Abstract The serine-rich repeat glycoprotein Srr1 of Streptococcus agalactiae (GBS) is thought to be an important adhesin for the pathogenesis of meningitis. Although expression of Srr1 is associated with increased binding to human brain microvascular endothelial cells (hBMEC), the molecular basis for this interaction is not well defined. We now demonstrate that Srr1 contributes to GBS attachment to hBMEC via the direct interaction of its binding region (BR) with human fibrinogen. When assessed by Far Western blotting, Srr1 was the only protein in GBS extracts that bound fibrinogen. Studies using recombinant Srr1-BR and purified fibrinogen in vitro confirmed a direct protein-protein interaction. Srr1-BR binding was localized to amino acids 283–410 of the fibrinogen Aα chain. Structural predictions indicated that the conformation of Srr1-BR is likely to resemble that of SdrG and other related staphylococcal proteins that bind to fibrinogen through a “dock, lock, and latch” mechanism (DLL). Deletion of the predicted latch domain of Srr1-BR abolished the interaction of the BR with fibrinogen. In addition, a mutant GBS strain lacking the latch domain exhibited reduced binding to hBMEC, and was significantly attenuated in an in vivo model of meningitis. These results indicate that Srr1 can bind fibrinogen directly likely through a DLL mechanism, which has not been described for other streptococcal adhesins. This interaction was important for the pathogenesis of GBS central nervous system invasion and subsequent disease progression.

Author Summary Streptococcus agalactiae (Group B streptococcus, GBS) is a leading cause of meningitis in newborns and infants. This life-threatening infection of the brain and surrounding tissues continues to result in a high incidence of morbidity and mortality, despite antibiotic therapy. A key factor in disease production is the ability of this organism to invade the central nervous system, via the bloodstream. We now report that a GBS surface protein called Srr1 binds fibrinogen, a major protein in human blood. This interaction enhances the attachment of GBS to brain vascular endothelial cells, and contributes to the development of meningitis. A mutation in Srr1 that specifically disrupted binding to fibrinogen significantly reduced GBS attachment to brain endothelium, and markedly reduced virulence in an in vivo model of GBS disease. These studies have identified a new mechanism by which Srr1 contributes to GBS invasion of the central nervous system and may provide a basis for novel therapies targeting Srr1 binding.

Citation: Seo HS, Mu R, Kim BJ, Doran KS, Sullam PM (2012) Binding of Glycoprotein Srr1 of Streptococcus agalactiae to Fibrinogen Promotes Attachment to Brain Endothelium and the Development of Meningitis. PLoS Pathog 8(10): e1002947. https://doi.org/10.1371/journal.ppat.1002947 Editor: Michael R. Wessels, Children's Hospital Boston, United States of America Received: May 25, 2012; Accepted: August 20, 2012; Published: October 4, 2012 This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Funding: This study was supported by the Department of Veterans Affairs and the VA Merit Review program, the Northern California Institute for Research and Education, grants R01-AI41513 (P.M.S.), R01-AI057433 (P.M.S.), a Fellowship Award from the American Heart Association, Western Affiliate (H.S.S), and R01-NS051247 (K.S.D.) from the National Institutes of Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.

Introduction The serine-rich repeat (SRR) glycoproteins are a large and diverse family of adhesins found in Gram-positive bacteria [1], [2]. Each SRR protein is encoded within a large locus that also contains genes encoding proteins responsible for glycosylating the SRR protein, as well as an accessory Sec system that is dedicated to the export of the adhesin. The SRR proteins have a highly conserved domain organization, including a long and specialized signal sequence, two extensive serine-rich repeat regions that undergo glycosylation, and a typical LPXTG cell wall anchoring motif [3], [4]. The N-termini also contain a binding region that varies considerably, both in terms of structure and adherence properties (Figure 1). PPT PowerPoint slide

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larger image TIFF original image Download: Figure 1. Schematic diagram of three staphylococcal fibrinogen binding proteins (ClfA, SdrG and ClfB) and the serine rich repeat proteins Srr1 and GspB. Level of identity (%) between regions is indicated. *S: signal sequence; N1, N2, and N3: DEv-IgG domains; B1 and B2, repeats of unknown function; SD: serine and aspartic acid rich region; SRR1 and SRR2: serine rich regions; CNA: IgG fold domain; Siglec: sialic acid binding domain; LPxTG: cell wall anchoring motif. https://doi.org/10.1371/journal.ppat.1002947.g001 Among the best-characterized is GspB of Streptococcus gordonii, which binds human platelets through its interaction with sialyl-T antigen on the platelet receptor GPIbα [2], [5]. This appears to be an important event in the pathogenesis of infective endocarditis, since disruption of Siglec-mediated binding results in reduced virulence, as measured by an animal model of endocardial infection [3], [4]. A number of other SRR proteins have been shown to contribute to virulence, including SraP of Staphylococcus aureus, PsrP of Streptococcus pneumoniae, and the two SRR proteins (Srr1 and Srr2) of GBS [6]–[11]. However, the molecular basis for binding by these other adhesins is less defined. Their binding regions have no homology to that of GspB, indicating that they are not Siglec-like adhesins. Although SraP mediates binding to platelets, the receptor for this SRR protein has not been identified [6]. PsrP binds cytokeratin 10 in vitro, which appears to be important for binding to pulmonary epithelial cells and subsequent pneumonia [12]. Expression of Srr1 or Srr2 by GBS has been shown to contribute to virulence in models of meningitis [7], [8]. Srr1 mediates binding to several types of human epithelial cell lines, as well as human brain microvascular endothelial cells (hBMEC) [7], [13]. Binding of these cells appears to be important for both colonization and invasion. In vitro studies have indicated that one ligand for Srr1 is human keratin 4, which may facilitate attachment to cervical, vaginal, and pharyngeal cells [13], [14]. We now report, however, that Srr1 also binds human fibrinogen directly through its interaction with the Aα chain of the heteromultimeric protein. This interaction mediates the binding of GBS both to fibrinogen and to hBMEC, and appears to be important for virulence in the setting of meningitis.

Discussion The SRR proteins of GBS are thought to be important both for colonization of the female genital tract, and for the pathogenesis of invasive diseases, such as sepsis and meningitis. Expression of Srr1 has been shown to enhance the attachment of bacteria to vaginal and cervical epithelial cells in vitro, and to facilitate genital colonization in mice [30]. These interactions may be mediated in part by the binding of Srr1 to cytokeratin 4 on the surface of these epithelial cells. Studies in vitro indicate that the Srr1 interacts with cytokeratin 4 to promote bacterial attachment to the cell surface [14], [30]. However, binding can be blocked by sWGA, suggesting that the glycosylated serine-rich domains may also be involved in the interaction of Srr1 with cytokeratin 4 [14]. Strains expressing Srr1 are also more virulent in animal models of meningitis, as compared with their isogenic, srr1-deleted variants [7], [8]. Expression of Srr1 enhances GBS binding to hBMEC, which is likely to be an essential step for initiating central nervous system invasion and meningitis [7]. Our results now demonstrate that Srr1 promotes the adherence of GBS to human fibrinogen, and that this process is likely to be important for the pathogenesis of meningitis. Binding occurs via the interaction of Srr1-BR with the C-terminus of the fibrinogen Aα chain. This appears to be a specific event, requiring the entire Srr1-BR, and amino acids 283–410 of the Aα chain. Although Srr1 has limited primary sequence similarity to other known fibrinogen binding proteins, our secondary structure analyses indicate that Srr1-BR is likely to have a conformation resembling that of ClfA and possibly other related proteins, such as SdrG of Staphylococcus epidermidis. These and a number of other Gram-positive bacterial adhesins are thought to bind fibrinogen through a “dock, lock, and latch” (DLL) mechanism [24]–[26], as described above. Deletion of the predicted latch-like domain of Srr1 significantly reduced fibrinogen binding by the recombinant protein, as well as by bacteria, suggesting that Srr1 binding occurred by a comparable mechanism. If so, this would be the first example of a streptococcal DLL adhesin. Notwithstanding these similarities, there are some notable differences between Srr1 and its staphylococcal counterparts. For example, while Srr1 binds the Aα chain of fibrinogen, ClfA recognizes the C-terminus of the γ chain, and SdrG binds the N-terminus of the β chain [24], [25], [27]. Although both Srr1 and ClfB bind the C-terminus of the Aα chain, their binding sites on fibrinogen appear to differ [27], [28], [31]. A recombinant peptide representing the Aα chain binding site for ClfB (AA283–347) did not inhibit Srr1-BR binding to fibrinogen (Figure S7). Conversely, a peptide containing Aα chain residues 348–410 effectively blocked Srr1-BR binding, but no effect on ClfB binding to fibrinogen. These findings suggest that, while the binding of Srr1 to the Aα chain has some features in common with ClfB, the interactions of these adhesins with fibrinogen must also differ significantly. Further understanding of the precise basis for Srr1 binding to fibrinogen, and whether it occurs via a DLL mechanism, will require solution of its crystal structure. Srr1 binding to fibrinogen was also important for the attachment of GBS to hBMEC in vitro. Binding of GBS to brain endothelium was reduced by deletion of the putative latch domain of Srr1, and was significantly enhanced by adding human fibrinogen, at concentrations (20 µg/ml) well within those found in whole blood (2–4 mg/ml) [32]. These findings indicate that the Srr1-fibrinogen binding is a relevant process for CNS invasion, and indeed we found that in mice with experimental meningitis, the latch deletion was also associated with significantly reduced levels of bacteria, mortality, and inflammation within the CNS. Of note, levels of the bacteria within the bloodstream were not altered by the above mutation, further indicating that the virulence properties associated with Srr1 and fibrinogen binding are specific to CNS infection. FbsA and FbsB are two additional fibrinogen binding proteins of GBS that have been characterized [33], [34]. These proteins appear to be structurally unrelated to Srr1 or other known fibrinogen binding proteins. FbsA and FbsB can bind fibrinogen directly in vitro, although their binding sites on fibrinogen have not been identified. FbsA can also enhance the attachment of GBS to hBMEC [35]. However, FbsA alone is not sufficient for cell invasion, but appears to require FbsB for this process [36]. The contribution of FbsA and FbsB, and their interactions with fibrinogen to virulence is not well-defined. Neither protein has been examined for its role in the pathogenesis of meningitis. Deletion of fbsA was associated with decreased virulence in an animal model of septic arthritis and septicemia [37]. However, neither active nor passive immunization with FbsA or FbsA-specific antibodies resulted in protection against subsequent infection [37], suggesting that the virulence properties of FbsA may be unrelated to fibrinogen binding. Two other GBS proteins (the fibronectin binding protein Fib and a predicted ABC transport protein SAG0242) have been shown to bind fibrinogen, but neither the mechanisms for protein binding, nor the biologic importance of these interactions, have been addressed [33]. In summary, our results show that Srr1 mediates the binding of GBS to fibrinogen, and that this interaction is likely to occur via a DLL-like mechanism, involving the C-terminus of the fibrinogen Aα chain. It is the first streptococcal adhesin for which this type of binding has been identified, indicating that DLL binding may be a generalized mechanism for attachment by Gram-positive organisms. In addition, Srr1-fibrinogen binding appears to be important for the adherence to brain endothelium and the development of meningitis Given that Srr1 or its homolog Srr2 appear to be expressed by most clinical isolates of GBS, this interaction may prove to be a promising candidate for novel therapies targeting bacterial virulence.

Materials and Methods Ethics statement This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Institutional Animal Care and Use Committee of San Diego State University (Animal Welfare Assurance Number: A3728-01). All efforts were made to minimize suffering of animals employed in this study. Reagents Purified human fibrinogen was obtained from Haematologic Technologies. Rabbit anti-fibrinogen IgG was purchased from Aniara. Rabbit anti-Srr1 IgG was generated using purified Srr1-BR protein (NeoPeptide). Strains and growth conditions The bacteria and plasmids used in this study are listed in Table S1 and S2. S. agalatiae strains were grown in Todd-Hewitt broth (Difco) supplemented with 0.5% yeast extract (THY). All mutant strains grow comparably well in vitro (data not shown). Escherichia coli strains DH5α, BL21 and BL21(DE3) were grown at 37°C under aeration in Luria broth (LB; Difco). Appropriate concentrations of antibiotics were added to the media, as required. Cloning and expression of Srr1-BR Genomic DNA was isolated from GBS NCTC 10/84, using Wizard Genomic DNA purification kits (Promega), according to the manufacturer's instructions. PCR products were purified, digested, and ligated into pET28 FLAG to express FLAG-tagged versions of Srr1-BR (amino acids [AA] 303–641), the amino terminus of Srr1-BR (AA 303–479), the carboxy terminus of Srr1-BR (AA480–641) or the latch deletion of Srr1-BR (AA 303–628). Untagged Srr1-BR and Srr1-BRΔlatch were cloned into pET22b(+) (Novagen). The plasmids were then introduced to E. coli BL21(DE3) for over-expression. Proteins were purified by either Ni-NTA (Promega) or anti-FLAG M2 agarose affinity chromatography (Sigma-Aldrich), according to the manufacturers' instructions. Cloning and expression of fibrinogen chains cDNAs encoding the Aα-, Bβ- and γ-chains of human fibrinogen were generously provided by Professor Susan Lord (University of North Carolina at Chapel Hill) [38]–[40]. The full length and truncated forms of chains were amplified and cloned into pMAL-C2X (New England Laboratory) to express MalE-tagged versions of the chains. Plasmids were then introduced to E. coli BL21 by transformation. All recombinant proteins were purified by affinity chromatography with amylose resin, according to the manufacturer's instructions (New England Biolabs). Analysis of Srr1-BR binding to fibrinogen by Far Western blotting Purified human fibrinogen and recombinant fibrinogen chains were separated by electrophoresis through 4–12% NuPAGE Tris-Acetate gels (Invitrogen) and transferred onto nitrocellulose membranes. The membranes were treated with casein-based blocking solution (Western Blocking Reagent; Roche) at room temperature, and then incubated for 1 h with FLAG-tagged Srr1-BR (0.5 µM) suspended in PBS-0.05% Tween 20 (PBS-T). The membranes were then washed three times for 15 min in PBS-T, and bound proteins were detected with mouse anti-FLAG antibody (Sigma-Aldrich). Analysis of Srr1-BR binding to fibrinogen by enzyme linked immunosorbent assay (ELISA) Purified fibrinogen (0.1 µM) was immobilized in 96-well microtiter dishes by overnight incubation at 4°C. The wells were washed twice with PBS and blocked with 300 µl of a casein-based blocking solution for 1 h at room temperature [41], [42]. The plates were washed three times with PBS-T, and FLAG Srr1-BR, FLAG Srr1-BR-N, FLAG Srr1-BR-C or FLAG Srr1-BRΔlatch in PBS-T was added over a range of concentrations. The plates were then incubated for 1 h at 37°C. Unbound protein was removed by washing with PBS-T, and the plates were incubated with mouse anti-FLAG antibodies diluted 1∶4000 in PBS-T for 1 h at 37°C. Wells were washed and incubated with HRP-conjugated rabbit anti-mouse IgG diluted 1∶5000 in PBS-T for 1 h at 37°C. The dissociation constant K D for Srr1 binding was calculated using Prism software v. 4.0 (GraphPad). For inhibition assays, the wells containing immobilized with fibrinogen (0.1 µM) were pretreated with rabbit anti-fibrinogen or rabbit IgG for 30 min, followed by washing to remove unbound antibody prior to the addition of FLAG Srr1-BR. In addition, FLAG Srr1-BR was coincubated with anti-Srr1 IgG or purified untagged Srr1-BR proteins on the wells immobilized with fibrinogen. After washing out unbound proteins, bound FLAG Srr1-BR was then assessed as described above. Fluorescent microscopy hBMEC were fixed with 4% paraformaldehyde and fibrinogen was stained with rabbit anti-fibrinogen IgG (1∶1000) and Alexa Fluor 488 conjugated goat anti-rabbit IgG (Invitrogen). Coverslips were mounted on glass slides using Vectashield (Vector labs) and visualized with a confocal laser scanning microscope (Leica Microsystems). Binding of GBS to immobilized fibrinogen Overnight cultures of GBS were harvested by centrifugation and adjusted to a concentration of 106 CFU/ml in PBS. Purified fibrinogen (0.1 µM) was immobilized in 96-well microtiter plates as described above, and then incubated with 100 µl of GBS suspension for 30 min at 37°C. The wells were then washed to remove unbound bacteria, and then treated with 100 µl of trypsin (2.5 mg/ml) for 10 min at 37°C to release the attached bacteria. The number of bound bacteria was determined by plating serial dilutions of the recovered bacteria onto THB agar plates as previously described [41]. Cell lines and infection assay The human brain microvascular endothelial cell line (hBMEC) was developed and kindly provided by Kwang Sik Kim (Johns Hopkins University) [43], [44] and cultured as previously described [45]. Bacterial adherence assays were performed as described [46]. In brief, bacteria were grown to mid-log phase and then added to confluent hBMEC monolayers at a multiplicity of infection (MOI) of 0.1. After 30 min incubation, monolayers were washed 6 times with PBS to remove non-adherent bacteria, lysed and plated on THB agar to enumerate the bacteria. Bacterial adherence was calculated as (recovered CFU/initial inoculum CFU)×100%. In indicated experiments exogenous fibrinogen (20 µg/ml) was added directly to bacteria and incubated 1.5 hours with rotation at 37°C prior to addition to hBMEC monolayers. Western blot and lectin blot analysis of GBS Srr1 GBS cell wall extracts were prepared by treatment with spheroplasting buffer (500 units/ml mutanolysin, 20 mM Tris, 10 mM MgCl 2 ·6H 2 O, and 0.5 M raphinose), as described previously [47], [48]. Proteins were separated by SDS-PAGE with 3–8% Tris-Acetate gels (Invitrogen) under reducing conditions and then were transferred to nitrocellulose membranes. After blocking with casein based blocking reagent (Roche), the membranes incubated with either 1) anti-Srr1-BR IgG (1∶3000) following by incubation with anti-rabbit IgG (1∶10,000); or 2) biotin conjugated wheat germ agglutinin (WGA; Vector Labs) (0.2 µg/ml) followed by incubation with HRP conjugated streptavidin (0.2 µg/ml). Mouse model of meningitis A murine model of hematogenous GBS meningitis has been described previously [46]. Outbred 6- to 8-week old male CD-1 mice (Charles River Laboratories; 10 mice per group) were injected via the tail vein with 5×107 CFU WT GBS (NCTC 10/84) or GBSΔlatch mutant. At 24 h post GBS injection, blood was collected via tail vein (20 µl) and plated on THB agar to determine the bacterial load in the bloodstream. Mouse survival was accessed over time. At the time of death, or at 78 h post infection, blood and brain tissue were collected aseptically from mice after euthanasia. Bacterial counts were in blood and tissue homogenates were determined by plating serial 10-fold dilutions on THB agar. Brain sections were also embedded in paraffin and stained with hematoxylin and eosin (H&E). Bioinformatic analysis Amino acid similarity was compared using PSI-BLAST and secondary structure was determined by the prediction servers (PHYRE and HHPRED) [19], [49], [50]. Data analysis Data were expressed as means ± standard deviations and were compared for statistical significance by the unpaired t test.

Acknowledgments We thank Barbara Bensing, Ravin Seepersaud, and Tina Iverson (Vanderbilt University) for their helpful scientific and editorial advice, Nai-Yu Wang for technical assistance, Kwang Sik Kim (Johns Hopkins) for providing hBMEC, Craig Rubens (University of Washington at Seattle) for providing GBS isolates, and Susan Lord (University of North Carolina at Chapel Hill) for providing us with fibrinogen plasmids.

Author Contributions Conceived and designed the experiments: HSS RM BJK KSD PMS. Performed the experiments: HSS RM BJK. Analyzed the data: HSS RM BJK KSD PMS. Contributed reagents/materials/analysis tools: HSS KSD PMS. Wrote the paper: HSS KSD PMS.