Allelic dimorphism in a surface antigen gene of the malaria parasite Plasmodium falciparum

Merozoites of the malaria parasite Plasmodium falciparum carry surface proteins processed from a precursor termed p190 or p195. Polymorphism has been reported in this protein. Since the protein is a candidate for a malaria vaccine, it is important to understand the nature of this polymorphism. We have determined the complete nucleotide sequence of the p190 gene from the MAD20 strain (a Papua New Guinea isolate). Comparisons of the gene with that from other strains of P. falciparum allowed us to study the genetic basis of the antigen's polymorphism. The gene consists of sequences distributed in variable blocks, which are separated by conserved or semi-conserved sequences. Variable sequences occur both in regions that code for tripeptide repeats and in regions with no apparent repeats. Interestingly, according to the present data, variable sequences are not widely polymorphic but fall into two distinct types. We argue that the p190 protein is encoded by dimorphic alleles capable of limited genetic exchange and present evidence at the nucleotide level documenting intragenic recombination in Plasmodium.     

Mitosis in the human malaria parasite Plasmodium falciparum

Malaria is caused by intraerythrocytic protozoan parasites belonging to Plasmodium spp. (phylum Apicomplexa) that produce significant morbidity and mortality, mostly in developing countries. Plasmodium parasites have a complex life cycle that includes multiple stages in anopheline mosquito vectors and vertebrate hosts. During the life cycle, the parasites undergo several cycles of extreme population growth within a brief span, and this is critical for their continued transmission and a contributing factor for their pathogenesis in the host. As with other eukaryotes, successful mitosis is an essential requirement for Plasmodium reproduction; however, some aspects of Plasmodium mitosis are quite distinct and not fully understood. In this review, we will discuss the current understanding of the architecture and key events of mitosis in Plasmodium falciparum and related parasites and compare them with the traditional mitotic events described for other eukaryotes.     

Proteomics of the human malaria parasite Plasmodium falciparum

The lethal species of malaria parasite, Plasmodium falciparum, continues to exact a huge toll of mortality and morbidity, particularly in sub-Saharan Africa. Completion of the genome sequence of this organism and advances in proteomics and mass spectrometry have opened up unprecedented opportunities for understanding the complex biology of this parasite and how it responds to drug challenge and other interventions. This review describes recent progress that has been made in applying proteomics technology to this important pathogen and provides a look forward to likely future developments.     

Identification of proteases that regulate erythrocyte rupture by the malaria parasite Plasmodium falciparum

Newly replicated Plasmodium falciparum parasites escape from host erythrocytes through a tightly regulated process that is mediated by multiple classes of proteolytic enzymes. However, the identification of specific proteases has been challenging. We describe here a forward chemical genetic screen using a highly focused library of more than 1,200 covalent serine and cysteine protease inhibitors to identify compounds that block host cell rupture by P. falciparum. Using hits from the library screen, we identified the subtilisin-family serine protease PfSU B1 and the cysteine protease dipeptidyl peptidase 3 (DPAP3) as primary regulators of this process. Inhibition of both DPAP3 and PfSUB1 caused a block in proteolytic processing of the serine repeat antigen (SERA) protein SERA5 that correlated with the observed block in rupture. Furthermore, DPAP3 inhibition reduced the levels of mature PfSUB1. These results suggest that two mechanistically distinct proteases function to regulate processing of downstream substrates required for efficient release of parasites from host red blood cells.     

Genetic mapping in the human malaria parasite Plasmodium falciparum

The Plasmodium falciparum genome sequence has boosted hopes for a new era of malaria research and for the application of comprehensive molecular knowledge to disease control, but formidable obstacles remain: approximately 60% of the predicted P. falciparum proteins have no known functions or homologues, and most life cycle stages of this haploid eukaryotic parasite are relatively intractable to cultivation and biochemical manipulation. Genetic mapping based on high-resolution maps saturated with single-nucleotide polymorphisms or microsatellites is now providing effective strategies for discovering candidate genes determining important parasite phenotypes. Here we review classical linkage studies using laboratory crosses and population associations that are now amenable to genome-wide approaches and are revealing multiple candidate genes involved in complex drug responses. Moreover, mapping by linkage disequilibrium is practicable in cases where chromosomal segments flanking drug-selected genes have been preserved in populations during relatively recent P. falciparum evolution. We discuss the advantages and limitations of these various genetic mapping strategies, results from which offer complementary insights to those emerging from gene knockout experiments and/or high-throughput genomic technologies.     

Transfection of the human malaria parasite Plasmodium falciparum.

In the past few years, methods have been developed which allow the introduction of exogenous DNA into the human malaria parasite Plasmodium falciparum. This important technical advance known as parasite transfection, provides powerful new tools to study the function of Plasmodium proteins and their roles in biology and disease. Already it has allowed the analysis of promoter function and has been successfully applied to establish the role of particular molecules and/or mutations in the biology of this parasite. This review summarises the current state of the technology and how it has been applied to dissect the function of the P. falciparum genome.     

Redox and antioxidant systems of the malaria parasite Plasmodium falciparum

The malaria parasite Plasmodium falciparum is highly adapted to cope with the oxidative stress to which it is exposed during the erythrocytic stages of its life cycle. This includes the defence against oxidative insults arising from the parasite's metabolism of haemoglobin which results in the formation of reactive oxygen species and the release of toxic ferriprotoporphyrin IX. Central to the parasite's defences are superoxide dismutases and thioredoxin-dependent peroxidases; however, they lack catalase and glutathione peroxidases. The vital importance of the thioredoxin redox cycle (comprising NADPH, thioredoxin reductase and thioredoxin) is emphasized by the confirmation that thioredoxin reductase is essential for the survival of intraerythrocytic P. falciparum. The parasites also contain a fully functional glutathione redox system and the low-molecular-weight thiol glutathione is not only an important intracellular thiol redox buffer but also a cofactor for several redox active enzymes such as glutathione S-transferase and glutaredoxin. Recent findings have shown that in addition to these cytosolic redox systems the parasite also has an important mitochondrial antioxidant defence system and it is suggested that lipoic acid plays a pivotal part in defending the organelle from oxidative damage.     

Large-scale growth of the Plasmodium falciparum malaria parasite in a wave bioreactor

We describe methods for the large-scale in vitro culturing of synchronous and asynchronous blood-stage Plasmodium falciparum parasites in sterile disposable plastic bioreactors controlled by wave-induced motion (wave bioreactor). These cultures perform better than static flask cultures in terms of preserving parasite cell cycle synchronicity and reducing the number of multiple-infected erythrocytes. The straight-forward methods described here will facilitate the large scale production of malaria parasites for antigen and organelle isolation and characterisation, for the high throughput screening of compound libraries with whole cells or extracts, and the development of live- or whole-cell malaria vaccines under good manufacturing practice compliant standards.     

Immune evasion by Babesia bovis and Plasmodium falciparum: cliff-dwellers of the parasite world

Erythrocyte-dwelling parasites, such as Babesia bovis and Plasmodium falciparum, are not accessible to the host immune system during most of their asexual reproductive cycle because they are intracellular. While intracellular, the host immune response must be directed toward the surface of the infected erythrocyte. Immune individuals mount protective antibody and cell-mediated responses which eliminate most of the parasites, yet some survive to establish chronic infections. In this review, David Allred discusses some of the mechanisms used by these parasites to evade individual immune mechanisms targeting the infected erythrocyte to survive in the hostile environment of an effective immune response.     

Adenylyl and guanylyl cyclases from the malaria parasite Plasmodium falciparum

Completion of several malaria parasite genome sequences and advances in Plasmodium gene manipulation technology, will lead to significant advances in our knowledge of the biology of these organisms. Biochemical analysis of the cyclic nucleotide signalling pathways of P. falciparum has provided important information on malaria parasite development. The Plasmodium purine nucleotide cyclase enzymes have extremely unusual structures and the regulatory mechanisms controlling parasite enzyme activity are distinct from those operating on the analogous host molecules. Study of these enzymes could therefore lead to novel strategies for anti-malarial intervention in addition to providing unique insights into the intriguing biology of the parasite.     

Circumsporozoite protein gene from Plasmodium reichenowi, a chimpanzee malaria parasite evolutionarily related to the human malaria parasite Plasmodium falciparum

We have cloned and sequenced the gene encoding the circumsporozoite (CS) protein of Plasmodium reichenowi a Plasmodium falciparum-like malaria parasite of chimpanzees. Comparison of the two CS proteins reveals both similarities and differences in these two evolutionarily related parasites that have adapted to different hosts. The P. reichenowi CS protein has a new repeat sequence, NVNP, in addition to the P. falciparum-like NANP and NVDP repeats. In the immunodominant TH2R and TH3R regions of the CS protein, the amino acid sequences are similar in both parasite proteins. The differences in the two proteins exist in domains around the conserved regions, Region I and Region II, which are otherwise conserved in the CS proteins of P. falciparum analyzed to date. Studies of parasite protein genes of evolutionarily related malaria parasites, together with other immunologic and biologic characteristics, will help better understand the evolution and host parasite relationship of malaria parasites and may provide a tool for identifying protein determinants for malaria vaccine development.     

Variation in the gene encoding a major merozoite surface antigen of the human malaria parasite Plasmodium falciparum.

Plasmodium falciparum merozoites have a variable surface protein of about 195,000 molecular weight which may be involved in strain-specific immunity. We have cloned and sequenced a major portion of the gene encoding this antigen from the CAMP strain and have located sites of preferred mung bean nuclease cleavage around the gene. These sites depend on reaction conditions, but at 40% formamide and 2 units of mung bean nuclease per microgram DNA, the intact gene was excised from the chromosome. Comparison of the CAMP strain gene with the same gene from other strains of P. falciparum by matching available DNA sequences and by DNA hybridization revealed five regions of homology separated by divergent segments. Two of the variable regions encoded three amino acid repeats, predominantly Ser-Gly-Thr and Thr-Glu-Glu. Implications of these findings on the function of the antigen, and possible mechanisms for generation of variants are discussed.     

Polyamine synthesis and salvage pathways in the malaria parasite Plasmodium falciparum

We demonstrate, for the first time, a functional polyamine biosynthetic pathway in the malaria parasite Plasmodium falciparum that culminates in the synthesis of spermine. Additionally, we also report putrescine and spermidine salvage in the malaria parasite. Putrescine and spermidine transport in P. falciparum infected red blood cells is a highly specific, carrier mediated and active process, mediated by new transporters that differ from the transporters of uninfected red blood cells in their kinetic parameters, Vmax and km, as well as in their activation energy.     

The membrane potential of the intraerythrocytic malaria parasite Plasmodium falciparum

The membrane potential (Deltapsi) of the mature asexual form of the human malaria parasite, Plasmodium falciparum, isolated from its host erythrocyte using a saponin permeabilization technique, was investigated using both the radiolabeled Deltapsi indicator tetraphenylphosphonium ([(3)H]TPP(+)) and the fluorescent Deltapsi indicator DiBAC(4)(3) (bis-oxonol). For isolated parasites suspended in a high Na(+), low K(+) solution, Deltapsi was estimated from the measured distribution of [(3)H]TPP(+) to be -95 +/- 2 mV. Deltapsi was reduced by the specific V-type H(+) pump inhibitor bafilomycin A(1), by the H(+) ionophore CCCP, and by glucose deprivation. Acidification of the parasite cytosol (induced by the addition of lactate) resulted in a transient hyperpolarization, whereas a cytosolic alkalinization (induced by the addition of NH(4)(+)) resulted in a transient depolarization. A decrease in the extracellular pH resulted in a membrane depolarization, whereas an increase in the extracellular pH resulted in a membrane hyperpolarization. The parasite plasma membrane depolarized in response to an increase in the extracellular K(+) concentration and hyperpolarized in response to a decrease in the extracellular K(+) concentration and to the addition of the K(+) channel blockers Ba(2+) or Cs(+) to the suspending medium. The data are consistent with Deltapsi of the intraerythrocytic P. falciparum trophozoite being due to the electrogenic extrusion of H(+) via the V-type H(+) pump at the parasite surface. The current associated with the efflux of H(+) is countered, in part, by the influx of K(+) via Ba(2+)- and Cs(+)-sensitive K(+) channels in the parasite plasma membrane.     

RAP1 controls rhoptry targeting of RAP2 in the malaria parasite Plasmodium falciparum

Rhoptry associated protein 1 (RAP1) and 2 (RAP2), together with a poorly described third protein RAP3, form the low molecular weight complex within the rhoptries of Plasmodium falciparum. These proteins are thought to play a role in erythrocyte invasion by the extracellular merozoite and are important vaccine candidates. We used gene-targeting technology in P.falciparum blood-stage parasites to disrupt the RAP1 gene, producing parasites that express severely truncated forms of RAP1. Immunoprecipitation experiments suggest that truncated RAP1 species did not complex with RAP2 and RAP3. Consistent with this were the distinct subcellular localizations of RAP1 and 2 in disrupted RAP1 parasites, where RAP2 does not traffic to the rhoptries but is instead located in a compartment that appears related to the lumen of the endoplasmic reticulum. These results suggest that RAP1 is required to localize RAP2 to the rhoptries, supporting the hypothesis that rhoptry biogenesis is dependent in part on the secretory pathway in the parasite. The observation that apparently host-protective merozoite antigens are not essential for efficient erythrocyte invasion has important implications for vaccine design.     

Intranuclear structures in pyrimethamine-resistant isolates of the malaria parasite Plasmodium falciparum

The ultrastructure of the malaria parasite Plasmodium falciparum is well known, both from natural infections and from culture material ( Aikawa , 1977, Langreth et al., 1978). It is noteworthy that all of these studies were done with pyrimethamine-sensitive strains, e.g. FCR-3/Gambia. Except for spindle microtubules during schizogony, no intranuclear structures have been described in any of the asexual erythrocytic stages. In the course of isolating clones from the pyrimethamine-resistant strain Honduras I/CDC (V.K. Bhasin and W. Trager , in print) and checking by electron microscopy for the presence or absence of knobs, we noticed intranuclear structures that might be correlated with pyrimethamine resistance. For comparison, we then examined the multi-drug-resistant strain Indochina 1. We present here a first report on these structures as a basis for further studies.     

Spatial and temporal mapping of the PfEMP1 export pathway in Plasmodium falciparum

The human malaria parasite, Plasmodium falciparum, modifies the red blood cells (RBCs) that it infects by exporting proteins to the host cell. One key virulence protein, P. falciparum Erythrocyte Membrane Protein‐1 (PfEMP1), is trafficked to the surface of the infected RBC, where it mediates adhesion to the vascular endothelium. We have investigated the organization and development of the exomembrane system that is used for PfEMP1 trafficking. Maurer's cleft cisternae are formed early after invasion and proteins are delivered to these (initially mobile) structures in a temporally staggered and spatially segregated manner. Membrane‐Associated Histidine‐Rich Protein‐2(MAHRP2)‐containing tether‐like structures are generated as early as 4 h post invasion and become attached to Maurer's clefts. The tether/Maurer's cleft complex docks onto the RBC membrane at ～ 20 h post invasion via a process that is not affected by cytochalasin D treatment. We have examined the trafficking of a GFP chimera of PfEMP1 expressed in transfected parasites. PfEMP1B‐GFP accumulates near the parasite surface, within membranous structures exhibiting a defined ultrastructure, before being transferred to pre‐formed mobile Maurer's clefts. Endogenous PfEMP1 and PfEMP1B‐GFP are associated with Electron‐Dense Vesicles that may be responsible for trafficking PfEMP1 from the Maurer's clefts to the RBC membrane.