Chloroquine-resistant isoforms of the Plasmodium falciparum chloroquine resistance transporter acidify lysosomal pH in HEK293 cells more than chloroquine-sensitive isoforms

The emergence of chloroquine-resistant Plasmodium falciparum malaria imperils the lives of millions of people in Africa, Southeast Asia and South America. Chloroquine resistance is associated with mutations in the P. falciparum chloroquine resistance transporter (PfCRT). We expressed chloroquine-sensitive (HB3) and resistant (Dd2) pfcrt alleles in HEK293 human embryonic kidney cells. PfCRT localized to the lysosomal limiting membrane and was not detected in the plasma membrane. We observed significant acidification of lysosomes containing PfCRT HB3 and Dd2, with Dd2 acidifying significantly more than HB3. A mutant HB3 allele expressing the K76T mutation (earlier found to be key for chloroquine resistance) acidified to the same extent as Dd2, whereas the acidification by a Dd2 allele expressing the T76K “back mutation” was significantly less than Dd2. Thus, the amino acid at position 76 is both an important determinant of chloroquine resistance in parasites and of lysosomal acidification following heterologous expression. PfCRT may be capable of modulating the pH of the parasite digestive vacuole, and thus chloroquine availability. Chloroquine accumulation and glycyl-phenylalanine-2-naphthylamide-induced release of lysosomal Ca²⁺ stores were unaffected by PfCRT expression. Cytoplasmic domain mutations did not alter PfCRT sorting to the lysosomal membrane. This heterologous expression system will be useful to characterize PfCRT protein structure and function, and elucidate its molecular role in chloroquine resistance.     

Structure and expression of the gene for Pv200, a major blood-stage surface antigen of Plasmodium vivax.

Molecular cloning and structure analysis of the gene encoding the Pv200 protein of the Sal-1 strain of Plasmodium vivax revealed an overall identity of 34–37% when the deduced amino acid sequence was compared with the sequences of various major merozoite surface antigens of Plasmodium falciparum, Plasmodium yoelii and Plasmodium chabaudi. When the Sal-1 Pv200 sequence was compared with the corresponding sequence from the Belèm strain of P. vivax, it was found that the two merozoite surface antigens were relatively well conserved with an overall amino acid sequence identity of 81%. A region of 23 repeated glutamine residues, found in the sequence of the Belèm isolate was not found, however, in the Sal-1 sequence. Amino-and carboxy-terminal domains of the Pv200 protein were expressed in the yeast Saccharomyces cerevisiae. Each recombinant protein was shown to react with antibodies in sera from splenectomized Bolivian Saimiri monkeys that had been infected previously with P. vivax, and in human sera from individuals with a history of exposure to vivax malaria. The availability of recombinant DNA-derived Pv200 proteins will now allow a full assessment of their utility in the diagnosis and immunoprophylaxis of the benign tertian malaria associated with P. vivax infection.     

Extense variant gene family repertoire overlap in Western Amazon Plasmodium falciparum isolates

In order to find a molecular basis for observations of relatively fast developing immunity to malarial infections in the Western Amazon region, the partial var, stevor and rif gene repertoires of nine different Plasmodium falciparum isolates collected in 1985 and 2000–2004 were evaluated. In contrast to previous results from South East Asia, the variant gene repertoire in Brazilian isolates is rather small and redundant. While the individual var repertoire sizes of Brazilian strains did not differ from Southeast Asian/African isolates, we found an over three times higher overlap of var sequence repertoires in Amazonian strains which was also conserved over time, suggesting the ongoing circulation of a similar var gene repertoire. Coincidently, almost 40% of the sequences identified herein showed the highest degree of similarity to var genes from either Brazilian or Venezuelan isolates, indicating a limited var repertoire of P. falciparum in the Amazon Basin as a whole. The intrastrain similarities of var genes were slightly but significantly lower than in Southeast Asian/African samples suggesting a higher selective pressure for diversification in Amazonian isolates. Despite of higher copy numbers per genome, rif genes also showed a significant repertoire overlap. stevor genes, which share the same predominant subtelomeric localization as var and rif genes, showed a still higher repertoire overlap and were highly similar to 3D7 stevor genes, indicating stronger functional conservation than var and rif genes. This is the first study that reveals that P. falciparum variant gene repertoires of certain areas can be limited. This has important implications for the strain-specific immunity against variant antigens occurring in these areas.     

Development of diagnostic reagents: raising antibodies against synthetic peptides of PfHRP-2 and LDH using microsphere delivery

Malaria causes significant morbidity and mortality worldwide, including countries with mainly imported malaria. In developing nations, scarce resources lead to inadequate diagnostic procedures. Microscopy of Giemsa-stained thick and thin films remains the current gold standard for diagnosis. Although it has good sensitivity and allows species identification, it is time consuming, requires microscopical expertise and maintenance of equipment. Antigen tests are promising tools for the diagnosis of malaria. Two such antigens are Plasmodium falciparum histidine rich protein (pfHRP-2) and lactate dehydrogenase (LDH). The present study was aimed to develop indigenous, rapid and sensitive immunodiagnostic method based on detection of PfHRP-2 and LDH antigen in the blood. Unique peptide sequences of PfHRP-2 (two regions) and LDH (three regions) antigen were synthesized by solid phase technique and purified to homogeneity. The antibodies raised against these sequences were raised in mice as well as rabbit using microspheres (PLGA) to generate high titre and affinity antibodies. The peptide-specific peak titres varied from 25,000 to 50,000 and affinity of the antibodies produced was found to be in order of 0.73–5.3 nM. The antibodies generated using microspheres were able to detect the PfHRP-2 and LDH antigen in the culture supernatant and parasitized RBC lysate of P. falciparum respectively by sandwich ELISA up to 0.002% parasitaemia level. The assay allowed the detection of parasite infections of 0.08–2.68% parasitaemia with a sensitivity of 100% in the whole blood of P. falciparum positive patients. No cross-reactivity was observed with P. vivax infected patients.     

The shape and size of hemozoin crystals distinguishes diverse Plasmodium species

All Plasmodium species produce a brown birefringent crystal known as malarial pigment or hemozoin. This work compares the morphology of hemozoin from P. falciparum, P. vivax, P. ovale, P. malariae, P. knowlesi, P. brasilianum, P. yoelii and P. gallinaceum. The human, primate and mouse hemozoins have a regular, flat-faced cuboidal morphology with modest size differences in contrast to the larger, regularly irregular barrel shape with a waffle surface of the avian, P. gallinaceum, pigment. Hydrogen peroxide (H₂O₂), as a biochemical test reagent, can distinguish the hemozoins by different concentrations to degrade half of the crystals. A surface area to volume ratio explains both the appearance and susceptibility to H₂O₂ degradation. The hemozoin from each species is able to be a template for hemozoin extension inhibitable by the quinolines. P. gallinaceum hemozoin more closely resembles the hemozoin from another avian apicomplexan, Haemoproteus, rather than the hemozoin from the mammalian malaria species. These distinct morphological characteristics between mammalian and avian crystals suggest different biochemical environments that affect morphology.     

Molecular cloning, expression, characterization and mutation of Plasmodium falciparum guanylate kinase

The present work describes cloning, expression, purification, characterization, and mutation of Plasmodium falciparum guanylate kinase (PlasmoDB ID PFI1420w). Amino-acid sequence alignment revealed important differences especially in K42-V51, Y73-A77, and F100-L110, which include residues important for kinase activity, and at helix 3, which is important for domain movements. The catalytic efficiency for dGMP was 22-fold lower than that for GMP, whose value is the lowest among known guanylate kinases. dGMP was found to a competitive inhibitor for GMP with K_i = 0.148 mM and a mixed-type inhibitor with regard to ATP with measured K_i = 0.4 mM. The specificity constant (K_cat/K_m) of the four examined mutants varied for natural substrate GMP/dGMP, indicating the involvement of different mechanisms in substrate recognition and subsequent loop-domain movement. These results show that P. falciparum guanylate kinase is structurally and biochemically distinct from other guanylate kinases and could be a possible target in drug development.     

Abundance of intrinsically unstructured proteins in P. falciparum and other apicomplexan parasite proteomes

Preliminary sequence analysis of Plasmodium falciparum has shown that the proteome of this organism is enriched in intrinsically unstructured proteins (IUPs), which are either completely disordered or contain large disordered regions. IUPs have been characterized as a unique class of proteins that plays an important role in biology and disease. In this study, the IUP contents in the proteomes of apicomplexan parasites, especially the proteome of P. falciparum and its various life cycle stages, have been evaluated with DisEMBL-1.4. Compared with other proteomes, apicomplexan species are extremely abundant in proteins containing long disordered regions, and the IUP contents in mammalian Plasmodium species are higher than in most other apicomplexan parasites. The proteome of the P. falciparum sporozoite appears to be distinct from the other life cycle stages in having an even higher content of disordered proteins. The abundance of IUPs in the P. falciparum proteome correlates with its enrichment in repetitive sequences. The structural plasticity of IUPs, which allows promiscuous binding interactions, may favour parasite survival both by inhibiting the generation of effective high affinity antibody responses and by facilitating the interactions with host molecules necessary for attachment and invasion of host cells.     

Identification and biochemical characterisation of a protein phosphatase 5 homologue from Plasmodium falciparum

We report the identification of a new serine/threonine phosphatase from Plasmodium falciparum at the DNA and protein levels. A 1.8 kb cDNA fragment encoding the protein phosphatase was identified via PCR amplification. The sequence has a coding capacity of 594 amino acids. Immunoblot analysis of P. falciparum extracts showed that antibodies generated against the His₆-fusion protein recognise a protein of approximately 80 kDa. The deduced amino acid sequence shares 55% identity with a mouse protein, identified as Protein Phosphatase 5 (PP5). We show that the P. falciparum PP5 homologue (PfPP5) has all structural and functional characteristics of this class of enzymes. It contains three tetratricopeptide repeats (TPR) and a nuclear targeting sequence at its N-terminus and a highly conserved C-terminal catalytic domain. Southern blot results are compatible with the existence of PfPP5 as a single copy gene. Purified recombinant protein, like the native protein enriched from P. falciparum extracts exhibited phosphatase activity that can be enhanced by both arachidonic and oleic acids, but not by myristic or stearic acid. In addition, the activity is inhibited by okadaic acid (OA) with an IC₅₀ of 4 nM. Immunofluorescence microscopy has localised PfPP5 preferentially to the nucleus. The function of PfPP5 is presently unclear, but like other PP5s of many eukaryotic organisms, it may have important regulatory functions in the parasite cell cycle.     

Expression of Plasmodium falciparum trimeric G proteins and their involvement in switching to sexual development

Both cholera and pertussis toxins were used to label and study the expression of heterotrimeric G protein subunits in Plasmodium falciparum extracts. Expression of these proteins is developmentally regulated throughout the erythrocytic cycle with peak expression during early asexual development and in mature sexual stages. Treatment of P. falciparum cultures with cholera toxin causes an increase in conversion to sexual development, and at the same concentration has a marginal inhibitory effect on asexual growth and division. Through precise synchronisation of the parasites’ asexual cell cycle, we have defined the period of sensitivity to this induction at around the time of invasion, one cycle before the development of the sexual form. Fluorescent microscopy confirmed that access of the toxin to the parasite is limited to the invasive form — the free merozoite, while further labelling studies revealed expression of a single G protein subunit in these stages. These observations are consistent with the view that a G protein-dependent signal transduction pathway is involved in coupling the parasite's environment to commitment to sexual development (gametocytogenesis). This means of artificially stimulating the pathways leading to sexual development can now be used to biochemically follow the activation of the signalling pathways involved.     

Pfcrk-1, a developmentally regulated cdc2-related protein kinase of Plasmodium falciparum

A gene encoding a novel cdc2-related protein kinase has been identified in Plasmodium falciparum, using degenerate oligonucleotides designed to hybridise to regions that are conserved in members of the cdc2 gene family. This gene, called Pfcrk-1, is located on chromosome 4. It is most closely related to the p58^GTA gene family, members of which are negative regulators of cell growth in vertebrates. Pfcrk-1 is developmentally regulated, as indicated by stage-specific accumulation of mRNA in gametocytes.     

Characterization of the 175-kilodalton erythrocyte binding antigen of Plasmodium falciparum

EBA-175 is a soluble 175-kDa Plasmodium falciparum antigen that is released into culture supernatants during rupture of schizont-infected erythrocytes. EBA-175 binds to erythrocytes and binding is sialic acid-dependent. A clone expressing the gene encoding EBA-175 was obtained previously by screening a genomic DNA expression library with antibodies that had been affinity-purified from EBA-175. Antibodies were raised against a 43-amino-acid peptide (EBA-peptide 4) synthesized according to the deduced amino acid sequence. Antibodies to peptide 4 and affinity-purified antibodies specific for EBA-175 were used to characterize further EBA-175 giving the following results: (1) EBA-175 differs biochemically and immunologically from other reported malarial antigens; (2) the EBA-175s from six geographical isolates of P. falciparum are antigenically conserved; (3) EBA-175 is expressed during schizogony as a 190-kDa protein which is larger than the culture supernatant form of the antigen. The 190-kDa form of the protein is recovered from the cell pellet in schizont-infected erythrocytes and partitions to the soluble fraction when extracted with detergent; (4) release of soluble EBA-175 into the culture supernatant coincides with schizont rupture; (5) there was no observable change in pI (pI=6.86) by isoelectric focusing between the cellular and supernatant species of the protein; and (6) release of EBA-175 into the culture supernatant is inhibited by the addition of chymostatin and leupeptin. The continued research into the role of EBA-175 during erythrocyte invasion may aid in vaccine development for malaria.     

Metabolic labelling and characterisation of S-antigens, the heat-stable, strain-specific antigens of Plasmodium falciparum

The C-10 clone of Plasmodium falciparum was metabolically labelled with [³H]glycine. Analysis by sodium dodecylsulphate/polyacrylamide gel electrophoresis (SDS/PAGE) revealed that S-antigens were the only significantly labelled products released into culture supernatants by the end of the asexual cycle. This result indicates conclusively that heat-stable, strain-specific antigens (the ‘S-antigens’) emanate from the parasite and not the host. The S-antigen of clone C-10 was resolved on SDS/PAGE as two labelled products with molecular weights of 156 000 ± 5 000 and 130 000 ± 2 000. The two components were differentially susceptible to proteolysis with trypsin. In other experiments, boiled plasma from Aotus monkeys infected with a different isolate of P. falciparum was partially purified by isoelectric focusing and the fraction containing S-antigen was iodinated by the Iodogen procedure. Analysis by SDS/PAGE of immunoprecipitated material revealed an iodinated component corresponding to the lower molecular weight band of the metabolically labelled S-antigen.     

Repeat structures in a Plasmodium falciparum protein (MESA) that binds human erythrocyte protein 4.1

The mature-parasite-infected erythrocyte surface antigen (MESA, also known as PfEMP-2 and pp300) of Plasmodium falciparum is a phosphoprotein of approx. 250 300 kDa that is exported from the parasite to the erythrocyte membrane skeleton where it binds to protein 4.1. Determination of the primary sequence of MESA reveals that it is encoded by 2 exons, a structure common to other exported proteins of P. falciparum. The MESA protein is heavily charged and contains 7 distinct repeat regions that compose over 60% of the protein. The predicted secondary structure suggests that MESA is a fibrillar protein and it shows similarity to a number of cytoskeletal and neurofilament proteins, including myosin, a protein that itself binds to protein 4.1.     

The virulent Saimiri-adapted Palo Alto strain of Plasmodium falciparum does not express the ring-infected erythrocyte surface antigen

The Palo Alto strain of Plasmodium falciparum is highly virulent for the Saimiri scieureus monkey. We have observed that these parasites do not express the Ring-infected erythrocyte surface antigene (RESA) gene. Immunoblots indicated that the Pf155/RESA protein was absent. The RESA mRNA could not be detected. Polymerase chain reaction and Southern blot analysis demonstrated that this lack of expression is due to gene rearrangements. The majority of the Palo Alto parasites have a deletion of the entire RESA gene, whereas in a minor fraction the RESA sequences remain detectable, but the 5′ miniexon 1 is inverted. These data show that the RESA protein is dispensable for in vivo parasite growth, at least in Saimiri monkeys.     

Characterisation and translation studies of messenger RNA from the human malaria parasite Plasmodium falciparum and construction of a cDNA library

RNA was isolated from trophozoites, schizonts and mixed populations of Plasmodium falciparum. 5% of the total was poly(A⁺) message, of average length 1.2 kb (10–12 kb maximum) and a poly(A) content of 10%. The mRNA fractions could be translated in vitro by reticulocyte lysates supplemented either with exogenous or P. falciparum tRNA. The patterns from two independent isolates, one cloned (T9-94) and one uncloned (K1) were virtually identical. Major translation products from 16–230 kDa have been measured. The most abundant is lactate dehydrogenase (34.8 kDa). Trophozoite mRNA codes principally for proteins of 93 kDa, while schizont mRNA codes for additional proteins of higher molecular mass. There are marked similarities between the in vitro translation products and proteins synthesised in vivo in synchronous cultures. A number of schizont mRNA translation products (principally those of 230, 203, 185, 170, 115, 101 and 71 kDa) are specifically precipitated without post-translational modification by sera from humans exposed to malaria. A cDNA library has been constructed in phage λ from total poly(A⁺) RNA and partially characterised. About 10% of the clones derive from abundant mRNA sequences. Putative actin clones have been isolated from this library and the parasite actin mRNA sized at approx. 2.8 kb.     

Transient supplementation of superoxide dismutase protects endothelial cells against Plasmodium falciparum-induced oxidative stress

The pathogenesis of cerebral malaria, a major complication of Plasmodium falciparum infection, relies on mechanisms such as cytokine production and cytoadherence of parasitized red blood cells (PRBCs) on microvascular endothelial cells. In this way parasites avoid spleen clearance by sequestration in post-capillary venules of various organs including the brain. Infected erythrocytes adhesion has also been shown to have molecular signaling consequences providing insight on how tissue homeostasis could be comprised by endothelium perturbation. Our previous work demonstrated that PRBCs adhesion to human lung endothelial cells (HLEC) induces caspases activation, oxidative stress and apoptosis. Cytoplasmic Cu/Zn superoxide dismutase (SOD1), which provides the first line of defense against oxidative stress within a cell, is now used as a treatment of numerous diseases including traumatic brain injury and ischemic stroke. In this report, we demonstrated that transient supplementation of SOD1 protects endothelial cells against P. falciparum induced oxidative stress and apoptosis. We also showed a significant decrease in PRBCs cytoadherence through a downregulation of ICAM-1 and an induction of iNOS. Protection of endothelium via antioxidant delivery may constitute a relevant strategy in cerebral malaria treatment.     

Antibody reactivity to conserved linear epitopes of Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1)

The Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family of protein antigens are involved in adhesion of P. falciparum infected erythrocytes to the capillary endothelium of the host. Antibodies to variable regions of these proteins, measured by agglutination, correlates with clinical protection against falciparum malaria. In this study we investigated the occurrence of antibodies to conserved sequences of these very variable proteins in a population living in an area endemic for falciparum malaria. Using the ELISA method, we were able to measure an antibody response to three synthetic peptides derived from conserved regions of PfEMP1. The antibody responses to these peptides increased with age and were higher in individuals with asymptomatic P. falciparum infection compared to individuals presenting with fever attributable to falciparum malaria. This indicates that antibodies recognising the conserved regions of PfEMP1 arise upon exposure to the parasite, and that these may be involved in the development of protection against malaria. Antibodies to the Pfalhesin peptide of the human aniontransporter, band3, were measured by the same method. The magnitude of this antibody response did not correlate with neither age nor clinical protection.