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Functional studies of a fibrinogen binding protein from Staphylococcus epidermidis - PubMed

Functional studies of a fibrinogen binding protein from Staphylococcus epidermidis

L Pei et al. Infect Immun. 1999 Sep.

Abstract

A gene encoding a fibrinogen binding protein from Staphylococcus epidermidis was previously cloned, and the nucleotide sequence was determined. A portion of the gene encompassing the fibrinogen binding domain has now been subcloned in an expression-fusion vector. The fusion protein can bind to fibrinogen in a capture enzyme-linked immunosorbent assay and can be purified by fibrinogen affinity chromatography. This protein can completely inhibit the adherence of S. epidermidis to immobilized fibrinogen, suggesting that the adherence of S. epidermidis to fibrinogen is mainly due to this protein. Antibodies against this fibrinogen binding protein were also found to efficiently block the adherence of S. epidermidis to immobilized fibrinogen. Despite homology with clumping factors A and B from S. aureus (cell surface-associated proteins binding to fibrinogen), binding involved the beta chain of fibrinogen rather than the gamma chain, as in clumping factor A.

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Figures

FIG. 1
FIG. 1

(A) SDS-PAGE. Lane 1, Molecular size markers (94, 67, 43, 30, 20, and 14 kDa); lane 2, GST-Fbe purified by glutathione-Sepharose 4B affinity chromatography; lane 3, GST-Fbe purified by glutathione-Sepharose 4B affinity chromatography and cleaved with thrombin; lane 4, GST-Fbe purified by glutathione-Sepharose 4B affinity chromatography, cleaved with thrombin, and recovered as Fbe by FPLC; lane 5, GST-Fbe purified by Fg-Sepharose 4B affinity chromatography; lane 6, GST-Fbe purified by Fg-Sepharose 4B affinity chromatography and cleaved with thrombin; lane 7, GST-Fbe purified by Fg-Sepharose 4B affinity chromatography, cleaved with thrombin, and recovered as Fbe by FPLC; lane 8, GST purified from a culture induced with plasmid pGEX-4T3. (B) Western affinity blotting. Proteins from SDS-PAGE were transferred to nitrocellulose filters. The filters were probed with Fg followed by HRP-conjugated anti-Fg antibodies. Lanes are as in panel A.

FIG. 2
FIG. 2

(A) Capture of GST-Fbe or GST by Fg. Microtiter plate wells were coated with the indicated concentrations (conc.) of Fg and then coated with BSA. GST-Fbe (open squares) or purified GST (closed squares) was added at 25 μg/ml and incubated. Detection of GST-Fbe or GST capture was done with antibodies raised against GST-Fbe in a rat, followed by HRP-conjugated antibodies against rat IgG. (B) Capture of Fg by GST-Fbe or GST. Microtiter plate wells were coated with the indicated concentrations of GST-Fbe (open squares) or purified GST (closed squares) and then coated with BSA. Fg was added at 20 μg/ml and incubated. Detection of Fg capture was done with HRP-conjugated antibodies against Fg.

FIG. 3
FIG. 3

Effect of Ca2+ on the interaction between GST-Fbe and Fg. The binding of GST-Fbe and of Fbe to Fg was measured at different concentrations (conc.) of Ca2+ essentially as described in the legend to Fig. 2A. Likewise, the binding of Fg to GST-Fbe and to Fbe was measured at different concentrations of Ca2+. All proteins were dialyzed against EDTA to remove endogenous Ca2+. Symbols: ■, binding of Fg to GST-Fbe; □, binding of Fg to Fbe; ●, binding of GST-Fbe to Fg; ○, binding of Fbe to Fg. The experiment shown is representative of at least three experiments.

FIG. 4
FIG. 4

Inhibition of S. epidermidis adherence to immobilized Fg. To microtiter plate wells coated with Fg, inhibiting proteins at the indicated concentrations (conc.) were added. Immediately thereafter, radiolabeled cells of S. epidermidis were added and allowed to adhere. After the removal of nonadherent bacteria, bound bacteria were quantified by scintillation counting. Symbols: ○, GST; □, GST-Fbe; ◊, Fbe FPLC purified after thrombin cleavage; ▵, ClfA. The experiment shown is representative of at least three experiments.

FIG. 5
FIG. 5

Competition in a capture ELISA with ClfA or GST-Fbe. Microtiter plate wells were coated with GST-Fbe or ClfA and then with BSA. The indicated concentrations (conc.) of GST-Fbe or ClfA as an inhibitor were added immediately before the addition of Fg. The relative amount of bound Fg was determined with HRP-conjugated antibodies against Fg. Symbols: □, GST-Fbe inhibition of Fg binding to GST-Fbe; ○, ClfA inhibition of Fg binding to ClfA; ▵, inability of GST-Fbe to inhibit Fg binding to ClfA. The experiment shown is representative of at least three experiments.

FIG. 6
FIG. 6

(a) Western blot of separated Fg chains. Fg from SDS-PAGE was transferred to nitrocellulose filters. The filters were probed with either GST-Fbe followed by rat anti–GST-Fbe antibodies or ClfA followed by rat anti-ClfA antibodies and, in both cases, by anti-rat IgG antibodies conjugated with HRP. Lanes (Coomassie blue-stained gel in lanes 1 and 2 and Western blot strips in lanes A to D): 1, molecular size markers (94, 67, and 43 kDa, from the top); 2, α, β, and γ chains of Fg; A, membrane probed with GST-Fbe and anti–GST-Fbe serum; B, as in A, but without GST-Fbe; C, membrane probed with ClfA and anti-ClfA serum; D, as in C, but without ClfA. (b) Western blot of purified Fg chains. Purified α, β, and γ chains (lanes 2, 3, and 4, respectively) from SDS-PAGE were transferred to nitrocellulose filters. (B) The filters were probed with GST-Fbe followed by rat anti–GST-Fbe antibodies and anti-rat IgG antibodies conjugated with HRP. (C) As in B, but without GST-Fbe. (A) Coomassie blue-stained gel. Lanes 1, molecular size markers.

FIG. 7
FIG. 7

Binding of GST-Fbe to the β chain of Fg. Microtiter plate wells were coated with the indicated concentrations (conc.) of the β (□) or γ (■) chain of Fg and then coated with BSA. GST-Fbe was added at 10 μg/ml and incubated. Detection of GST-Fbe capture was done with antibodies raised against GST-Fbe in a rat, followed by HRP-conjugated antibodies against rat IgG. Mean values from quadruplicate experiments are shown, with bars indicating standard errors.

FIG. 8
FIG. 8

Inhibition of S. epidermidis adherence to immobilized Fg by antibodies. To microtiter plate wells coated with Fg, radiolabeled cells of S. epidermidis which had been preincubated with purified IgG at the indicated concentrations (conc.) were added and allowed to adhere. After the removal of nonadherent bacteria, bound bacteria were quantified by scintillation counting. Mean values from triplicate experiments are shown, with bars indicating standard errors. Symbols: □, IgG against GST-Fbe; ○, IgG against Fbe cleaved from GST-Fbe by thrombin; ◊, IgG against GST; ▵, serum taken before immunization.

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