VAR2CSA signatures of high Plasmodium falciparum parasitemia in the placenta

Abstract

Plasmodium falciparum infected erythrocytes (IE) accumulate in the placenta through the interaction between Duffy-binding like (DBL) domains of parasite-encoded ligand VAR2CSA and chondroitin sulphate-A (CSA) receptor. Polymorphisms in these domains, including DBL2X and DBL3X, may affect their antigenicity or CSA-binding affinity, eventually increasing parasitemia and its adverse effects on pregnancy outcomes. A total of 373 DBL2X and 328 DBL3X sequences were obtained from transcripts of 20 placental isolates infecting Mozambican women, resulting in 176 DBL2X and 191 DBL3X unique sequences at the protein level. Sequence alignments were divided in segments containing combinations of correlated polymorphisms and the association of segment sequences with placental parasite density was tested using Bonferroni corrected regression models, taking into consideration the weight of each sequence in the infection. Three DBL2X and three DBL3X segments contained signatures of high parasite density (P<0.003) that were highly prevalent in the parasite population (49-91%). Identified regions included a flexible loop that contributes to DBL3X-CSA interaction and two DBL3X motifs with evidence of positive natural selection. Limited antibody responses against signatures of high parasite density among malaria-exposed pregnant women could not explain the increased placental parasitemia. These results suggest that a higher binding efficiency to CSA rather than reduced antigenicity might provide a biological advantage to parasites with high parasite density signatures in VAR2CSA. Sequences contributing to high parasitemia may be critical for the functional characterization of VAR2CSA and the development of tools against placental malaria.

Figure 1. Variability in DBL2X and DBL3X amino acid sequences.

(A) VAR2CSA domain structure and regions covered by sequencing in the reference strain A4 (NTS: N-terminal segment; DBL, Duffy-binding like; CIDR, cysteine-rich inter-domain region, TM: trans-membrane; ATS: acidic-terminal segment). (B, C) Shannon entropy values were calculated on the multiple sequence alignment of 176 DBL2X (and Dataset S1) and 191 DBL3X amino acid sequences (and Dataset S2) transcribed by placental parasites. Dotted horizontal lines indicate median of all positive entropy values (H_DBL2X = 0.48 and H_DBL3X = 0.14). Arrows indicate DBL sub-domain boundaries . White boxes delimit segments with residues below the entropy threshold; colored boxes delimit variable segments carrying signatures not associated (yellow) or associated with high placental parasite density (orange, Bonferroni corrected Wald test: P[S2A] = 0.0020, P[S2B, S2C, S3A and S3B]<0.001; P[S3C] = 0.0032). Regions corresponding to previously reported parity-linked motifs (P1, P2) , and variable blocks (VB) are underlined.

Figure 2. Surface mapping of segments containing high parasite density signatures.

(A) 3D model for DBL2X domain. (B) Crystal structure of DBL3X domain (PDB 3BQK). Segments with BepiPred scores ≥0.9 are shown in dark grey. Residues predicted to interact with CSA sulfate groups by Higgins and Singh et al. are colored in blue (R1467, R1503, K1504, K1507, K1510), or orange for those that are also part of the S3B segment (K1324, K1327, G1329). (C) Detail of predicted CSA binding amino acids ,. (D) Ribbon visualization of the DBL3X loop containing segment S3B and K1328 in the presence (PDB 3BQK, left) or absence (PDB 3BQI, right) of sulfate groups .

Figure 3. Recognition of DBL2X and DBL3X peptides by plasma IgG from 100 pregnant women.

Seroprevalence is represented by bars as the % of responders. Peptides representative of signatures of high (HDS) and low (LDS) parasite density (Table 2 and Materials and Methods) and their optical density thresholds for seroprevalence (mean plus 3 standard deviations of OD in negative controls) were: HDS, S2A.1 = 0.690, S2B.1 = 0.210, S2C.1 = 0.266, S3A.1 = 0.293, S3B.1 = 0.140, S3C.1 = 0.295; LDS, S2A.2 = 0.450, S2B.4 = 0.266, S2C.3 = 0.480, S3A.3 = 0.366, S3B.2 = 0.151, S3C.3 = 0.195. P-values were calculated using McNemar's test (tests for S2A and S3A were not applicable due to low seroprevalence).

Figure 4. VAR2CSA sequences with high parasite density signatures in different P. falciparum populations.

Colored bars show the frequency of the HDS sequence types identified among Mozambican isolates (Table 2) in DBL2X (A) and DBL3X (B) sequence sets from other geographical origins. Likewise, white bars show the frequency of LDS sequences. Plain columns indicate cDNA sequences, dotted columns indicate gDNA sequences. Columns ‘A+B+C’ show prevalence of VAR2CSA sequences with 3 signatures of high parasite density.