Defining the Erythrocyte Binding Domains of Plasmodium vivax Tryptophan Rich Antigen 33.5

Tryptophan-rich antigens play important role in host-parasite interaction. One of the Plasmodium vivax tryptophan-rich antigens called PvTRAg33.5 had earlier been shown to be predominantly of alpha helical in nature with multidomain structure, induced immune responses in humans, binds to host erythrocytes, and its sequence is highly conserved in the parasite population. In the present study, we divided this protein into three different parts i.e. N-terminal (amino acid position 24–106), middle (amino acid position 107–192), and C-terminal region (amino acid position 185–275) and determined the erythrocyte binding activity of these fragments. This binding activity was retained by the middle and C-terminal fragments covering 107 to 275 amino acid region of the PvTRAg33.5 protein. Eight non-overlapping peptides covering this 107 to 275 amino acid region were then synthesized and tested for their erythrocyte binding activity to further define the binding domains. Only two peptides, peptide P4 (at 171–191 amino acid position) and peptide P8 (at 255–275 amino acid position), were found to contain the erythrocyte binding activity. Competition assay revealed that each peptide recognizes its own erythrocyte receptor. These two peptides were found to be located on two parallel helices at one end of the protein in the modelled structure and could be exposed on its surface to form a suitable site for protein-protein interaction. Natural antibodies present in the sera of the P. vivax exposed individuals or the polyclonal rabbit antibodies against this protein were able to inhibit the erythrocyte binding activity of PvTRAg33.5, its fragments, and these two synthetic peptides P4 and P8. Further studies on receptor-ligand interaction might lead to the development of the therapeutic reagent.     

Plasmodium vivax: immunological properties of tryptophan-rich antigens PvTRAg 35.2 and PvTRAg 80.6

Need for malaria vaccine necessitates the characterization of potential antigens of the Plasmodium parasite. Recently, we have identified several Plasmodium vivax tryptophan-rich antigens (PvTRAgs). Here, we describe the immunological characterization of hitherto undescribed two such antigens PvTRAg 35.2 and PvTRAg 80.6 which are respective homologue of Plasmodium falciparum merozoite associated tryptophan-rich antigen (PfMaTrA) and P. falciparum tryptophan and threonine rich antigen (PfTryThrA) involved in erythrocyte invasion. Each of the pvtrag genes is comprised of two exons where exon 2 encodes for major part of the protein. PvTRAg 35.2 and PvTRAg 80.6 showed 97.06% and 94.12% (n = 34) seropositivity rates, and 92.3% (n = 13) and 100% (n = 29) lymphoproliferative responses, respectively, among P. vivax exposed individuals. Geometric mean values of IL-12, IFN-γ, TNF-α, IL-4 and IL-10 in PBMC culture supernatants of P. vivax exposed individuals were 182.02, 60.3, 62.84, 196.01 and 177.17 pg/ml against PvTRAg 35.2 and 185.27, 58.15, 64.56, 142.01 and 157.2 pg/ml against PvTRAg 80.6 showing mixed immune response with distinct biased towards anti-inflammatory Th2 phenotype. The pvtrag 35.2 gene was highly conserved in the parasite population whereas pvtrag 80.6 showed minor variations in the N-terminal region but highly conserved in the C-terminal region containing tryptophan-rich domain.     

Ultrastructure of the Erythrocytic Stages of Plasmodium malariae1

This report describes the fine structure of the erythrocytic stages of Plasmodium malariae. Erythrocytic parasites from a naturally acquired human infection and an experimentally infected chimpanzee were morphologically indistinguishable and structurally similar to other primate malarias. New findings included observations of highly structured arrays of merozoite surface coat proteins in the cytoplasm of early schizonts and on the surface of budding merozoites and the presence of knobs in the membranes of Maurer's clefts. Morphological evidence is presented suggesting that proteins are transported between the erythrocyte surface and intracellular parasites via two routes: one associated with Maurer's clefts for transport of membrane-associated knob material and a second associated with caveolae in the host cell membrane for the import or export of host- or parasite-derived substances through the erythrocyte cytoplasm.     

Fine Structural Observations of the Erythrocytic Stages of Plasmodium chabaudi Landau, 1965*

SYNOPSIS. Electron microscopic examination of Plasmodium chabaudi in mouse erythrocytes revealed many characteristics resembling those observed in other mammalian malarial parasites. A double unit membrane surrounds the trophozoite cytoplasm which contains many ribonucleoprotein particles, a limited amount of endoplasmic reticulum and membraned organelles including sausage-shaped vacuoles and multilaminated membraned bodies. More or less circular double membraned vacuoles, possibly cross sections of the sausage-shaped vacuoles, are common. Typical protozoan mitochondria are lacking. The limiting membrane of the merozoites is triple-layered. Paired organelles and small dense bodies are found in the merozoites along with dense granular masses in the nuclei. Trophozoites have cytostomal structures as well as invaginations of the plasma membrane at sites where no cytostomes are evident. Digestion appears to occur in single membrane-bound vesicles which contain one to several pigment grains. P. chabaudi frequently contains multiple food vacuoles and has polymorphism manifested in part by the presence of cytoplasmic extensions and of nuclei with a variety of shapes. Several apparently free forms are noted, often accompanied by a thin rim of host cytoplasm. “Appliqué” forms are common among the trophozoites as are forms in which 2 or more trophozoites are joined together. Finally, alterations in the host cytoplasm resembling the socalled Maurer's clefts are frequent. Ferritin-containing vacuoles also appear in the host cell.     

Accessible Mutational Trajectories for the Evolution of Pyrimethamine Resistance in the Malaria Parasite Plasmodium vivax

Antifolate antimalarials, such as pyrimethamine, have experienced a dramatic reduction in therapeutic efficacy as resistance has evolved in multiple malaria species. We present evidence from one such species, Plasmodium vivax, which has experienced sustained selection for pyrimethamine resistance at the dihydrofolate reductase (DHFR) locus since the 1970s. Using a transgenic Saccharomyces cerevisiae model expressing the P. vivax DHFR enzyme, we assayed growth rate and resistance of all 16 combinations of four DHFR amino acid substitutions. These substitutions were selected based on their known association with drug resistance, both in natural isolates and in laboratory settings, in the related malaria species P. falciparum. We observed a strong correlation between the resistance phenotypes for these 16 P. vivax alleles and previously observed resistance data for P. falciparum, which was surprising since nucleotide diversity levels and common polymorphic variants of DHFR differ between the two species. Similar results were observed when we expressed the P. vivax alleles in a transgenic bacterial system. This suggests common constraints on enzyme evolution in the orthologous DHFR proteins. The interplay of negative trade-offs between the evolution of novel resistance and compromised endogenous function varies at different drug dosages, and so too do the major trajectories for DHFR evolution. In simulations, it is only at very high drug dosages that the most resistant quadruple mutant DHFR allele is favored by selection. This is in agreement with common polymorphic DHFR data in P. vivax, from which this quadruple mutant is missing. We propose that clinical dosages of pyrimethamine may have historically been too low to select for the most resistant allele, or that the fitness cost of the most resistant allele was untenable without a compensatory mutation elsewhere in the genome.     

Molecular Evidence of Plasmodium vivax Mono and Mixed Malaria Parasite Infections in Duffy-Negative Native Cameroonians

The malaria parasite Plasmodium vivax is known to be majorly endemic to Asian and Latin American countries with no or very few reports of Africans infected with this parasite. Since the human Duffy antigens act as receptors for P. vivax to invade human RBCs and Africans are generally Duffy-negative, non-endemicity of P. vivax in Africa has been attributed to this fact. However, recent reports describing P. vivax infections in Duffy-negative Africans from West and Central parts of Africa have been surfaced including a recent report on P. vivax infection in native Cameroonians. In order to know if Cameroonians living in the southern regions are also susceptible to P. vivax infection, we collected finger-prick blood samples from 485 malarial symptomatic patients in five locations and followed PCR diagnostic assays with DNA sequencing of the 18S ribosomal RNA gene. Out of the 201 malaria positive cases detected, 193 were pure P. falciparum, six pure P. vivax and two mixed parasite infections (P. falciparum + P. vivax). The eight P. vivax infected samples (six single + two mixed) were further subjected to DNA sequencing of the P. vivax multidrug resistance 1 (pvmdr1) and the P.vivax circumsporozoite (pvcsp) genes. Alignment of the eight Cameroonian pvmdr1 sequences with the reference sequence showed high sequence similarities, reconfirming P. vivax infection in all the eight patients. DNA sequencing of the pvcsp gene indicated all the eight P. vivax to be of VK247 type. Interestingly, DNA sequencing of a part of the human Duffy gene covering the promoter region in the eight P. vivax-infected Cameroonians to identify the T-33C mutation revealed all these patients as Duffy-negative. The results provide evidence of single P. vivax as well as mixed malaria parasite infection in native Cameroonians and add knowledge to the growing evidences of P. vivax infection in Duffy-negative Africans.     

Asexual and Sexual Stages of a Malaria Parasite in the Thrombocytes of Tropidurus torquatus (Iguanidae) Infected with Plasmodium tropiduri*

SYNOPSIS. Sexual and asexual stages of a parasite found in the thrombocyte-like cells of some Tropidurus torquatus infected with Plasmodium tropiduri are described. The strong ultrastructural similarities between the gametocytes of this parasite and the gametocytes of P. tropiduri, and the finding of this parasite in lizards inoculated with P. tropiduri suggest that this malaria parasite can develop both in erythrocytes and thrombocytes. Evidence in favor of this hypothesis is discussed.     

Host Cell and Malarial Targets for Docetaxel (Taxotere™) during the Erythrocytic Development of Plasmodium falciparum

The microtubular stabilizing agent docetaxel (Taxotere™) is known to inhibit the intraerythrocytic development of Plasmodium falciparum. To investigate the mechanism(s) of inhibition, we analyzed the structural organization of the mitotic spindle by immunofluorescence and electron microscopy. When 30 μM docetaxel was applied for five hours on ring forms, alterations in the mitotic spindles leading to abnormal nuclear divisions were observed. At the trophozoite- and schizont-stage, docetaxel pulses prevent mitosis by stabilizing microtubular structures associated with the mitotic apparatus, giving abnormal spindles. However, this inhibition did not interfere with parasite DNA synthesis indicating the absence of a checkpoint that couples exit from mitosis with proper spindle assembly as observed in higher eukaryotic cells. In parallel, intraerythrocytic concentration of docetaxel was measured in parasitized erythrocytes, after incubation of cells with ³H-docetaxel for five hours. It was found to be 14-fold increased at the ring-stage of infected erythrocytes compared to normal ones, 170-fold increased at the trophozoite-stage and 1,500-fold increased at the schizont-stage. Our data show that, even though the overall intracellular concentration of docetaxel is low in docetaxel-pulsed rings, the agent might be sufficient to disturb the spindle organization. However, the existence of targets for docetaxel other than mitotic spindle microtubules. i.e. erythrocyte membrane components, could interfere with mitotic spindle formation     

Plasmodium simium,a Plasmodium vivax-Related Malaria Parasite: Genetic Variability of Duffy Binding Protein II and the Duffy Antigen/Receptor for Chemokines

Plasmodium simium is a parasite from New World monkeys that is most closely related to the human malaria parasite Plasmodium vivax; it also naturally infects humans. The blood-stage infection of P. vivax depends on Duffy binding protein II (PvDBPII) and its cognate receptor on erythrocytes, the Duffy antigen receptor for chemokines (hDARC), but there is no information on the P. simium erythrocytic invasion pathway. The genes encoding P. simium DBP (PsDBP_II) and simian DARC (sDARC) were sequenced from Southern brown howler monkeys (Alouatta guariba clamitans) naturally infected with P. simium because P. simium may also depend on the DBPII/DARC interaction. The sequences of DBP binding domains from P. vivax and P. simium were highly similar. However, the genetic variability of PsDBPII was lower than that of PvDBPII. Phylogenetic analyses demonstrated that these genes were strictly related and clustered in the same clade of the evolutionary tree. DARC from A. clamitans was also sequenced and contained three new non-synonymous substitutions. None of these substitutions were located in the N-terminal domain of DARC, which interacts directly with DBPII. The interaction between sDARC and PvDBPII was evaluated using a cytoadherence assay of COS7 cells expressing PvDBPII on their surfaces. Inhibitory binding assays in vitro demonstrated that antibodies from monkey sera blocked the interaction between COS-7 cells expressing PvDBPII and hDARC-positive erythrocytes. Taken together, phylogenetic analyses reinforced the hypothesis that the host switch from humans to monkeys may have occurred very recently in evolution, which sheds light on the evolutionary history of new world plasmodia. Further invasion studies would confirm whether P. simium depends on DBP/DARC to trigger internalization into red blood cells.     

Species-specific features of DARC, the primate receptor for Plasmodium vivax and Plasmodium knowlesi

The DARC (Duffy antigen/receptor for chemokines) gene, also called Duffy or FY, encodes a membrane-bound chemokine receptor. Two malaria parasites, Plasmodium vivax and Plasmodium knowlesi, use DARC to trigger internalization into red blood cells. Although much has been reported on the evolution of DARC null alleles, little is known about the evolution of the coding portion of this gene or the role that protein sequence divergence in this receptor may play in disease susceptibility or zoonosis. Here, we show that the Plasmodium interaction domain of DARC is nearly invariant in the human population, suggesting that coding polymorphism there is unlikely to play a role in differential susceptibility to infection. However, an analysis of DARC orthologs from 35 simian primate species reveals high levels of sequence divergence in the Plasmodium interaction domain. Signatures of positive selection in this domain indicate that species-specific mutations in the protein sequence of DARC could serve as barriers to the transmission of Plasmodium between primate species.     

Structural Analysis of the Synthetic Duffy Binding Protein (DBP) Antigen DEKnull Relevant for Plasmodium vivax Malaria Vaccine Design

The Plasmodium vivax vaccine candidate Duffy Binding Protein (DBP) is a protein necessary for P. vivax invasion of reticulocytes. The polymorphic nature of DBP induces strain-specific immune responses that pose unique challenges for vaccine development. DEKnull is a synthetic DBP based antigen that has been engineered through mutation to enhance induction of blocking inhibitory antibodies. We determined the x-ray crystal structure of DEKnull to identify if any conformational changes had occurred upon mutation. Computational and experimental analyses assessed immunogenicity differences between DBP and DEKnull epitopes. Functional binding assays with monoclonal antibodies were used to interrogate the available epitopes in DEKnull. We demonstrate that DEKnull is structurally similar to the parental Sal1 DBP. The DEKnull mutations do not cause peptide backbone shifts within the polymorphic loop, or at either the DBP dimerization interface or DARC receptor binding pockets, two important structurally conserved protective epitope motifs. All B-cell epitopes, except for the mutated DEK motif, are conserved between DEKnull and DBP. The DEKnull protein retains binding to conformationally dependent inhibitory antibodies. DEKnull is an iterative improvement of DBP as a vaccine candidate. DEKnull has reduced immunogenicity to polymorphic regions responsible for strain-specific immunity while retaining conserved protein folds necessary for induction of strain-transcending blocking inhibitory antibodies.     

Alterations in cytokines and haematological parameters during the acute and convalescent phases of Plasmodium falciparum and Plasmodium vivax infections

Haematological and cytokine alterations in malaria are a broad and controversialsubject in the literature. However, few studies have simultaneously evaluated variouscytokines in a single patient group during the acute and convalescent phases ofinfection. The aim of this study was to sequentially characterise alterations inhaematological patters and circulating plasma cytokine and chemokine levels inpatients infected with Plasmodium vivax or Plasmodium falciparum from a Brazilianendemic area during the acute and convalescent phases of infection. During the acutephase, thrombocytopaenia, eosinopaenia, lymphopaenia and an increased number of bandcells were observed in the majority of the patients. During the convalescent phase,the haematologic parameters returned to normal. During the acute phase, P. vivax andP. falciparum patients had significantly higher interleukin (IL)-6, IL-8, IL-17,interferon-γ, tumour necrosis factor (TNF)-α, macrophage inflammatory protein-1β andgranulocyte-colony stimulating factor levels than controls and maintained high levelsduring the convalescent phase. IL-10 was detected at high concentrations during theacute phase, but returned to normal levels during the convalescent phase. PlasmaIL-10 concentration was positively correlated with parasitaemia in P. vivax and P.falciparum-infected patients. The same was true for the TNF-α concentration in P.falciparum-infected patients. Finally, the haematological and cytokine profiles weresimilar between uncomplicated P. falciparum and P. vivax infections.     

Polymorphism at the Apical Membrane Antigen 1 Gene (AMA1) of the Malaria Parasite Plasmodium falciparum in a Vietnamese Population

The patterns of molecular evolution of the most diverse region of the apical membrane antigen 1 (AMA1) gene in Plasmodium falciparum from a Vietnamese subpopulation (Bao Loc) were investigated. Within the Bao Loc population, the sequenced gene region showed relatively high allelic and nucleotide diversity (0.985 and 0.02694, respectively). Further, the level of population recombination was substantial, resulting in a significant decay of linkage disequilibrium along the gene region. The results suggest that AMA1 is a useful genetic marker for studying the relationships between adaptation of parasite populations (to the human host immune system) and malaria epidemiology.     

Preferential Invasion by Plasmodium Merozoites and the Self-Regulation of Parasite Burden

The preferential invasion of particular red blood cell (RBC) age classes may offer a mechanism by which certain species of Plasmodia regulate their population growth. Asexual reproduction of the parasite within RBCs exponentially increases the number of circulating parasites; limiting this explosion in parasite density may be key to providing sufficient time for the parasite to reproduce, and for the host to develop a specific immune response. It is critical that the role of preferential invasion in infection is properly understood to model the within-host dynamics of different Plasmodia species. We develop a simulation model to show that limiting the range of RBC age classes available for invasion is a credible mechanism for restricting parasite density, one which is equally as important as the maximum parasite replication rate and the duration of the erythrocytic cycle. Different species of Plasmodia that regularly infect humans exhibit different preferences for RBC invasion, with all species except P. falciparum appearing to exhibit a combination of characteristics which are able to self-regulate parasite density.     

Global Population Structure of the Genes Encoding the Malaria Vaccine Candidate,Plasmodium vivax Apical Membrane Antigen 1 (PvAMA1)

Background

The Plasmodium vivax Apical Membrane Antigen 1 (PvAMA1) is a promising malaria vaccine candidate, however it remains unclear which regions are naturally targeted by host immunity and whether its high genetic diversity will preclude coverage by a monovalent vaccine. To assess its feasibility as a vaccine candidate, we investigated the global population structure of PvAMA1.

Methodology and Principal Findings

New sequences from Papua New Guinea (PNG, n = 102) were analysed together with published sequences from Thailand (n = 158), India (n = 8), Sri Lanka (n = 23), Venezuela (n = 74) and a collection of isolates from disparate geographic locations (n = 8). A total of 92 single nucleotide polymorphisms (SNPs) were identified including 22 synonymous SNPs and 70 non-synonymous (NS) SNPs. Polymorphisms and signatures of balancing (positive Tajima''s D and low F_ST values) selection were predominantly clustered in domain I, suggesting it is a dominant target of protective immune responses. To estimate global antigenic diversity, haplotypes comprised of (i) non-singleton (n = 40) and (ii) common (≥10% minor allele frequency, n = 23) polymorphic amino acid sites were then analysed revealing a total of 219 and 210 distinct haplotypes, respectively. Although highly diverse, the 210 haplotypes comprised of only common polymorphisms were grouped into eleven clusters, however substantial geographic differentiation was observed, and this may have implications for the efficacy of PvAMA1 vaccines in different malaria-endemic areas. The PNG haplotypes form a distinct group of clusters not found in any other geographic region. Vaccine haplotypes were rare and geographically restricted, suggesting potentially poor efficacy of candidate PvAMA1 vaccines.

Conclusions

It may be possible to cover the existing global PvAMA1 diversity by selection of diverse alleles based on these analyses however it will be important to first define the relationships between the genetic and antigenic diversity of this molecule.     

Kinetic Mechanism and the Rate-limiting Step of Plasmodium vivax Serine Hydroxymethyltransferase

Serine hydroxymethyltransferase (SHMT) is a pyridoxal 5′-phosphate (PLP)-dependent enzyme that catalyzes a hydroxymethyl group transfer from l-serine to tetrahydrofolate (H₄folate) to yield glycine and 5,10-methylenetetrahydrofolate (CH₂-H₄folate). SHMT is crucial for deoxythymidylate biosynthesis and a target for antimalarial drug development. Our previous studies indicate that PvSHMT catalyzes the reaction via a ternary complex mechanism. To define the kinetic mechanism of this catalysis, we explored the PvSHMT reaction by employing various methodologies including ligand binding, transient, and steady-state kinetics as well as product analysis by rapid-quench and HPLC/MS techniques. The results indicate that PvSHMT can bind first to either l-serine or H₄folate. The dissociation constants for the enzyme·l-serine and enzyme·H₄folate complexes were determined as 0.18 ± 0.08 and 0.35 ± 0.06 mm, respectively. The amounts of glycine formed after single turnovers of different preformed binary complexes were similar, indicating that the reaction proceeds via a random-order binding mechanism. In addition, the rate constant of glycine formation measured by rapid-quench and HPLC/MS analysis is similar to the k_cat value (1.09 ± 0.05 s⁻¹) obtained from the steady-state kinetics, indicating that glycine formation is the rate-limiting step of SHMT catalysis. This information will serve as a basis for future investigation on species-specific inhibition of SHMT for antimalarial drug development.     

A Bacterial Phosphatase-Like Enzyme of the Malaria Parasite Plasmodium falciparum Possesses Tyrosine Phosphatase Activity and Is Implicated in the Regulation of Band 3 Dynamics during Parasite Invasion

Eukaryotic parasites of the genus Plasmodium cause malaria by invading and developing within host erythrocytes. Here, we demonstrate that PfShelph2, a gene product of Plasmodium falciparum that belongs to the Shewanella-like phosphatase (Shelph) subfamily, selectively hydrolyzes phosphotyrosine, as shown for other previously studied Shelph family members. In the extracellular merozoite stage, PfShelph2 localizes to vesicles that appear to be distinct from those of rhoptry, dense granule, or microneme organelles. During invasion, PfShelph2 is released from these vesicles and exported to the host erythrocyte. In vitro, PfShelph2 shows tyrosine phosphatase activity against the host erythrocyte protein Band 3, which is the most abundant tyrosine-phosphorylated species of the erythrocyte. During P. falciparum invasion, Band 3 undergoes dynamic and rapid clearance from the invasion junction within 1 to 2 s of parasite attachment to the erythrocyte. Release of Pfshelph2 occurs after clearance of Band 3 from the parasite-host cell interface and when the parasite is nearly or completely enclosed in the nascent vacuole. We propose a model in which the phosphatase modifies Band 3 in time to restore its interaction with the cytoskeleton and thus reestablishes the erythrocyte cytoskeletal network at the end of the invasion process.     

Structure of lactate dehydrogenase from Plasmodium vivax: complexes with NADH and APADH

Malaria caused by Plasmodium vivax is a major cause of global morbidity and, in rare cases, mortality. Lactate dehydrogenase is an essential Plasmodium protein and, therefore, a potential antimalarial drug target. Ideally, drugs directed against this target would be effective against both major species of Plasmodium, P. falciparum and P. vivax. In this study, the crystal structure of the lactate dehydrogenase protein from P. vivax has been solved and is compared to the equivalent structure from the P. falciparum enzyme. The active sites and cofactor binding pockets of both enzymes are found to be highly similar and differentiate these enzymes from their human counterparts. These structures suggest effective inhibition of both enzymes should be readily achievable with a common inhibitor. The crystal structures of both enzymes have also been solved in complex with the synthetic cofactor APADH. The unusual cofactor binding site in these Plasmodium enzymes is found to readily accommodate both NADH and APADH, explaining why the Plasmodium enzymes retain enzymatic activity in the presence of this synthetic cofactor.