Immune evasion by acquisition of complement inhibitors: the mould Aspergillus binds both factor H and C4b binding protein

Abstract

Pathogenic fungi represent a major threat particularly to immunocompromised hosts, leading to severe, and often lethal, systemic opportunistic infections. Although the impaired immune status of the host is clearly the most important factor leading to disease, virulence factors of the fungus also play a role. Factor H (FH) and its splice product FHL-1 represent the major fluid phase inhibitors of the alternative pathway of complement, whereas C4b-binding protein (C4bp) is the main fluid phase inhibitor of the classical and lectin pathways. Both proteins can bind to the surface of various human pathogens conveying resistance to complement destruction and thus contribute to their pathogenic potential. We have recently shown that Candida albicans evades complement by binding both Factor H and C4bp. Here we show that moulds such as Aspergillus spp. bind Factor H, the splicing variant FHL-1 and also C4bp. Immunofluorescence and flow cytometry studies show that the binding of Factor H and C4bp to Aspergillus spp. appears to be even stronger than to Candida spp. and that different, albeit possibly nearby, binding moieties mediate this surface attachment.

Fig. 1

Immunofluorescence analyses of Factor H (FH) binding to Aspergillus species. A. fumigatus (A–D) and A. terreus (E–H). Interactions were shown for Factor H and FHL-1 from plasma using antiserum specific for Factor H (A and E), Factor H/FHL-1 SCRs 1–4 (B and F) and C-terminal Factor H SCRs 19–20 (C and G). D, H: plasma, followed by goat IgG control. The results obtained after staining with rabbit IgG, instead of anti-FH SCRs 1–4 or anti-FH SCRs 19–20 were similar to the control (not shown). Representative examples of quadruplicates experiments are shown.

Fig. 2

Determination of Factor H (FH) binding to the surface of A. fumigatus (A–C) and A. terreus by flow cytometry (D–F). Conidia were incubated with Factor H (A and D), FHL-1 (B and E) or Factor H SCRs 8–20 (C and F). Bound regulators were detected with the specific Factor H antisera. Cells incubated in buffer only were used as control. The results obtained after staining with goat IgG or rabbit IgG, instead of anti-Factor H antiserum or anti-FH SCRs 1–4 or anti-FH SCRs 19–20, respectively, were similar to the buffer control (not shown). Representative examples of quadruplicates experiments are shown.

Fig. 3

Immunofluorescence analyses of C4bp binding to A. fumigatus (A) and A. terreus (C), as detected by using C4bp followed by polyclonal anti-C4bp. (B and D): C4bp, followed by rabbit IgG control. The binding patterns were identical when the C4bp construct without β-chain was used for both antibodies (not shown). Representative examples of quadruplicates experiments are shown.

Fig. 4

Determination by flow cytometry of C4bp surface bound to A. fumigatus (A and C) and A. terreus (B and D). Conidia were incubated with C4bp and monoclonal IgG (A and B) or the C4bp construct without β-chain and the monoclonal anti-C4bp (C and D). The binding patterns were identical when the polyclonal anti-C4bp IgG was used for detection (not shown). Cells incubated in buffer instead of C4bp or its construct was used as control. The application of mouse IgG or rabbit IgG was comparable to the control (not shown). Representative examples of quadruplicates experiments are shown.

Fig. 5

Interference of C4bp binding to A. fumigatus (A) after preabsorption with FHL-1 (B) or Factor H (FH) (C) and influence of FHL-1 or Factor H binding to A. fumigatus (D and F) after preabsorption with C4bp (E and G). Determination by polyclonal anti-C4bp (A–C) or polyclonal anti-Factor H (D–G). Cells incubated in buffer were used as control. Representative examples of quadruplicates experiments are shown.

Fig. 6

Evaluation of Factor H (A and C) or C4bp (B and D) binding to A. fumigatus (A and B) in comparison to C. albicans (C and D). Determination by polyclonal anti-Factor H (A and C) or polyclonal anti-C4bp (B and D). Cells incubated in buffer were used as control. Representative examples of quadruplicates experiments are shown.