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, rhoptry-associated 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.

FIG. 1.

Sequence alignment of PfRAP2, PfRAP3, and the RAP2/3 homologues from P. yoelii, P. berghei, P. knowlesi, and P. vivax. Identical residues are shown in black, whereas conserved residues are shown in gray. Conserved cysteine residues are indicated by asterisks. The putative signal cleavage site is indicated by an arrow. PLAFA, P. falciparum; PLAYO, P. yoelii; PLABE, P. berghei; PLAKN, P. knowlesi; PLAVI, P. vivax.

FIG. 2.

Organization of the chromosomal region around the RAP2, RAP3, and RAP2/3 genes of P. falciparum and P. yoelii. The nuc2+ gene encodes a nuclear scaffold-like protein of fission yeast. The FIRA gene encodes the P. falciparum interspersed repeat antigen. The sbp1 gene encodes skeleton binding protein 1.

FIG. 3.

Disruption of the RAP3 gene. (A) Integration of the pHH1ΔRAP3 plasmid by single-site homologous recombination produces a “pseudodiploid,” in which the upstream copy of RAP3 is truncated while the downstream copy lacks a promoter element and has a mutated start codon. Two copies of the plasmid have been integrated as shown. E, EcoRI; B, BsrGI. (B) Hybridization of a pulsed-field gel electrophoresis blot, containing separated P. falciparum chromosomes, with pGem plasmid sequences alone (left panel) reveals multiple transgenic sequences in W2mefΔRAP3/0, corresponding to episomal plasmid DNA. After cycling of cultures with or without WR99210, stable parasite line W2mefΔRAP3/1 exhibits a band that comigrates with chromosome 5 along with a more slowly migrating band that does not correspond to any chromosome. Two independent clonal isolates derived from the W2mefΔRAP3/1 transfectants, W2mefΔRAP3c1 and W2mefΔRAP3c2, exhibit a single band that comigrates with chromosome 5. The upper band on both blots corresponds to DNA remaining in the wells. Hybridization of an identical blot with labeled RAP3 (right) confirms that the pGem sequences are present on the same chromosome as RAP3. (C) Digestion of genomic DNA with EcoRI or BsrGI confirms that the RAP3 gene has been disrupted in W2mefΔRAP3c1 and W2mefΔRAP3c2, yielding the expected restriction pattern.

FIG. 4.

Transcription of the RAP3 gene is disrupted in W2mefΔRAP3c1 and W2mefΔRAP3c2. (A) Schematic of the wild-type RAP3 gene, showing the locations of the wild-type-specific primers and the expected 916-bp transcript as well as the locations of the mutant-specific primers and the expected 1,044-bp transcript. Restriction sites shown: E, EcoRI; B, BamHI. (B) PCR products obtained from W2mef wild-type cDNA and genomic DNA show that the RAP3 gene is transcribed in W2mef parasites. Lanes 1, 4, and 5, wild-type primers; lanes 2 and 6, mutant primers; lane 3, DHPS control primers. (C) W2mefΔRAP3c1 cDNA and genomic DNA PCR products indicate that wild-type RAP3 transcripts are not made in ΔRAP3 mutant parasites. Instead, a mutant transcript is produced, which suggests that a truncated RAP3 protein may be expressed. Lanes 1 and 5, wild-type primers; lanes 2, 4, and 6, mutant primers; lane 3, DHPS control primers. (D) PCR products from W2mefΔRAP3c2 cDNA and genomic DNA also show that a mutant transcript but not a wild-type transcript is produced. Lanes 1 and 5, wild-type primers; lanes 2, 4, and 6, mutant primers; lane 3, DHPS control primers. gDNA, genomic DNA; RT, reverse transcriptase.

FIG. 5.

Immunoprecipitation of the RAP complex from W2mef, W2mefΔRAP3c1, and W2mefΔRAP3c2. Purified trophozoites were metabolically labeled for 6 h with [³⁵S]methionine and immunoprecipitated with monoclonal antibody 7H8/50 (left panel); background binding to protein G beads alone is shown (center panel), and total labeled protein is also shown (right panel). Both RAP2 (42 kDa) and RAP3 (40 kDa) were coprecipitated with RAP1 (82 and 67 kDa) in wild-type W2mef parasites. In the ΔRAP3 mutant parasites the 40-kDa RAP3 protein no longer coprecipitates with RAP1 and RAP2.

FIG. 6.

Subcellular localization of RAP1 and RAP2 in W2mef and W2mefΔRAP3c1. Antibodies to RAP1 (green) or RAP2 (red) were incubated with either W2mef or W2mefΔRAP3c1. The yellow in the merged image indicates colocalization.

FIG. 7.

Phylogenetic analysis of the RAP2/3 proteins from Plasmodium spp. A phylogram was generated by using the neighbor-joining algorithm implemented in ClustalX 1.81 of the multiple alignment depicted in Fig. 1. The bootstrap values are shown on the branches and indicate the number of times out of 1,000 replications that the branching was supported. This tree is unrooted, as no outgroup homologous protein exist.