RALP1 Is a Rhoptry Neck Erythrocyte-Binding Protein of Plasmodium falciparum Merozoites and a Potential Blood-Stage Vaccine Candidate Antigen

Abstract

f Erythrocyte invasion by merozoites is an obligatory stage of Plasmodium infection and is essential to disease progression. Proteins in the apical organelles of merozoites mediate the invasion of erythrocytes and are potential malaria vaccine candidates. Rhoptry-associated, leucine zipper-like protein 1 (RALP1) of Plasmodium falciparum was previously found to be specifically expressed in schizont stages and localized to the rhoptries of merozoites by immunofluorescence assay (IFA). Also, RALP1 has been refractory to gene knockout attempts, suggesting that it is essential for blood-stage parasite survival. These characteristics suggest that RALP1 can be a potential blood-stage vaccine candidate antigen, and here we assessed its potential in this regard. Antibodies were raised against recombinant RALP1 proteins synthesized by using the wheat germ cell-free system. Immunoelectron microscopy demonstrated for the first time that RALP1 is a rhoptry neck protein of merozoites. Moreover, our IFA data showed that RALP1 translocates from the rhoptry neck to the moving junction during merozoite invasion. Growth and invasion inhibition assays revealed that anti-RALP1 antibodies inhibit the invasion of erythrocytes by merozoites. The findings that RALP1 possesses an erythrocyte-binding epitope in the C-terminal region and that anti-RALP1 antibodies disrupt tight-junction formation, are evidence that RALP1 plays an important role during merozoite invasion of erythrocytes. In addition, human sera collected from areas in Thailand and Mali where malaria is endemic recognized this protein. Overall, our findings indicate that RALP1 is a rhoptry neck erythrocyte-binding protein and that it qualifies as a potential blood-stage vaccine candidate.

Antibodies against a short region of PfRipr inhibit Plasmodium falciparum merozoite invasion and PfRipr interaction with Rh5 and SEMA7A

“…The proteins were expressed with WGCFS (CellFree Sciences) as N-terminal glutathione S-transferase (GST) fusion proteins with C-terminal His-tags as described 49 . Ecto-PfRipr and PfRipr truncate recombinant proteins were purified using a Ni 2+ Sepharose affinity column (GE Healthcare) as described 50 .…”

“…schizont-rich parasite pellets from P. falciparum 3D7 strain was conducted as described 50 . Additional details are included in the Supplementary Methods.…”

Host Cell Invasion by Apicomplexan Parasites: The Junction Conundrum

“…This is the case of several members of the micronemal erythrocyte binding like (EBL) family of proteins, such as Plasmodium falciparum erythrocyte-binding antigen 175 (EBA175) [35] and P. knowlesi Duffy binding protein (DBP) [36]. This is also the case of several rhoptry proteins, including the P. falciparum reticulocyte-binding like (RBL) homologue PfRh1 [37], PfRh2a [38], and PfRh5 [39], and of the rhoptry-associated leucine zipper-like protein 1 (RALP1) [40]. However, function is difficult to assess in the Plasmodium merozoite, typically relying on antibody inhibition and negative transfection experiments.…”

Immunoscreening of Plasmodium falciparum proteins expressed in a wheat germ cell-free system reveals a novel malaria vaccine candidate

“…As previously described35, we generated rabbit polyclonal antisera against LSA3-C (V 750 to K 1433 of the 3D7 sequence; PF3D7_0220000) and mouse antisera against AMA1 (Q 25 to K 546 ; PF3D7_1133400), RAP1 (M 1 to D 782 ; PF3D7_1410400), and RON2 (K 84 to Q 968 ; PF3D7_1452000). All antigens were synthesized by WGCFS and purified by GST or hexa-histidine (His) protein-tag fused with the N-terminus of the recombinant proteins.…”

“…IFA was conducted as previously described35. The samples were stained with primary antibodies diluted at the following concentrations in blocking solution at 37 °C for 1 h: human anti-LSA3-C antibody, 0.173 μg/ml; rabbit anti-LSA3-C antibody, 1:500; mouse anti-AMA1 antibody, 1:100; mouse anti-RAP1 antibody, 1:1,000; mouse anti-RON2 antibody, 1:100; mouse anti-RESA monoclonal antibody, 1:100.…”