Interaction between Plasmodium falciparum apical membrane antigen 1 and the rhoptry neck protein complex defines a key step in the erythrocyte invasion process of malaria parasites

Abstract

Invasion of host cells by apicomplexan parasites, including Plasmodium falciparum and Toxoplasma gondii, is a multistep process. Central to invasion is the formation of a tight junction, an aperture in the host cell through which the parasite pulls itself before settling into a newly formed parasitophorous vacuole. Two protein groups, derived from different secretory organelles, the micronemal protein AMA1 and the rhoptry proteins RON2, RON4, and RON5, have been shown to form part of this structure, with antibodies targeting P. falciparum AMA1 known to inhibit invasion, probably via disruption of its association with the PfRON proteins. Inhibitory AMA1-binding peptides have also been described that block P. falciparum merozoite invasion of the erythrocyte. One of these, R1, blocks invasion some time after initial attachment to the erythrocyte and reorientation of the merozoite to its apical pole. Here we show that the R1 peptide binds the PfAMA1 hydrophobic trough and demonstrate that binding to this region prevents its interaction with the PfRON complex. We show that this defined association between PfAMA1 and the PfRON complex occurs after reorientation and engagement of the actomyosin motor and argue that it precedes rhoptry release. We propose that the formation of the AMA1-RON complex is essential for secretion of the rhoptry contents, which then allows the establishment of parasite infection within the parasitophorous vacuole.

FIGURE 1.

The R1 invasion inhibitory peptide binds the AMA1 hydrophobic groove. A, ¹H-¹³C HSQC of [¹³C]methyl l-methionine labeled PfAMA1 ectodomain in the presence (red) and absence (blue) of a molar excess of R1. The peak assigned as the Met-273 methyl resonance is labeled, and the peaks corresponding to the cluster of methionine residues (Met-224, Met-190, Met-193, Met-189) are marked with asterisks. B, domains I and II of PfAMA1 (PDB code 1Z40), showing the location of methionine methyl groups (red spheres), the hydrophobic trough (green), and the 1F9 epitope (blue). Four additional methionine residues present in domain III are not shown. The figure was prepared with PyMOL (DeLano Scientific).

FIGURE 2.

The PfRON4 and PfRON5 proteins localize to the rhoptry neck. A, antibodies raised against PfRON4 and PfRON5 recognize specific protein products in schizont parasite extracts. B, immunofluorescence assays using the anti-PfRON antibodies reveal staining of the apical tip of free merozoite, in close apposition with the rhoptry bulb markers PfRAP1 and PfRAMA, suggesting a rhoptry neck localization. Scale bar: 0.2 μm. C, immunoelectron microscopy confirms that PfRON4 localizes to the rhoptry neck. Scale bar: 0.1 μm.

FIGURE 3.

The PfRON4 and PfRON5 proteins form a complex with PfAMA1 in schizonts. Immunoprecipitation using the anti-PfRON4 probed by Western blot with antibodies against the different PfRONs and PfAMA1 is shown. NRS, normal rabbit serum; NMS, normal mouse serum; NR, non-reduced.

FIGURE 4.

R1 prevents the association between PfAMA1 and the PfRON complex. A, PfAMA1 immunoprecipitation on schizont stage parasites solubilized in increasing concentrations of the R1 peptide shows a dose-dependent decrease in the amount of PfRON4 and PfRON5 pulled down. An unrelated peptide (X1) does not have any effect on the PfAMA1-RON interaction. B, relative amounts of each RON pulled down by AMA1 under increasing amounts of R1 or X1 peptide.

FIGURE 5.

F1 prevents the association between PfAMA1 and the PfRON complex. A, PfAMA1 immunoprecipitation on schizont stage parasites solubilized in increasing concentrations of the F1 peptide shows a dose-dependent decrease in the amount of PfRON4 and PfRON5 pulled down. A scrambled version of F1 (F1s) does not have any effect on the PfAMA1-RON interaction. B, relative amounts of each RON pulled down by AMA1 under increasing amounts of F1 or F1s peptide.

FIGURE 6.

A model for the early step of merozoite invasion of erythrocytes. 1, after initial contact and reorientation of the merozoite, its apical tip comes in contact with the erythrocyte membrane, leading to the formation of a tight junction and translocation of the RON complex across the host cell membrane. 2a, interaction between the RON complex and AMA1 triggers secretion of the rhoptry bulb contents, which are then used in the generation of the nascent parasitophorous vacuole. The parasite can then pull itself through the tight junction powered by its actomyosin motor. 2b, binding of the R1 peptide to the AMA1 hydrophobic trough prevents its interaction with the RON complex and subsequent rhoptry secretion. However, because the tight junction and the invasion motor are established, the merozoite is able to pull on the red blood cell, which ends up wrapping around the parasite because of the absence of a nascent parasitophorous vacuole. MSPs refers to merozoite surface proteins.