Hijacking the human complement inhibitor C4b-binding protein by the sporozoite stage of the Plasmodium falciparum parasite

Abstract

The complement system is considered the first line of defense against pathogens. Hijacking complement regulators from blood is a common evasion tactic of pathogens to inhibit complement activation on their surfaces. Here, we report hijacking of the complement C4b-binding protein (C4bp), the regulator of the classical and lectin pathways of complement activation, by the sporozoite (SPZ) stage of the Plasmodium falciparum parasite. This was shown by direct binding of radiolabeled purified C4bp to live SPZs as well as by binding of C4bp from human serum to SPZs in indirect immunofluorescence assays. Using a membrane-bound peptide array, peptides from the N-terminal domain (NTD) of P. falciparum circumsporozoite protein (CSP) were found to bind C4bp. Soluble biotinylated peptide covering the same region on the NTD and a recombinantly expressed NTD also bound C4bp in a dose-dependent manner. NTD-binding site on C4bp was mapped to the CCP1-2 of the C4bp α-chain, a common binding site for many pathogens. Native CSP was also co-immunoprecipitated with C4bp from human serum. Preventing C4bp binding to the SPZ surface negatively affected the SPZs gliding motility in the presence of functional complement and malaria hyperimmune IgG confirming the protective role of C4bp in controlling complement activation through the classical pathway on the SPZ surface. Incorporating the CSP-C4bp binding region into a CSP-based vaccine formulation could induce vaccine-mediated immunity that neutralizes this immune evasion region and increases the vaccine efficacy.

Keywords: C4b binding protein; Plasmodium; circumsporozoite protein; complement evasion; sporozoites.

Figure 1

Binding of C4bp to SPZs. (A) Radiolabeled C4bp was incubated with Fusobacterium necrophorum (FN) bacteria (positive control) and with different amounts of P. falciparum SPZs or insoluble fraction of mosquito salivary glands (SG) (negative control). (B) Binding of serum C4bp to SPZs fixed to the wells of microtiter plates was tested in a whole-SPZ ELISA. Bound C4bp was detected using sheep polyclonal anti-human C4bp, donkey anti-sheep IgG/HRP. Comparisons between groups were analyzed by ANOVA followed by post-hoc Tukey’s multiple pairwise comparisons test. The asterisks represent a significant difference between groups. (C) Binding of serum C4bp to the SPZ surface in indirect immunofluorescence assays (IFA). SPZs were incubated with either HIS (A, C) or VBS++ (B, D) and subjected to IFA. (A, B) The α-chain of C4bp (C4bp-α) was labeled on the SPZ surface using primary antibodies (1°) against C4bp-α and detected with Alexa Fluor488-labeled (green) secondary antibody (2°). (C, D) As a secondary antibody control, SPZs were incubated with secondary antibody in the absence of primary antibody. Nuclei were detected by DAPI staining (blue). Asterisks indicate the p values: p < 0.05 = *; p < 0.01 = **; p < 0.001 = ***, and p < 0.0001 = ****. Scale bar, 3 µm.

Figure 2

Binding of C4bp to CSP peptides. (A) Schematic representation of P. falciparum CSP. SP, signal peptide; NTD, N-terminal domain; CTD, C-terminal domain; RI, region I; RII+, region II plus; GPI, GPI anchor sequence; hyphen symbols denote locations of C4bp binding signals. (B) PepSpot membrane showing C4bp binding signals to CSP peptides. Circles drawn on membrane indicates locations of the 92 overlapping peptides. (C) C4bp binding signals delineated by rectangles on the CSP amino acid sequence. Dash, single, and dash long line under specific amino acid sequences indicates SP, RI and RII+ regions, respectively. (D) Synthesized biotinylated CSP peptides delineated on the FL-CSP polypeptide sequence. Number 1, 3, and 5 above the sequence denote Pep1, 3, and 5 that showed binding to C4bp in the PepSpot assay, respectively, and number 2 and 4 denote the control peptides Pep2 and 4, respectively.

Figure 3

Binding of CSP biotinylated peptides to C4bp and CCP1-2 domains of the C4bp α-chain. (A) Binding of CSP Pep1, 3 and 5 (representing the 3 positive binding signals observed in the peptide array analysis) and Pep2 and 4 (negative control peptides) to C4bp. (B) Binding of the CSP peptides to CCP1-2 domains. (C) Inhibition of Pep1 binding to C4bp by CCP1-2 domains.

Figure 4

A dose response analysis of NTD-H binding to C4bp and to the alternative pathway regulator FH. The binding experiment was done by ELISA. Comparison between the two data sets was analyzed by the t test. p < 0.01 = **.

Figure 5

Native CSP from SPZs interacts with C4bp. SPZs were incubated with either HIS or VBS⁺⁺. SPZs were then thoroughly washed and lysed with a lysis buffer. C4bp bound to SPZ surface proteins was immunoprecipitated from the lysate using anti-C4bp and immunoprecipitated proteins were subjected to SDS-PAGE under reducing conditions and Western blotting. The horizontal line represents the membrane separation point for the membrane pieces probed with anti-C4bp (upper piece) and anti-CSP antibodies (lower piece). HIS: heat inactivated serum; Input: starting material; SN: supernatant; IP: immunoprecipitates.

Figure 6

C4bp enhances SPZ resistance to malaria hyperimmune IgG -mediated complement activity. Salivary gland SPZs were pre-incubated with NHS or NHSΔC4bp (C4bp-depleted NHS), both in the presence of hyperimmune IgG, NHSΔC4bp supplemented with physiological level of C4bp in addition to hyperimmune IgG, HIS or HISΔC4bp, both in the presence of hyperimmune IgG and subjected to a gliding motility assay. The number of fluorescent circles produced by each SPZ was counted for a total of 100 SPZ per test condition and the percentages of SPZs with 1, 2-10 or >10 trail circles are shown. Comparisons between groups were analyzed by ANOVA followed by post-hoc Tukey’s multiple pairwise comparisons test. p < 0.001 = ***, p < 0.0001 = **** and not significant = ns.

Figure 7

Variable quantities of NTD of CSP are displayed on the surface of SPZs in the in-vitro assays. CSP (green) displayed on the surface of SPZs was stained with antibodies generated against: (A) the NTD, (B) the full length CSP (FL-CSP) and (C) the CSP repeats. Nuclei were stained with by DAPI (blue). Scale bar, 10 µm.

Figure 8

Antibody response against CSP peptides. Mice were immunized with either FL-CSP or NTD-GST. (A) Sequences of CSP peptides tested in the ELISA assays delineated on the amino-acid sequence of CSP. (B) Antibody levels in a serum pool of 4 mice immunized with FL-CSP against CSP peptides representing the NTD (Pep1, Pep2, Pep3, and Pep4), junction region (NPDP15 and NPDP19), central repeat region (NANP24) and the CTD (Pep5). (C) Antibody levels in a serum pool of 4 mice immunized with NTD-GST.