An EGF-like protein forms a complex with PfRh5 and is required for invasion of human erythrocytes by Plasmodium falciparum

Abstract

Invasion of erythrocytes by Plasmodium falciparum involves a complex cascade of protein-protein interactions between parasite ligands and host receptors. The reticulocyte binding-like homologue (PfRh) protein family is involved in binding to and initiating entry of the invasive merozoite into erythrocytes. An important member of this family is PfRh5. Using ion-exchange chromatography, immunoprecipitation and mass spectroscopy, we have identified a novel cysteine-rich protein we have called P. falciparumRh5 interacting protein (PfRipr) (PFC1045c), which forms a complex with PfRh5 in merozoites. Mature PfRipr has a molecular weight of 123 kDa with 10 epidermal growth factor-like domains and 87 cysteine residues distributed along the protein. In mature schizont stages this protein is processed into two polypeptides that associate and form a complex with PfRh5. The PfRipr protein localises to the apical end of the merozoites in micronemes whilst PfRh5 is contained within rhoptries and both are released during invasion when they form a complex that is shed into the culture supernatant. Antibodies to PfRipr1 potently inhibit merozoite attachment and invasion into human red blood cells consistent with this complex playing an essential role in this process.

Figure 1. The processed 45 kDa PfRh5 C-terminal domain exists as a larger complex.

(A) PfRh5 partially purified from parasite culture supernatant was analysed by size exclusion chromatography on a Superdex 200 analytical column. PfRh5 was eluted from the column as a ∼150–200 kDa species. Molecular weight of eluted protein fractions are indicated by arrows as shown using standard proteins of known size. PfRh5 was detected using 2F1, a specific monoclonal antibody to the protein. (B) Blue native gel electrophoresis confirmed that PfRh5 migrates as a ∼150–200 kDa species. The processed PfRh1 fragment of 110 kDa is included as a control. (C) 300 µl of the PfRh5-containing fraction, isolated from culture supernatant, was loaded onto a Superdex 200 analytical column and eluted with PBS. (D) An identical 300 µl sample was pre-incubated with 25 µg of monoclonal anti-PfRh5 antibody before loading onto the same Superdex 200 column. The peak of the eluted PfRh5 protein lies at two preceding fractions as compared to panel C, corresponding to an increase in molecular weight of ∼150 kDa indicating that one antibody molecule was bound to the PfRh5-containing species. The monoclonal antibody-bound PfRh5 is indicated by an arrow. PfRh5 and the immunoglobulin heavy and light chain of antibody seen in fraction #22–25 are the free PfRh5 complex and the excess antibody. The asterisks refer to cross-hybridising bands corresponding to the immunoglobulin heavy and light chain.

Figure 2. PfRh5 and PfRipr form a complex.

(A) A triple Haemaglutinin (HA) tag was added to the C-terminus of PfRipr by 3′ single homologous crossover recombination. (B) HA-tagged PfRiprHA was detected in the parasite membrane pellet and in the culture supernatant of 3D7PfRiprHA line, confirming successful tagging. (C) PfRiprHA was analysed by SDS-PAGE under reducing and non-reducing conditions. Similar mobility, of the 65 kDa C-terminal fragment under both conditions, indicates that the N-terminal and C-terminal of PfRipr was not linked by disulphide bonds after processing. (D) Co-immunoprecitation of PfRh5 with PfRiprHA was performed using proteins solubilized from saponin pellets of 3D7-PfRiprHA schizont-infected erythrocytes. Immunoprecipitation of HA-tagged PfRipr by anti-HA antibody co-immunoprecipitated PfRh5. (E) Immunoprecitation with anti-PfRh5 antibody using the same schizont materials co-immunoprecipited PfRiprHA but not PfRh2a/b. (F) Co-immnuoprecipitation of PfRh5 with PfRiprHA from culture supernatants using anti-HA-Sepharose bead. Detection of PfRh5 but not PfRh2a or PfRh2b in the anti-HA bead bound material confirmed that PfRh5 was specifically co-immunoprecipitated with PfRiprHA. (G) Co-immunoprecipitation of PfRiprHA with PfRh5 from culture supernatants using monoclonal anti-PfRh5 antibody coupled to Mini-beads. Probing of the bound material with anti-HA antibody detected PfRiprHA only from the 3D7-PfRiprHA parasites and no PfRh2a/b was detected in the bound materials, indicating that PfRiprHA was specifically co-immunoprecipitated with PfRh5. (H) Immnunoprecipitation of culture supernatant from PfRiprHA parasites with rabbit anti-PfRh2a/b antibodies that recognize 85 kDa processed PfRh2a/b fragment did not pull-down PfRh5 or PfRipr. In all cases, the asterisk indicates a cross-hybridizing band corresponding to the heavy chain of IgG eluted from the antibody affinity beads. The arrows in Figure 2E, F and G indicate where 85 kDa PfRh2a/b band should appear.

Figure 3. PfRipr protein has ten EGF-like domains.

(A) Diagram shows the characteristics of PfRipr and the region used to generate the recombinant proteins. The arrow corresponds to the approximate position of the PfRipr cleavage site. (B) Alignment of the ten EGF-like domains within PfRipr.

Figure 4. The N-terminal and C-terminal cleaved polypeptides remain associated after cleavage.

(A) Amino acids 238-368 and 791-900 of PfRipr that encompassed the N-terminal first two EGF-like domains and C-terminal two EGF-like domains respectively were expressed as recombinant proteins in E. coli and purified for immunizing rabbits. The proteins were stained with Coomassie blue. (B) Rabbit polyclonal antibodies (anti-PfRipr/1 and anti-PfRipr/2) raised against the recombinant protein recognize native PfRipr. (C) Rat anti-HA antibody affinity beads were used to specifically immuno-precipitate proteins solubilized from schizont stages parasites of 3D7 and 3D7RiprHA lines and the bound materials immunoblotted and probed with rabbit anti-PfRipr/3 antibodies. (D) Rabbit anti-PfRipr/3 antibodies, in conjunction with protein G Sepharose, was used to specifically immuno-precipitate the proteins solubilized from schizont stages of 3D7 (panel 1) or culture supernatants (panel 2) and then the bound material was immunoblotted and probed with mouse anti-HA antibodies. The IgG from the pre-bleed serum of the same rabbit was used as a control. (E) Rabbit anti-PfRipr/3 antibodies, in conjunction with protein G Sepharose, was used to specifically immuno-precipitate the proteins from culture supernatant of 3D7 and 3D7RiprHA parasites and the bound material immunoblotted and probed with mouse anti-HA antibodies. The protein marked with a * in the panel D and E is protein G eluted from the beads that cross-reacts with the anti-HA antibodies.

Figure 5. Protein expression patterns of PfRh5 and PfRipr are consistent with the complex formation.

(A) Both anti-PfRh5 and anti-PfRipr antibodies recognize the same complex resolved by blue native gel electrophoresis. (B) Both PfRh5 and PfRipr are expressed late in the blood stage life cycle of parasite development. Synchronized PfRiprHA parasites were distributed into six 5 ml cultures. One was harvested immediately after synchronization (8 – 12 hr ring stage). The second was harvested 16 hr post synchronization, and the third 8 hr later. The rest of the time points were harvested every 6 hr until the end of growth cycle. Proteins were extracted from saponin pellets, separated by SDS-PAGE and transferred to nitrocellulose membrane. The membrane was probed with monoclonal anti-HA antibody (top panel) to detect PfRiprHA and then stripped and re-probed to detect PfRh5 (middle panel) and PfHsp70 (bottom panel). Both PfRh5 and PfRipr proteins are expressed primarily in late schizonts.

Figure 6. PfRipr and PfRh5 are peripherally associated with parasite membrane.

(A) Differential solubilization of proteins from pellet prepared by hypotonic lysis of red blood cells that had been infected by the late schitzont stage 3D7-RiprHA parasite. (B) Differential solubilization of proteins from saponin pellets of late schitzont stage 3D7RiprHA parasites.

Figure 7. PfRipr localizes to the apical end of merozoites.

a) Rabbit polyclonal anti-PfRip antibody (anti-PfRipr/1) recognizes HA-tagged PfRiprHA. b) PfRipr does not co-localize with the rhoptry neck protein RON4 in schizonts. c) PfRipr does not co-localize with the rhoptry neck protein RON4 in merozoites. d) PfRipr does not co-localize with the rhoptry bulb protein, RAP1 in schizonts. e) PfRipr does not co-localize with the rhoptry bulb protein, RAP1 in merozoites. f) PfRipr partially co-localizes with PfRh5 in the schizonts. g) PfRipr mainly co-localizes with PfRh5 in purified merozoites. h) PfRipr co-localizes with the micronemal marker, EBA175, in schizonts. i) PfRipr does not co-localize with the micronemal marker, EBA175, in merozoites.

Figure 8. Subcellular localization of PfRipr and PfRh5 by immuno-electron microscopy.

(A) Localisation of HA-tagged PfRiprHA using anti-HA antibodies. The protein localizes to electron dense structures at the apical end of the merozoite that resemble micronemes (arrows). Mn, micronemes: Rh, rhoptries. (B) PfRiprHA can be detected at the leading edge of the tight junction formed between the erythrocyte and merozoite in a merozoite in the process of invading a red blood cell. Nu, nucleus: TJ, tight junction. (C) PfRiprHA can be observed concentrating in structures at the apical end at the periphery of the parasite. Rh, rhoptries. (D) Some merozoites show labeling of PfRipHA on the merozoite surface. (E) and (F) Localisation of PfRh5 using monoclonal anti-PfRh5 antibody. PfRh5 localises to rhoptries but was also observed at the apical surface, suggesting it was being released in free merozoites. Rh, Rhoptries. Scale bar is 200 nm.

Figure 9. Antibodies to PfRipr inhibit attachment of merozoites to erythrocytes and parasite growth.

(A) Anti-PfRipr/1 antibodies inhibit invasion of P. falciparum strains into erythrocytes. Shown are growth inhibition assays of the parasite strains FCR3, W2mef, T994, CSL2, E8B, MCAMP, 7G8, D10, HB3 and 3D7. The final antibody concentration is 2 mg/ml. (B) Titration of anti-PfRipr/1 antibodies in growth inhibition assays of the FCR3 strain. (C) Titration of anti-PfRipr/1 antibodies in growth inhibition assays of the 3D7 strain. (D) Pre-incubation of purified merozoites from 3D7 strain (left panel) and FCR3 strain (right panel) with protein-A purified antibodies raised against recombinant PfRipr inhibited merozoite attachment to red blood cells. Protein-A purified antibodies from normal serum were used as a negative control. The final antibody concentration is 2 mg/ml. (E) Various combinations of anti-PfRipr/1, EBA-175, PfRh2a/b and PfRh4 antibodies increase inhibitory activity for the 3D7 strain of P. falciparum. The final concentration of each antibody is 1 mg/ml. In all the cases, each graph represents three independent experiments done in triplicate with each normalised to the negative control (Protein A purified IgG from normal rabbit serum). The error bars represent standard error of the mean of the three independent experiments.