The high molecular mass rhoptry protein, RhopH1, is encoded by members of the clag multigene family in Plasmodium falciparum and Plasmodium yoelii.

Malarial merozoite rhoptries contain a high molecular mass protein complex called RhopH. RhopH is composed of three polypeptides, RhopH1, RhopH2, and RhopH3, encoded by distinct genes. Using monoclonal antibody-purified protein complex from both Plasmodium falciparum and Plasmodium yoelii, peptides were obtained by digestion of RhopH1 and their sequence determined either by mass spectrometry or Edman degradation. In both species the genes encoding RhopH1 were identified as members of the cytoadherence linked asexual gene (clag) family. In P. falciparum the family members on chromosome 3 were identified as encoding RhopH1. In P. yoelii two related genes were identified and sequenced. One of the genes, pyrhoph1a, was positively identified as encoding RhopH1 by the peptide analysis and the other gene, pyrhoph1a-p, was at least transcribed. Genes in the clag family present in both parasite species have a number of conserved features. The size and location of the P. yoelii protein complex in the rhoptries was confirmed. The first clag gene identified on chromosome 9 was implicated in cytoadherence, the binding of infected erythrocytes to host endothelial cells; this study shows that other members of the family encode merozoite rhoptry proteins, proteins that may be involved in merozoite-erythrocyte interactions. We propose that the family should be renamed as rhoph1/clag.     

Orthologous gene sequences of merozoite surface protein 1 (MSP1) from Plasmodium reichenowi and P. gallinaceum confirm an ancient divergence of P. falciparum alleles

Merozoite surface protein 1 (MSP 1) of Plasmodium falciparum has a major allelic dimorphism in the majority of its sequence, the origin and significance of which is obscure. Here, the cloning and sequencing of the msp1 gene from P. reichenowi (a chimpanzee parasite that is the nearest relative of P. falciparum) and P. gallinaceum (a malaria parasite of birds) is reported. P. reichenowi msp1 is most closely related to one allelic type (K1) of P. falciparum. The other P. falciparum major allelic type (MAD20) is very divergent from these sequences, although not as divergent as msp1 of P. gallinaceum. Assuming a date of 6 million years ago (mya) for the divergence of the P. falciparum K1 and the P. reichenowi msp1 genes (on the basis of previous estimates for these parasite species as well as host divergence times), the most recent common ancestor of the dimorphic region of msp1 would date to approximately 27mya. Thus, the P. falciparum msp1 dimorphism is confirmed as one of the oldest polymorphisms known with the exception of self-incompatibility S genes in Solanaceae. In contrast with the major allelic dimorphism, the polymorphisms present in the relatively conserved C terminus of P. falciparum msp1 appear to have arisen since the divergence of the P. falciparum and P. reichenowi msp1 genes.     

Global genetic diversity and evolution of var genes associated with placental and severe childhood malaria

In Plasmodium falciparum, var genes encode adhesive proteins that are transported to the surface of infected erythrocytes and act as major virulence determinants for infected erythrocyte binding and immune evasion. Var genes are highly diverse and can be classified into five major groups (UpsA, B, C, D, and E). Previous serological studies have suggested that the UpsA var group may contain common antigenic types that have important roles in severe childhood malaria. Here, our analysis found that UpsA vars are highly diverse between 22 world-wide parasite isolates, although they could be grouped into two broad clusters that may be separately recombining. By comparison, orthologs of the UpsA-linked Type 3 var and UpsE-linked var2csa were detected in nearly all parasite isolates, and a var2csa ortholog was also present in a chimpanzee malaria P. reichenowi that diverged from P. falciparum approximately 5-7 million years ago. Although the specific function of Type 3 var genes is unknown, var2csa is a leading candidate for a pregnancy associated malaria vaccine. Compared to typical var genes, var2csa is unusually conserved but still had only 54-94% amino acid identity in extracellular binding regions. However, var2csa alleles have extensive gene mosaicism within polymorphic blocks that are shared between world-wide parasite isolates and recognizable in P. rechenowi suggesting a high rate of self-self recombination and an ancient and globally-related pool of var2csa polymorphism. These studies aid our understanding of the evolutionary mechanisms that shape var diversity and will be important to the development of vaccines against pregnancy associated malaria and severe malaria.     

Characterisation of the rhoph2 gene of Plasmodium falciparum and Plasmodium yoelii

The high molecular mass protein complex (RhopH) in the rhoptries of the malaria parasite consists of three distinct polypeptides with estimated sizes in Plasmodium falciparum of 155kDa (PfRhopH1), 140kDa (PfRhopH2) and 110kDa (PfRhopH3). Using a number of reagents, including a new mAb 4E10 that is specific for the PfRhopH complex, it was shown that the RhopH complex is synthesised during schizogony and transferred intact to the ring stage in newly invaded erythrocytes. The genes encoding RhopH1 and RhopH3 have already been identified and characterised in both P. falciparum and Plasmodium yoelii. In this report, we describe the identification of the gene for RhopH2 in both these parasite species. Peptide sequences were obtained from purified RhopH2 proteins and used to generate oligonucleotide primers and search malaria sequence databases. In a parallel approach, mAb 4E10 was used to identify a clone coding for RhopH2 from a P. falciparum cDNA library. The sequences of both P. falciparum and P. yoelii genes for RhopH2 were completed and compared. They both contain nine introns and there is a high degree of similarity between the deduced amino acid sequences of the two proteins. The P. falciparum gene is a single copy gene located on chromosome 9, and is transcribed in schizonts.     

Structural polymorphism and diversifying selection on the pregnancy malaria vaccine candidate VAR2CSA

VAR2CSA is the main candidate for a pregnancy malaria vaccine, but vaccine development may be complicated by sequence polymorphism. Here, we obtained partial or full-length var2CSA sequences from 106 parasites and applied novel computational methods and three-dimensional modeling to investigate VAR2CSA geographic variation and selection pressure. Our analysis reveals structural patterns of VAR2CSA sequence variation in which polymorphic sites group into segments of limited diversity. Within these segments, two or three basic types characterize a substantial majority of the parasite samples. Comparison to the primate malaria Plasmodium reichenowi shows that these basic types have ancient origins. Globally, var2CSA genes are comprised of a mosaic of these ancestral polymorphic segments that have recombined extensively between var2CSA alleles. Three-dimensional modeling reveals that polymorphic segments concentrate in flexible loops at characteristic locations in the six VAR2CSA Duffy binding-like (DBL) adhesion domains. Individual DBL domain surfaces have distinct patterns of diversifying selection, suggesting that limited and differing portions of each DBL domain are targeted by host antibody. Since standard phylogenetic tree analysis is inadequate for highly recombining genes like var2CSA, we developed a novel phylogenetic approach that incorporates recombination and tracks new mutations in segment types. In the resulting tree, P. reichenowi is confirmed as an outlier and African and Asian P. falciparum isolates have slightly diverged. These findings validate a new approach to modeling protein evolution in the presence of frequent recombination and provide a clearer understanding of how var gene products function as immunoevasive binding ligands.     

Evidence of non-neutral polymorphism in Plasmodium falciparum gamete surface protein genes Pfs47 and Pfs48/45

Targeted disruption of particular members of the Plasmodium 6-cys protein gene family, including Ps47, Ps48/45 and Ps230, is known to dramatically affect parasite fertility. Because loci critical to fertility in many eukaryote species have been shown to be under strong positive selection, we examined sequence variation in four members of the 6-cys protein gene family in Plasmodium falciparum (Pfs36, Pfs38, Pfs47 and Pfs48/45) to determine whether genetic variation in these loci may be of functional significance. Sequence polymorphism among 11 laboratory isolates of P. falciparum was compared with divergence from the respective orthologues in the closely related species P. reichenowi, showing an almost significant skew towards within-species non-synonymous polymorphism in Pfs47 and Pfs48/45 (by the McDonald-Kreitman test) but clearly non-significant results for Pfs36 and Pfs38. A preliminary analysis of Pfs47 sequence polymorphism in field isolates of P. falciparum showed exceptionally high fixation indices (F(ST)) among geographically distinct populations, similar to results seen previously for Pfs48/45. Therefore, both Pfs47 and Pfs48/45 were further analysed by sequencing polymorphic parts of the genes from a Tanzanian population sample of oocysts (a means of analysing diploid genotypes). Both genes displayed higher inbreeding coefficients (F(IS)) compared with the average of 11 unlinked microsatellite loci. These results suggest that allelic variation in these two genes may be functionally significant in influencing mating interactions, a hypothesis that could be tested by fertilization experiments with targeted allelic replacement.     

Polymorphism in the gene encoding the Pfs48/45 antigen of Plasmodium falciparum. XI. Asembo Bay Cohort Project.

We have investigated the genetic diversity of the gene encoding the transmission-blocking vaccine antigen Pfs48/45 of Plasmodium falciparum parasites from western Kenya and compared it with parasite populations from Thailand, India, and Venezuela. We report 44 complete new sequences. Overall, the antigen is less polymorphic as compared with other pre-erythrocytic and blood stage antigens. Contrary to other P. falciparum antigens, the number of synonymous substitutions per synonymous site exceeds the number of non-synonymous substitutions per non-synonymous site. We have found that the Pfs48/45 gene of Kenyan parasites is more polymorphic than parasites from other geographic origins. Our analysis reveals that positive natural selection is involved in the maintenance of the observed polymorphism. No evidence of intragenic recombination was found. F(st) values reveal high levels of gene flow between India and Thailand, however, there are strong constraints in gene flow among Kenyan, Southeast Asian, and Venezuelan parasites. No alleles could be linked to a specific geographic region. The results of this study suggest that this gametocyte antigen, like other asexual blood stage antigens, is under selection pressure.     

Molecular characterisation of Plasmodium reichenowi apical membrane antigen-1 (AMA-1), comparison with P. falciparum AMA-1, and antibody-mediated inhibition of red cell invasion

Apical membrane antigen 1 is a candidate vaccine component for malaria. It is encoded by a single copy gene and has been characterised in a number of malaria species as either an 83-kDa de novo product (Plasmodium falciparum; Pf AMA-1) or a 66-kDa product (all other species). All members of the AMA-1 family are expressed during merozoite formation in maturing schizonts and are initially routed to the rhoptries. Processed forms may subsequently be associated with the merozoite surface. Because of the unique occurrence of the 83-kDa form in P. falciparum we were interested to determine whether the phylogenetically closely related chimpanzee malaria Plasmodium reichenowi shared characteristics with Pf AMA-1. Here we show that the molecular structure, the localisation and processing are similar to that of Pf AMA-1 and that in vitro growth inhibitory mAbs reactive with Pf AMA-1 also inhibit P. reichenowi growth in an in vitro assay. Polymorphism in the 83-kDa AMA-1 family was analysed through comparison of Pr ama-1 with Pf ama-1 alleles, which showed the most significant evidence for selection maintaining polymorphism in Domains I-III of AMA-1 in P. falciparum. The most substantial divergence between Pr AMA-1 and Pf AMA-1 sequences was in the N-terminal region unique to the 83-kDa form of AMA-1. It was confirmed that the specific Pr ama-1-type allele was not present among P. falciparum parasites in an African population, and an allele coding for lysine at amino acid 187 was uniquely associated with field isolates in this population.     

Patterns of polymorphism in genomic regions flanking three highly polymorphic surface antigens in Plasmodium falciparum

Many surface antigens of the human malaria parasite Plasmodium falciparum show extraordinary diversity, with different alleles being so divergent as to be unalignable in some coding regions. To better understand the population history and modes of selection on such loci, we sequenced genomic regions flanking the highly polymorphic genes merozoite surface protein-1, merozoite surface protein-2, and circumsporozoite protein, from reference isolates of P. falciparum. Diversity was much lower in genomic flanking regions than in the coding sequences. Average pairwise nucleotide diversity for these regions was 0.00088, similar to other genomic regions not thought to be evolving under balancing selection, suggesting against balancing selection acting on promoter regions of these genes. Most observed polymorphisms were singletons. A higher ratio of SNPs to indels than previously reported for P. falciparum was observed. An 11 bp repeat upstream of msp2 showed an intriguing pattern of polymorphism possibly suggestive of purifying selection on total allele length.     

Polymorphism study of rhoptry associated membrane antigen (RAMA) gene of Plasmodium falciparum--a putative vaccine candidate

The antigenic diversity of Plasmodium falciparum is one of the major obstacles to antimalarial vaccine development. Thus, it becomes obvious to search for a protein that is fairly conserved and is necessary for the parasite to survive in the host cell. Rhoptry associated membrane antigen (RAMA) plays a potential role in parasite biology and invasion. The C-terminal end of RAMA is shown to have protective immune responses and binds to the erythrocyte membrane. In this work, we have studied the polymorphism of RAMA gene in the C-terminal end from 1525 to 3196 nucleotide (nt) in 230 samples. The presence of few variants suggests RAMA to be under a balancing selection. The above criterion of restricted antigenic diversity and a protective immune response towards the C-terminal end makes RAMA a strong vaccine candidate.     

Recent independent evolution of msp1 polymorphism in Plasmodium vivax and related simian malaria parasites

The Plasmodium MSP-1 is a promising malaria vaccine candidate. However, the highly polymorphic nature of the MSP-1 gene (msp1) presents a potential obstacle for effective vaccine development. To investigate the evolutionary history of msp1 polymorphism in P. vivax, we construct phylogenetic trees of msp1 from P. vivax and related monkey malaria parasite species. All P. vivax msp1 alleles cluster in the P. vivax lineage and are not distributed among other species. Similarly, all P. cynomolgi msp1 alleles cluster in the P. cynomolgi lineage. This suggests that, in contrast to presumed ancient origin of P. falciparum msp1 polymorphism, the origin of P. vivax msp1 polymorphism is relatively recent. We observed positive selection in the P. vivax lineage but not in P. cynomolgi. Also, positive selection acts on different regions of msp1 in P. vivax and P. falciparum. This study shows that the evolutionary history of msp1 differs greatly among parasite lineages.     

The sequence of a 200 kb portion of a Plasmodium vivax chromosome reveals a high degree of conservation with Plasmodium falciparum chromosome 3.

Within a 199,866 base pair (bp) portion of a Plasmodium vivax chromosome we identified a conserved linkage group consisting of at least 41 genes homologous to Plasmodium falciparum genes located on chromosome 3. There were no P. vivax homologues of the P. falciparum cytoadherence-linked asexual genes clag 3.2, clag 3.1 and a var C pseudogene found on the P. vivax chromosome. Within the conserved linkage group, the gene order and structure are identical to those of P. falciparum chromosome 3. This conserved linkage group may extend to as many as 190 genes. The subtelomeric regions are different in size and the P. vivax segment contains genes for which no P. falciparum homologues have been identified to date. The size difference of at least 900 kb between the homologous P. vivax chromosome and P. falciparum chromosome 3 is presumably due to a translocation. There is substantial sequence divergence with a much higher guanine+cytosine (G+C) content in the DNA and a preference for amino acids using GC-rich codons in the deduced proteins of P. vivax. This structural conservation of homologous genes and their products combined with sequence divergence at the nucleotide level makes the P. vivax genome a powerful tool for comparative analyses of Plasmodium genomes.     

Origin of Plasmodium falciparum malaria is traced by mitochondrial DNA

The origin and geographical spread of Plasmodium falciparum is here determined by analysis of mitochondrial DNA sequence polymorphism and divergence from its most closely related species P. reichenowi (a rare parasite of chimpanzees). The complete 6 kb mitochondrial genome was sequenced from the single known isolate of P. reichenowi and from four different cultured isolates of P. falciparum, and aligned with the two previously derived P. falciparum sequences. The extremely low synonymous nucleotide polymorphism in P. falciparum (pi=0.0004) contrasts with the divergence at such sites between the two species (kappa=0.1201), and supports a hypothesis that P. falciparum has recently emerged from a single ancestral population. To survey the geographical distribution of mitochondrial haplotypes in P. falciparum, 104 isolates from several endemic areas were typed for each of the identified single nucleotide polymorphisms. The haplotypes show a radiation out of Africa, with unique types in Southeast Asia and South America being related to African types by single nucleotide changes. This indicates that P. falciparum originated in Africa and colonised Southeast Asia and South America separately.     

Polymorphism in the gene encoding the apical membrane antigen-1 (AMA-1) of Plasmodium falciparum. X. Asembo Bay Cohort Project

We have investigated the genetic diversity of the gene encoding the apical membrane antigen-1 (AMA-1) in natural populations of Plasmodium falciparum from western Kenya and compared it with parasite populations from other geographic regions. A total of 28 complete sequences from Kenya, Thailand, India, and Venezuela field isolates were obtained. The genetic polymorphism is not evenly distributed across the gene, which is in agreement with the pattern reported in earlier studies. The alleles from Kenya exhibit 20 and 30% more polymorphism than that found in Southeast Asia and Venezuelan alleles, respectively. Based on the gene genealogies derived from sequencing data, no evidence for allele families was found. We have found evidence supporting limited gene flow between the parasite populations, specifically, between the Southeast Asian and Venezuelan isolates; however, no alleles could be linked to a specific geographic region. This study reveals that positive natural selection is an important factor in the maintenance of genetic diversity for AMA-1. We did not find conclusive evidence indicating intragenic recombination is important in the generation of the AMA-1 allelic diversity. The study provides information on the genetic diversity of the AMA-1 gene that would be useful in vaccine development and testing, as well as in assessing factors that are involved in the generation and maintenance of the genetic diversity in P. falciparum.     

The RhopH complex is transferred to the host cell cytoplasm following red blood cell invasion by Plasmodium falciparum

The high-molecular mass rhoptry protein complex (PfRhopH), which comprises three distinct gene products, RhopH1, RhopH2, and RhopH3, is known to be secreted and transferred to the parasitophorous vacuole membrane upon invasion of a red blood cell by the malaria parasite Plasmodium falciparum. Here we show that the merozoite-acquired RhopH complex is also transferred to defined domains of the red blood cell cytoplasm, and possibly transiently associated with Maurer's clefts. This is the first report of trafficking in the host cell cytoplasm for P. falciparum rhoptry proteins secreted upon red blood cell invasion. Based on its newly identified sub-cellular location and the phenotype of RhopH1 mutants, we propose that the RhopH complex participate in the assembly of the cytoadherence complex.     

In vitro studies with recombinant Plasmodium falciparum apical membrane antigen 1 (AMA1): production and activity of an AMA1 vaccine and generation of a multiallelic response

Apical membrane antigen 1 (AMA1) is regarded as a leading malaria blood-stage vaccine candidate. While the overall structure of AMA1 is conserved in Plasmodium spp., numerous AMA1 allelic variants of P. falciparum have been described. The effect of AMA1 allelic diversity on the ability of a recombinant AMA1 vaccine to protect against human infection by different P. falciparum strains is unknown. We characterize two allelic forms of AMA1 that were both produced in Pichia pastoris at a sufficient economy of scale to be usable for clinical vaccine studies. Both proteins were used to immunize rabbits, singly and in combination, in order to evaluate their immunogenicity and the ability of elicited antibodies to block the growth of different P. falciparum clones. Both antigens, when used alone, elicited high homologous anti-AMA1 titers, with reduced strain cross-reactivity. Similarly, sera from rabbits immunized with a single antigen were capable of blocking the growth of homologous parasite strains at levels theoretically sufficient to clear parasite infections. However, heterologous inhibition was significantly reduced, providing experimental evidence that AMA1 allelic diversity is a result of immune pressure. Encouragingly, rabbits immunized with a combination of both antigens exhibited titers and levels of parasite inhibition as good as those of the single-antigen-immunized rabbits for each of the homologous parasite lines, and consequently exhibited a broadening of allelic diversity coverage.     

Rapid evolution of an erythrocyte invasion gene family: the Plasmodium reichenowi Reticulocyte Binding Like (RBL) genes

Malarial merozoites use an array of ligands, including members of the Reticulocyte Binding Like (RBL) super-family of invasion proteins, to identify and invade erythrocytes. RBL family members are large Type I membrane anchored proteins expressed at the invasive end of merozoites that share homology with the Reticulocyte Binding Proteins 1 and 2 (PvRBP1 and 2) of Plasmodium vivax. Plasmodium species vary widely both in the number and sequence of their RBL genes, with the recently completed Plasmodium falciparum genome containing five RBL genes. Of these, three encode proteins shown to be involved in erythrocyte invasion, a fourth is a pseudogene, and the role of the fifth is as yet unclear. In order to identify sequence similarities and differences that may have functional implications for erythrocyte invasion as well as to gain insights into the recent evolutionary history of the P. falciparum RBL genes, we have sequenced all five corresponding RBL genes from the chimpanzee parasite Plasmodium reichenowi, which is the closest phylogenetic relative of P. falciparum, yet is unable to invade human erythrocytes. Two of the five P. falciparum RBL genes have highly conserved complete open reading frames in both species, while the other three genes show evidence of gene conversion and rapid evolution. The RBL super-family, therefore, appears to be surprisingly dynamic and divergent, implying that it is involved in species-specific aspects of erythrocyte recognition and invasion.     

Genes for glycosylphosphatidylinositol toxin biosynthesis in Plasmodium falciparum

About 2.5 million people die of Plasmodium falciparum malaria every year. Fatalities are associated with systemic and organ-specific inflammation initiated by a parasite toxin. Recent studies show that glycosylphosphatidylinositol (GPI) functions as the dominant parasite toxin in the context of infection. GPIs also serve as membrane anchors for several of the most important surface antigens of parasite invasive stages. GPI anchoring is a complex posttranslational modification produced through the coordinated action of a multicomponent biosynthetic pathway. Here we present eight new genes of P. falciparum selected for encoding homologs of proteins essential for GPI synthesis: PIG-A, PIG-B, PIG-M, PIG-O, GPI1, GPI8, GAA-1, and DPM1. We describe the experimentally verified mRNA and predicted amino acid sequences and in situ localization of the gene products to the parasite endoplasmic reticulum. Moreover, we show preliminary evidence for the PIG-L and PIG-C genes. The biosynthetic pathway of the malaria parasite GPI offers potential targets for drug development and may be useful for studying parasite cell biology and the molecular basis for the pathophysiology of parasitic diseases.