Complement C1s cleaves PfEMP1 at interdomain conserved sites inhibiting Plasmodium falciparum cytoadherence

Abstract

Cytoadhesion of Plasmodium falciparum-infected erythrocytes (IEs) to the endothelial lining of blood vessels protects parasites from splenic destruction, but also leads to detrimental inflammation and vessel occlusion. Surface display of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) adhesion ligands exposes them to host antibodies and serum proteins. PfEMP1 are important targets of acquired immunity to malaria, and through evolution, the protein family has expanded and diversified to bind a select set of host receptors through antigenically diversified receptor-binding domains. Here, we show that complement component 1s (C1s) in serum cleaves PfEMP1 at semiconserved arginine motifs located at interdomain regions between the receptor-binding domains, rendering the IE incapable of binding the two main PfEMP1 receptors, CD36 and endothelial protein C receptor (EPCR). Bioinformatic analyses of PfEMP1 protein sequences from 15 P. falciparum genomes found the C1s motif was present in most PfEMP1 variants. Prediction of C1s cleavage and loss of binding to endothelial receptors was further corroborated by testing of several different parasite lines. These observations suggest that the parasites have maintained susceptibility for cleavage by the serine protease, C1s, and provides evidence for a complex relationship between the complement system and the P. falciparum cytoadhesion virulence determinant.

Keywords: C1s; EPCR; PfEMP1; cytoadhesion; malaria.

Fig. 1.

Serum fractions B11 and C3 inhibit IT4var19 IE binding to brain endothelial cells. (A) Chromatographic fractionation of serum by size exclusion chromatography. IgG-depleted serum was run on a Superdex 200 Increase 10/300 GL column. Protein fractions were eluted using a linear gradient and the absorbance (mAbs) monitored at 280 nm. The arrows show the binding inhibitory fractions B11 and C3. (B) Binding of IT4var19 IEs to HBEC-5i in binding medium with 10% human serum, IgG-depleted human serum, or serum fractions (A6-C6). A no-serum control was included. Each data point is from two independent experiments done in duplicate, and the mean and SEM are shown for each fraction. Fractions B11 and C3 inhibited the binding the most. These fractions were run on MS/MS, and the components were run to determine their effect on binding as shown in C to identify potential inhibitors for binding. (C) Binding of IT4var19 IEs to HBEC-5i in binding medium with proteins identified by MS/MS in the serum inhibitory B11 and C3 fractions (attractin, C1s, C4b, factor H, IgD, ITIH1, ITIH2, and kappa light chain). Data are mean ± SEM, and each data point is from an independent experiment. **P < 0.01; ***P < 0.001.

Fig. 2.

C1s inhibits binding of IT4var19 IEs to EPCR but does not reverse binding of bound IEs. (A) Binding of IT4var19 IEs to EPCR in the absence or presence of C1s (0.03 to 30 μg/mL). (B) No evidence of the reversal of IT4var19 IEs bound to EPCR in the presence of C1s (30 to 100 μg/mL). The mean and SEM are shown, and each data point is from an independent experiment. All mean values were log-transformed, and a two-tailed unpaired t test was conducted to compare C1s-treated groups to the positive control (EPCR only). **P < 0.01; ***P < 0.001.

Fig. 3.

C1s cleaves the native PfEMP1 on the surface of IT4var19 IEs. Staining of (A) IT4var19 IEs, (B) C1s-treated IT4var19 IEs, (C) FCR3 (VAR2CSA), and (D) C1s-treated FCR3 (VAR2CSA) with rabbit polyclonal IgG to IT4var19 NTS.DBLα or rat polyclonal IgG to FCR3 full-length VAR2CSA. IEs were identified by DAPI staining of parasite nuclei (blue). IT4var19 PfEMP1 staining of the IEs is shown in green, and FCR3 VAR2CSA PfEMP1 staining is shown in red.

Fig. 4.

C1s inhibits binding of IT4var20 IE to EPCR and IT4var13 IE binding to CD36 but not ICAM-1. (A) Binding of IT4var20 IEs to EPCR in the absence or presence of C1s (100 μg/mL). Recombinant EPCR (rEPCR) was included as a control and indicator of how well C1s treatment abrogated IT4var20 binding to EPCR. (B) Binding of IT4var13 IEs to CD36 or ICAM-1 in the absence or presence of C1s (100 μg/mL). In both binding assays, bovine serum albumin (BSA) was included as a negative control. The mean and SEM are shown, and each data point is from an independent experiment. For statistical testing, all mean values were log-transformed, and a two-tailed unpaired t test was conducted to compare experimental groups to the positive control (EPCR only). (C) Var gene transcript levels relative to the housekeeping control gene, seryl-t-transferase, confirming the major var types being expressed as IT4var20 and IT4var13. *P < 0.05; **P < 0.01; ns, not significant.

Fig. 5.

Analysis of observed and predicted C1s cleavage sites in PfEMP1. (A) SDS/PAGE showing C1s cleaves the full-length recombinant ectodomains of IT4var13 and IT4var20 PfEMP1 into two major bands (arrows, numbered 1–4). (B) Protein schematics of the IT4var13 and IT4var20 ectodomains showing the C1s cleavage products (black arrows, above the protein schematic from A). The boxed Insets show the interdomain sequences where C1s cleavage sites were identified in IT4var13 and IT4var20 by LS/MS and N-terminal protein sequencing analyses. C1s cleaves at one site in IT4var13 and three sites in IT4var20 (red arrows) at residues (large amino acid letters) resembling C1s-motifs. Of the four C1s-like sequence motifs found in IT4var20, three (red arrows) in B were experimentally confirmed and the fourth site (green arrow) in B may also have been cleaved but could not be validated due to its presence in a small peptide product lost in gel electrophoresis. (C) C1s cleavage sites in PfEMP1 resemble previously identified semiconserved sequence motifs, known as homology blocks HB49, HB134, and HB241 (11), found in the otherwise low sequence complexity interdomain regions of PfEMP1. (D) Reanalysis of conserved sequence motifs in PfEMP1 interdomains from 15 long-read sequenced P. falciparum genomes using MEME confirms the presence of C1s-like cleavage motifs (sequence LOGOs shown at Left). The right side shows 275 representative full-length PfEMP1 from 15 PacBio long-read sequenced P. falciparum genomes (44). The different PfEMP1 are stacked from Top to Bottom in three columns and aligned by their domain composition (not scaled to sequence length). Each domain type is color-coded, and white gaps between domains represent interdomain regions, which vary from 10 to 225 amino acids in length. Interdomain regions containing one or more predicted C1s-like cleavage site(s) (MEMEs 1 and 2) are indicated by red crosses. ATS, acidic terminal segment or cytoplasmic tail; NTS, N-terminal segment at the beginning of protein.

Fig. 6.

Analysis of C1s-treated recombinant PfEMP1. (A) SDS/PAGE gel showing two representative PfEMP1 CIDR domains (EPCR-binding IT4var19 CIDRα1 and CD36-binding CIDRα3) and two EPCR-binding head-structure domain complexes (PFD1235w [NTSA-DBLα1-CIDRα1] and IT4var20 [NTSA-DBLα1-CIDRα1]) before and after treatment with 50 nM C1s at 1:1 molar ratio for 1 h at 37 °C. No proteolysis was seen. (B) ELISA data showing the effect of 50 nM C1s pretreatment on binding of recombinant PfEMP1 proteins to coated EPCR, CD36, or ICAM-1 (filled boxes) versus EPCR binding to coated recombinant PfEMP1 (hashed boxes). Whereas C1s treatment reduced binding activity of full-length PfEMP1 ectodomains, recombinant PfEMP1 head structure domains are insensitive to C1s treatment, indicating that C1s cleavage sites are located in the interdomain regions, consistent with MS analysis. As a control, C1s treatment of EPCR did not affect its binding to full-length PfEMP1 ectodomains (hashed boxes).