Antibody response to fibronectin-binding adhesin FnbpA in patients with Staphylococcus aureus infections

Abstract

We have analyzed antibody reactivity to a fibronectin-binding microbial surface component that recognizes adhesive matrix molecules (MSCRAMM) in blood plasma collected from patients with staphylococcal infections. All patients had elevated levels of anti-MSCRAMM antibodies compared to those of young children who, presumably, had not been exposed to staphylococcal infections. The anti-MSCRAMM antibodies preferentially reacted with the ligand-binding repeat domain of the adhesin. However, these antibodies did not inhibit fibronectin binding. Essentially, all patients had antibodies which specifically recognized the fibronectin-MSCRAMM complex but not the isolated components. Epitopes recognized by these anti-ligand-induced binding sites antibodies were found in each repeat unit of the MSCRAMM. These results demonstrate that staphylococci have bound fibronectin some time during infection and that each repeat unit in the MSCRAMM can engage in ligand binding. Furthermore, our previously proposed model, suggesting that an unordered structure in the MSCRAMM undergoes a conformational change upon ligand binding (K. House-Pompeo, Y. Xu, D. Joh, P. Speziale, and M. Höök, J. Biol. Chem. 271:1379-1384, 1996), is presumably operational in patients during infections.

FIG. 1

Schematic representation of recombinant proteins containing fragments of MSCRAMM FnbpA. All the segments were expressed in fusion with GST carrier. Fn-binding repeat units are indicated by Du, D1, D2, D3, and D4; S, signal sequence; W, cell wall spanning region; M, membrane-spanning region.

FIG. 2

Mapping of epitopes in GST-FnbpA fusion protein derivatives. GST-FnbpA polypeptides GST-UA, GST-UB, GST-UC and GST-Du1234 (1 μg in 100 μl) were immobilized in microtiter wells and probed with IgG preparations (2 μg in 100 μl). Bound antibody was detected as described in Materials and Methods. Pooled sera from 2-year-old children were used as the control (∗). Results are expressed as means ± standard deviations (error bars). Each sample point was done in duplicate.

FIG. 3

Antibody preparations from patients do not inhibit Fn binding to FnbpA. (A) Cells of S. aureus Cowan 1 (10⁸) were incubated with ¹²⁵I-labeled N29 (5 × 10⁴) in the presence of 50 μg of each IgG preparation, and the radioactivity bound to the cells was quantitated. (B) GST-Du1234 was immobilized onto microtiter wells (1 μg/well) and probed with 8 × 10⁴ cpm of ¹²⁵I-labeled N29 in the presence of 2 μg of each IgG preparation. After being washed extensively with PBS containing 0.1% Tween 20, the plates were incubated with 200 μl of 2% SDS at 37°C for 30 min, and the radioactivity released from the wells was quantitated with a gamma counter. Controls are reported when bacteria or GST-Du1234-coated plates were incubated without antibodies (∗) or with antibodies isolated from pooled sera from young children (∗∗). Data are reported as means ± standard deviations (error bars).

FIG. 4

Specific formation of LIBS epitopes induced by Fn. GST-Du1234 recombinant protein was immobilized onto microtiter wells (1 μg in 100 μl) and probed with each IgG preparation (2 μg in 100 μl) in the presence of Fn, the N-terminal Fn fragment (N29), collagen, or fibrinogen. Binding of antibodies to the recombinant protein in absence of ligand is also shown. Bound antibody was detected as described in Materials and Methods. Bars represent means ± standard deviations with duplicate testing. Controls (∗) represent immobilized GST-Du1234 incubated with antibodies from young children.

FIG. 5

Screening of the whole panel of IgG preparations for anti-LIBS activity. Microtiter wells were coated with GST-Du1234 (1 μg in 100 μl) and incubated with each IgG preparation (2 μg in 100 μl) either in the absence (□) or presence (■) of 0.5 μg of N29. Bound antibody was detected as described in Materials and Methods. Data are expressed as means ± standard deviations (error bars) of duplicate testing.

FIG. 6

ELISA of anti-LIBS antibodies isolated by affinity chromatography on GST-Du1234. A preparation of IgG obtained from patient 5 was fractionated onto a GST-Du1234–Sepharose column as described in Materials and Methods. GST-Du1234 protein was immobilized onto microtiter wells (1 μg in 100 μl) and assayed by ELISA with increasing concentrations of IgG from the flowthrough of the column (B) or with antibodies bound and eluted from the affinity matrix (C). Reactivity of the unfractionated IgG preparation is shown in panel A. Assays were carried out in the absence or presence of N29. Bound antibody was detected as described in Materials and Methods.

FIG. 7

Localization of LIBS epitopes in the individual Fn-binding motifs. The Fn-binding motifs Du, D1, D2, D3, and D4 fused to the GST carrier were immobilized onto microtiter wells (1 μg in 100 μl) and probed with the panel of IgG preparations either in the absence (□) or presence (■) of the N-terminal fragment (N29) of Fn. Bound antibody was detected as described in Materials and Methods.

FIG. 8

Localization of LIBS epitopes in the subfragments of the D3 motif. Microtiter wells were coated with recombinant subfragments GST-D3_1–21 and GST-D3_15–36 (1 μg in 100 μl) and incubated with IgG preparations in either the absence (□) or presence (■) of N29 (see Results). Bound antibody was detected as described in Materials and Methods.