The direct binding of Plasmodium vivax AMA1 to erythrocytes defines a RON2-independent invasion pathway

Abstract

We used a transgenic parasite in which Plasmodium falciparum parasites were genetically modified to express Plasmodium vivax apical membrane antigen 1 (PvAMA1) protein in place of PfAMA1 to study PvAMA1-mediated invasion. In P. falciparum, AMA1 interaction with rhoptry neck protein 2 (RON2) is known to be crucial for invasion, and PfRON2 peptides (PfRON2p) blocked the invasion of PfAMA1 wild-type parasites. However, PfRON2p has no effect on the invasion of transgenic parasites expressing PvAMA1 indicating that PfRON2 had no role in the invasion of PvAMA1 transgenic parasites. Interestingly, PvRON2p blocked the invasion of PvAMA1 transgenic parasites in a dose-dependent manner. We found that recombinant PvAMA1 domains 1 and 2 (rPvAMA1) bound to reticulocytes and normocytes indicating that PvAMA1 directly interacts with erythrocytes during the invasion, and invasion blocking of PvRON2p may result from it interfering with PvAMA1 binding to erythrocytes. It was previously shown that the peptide containing Loop1a of PvAMA1 (PvAMA1 Loop1a) is also bound to reticulocytes. We found that the Loop1a peptide blocked the binding of PvAMA1 to erythrocytes. PvAMA1 Loop1a has no polymorphisms in contrast to other PvAMA1 loops and may be an attractive vaccine target. We thus present the evidence that PvAMA1 binds to erythrocytes in addition to interacting with PvRON2 suggesting that the P. vivax merozoites may exploit complex pathways during the invasion process.

Keywords: Plasmodium vivax; apical membrane antigen 1; transgenic parasites.

Fig. 1.

PvAMA1 binds to both normocytes and reticulocytes. Schematic structure of AMA1 and RON2p in P. vivax and P. falciparum, and the corresponding recombinant domains 1 and 2 of PvAMA1 [rPvAMA1; amino acids (aa) 43 to 385] and PfAMA1 (rPfAMA1; aa 93 to 442). PvAMA1 (aa 562) and PfAMA1 (aa 622) have one signal peptide (SP), transmembrane (TM) domain and Pro (prodomain). PvRON2 (aa 2203) and PfRON2 (aa 2189) have one SP, TM domain and two cysteine-rich domains. Peptides and proteins made for this study are highlighted in red. PfAMA1 (Pf) and PvAMA1 (Pv) recombinant proteins as indicated by the red line in (A) were expressed in mammalian cells with a 6xHis tag at the C-terminal. The recombinant proteins were visualized in Coomassie Blue staining (B) and Western blot with anti-His antibodies (C). (D) The purified recombinant proteins (40 µg/mL) were incubated with reticulocyte-enriched erythrocyte samples to evaluate binding activity. Erythrocytes were stained with TO to distinguish reticulocytes from normocytes. Values are means of duplicate for the same sample studied (upper and lower end of lines). (E) Binding of rPvAMA1 protein in varying concentration to erythrocytes was determined. The binding results are shown in three independent experiments (Fig. 1 D and E and SI Appendix, Figs. S1 and S2).

Fig. 2.

Invasion inhibition studies of PfAMA1 and transgenic PvAMA1 parasites and erythrocyte binding of rPvAMA1 in the presence of PfRON2p. PfRON2p inhibits parasites expressing PfAMA1, but not PvAMA1. Purified late-stage (A) PfAMA1 and (B) PvAMA1-expressing parasites were mixed with CTFR-labelled erythrocytes and exposed to varying concentrations of PfRON2p (0 µg/mL to 100 µg/mL). Parasites were allowed to undergo egress and invade CTFR⁺ cells overnight. Cells were stained with HO (DNA stain) and double-positive cells were identified (HO⁺CTFR⁺) and measured by flow cytometry. (C) Different concentrations of PfRON2p (0 to 20 µg/mL) and rPvAMA1 (40 µg/mL) were co-incubated for 30 min at room temperature prior to erythrocyte binding assays to evaluate if PfRON2p inhibited the binding of rPvAMA1 to erythrocytes. Dot plot showing the binding inhibition studies is shown (SI Appendix, Fig. S4). Data from three independent experiments were normalized to control (0 µg/mL). Values are means ± 95% CI. Statistical significance was determined by repeated-measures ANOVA, where P < 0.05 is considered significant (ns, not significant; **P < 0.01). (A) p = 0.0003, F = 337.6, df = 5, R² = 0.9941; (B) p = 0.5133, F = 0.6556, df = 5, R² = 0.2469; (C) Reticulocytes: p = 0.7397, F = 0.1614, df = 4, R² = 0.07468, Normocytes: p = 0.5215, F = 0.7516, df = 4, R² = 0.2731. For pairwise comparison, refer to SI Appendix Tables S1 and S2.

Fig. 3.

Invasion inhibition studies of PfAMA1 and transgenic PvAMA1 parasites and erythrocyte binding of rPvAMA1 in the presence of PvRON2p. PvRON2p inhibits parasites expressing PvAMA1, but not PfAMA1. Purified late-stage (A) PfAMA1 and (B) PvAMA1 expressing parasites were mixed with CTFR-labelled erythrocytes and exposed to varying concentrations of PvRON2p (0 µg/mL to 200 µg/mL). Parasites were allowed to undergo egress and invade CTFR⁺ cells overnight. Cells were stained with HO (DNA stain) and double-positive cells were identified (HO⁺CTFR⁺) and measured by flow cytometry. (C) Serial dilutions of PvRON2p (0 to 20 µg/mL) were co-incubated with rPvAMA1 protein (40 µg/mL) for 30 min at room temperature prior to incubation with erythrocytes to evaluate whether PvRON2p inhibited the binding of rPvAMA1 to erythrocytes. After incubation, erythrocyte binding by PvAMA1 was measured by flow cytometry. Dot plot showing the binding inhibition studies is shown (SI Appendix, Fig. S4). Data from three independent experiments were normalized to control (0 µg/mL). Values are means ± 95% CI. Statistical significance was determined by repeated measures ANOVA, where P < 0.05 is considered significant (ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001). (A) p = 0.1225, F = 5.602, df = 5, R² = 0.7369; (B) p = 0.0209, F = 29.07, df = 5, R² = 0.9356; (C) Reticulocytes: p = 0.0503, F = 15.73, df = 4, R² = 0.8872, Normocytes: p = 0.0147, F = 18.96, df = 4, R² = 0.9046. For pairwise comparison (SI Appendix Tables S1 and S2).

Fig. 4.

P. vivax AMA1 interaction with the peptide PvRON2p and the effect of PvAMA1 Loop 1a on invasion and erythrocyte binding. (A) Molecular surface of PvAMA1 with PvRON2L-interacting loops (labeled 1a–1f) (23) is shown in ribbon format. Loops 1a (pink), 1b (tan), 1c (yellow), 1d (blue), 1e (cyan) and 1f (green) surround the bound RON2L peptide (purple). Side chains of PvAMA1 Loop 1a that contact the PvRON2L ligand are shown in stick representation; Arg 88 and Tyr 87 are labeled. (B) Space-filling representation of PvAMA1 Loop 1a (carbons colored pink) and the PvRON2L peptide (carbons colored purple) showing the intimate association of the loop with the peptide. Residues Tyr 87 from PvAMA1 and Pro 2047 from RON2L are labeled. Oxygen atoms are colored red, nitrogen atoms blue and sulfur atoms yellow. (C) A peptide based on PvAMA1 Loop 1a inhibits binding of rPvAMA1 to normocytes and reticulocytes. Serial dilutions of PvAMA1 Loop 1a peptide and rPvAMA1 protein (40 µg/mL) were incubated with erythrocytes and binding of PvAMA1 was quantified. (D) Purified late-stage parasites expressing PvAMA1 were mixed with CTFR-labelled erythrocytes in the presence of varying concentrations of PvAMA1 Loop 1a peptide (0 µg/mL to 100 µg/mL). Parasites were allowed to undergo egress and invade CTFR⁺ cells overnight. Cells were stained with HO (DNA stain) and double-positive cells were identified (HO⁺CTFR⁺) and measured by flow cytometry. Data from three independent experiments were normalized to control (0 µg/mL). Values are means ± 95% CI. Statistical significance was determined by repeated measures ANOVA, where P < 0.05 is considered significant (ns, not significant). (C) Reticulocytes: p = 0.0281, F = 32.13, df = 3, R² = 0.9414, Normocytes: p = 0.2644, F = 2.311, df = 3, R² = 0.5361; (D) p = 0.3299, F = 1.568, df = 5, R² = 0.4395. For pairwise comparison (SI Appendix Tables S1 and S2).