Complement Factor H-Related Proteins CFHR2 and CFHR5 Represent Novel Ligands for the Infection-Associated CRASP Proteins of Borrelia burgdorferi

Background: One virulence property of Borrelia burgdorferi is its resistance to innate immunity, in particular to complement-mediated killing. Serum-resistant B. burgdorferi express up to five distinct complement regulator-acquiring surface proteins (CRASP) which interact with complement regulator factor H (CFH) and factor H-like protein 1 (FHL1) or factor H-related protein 1 (CFHR1). In the present study we elucidate the role of the infection-associated CRASP-3 and CRASP-5 protein to serve as ligands for additional complement regulatory proteins as well as for complement resistance of B. burgdorferi. Methodology/Principal Findings: To elucidate whether CRASP-5 and CRASP-3 interact with various human proteins, both borrelial proteins were immobilized on magnetic beads. Following incubation with human serum, bound proteins were eluted and separated by Glycine-SDS-PAGE. In addition to CFH and CFHR1, complement regulators CFHR2 and CFHR5 were identified as novel ligands for both borrelial proteins by employing MALDI-TOF. To further assess the contributions of CRASP-3 and CRASP-5 to complement resistance, a serum-sensitive B. garinii strain G1 which lacks all CFH-binding proteins was used as a valuable model for functional analyses. Both CRASPs expressed on the B. garinii outer surface bound CFH as well as CFHR1 and CFHR2 in ELISA. In contrast, live B. garinii bound CFHR1, CFHR2, and CFHR5 and only miniscute amounts of CFH as demonstrated by serum adsorption assays and FACS analyses. Further functional analysis revealed that upon NHS incubation, CRASP-3 or CRASP-5 expressing borreliae were killed by complement. Conclusions/Significance: In the absence of CFH and the presence of CFHR1, CFHR2 and CFHR5, assembly and integration of the membrane attack complex was not efficiently inhibited indicating that CFH in co-operation with CFHR1, CFHR2 and CFHR5 supports complement evasion of B. burgdorferi

Human factor H-related protein 2 (CFHR2) regulates complement activation.

Mutations and deletions within the human CFHR gene cluster on chromosome 1 are associated with diseases, such as dense deposit disease, CFHR nephropathy or age-related macular degeneration. Resulting mutant CFHR proteins can affect complement regulation. Here we identify human CFHR2 as a novel alternative pathway complement regulator that inhibits the C3 alternative pathway convertase and terminal pathway assembly. CFHR2 is composed of four short consensus repeat domains (SCRs). Two CFHR2 molecules form a dimer through their N-terminal SCRs, and each of the two C-terminal ends can bind C3b. C3b bound CFHR2 still allows C3 convertase formation but the CFHR2 bound convertases do not cleave the substrate C3. Interestingly CFHR2 hardly competes off factor H from C3b. Thus CFHR2 likely acts in concert with factor H, as CFHR2 inhibits convertases while simultaneously allowing factor H assisted degradation by factor I

CFHR2 inhibits terminal pathway activation.

<p>(<b>A</b>) CFHR2 (blue column), factor H (gray column) and vitronectin (Vn, gray column) were preincubated with purified TCC components C7, C8,C9 and added to C5b-6 loaded SRBC. TCC mediated erythrocyte lysis was monitored by measuring the absorbance of the supernatant at 414 nm. Reduced absorbance correlates with inhibition of the TCC formation on SRBC. (<b>B</b>) CFHR2 inhibited TCC formation via the N-terminal SCRs 1–2 in a dose dependent manner. CFHR2 proteins were preincubated with TCC components and lysis of SRBC was measured. Data in (<b>A</b>) and (<b>B</b>) represent mean values ± SD of three independent experiments (^***p≤0.001).</p

Scematic overview of CFHR2 functions in regulation of the AP.

<p>CFHR2 inhibits the amplification loop by inhibiting C3 cleavage by C3 convertases and acts on the assembly of the terminal complement complex.</p

CFHR2 inhibits the AP C3 convertase.

<p>CFHR2 inhibited <i>in vitro</i> assembled AP C3 convertase as measured by C3a generation. The corresponding densitometric analysis of the C3a bands of the Western blot is shown. C3a generation by C3 convertase alone was normalized to 100%. A representative analysis of three experiments is shown.</p

CFHR2 regulates AP complement activation.

<p>(<b>A</b>) CFHR2 inhibits AP activation in NHS. (<b>B</b>) CFHR2 had no effect on CP activation in NHS. TCC deposition in untreated NHS was set as 100%. C4bp is a classical pathway inhibitor. (<b>C</b>) CFHR2 and CFHR2/1–2 but not CFHR2/3–4 inhibited C3b deposition. (<b>D</b>) C3a generation in NHS activated via AP (ELISA). C3b depositon in untreated NHS was set as 100%. (<b>E</b>) CFHR2 together with factor H increased inhibition of C3b deposition in NHS as compared to CFHR2 or factor H alone. Data in (<b>A–E</b>) represent the mean values ± SD of three independent experiments (^*p≤0.05, ^***p≤0.001). Control (ctrl) indicates binding of the detection Ab to the plate.</p

CFHR2 exerts neither decay acceleration nor cofactor activity.

<p>(<b>A</b>) CFHR2 can not dissociate factor B from the <i>in vitro</i> assembled convertase C3bBb, but factor H efficiently decays factor Bb fragment from C3b. (<b>B</b>) CFHR2 does not influence cleavage of C3b bound factor B by factor D. CFHR2 preincubated NHS, which was activated by surface immobilized IgM via the AP and Ba cleavage was followed by ELISA. A representative experiment is shown. (<b>C</b>) CFHR2 alone lacks cofactor activity for factor I to degrade C3b (lanes 6–8). CFHR2 does not interfere with factor H cofactor activity (lanes 2–5). Increasing amounts of CFHR2 (1–100 µg/ml) were incubated with C3b and factor I in the presence or absence of factor H. Reaction mixtures were separated by SDS PAGE and immunoblotted using a C3 polyclonal antibody.</p

CFHR2 binds via the C-terminus to C3b and C3d.

<p>(<b>A</b>) CFHR2 SCR domains are aligned against the highly related SCR domains of CFHR1 and factor H. The number above each SCR indicates the identity to the corresponding domain in CFHR1 respectively factor H. The regulatory region (yellow), SCRs 6–7 (green), and the surface recognition site (blue) of factor H are marked. (<b>B</b>) CFHR2 binds to C3b, C3dg and C3d, but not to C3c. In parallel factor H binding was determined. Equimolar amounts of CFHR2 and factor H were used. Data represent mean values ± SD of four independent ELISA experiments. Background binding of the antibodies to ligand C3b alone (lig ctrl) or without ligand C3b (Ab ctrl) is shown. (<b>C</b>) CFHR2 binds via the two C-terminal domains to C3b and C3d. The proteins were used in equimolar amounts. Data represent mean values ± SD of three independent experiments. Background binding of the antibodies to ligand C3b or C3d alone (ctrl) is shown.</p