Multiple Binding Sites in the Interaction between an Extracellular Fibrinogen-binding Protein from Staphylococcus aureus and Fibrinogen*

Efb (previously Fib) is a fibrinogen-binding protein secreted by Staphylococcus aureus. It has previously been shown that it plays a role in a wound infection model in the rat and that antibodies against Efb reduce the number of recovered bacteria from the mammary glands in a mouse mastitis model. Efb binds to the α-chain of fibrinogen and does not participate in bacterial adherence to fibrinogen. The binding of Efb to fibrinogen is divalent, with one binding site within the two repeat regions in Efb at the N terminus and one binding site at the C terminus. The divalent binding nature leads to precipitation of Efb-fibrinogen complex when the proteins are added to each other at a 1:1 molar ratio. The interaction between Efb and fibrinogen is strongly enhanced by Ca2+ or Zn2+ but not by Mg2.

coagulase is produced early, and the 60-kDa protein is turned on at late log phase.
The fourth FgBP has been shown to be the major cause of adhesion of S. aureus to Fg and is termed the clumping factor (Clf) (3,7). It is cell surface-associated as a result of an LPXTG motif anchoring Clf to the peptidoglycan (8). The fifth FgBP is a cell surface-associated coagulase with unknown function (4).
The nucleotide sequence of Efb revealed two highly homologous 22-amino acid repeats in the N-terminal region separated by a 9-amino acid spacer. These repeats are homologous to the five (9) or eight (10) 27-amino acid repeats found at the Cterminal end of coagulase. In coagulase, these repeats have been suggested to be responsible for the Fg binding activity, whereas the N-terminal end of coagulase is responsible for the coagulase activity and also includes a prothrombin binding domain (9,(11)(12)(13).
An allele replacement mutant of Efb has been constructed where the efb gene was replaced with a gene encoding gentamycin resistance (14). The adherence of the efb negative mutant strain to immobilized Fg was unaltered compared with the parental strain, indicating that adherence is not directly dependent on Efb.
The Efb negative mutant was used in an experimental wound infection model in the rat. Compared with the isogenic parental strain, the Efb negative mutant resulted in significantly reduced severity of signs of infection (14). Furthermore, in a vaccination study using a mouse mastitis experimental infection model, antibodies against Efb were shown to significantly reduce the number of bacteria recovered from the mammary glands, and the level of histopathological signs of infection was reduced (15). These two findings together and the high incidence of the Efb protein among S. aureus isolates suggest that Efb is an important virulence factor worth further investigation.
The biological function of Efb is not known, but the observation that the severity of S. aureus-infected wounds is influenced by Efb implies that wound healing is delayed due to its binding to Fg, probably affecting the clotting process.
We have here compared Efb and Clf and found that Efb binds to Fg in a different way than Clf and that Efb has two binding sites for Fg leading to precipitation of the Efb⅐Fg complex.

EXPERIMENTAL PROCEDURES
Bacterial Strain and Culture Conditions-S. aureus strain Newman was radiolabeled by growing in Luri-Bertani (LB) medium for 5 h at 37°C in the presence of 50 Ci of [ 3 H]thymidin (specific activity 80 mCi/mmol). The cells were washed with phosphate-buffered saline (PBS) and then resuspended in PBS containing 0.05% Tween 20 (PBST) to A 600 ϭ 1.0.
Purification of Efb and Clumping Factor-One liter of S. aureus strain Newman was grown for 19 h at 37°C in LB. The culture was centrifuged, and FgBPs from the supernatant were isolated by affinity * This work was supported by Medical Research Council Grants K97Ϫ16X-12218-01A and K96Ϫ16V-11799Ϫ01. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Dept. of Immunology, Microbiology, Pathology, and Infectious Diseases, Karolinska Institutet, Huddinge University Hospital, F82, S-141 86 Huddinge, Sweden. Tel.: 46 8 58581169; E-mail: jan-ingmar.flock@impi.ki.se. 1 The abbreviations used are: FgBP, fibrinogen-binding protein; Efb (previously Fib), the extracellular fibrinogen-binding protein studied here; GST-RR, the N-terminal repeat regions of Efb as a fusion to glutathione S-transferase; Efb210, the C-terminal part of Efb as a fusion to His 6 ; Clf, clumping factor; PBS, phosphate-buffered saline; PBST, PBS containing 0.05% Tween 20; PAGE, polyacrylamide gel electrophoresis; HRP, horseradish peroxidase; RT, room temperature; ELISA, enzyme-linked immunosorbent assay. chromatography on Fg-Sepharose (Amersham Pharmacia Biotech) as described before (16). Proteins were eluted with 0.7% acetic acid, dialyzed against 40 mM phosphate buffer, pH 6.5 (buffer A), and subjected to fast protein liquid chromatography on a Mono S column using a gradient of 0 to 100% buffer B (1 M NaCl in buffer A). Three peaks of protein were eluted, and the second one, which eluted at a salt concentration of 0.35-0.45 M NaCl, contained Efb.
This plasmid was kindly supplied to us by T. J. Foster (Dublin, Ireland) (7). The His 6 -Clf fusion protein was purified using nickel chelator according to the instructions provided by Qiagen.
Construction of GST-RR and Efb210 Protein-The 5Ј part of the efb gene encoding the two repeat regions (amino acids 8 -69) was amplified by PCR using oligonucleotide primers, efb R.U. with the sequence 5Ј-TGAGGGGATCCCACAATATCGTAGAG-3Ј and efb R.L. 5Ј-AGTTC-CCCGGGTTTTCGCTGCTGGTT-3Ј (obtained from CyberGen AB, Huddinge, Sweden). The plasmid pBFibIII (2) was used as a template, and the reaction contained 5 pmol of each primer, 0.2 mM dNTPs, 0.5 units of Taq polymerase, and the thermal step program included denaturation at 94°C for 10 s, annealing at 50°C for 20 s, and extension at 72°C for 30 s. The DNA fragment was digested with BamHI and SmaI (efb R.U. has the BamHI site and efb R.L. has the SmaI site incorporated) and ligated into the BamHI-SmaI sites of the vector pGEX (Amersham). For cloning, E. coli strain XL1-Blue was used, and colonies with a recombinant plasmid containing the two repeat regions were detected by PCR using the primers efb R.U. and efb R.L. For expression of the recombinant protein (GST-RR), the E. coli strain BL21 was used. GST-RR protein was affinity-purified by glutathione-Sepharose using the procedure recommended by the manufacturer (Amersham).
A recombinant protein containing the C-terminal part of Efb (Efb210; in Ref. 2, called Fib210) (amino acids 70 -136) was purified from E. coli using the previously described plasmid pQFibC210 derived from pQE12 (Qiagen) (2). The pQE12 vector contains an affinity tag of His 6 fused to the C-terminal end of the recombinant protein. Efb210 has been purified both on the nickel nitrilotriacetic acid resin and on Fg-Sepharose. Both the Efb210 peptide and the GST-RR were analyzed by SDS-PAGE and by immunoblot probed with Fg (Sigma) followed by horseradish peroxidase (HRP)-conjugated anti-Fg antibodies (DAKO, Glostrup, Denmark).
Adherence of S. aureus to Fg-A 96-well mitrotiter plate (Falcon; Becton Dickinson and Co., New Jersey) was coated with 100 l of 2 g/ml Fg overnight at room temperature (RT). The wells were then blocked by incubation with 2% bovine serum albumin (Sigma) in PBS for 1 h at 37°C. After washing three times with PBST, 50 l of a 3 H-labeled S. aureus suspension (10 7 cells in PBST) and 50 l of a solution containing the protein used as an inhibitor (Efb or Clf) were added. After a 2-h incubation at 37°C, the wells were washed three times with PBST, and bound bacteria were released by the addition of 2 ϫ 50 l of 3% SDS for 2 ϫ 30 min at RT. The amount of bound bacteria was measured by scintillation counting.
Binding of Fg to Clf and Efb-Microtiter plates (96 wells, Falcon) were coated with Efb or Clf (10 g/ml) in 100 l of PBS at room temperature overnight, and nonspecific binding sites were blocked with 2% bovine serum albumin for 1 h at 37°C. A series of different amounts of Clf in 50 l of PBST (0.01-30 g) and a constant amount of Fg (10 ng) in 50 l of PBST were added to the wells followed by a 2-h incubation at room temperature. Bound Fg was subsequently detected by HRPconjugated rabbit immunoglobulins against Fg diluted 1:1000 in PBST. The plate was washed with PBST after every incubation.
Precipitation Assay of Efb-Fg-Efb-Fg precipitation in solution was measured in microplate wells. One hundred l of PBS solutions with Fg (8.6, 4.3, 2.1, or 0 mg/ml) were added to columns 1, 2, 3, and 4. One hundred l of PBS with Efb (600, 300, 150, 75, or 37 g/ml) were added to wells in rows A, B, C, D, and E, respectively. The plate was incubated 30 min at room temperature, and the increase in absorbance caused by the precipitation was measured at 405 nm using a microplate reader. The same experiment with the same concentrations was also done with Clf or coagulase mixed with Fg.
Efb-Fg precipitation was also measured in 1.2% agarose in a Petri dish into which wells were cut. In the central well was added 20 l of Fg in PBS, and in the peripheral wells were added 5 l of different concentrations of Efb (from 600 g/ml and 2-step dilutions). Different concentrations of Fg were tested from 1 to 10 mg/ml. The Petri dish was stored in a humid chamber overnight for 2 days at 4°C.
Stimulation of Precipitation of Efb-Fg by Divalent Cations-To mi-croplate wells were added 100 l of Fg (5 mg/ml) and 100 l of Efb (300 g/ml) in PBS. Different amounts of divalent cations (Ca 2ϩ , Mg 2ϩ , or Zn 2ϩ ) were added immediately before the addition of Efb. Fg and Efb were each dialyzed before the experiment against 10 mM EDTA to remove divalent cations. The amount of precipitation was measured after 30 min at 405 nm using a microplate reader.
Binding of Fg to the C-and N-terminal Portions of Efb-Microplates with 96 wells were coated overnight at RT with 100 l of different concentrations of Efb210 (2) or GST-RR in PBS. The wells were then blocked by incubation with 2% bovine serum albumin for 1 h at 37°C. After washing three times with PBST, 100 l of Fg (0.1 g/ml) was added to each well, and the microtiter plate was incubated for 1 h at 37°C. Bound Fg was detected using antibodies (1:1000) against Fg conjugated with HRP. The plate was developed with 1,2-phenylediamime (DAKO, Glostrup, Denmark) and then read at 492 nm.
SDS-PAGE and Western Ligand Immunoblotting-SDS-PAGE was run using the Phast System (Amersham). The three different chains of Fg were separated by a 8 to 25% gradient or 7.5% homogenous Phast gels. The proteins were transferred to a nitrocellulose filter that was then blocked by 1% Tween for 20 min at RT. The filter was incubated with 10 g/ml purified Efb in PBST for 2 h at RT. The bound Efb was detected by rabbit anti-Efb antiserum diluted 1:1000 in PBST followed by HRP-conjugated swine anti-rabbit immunoglobulin G antibodies (DAKOpatts) diluted 1:1000 and incubated with the filter for 1 h at RT. The nitrocellulose filter was washed three times with PBST after every incubation, and the color reaction was developed using 4-chloro-1-naphthol tablets (Sigma).

Comparison of Binding Characteristics of Clf and Efb-The
Fg-binding protein clumping factor (Clf), which is present on the bacterial cell surface, has been shown to mediate adherence of S. aureus to Fg (3). In contrast, a mutant strain lacking the efb gene has been shown to have unaltered adherence properties (14). This result was not surprising, since the Efb protein is secreted into the medium and is not present on the bacterial cell surface. However, it seemed possible that Efb might influence adherence by competition with Clf for a common binding site on Fg. The adherence of radiolabeled S. aureus to immobilized Fg was measured in the presence of Clf or Efb. The presence of Clf decreased bacterial adherence by up to 50%. In contrast, the presence of Efb did not reduce the adherence as shown in Fig. 1 between Fg and immobilized Efb. In contrast, soluble Clf inhibited up to 90% of the binding of Fg to immobilized Clf as shown in Fig. 2. In a similar experiment where various concentrations of Efb were added to assess its inhibitory activity on Efb to Fg binding, a precipitate was formed between Efb and Fg (see below).
To further establish that Clf and Efb have different binding sites on Fg, the ␣-, ␤-, and ␥-chains of Fg were separated by SDS-PAGE and subjected to a Western affinity blot using Efb or Clf as probes followed by the respective antibodies. Fig. 3 shows that Clf bound to the ␥-chains, as shown before (17,18), whereas Efb bound to the ␣-chain.
Precipitation of Efb and Fg-Precipitation of antibody-antigen complexes is often visualized in a double diffusion test in agarose. A precipitation line is formed between the wells containing antibody or antigen at a position where the concentrations of each are optimal for precipitation. The same method was used here to demonstrate that the interaction between Fg and Efb leads to precipitation (Fig. 4).
Precipitation could also be visualized in solution. Efb was added at various concentrations to solutions of Fg in microtiter wells using three different concentrations of Fg. A precipitate was formed that could be determined by the increased light absorbance. In Fig. 5A it is seen that the higher the Fg concentration that was used, the higher the Efb concentration that was required to obtain maximal precipitation; increasing the Fg concentration twice or four times gave the most precipitation at twice or four times the concentration of Efb. An excess of either Efb or Fg resulted in less precipitation. It was calculated that maximal precipitation was obtained at a molar ratio close to 1:1. The precipitate formed was threadlike, and the solution never became solid as is the case in coagulation. Purified coagulase was used in parallel experiments for comparison using whole plasma. Mixtures of coagulase and purified Fg gave only weak coagulation after a long time, most likely due to trace amounts of contaminating prothrombin. No optimal ratio for clotting was found for coagulase and Fg. Instead, less coagulase just required more time to produce clotting. Furthermore, the Efb/Fg precipitate was washed extensively in PBS, boiled in loading buffer, run on SDS-PAGE, and Commassie-stained. The three ␣-, ␤-, and ␥-chains were seen together with Efb in what appeared to be equimolar amounts (data not shown).
Each molecule of Fg has been shown to bind three Ca 2ϩ ions, which is important for the structure and functional role of Fg during coagulation (19). Therefore, the interaction between Fg and Efb was tested for its dependence on divalent cations. Fg and Efb were dialyzed separately against EDTA to remove metals associated with the proteins, and a determination of the effect of various concentrations of divalent cations on the precipitation of Fg/Efb mixtures was tested in microtiter wells. Enhancement of precipitation was obtained by the addition of Ca 2ϩ and Zn 2ϩ but not by Mg 2ϩ (Fig. 5B).
Fg Interaction with the C-and N-terminal Portions of Efb-To cause precipitation of Fg, multiple binding sites in Efb would be required. To test this, the N-terminal part of Efb containing the two repeat regions was expressed as a fusion protein with glutathione S-transferase. This protein was designated GST-RR. Also the C-terminal part was expressed separately as a His 6 fusion protein, Efb210, as described previously (2). Fig. 6 shows that Fg was able to bind to each of these fusion proteins in a capture ELISA. As negative controls, GST alone and another His 6 fusion protein were tested for capture of Fg. Fg could not bind to any of these proteins (data not shown).
Both Clf, GST-RR (the Efb repeat regions) and the Efb210 protein (the C-terminal portion) were tested in both precipitation assays, but no precipitation was seen with these proteins (data not shown).
Both the purified Efb210 protein and GST-RR could be further purified by fibrinogen-Sepharose, demonstrating affinity for both parts of Efb for fibrinogen. This confirms earlier findings (2) (not shown here). DISCUSSION We have shown here that the binding of Efb to Fg involves two separate binding sites on Efb. The two repeat regions at the N terminus of Efb (amino acids 8 -69), which are homologous to the repeat regions at the C terminus of coagulase, constitute one binding region. This is in agreement with suggestions that the repeat regions in coagulase bind to Fg. Another binding domain is located at the C terminus of Efb (amino acids 70 -136). The Fg binding to the C-and N-terminal ends of Efb was demonstrated by capture ELISA and by the ability to purify the protein on Fg-Sepharose.
Binding of Efb to Fg clearly takes place to the ␣-chain of Fg as compared with the binding of Clf, which is to the ␥-chain, shown here and previously (18). It is not yet clear if the binding of Efb to the ␣-chain is due to the binding of the repeat regions or due to the C-terminal part or if both binding domains of Efb recognize the ␣-chain. The exact binding site(s) on Fg is presently under investigation.
Efb was unable to compete with S. aureus for adherence to Fg. Combined with our previous finding that a mutant lacking the efb gene retained wild type adherence properties (14), it is likely that Efb is not involved in adherence. No enzymatic function of Efb has been shown, and its binding to Fg seems, therefore, to be the only function of Efb. In the case of coagulase, the binding to Fg is logical, since coagulase forms a complex with prothrombin that binds and converts Fg to fibrin, resulting in clot formation. A biological function of Efb, with no other activity than the ability to bind to Fg, is not obvious and can only be speculative.
We have demonstrated earlier that Efb plays an important role in a wound infection model in the rat. Fibrin is a major component of blood clots in wounds. A possible beneficial effect of Efb for the bacterium, by binding to Fg and affecting the conversion of Fg to fibrin, thereby delaying the wound healing process, is under investigation.
Fg is a symmetric protein with two chains each of ␣, ␤, and ␥, and each ␣-chain has a recognition site for Efb. Therefore, there are at least two recognition sites for Efb on each Fg molecule. Also, both the N-and C-terminal halves of Efb can bind Fg. This would permit the potential formation of three different combinations of two Efb with one Fg and also the formation of larger aggregates of these complexes. The resulting aggregate might be the precipitate that is observed during Efb-Fg interactions. Such a precipitate structure is supported by the finding that the optimal molar ratio between Efb and Fg to get precipitation is 1:1.
In addition, the presence of Ca 2ϩ or Zn 2ϩ enhances the precipitation of the proteins from equimolar mixtures of Efb and fibrinogen. Ca 2ϩ is known to bind to Fg (19,20) and is involved in blood clotting and wound healing. Fg and possibly also Efb contain Ca 2ϩ binding sites, Fg at the C-terminal part of the ␥-chain (DNDNDKFEGNC), and a putative Ca 2ϩ binding site is found in the second repeat region of Efb. Ca 2ϩ may facilitate the interaction between Fg and Efb, possibly by causing some conformational change in one or both of the molecules.