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Evidence for negative selection on the gene encoding rhoptry-associated protein 1 (RAP-1) in Plasmodium spp.

ABSTRACT: Assessing how natural selection, negative or positive, operates on genes with low polymorphism is challenging. We investigated the genetic diversity of orthologous genes encoding the rhoptry-associated protein 1 (RAP-1), a low polymorphic protein of malarial parasites that is involved in erythrocyte invasion. We applied evolutionary genetic methods to study the polymorphism in RAP-1 from Plasmodium falciparum (n=32) and Plasmodium vivax (n=6), the two parasites responsible for most human malaria morbidity and mortality, as well as RAP-1 orthologous in closely related malarial species found in non-human primates (NHPs). Overall, genes encoding RAP-1 are highly conserved in all Plasmodium spp. included in this investigation. We found no evidence for natural selection, positive or negative, acting on the gene encoding RAP-1 in P. falciparum or P. vivax. However, we found evidence that the orthologous genes in non-human primate parasites (Plasmodium cynomolgi, Plasmodium inui, and Plasmodium knowlesi) are under purifying (negative) selection. We discuss the importance of considering negative selection while studying genes encoding proteins with low polymorphism and how selective pressures may differ among orthologous genes in closely related malarial parasites species.

SUBMITTER: Pacheco MA

PROVIDER: S-EPMC2881667 | biostudies-literature | 2010 Jul

REPOSITORIES: biostudies-literature

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Evidence for negative selection on the gene encoding rhoptry-associated protein 1 (RAP-1) in Plasmodium spp.

Pacheco M Andreína MA Ryan Elizabeth M EM Poe Amanda C AC Basco Leonardo L Udhayakumar Venkatachalam V Collins Williams E WE Escalante Ananias A AA

Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases 20100402 5

Assessing how natural selection, negative or positive, operates on genes with low polymorphism is challenging. We investigated the genetic diversity of orthologous genes encoding the rhoptry-associated protein 1 (RAP-1), a low polymorphic protein of malarial parasites that is involved in erythrocyte invasion. We applied evolutionary genetic methods to study the polymorphism in RAP-1 from Plasmodium falciparum (n=32) and Plasmodium vivax (n=6), the two parasites responsible for most human malaria ...[more]

PMID: 20363375

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Project description:The Plasmodium rhoptry-associated protein 1 (RAP-1) plays a role in the formation of the parasitophorous vacuole following the parasite's invasion of red blood cells. Although there is some evidence that the protein is recognized by the host's immune system, study of Plasmodium falciparum RAP-1 (PfRAP-1) suggests that it is not under immune pressure. A previous study on five old (1953-1962) P. knowlesi strains suggested that RAP-1 has limited genetic polymorphism and might be under negative selection. In the present study, 30 recent P. knowlesi isolates were studied to obtain a better insight into the polymorphism and natural selection of PkRAP-1.Blood samples from 30 knowlesi malaria patients were used. These samples were collected between 2010 and 2014. The PkRAP-1 gene, which contains two exons, was amplified by PCR, cloned into Escherichia coli and sequenced. Genetic diversity and phylogenetic analyses were performed using MEGA6 and DnaSP ver. 5.10.00 programs.Thirty PkRAP-1 sequences were obtained. The nucleotide diversity (?) of exons 1, 2 and the total coding region (0.00915, 0.01353 and 0.01298, respectively) were higher than those of the old strains. Further analysis revealed a lower rate of non-synonymous (dN) than synonymous (dS) mutations, suggesting negative (purifying) selection of PkRAP-1. Tajima's D test and Fu and Li's D test values were not significant. At the amino acid level, 22 haplotypes were established with haplotype H7 having the highest frequency (7/34, 20.5 %). In the phylogenetic analysis, two distinct haplotype groups were observed. The first group contained the majority of the haplotypes, whereas the second had fewer haplotypes.The present study found higher genetic polymorphism in the PkRAP-1 gene than the polymorphism level reported in a previous study. This observation may stem from the difference in sample size between the present (n = 30) and the previous (n = 5) study. Synonymous and non-synonymous mutation analysis indicated purifying (negative) selection of the gene. The separation of PkRAP-1haplotypes into two groups provides further evidence to the postulation of two distinct P. knowlesi types or lineages.

Project description:Immune responses to Plasmodium falciparum rhoptry-associated protein 1 (RAP-1), RAP-2, and RAP-3 appear to contribute to protection against infection by this human malarial parasite. This conclusion is suggested by results of monkey immunization trials and of cell culture studies showing antibody-dependent inhibition of erythrocyte invasion. In the present study, splenectomized owl monkeys were infected with P. falciparum in order to monitor anti-RAP-1 antibody production as antiparasite immunity developed. The monkeys responded to a primary infection with the production of antibodies to a fragment of RAP-1 containing amino acids 1 to 294 (RAP-1(1-294)). After drug cure and reinfection, the monkeys had a prolonged prepatent period, indicating they had already developed partial immunity to the parasite. Sera from these animals showed major increases in anti-RAP-1(1-294) antibodies. In contrast, only low levels of antibodies to inhibitory B-cell epitope 1 (iB-1), an inhibitory epitope in RAP-1(1-294) with the sequence N200TLTPLEELYPT211, was observed after the initial parasite infection, and the anti-iB-1 antibodies were not readily boosted upon reinfection. These results suggest that iB-1 is an immunogenic but not immunodominant epitope and that anti-iB-1 antibodies do not substantially contribute to early stages of naturally acquired immunity in the owl monkey model. To identify additional epitopes bound by inhibitory antibodies, mouse monoclonal antibodies were produced with a recombinant fusion protein containing RAP-1(1-294). Monoclonal antibody 1D6 inhibited parasite invasion of erythrocytes in vitro. 1D6 did not bind peptide iB-1 but rather bound a second inhibitory epitope called iB-2. iB-2, like iB-1, is found near the amino terminus of p67, a RAP-1 processing product thought to be involved in merozoite invasion of erythrocytes. Since anti-iB-1 antibodies were not readily produced during parasite infection, it may be desirable to direct antibody responses to particular epitopes in RAP-1, such as iB-1 and iB-2.

Project description:Evidence for natural selection, positive or negative, on gene encoding antigens may indicate variation or functional constraints that are immunologically relevant. Most malaria surface antigens with high genetic diversity have been reported to be under positive-diversifying selection. However, antigens with limited genetic variation are usually ignored in terms of the role that natural selection may have in generating such patterns. We investigated orthologous genes encoding two merozoite proteins, MSP8 and MSP10, among several mammalian Plasmodium spp. These antigens, together with MSP1, are among the few MSPs that have two epidermal growth factor-like domains (EGF) at the C-terminal. Those EGF are relatively conserved (low levels of genetic polymorphism) and have been proposed to act as ligands during the invasion of RBCs. We use several evolutionary genetic methods to detect patterns consistent with natural selection acting on MSP8 and MSP10 orthologs in the human parasites Plasmodium falciparum and P. vivax, as well as closely related malarial species found in non-human primates (NHPs). Overall, these antigens have low polymorphism in the human parasites in comparison with the orthologs from other Plasmodium spp. We found that the MSP10 gene polymorphism in P. falciparum only harbor non-synonymous substitutions, a pattern consistent with a gene under positive selection. Evidence of purifying selection was found on the polymorphism observed in both orthologs from P. cynomolgi, a non-human primate parasite closely related to P. vivax, but it was not conclusive in the human parasite. Yet, using phylogenetic base approaches, we found evidence for purifying selection on both MSP8 and MSP10 in the lineage leading to P. vivax. Such antigens evolving under strong functional constraints could become valuable vaccine candidates. We discuss how comparative approaches could allow detecting patterns consistent with negative selection even when there is low polymorphism in the extant populations.

Project description:BACKGROUND: Rhoptries are specialized organelles from parasites belonging to the phylum Apicomplexa; they secrete their protein content during invasion of host target cells and are sorted into discrete subcompartments within rhoptry neck or bulb. This distribution is associated with these proteins' role in tight junction (TJ) and parasitophorous vacuole (PV) formation, respectively. METHODS: Plasmodium falciparum RON2 amino acid sequence was used as bait for screening the codifying gene for the homologous protein in the Plasmodium vivax genome. Gene synteny, as well as identity and similarity values, were determined for ron2 and its flanking genes among P. falciparum, P. vivax and other malarial parasite genomes available at PlasmoDB and Sanger Institute databases. Pvron2 gene transcription was determined by RT-PCR of cDNA obtained from the P. vivax VCG-1 strain. Protein expression and localization were assessed by Western blot and immunofluorescence using polyclonal anti-PvRON2 antibodies. Co-localization was confirmed using antibodies directed towards specific microneme and rhoptry neck proteins. RESULTS AND DISCUSSION: The first P. vivax rhoptry neck protein (named here PvRON2) has been identified in this study. PvRON2 is a 2,204 residue-long protein encoded by a single 6,615 bp exon containing a hydrophobic signal sequence towards the amino-terminus, a transmembrane domain towards the carboxy-terminus and two coiled coil ?-helical motifs; these are characteristic features of several previously described vaccine candidates against malaria. This protein also contains two tandem repeats within the interspecies variable sequence possibly involved in evading a host's immune system. PvRON2 is expressed in late schizonts and localized in rhoptry necks similar to what has been reported for PfRON2, which suggests its participation during target cell invasion. CONCLUSIONS: The identification and partial characterization of the first P. vivax rhoptry neck protein are described in the present study. This protein is homologous to PfRON2 which has previously been shown to be associated with PfAMA-1, suggesting a similar role for PvRON2.

Dataset Information

Evidence for negative selection on the gene encoding rhoptry-associated protein 1 (RAP-1) in Plasmodium spp.

Publications

Evidence for negative selection on the gene encoding rhoptry-associated protein 1 (RAP-1) in Plasmodium spp.

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