Plasmodium falciparum Pf34, a novel GPI-anchored rhoptry protein found in detergent-resistant microdomains

Abstract

Apicomplexan parasites are characterised by the presence of specialised organelles, such as rhoptries, located at the apical end of invasive forms that play an important role in invasion of the host cell and formation of the parasitophorous vacuole. In this study, we have characterised a novel Plasmodium falciparum rhoptry protein, Pf34, encoded by a single exon gene located on chromosome 4 and expressed as a 34kDa protein in mature asexual stage parasites. Pf34 is expressed later in the life cycle than the previously described rhoptry protein, Rhoptry Associated Membrane Antigen (RAMA). Orthologues of Pf34 are present in other Plasmodium species and a potential orthologue has also been identified in Toxoplasma gondii. Indirect immunofluorescence assays show that Pf34 is located at the merozoite apex and localises to the rhoptry neck. Pf34, previously demonstrated to be glycosyl-phosphatidyl-inositol (GPI)-anchored [Gilson, P.R., Nebl, T., Vukcevic, D., Moritz, R.L., Sargeant, T., Speed, T.P., Schofield, L., Crabb, B.S. (2006) Identification and stoichiometry of GPI-anchored membrane proteins of the human malaria parasite Plasmodium falciparum. Mol. Cell. Proteomics 5, 1286-1299.], is associated with parasite-derived detergent-resistant microdomains (DRMs). Pf34 is carried into the newly invaded ring, consistent with a role for Pf34 in the formation of the parasitophorous vacuole. Pf34 is exposed to the human immune system during infection and is recognised by human immune sera collected from residents of malaria endemic areas of Vietnam and Papua New Guinea.

Fig. 1

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).

Fig. 2

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.

Fig. 3

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.

Fig. 4

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.