PMC

Reactivity of human immune sera and stage-specific expression of Pf34. (A) The recombinant proteins Pf34-A (lane A), Pf34-B (lane B) and Pf34-C (lane C) and glutathione-S-transferase alone (lane D) were resolved under denaturing conditions. Shown are Coomassie-stained samples and those immunoblotted with human immune sera from endemic areas of Vietnam and Papua New Guinea, and non-immune sera from Australia. (B) Stage-specific expression of Pf34. Synchronised parasite culture was sampled at various time points (indicated in hours) and lysates resolved by SDS-PAGE. All samples were immunoblotted with antisera raised against Pf34 fragments A, C and RAMA. Anti-GRP(BiP) sera was used as a loading control. Pf34 expression is not convincingly demonstrable at 22–28 h.

Pf34 is associated with detergent-resistant microdomains (DRMs). (A) Asynchronous parasites were saponin treated (C) and resuspended in TX-100 at 4°C or 37°C. After incubation the soluble (S) and pellet (P) fractions were resolved by SDS-PAGE. Enhanced solubility at 37°C is indicative of proteins associated with DRMs. The samples were immunoblotted with anti-Pf34-A sera. (B) Immunoblot detection of sucrose density gradient samples of Plasmodium falciparum parasites. Asynchronous parasites were extracted in cold TX-100 and subjected to sucrose density gradient flotation. Six gradient fractions were obtained (1–6), where fractions 2–4 contain floating material, and thus, DRMs. Fractions 5 and 6 represent the high density loading fraction. The samples were immunoblotted with anti-RAMA (positive control), anti-HSP70 (negative control) and anti-Pf34-A and C antibodies.

Localisation of Pf34 to the rhoptries. (A) Rhoptry localisation was demonstrated in late stage parasites by immunostaining infected blood smears with anti-Pf34 and co-localising with either anti-RAMA or anti-RhopH3 (both rhoptry markers) or anti-AMA-1 (microneme marker) antibodies. (B) A mature schizont (i) and two extracellular merozoites (ii) showing the close apposition of Pf34-positive (green) and RAMA-positive (red) structures in the apex of the merozoite. Panels (iii) and (iv) show the apex of merozoites and the two rhoptries connected to the Pf34-positive structure at the anterior when visualised by confocal microscopy. N – nucleus; R – rhoptry. Bars represent 1 μm in (i) and (ii) and 500 nm in (iii) and (iv). (C) Late stage parasites were stained with anti-Pf34 (green) and anti-RAMA (red) (i) or anti-Pf34 (green) and anti-AMA-1 (red) (ii) antibodies, highlighting the differential expression of the rhoptry proteins (Pf34 and RAMA) when compared with AMA-1 (microneme protein). (D) Ring stage expression of Pf34 was shown using anti-Pf34 co-localised with anti-RAMA.

Structure, features and conservation of Pf34. (A) Schematic representation of full-length Pf34, a 325 residue protein. The N-terminus contains a predicted signal peptide (residues 1 – 23), whereas the C-terminus contains a probable glycosylphosphatidylinositol (GPI) attachment site (residue 306), followed by a hydrophobic anchor sequence (residues 307 – 325). Pf34 also contains a central domain (residues 140 - 248) that is highly conserved in orthologues identified in Plasmodium falciparum, Plasmodium vivax and Plasmodium knowlesi (shown in bold in Fig. 1B). Fragments corresponding to residues 24 – 110 (Pf34-A), 110 – 256 (Pf34-B) and 260 – 307 (Pf34-C) were produced as glutathione-S-transferase fusion proteins. (B) Sequence similarity between P. falciparum Pf34 and orthologues in P. vivax and P. knowlesi. Identical (*), highly similar (:) and similar (.) residues are indicated. The central highly conserved region is shown in bold type. Probable GPI attachment sites are indicated by the di-serine motif (underlined).