In vitro biosynthesis and membrane translocation of the serine rich protein of Plasmodium falciparum

The serine-rich protein (SERP) of Plasmodium falciparum is found within the parasitophorous vacuole. Exons 1 and 2 of the SERP gene were combined to a continuous open reading frame and expressed in a cell free translation/translocation system to study translocation of the protein across membranes. The protein was found to be translocated co-translationally across canine pancreatic microsomes. This process required the presence of the signal recognition particle, and it was accompanied by cleavage of a signal peptide. We conclude that the authentic SERP is exported from the parasite cell via the endoplasmic reticulum.     

Genes for glycosylphosphatidylinositol toxin biosynthesis in Plasmodium falciparum

About 2.5 million people die of Plasmodium falciparum malaria every year. Fatalities are associated with systemic and organ-specific inflammation initiated by a parasite toxin. Recent studies show that glycosylphosphatidylinositol (GPI) functions as the dominant parasite toxin in the context of infection. GPIs also serve as membrane anchors for several of the most important surface antigens of parasite invasive stages. GPI anchoring is a complex posttranslational modification produced through the coordinated action of a multicomponent biosynthetic pathway. Here we present eight new genes of P. falciparum selected for encoding homologs of proteins essential for GPI synthesis: PIG-A, PIG-B, PIG-M, PIG-O, GPI1, GPI8, GAA-1, and DPM1. We describe the experimentally verified mRNA and predicted amino acid sequences and in situ localization of the gene products to the parasite endoplasmic reticulum. Moreover, we show preliminary evidence for the PIG-L and PIG-C genes. The biosynthetic pathway of the malaria parasite GPI offers potential targets for drug development and may be useful for studying parasite cell biology and the molecular basis for the pathophysiology of parasitic diseases.     

Plasmodium falciparum malaria vaccines in development

The development and implementation of a malaria vaccine would constitute a major breakthrough for global health. Recently, numerous new candidates have entered clinical testing, following strategies that are as diverse as the malaria cycle is complex. While promising results have been obtained, some candidate vaccines have not fulfilled expectations. The challenges are not merely scientific; further progresses will require the development of competent investigator networks, partnerships between academics, industry and funding agencies, and continuous political commitment. In this review, we present the developmental status of all malaria vaccine candidates that are currently in human clinical testing against Plasmodium falciparum, as well as selected malaria vaccine candidates at preclinical development stage, and discuss the main challenges facing the field of malaria vaccine development.     

The putative gene for the first enzyme of glutathione biosynthesis in Plasmodium berghei and Plasmodium falciparum

The putative gene for gamma-glutamylcysteine synthetase, the rate-limiting enzyme in glutathione biosynthesis, has been characterized both in Plasmodium berghei and Plasmodium falciparum. Protein sequence comparison between these two species reveals large conserved regions sharing more than 80% similarity, separated by less conserved portions. When the comparison is extended to known gamma-glutamylcysteine synthetases from other eukaryotes, a number of high similarity blocks are observed which may help in identifying sequence essential for protein function.     

Peptidases from Plasmodium falciparum cultured in vitro

An acid peptidase that degrades hemoglobin optimally at pH 3.5, a neutral aminopeptidase and an alkaline endopeptidase that acts on an alpha-N-blocked synthetic substrate have been demonstrated in Plasmodium falciparum in culture. The enzymes were shown to be distinct by anion exchange chromatography, gel filtration on Sephadex G-200 and isoelectric focusing. The activities of the acid peptidase and the aminopeptidase were inhibited by antimalarial compounds.     

Obtaining hepatic stages of Plasmodium falciparum in vitro

Sporozoites of Plasmodium falciparum, obtained by membrane feeding of Anopheles freeborni or A. stephensi with cultured gametocytes, were used to infect monolayers of human hepatocytes. Fluorescent labelling with an African serum as well as Giemsa staining performed from day one to day 7 of cultures, demonstrated the presence of numerous hepatic schizonts measuring up to 40 micron.     

In vitro inhibition of Plasmodium falciparum by pyrazofurin, an inhibitor of pyrimidine biosynthesis de novo

The effect of pyrazofurin, an inhibitor of UMP synthesis, on Plasmodium falciparum growth in vitro has been studied. ID50 values (concentration of compound causing 50% inhibition of [3H]hypoxanthine incorporation) for the FCQ-27, FCI-1 and K-1 (chloroquine-resistant) isolates were 10 +/- 8.7, 6.4 +/- 5.3 and 6.3 +/- 0.5 microM, respectively. Comparative ID50 values for chloroquine were 13.5 +/- 4.2, 22.8 +/- 7.6 and 343 +/- 114 microM, respectively. Over the 48-h intraerythrocytic cycle of tightly synchronized parasites, pyrazofurin both reduced the parasitemia and retarded the maturation of trophozoites and schizonts. Addition of uracil or uridine to the in vitro culture did not decrease the anti-parasitic activity of pyrazofurin. Chloroquine reduced the parasitemia, but did not retard development of the remaining viable parasites. Pyrazofurin (20 microM) caused a 50% inhibition of parasite orotate phosphoribosyltransferase (E.C. 2.4.2.10) and, in the presence of adenosine kinase and ATP, a 73% inhibition of orotidine-5'-phosphate decarboxylase (E.C. 4.1.1.23).     

Assessing the polyamine metabolism of Plasmodium falciparum as chemotherapeutic target

More than 30 years ago the potent ornithine decarboxylase inhibitor difluoromethylornithine (DFMO) was designed as new anticancer drug. Its efficacy was not as expected since the polyamine metabolism in mammalian cells seemed to be far more complex. However when DFMO was applied to African trypanosomes its effect on this protozoan parasite was highly convincing. Thenceforward many researchers tested DFMO and also other polyamine synthesis inhibitors against different parasites among them the causative agent of malaria Plasmodium. This review recapitulates the different attempts to interfere chemically with the plasmodial polyamine metabolism, the impact on the disease as well as its biochemical and molecular background. It will show that this fast proliferating organism depends for growth on high amounts of polyamines and that Plasmodium has its own and unique polyamine synthesis, differing highly from the mammalian one mainly in the arrangement of the key enzymes, S-adenosylmethionine decarboxylase and ornithine decarboxylase (AdoMetDC/ODC), on a bifunctional protein.     

Effect of protease inhibitors on exflagellation in Plasmodium falciparum

Enzymes involved in sexual differentiation and fertilization of the human malaria parasite Plasmodium falciparum represent potential targets for transmission blocking strategies. Parasite proteases are putatively involved in several steps during fertilization, but the types of proteases, their targets and modes of action remain hitherto unknown. We investigated the involvement of proteases in gametogenesis via exflagellation and immunofluorescence assays, using a variety of commercially available as well as newly designed protease inhibitors. The assays revealed a blockade of microgamete formation by the cysteine/serine protease inhibitors TLCK and TPCK. The serine protease inhibitor PMSF, the falcipain-targeting inhibitor RV112D, and the aspartic protease inhibitor EPNP also significantly decreased formation of microgametes. The metalloprotease inhibitor 1,10-phenanthroline, on the other hand, inhibited exflagellation by interfering with microgamete motility. Furthermore, EPNP reduced the activation of male and female gametocytes. Our data point to a major involvement of serine proteases and a non-thermolysin-like zinc metalloprotease in microgametocyte exflagellation.     

Antibody-independent inhibition of Plasmodium falciparum in vitro cultures.

The sera of 100 Colombian individuals of African origin living in a malaria-endemic area of the Pacific coast were studied with regard to their capacity to inhibit Plasmodium falciparum cultures in vitro. Antimalarial antibody levels determined by indirect immunofluorescence were higher in the group of infected individuals than in the noninfected individuals, and inhibitory activity assessed by the inhibition of parasite incorporation of 3H-hypoxanthine in vitro was present in the sera of both the infected and noninfected patients. We believe that the noninfected patients were probably immune. The sera of some of the infected patients had high inhibitory capacities for the P. falciparum FCB-1 isolate. When the inhibitory effects of some of the sera were tested by using four parasite isolates from different regions of the world, striking differences among them were found.     

Clinical correlates of in vitro Plasmodium falciparum cytoadherence.

To determine whether isolates of Plasmodium falciparum have intrinsically different cytoadherent properties and whether these differences contribute to the clinical severity of human falciparum malaria, we studied the cytoadherence to C32 melanoma cells in vitro of 59 parasite isolates from patients with naturally acquired infections in Thailand. Parasitized erythrocytes adhere to these melanoma cells principally via the glycoprotein CD36, which is also expressed on most vascular endothelium. In vitro cytoadherence was significantly greater for isolates from patients with biochemical evidence of severe malaria. The cytoadherent properties of P. falciparum parasites may thus be a virulence factor in human falciparum malaria. However, there was no correlation between the degree of in vitro cytoadherence and cerebral symptoms, which suggests that other receptors and/or host factors may be important in the adherence of malaria parasites to cerebral vascular endothelium. The cytokines tumor necrosis factor, interleukin-1, and gamma interferon, which have been implicated in the pathogenesis of cerebral malaria and are known to promote intercellular adhesion in other systems, did not enhance the cytoadherence of P. falciparum isolates to C32 melanoma cells.     

Thalassemic erythrocytes inhibit in vitro growth of Plasmodium falciparum

Blood specimens from 100 thalassemic patients were screened in vitro for inhibitory effects on growth and multiplication of Plasmodium falciparum. The culture medium mixture designated REM consisted of 9 volumes of minimum essential medium (GIBCO Laboratories, Grand Island, N.Y.) and 1 volume of RPMI 1640 (GIBCO) supplemented with 10% heat-inactivated human serum. Parasite multiplication in erythrocytes containing normal hemoglobin cultured in RPMI or REM was similar. Significant reduction in parasite multiplication rates was observed in erythrocytes containing abnormal hemoglobin when these were cultured in REM. The degree of reduction in five types of thalassemic erythrocytes was in the following descending order: hemoglobin H disease with Hb Constant Spring, classical hemoglobin H disease, beta(0)-thalassemia-hemoglobin E in which blood harbored a high percentage of hemoglobin F-containing cells, beta (0)-thalassemia-hemoglobin E in which blood harbored few hemoglobin F-containing cells, and beta-thalassemia heterozygous variant.     

Improved synchronous production of Plasmodium falciparum gametocytes in vitro

The sexual stages of the Plasmodium falciparum life cycle are attractive targets for vaccines and transmission blocking drugs. Difficulties in culturing and obtaining large amounts of sexual stage P. falciparum parasites, particularly early stages, have often limited research progress in this area. We present a new protocol which simplifies the process of stimulating gametocytogenesis leading to improved synchronous gametocyte production. This new method can be adapted to enrich for early stage gametocytes (I and II) with a higher degree of purity than has previously been achieved, using MACS magnetic affinity columns. The protocol described lends itself to large scale culturing and harvesting of synchronous parasites suitable for biochemical assays, northern blots, flow cytometry, microarrays and proteomic analysis.     

Cytostatic effect of DL-α-difluoromethylornithine against Plasmodium falciparum and its reversal by diamines and spermidine

DL--difluoromethylornithine (DFMO) inhibited ornithine decarboxylase (ODC) activity and arrested the growth of Plasmodium falciparum at the early trophozoite stage. The inhibition of ODC activity did not result in the formation of an alternative diamine such as cadaverine. When putrescine or spermidine were added to the parasites grown in culture, the arrest was reversed, and normal schizogony was completed even in the presence of DFMO. Some reversal of the inhibition was achieved with cadaverine at high concentrations, while 1,3-diaminopropane and spermine failed to restore the development. Resumption of growth could be detected when putrescine was added even after 67 h of DFMO treatment. Electron microscopy did not reveal any changes in the morphology of parasites treated for 47 h, while 73 h of treatment with DFMO induced massive accumulation of pigment. Death was observed a few hours later. These results suggest that DFMO acts as a cytostatic rather than as a cytocidal agent. The four carbon diamine restored cell growth while the shorter or the longer homologous compounds showed little activity.     

Filling the gap of intracellular dephosphorylation in the Plasmodium falciparum vitamin B1 biosynthesis

Thiamine pyrophosphate (TPP), the active form of vitamin B1, is an essential cofactor for several enzymes. Humans depend exclusively on the uptake of vitamin B1, whereas bacteria, plants, fungi and the malaria parasite Plasmodium falciparum are able to synthesise thiamine monophosphate (TMP) de novo. TMP has to be dephosphorylated prior to pyrophosphorylation in order to obtain TPP. In P. falciparum the phosphatase capable to catalyse this reaction has been identified by analysis of the substrate specificity. The recombinant enzyme accepts beside vitamin B1 also nucleotides, phosphorylated sugars and the B6 vitamer pyridoxal 5'-phosphate. Vitamin B1 biosynthesis is known to occur in the cytosol. The cytosolic localisation of this phosphatase was verified by transfection of a GFP chimera construct. Stage specific Northern blot analysis of the phosphatase clearly identified an expression profile throughout the entire erythrocytic life cycle of P. falciparum and thereby emphasises the importance of dephosphorylation reactions within the malaria parasite.     

De novo and salvage biosynthesis of pteroylpentaglutamates in the human malaria parasite, Plasmodium falciparum

Plasmodium falciparum was shown to synthesize pteroylpolyglutamate de novo from guanosine 5'-triphosphate (GTP), p-aminobenzoate (PABA), and L-glutamate (L-Glu). The parasite also had the capacity to synthesize pteroylpolyglutamate from both intact and degradation moieties (p-aminobenzoylglutamate and pterin-aldehyde) of exogenous folate added into the growth medium. The major product was identified as 5-methyl-tetrahydroteroylpentaglutamate following exposure to pteroylpolyglutamate hydrolase and oxidative degradation of the C9-N10 bond in the molecule and identification of products by reversed-phase high performance liquid chromatography. Inhibition of pteroylpentaglutamate synthesis from the radiolabelled metabolic precursors (GTP, PABA, L-Glu) and folate by the antifolate antimalarials, pyrimethamine and sulfadoxine at therapeutic concentrations, may suggest the existence of a unique biosynthetic pathway in the malaria parasite.     

In vitro sensitivity pattern of chloroquine and artemisinin in Plasmodium falciparum

Artemisinin (ART) and its derivatives form the mainstay of antimalarial therapy. Emergence of resistance to them poses a potential threat to future malaria control and elimination on a global level. It is important to know the mechanism of action of drug and development of drug resistance. We put forwards probable correlation between the mode of action of chloroquine (CQ) and ART. Modified trophozoite maturation inhibition assay, WHO Mark III assay and molecular marker study for CQ resistance at K76T codon in Plasmodium falciparum CQ-resistant transporter gene were carried out on cultured P. falciparum. On comparing trophozoite and schizont growth for both CQ-sensitive (MRC-2) and CQ-resistant (RKL-9) culture isolates, it was observed that the clearance of trophozoites and schizonts was similar with both drugs. The experiment supports that CQ interferes with heme detoxification pathway in food vacuoles of parasite, and this may be correlated as one of the plausible mechanisms of ART.     

Cytokine-induced inhibition of Plasmodium falciparum erythrocytic growth in vitro.

The addition of recombinant cytokines to Plasmodium falciparum in vitro cultures retarded the growth of the parasite with the effect of recombinant IL-2 (rIL-2) > interferon-gamma (IFN-gamma) > tumour necrosis factor-beta (TNF-beta). The process was concentration dependent, being greatest at 30,000 U/ml and required a 72-h period of continuous exposure for maximum effect. Growth inhibition, as determined morphologically and radiometrically, was a consequence of defective schizont maturation rather than inhibition of merozoite invasion. It was cumulative and detectable within one erythrocytic (48 h) growth cycle.     

Characterization of Plasmodium falciparum sexual stage antigens and their biosynthesis in synchronised gametocyte cultures

Synchronised gametocyte cultures were used to study the biosynthesis of the sexual stage target antigens (Mr 230 000, 48 000 and 25 000) for anti gamete/zygote antibodies. These antigens were shown to be synthesized during gametocyte development from day 2-3 onwards until gametogenesis occurred. After gametogenesis a 25 kDa protein was predominantly synthesized, whereas synthesis of the other target proteins was hardly detectable. The 48, 45, and 25 kDa proteins appeared to be glycosylated, in addition the 25 kDa was also acylated in that it bound [3H]palmitic acid covalently. The iso-electric point (pI) of these proteins was assessed as being 6.0 +/- 0.1 (for both 48 and 45 kDa) and 5.6 +/- 0.1 (for 25 kDa).