Vital functions of the malarial ookinete protein, CTRP, reside in the A domains

The transformation of malaria ookinetes into oocysts occurs in the mosquito midgut and is a major bottleneck for parasite transmission. The secreted ookinete surface protein, circumsporozoite- and thrombospondin-related adhesive protein (TRAP)-related protein (CTRP), is essential for this transition and hence constitutes a potential target for malaria transmission blockade. CTRP is a modular multidomain protein containing six tandem von Willebrand factor A-like (A) domains and seven tandem thrombospondin type I repeat-like (TS) domains. Here we present, to our knowledge, the first structure-function analysis of CTRP using genetically modified Plasmodium berghei parasites expressing mutant versions of the ctrp gene. Our data show that the A domains of CTRP are critical for ookinete gliding motility and oocyst formation whilst, unexpectedly, its TS domains are fully redundant. These results may have important implications for the design of CTRP-based transmission blocking strategies.     

Diversity in the thrombospondin-related adhesive protein gene (TRAP) of Plasmodium vivax

We analyzed 22 clinical isolates of Plasmodium vivax from Thailand and 17 from Brazil to investigate the extent of sequence variation in the thrombospondin-related adhesive protein of Plasmodium vivax (PvTRAP), a homologue of P. falciparum TRAP (PfTRAP) which has been considered to be a promising vaccine candidate. In total 54 haplotypes were identified from 73 distinct gene clones. Coexistence of different PvTRAP in circulation occurred in 10 and 13 isolates from Thailand and Brazil, respectively. Forty out of 48 substituted nucleotides are non-synonymous changes. Most of the substituted residues reside in the von Willebrand factor type A-domain (region II), a sulfated glycosaminoglycan-binding domain (region III) and a proline-rich region (region IV). All nucleotide substitutions are dimorphic. Two haplotypes from Thailand contain an inserted sequence encoding aspartic acid-serine-proline in the proline-rich region. Sequence analysis has revealed that nucleotide diversity in PvTRAP is low although Brazilian isolates display a higher degree of variation than those from Thailand. Phylogenetic construction using the neighbor joining method has shown that most of the Thai and the Brazilian isolates appear to be mainly clustered into distinct groups. Significantly greater than expected values of the mean number of non-synonymous (d(n)) than synonymous (d(s)) nucleotide substitutions per site were observed in regions II and III of PvTRAP. Analysis of the published PfTRAP sequences has shown a similar finding in regions II and IV suggesting that positive selection operates on the regions. Hence, different regions in PvTRAP and PfTRAP could be under different pressures in terms of immune selection, structural and/or functional constraints.     

The Plasmodium vivax rhoptry-associated protein 1

Rhoptries are cellular organelles localized at the apical pole of apicomplexan parasites. Their content is rich in lipids and proteins that are released during target cell invasion. Plasmodium falciparum rhoptry-associated protein 1 (RAP1) has been the most widely studied among this parasite species' rhoptry proteins and is considered to be a good anti-malarial vaccine candidate since it displays little polymorphism and induces antibodies in infected humans. Monoclonal antibodies directed against RAP1 are also able to inhibit target cell invasion in vitro and protection against P. falciparum experimental challenge is induced when non-human primates are immunized with this protein expressed in its recombinant form. This study describes identifying and characterizing RAP1 in Plasmodium vivax, the most widespread parasite species causing malaria in humans, producing more than 80 million infections yearly, mainly in Asia and Latin America. This new protein is encoded by a two-exon gene, is proteolytically processed in a similar manner to its falciparum homologue and, as observed by microscopy, the immunofluorescence pattern displayed is suggestive of its rhoptry localization. Further studies evaluating P. vivax RAP1 protective efficacy in non-human primates should be carried out taking into account the relevance that its P. falciparum homologue has as an anti-malarial vaccine candidate.     

Targeting the Plasmodium vivax Duffy-binding protein

Plasmodium vivax requires interaction with the Duffy antigen receptor for chemokines (DARC) to enable its invasion of human erythrocytes. Interaction with DARC is mediated by the P. vivax Duffy-binding protein (PvDBP) and is essential for junction formation, which is a key step in the invasion process. The receptor-binding domain of PvDBP maps to a conserved cysteine-rich region, referred to as region II (PvRII). Here, we review data on the interaction of PvRII with DARC and explore the potential of targeting this crucial receptor-ligand interaction to develop new intervention strategies against P. vivax.     

A calcium-dependent protein kinase regulates Plasmodium ookinete access to the midgut epithelial cell

Plasmodium parasites are fertilized in the mosquito midgut and develop into motile zygotes, called ookinetes, which invade the midgut epithelium. Here we show that a calcium-dependent protein kinase, CDPK3, of the rodent malarial parasite (Plasmodium berghei) is produced in the ookinete stage and has a critical role in parasite transmission to the mosquito vector. Targeted disruption of the CDPK3 gene decreased ookinete ability to infect the mosquito midgut by nearly two orders of magnitude. Electron microscopic analyses demonstrated that the disruptant ookinetes could not access midgut epithelial cells by traversing the layer covering the cell surface. An in vitro migration assay showed that these ookinetes lack the ability to migrate through an artificial gel, suggesting that this defect caused their failure to access the epithelium. In vitro migration assays also suggested that this motility is induced in the wild type by mobilization of intracellular stored calcium. These results indicate that a signalling pathway involving calcium and CDPK3 regulates ookinete penetration of the layer covering the midgut epithelium. Because humans do not possess CDPK family proteins, CDPK3 is a good target for blocking malarial transmission to the mosquito vector.     

Identification and characterisation of the Plasmodium vivax rhoptry-associated protein 2

Plasmodium vivax is currently the most widespread of the four parasite species causing malaria in humans around the world. It causes more than 75 million clinical episodes per year, mainly on the Asian and American continents. Identifying new antigens to be further tested as anti-P. vivax vaccine candidates has been greatly hampered by the difficulty of maintaining this parasite cultured in vitro. Taking into account that one of the most promising vaccine candidates against Plasmodium falciparum is the rhoptry-associated protein 2, we have identified the P. falciparum rhoptry-associated protein 2 homologue in P. vivax in the present study. This protein has 400 residues, having an N-terminal 21 amino-acid stretch compatible with a signal peptide and, as occurs with its falciparum homologue, it lacks repeat sequences. The protein is expressed in asexual stage P. vivax parasites and polyclonal sera raised against this protein recognised a 46 kDa band in parasite lysate in a Western blot assay.     

Identifying and characterising the Plasmodium falciparum merozoite surface protein 10 Plasmodium vivax homologue

Plasmodium vivax malaria is one of the most prevalent parasitic diseases in Asia and Latin-America. The difficulty of maintaining this parasite culture in vitro has hampered identifying and characterising proteins implied in merozoite invasion of red blood cells. We have been able to identify an open reading frame in P. vivax encoding the Plasmodium falciparum merozoite surface protein 10 homologous protein using the partial sequences from this parasite's genome reported during 2004. This new protein contains 479 amino-acids, two epidermal growth factor-like domains, hydrophobic regions at the N- and C-termini, being compatible with a signal peptide and a glycosylphosphatidylinositol anchor site, respectively. The protein is expressed during the parasite's asexual stage and is recognised by polyclonal sera in parasite lysate using Western blot. P. vivax-infected patients' sera highly recognised recombinant protein by ELISA.     

The paroxysm of Plasmodium vivax malaria

The paroxysms of Plasmodium vivax malaria are antiparasite responses that, although distressing to the human host, almost never impart serious acute pathology. Using plasma and blood cells from P. vivax patients, the cellular and noncellular mediators of these events have been studied ex vivo. The host response during a P. vivax paroxysm was found to involve T cells, monocytes and neutrophils, and the activity, among others, of the pyrogenic cytokines tumor necrosis factor alpha and interleukin 2 in addition to granulocyte macrophage-colony stimulating factor. However, interferon gamma activity, associated with serious acute pathogenesis in other studies on malaria, was absent. Induction of the cytokines active during a P. vivax paroxysm depends upon the presence of parasite products, which are released into the plasma before the paroxysm. Chemical identification of these natural parasite products will be important for our understanding of pathogenesis and protection in malaria.     

Intracellular calcium levels in the Plasmodium berghei ookinete

Ookinetes are the motile and invasive stages of Plasmodium parasites in the mosquito host. Here we explore the role of intracellular Ca2+ in ookinete survival and motility as well as in the formation of oocysts in vitro in the rodent malaria parasite Plasmodium berghei. Treatment with the Ca2+ ionophore A23187 induced death of the parasite, an effect that could be prevented if the ookinetes were co-incubated with insect cells before incubation with the ionophore. Treatment with the intracellular calcium chelator BAPTA/AM resulted in increased formation of oocysts in vitro. Calcium imaging in the ookinete using fluorescent calcium indicators revealed that the purified ookinetes have an intracellular calcium concentration in the range of 100 nm. Intracellular calcium levels decreased substantially when the ookinetes were incubated with insect cells and their motility was concomitantly increased. Our results suggest a pleiotropic role for intracellular calcium in the ookinete.     

Molecular cloning of Plasmodium vivax calcium-dependent protein kinase 4

A family of calcium-dependent protein kinases (CDPKs) is a unique enzyme which plays crucial roles in intracellular calcium signaling in plants, algae, and protozoa. CDPKs of malaria parasites are known to be key regulators for stage-specific cellular responses to calcium, a widespread secondary messenger that controls the progression of the parasite. In our study, we identified a gene encoding Plasmodium vivax CDPK4 (PvCDPK4) and characterized its molecular property and cellular localization. PvCDPK4 was a typical CDPK which had well-conserved N-terminal kinase domain and C-terminal calmodulin-like structure with 4 EF hand motifs for calcium-binding. The recombinant protein of EF hand domain of PvCDPK4 was expressed in E. coli and a 34 kDa product was obtained. Immunofluorescence assay by confocal laser microscopy revealed that the protein was expressed at the mature schizont of P. vivax. The expression of PvCDPK4-EF in schizont suggests that it may participate in the proliferation or egress process in the life cycle of this parasite.     

A reticulocyte-binding protein complex of Plasmodium vivax merozoites.

Plasmodium vivax merozoites primarily invade reticulocytes. The basis of this restricted host cell preference has been debated. Here we introduce two novel P. vivax proteins that comigrate on reducing SDS-polyacrylamide gels, colocalize at the apical pole of merozoites, and adhere specifically to reticulocytes. The genes encoding these proteins, P. vivax reticulocyte-binding proteins 1 and 2 (PvRBP-1 and PvRBP-2), have been cloned and analyzed. Homologous genes are evident in the closely related simian malaria parasite, P. cynomolgi, which also prefers to invade reticulocytes, but are not evident in the genome of another related simian malaria parasite, P. knowlesi, which invades all red blood cell subpopulations. Native PvRBP-1 is likely a transmembrane-anchored disulfide-linked protein, and along with PvRBP-2 may function as an adhesive protein complex. We propose that the RBPs of P. vivax, and homologous proteins of P. cynomolgi, function to target the reticulocyte subpopulation of red blood cells for invasion.     

Positive diversifying selection on Plasmodium vivax RON2 protein

Plasmodium rhoptry neck protein 2 (RON2), which is released from the neck portion of the merozoite rhoptries and interacts with the microneme protein Apical Membrane Antigen 1 (AMA1), plays a crucial role in erythrocyte invasion. In this study, we sequenced the Plasmodium vivax RON2 gene from 19 P. vivax isolates collected in central China in order to establish whether this protein is under positive diversifying selection, which may occur as a result of protective host immune pressure?. In comparison with the P. vivax Sal-1 reference line, we found 10 amino acid substitutions dispersed throughout the open reading frame as well as indels caused by polymorphism in a repeat unit (21-23 repeats of (Q/E/K/N/H)(G/D)G(H/L/Y/P)G) in the second tandem repeat region located at amino acid positions 541-650. A McDonald-Kreitman test with RON2 sequences from the primate malaria parasite Plasmodium knowlesi, detected significant departure from neutrality in the PvRON2 3' region (nucleotide positions 2668-6609). These results suggest that the PvRON2 gene has evolved under positive diversifying selection.     

The Plasmodium vivax genome sequencing project

With the successful completion of the project to sequence the Plasmodium falciparum genome, researchers are now turning their attention to other malaria parasite species. Here, an update on the Plasmodium vivax genome sequencing project is presented, as part of the Trends in Parasitology series of reviews expanding on various aspects of P. vivax research.     

The genetic diversity of Plasmodium vivax populations

Little is known of the genetic diversity and population structure of Plasmodium vivax, a debilitating and highly prevalent malaria parasite of humans. This article reviews the known polymorphic genetic markers, summarizes current data on the population structure of this parasite and discusses future prospects for using knowledge of the genetic diversity to improve control measures.     

Sequence polymorphism in two novel Plasmodium vivax ookinete surface proteins, Pvs25 and Pvs28, that are malaria transmission-blocking vaccine candidates.

BACKGROUND: For many malarious regions outside of Africa, development of effective transmission-blocking vaccines will require coverage against both Plasmodium falciparum and P. vivax. Work on P. vivax transmission-blocking vaccines has been hampered by the inability to clone the vaccine candidate genes from this parasite. Materials and METHODS: To search for genes encoding the ookinete surface proteins from P. vivax, the DNA sequences of the eight known proteins in the P25 subfamily (Pfs25, Pgs25, Pys25, Pbs25) and in the P21/28 subfamily (Pfs28, Pgs28, Pys21, Pbs21) of zygote/ookinete surface proteins were aligned. Regions of highest identity were used to design degenerate PCR oligonucleotides. Genomic DNA from the Sal I strain of P. vivax and genomic and splinkerette DNA libraries were used as PCR templates. To characterize the polymorphisms of Pvs25 and Pvs28, these two genes were PCR amplified and the DNA sequences were determined from genomic DNA extracted from patients infected with P. vivax. RESULTS: Analysis of the deduced amino acid sequence of Pvs28 revealed a secretory signal sequence, four epidermal growth factor (EGF)-like domains, six copies of the heptad amino acid repeat (GSGGE/D), and a short hydrophobic region. Because the fourth EGF-like domain has four rather than six cysteines, the gene designated Pvs28 is the presumed homologue of P21/28 subfamily members. Analysis of the deduced amino acid sequence of Pvs25 revealed a similar structure to that of Pvs28. The presence of six rather than four cysteines in the fourth EGF-like domain suggested that Pvs25 is the homologue of P25 subfamily members. Several regions of genetic polymorphisms in Pvs25 and Pvs28 were identified in field isolates of P. vivax. CONCLUSIONS: The genes encoding two ookinete surface proteins, Pvs28 and Pvs25, from P. vivax have been isolated and sequenced. Comparison of the primary structures of Pvs25, Pvs28, Pfs25, and Pfs28 suggest that there are regions of genetic polymorphism in the P25 and P21/28 subfamilies.     

Cultivation of Plasmodium vivax

Establishment of a continuous line of Plasmodium vivax parasite is crucial to understand the parasite's biology; however, this has not yet been achieved. Beginning in the 19th century, there were several efforts to cultivate this malaria parasite but without much success until the late 1980s. In addition, to date, only minor modifications of the methodology have been investigated, which has resulted in extending the cultivation period to around four weeks by supplying reticulocytes obtained from normal blood or rare hemochromatotic blood. However, the use of laboratory-produced erythroblasts to cultivate P. vivax enables maintenance of a continuous line of the parasite stably in the laboratory. Here, we summarize and compare the available methodologies and conditions for the in vitro cultivation of P. vivax.     

The origin and age of Plasmodium vivax

The evolutionary history of Plasmodium vivax has recently been addressed in terms of its origin as a parasite of humans and the age of extant populations. The consensus is that P. vivax originated as a result of a host switch from a non-human primate to hominids and that the extant populations did not originate as recently as previously proposed. Here, we show that, in a comparison of parasite isolates from across the world, Asian populations of P. vivax are the oldest. We discuss how this result, together with the phylogenetic evidence that P. vivax derived from Plasmodium found in Southeast Asian macaques, is most simply explained by assuming an Asian origin of this parasite. Nevertheless, the available data show only the tip of the iceberg. We discuss how sampling might affect time estimates to the most recent common ancestor for P. vivax populations and suggest that spatially explicit estimates are needed to understand the demographic history of this parasite better.     

Antigenic analysis of the repeat domain of the circumsporozoite protein of Plasmodium vivax

In the present study we analyzed the fine specificity of mouse monoclonal and human polyclonal antibodies directed against the repeat domain of the circumsporozoite (CS) protein of the human malaria parasite, Plasmodium vivax. Five synthetic peptides, representing monomeric and dimeric repeats of this malarial antigen, were assayed for their capacity to inhibit the binding of these antibodies to a yeast-derived recombinant CS protein. The results revealed the existence of at least two distinct repeated overlapping epitopes in the CS protein of P. vivax. Furthermore, polyclonal sera contain antibodies which recognize additional determinants not represented by the synthetic repeat peptides. Some of these sera contain antibodies recognizing a region flanking the repeat domain (region I). The present findings are in contrast with the antibody response in rodents and humans to the Plasmodium falciparum CS protein, which is directed against a single repeated immunodominant epitope.