Independent translocation of two micronemal proteins in developing Plasmodium falciparum merozoites

Abstract

Apical membrane antigen 1 of Plasmodium falciparum (PfAMA1) contains an N-terminal propeptide that is removed prior to the translocation of the mature protein onto the merozoite surface. We localized unprocessed PfAMA1 to the microneme organelles of the intraerythrocytic schizont. The results have suggested that the processed form of PfAMA1 translocates from the microneme compartment independently of another microneme protein, EBA175, which is also involved in the invasion of human erythrocytes.

FIG. 1.

Analysis of recognition patterns of anti-P. falciparum antisera by Western blotting. Saponin-lysed mature 3D7 schizonts were probed with rabbit polyclonal antisera raised against EBA175 (lane 1) and PfAMA1 ectodomain (strain 3D7) (lane 3). The reactivities of mouse MAbs 5G8 (lane 2) against the AMA1 pro-region and 1F9 (lane 4) against AMA1 domain 1 are also shown. The parasite samples in lanes 1 and 2 were treated with 2-mercaptoethanol as a reducing agent, whereas those in lanes 3 and 4 were run under nonreducing conditions, as the epitope for MAb 1F9 has been shown to be reduction sensitive.

FIG. 2.

Single and dual indirect immunofluorescence images showing the same subcellular localization of AMA1 and EBA-175 in two mature schizonts of P. falciparum. (a to d) FITC-labeled MAb 5G8 against the PfAMA1 pro-region (a and b) and rhodamine-labeled anti-EBA175 (c and d). (e and f) Merged images show full-length AMA1 colocalized with EBA175. (g and h) DAPI-stained parasites included to show the stage of merozoite development.

FIG. 3.

Single and dual indirect immunofluorescence images showing incomplete colocalization of EBA175 and processed AMA1 in mature schizonts of P. falciparum. (a to d) FITC-labeled MAb 1F9 against PfAMA1 domain 1 (a and b) and rhodamine-labeled rabbit anti-EBA175 (c and d). (e and f) Merged images of AMA1 and EBA175 show differential localization of the two proteins. (g and h) DAPI-stained parasites included to show the stage of merozoite development.

FIG. 4.

Single and dual indirect immunofluorescence images showing two subpopulations of intramerozoite AMA1 within P. falciparum schizonts. (a and b) FITC-labeled MAb 5G8 against PfAMA1 pro-region showing micronemal localization. (c to f) Rhodamine-labeled rabbit polyclonal serum against AMA1 (c and d) showing apical localization and merged images (e and f) showing incomplete colocalization of the two AMA1 antibodies. (g and h) DAPI-stained parasites included to show the stage of merozoite development.

FIG. 5.

Single and dual indirect immunofluorescence images of two AMA1-specific antibodies in late P. falciparum schizonts. (a and b) FITC-labeled anti-AMA1 domain 1-specific MAb 1F9 showing apical fluorescence. (c and d) Rhodamine-labeled rabbit polyclonal serum against AMA1 showing apical localization. (e and f) Merged images show colocalization of the two AMA1 antibodies. (g and h) DAPI-stained parasites included to show the stage of merozoite development.

FIG. 6.

Immunogold localization of AMA1 and EBA175 in late schizonts of P. falciparum 3D7. (a and b) Localization of AMA1 with 15-nm-diameter colloidal gold. Labeling is confined to micronemes (arrows) and absent from rhoptries (Rh). (c and d) Localization of EBA175 with 12-nm-diameter colloidal gold. Labeling is confined to micronemes and absent from rhoptries (Rh), nuclei (Nu), and other structures. (e to h) Double-labeling immunogold for AMA1 (15-nm-diameter gold [small arrow]) and EBA175 (10-nm-diameter gold [large arrows]). Magnification, ×48,000 (a), ×49,000 (b), ×58,000 (c), ×55,000 (d), ×62,000 (e), ×87,000 (f), ×64,000 (g), and ×78,000 (h).

FIG. 7.

Schematic model of AMA1-processing events in P. falciparum merozoites. AMA1 is synthesized as an 83-kDa precursor protein (A) with signal peptide to allow transport through the endoplasmic reticulum (ER), where removal of the signal peptide presumably occurs. From there it traffics to the micronemes (Mn). The N-terminal pro-region segment is proteolytically cleaved there (B), and the mature 62-kDa polypeptide is translocated out of the micronemes (C) via the neck of the rhoptry (Rn) to the merozoite surface, where it undergoes further C-terminal processing (D), releasing part of the ectodomain off the surface membrane. The domains of AMA1 are represented as follows: signal peptide, red; pro-domain, blue; ectodomains 1, 2, and 3, pink; transmembrane domain, black; and cytoplasmic tail, green.