antigen I/II (AgI/II) family polypeptides SspA and SspB and a sialic acid-binding surface protein designated Hsa or GspB. In this study we determined the relative functions of the AgI/II polypeptides and Hsa in interactions of S. gordonii DL1 (Challis) with host receptors. For an isogenic mutant with the sspA and sspB genes deleted the levels of adhesion to surface-immobilized gp340 were reduced 40%, while deletion of the hsa gene alone resulted in >80% inhibition of bacterial cell adhesion to gp340. Adhesion of S. gordonii DL1 cells to gp340 was sialidase sensitive, verifying that Hsa has a major role in mediating sialic acid-specific adhesion to gp340. Conversely, aggregation of S. gordonii cells by fluid-phase gp340 was not affected by deletion of hsa but was eliminated by deletion of the sspA and sspB genes. Deletion of the AgI/II polypeptide genes had no measurable effect on hsa mRNA levels or Hsa surface protein expression, and deletion of hsa did not affect AgI/II polypeptide expression. Further analysis of mutant phenotypes showed that the Hsa and AgI/II proteins mediated adhesion of S. gordonii DL1 to human HEp-2 epithelial cells. Hsa was also a principal streptococcal cell surface component promoting adhesion of human platelets to immobilized streptococci, but Hsa and AgI/II polypeptides acted in concert in mediating streptococcal cell-platelet aggregation. The results suggest that Hsa directs primary adhesion events for S. gordonii DL1 (Challis) with immobilized gp340, epithelial cells, and platelets. AgI/II polypeptides direct gp340-mediated aggregation, facilitate multimodal interactions necessary for platelet aggregation, and modulate S. gordonii-host engagements into biologically productive phenomena.
The accessory Sec (SecA2/Y2) systems of streptococci and staphylococci are dedicated to the transport of large serine-rich repeat (SRR) glycoproteins to the bacterial cell surface. The means by which the glycosylated preproteins are selectively recognized by the accessory Sec system have not been fully characterized. In Streptococcus gordonii, the SRR glycoprotein GspB has a 90-residue amino-terminal signal sequence that is essential for transport by SecA2/Y2 but is not sufficient to mediate the transport of heterologous proteins by this specialized transporter. We now report that a preprotein must remain at least partially unfolded prior to transport by the accessory Sec system. In addition, a region of approximately 20 residues from the amino-terminal end of mature GspB (the accessory Sec transport or AST domain) is essential for SecA2/Y2-dependent transport. The replacement of several AST domain residues with glycine strongly interferes with export, which suggests that a helical conformation may be important. Analysis of GspB variants with alterations in the AST domain, in combination with the results with a SecY2 variant, indicates that the AST domain is essential both for targeting to the SecA2/Y2 translocase and for initiating translocation through the SecY2 channel. The combined results suggest a unique mechanism that ensures the transport of a single substrate by the SecA2/Y2 system.
The Pneumococcal serine-rich repeat protein (PsrP) is a pathogenicity island encoded adhesin that has been positively correlated with the ability of Streptococcus pneumoniae to cause invasive disease. Previous studies have shown that PsrP mediates bacterial attachment to Keratin 10 (K10) on the surface of lung cells through amino acids 273–341 located in the Basic Region (BR) domain. In this study we determined that the BR domain of PsrP also mediates an intra-species interaction that promotes the formation of large bacterial aggregates in the nasopharynx and lungs of infected mice as well as in continuous flow-through models of mature biofilms. Using numerous methods, including complementation of mutants with BR domain deficient constructs, fluorescent microscopy with Cy3-labeled recombinant (r)BR, Far Western blotting of bacterial lysates, co-immunoprecipitation with rBR, and growth of biofilms in the presence of antibodies and competitive peptides, we determined that the BR domain, in particular amino acids 122–166 of PsrP, promoted bacterial aggregation and that antibodies against the BR domain were neutralizing. Using similar methodologies, we also determined that SraP and GspB, the Serine-rich repeat proteins (SRRPs) of Staphylococcus aureus and Streptococcus gordonii, respectively, also promoted bacterial aggregation and that their Non-repeat domains bound to their respective SRRPs. This is the first report to show the presence of biofilm-like structures in the lungs of animals infected with S. pneumoniae and show that SRRPs have dual roles as host and bacterial adhesins. These studies suggest that recombinant Non-repeat domains of SRRPs (i.e. BR for S. pneumoniae) may be useful as vaccine antigens to protect against Gram-positive bacteria that cause infection.
Serine-rich repeat proteins (SRRPs) are a family of surface-expressed proteins found in numerous Gram-positive pathogens, including Staphylococcus aureus, Streptococcus pneumoniae, Group B streptococci, and the oral streptococci that cause infective endocarditis. For all of these bacteria, SRRPs have been demonstrated to play pivotal roles in adhesion to tissues and the development of invasive disease. It is now known that biofilm formation is an important step for bacterial pathogenesis. Bacteria in biofilms have been shown to have differences in metabolism, gene expression, and protein production that contribute to enhanced surface adhesion and the persistence of an infection. Herein we describe a novel role for PsrP, the S. pneumoniae SRRP, as an intra-species bacterial adhesin that promotes bacterial aggregation in the lungs of infected mice during pneumonia. In vitro we show that the Basic Region domain of PsrP promotes self-interactions that result in denser biofilms, greater biofilm biomass, and altered architectures of surface grown cultures; these interactions could be neutralized by antibodies to PsrP that are protective against pneumococcal infection. We also demonstrate that the SRRPs of S. aureus and Streptococcus gordonii also function as intra-species bacterial adhesins. Therefore we conclude that SRRPs have dual roles as host-cell and intra-species bacterial adhesins.
The accessory Sec system of Streptococcus gordonii is comprised of SecY2, SecA2, and five proteins (Asp1 through -5) that are required for the export of a serine-rich glycoprotein, GspB. We have previously shown that a number of the Asps interact with GspB, SecA2, or each other. To further define the roles of these Asps in export, we examined their subcellular localization in S. gordonii and in Escherichia coli expressing the streptococcal accessory Sec system. In particular, we assessed how the locations of these accessory Sec proteins were altered by the presence of other components. Using fluorescence microscopy, we found in E. coli that SecA2 localized within multiple foci at the cell membrane, regardless of whether other accessory Sec proteins were expressed. Asp2 alone localized to the cell poles but formed a similar punctate pattern at the membrane when SecA2 was present. Asp1 and Asp3 localized diffusely in the cytosol when expressed alone or with SecA2. However, these proteins redistributed to the membrane in a punctate arrangement when all of the accessory Sec components were present. Cell fractionation studies with S. gordonii further corroborated these microscopy results. Collectively, these findings indicate that Asp1 to -3 are not integral membrane proteins that form structural parts of the translocation channel. Instead, SecA2 serves as a docking site for Asp2, which in turn attracts a complex of Asp1 and Asp3 to the membrane. These protein interactions may be important for the trafficking of GspB to the cell membrane and its subsequent translocation.
Streptococcus pneumoniae is a leading cause of community-acquired pneumonia and gram-positive sepsis. While multiple virulence determinants have been identified, the combination of features that determines the propensity of an isolate to cause invasive pneumococcal disease (IPD) remains unknown. In this study, we determined the genetic composition of 42 invasive and 30 noninvasive clinical isolates of serotypes 6A, 6B, and 14 by comparative genomic hybridization. Comparison of the present/absent gene matrix (i.e., comparative genomic analysis [CGA]) identified a candidate core genome consisting of 1,553 genes (73% of the TIGR4 genome), 154 genes whose presence correlated with the ability to cause IPD, and 176 genes whose presence correlated with the noninvasive phenotype. Genes identified by CGA were cross-referenced with the published signature-tagged mutagenesis studies, which served to identify core and IPD-correlated genes required for in vivo passage. Among these, two pathogenicity islands, region of diversity 8a (RD8a), which encodes a neuraminidase and V-type sodium synthase, and RD10, which encodes PsrP, a protein homologous to the platelet adhesin GspB in Streptococcus gordonii, were identified. Mice infected with a PsrP mutant were delayed in the development of bacteremia and demonstrated reduced mortality versus wild-type-infected controls. Finally, the presence of seven RDs was determined to correlate with the noninvasive phenotype, a finding that suggests some RDs may contribute to asymptomatic colonization. In conclusion, RDs are unequally distributed between invasive and noninvasive isolates, RD8a and RD10 are correlated with the propensity of an isolate to cause IPD, and PsrP is required for full virulence in mice.
Type II secretion systems (T2SSs) are critical for secretion of many proteins from Gram-negative bacteria. In the T2SS, the outer membrane secretin GspD forms a multimeric pore for translocation of secreted proteins. GspD and the inner membrane protein GspC interact with each other via periplasmic domains. Three different crystal structures of the homology region domain of GspC (GspCHR) in complex with either two or three domains of the N-terminal region of GspD from enterotoxigenic Escherichia coli show that GspCHR adopts an all-β topology. N-terminal β-strands of GspC and the N0 domain of GspD are major components of the interface between these inner and outer membrane proteins from the T2SS. The biological relevance of the observed GspC–GspD interface is shown by analysis of variant proteins in two-hybrid studies and by the effect of mutations in homologous genes on extracellular secretion and subcellular distribution of GspC in Vibrio cholerae. Substitutions of interface residues of GspD have a dramatic effect on the focal distribution of GspC in V. cholerae. These studies indicate that the GspCHR–GspDN0 interactions observed in the crystal structure are essential for T2SS function. Possible implications of our structures for the stoichiometry of the T2SS and exoprotein secretion are discussed.
Many bacterial pathogens affecting humans, animals and plants export diverse proteins across the cell membranes into the medium surrounding the bacteria. Some of these secreted proteins are involved in pathogenesis. One example is cholera toxin secreted by the bacterium Vibrio cholerae, a causative agent of cholera. The sophisticated type II secretion system is responsible for moving this toxin, and several other proteins, across the outer membrane. Here, we studied the interaction between the outer membrane pore of the type II secretion system, the secretin GspD, and the inner membrane protein GspC. We have solved three crystal structures of complexes between the interacting domains and identified critical contacts in the GspC–GspD interface. We also showed the importance of these contacts for assembly of the secretion system and for secretion of proteins by V. cholerae. Our studies provide a major piece in the puzzle of how the type II secretion system is assembled and how it functions. One day this knowledge might allow us to design compounds which interfere with this secretion process. Such compounds would be useful in the battle against bacteria affecting human health.
The accessory Sec system of Streptococcus gordonii is essential for transport of the glycoprotein GspB to the bacterial cell surface. A key component of this dedicated transport system is SecA2. The SecA2 proteins of streptococci and staphylococci are paralogues of SecA and are presumed to have an analogous role in protein transport, but they may be specifically adapted for the transport of large, serine-rich glycoproteins. We used a combination of genetic and biochemical methods to assess whether the S. gordonii SecA2 functions similarly to SecA. Although mutational analyses demonstrated that conserved amino acids are essential for the function of SecA2, replacing such residues in one of two nucleotide binding folds had only minor effects on SecA2 function. SecA2-mediated transport is highly sensitive to azide, as is SecA-mediated transport. Comparison of the S. gordonii SecA and SecA2 proteins in vitro revealed that SecA2 can hydrolyze ATP at a rate similar to that of SecA and is comparably sensitive to azide but that the biochemical properties of these enzymes are subtly different. That is, SecA2 has a lower solubility in aqueous solutions and requires higher Mg2+ concentrations for maximal activity. In spite of the high degree of similarity between the S. gordonii paralogues, analysis of SecA-SecA2 chimeras indicates that the domains are not readily interchangeable. This suggests that specific, unique contacts between SecA2 and other components of the accessory Sec system may preclude cross-functioning with the canonical Sec system.
Bidirectional movement of proteins and RNAs across the nuclear envelope requires Ran, a Ras-like GTPase. A genetic screen of the yeast Saccharomyces cerevisiae was performed to isolate conditional alleles of GSP1, a gene that encodes a homolog of Ran. Two temperature-sensitive alleles, gsp1-1 and gsp1-2, were isolated. The mutations in these two alleles map to regions that are structurally conserved between different members of the Ras family. Each mutant strain exhibits various nuclear transport defects. Both biochemical and genetic experiments indicate a decreased interaction between Ntf2p, a factor which is required for protein import, and the mutant GSP1 gene products. Overexpression of NTF2 can suppress the temperature sensitive phenotype of gsp1-1 and gsp1-2 and partially rescue nuclear transport defects. However, overexpression of a mutant allele of NTF2 with decreased binding to Gsp1p cannot rescue the temperature sensitivity of gsp1-1 and gsp1-2. Taken together, these data demonstrate that the interaction between Gsp1p and Ntf2p is critical for nuclear transport.
The N-terminal receptor-binding domain of the FedF adhesin from enterotoxigenic E. coli has been crystallized. This required the deletion of its first 14 residues, which are also cleaved off naturally.
FedF is the two-domain tip adhesin of F18 fimbriae from enterotoxigenic Escherichia coli. Bacterial adherence, mediated by the N-terminal receptor-binding domain of FedF to carbohydrate receptors on intestinal microvilli, causes diarrhoea and oedema disease in newly weaned piglets and induces the secretion of Shiga toxins. A truncate containing only the receptor-binding domain of FedF was found to be further cleaved at its N-terminus. Reconstruction of this N-terminal truncate rendered FedF amenable to crystallization, resulting in crystals with space group P212121 and unit-cell parameters a = 36.20, b = 74.64, c = 99.03 Å that diffracted to beyond 2 Å resolution. The binding specificity of FedF was screened for on a glycan array, exposing 264 glycoconjugates, to identify specific receptors for cocrystallization with FedF.
The βC-S lyases from two oral bacteria, Streptococcus anginosus and S. gordonii, were cloned, overproduced, purified and crystallized. The obtained crystals were characterized by X-ray diffraction.
Hydrogen sulfide, which causes oral malodour, is generally produced from l-cysteine by the action of βC–S lyase from oral bacteria. The βC–S lyases from two oral bacteria, Streptococcus anginosus and S. gordonii, have been cloned, overproduced, purified and crystallized. X-ray diffraction data were collected from the two types of crystals using synchrotron radiation. The crystal of S. anginosus βC–S lyase belonged to the orthorhombic space group P212121, with unit-cell parameters a = 67.0, b = 111.1, c = 216.4 Å, and the crystal of S. gordonii βC–S lyase belonged to the same space group, with unit-cell parameters a = 58.0, b = 73.9. c = 187.6 Å. The structures of the βC–S lyases were solved by molecular-replacement techniques.
βC–S lyases; Streptococcus anginosus; Streptococcus gordonii
Ca2+-bound and Zn2+-bound forms of ALG-2 were crystallized and X-ray diffraction data were collected to 3.1 and 3.3 Å resolution, respectively.
ALG-2 (apoptosis-linked gene 2) is an apoptosis-linked calcium-binding protein with five EF-hand motifs in the C-terminal region. N-terminally truncated ALG-2 (des3-23ALG-2) was crystallized by the vapour-diffusion method in buffer consisting of either 50 mM MES pH 6.5, 12.5%(v/v) 2-propanol and 150 mM calcium acetate or 100 mM MES pH 6.0, 15%(v/v) ethanol and 200 mM zinc acetate. Crystals of the Ca2+-bound form belonged to space group P212121, with unit-cell parameters a = 54.8, b = 154.4, c = 237.7 Å, α = β = γ = 90°, and diffracted to 3.1 Å resolution. Crystals of the Zn2+-bound form belonged to space group P212121, with unit-cell parameters a = 52.8, b = 147.5, c = 230.7 Å, α = β = γ = 90°, and diffracted to 3.3 Å resolution. The structures of the Ca2+-bound form and the Zn2+-bound form were solved by the molecular-replacement method. Although both crystals contained eight ALG-2 molecules per asymmetric unit, the metal-ion locations and octameric arrangements were found to be significantly different.
apoptosis; calcium-binding protein; penta-EF-hand