Article Figures & Data
Figures
- FIG. 1.
Immunofluorescence analysis of C4BP and factor H bound to candida cells and hyphal forms. Cells and hyphal forms of C. albicans were incubated in NHS-EDTA. The samples were washed and incubated further with a mouse MAb (MAb70) detecting CCP1 of the C4BP α-chain and a secondary rabbit anti-mouse antibody labeled with FITC (A and B) or Alexa 488, producing red fluorescence (C, E, and G). Factor H was detected with an anti-mouse serum and a secondary goat anti-mouse antiserum labeled with Alexa 647 for green fluorescence (C, F, and G). Parallel binding of C4BP and factor H is shown in panels C and G.
- FIG. 2.
Binding of C4BP to yeast and hyphal forms of C. albicans determined by flow cytometry. Cells incubated in NHS or NHS depleted of C4BP (A) and hyphal forms incubated in NHS (B) or purified C4BP (C) were used. The samples were washed and incubated with a C4BP-specific MAb70. After being washed, bound antibodies were detected with an FITC-conjugated rabbit anti-mouse antibody and stained cells were subjected to flow cytometry. Cells incubated in buffer were used as controls.
- FIG. 3.
Binding of C4BP to intact cells and hyphae of C. albicans. Cells or hyphae (109) were incubated in NHS and washed extensively, and bound proteins were eluted with 3 M KSCN. The wash (W) and eluate (E) fractions were separated by SDS-PAGE under reducing conditions and analyzed by Western blotting with anti-C4BP MAb104 detecting CCP1 of the C4BP α-chain. A control where C4BP from NHS is detected with the same antiserum is shown in lane 5. The mobility of the size markers is indicated on the left and indicated in kilodaltons.
- FIG. 4.
Binding of C4BP to intact cells of different species and strains of Candida. Cells (109) of the indicated species were incubated in NHS and washed extensively, and bound proteins were eluted with 3 M KSCN. The wash (W) and eluate (E) fractions were separated by SDS-PAGE under reducing conditions and analyzed by Western blotting with MAb104 detecting CCP1 of the C4BP α-chain. The mobility of the size markers is indicated on the left and indicated in kilodaltons.
- FIG. 5.
Dose-dependent binding of C4BP to C. albicans and inhibition of binding with different antibodies. ELISA plates were coated with C. albicans hyphal forms. Purified native or recombinant C4BP was added to the cells in increasing concentrations. BSA was used as a control (A). For inhibition of binding of C4BP to the C. albicans hyphae, the indicated antibodies were added to the reaction mixture (B). After being washed, bound proteins were detected with a polyclonal anti-C4BP antibody. A positive control, where hyphae were incubated with C4BP alone, was considered to have 100% binding. The results show the mean values with indicated standard deviations of the results from five independent experiments. OD 420, optical density at 420 nm.
- FIG. 6.
Localization of the binding region in C4BP. C. albicans hyphae were coated on ELISA plates, and purified C4BP, intact rC4BP, and recombinant deletion mutants of C4BP were added. After being washed, bound proteins were detected with a polyclonal anti-C4BP antibody. The individual deletion mutants are represented as follows: ΔCCP1, D1; ΔCCP2, D2; ΔCCP3, D3; ΔCCP4, D4; ΔCCP5, D5; ΔCCP6, D6; ΔCCP7, D7; ΔCCP8, D8. The results show the mean values with indicated standard deviations of the results from four independent experiments.
- FIG. 7.
Effect of salt on the binding of C4BP to hyphae of C. albicans. ELISA plates were coated with C. albicans hyphal forms and native C4BP or rC4BP was added to the buffer containing the indicated concentrations of NaCl. After being washed, bound proteins were detected with a polyclonal anti-C4BP antibody.
- FIG. 8.
Inhibition of binding of C4BP to C. albicans cells with FHL-1, factor H, and C4BP (A) and with C4b, heparin, and BSA (B). C. albicans hyphal forms (109/assay) were used in direct binding assays. Hyphae were incubated with 125I-C4BP at the indicated concentrations of purified proteins in the presence or absence of the indicated inhibitors. After 20 min of incubation, the bound protein was separated from the unbound protein by centrifugation through a sucrose column. Binding is expressed as a percentage of bound protein compared to the total protein input. The 100% value is indicated by the dotted line.
- FIG. 9.
Cofactor activity of surface-bound C4BP. C. albicans cells were preincubated in NHS (lanes 2 and 3), serum depleted of C4BP (lane 4), C4BP purified from human serum (lane 5), or rC4BP (lane 6). The substrate of C4BP-purified C4b is separated in lane 1, and the three chains of the protein (α, β, and γ) are indicated. After extensive washing, biotin-labeled C4b and factor I were added, the mixture was incubated for 30 min, the products were separated by SDS-PAGE, and C4b and its cleavage products were visualized by luminescence. Inactivation of C4b is observed by the disappearance of the α′ band and the appearance of the 45-kDa C4d and the 20-kDa α3 fragment, as seen in the positive control in lane 2. Cofactor activity of C4BP bound from human serum was detected (lanes 2 and 3), but human serum depleted of C4BP showed no cofactor activity (lane 4). Purified C4BP and rC4BP used at lower concentrations also showed cofactor activity (lanes 5 and 6).
- FIG. 10.
C4BP attached to the C. albicans surface mediates adhesion to human endothelial cells. HUVEC cells grown on microtiter plates were incubated with C. albicans cells preincubated in native C4BP purified from human plasma or rC4BP. The results show the mean values of the results from three independent experiments. The standard deviations are indicated. WT, wild type.
