Identification and Disruption of the Gene Encoding the Third Member of the Low-Molecular-Mass Rhoptry Complex in Plasmodium falciparum

Abstract

The low-molecular-mass rhoptry complex of Plasmodium falciparum consists of three proteins, rhoptryassociated protein 1 (RAP1), RAP2, and RAP3. The genes encoding RAP1 and RAP2 are known; however, the RAP3 gene has not been identified. In this study we identify the RAP3 gene from the P. falciparum genome database and show that this protein is part of the low-molecular-mass rhoptry complex. Disruption of RAP3 demonstrated that it is not essential for merozoite invasion, probably because RAP2 can complement the loss of RAP3. RAP3 has homology with RAP2, and the genes are encoded on chromosome 5 in a head-to-tail fashion. Analysis of the genome databases has identified homologous genes in all Plasmodium spp., suggesting that this protein plays a role in merozoite invasion. The region surrounding the RAP3 homologue in the Plasmodium yoelii genome is syntenic with the same region in P. falciparum; however, there is a single gene. Phylogenetic comparison of the RAP2/3 protein family from Plasmodium spp. suggests that the RAP2/3 duplication occurred after divergence of these parasite species.The merozoite form of the asexual life cycle in the blood stage of Plasmodium spp. attaches to the surface of the erythrocyte, thus initiating the invasion process (23). Ultrastructural and biochemical studies of merozoites have suggested that the rhoptries play an essential role in the invasion process. These electron-dense organelles, part of the apical complex, are connected to the surface of the apical end of the merozoite by a duct-like structure, and their contents are expelled during erythrocyte invasion (1). Proteins located on the surface of merozoites and those released from the apical organelles, the rhoptries and micronemes, are exposed to the immune system. Consequently, these proteins are potential vaccine candidates, and there is considerable interest in the identification and characterization of proteins in these organelles (2).A number of proteins located in the rhoptries have been identified and characterized, such as the low-molecular-mass rhoptry protein complex of Plasmodium falciparum (28). This complex consists of various polypeptides, ranging in size from 86 to 37 kDa, that are immunoprecipitated by antibodies to rhoptry-associated protein 1 (RAP1) (7,11,29,43). Several studies established that the largest product, reported to be 86 kDa, is a short-lived precursor of the 82-kDa RAP1 protein and that this 82-kDa protein is further processed to a 67-kDa protein (7,26,27,29). The 42-kDa protein (also reported as 39 kDa) was found to be the product of a second gene, RAP2 (7,11,28,39,42). Debate remained regarding the origin of the 40-kDa (or 37-kDa) protein also immunoprecipitated as a part of the RAP1 complex. It was suggested that this protein was a breakdown product of either RAP1 or RAP2 (41). It was subsequently shown, however, that RAP1, RAP2, and RAP3 yield different V8 protease cleavage patterns and are thus products of three distinct genes (28). The RAP2 and RAP3 proteins, in particular, were hypothesiz...

Proteomic Analysis of Rhoptry Organelles Reveals Many Novel Constituents for Host-Parasite Interactions in Toxoplasma gondii

“…Among the 38 novel Toxoplasma rhoptry constituents identified in this study, we failed to detect clear homologues for many known rhoptry proteins of Plasmodium, including RhopH2-3, RAP1-3, PF148, and RAMA1 (42,45,70,74). Consistent with this, homologues for these genes are not apparent in the near-complete Toxoplasma genome, and so they are likely to be truly restricted to Plasmodium and its close relatives.…”

Emerging Rules for Subunit-Based, Multiantigenic, Multistage Chemically Synthesized Vaccines

“…Other equally important rhoptry-localized proteins (found to be invasion mediators) are the rhoptry proteins (Rhop-1-3), 15 rhoptry-associated proteins (RAP1-3), 16 rhoptry H1-148/ FIGURE 1. (A) The P. falciparum malaria-parasite life cycle in its human and mosquito hosts and (B) the merozoite structure with its most important cellular compartments and organelles, as well as the most important molecules involved in invasion of RBCs.…”

Plasmodium falciparum rhoptry protein RSP2 triggers destruction of the erythroid lineage

“…Similar results were obtained for FCR3 CD36 , 10,11 C1-1 var1CSA knockout and C1 CSA , 12 placental strain no. 193, 13 D10, 14 W2mef, 16 and W2mef⌬RAP3, 17 but not with parasites unable to tag cells with RSP2 (D10⌬RAP1) 15 (data not shown). Thus, the C3 labeling of erythroblasts required tagging with a cytophilic anti-RSP2 antibody.…”

“…The parasites used in this study were FCR3 CSA , FCR3 CD36 , 10,11 C1-1 var1CSA knockout, C1 CSA , 12 placental strain no. 193 from Cameroon, 13 D10, 14 D10⌬RAP1 (lacking RAP1 and RAP2 on its surface), 15 W2mef, 16 and W2mef⌬RAP3 (lacking only RAP3) 17 (a gift from A. F. Cowman, The Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia).…”