Figure 1.
Adsorption of Factor H and FHR-1 to intact S. aureus.
Cells of S. aureus strain H591 were incubated in human serum (A) or with purified Factor H (B). After extensive washing, bound proteins were eluted, separated by SDS-PAGE and analyzed by Western blotting using polyclonal Factor H antiserum. (A) In the eluted fraction (lane 2) polyclonal Factor H antiserum reacted with three bands of 150, 43 and 37 kDa representing Factor H, FHR-1β and FHR-1α, respectively. The same proteins were also identified in human serum (lane 3). (B) Purified Factor H bound to the bacteria and was detected in the elute fraction (lane 2). The mobility of the marker proteins is indicated. NHS, normal human serum.
Figure 2.
Sbi constructs and antibody binding to Sbi.
(A) Schematic structure of Sbi and of Sbi deletion constructs used in the experiments. The IgG binding domains Sbi-I and Sbi-II are shown in white and the non IgG binding, but Factor H, C3 binding domains (domains III and IV) are shown in black. In addition the position of the signal peptide (SP), the prolin-rich (P) and the tyrosine-rich (Y) regions are indicated. (B) Sbi deletion constructs expressed in E. coli were purified by nickel chromatography, separated by SDS PAGE and identified by silver staining. Based on mobility of the marker proteins the molecular mass of the fragments is as follows. Sbi-E 34 kDa; Sbi-I 9.7 kDa; Sbi-III/IV 17 kDa and Sbi-IV 11 kDa. (C) The IgG binding fragments Sbi-E and Sbi-I mediate unspecific binding of the Factor H∶IgG complex. O binding of purified Factor H is detected to the Non-IgG binding domains Sbi-III/IV and Sbi-IV. Factor H was immobilized and used a positive control. Binding of Factor H to immobilized recombinant constructs Sbi-E, Sbi-I, Sbi-III/IV and Sbi-IV was identified with polyclonal Factor H antiserum (anti-SCR 1–4) and specific mABs B22 and C18 by ELISA.
Figure 3.
Binding of Factor H and FHR-1 to Sbi.
(A) CEWA was used to analyze interaction of host complement regulators with the IgG binding Sbi protein. Sbi-E, Sbi-I, Sbi-III/IV and Sbi-IV were immobilized onto the surface of a microtiter plate and human serum was applied. After extensive washing bound proteins were eluted, separated by SDS-PAGE and identified by Western blotting based on their mobility and their reactivity with mAB C18 that is specific for the C-terminal SCR domain of Factor H and FHR-1. The borrelial Factor H binding CRASP-1, the Factor H/FHR-1 binding CRASP-5 and BSA were used as controls. The mobility of the marker proteins is indicated. (B) Purified Factor H was used in the same assay.
Figure 4.
Binding of Factor H and C3 to Sbi.
(A) Sbi-E, Sbi-I, Sbi-III/IV and Sbi-IV, CRASP-1 and CRASP-5 of Borrelia burgdorferi and BSA were immobilized and incubated with Factor H together with C3b or C3d. After washing bound proteins were eluted from the wells, separated by SDS-PAGE and analyzed by Western blotting using the mAB C18. In the presence of C3b or C3d Factor H bound to Sbi-E, Sbi-III/IV, Sbi-IV, CRASP-1 and to CRASP-5. (B) Sbi-III/IV, Sbi-IV, CRASP-1 and BSA were immobilized and binding of Factor H alone, Factor H and C3b, or Factor H and C3d was assayed by ELISA using polyclonal Factor H antiserum. Again in the presence of C3b or C3d Factor H bound to Sbi-III/IV, to Sbi-IV weakly and to CRASP-1. Factor H alone did bind to CRASP-1. (C) Western blot showing C3 and its degradation products which were used for the CEWA approach. (D) Binding of Sbi to C3 and various degradation. Sbi-E was immobilized and the indicated C3 forms were assayed for binding in the presence of Factor H. Afterward protein were eluted, separated by SDS-Page, transferred to a membrane and C3 fragments were visualized with specific antiserum. C3, C3b and C3d bound to Sbi-E (lanes 1, 3 and 5); and iC3b, C3c and C3a did not bind (lanes 2, 4 and 7). In addition NHS derived C3 bound to Sbi-E (lane 6). BSA was used as negative control (lane 8). (E) Sbi-III/IV was immobilized and binding of Factor H in combination with either C3, C3b, C3d, C3a and C3c, or Factor H alone was measured using polyclonal Factor H antiserum.
Figure 5.
Analyzing Sbi∶C3 interaction by surface plasmon resonance.
(A) Sbi-E was immobilized and C3, C3b and C3d were applied in fluid phase. C3 showed strong association and after removal of the analyte a relative fast dissociation (dash-dotted line). C3b showed a slower association profile as compared to unprocessed C3, however the complex was rather stable (dotted line). In addition C3d showed a prominent association profile and the complex was rather stable (dashed line). (B) Dose-dependent interaction of Sbi-E to immobilized C3d. (C) Effect of C3b on Factor H-Sbi interaction. Sbi-E was immobilized and binding of the single components Factor H or C3b, or of a combination of the two proteins Factor H and C3b was analyzed. (D) Effect of C3d on Factor H-Sbi interaction. Sbi-E was immobilized and binding of the single components Factor H or C3d, or of a combination of the two proteins Factor H and C3d was analyzed. As identical concentrations were used the binding profiles can be directly compared and thus demonstrate stronger interaction of the complex in the presence of C3d as compared to C3b. (E) The various fragments Sbi-E, Sbi-I and Sbi-III/IV were coupled to the surface of a sensor chip and binding of proteins Factor H alone or C3d alone or in combination was analyzed. C3d bound to Sbi-E and Sbi-III/IV with comparable intensities, but did not bind to Sbi-I. The Factor H∶C3d complex showed stronger binding to Sbi-E and Sbi-III/IV, but did not bind to Sbi-I. (F) Subsequent association of Sbi-E, C3d and Factor H to an NTA-chip forming a stable Sbi-E∶C3d∶Factor H complex.
Figure 6.
Localization of the Sbi binding regions within Factor H.
The indicated Factor H deletion constructs were immobilized to the surface of a microtiter plate and the Sbi deletion constructs together with C3b were added. Binding was assayed by ELISA using polyclonal Factor H antiserum. SCRs 15–20 and SCRs 19–20 bound to Sbi-E and Sbi-III/IV but not to Sbi-I. The additional Factor H deletion mutants did not bind to Sbi.
Figure 7.
Factor H retains cofactor activity within the Sbi∶C3b∶Factor H complex.
Sbi-E, Sbi-I, Sbi-III/IV and Sbi-IV, and the borrelial CRASP-1 were immobilized to the surface of a microtiter plate. Factor H and C3b were added and following extensive washing Factor I was applied. After 30 min incubation the mixture was harvested and separated by SDS-PAGE. C3b degradation was analyzed by Western blotting using a polyclonal C3 antiserum. The mobility of the α' and β' chain of C3b and the cleavage products α'68 and α'43 are indicated. Factor H mediated cofactor activity is detected when the complex is coupled to Sbi-E and Sbi-III/IV (lanes 1 and 3). The borrelial CRASP-1 protein and BSA were used as controls (lane 5 and 6). In the absence of Factor H C3b remains intact (lanes 7 and 8).
Figure 8.
Characterization of the tripartite Sbi∶Factor H∶C3 complex.
(A) Polyclonal Factor H antiserum blocks the formation of the Sbi∶C3b∶Factor H complex. Sbi-E, Sbi-I, Sbi-III/IV and Sbi-IV were immobilized onto the surface of a microtiter plate and Factor H and C3b (upper panel) or Factor H, C3b preincubated with polyclonal Factor H antiserum (lower panel) were added. After extensive washing the bound proteins were eluted, separated by SDS-PAGE and Factor H was identified by Western blotting using polyclonal SCR 1–4 antiserum. Polyclonal Factor H antiserum inhibited formation of the tripartite complex. CRASP-1 of B. burgdorferi and BSA were used as controls. (B) Formation of the tripartite Sbi-III/IV∶C3d∶Factor H complex was studied by ELISA. In order to determine the minimal amount of C3d required for increased Factor H binding Sbi-III/IV was immobilized and Factor H in combination with increasing amounts of C3d were added. Binding of Factor H was recorded. (C) Sbi-III/IV and BSA were immobilized, incubated with C3d and increasing dilutions of C3d antiserum was added. After washing Factor H was added and binding was assayed. (D) Saturating concentrations of C3d (0.2 µg) were added to immobilized Sbi-III/IV and BSA. Simultaneously Factor H was preincubated with increasing amounts of C3d and the protein mixture was added to the immobilized Sbi-III/IV∶C3d complexes. Subsequently, Factor H binding was measured.
Figure 9.
Sbi is a complement inhibitor.
(A) Alternative pathway-mediated hemolysis of rabbit erythrocytes was assayed in the presence of increasing amounts of Sbi-E (columns 3–8) or Sbi-III/IV (columns 9–14). Hemolysis of rabbit erythrocytes with ddH2O is shown in column 1; hemolysis in human serum is shown in column 2. (B) Hemolysis of rabbit erythrocytes following incubation in increasing concentrations of complement active human serum in the absence (open squares) or the presence of Sbi-E (filled rhombi). (C) Lysis of rabbit erythrocytes in serum (30%) derived from the indicated species; i.e. human, mouse, guinea pig, dog, goat and sheep. (D) Comparing the effect of Sbi on alternative pathway (AP)−, alternative, classical and lectin pathway (AP+CP/LP)−, as well as classical and lectin pathway (CP/LP)-mediated hemolysis of human serum on rabbit erythrocytes. Background lysis was subtracted for (A) and (C). NHS, normal human serum, ΔFB-HS, Factor B depleted human serum. (E) Sbi inhibits opsonophagocytosis of S. aureus by THP-1 macrophage. Bacteria were incubated in 40% NHS in the presence or absence of Sbi for 20 min. Bacteria were added to activated human THP1 macrophages and at the indicated times bacteria were recovered and the number of live bacteria was quantitated. bacteria were plated on agar and the colony formation was determined (CFU).