Binding of Plasmodium merozoite proteins RON2 and AMA1 triggers commitment to invasion

Abstract

The commitment of Plasmodium merozoites to invade red blood cells (RBCs) is marked by the formation of a junction between the merozoite and the RBC and the coordinated induction of the parasitophorous vacuole. Despite its importance, the molecular events underlying the parasite's commitment to invasion are not well understood. Here we show that the interaction of two parasite proteins, RON2 and AMA1, known to be critical for invasion, is essential to trigger junction formation. Using antibodies (Abs) that bind near the hydrophobic pocket of AMA1 and AMA1 mutated in the pocket, we identified RON2's binding site on AMA1. Abs specific for the AMA1 pocket blocked junction formation and the induction of the parasitophorous vacuole. We also identified the critical residues in the RON2 peptide (previously shown to bind AMA1) that are required for binding to the AMA1 pocket, namely, two conserved, disulfide-linked cysteines. The RON2 peptide blocked junction formation but, unlike the AMA1-specific Ab, did not block formation of the parasitophorous vacuole, indicating that formation of the junction and parasitophorous vacuole are molecularly distinct steps in the invasion process. Collectively, these results identify the binding of RON2 to the hydrophobic pocket of AMA1 as the step that commits Plasmodium merozoites to RBC invasion and point to RON2 as a potential vaccine candidate.

Fig. 1.

Localization of Pf AMA1, RON2, and RON4 at the moving junction of invading merozoites. Confocal microscopy images of merozoite invading RBC are shown. (A) Pf AMA1, RON2, and RON4 colocalize during invasion. (B) AMA1 is present at the moving junction (Top) with RON4 colocalizing with AMA1 at the junction (Middle). RON4 marks the sealing of the junction after invasion (Bottom). Scale bars represent 1μM.

Fig. 2.

RON2L binds the hydrophobic pocket of AMA1. (A) RON2L binding to AMA1 is inhibited by mAbs 4G2 and 1F9 that bind near or in the hydrophobic pocket, respectively. (B) Prebound RON2L was displaced by AMA1 Abs. RON2L (20 pmol) was preincubated with recombinant AMA1 for 1 h before adding mAbs 4G2 (5 μg/mL), 4F2 (5 μg/mL), and 1F9 (2.5 μg/mL). (C) Mutating Tyr-251 to Ala (Y251A) in the hydrophobic pocket of AMA1 abolishes binding of RON2L. Results in A and B are pooled data from three independent experiments, and C is from two independent experiments. Data are represented as mean ± SEM.

Fig. 3.

Abs to RON2L inhibit merozoite invasion of RBCs. Affinity purified anti-RON2L IgG from two rabbits (black and gray bars) inhibit merozoite invasion of 3D7 and FVO parasites as determined by standard growth inhibition assay. Anti-AMA1 rabbit IgG to a combination of FVO and 3D7 (white bars) was used as a positive control. Purified IgG from preimmune rabbit sera or sample from which RON2L IgG was depleted (using protein G) was used as negative control. Numbers in parentheses indicate the amount of IgG used (mg/mL) in this assay. Results shown are mean ± SEM from pooled data. Numbers above each bar (n) indicate the number of independent experiments.

Fig. 4.

RON2 binding to AMA1 is required for triggering junction formation. (A) Transmission electron microscopy of merozoites treated with 2 μM cytochalasin D. (a–c) Merozoite attachment (a), reorientation and junction formation (b), and the presence of vacuoles (c), indicative of rhoptry bulb secretion. (d–i) The processes affected by the presence of invasion inhibitory mAb 4G2 (d–f) and RON2L peptide (g–i). Black arrows show the junction, and white arrows show the secreted vacuoles. R, rhoptry; M, microneme. (B) The percentage of merozoites in transmission electron microscopy that are attached to RBCs in each category and that are apical, not apical, and indeterminate. (C) The percentage of apically oriented merozoites in each category that form a junction and RBCs with vacuoles (indicative of rhoptry bulb secretion). Data shown for mAb 4G2 and RON2L peptide are pooled from two and three independent experiments, respectively. Numbers within each bar represent the number of merozoite-attached RBCs in each category. The number 0 indicates that no merozoites were found in that category. Scale bars represent 250 nm.

Fig. 5.

AMA1 cytoplasmic tail binds aldolase. (A) Sequence of the native cytoplasmic tail (Wt) of AMA1. Mutations analyzed in the current study are underlined. pS, phosphorylated Ser (S610). The sequence of Plasmodium TRAP and the subterminal Trp residue in TRAP mutated to Ala is also shown. (B) Identifying the critical residues in AMA1 cytoplasmic tail for aldolase binding. Wt AMA1 cytoplasmic tail binds recombinant rabbit muscle aldolase (lane 1). Ser-610 in the AMA1 cytoplasmic tail (lane 6) or its phosphorylation (lane 2) is not critical for binding, whereas the central Phe, Trp (F602W603), and the terminal Tyr (Y621 and Y622) are critical for aldolase binding (lanes 3 and 4). (C) Cytoplasmic tail of AMA1 interacts with the aldolase substrate binding pocket. Binding of aldolase to Wt and pS AMA1 cytoplasmic tails was inhibited by the aldolase substrate fructose-1,6-biphosphate (FBP; lanes 2 and 4). Binding of TRAP (lane 5) was also inhibited by FBP (lane 6) as well as by mutation of the subterminal Trp (lane 7). All of the lanes were from the same gel with the empty lane removed (white dotted line) for showing lanes side by side.

Fig. 6.

Schematic model of the steps involved in Pf merozoite (Mrz) invasion. (A) See text for details. (B) Cytochalasin-treated merozoites are able to reorient and form the junction as well as induce membrane flow (1). In the absence of the RBC receptor (e.g., GlycophorinA) the merozoite does not come into close apposition with the RBC membrane and cannot form a junction (2). In the presence of RON2L peptide (3) or AMA1-specific mAb 4G2 that competes with parasite secreted RON2 for binding AMA1 (4), junction formation is blocked. In the presence of mAb 4G2 no vacuoles are seen inside the RBC (4). However, binding of RON2L peptide to parasite AMA1 does not block rhoptry bulb secretion into the RBC (3).