Determination of the Plasmodium vivax schizont stage proteome

With the genome of the malaria parasite Plasmodium vivax sequenced, it is important to determine the proteomes of the parasite in order to assist efforts in antigen and drug target discovery. Since a method for continuous culture of P. vivax parasite is not available, we tried to study the proteome of the erythrocytic stages using fresh parasite isolates from patients. In schizont-enriched samples, 316 proteins were confidently identified by tandem mass spectrometry. Almost 50% of the identified proteins were hypothetical, while other major categories include proteins with binding function, protein fate, protein synthesis, metabolism and cellular transport. To identify proteins that are recognized by host humoral immunity, parasite proteins were separated by two-dimensional gel electrophoresis and screened by Western blot using an immune serum from a P. vivax patient. Mass spectrometry analysis of protein spots recognized by the serum identified four potential antigens including PV24. The recombinant protein PV24 was recognized by antibodies from vivax malaria patients even during the convalescent period, indicating that PV24 could elicit long-lasting antibody responses in P. vivax patients.     

Plasmodium vivax: Merozoites, invasion of reticulocytes and considerations for malaria vaccine development

Several Plasmodium vivax merozoite proteins have been characterized over the past few years, including two that bind specifically to reticulocytes. Here, Mare Galinski and John Barnwell examine P. vivax merozoites and constituent molecules that are involved in host cell selection and invasion, and that also are viewed as malaria vaccine candidates. They also discuss how knowledge of the reticulocyte-binding proteins furthers the development of a conceptual framework for malaria merozoite invasion at the molecular level, not only for P. vivax, but for all species of the 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.     

Serum proteome analysis of vivax malaria: An insight into the disease pathogenesis and host immune response

Vivax malaria is the most widely distributed human malaria resulting in 80-300 million clinical cases every year. It causes severe infection and mortality but is generally regarded as a benign disease and has not been investigated in detail. The present study aimed to perform human serum proteome analysis in a malaria endemic area in India to identify potential serum biomarkers for vivax malaria and understand host response. The proteomic analysis was performed on 16 age and gender matched subjects (vivax patients and control) in duplicate. Protein extraction protocols were optimized for large coverage of the serum proteome and to obtain high-resolution data. Identification of 67 differentially expressed and statistically significant (Student's t-test; p<0.05) protein spots was established by MALDI-TOF/TOF mass spectrometry. Many of the identified proteins such as apolipoprotein A and E, serum amyloid A and P, haptoglobin, ceruloplasmin, and hemopexin are interesting from a diagnostic point of view and could further be studied as potential serum biomarkers. The differentially expressed serum proteins in vivax malaria identified in this study were subjected to functional pathway analysis using multiple software, including Ingenuity Pathway Analysis (IPA), Protein ANalysis THrough Evolutionary Relationships (PANTHER) and Database for Annotation, Visualization and Integrated Discovery (DAVID) functional annotation tool for better understanding of the biological context of the identified proteins, their involvement in various physiological pathways and association with disease pathogenesis. Functional pathway analysis of the differentially expressed proteins suggested the modulation of multiple vital physiological pathways, including acute phase response signaling, complement and coagulation cascades, hemostasis and vitamin D metabolism pathway due to this parasitic infection. This article is part of a Special Issue entitled: Proteomics: The clinical link.     

Mapping the genome of Plasmodium falciparum on the drug-like chemical space reveals novel anti-malarial targets and potential drug leads

The parasite Plasmodium falciparum is the main agent responsible for malaria. In this study, we exploited a recently published chemical library from GlaxoSmithKline (GSK) that had previously been confirmed to inhibit parasite growth of the wild type (3D7) and the multi-drug resistance (D2d) strains, in order to uncover the weak links in the proteome of the parasite. We predicted 293 proteins of P. falciparum, including the six out of the seven verified targets for P. falciparum malaria treatment, as targets of 4645 GSK active compounds. Furthermore, we prioritized druggable targets, based on a number of factors, such as essentiality for growth, lack of homology with human proteins, and availability of experimental data on ligand activity with a non-human homologue of a parasite protein. We have additionally prioritized predicted ligands based on their polypharmacology profile, with focus on validated essential proteins and the effect of their perturbations on the metabolic network of P. falciparum, as well as indication of drug resistance emergence. Finally, we predict potential off-target effects on the human host with associations to cancer, neurological and dermatological disorders, based on integration of available chemical-protein and protein-protein interaction data. Our work suggests that a large number of the P. falciparum proteome is potentially druggable and could therefore serve as novel drug targets in the fight against malaria. At the same time, prioritized compounds from the GSK library could serve as lead compounds to medicinal chemists for further optimization.     

An update on the search for a Plasmodium vivax vaccine

Although Plasmodium falciparum is the leading cause of morbidity and mortality due to malaria worldwide, nearly 2.5 billion people, mostly outside Africa, are also at risk from malaria caused by Plasmodium vivax infection. Currently, almost all efforts to develop a malaria vaccine have focused on P. falciparum. For example, there are 23 P. falciparum vaccine candidates undergoing advanced clinical studies and only two P. vivax vaccine candidates being tested in preliminary (Phase I) clinical trials, with few others being assessed in preclinical studies. More investment and a greater effort toward the development of P. vivax vaccine components for a multi-species vaccine are required. This is mainly because of the wide geographical coexistence of both parasite species but also because of increasing drug resistance, recent observations of severe and lethal P. vivax cases and relapsing parasite behaviour. Availability of the P. vivax genome has contributed to antigen discovery but new means to test vaccines in future trials remain to be designed.     

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.     

Plasmodium vivax: in vitro susceptibility of blood stages to synthetic trioxolane compounds and the diamidine DB75

Plasmodium vivax is an important human pathogen causing malaria in more temperate climates of the world. Similar to Plasmodium falciparum, the causative agent for malaria tropica, drug resistance is beginning to emerge for this parasite species and this hampers adequate treatment of infection. We have used a short-term ex vivo drug assay to monitor activity of OZ277 (RBx-11160), a fully synthetic anti-malarial peroxide, and the diamidine DB75 against P. vivax. For both compounds as well as the anti-malarial reference compounds artesunate, artemether, and chloroquine, the in vitro IC(50) values were determined in one-cycle hypoxanthine incorporation assays. Results from such assays were found to be very similar compared to IC(50) values obtained from one-cycle P. falciparum hypoxanthine assays. We demonstrate the anti-parasite activity of OZ277 and the reference compounds to be faster than that of DB75. These data warrant clinical testing of OZ277 against P. vivax malaria and support recent data on clinical activity against P. vivax for DB75.     

Variant genes and the spleen in Plasmodium vivax malaria

It is generally accepted that Plasmodium vivax, the most widely distributed human malaria, does not cytoadhere in the deep capillaries of inner organs and thus this malaria parasite must have evolved splenic evasion mechanism in addition to sequestration. The spleen is a uniquely adapted lymphoid organ whose central function is the selective clearance of cell and other particles from the blood, and microbes including malaria. Splenomegaly is a hallmark of malaria and no other disease seems to exacerbate this organ as this disease does. Besides this major selective clearance function however, the spleen is also an erythropoietic organ which, under stress conditions, can be responsible for close to 40% of the RBC populations. Data obtained in experimental infections of human patients with P. vivax showed that anaemia is associated with acute and chronic infections and it has been postulated that the continued parasitemia might have been sufficient to infect and destroy most circulating reticulocytes. We review here the basis of our current knowledge of variant genes in P. vivax and the structure and function of the spleen during malaria. Based on this data, we propose that P. vivax specifically adhere to barrier cells in the human spleen allowing the parasite to escape spleen-clearance while favouring the release of merozoites in an environment where reticulocytes, the predominant, if not exclusive, host cell of P. vivax, are stored before their release into circulation to compensate for the anaemia associated with vivax malaria.     

Host switch leads to emergence of Plasmodium vivax malaria in humans

The geographical origin of Plasmodium vivax, the most widespread human malaria parasite, is controversial. Although genetic closeness to Asian primate malarias has been confirmed by phylogenetic analyses, genetic similarities between P. vivax and Plasmodium simium, a New World primate malaria, suggest that humans may have acquired P. vivax from New World monkeys or vice versa. Additionally, the near fixation of the Duffy-negative blood type (FY x B(null)/FY x B(null)) in West and Central Africa, consistent with directional selection, and the association of Duffy negativity with complete resistance to vivax malaria suggest a prolonged period of host-parasite coevolution in Africa. Here we use Bayesian and likelihood methods in conjunction with cophylogeny mapping to reconstruct the genetic and coevolutionary history of P. vivax from the complete mitochondrial genome of 176 isolates as well as several closely related Plasmodium species. Taken together, a haplotype network, parasite migration patterns, demographic history, and cophylogeny mapping support an Asian origin via a host switch from macaque monkeys.     

The genetic diversity of Plasmodium vivax--a review

The genetic diversity of Plasmodium vivax has been investigated in several malaria-endemic areas, including the Brazilian Amazon region, where this is currently the most prevalent species causing malaria in humans. This review summarizes current views on the use of molecular markers to examine P. vivax populations, with a focus on studies performed in Brazilian research laboratories. We emphasize the importance of phylogenetic studies on this parasite and discuss the perspectives created by our increasing understanding of genetic diversity and population structure of this parasite for the development of new control strategies, including vaccines, and more effective drugs for the treatment of P. vivax malaria.     

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.     

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.     

Variant proteins of Plasmodium vivax are not clonally expressed in natural infections

Plasmodium vivax is the most widely distributed human malaria parasite and responsible for 70-80 million clinical cases each year and a large socio-economical burden. The sequence of a chromosome end from P. vivax revealed the existence of a multigene superfamily, termed vir (P. vivax variant antigens), that can be subdivided into different subfamilies based on sequence similarity analysis and which represents close to 10-20% of the coding sequences of the parasite. Here we show that there is a vast repertoire of vir genes abundantly expressed in isolates obtained from human patients, that different vir gene subfamilies are transcribed in mature asexual blood stages by individual parasites, that VIR proteins are not clonally expressed and that there is no significant difference in the recognition of VIR-tags by immune sera of first-infected patients compared with sera of multiple-infected patients. These data provide to our knowledge the first comprehensive study of vir genes and their encoding variant proteins in natural infections and thus constitute a baseline for future studies of this multigene superfamily. Moreover, whereas our data are consistent with a major role of vir genes in natural infections, they are inconsistent with a predominant role in the strict sense of antigenic variation.     

Plasmodium vevax and P. falciparum: Biological interactions and the possibility of cross-species immunity

The question of whether infection of humans with one species of malaria parasite alters the course of infection with another has been largely ignored because no such interaction was found during studies of induced malaria in patients with neurosyphilis. However, in animal model systems some degree of cross-species interaction is the rule rather than the exception. Furthermore, recent epidemiological observations in Vanuatu in the South Pacific have suggested a biological interaction between the dominant species, Plasmodium vivax, and P. falciparum. Kathryn Maitland, Tom Williams and Chris Newbold here speculate on the basis of these observations and other published findings that infection with P. vivax may result in the development of immunity sufficient to ameliorate the clinical course of subsequent infections with the potentially lethal parasite P. falciparum.     

Quantitative isolation and in vivo imaging of malaria parasite liver stages

The liver stages of Plasmodium, the causative agent of malaria, are the least explored forms in the parasite's life cycle despite their recognition as key vaccine and drug targets. In vivo experimental access to liver stages of human malaria parasites is practically prohibited and therefore rodent model malaria parasites have been used for in vivo studies. However, even in rodent models progress in the analysis of liver stages has been limited, mainly due to their low abundance and associated difficulties in visualisation and isolation. Here, we present green fluorescent protein (GFP)-tagged Plasmodium yoelii rodent malaria parasite liver infections in BALB/c mice as an excellent quantitative model for the live visualisation and isolation of the so far elusive liver stages. We believe P. yoelii GFP-tagged liver stages allow, for the first time, the efficient quantitative isolation of intact early and late liver stage-infected hepatocyte units by fluorescence activated cell sorting. GFP-tagged liver stages are also well suited for intravital imaging, allowing us for the first time to visualise them in real time. We identify previously unrecognised features of liver stages including vigorous parasite movement and expulsion of 'extrusomes'. Intravital imaging thus reveals new, important information on the malaria parasite's transition from tissue to blood stage.     

Transmission of Plasmodium vivax in south-western Uganda: report of three cases in pregnant women

Plasmodium vivax is considered to be rare in the predominantly Duffy negative populations of Sub-Saharan Africa, as this red blood cell surface antigen is essential for invasion by the parasite. However, despite only very few reports of molecularly confirmed P. vivax from tropical Africa, serological evidence indicated that 13% of the persons sampled in Congo had been exposed to P. vivax. We identified P. vivax by microscopy in 8 smears from Ugandan pregnant women who had been enrolled in a longitudinal study of malaria in pregnancy. A nested polymerase chain reaction (PCR) protocol was used to detect and identify the Plasmodium parasites present. PCR analysis confirmed the presence of P. vivax for three of the women and analysis of all available samples from these women revealed clinically silent chronic low-grade vivax infections for two of them. The parasites in one woman carried pyrimethamine resistance-associated double non-synonymous mutations in the P. vivax dihydrofolate reductase gene. The three women found infected with P. vivax were Duffy positive as were nine of 68 women randomly selected from the cohort. The data presented from these three case reports is consistent with stable transmission of malaria in a predominantly Duffy negative African population. Given the substantial morbidity associated with vivax infection in non-African endemic areas, it will be important to investigate whether the distribution and prevalence of P. vivax have been underestimated in Sub-Saharan Africa. This is particularly important in the context of the drive to eliminate malaria and its morbidity.