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.     

The plastid DNA of the malaria parasite Plasmodium falciparum is replicated by two mechanisms

In common with other apicomplexan parasites, Plasmodium falciparum, a causative organism of human malaria, harbours a residual plastid derived from an ancient secondary endosymbiotic acquisition of an alga. The function of the 35 kb plastid genome is unknown, but its evolutionary origin and genetic content make it a likely target for chemotherapy. Pulsed field gel electrophoresis and ionizing radiation have shown that essentially all the plastid DNA comprises covalently closed circular monomers, together with a tiny minority of linear 35 kb molecules. Using two-dimensional gels and electron microscopy, two replication mechanisms have been revealed. One, sensitive to the topoisomerase inhibitor ciprofloxacin, appears to initiate at twin D-loops located in a large inverted repeat carrying duplicated rRNA and tRNA genes, whereas the second, less drug sensitive, probably involves rolling circles that initiate outside the inverted repeat.     

Protein tyrosine kinase activity in human malaria parasite Plasmodium falciparum.

Protein tyrosine kinases (PTKs) are believed to be implicated in the parasite growth, maturation and differentiation functions. Protein tyrosine kinase activity was found to be distributed in all the stages of P. falciparum parasite maturation. Membrane bound PTK activity was found to be increased during maturation process (ring stage to trophozoite stage) in chloroquine sensitive strains. In vivo conversion of the schizont stage to ring stage via release of merozoites was associated with a decrease in PTK activity. Chloroquine inhibited the membrane bound PTK activity in a dose dependent manner (IC50 = 45 microM). Kinetic studies show that chloroquine is a competitive inhibitor of PTK with respect to peptide substrate and noncompetitive with respect to ATP indicating that chloroquine inhibits PTK activity by binding with protein substrate binding site. The results suggest that maturation of malaria parasite is related to PTK and inhibition of this activity by chloroquine could provide a hypothesis to explain the mechanism of action of chloroquine.     

Molecular genetics of drug resistance in Plasmodium falciparum malaria

Resistance to dihydro folate reductase inhibitors and resistance to chloroquine have been mapped to single genetic loci in Plasmodium falciparum. Specific point mutations in the dihydro folate reductase gene confer different degrees of resistance to two dihydro folate inhibitors, cycloguanil and pyrimethamine, depending on the positions of the mutations and the residues involved. The chloroquine resistance locus has been mapped to a 400 kilobase (kb) segment of chromosome 7 in a P. falciparum cross. Identification and characterization of genes within this segment should lead to an understanding of the rapid drug efflux mechanism responsible for chloroquine resistance.     

Localisation of hypoxanthine phosphoribosyl transferase in the malaria parasite Plasmodium falciparum.

The enzyme hypoxanthine phosphoribosyl transferase of the human malaria parasite Plasmodium falciparum has been located in parasites and parasite-infected erythrocytes by antibody probing. The probe was a polyclonal rabbit antiserum made against the parasite enzyme made in Escherichia coli. The enzyme is associated with membrane-bound compartments in merozoites and asexual blood parasites. In particular, indirect immunofluorescence studies reveal the enzyme localized in vesicle-like structures within the cytoplasm of the infected erythrocyte. This is the first time a P. falciparum protein of defined metabolic function has been tracked to a site outside the parasite cytosol. Studies on the targeting of the enzyme using a cell-free system suggests that the protein reaches its destination via a route different from the normal secretory pathway.     

Genetic polymorphism and natural selection in the malaria parasite Plasmodium falciparum.

We have studied the genetic polymorphism at 10 Plasmodium falciparum loci that are considered potential targets for specific antimalarial vaccines. The polymorphism is unevenly distributed among the loci; loci encoding proteins expressed on the surface of the sporozoite or the merozoite (AMA-1, CSP, LSA-1, MSP-1, MSP-2, and MSP-3) are more polymorphic than those expressed during the sexual stages or inside the parasite (EBA-175, Pfs25, PF48/45, and RAP-1). Comparison of synonymous and nonsynonymous substitutions indicates that natural selection may account for the polymorphism observed at seven of the 10 loci studied. This inference depends on the assumption that synonymous substitutions are neutral, which we test by analyzing codon bias and G+C content in a set of 92 gene loci. We find evidence for an overall trend towards increasing A+T richness, but no evidence for mutation bias. Although the neutrality of synonymous substitutions is not definitely established, this trend towards an A+T rich genome cannot explain the accumulation of substitutions at least in the case of four genes (AMA-1, CSP, LSA-1, and PF48/45) because the Gleft and right arrow C transversions are more frequent than expected. Moreover, the Tajima test manifests positive natural selection for the MSP-1 and, less strongly, MSP-3 polymorphisms; the McDonald-Kreitman test manifests natural selection at LSA-1 and PF48/45. We conclude that there is definite evidence for positive natural selection in the genes encoding AMA-1, CSP, LSA-1, MSP-1, and Pfs48/45. For four other loci, EBA-175, MSP-2, MSP-3, and RAP-1, the evidence is limited. No evidence for natural selection is found for Pfs25.     

A single, bi-functional aquaglyceroporin in blood-stage Plasmodium falciparum malaria parasites.

The malaria parasite Plasmodium falciparum faces drastic osmotic changes during kidney passages and is engaged in the massive biosynthesis of glycerolipids during its development in the blood-stage. We identified a single aquaglyceroporin (PfAQP) in the nearly finished genome of P. falciparum with highest similarity to the Escherichia coli glycerol facilitator (50.4%), but both canonical Asn-Pro-Ala (NPA) motifs in the pore region are changed to Asn-Leu-Ala (NLA) and Asn-Pro-Ser (NPS), respectively. Expression in Xenopus oocytes renders them highly permeable for both water and glycerol. Sugar alcohols up to five carbons and urea pass the pore. Mutation analyses of the NLA/NPS motifs showed their structural importance, but the symmetrical pore properties were maintained. PfAQP is expressed in blood-stage parasites throughout the development from rings via trophozoites to schizonts and is localized to the parasite but not to the erythrocyte cytoplasm or membrane. Its unique bi-functionality indicates functions in the protection from osmotic stress and efficiently provides access to the serum glycerol pool for the use in ATP generation and primarily in the phospholipid synthesis.     

Experimental Plasmodium falciparum cerebral malaria in the squirrel monkey Saimiri sciureus.

Infection of the squirrel monkey, Saimiri sciureus, with several strains of Plasmodium falciparum leads in a proportion of animals to neurological symptoms with a fatal outcome. This first simian model for human cerebral malaria was studied with three strains of parasites, the uncloned Palo Alto(FUP-1) strain, the Palo AltoPLF3 clone MHB11, and the recently monkey-adapted P. falciparum strain IPC/RAY. Cerebral malaria could develop during primo infection of monkeys, whether the animals had been splenectomized or not. It did not occur in all animals and the appearance of neurological symptoms could not be predicted, as it was not related to the degree of parasitemia or duration of parasite infections.     

A comparison of thick smears, QBC malaria, PCR and PATH falciparum malaria test trip in Plasmodium falciparum diagnosis.

Blood samples from 182 patients presenting at the out-patient clinic in Richard-Toll. Senegal were analysed by Thick smear microscopy, the QBC, PCR and the new dipstick PATH Malaria assay which detects the histidine rich protein II antigen of Plasmodium falciparum. Thick smear microscopy was used as the reference method. Sensitivity, specificity, predictive positive and negative values were 100%, 83.6%, 93.4% and 100% QBC respectively; 100%, 72.7%, 89.4% and 100% for PCR; 96%, 92.7%, 96.8% and 91% for the PATH assay. PATH assay failed to detect one positive sample with Plasmodium malariae. Assays were also compared with regard to the expense of equipment and reagents and speed and ease of use. The rapid PATH assay can be performed with minimal training and may be specially useful in areas where P. falciparum is the predominant malaria species, in epidemic malaria regions, and where skilled microscopy is not readily available.     

The prognostic value of schizontaemia in imported Plasmodium falciparum malaria

ABSTRACT: BACKGROUND: In Plasmodium falciparum infection, peripheral parasite counts do not always correlate well with the sequestered parasite burden. As erythrocytes parasitized with mature trophozoites and schizonts have a high tendency to adhere to the microvascular endothelium, they are often absent in peripheral blood samples. The appearance of schizonts in peripheral blood smears is thought to be a marker of high sequestered parasite burden and severe disease. In the present study, the value of schizontaemia as an early marker for severe disease in nonimmune individuals with imported malaria was evaluated. METHODS: All patients in the Rotterdam Malaria Cohort diagnosed with P. falciparum malaria between 1 January 1999 and 1 January 2012 were included. Thick and thin blood films were examined for the presence of schizontaemia. The occurrence of WHO defined severe malaria was the primary endpoint. The diagnostic performance of schizontaemia was compared with previously evaluated biomarkers C-reactive protein and lactate. RESULTS: Schizonts were present on admission in 49 of 401 (12.2%) patients. Patients with schizontaemia were more likely to present with severe malaria, a more complicated course and had longer duration of admission in hospital. Schizontaemia had a specificity of 0.95, a sensitivity of 0.53, a negative predictive value of 0.92 and a positive predictive value of 0.67 for severe malaria. The presence of schizonts was an independent predictor for severe malaria. CONCLUSION: Absence of schizonts was found to be a specific marker for exclusion of severe malaria. Presence of schizonts on admission was associated with a high positive predictive value for severe malaria. This may be of help to identify patients who are at risk of a more severe course than would be expected when considering peripheral parasitaemia alone.     

Plasmodium falciparum: drug-resistant malaria complicating leukemias and lymphomas in children.

The present communication deals with drug-resistant Plasmodium falciparum malaria complicating hematologic malignancies (leukemias, n = 24, and lymphomas, n = 7) in children. Of 50 cases of hematologic malignancies, 31 patients were microscopically diagnosed as having P. falciparum infection (MP +). Initially, all the patients were treated with chloroquine. The results of primary treatment showed chloroquine resistance in 16 (51. 62%) cases. Of these 16 chloroquine-resistant cases, 13 were secondarily treated with a combination of pyrimethamine plus sulfamethopyrazine. The results of secondary treatment also revealed resistance to pyrimethamine plus sulfamethopyrazine in 6 of 13 (46. 10%) cases. The 6 pyrimethamine plus sulfamethopyrazine-resistant P. falciparum cases were finally cured by quinine therapy, against which no resistance was encountered. Conversely, in the control group comprising 38 cases of P. falciparum without malignancy, the incidence of chloroquine resistance was found in only 9 cases, which is rather low (23.70%). Of these 7 chloroquine-resistant cases, 5 were found to be sensitive to pyrimethamine plus sulfamethopyrazine treatment, while the 2 nonresponders were finally cured with quinine. The overall results of this study show a high prevalence of chloroquine resistance among clinical cases of falciparum malaria (25/69; 30.6%). Among the nonresponders (n = 20) 40% of cases were also resistant to the pyrimethamine plus sulfamethopyrazine combination. There was no resistance to quinine.     

Immune regulation of protection and pathogenesis in Plasmodium falciparum malaria

The immune mechanisms whereby malaria parasites are eliminated by the human host or how they may avoid the immune response are poorly understood. Individuals living in malaria-endemic areas gradually acquire immunity. It is well established that this immunity involves both cell-mediated and humoral mechanisms and that T cells are the major regulators in both these events. The existence of functionally distinct P. falciparum-specific CD4+ T-cell subsets in humans has been shown in several studies. However, in contrast to what is the case in murine models there is no definitive link between the activation of various T cells and the course of human P. falciparum blood-stage infection. In the present paper we will review recent findings which illustrate how the balance between functionally different T-cell subsets affects the development of malaria immunity but also may contribute to its pathogenicity. An example of the latter is the deposition of IgE-containing immune complexes in small vessels, probably leading to local overproduction of tumor-necrosis factor (TNF), a pathogenic factor in malaria.     

Plasmodium falciparum: DNA probe diagnosis of malaria in Kenya

We previously reported isolation of DNA probe which specifically recognizes Plasmodium falciparum and developed a simple method for its use. The sensitivity and specificity of this DNA probe method have now been extensively field tested in comparison with those of conventional microscopic examination of blood films in two separate studies in Malindi, Kenya, involving a total of 1179 patients. In the second study, which used improved techniques, sensitivity of the DNA probe was 89% when compared to microscopy. We conclude that the DNA probe method compares favorably with conventional microscopy in detecting parasite densities as low as 25 parasites per microliter of blood. A significant advantage of the DNA probe method is that it utilizes a standardized procedure which can simultaneously and reproducibly analyze a large number of samples without opportunity for significant reader bias.     

Towards a proteomic definition of CoArtem action in Plasmodium falciparum malaria

We have adopted a proteomic strategy to investigate the actions of the two active components of the new antimalarial CoArtem, artemether and lumefantrine, following pharmacologically relevant drug exposure in the human malaria parasite Plasmodium falciparum. Both drugs induced profound alterations in the parasite's proteome. Moreover, the pattern of proteome alteration was specific for the drug used. The two drugs induced opposing effects on key glycolytic enzymes while exerting similar influence of the expression of stress response proteins. These initial results demonstrate the power of this approach in the study of pleiomorphic mechanisms of drug action.     

The role of Plasmodium falciparum var genes in malaria in pregnancy

Sequestration of Plasmodium falciparum-infected erythrocytes in the placenta is responsible for many of the harmful effects of malaria during pregnancy. Sequestration occurs as a result of parasite adhesion molecules expressed on the surface of infected erythrocytes binding to host receptors in the placenta such as chondroitin sulphate A (CSA). Identification of the parasite ligand(s) responsible for placental adhesion could lead to the development of a vaccine to induce antibodies to prevent placental sequestration. Such a vaccine would reduce the maternal anaemia and infant deaths that are associated with malaria in pregnancy. Current research indicates that the parasite ligands mediating placental adhesion may be members of the P. falciparum variant surface antigen family PfEMP1, encoded by var genes. Two relatively well-conserved subfamilies of var genes have been implicated in placental adhesion, however, their role remains controversial. This review examines the evidence for and against the involvement of var genes in placental adhesion, and considers whether the most appropriate vaccine candidates have yet been identified.     

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.