The opportunistic human pathogenic fungus Aspergillus fumigatus evades the host complement system

Abstract

The opportunistic human pathogenic fungus Aspergillus fumigatus causes severe systemic infections and is a major cause of fungal infections in immunocompromised patients. A. fumigatus conidia activate the alternative pathway of the complement system. In order to assess the mechanisms by which A. fumigatus evades the activated complement system, we analyzed the binding of host complement regulators to A. fumigatus. The binding of factor H and factor H-like protein 1 (FHL-1) from human sera to A. fumigatus conidia was shown by adsorption assays and immunostaining. In addition, factor H-related protein 1 (FHR-1) bound to conidia. Adsorption assays with recombinant factor H mutants were used to localize the binding domains. One binding region was identified within N-terminal short consensus repeats (SCRs) 1 to 7 and a second one within C-terminal SCR 20. Plasminogen was identified as the fourth host regulatory molecule that binds to A. fumigatus conidia. In contrast to conidia, other developmental stages of A. fumigatus, like swollen conidia or hyphae, did not bind to factor H, FHR-1, FHL-1, and plasminogen, thus indicating the developmentally regulated expression of A. fumigatus surface ligands. Both factor H and plasminogen maintained regulating activity when they were bound to the conidial surface. Bound factor H acted as a cofactor to the factor I-mediated cleavage of C3b. Plasminogen showed proteolytic activity when activated to plasmin by urokinase-type plasminogen activator. These data show that A. fumigatus conidia bind to complement regulators, and these bound host regulators may contribute to evasion of a host complement attack.

FIG. 1.

Binding of factor H and FHL-1 to A. fumigatus conidia. Factor H, FHR-1, and FHL-1 were detected with polyclonal anti-factor H antiserum (for factor H and FHR-1) and mouse MAb directed against FHL-1. Conidia were incubated with either NHS or purified factor H and washed, and bound proteins were eluted with a chaotrope solution. Proteins in wash (W) and eluate (E) fractions were separated by SDS-PAGE, transferred to a membrane, and analyzed by Western blot analysis.

FIG. 2.

Binding of factor H to A. fumigatus conidia. Conidia were incubated with purified factor H, and binding was visualized by immunofluorescence with mouse MAb directed against the C terminus of factor H and an Alexa 488-coupled secondary antibody (green). The cell wall was stained with calcofluor (blue). (A) Immunofluorescence of conidia. (B) Three-dimensional rendering of the fluorescence of the conidia in panel C. (C) Higher magnification of two conidia. Note the patchy surface distribution of the fluorescence. (D) Negative control without factor H. (E) Negative control without the first antibody.

FIG. 3.

Localization of the binding site of factor H. Conidia were incubated with purified factor H mutant proteins, and bound proteins were eluted with a chaotrope solution. After SDS-PAGE separation, Western blot analysis was performed with polyclonal factor H antiserum. Incubation experiments were performed in the presence and absence of blocking solutions and showed similar results. (A) Binding of mutant proteins consisting of N-terminal SCRs; (B) binding of mutant proteins consisting of internal and C-terminal SCRs.

FIG. 4.

Heparin inhibits the binding of factor H to conidial surfaces. (A) A. fumigatus conidia (2 × 10⁹) were incubated with purified factor H (10 μg) in the presence or absence of heparin (800 IU). Bound proteins were eluted with a chaotrope solution, and wash (W) and eluate (E) fractions were subjected to SDS-PAGE and Western blot analysis. Fifty nanograms of purified factor H was used as a control. (B) Factor H was preincubated with increasing concentrations of heparin (0 to 640 IU/ml) and subsequently incubated with conidia (5 × 10⁶) in microtiter plates with a filter bottom. The binding of factor H to conidia was detected by enzyme-linked immunosorbent assay with a polyclonal antiserum directed against factor H.

FIG. 5.

Binding of plasminogen to A. fumigatus conidia. Conidia were incubated with NHS and washed, and bound proteins were eluted with a chaotrope solution. Wash (W) and eluate (E) fractions were subjected to SDS-PAGE and Western blot analysis. Plasminogen was detected with polyclonal antiplasminogen antiserum. Lane NHS, 1:10 diluted serum.

FIG. 6.

Binding of factor H and plasminogen to different developmental stages of A. fumigatus. Conidia, swollen conidia, and young hyphae were incubated with NHS and washed, and bound proteins were eluted with a chaotrope solution. Samples were separated by SDS-PAGE, and Western blot analysis was performed for the detection of factor H and FHR-1 (A), FHL-1 (B), and plasminogen (C). NHS as well as purified factor H and FHL-1 served as controls.

FIG. 7.

Activity of surface-bound host proteins. (A) Surface-attached factor H mediated complement-regulating activity. Conidia of A. fumigatus were incubated in purified factor H. After extensive washing, factor I (lane 1) or both factor I and C3b (lane 2) were added. Conidia incubated with factor I and C3b in the absence of factor H were used in lane 3. After incubation for 1 h, the supernatant was separated by SDS-PAGE and analyzed by Western blotting. The positions of the α′ and β chains of C3b are indicated as well as the positions of the α′ 68-kDa and the α′ 43-kDa cleavage products. (B) Plasminogen attached to the conidial surface is accessible to uPA for activation and mediates the cleavage of the chromogenic substrate S-2251. Cleavage of the substrate results in an increase in absorbance at 405 nm.