FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2000362347> ?p ?o ?g. }

• W2000362347 endingPage "35996" @default.
• W2000362347 startingPage "35982" @default.
• W2000362347 abstract "The serine-rich repeat glycoproteins of Gram-positive bacteria comprise a large family of cell wall proteins. Streptococcus agalactiae (group B streptococcus, GBS) expresses either Srr1 or Srr2 on its surface, depending on the strain. Srr1 has recently been shown to bind fibrinogen, and this interaction contributes to the pathogenesis of GBS meningitis. Although strains expressing Srr2 appear to be hypervirulent, no ligand for this adhesin has been described. We now demonstrate that Srr2 also binds human fibrinogen and that this interaction promotes GBS attachment to endothelial cells. Recombinant Srr1 and Srr2 bound fibrinogen in vitro, with affinities of KD = 2.1 × 10−5 and 3.7 × 10−6 m, respectively, as measured by surface plasmon resonance spectroscopy. The binding site for Srr1 and Srr2 was localized to tandem repeats 6–8 of the fibrinogen Aα chain. The structures of both the Srr1 and Srr2 binding regions were determined and, in combination with mutagenesis studies, suggest that both Srr1 and Srr2 interact with a segment of these repeats via a “dock, lock, and latch” mechanism. Moreover, properties of the latch region may account for the increased affinity between Srr2 and fibrinogen. Together, these studies identify how greater affinity of Srr2 for fibrinogen may contribute to the increased virulence associated with Srr2-expressing strains. The serine-rich repeat glycoproteins of Gram-positive bacteria comprise a large family of cell wall proteins. Streptococcus agalactiae (group B streptococcus, GBS) expresses either Srr1 or Srr2 on its surface, depending on the strain. Srr1 has recently been shown to bind fibrinogen, and this interaction contributes to the pathogenesis of GBS meningitis. Although strains expressing Srr2 appear to be hypervirulent, no ligand for this adhesin has been described. We now demonstrate that Srr2 also binds human fibrinogen and that this interaction promotes GBS attachment to endothelial cells. Recombinant Srr1 and Srr2 bound fibrinogen in vitro, with affinities of KD = 2.1 × 10−5 and 3.7 × 10−6 m, respectively, as measured by surface plasmon resonance spectroscopy. The binding site for Srr1 and Srr2 was localized to tandem repeats 6–8 of the fibrinogen Aα chain. The structures of both the Srr1 and Srr2 binding regions were determined and, in combination with mutagenesis studies, suggest that both Srr1 and Srr2 interact with a segment of these repeats via a “dock, lock, and latch” mechanism. Moreover, properties of the latch region may account for the increased affinity between Srr2 and fibrinogen. Together, these studies identify how greater affinity of Srr2 for fibrinogen may contribute to the increased virulence associated with Srr2-expressing strains. The serine-rich repeat (SRR) 2The abbreviations used are: SRRserine-rich repeatGBSgroup B streptococci (Streptococcus agalactiae)DLLdock, lock, and latchITCisothermal calorimetryhBMEChuman brain microvascular endothelial cellsMSCRAMMmicrobial surface components recognizing adhesive matrix moleculesCFUcolony-forming unitMBPmaltose-binding proteinSPRsurface plasmon resonanceBRbinding regionRUrepeating unitLS-CATLife Sciences Collaborative Access Team. glycoproteins of Gram-positive bacteria are a family of adhesins that are important virulence factors for their respective pathogens (1Bensing B.A. López J.A. Sullam P.M. The Streptococcus gordonii surface proteins GspB and Hsa mediate binding to sialylated carbohydrate epitopes on the platelet membrane glycoprotein Ibα.Infect. Immun. 2004; 72: 6528-6537Crossref PubMed Scopus (141) Google Scholar, 2Lizcano A. Sanchez C.J. Orihuela C.J. A role for glycosylated serine-rich repeat proteins in Gram-positive bacterial pathogenesis.Mol. Oral Microbiol. 2012; 27: 257-269Crossref PubMed Scopus (73) Google Scholar, 3Löfling J. Vimberg V. Battig P. Henriques-Normark B. Cellular interactions by LPxTG-anchored pneumococcal adhesins and their streptococcal homologues.Cell. Microbiol. 2011; 13: 186-197Crossref PubMed Scopus (41) Google Scholar). These bacterial surface components are encoded within large loci that also encode proteins mediating their glycosylation and export. Each SRR protein consists of a long and specialized signal sequence, a short serine-rich region (SRR1), a ligand binding region, a second lengthy SRR region, and a typical LPXTG cell wall anchoring motif at the C terminus (4Seo H.S. Mu R. Kim B.J. Doran K.S. Sullam P.M. Binding of glycoprotein Srr1 of Streptococcus agalactiae to fibrinogen promotes attachment to brain endothelium and the development of meningitis.PLoS Pathog. 2012; 8: e1002947Crossref PubMed Scopus (83) Google Scholar, 5Bensing B.A. Takamatsu D. Sullam P.M. Determinants of the streptococcal surface glycoprotein GspB that facilitate export by the accessory Sec system.Mol. Microbiol. 2005; 58: 1468-1481Crossref PubMed Scopus (62) Google Scholar). Although relatively few of the SRR proteins have been studied in detail, the binding regions of the SRR glycoproteins appear to vary significantly in predicted structure and binding properties. Among the best characterized SRR proteins is GspB of Streptococcus gordonii, which binds human platelets through its interaction with sialyl-T antigen on the platelet receptor GPIb (6Takamatsu D. Bensing B.A. Cheng H. Jarvis G.A. Siboo I.R. López J.A. Griffiss J.M. Sullam P.M. Binding of the Streptococcus gordonii surface glycoproteins GspB and Hsa to specific carbohydrate structures on platelet membrane glycoprotein Ibα.Mol. Microbiol. 2005; 58: 380-392Crossref PubMed Scopus (111) Google Scholar, 7Pyburn T.M. Bensing B.A. Xiong Y.Q. Melancon B.J. Tomasiak T.M. Ward N.J. Yankovskaya V. Oliver K.M. Cecchini G. Sulikowski G.A. Tyska M.J. Sullam P.M. Iverson T.M. A structural model for binding of the serine-rich repeat adhesin GspB to host carbohydrate receptors.PLoS Pathog. 2011; 7: e1002112Crossref PubMed Scopus (65) Google Scholar). This interaction appears to be an important event in the pathogenesis of endocarditis, because disruption of GspB binding is associated with a marked reduction in virulence, as tested by animal models of endocardial infection (7Pyburn T.M. Bensing B.A. Xiong Y.Q. Melancon B.J. Tomasiak T.M. Ward N.J. Yankovskaya V. Oliver K.M. Cecchini G. Sulikowski G.A. Tyska M.J. Sullam P.M. Iverson T.M. A structural model for binding of the serine-rich repeat adhesin GspB to host carbohydrate receptors.PLoS Pathog. 2011; 7: e1002112Crossref PubMed Scopus (65) Google Scholar, 8Xiong Y.Q. Bensing B.A. Bayer A.S. Chambers H.F. Sullam P.M. Role of the serine-rich surface glycoprotein GspB of Streptococcus gordonii in the pathogenesis of infective endocarditis.Microb. Pathog. 2008; 45: 297-301Crossref PubMed Scopus (83) Google Scholar). A number of other SRR proteins have been shown to contribute to virulence, including SraP of Staphylococcus aureus, PsrP of Streptococcus pneumoniae, and UafB of Staphylococcus saprophyticus (9Siboo I.R. Chambers H.F. Sullam P.M. Role of SraP, a serine-rich surface protein of Staphylococcus aureus, in binding to human platelets.Infect. Immun. 2005; 73: 2273-2280Crossref PubMed Scopus (189) Google Scholar, 10Sanchez C.J. Shivshankar P. Stol K. Trakhtenbroit S. Sullam P.M. Sauer K. Hermans P.W. Orihuela C.J. The pneumococcal serine-rich repeat protein is an intra-species bacterial adhesin that promotes bacterial aggregation in vivo and in biofilms.PLoS Pathog. 2010; 6: e1001044Crossref PubMed Scopus (148) Google Scholar, 11King N.P. Beatson S.A. Totsika M. Ulett G.C. Alm R.A. Manning P.A. Schembri M.A. UafB is a serine-rich repeat adhesin of Staphylococcus saprophyticus that mediates binding to fibronectin, fibrinogen, and human uroepithelial cells.Microbiology. 2011; 157: 1161-1175Crossref PubMed Scopus (31) Google Scholar), although the molecular basis for binding by these other adhesins is somewhat less well defined. serine-rich repeat group B streptococci (Streptococcus agalactiae) dock, lock, and latch isothermal calorimetry human brain microvascular endothelial cells microbial surface components recognizing adhesive matrix molecules colony-forming unit maltose-binding protein surface plasmon resonance binding region repeating unit Life Sciences Collaborative Access Team. Streptococcus agalactiae (group B Streptococcus, GBS) is a leading cause of neonatal sepsis, pneumonia, and meningitis (12Bohnsack J.F. Whiting A. Gottschalk M. Dunn D.M. Weiss R. Azimi P.H. Philips 3rd, J.B. Weisman L.E. Rhoads G.G. Lin F.Y. Population structure of invasive and colonizing strains of Streptococcus agalactiae from neonates of six U.S. Academic Centers from 1995 to 1999.J. Clin. Microbiol. 2008; 46: 1285-1291Crossref PubMed Scopus (89) Google Scholar, 13Melin P. Neonatal group B streptococcal disease: from pathogenesis to preventive strategies.Clin. Microbiol. Infect. 2011; 17: 1294-1303Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). In recent decades, this organism has also become a significant cause of invasive infections among adults (14Sunkara B. Bheemreddy S. Lorber B. Lephart P.R. Hayakawa K. Sobel J.D. Kaye K.S. Marchaim D. Group B Streptococcus infections in non-pregnant adults: the role of immunosuppression.Int. J. Infect. Dis. 2012; 16: e182-e186Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). GBS strains express either one of two SRR proteins, Srr1 or Srr2. Expression of Srr1 by GBS has been shown to contribute to colonization and virulence in models of infection (15van Sorge N.M. Quach D. Gurney M.A. Sullam P.M. Nizet V. Doran K.S. The group B streptococcal serine-rich repeat 1 glycoprotein mediates penetration of the blood-brain barrier.J. Infect. Dis. 2009; 199: 1479-1487Crossref PubMed Scopus (102) Google Scholar, 16Seifert K.N. Adderson E.E. Whiting A.A. Bohnsack J.F. Crowley P.J. Brady L.J. A unique serine-rich repeat protein (Srr-2) and novel surface antigen (ϵ) associated with a virulent lineage of serotype III Streptococcus agalactiae.Microbiology. 2006; 152: 1029-1040Crossref PubMed Scopus (96) Google Scholar, 17Sheen T.R. Jimenez A. Wang N.Y. Banerjee A. van Sorge N.M. Doran K.S. Serine-rich repeat proteins and pili promote Streptococcus agalactiae colonization of the vaginal tract.J. Bacteriol. 2011; 193: 6834-6842Crossref PubMed Scopus (72) Google Scholar). Srr1 mediates bacterial binding to cytokeratin 4, which is likely to be important for colonization of the female genital tract and is a risk factor for subsequent invasive disease (17Sheen T.R. Jimenez A. Wang N.Y. Banerjee A. van Sorge N.M. Doran K.S. Serine-rich repeat proteins and pili promote Streptococcus agalactiae colonization of the vaginal tract.J. Bacteriol. 2011; 193: 6834-6842Crossref PubMed Scopus (72) Google Scholar, 18Samen U. Eikmanns B.J. Reinscheid D.J. Borges F. The surface protein Srr-1 of Streptococcus agalactiae binds human keratin 4 and promotes adherence to epithelial HEp-2 cells.Infect. Immun. 2007; 75: 5405-5414Crossref PubMed Scopus (92) Google Scholar). In addition, we have recently shown that Srr1 binds to human fibrinogen via its interaction with the Aα chain of the protein. Srr1-mediated binding to fibrinogen is important for the attachment of GBS to human brain microvascular endothelial cells (hBMEC), where fibrinogen served as a bridging molecule between Srr1 and the endovascular surface (4Seo H.S. Mu R. Kim B.J. Doran K.S. Sullam P.M. Binding of glycoprotein Srr1 of Streptococcus agalactiae to fibrinogen promotes attachment to brain endothelium and the development of meningitis.PLoS Pathog. 2012; 8: e1002947Crossref PubMed Scopus (83) Google Scholar). Sequence comparisons and deletion mutagenesis studies (4Seo H.S. Mu R. Kim B.J. Doran K.S. Sullam P.M. Binding of glycoprotein Srr1 of Streptococcus agalactiae to fibrinogen promotes attachment to brain endothelium and the development of meningitis.PLoS Pathog. 2012; 8: e1002947Crossref PubMed Scopus (83) Google Scholar) suggest that the interaction between Srr1 and fibrinogen could employ the “dock, lock, and latch” (DLL) mechanism described for several other fibrinogen-binding adhesins, such as ClfB of S. aureus and SdrG of Staphylococcus epidermidis (19Xiang H. Feng Y. Wang J. Liu B. Chen Y. Liu L. Deng X. Yang M. Crystal structures reveal the multi-ligand binding mechanism of Staphylococcus aureus ClfB.PLoS Pathog. 2012; 8: e1002751Crossref PubMed Scopus (48) Google Scholar, 20Ganesh V.K. Barbu E.M. Deivanayagam C.C. Le B. Anderson A.S. Matsuka Y.V. Lin S.L. Foster T.J. Narayana S.V. Höök M. Structural and biochemical characterization of Staphylococcus aureus clumping factor B/ligand interactions.J. Biol. Chem. 2011; 286: 25963-25972Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 21Ponnuraj K. Bowden M.G. Davis S. Gurusiddappa S. Moore D. Choe D. Xu Y. Hook M. Narayana S.V. A “dock, lock, and latch” structural model for a staphylococcal adhesin binding to fibrinogen.Cell. 2003; 115: 217-228Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar). During this binding process, fibrinogen engages a cleft between two IgG-like folds (the N2 and N3 domains) of the binding region. This docking event results in a conformational change of the adhesin, such that the flexible C terminus of the N3 domain (the “latch”) forms a β-strand and completes a β-sheet within the N2 domain, thereby “locking” the ligand in place. Deletion of the latch region of Srr1 is associated with reduced GBS binding in vitro to fibrinogen and hBMEC and resulted in attenuated virulence in a mouse model of bacteremia and meningitis (4Seo H.S. Mu R. Kim B.J. Doran K.S. Sullam P.M. Binding of glycoprotein Srr1 of Streptococcus agalactiae to fibrinogen promotes attachment to brain endothelium and the development of meningitis.PLoS Pathog. 2012; 8: e1002947Crossref PubMed Scopus (83) Google Scholar). These findings indicate that fibrinogen binding via Srr1 may occur via a DLL mechanism and that this interaction enhances pathogenicity. As compared with Srr1, relatively little is known about the binding properties of Srr2 or its contribution toward GBS virulence. Srr2 has been detected in serotype III strains exclusively and only in isolates belonging to sequence multilocus sequence type 17 (ST-17), a genotype linked epidemiologically to increased invasive disease (16Seifert K.N. Adderson E.E. Whiting A.A. Bohnsack J.F. Crowley P.J. Brady L.J. A unique serine-rich repeat protein (Srr-2) and novel surface antigen (ϵ) associated with a virulent lineage of serotype III Streptococcus agalactiae.Microbiology. 2006; 152: 1029-1040Crossref PubMed Scopus (96) Google Scholar, 22Lamy M.C. Dramsi S. Billoët A. Réglier-Poupet H. Tazi A. Raymond J. Guérin F. Couvé E. Kunst F. Glaser P. Trieu-Cuot P. Poyart C. Rapid detection of the “highly virulent” group B Streptococcus ST-17 clone.Microbes Infect. 2006; 8: 1714-1722Crossref PubMed Scopus (98) Google Scholar, 23Bellais S. Six A. Fouet A. Longo M. Dmytruk N. Glaser P. Trieu-Cuot P. Poyart C. Capsular switching in group B Streptococcus CC17 hypervirulent clone: a future challenge for polysaccharide vaccine development.J. Infect. Dis. 2012; 206: 1745-1752Crossref PubMed Scopus (94) Google Scholar, 24Bisharat N. Crook D.W. Leigh J. Harding R.M. Ward P.N. Coffey T.J. Maiden M.C. Peto T. Jones N. Hyperinvasive neonatal group B streptococcus has arisen from a bovine ancestor.J. Clin. Microbiol. 2004; 42: 2161-2167Crossref PubMed Scopus (117) Google Scholar, 25Davies H.D. Jones N. Whittam T.S. Elsayed S. Bisharat N. Baker C.J. Multilocus sequence typing of serotype III group B streptococcus and correlation with pathogenic potential.J. Infect. Dis. 2004; 189: 1097-1102Crossref PubMed Scopus (57) Google Scholar, 26Jones N. Oliver K.A. Barry J. Harding R.M. Bisharat N. Spratt B.G. Peto T. Crook D.W. Enhanced invasiveness of bovine-derived neonatal sequence type 17 group B streptococcus is independent of capsular serotype.Clin. Infect. Dis. 2006; 42: 915-924Crossref PubMed Scopus (87) Google Scholar, 27Tazi A. Bellais S. Tardieux I. Dramsi S. Trieu-Cuot P. Poyart C. Group B Streptococcus surface proteins as major determinants for meningeal tropism.Curr. Opin. Microbiol. 2012; 15: 44-49Crossref PubMed Scopus (46) Google Scholar, 28Dramsi S. Morello E. Poyart C. Trieu-Cuot P. Epidemiologically and clinically relevant Group B Streptococcus isolates do not bind collagen but display enhanced binding to human fibrinogen.Microbes Infect. 2012; 14: 1044-1048Crossref PubMed Scopus (16) Google Scholar). In addition, strains expressing Srr2 were significantly more virulent in a mouse model of neonatal sepsis, as compared with Srr1-expressing strains (16Seifert K.N. Adderson E.E. Whiting A.A. Bohnsack J.F. Crowley P.J. Brady L.J. A unique serine-rich repeat protein (Srr-2) and novel surface antigen (ϵ) associated with a virulent lineage of serotype III Streptococcus agalactiae.Microbiology. 2006; 152: 1029-1040Crossref PubMed Scopus (96) Google Scholar), suggesting that this surface component may at least in part explain the increased virulence associated with ST-17 isolates. ST-17 strains also have higher levels of fibrinogen binding, but the molecular basis for this has not been well defined (28Dramsi S. Morello E. Poyart C. Trieu-Cuot P. Epidemiologically and clinically relevant Group B Streptococcus isolates do not bind collagen but display enhanced binding to human fibrinogen.Microbes Infect. 2012; 14: 1044-1048Crossref PubMed Scopus (16) Google Scholar). Delineating the molecular differences between Srr1 and Srr2 could improve our understanding of how Srr2 confers hypervirulence in S. agalactiae. We now report that both Srr1 and Srr2 bind to a specific tandem repeat region of fibrinogen Aα chain. Crystal structures and mutagenesis studies indicate that both proteins employ a DLL mechanism for host binding. Moreover, Srr2 has significantly higher binding affinity for fibrinogen as compared with Srr1, and analysis of their structures suggests that the physical positioning of the latch region may underlie this enhanced affinity. Purified human fibrinogen was obtained from Hematologic Technologies. Rabbit anti-fibrinogen IgG was purchased from Aniara. Rabbit anti-Srr2 IgG was generated by NeoPeptide, using purified recombinant protein corresponding to the binding region (BR) of Srr2. The bacteria and plasmids used in this study are listed in TABLE 1, TABLE 2. S. agalactiae strains were grown in Todd-Hewitt broth (Difco) supplemented with 0.5% yeast extract (THY). All mutant strains grew comparably well in vitro, as compared with parent strains (data not shown). Escherichia coli strains DH5α, BL21, and BL21(DE3) were grown at 37 °C under aeration in Luria broth (LB; Difco). Antibiotics were added to the media as required.TABLE 1StrainsStrain or plasmidGenotype or descriptionaErmR, erythromycin resistance; CmR, chloramphenicol resistance.SourceE. coliDH5αF−r−m+Ø80dlacZΔM15InvitrogenBL21 (DE3)Expression host, inducible T7 RNA polymeraseNovagenS. agalactiaeCOH31Serotype III67Wessels M.R. Haft R.F. Heggen L.M. Rubens C.E. Identification of a genetic locus essential for capsule sialylation in type III group B streptococci.Infect. Immun. 1992; 60: 392-400Crossref PubMed Google ScholarPS954COH31Δsrr1, CmR15van Sorge N.M. Quach D. Gurney M.A. Sullam P.M. Nizet V. Doran K.S. The group B streptococcal serine-rich repeat 1 glycoprotein mediates penetration of the blood-brain barrier.J. Infect. Dis. 2009; 199: 1479-1487Crossref PubMed Scopus (102) Google ScholarNCTC 10/84Serotype V, clinical isolate68Wilkinson H.W. Nontypable group B streptococci isolated from human sources.J. Clin. Microbiol. 1977; 6: 183-184Crossref PubMed Google ScholarPS2645NCTC 10/84Δsrr1, CmR15van Sorge N.M. Quach D. Gurney M.A. Sullam P.M. Nizet V. Doran K.S. The group B streptococcal serine-rich repeat 1 glycoprotein mediates penetration of the blood-brain barrier.J. Infect. Dis. 2009; 199: 1479-1487Crossref PubMed Scopus (102) Google ScholarCOH1Serotype III, clinical isolate, ST-1769Martin T.R. Rubens C.E. Wilson C.B. Lung antibacterial defense mechanisms in infant and adult rats: implications for the pathogenesis of group B streptococcal infections in the neonatal lung.J. Infect. Dis. 1988; 157: 91-100Crossref PubMed Scopus (85) Google ScholarPS2641COH1Δsrr2, CmR17Sheen T.R. Jimenez A. Wang N.Y. Banerjee A. van Sorge N.M. Doran K.S. Serine-rich repeat proteins and pili promote Streptococcus agalactiae colonization of the vaginal tract.J. Bacteriol. 2011; 193: 6834-6842Crossref PubMed Scopus (72) Google Scholar, 49Quach D. van Sorge N.M. Kristian S.A. Bryan J.D. Shelver D.W. Doran K.S. The CiaR response regulator in group B Streptococcus promotes intracellular survival and resistance to innate immune defenses.J. Bacteriol. 2009; 191: 2023-2032Crossref PubMed Scopus (64) Google ScholarPS2931PS2641/pDE-Srr1This studyPS2933PS2641/pDE-Srr2This studyH36BSerotype Ib70Lancefield R.C. McCarty M. Everly W.N. Multiple mouse-protective antibodies directed against group B streptococci. Special reference to antibodies effective against protein antigens.J. Exp. Med. 1975; 142: 165-179Crossref PubMed Scopus (239) Google ScholarNCTC 1/82Serotype IV4Seo H.S. Mu R. Kim B.J. Doran K.S. Sullam P.M. Binding of glycoprotein Srr1 of Streptococcus agalactiae to fibrinogen promotes attachment to brain endothelium and the development of meningitis.PLoS Pathog. 2012; 8: e1002947Crossref PubMed Scopus (83) Google ScholarJ48Serotype III, ST-1716Seifert K.N. Adderson E.E. Whiting A.A. Bohnsack J.F. Crowley P.J. Brady L.J. A unique serine-rich repeat protein (Srr-2) and novel surface antigen (ϵ) associated with a virulent lineage of serotype III Streptococcus agalactiae.Microbiology. 2006; 152: 1029-1040Crossref PubMed Scopus (96) Google ScholarNEM316Serotype III, ST-2371Glaser P. Rusniok C. Buchrieser C. Chevalier F. Frangeul L. Msadek T. Zouine M. Couvé E. Lalioui L. Poyart C. Trieu-Cuot P. Kunst F. Genome sequence of Streptococcus agalactiae, a pathogen causing invasive neonatal disease.Mol. Microbiol. 2002; 45: 1499-1513Crossref PubMed Scopus (386) Google Scholara ErmR, erythromycin resistance; CmR, chloramphenicol resistance. Open table in a new tab TABLE 2PlasmidsPlasmidDescriptionSourcepDE123Streptococcal shuttle vector, ErmR4Seo H.S. Mu R. Kim B.J. Doran K.S. Sullam P.M. Binding of glycoprotein Srr1 of Streptococcus agalactiae to fibrinogen promotes attachment to brain endothelium and the development of meningitis.PLoS Pathog. 2012; 8: e1002947Crossref PubMed Scopus (83) Google ScholarpDE123-srr1Vector for expression of Srr1, ErmR4Seo H.S. Mu R. Kim B.J. Doran K.S. Sullam P.M. Binding of glycoprotein Srr1 of Streptococcus agalactiae to fibrinogen promotes attachment to brain endothelium and the development of meningitis.PLoS Pathog. 2012; 8: e1002947Crossref PubMed Scopus (83) Google ScholarpDE123-srr2Vector for expression of Srr2, ErmRThis studypET22b(+)Expression vector, AmpRNovagenpET28-FLAGExpression vector with FLAG tag, KanR32Seo H.S. Xiong Y.Q. Mitchell J. Seepersaud R. Bayer A.S. Sullam P.M. Bacteriophage lysin mediates the binding of Streptococcus mitis to human platelets through interaction with fibrinogen.PLoS Pathog. 2010; 6: e1001047Crossref PubMed Scopus (48) Google ScholarpET22-Srr1-BRVector for expression of Srr1-BR, AmpR4Seo H.S. Mu R. Kim B.J. Doran K.S. Sullam P.M. Binding of glycoprotein Srr1 of Streptococcus agalactiae to fibrinogen promotes attachment to brain endothelium and the development of meningitis.PLoS Pathog. 2012; 8: e1002947Crossref PubMed Scopus (83) Google ScholarpET22-Srr2-BRVector for expression of Srr2-BR, AmpRThis studypET22-ClfA-BRVector for expression of ClfA-BR, AmpRThis studypET28-FLAG-Srr2-BRΔlatchVector for expression of FLAG-tagged Srr1 (303–627)This studypSET-5SStreptococcal thermosensitive suicide vector, CmR72Takamatsu D. Osaki M. Sekizaki T. Thermosensitive suicide vectors for gene replacement in Streptococcus suis.Plasmid. 2001; 46: 140-148Crossref PubMed Scopus (237) Google ScholarpSET-5S-srr2KOVector for deletion of srr2 gene, CmRThis studypMAL-C2XExpression vector with MBP fusion proteinNew England BiolabspMal-AαVector for expression of MBP-tagged Aα chain33Seo H.S. Sullam P.M. Characterization of the fibrinogen binding domain of bacteriophage lysin from Streptococcus mitis.Infect. Immun. 2011; 79: 3518-3526Crossref PubMed Scopus (12) Google ScholarpMal-BβVector for expression of MBP-tagged Bβ chain33Seo H.S. Sullam P.M. Characterization of the fibrinogen binding domain of bacteriophage lysin from Streptococcus mitis.Infect. Immun. 2011; 79: 3518-3526Crossref PubMed Scopus (12) Google ScholarpMal-γVector for expression of MBP-tagged γ chain33Seo H.S. Sullam P.M. Characterization of the fibrinogen binding domain of bacteriophage lysin from Streptococcus mitis.Infect. Immun. 2011; 79: 3518-3526Crossref PubMed Scopus (12) Google ScholarpMal-Aα(1–197)Vector for expression of MBP-tagged Aα variant4Seo H.S. Mu R. Kim B.J. Doran K.S. Sullam P.M. Binding of glycoprotein Srr1 of Streptococcus agalactiae to fibrinogen promotes attachment to brain endothelium and the development of meningitis.PLoS Pathog. 2012; 8: e1002947Crossref PubMed Scopus (83) Google ScholarpMal-Aα(198–610)Vector for expression of MBP-tagged Aα variant4Seo H.S. Mu R. Kim B.J. Doran K.S. Sullam P.M. Binding of glycoprotein Srr1 of Streptococcus agalactiae to fibrinogen promotes attachment to brain endothelium and the development of meningitis.PLoS Pathog. 2012; 8: e1002947Crossref PubMed Scopus (83) Google ScholarpMal-Aα(198–282)Vector for expression of MBP-tagged Aα variant4Seo H.S. Mu R. Kim B.J. Doran K.S. Sullam P.M. Binding of glycoprotein Srr1 of Streptococcus agalactiae to fibrinogen promotes attachment to brain endothelium and the development of meningitis.PLoS Pathog. 2012; 8: e1002947Crossref PubMed Scopus (83) Google ScholarpMal-Aα(283–410)Vector for expression of MBP-tagged Aα variant4Seo H.S. Mu R. Kim B.J. Doran K.S. Sullam P.M. Binding of glycoprotein Srr1 of Streptococcus agalactiae to fibrinogen promotes attachment to brain endothelium and the development of meningitis.PLoS Pathog. 2012; 8: e1002947Crossref PubMed Scopus (83) Google ScholarpMal-Aα(198–282 + 411–610)Vector for expression of MBP-tagged Aα variant4Seo H.S. Mu R. Kim B.J. Doran K.S. Sullam P.M. Binding of glycoprotein Srr1 of Streptococcus agalactiae to fibrinogen promotes attachment to brain endothelium and the development of meningitis.PLoS Pathog. 2012; 8: e1002947Crossref PubMed Scopus (83) Google ScholarpMal-Aα-RU1–10Vector for expression of MBP-tagged Aα variantThis studypMal-Aα-RU1–6Vector for expression of MBP-tagged Aα variantThis studypMal-Aα-RU1–7Vector for expression of MBP-tagged Aα variantThis studypMal-Aα-RU1–8Vector for expression of MBP-tagged Aα variantThis studypMal-Aα-RU1–9Vector for expression of MBP-tagged Aα variantThis study Open table in a new tab Genomic DNA was isolated from GBS NCTC 10/84 and COH1 using Wizard Genomic DNA purification kits (Promega), according to the manufacturer's instructions. PCR products were cloned into pET28-FLAG to express FLAG-tagged versions of Srr1-BR (amino acids 303–641), Srr2-BR (amino acids 303–641), or the latch deletion variant of Srr2-BR (amino acids 303–628). DNA encoding Srr1-BR, Srr2-BR, Srr1-BRΔlatch, Srr2-BRΔlatch, or ClfA-BR (N2N3) were cloned into pET22b(+) (Novagen) or pET28-FLAG. Proteins were purified by either nickel-nitrilotriacetic acid (Promega) or anti-FLAG M2-agarose affinity chromatography (Sigma), according to the manufacturers' instructions. DNA of each chain was amplified from cDNA encoding the Aα-, Bβ-, and γ-chains of human fibrinogen and cloned into pMAL-C2X (New England Laboratory) as described previously (29Lord S.T. Expression of a cloned human fibrinogen cDNA in Escherichia coli: synthesis of an Aα polypeptide.DNA. 1985; 4: 33-38Crossref PubMed Scopus (14) Google Scholar, 30Bolyard M.G. Lord S.T. High-level expression of a functional human fibrinogen γ chain in Escherichia coli.Gene. 1988; 66: 183-192Crossref PubMed Scopus (27) Google Scholar, 31Lord S.T. Strickland E. Jayjock E. Strategy for recombinant multichain protein synthesis: fibrinogen B β-chain variants as thrombin substrates.Biochemistry. 1996; 35: 2342-2348Crossref PubMed Scopus (46) Google Scholar). The recombinant proteins were purified by affinity chromatography with amylose resin, according to the manufacturer's instructions (New England Biolabs). Cysteine replacement mutations were made within latch and latching cleft domains of Srr-BRs by a two-stage PCR procedure. For codon conversion to cysteine in the latching cleft, overlapping primers were used with either primer 3006(NotI)/5003 (N423C) or 3003(N423C)/5006(XhoI) for Srr1-BR and either 3012(NotI)/5009 (N336C) or 3009(N339C)/5012(XhoI) for Srr2-BR to generate overlapping DNA fragments spanning the entire Srr1-BR and Srr2-BR. The two DNA fragments were combined for the second stage PCR and then amplified using primers 3006(NotI)/5006(XhoI, K639C) for the Srr1-BR and 3012(NotI)/5012(XhoI, N541C) for Srr2-BR. Amplified products were digested with the appropriate restriction enzymes and ligated into pET28-FLAG. The constructs were sequenced to confirm that the mutations were correctly positioned and then expressed in E. coli, as described above. Genomic DNA was isolated from COH1 and NCTC 10/84 strains, using Wizard Genomic DNA purification kits (Promega). Polymerase chain reaction (PCR) was performed with the primers (Srr1 forward, AAT CTA GAT AGA TTT CTA ATC ACT TAA TTT TAC, and Srr1 reverse, GCT CTA GAA GAA TTC AAA GTA GGT TTA GTC; Srr2 forward, TTT CTA GAT AGC ATT ATT TTT TAA ATA TGG, and Srr2 reverse, TTC TGC AGT TAA TCT TTT TTC TTC TTG C) to amplify srr1 or srr2 genes. PCR products were purified, digested, and ligated into pDE123 to express the full-" @default.
• W2000362347 created "2016-06-24" @default.
• W2000362347 creator A5016926827 @default.
• W2000362347 creator A5022472366 @default.
• W2000362347 creator A5030470781 @default.
• W2000362347 creator A5046083677 @default.
• W2000362347 creator A5056005181 @default.
• W2000362347 creator A5056811121 @default.
• W2000362347 creator A5057424046 @default.
• W2000362347 creator A5059776564 @default.
• W2000362347 creator A5079388251 @default.
• W2000362347 date "2013-12-01" @default.
• W2000362347 modified "2023-10-18" @default.
• W2000362347 title "Characterization of Fibrinogen Binding by Glycoproteins Srr1 and Srr2 of Streptococcus agalactiae" @default.
• W2000362347 cites W1500769488 @default.
• W2000362347 cites W1534209903 @default.
• W2000362347 cites W1535575371 @default.
• W2000362347 cites W1539727497 @default.
• W2000362347 cites W1539796472 @default.
• W2000362347 cites W1575019178 @default.
• W2000362347 cites W1974307260 @default.
• W2000362347 cites W1976700134 @default.
• W2000362347 cites W1980010909 @default.
• W2000362347 cites W1981054366 @default.
• W2000362347 cites W1981314849 @default.
• W2000362347 cites W1982346690 @default.
• W2000362347 cites W1983957332 @default.
• W2000362347 cites W1996504295 @default.
• W2000362347 cites W1997440923 @default.
• W2000362347 cites W2001326149 @default.
• W2000362347 cites W2004111638 @default.
• W2000362347 cites W2015308774 @default.
• W2000362347 cites W2023106107 @default.
• W2000362347 cites W2023183303 @default.
• W2000362347 cites W2023705783 @default.
• W2000362347 cites W2028962398 @default.
• W2000362347 cites W2029114752 @default.
• W2000362347 cites W2029531605 @default.
• W2000362347 cites W2029900219 @default.
• W2000362347 cites W2030904449 @default.
• W2000362347 cites W2038236709 @default.
• W2000362347 cites W2038840577 @default.
• W2000362347 cites W2048818416 @default.
• W2000362347 cites W2052620064 @default.
• W2000362347 cites W2054051063 @default.
• W2000362347 cites W2055763564 @default.
• W2000362347 cites W2057128960 @default.
• W2000362347 cites W2057659396 @default.
• W2000362347 cites W2067583120 @default.
• W2000362347 cites W2067803976 @default.
• W2000362347 cites W2068714412 @default.
• W2000362347 cites W2095102700 @default.
• W2000362347 cites W2096052499 @default.
• W2000362347 cites W2101203381 @default.
• W2000362347 cites W2101477346 @default.
• W2000362347 cites W2101634685 @default.
• W2000362347 cites W2103105107 @default.
• W2000362347 cites W2105048698 @default.
• W2000362347 cites W2109602941 @default.
• W2000362347 cites W2109819082 @default.
• W2000362347 cites W2112340752 @default.
• W2000362347 cites W2115864870 @default.
• W2000362347 cites W2116496714 @default.
• W2000362347 cites W2119722214 @default.
• W2000362347 cites W2120151328 @default.
• W2000362347 cites W2120685671 @default.
• W2000362347 cites W2121978062 @default.
• W2000362347 cites W2126573110 @default.
• W2000362347 cites W2126630915 @default.
• W2000362347 cites W2134201060 @default.
• W2000362347 cites W2135336860 @default.
• W2000362347 cites W2141423939 @default.
• W2000362347 cites W2144081223 @default.
• W2000362347 cites W2146582952 @default.
• W2000362347 cites W2147673959 @default.
• W2000362347 cites W2148271653 @default.
• W2000362347 cites W2148525793 @default.
• W2000362347 cites W2149396146 @default.
• W2000362347 cites W2150867769 @default.
• W2000362347 cites W2152896770 @default.
• W2000362347 cites W2161509463 @default.
• W2000362347 cites W2163341755 @default.
• W2000362347 cites W2165139231 @default.
• W2000362347 cites W2169161684 @default.
• W2000362347 cites W2330827545 @default.
• W2000362347 cites W4233838756 @default.
• W2000362347 doi "https://doi.org/10.1074/jbc.m113.513358" @default.
• W2000362347 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/3861647" @default.
• W2000362347 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/24165132" @default.
• W2000362347 hasPublicationYear "2013" @default.
• W2000362347 type Work @default.
• W2000362347 sameAs 2000362347 @default.
• W2000362347 citedByCount "76" @default.
• W2000362347 countsByYear W20003623472014 @default.
• W2000362347 countsByYear W20003623472015 @default.
• W2000362347 countsByYear W20003623472016 @default.
• W2000362347 countsByYear W20003623472017 @default.
• W2000362347 countsByYear W20003623472018 @default.

• next