Cyclosporin-binding proteins of Plasmodium falciparum

A number of cyclosporins, including certain non-immunosuppressive ones, are potent inhibitors of the intraerythrocytic growth of the human malarial parasite Plasmodium falciparum. The major cyclosporin-binding proteins of P. falciparum were investigated by affinity chromatography on cyclosporin-Affigel followed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, Western blotting, and peptide mass fingerprinting. The two bands obtained on gels were shown to correspond to cyclophilins, PfCyP-19A (formerly PfCyP-19) and PfCyP-19B, whose genes had been characterised previously. PfCyP-19B was an abundant protein of intraerythrocytic P. falciparum (up to 0.5% of parasite protein) that was present in the highest amounts in schizont-stage parasites. Unexpectedly, given its apparent signal sequence, it was located primarily in the cytosol of the parasite. The peptidyl-prolyl cis-trans isomerase activity of recombinant PfCyP-19B had the same profile of susceptibility to cyclosporin derivatives as the bulk isomerase activity of crude P. falciparum extracts. The binding of cyclosporins to cyclophilins may be relevant to the mechanism of action of the drug in the parasite.     

Rapid genotyping of loci involved in antifolate drug resistance in Plasmodium falciparum by primer extension

Current methods used to genotype point mutations in Plasmodium falciparum genes involved in resistance to antifolate drugs include restriction digestion of PCR products, allele-specific amplification or sequencing. Here we demonstrate that known point mutations in dihydrofolate reductase and dihydropteroate synthase can be scored quickly and accurately by single-nucleotide primer extension and detection of florescent products on a capillary sequencer. We use this method to genotype parasites in natural infections from the Thai-Myanmar border. This approach could greatly simplify large-scale screening of resistance mutations of the type required for evaluating and updating antimalarial drug treatment policies. The method can be easily adapted to other P. falciparum genes and will greatly simplify scoring of point mutations in this and other parasitic organisms.     

Cyclin-dependent kinase homologues of Plasmodium falciparum

The intraerythrocytic asexual cycle of the malarial parasite is complex and atypical: during schizogony the parasite undergoes multiple rounds of DNA replication and asynchronous nuclear division without cytokinesis. This cell cycle deviates from the classical eukaryotic cell cycle model where, 'DNA replicates only once per cell cycle'. A clear understanding of the molecular switches that control this unusual developmental cycle would be of great interest, both in terms of fundamental Plasmodium biology and in terms of novel potential drug target identification. In recent years considerable effort has been made to identify the malarial orthologues of the cyclin-dependent kinases, which are key regulators of the orderly progression of the eukaryotic cell cycle. This review focuses on the current state-of-knowledge of Plasmodium falciparum cyclin-dependent kinase-like kinases and their regulators.     

Dihydroorotase of human malarial parasite Plasmodium falciparum differs from host enzyme

Plasmodium falciparum, the causative agent of human malaria, is totally dependent on de novo pyrimidine biosynthetic pathway. A gene encoding P. falciparum dihydroorotase (pfDHOase) was cloned and expressed in Escherichia coli as monofunctional enzyme. PfDHOase revealed a molecular mass of 42 kDa. In gel filtration chromatography, the major enzyme activity eluted at 40 kDa, indicating that it functions in a monomeric form. This was similarly observed using the native enzyme purified from P. falciparum. Interestingly, kinetic parameters of the enzyme and inhibitory effect by orotate and its 5-substituted derivatives parallel that found in mammalian type I DHOase. Thus, the malarial enzyme shares characteristics of both type I and type II DHOases. This study provides the monofunctional property of the parasite DHOase lending further insights into its differences from the human enzyme which forms part of a multifunctional protein.     

Morphology and kinetics of the three distinct phases of red blood cell invasion by Plasmodium falciparum merozoites

The invasion of red blood cells (RBCs) is an essential event in the life cycle of all malaria-causing Plasmodium parasites; however, there are major gaps in our knowledge of this process. Here, we use video microscopy to address the kinetics of RBC invasion in the human malaria parasite Plasmodium falciparum. Under in vitro conditions merozoites generally recognise new target RBCs within 1 min of their release from their host RBC. Parasite entry ensues and is complete on average 27.6 s after primary contact. This period can be divided into two distinct phases. The first is an ∼11 s ‘pre-invasion’ phase that involves an often dramatic RBC deformation and recovery process. The second is the classical ‘invasion’ phase where the merozoite becomes internalised within the RBC in a ∼17 s period. After invasion, a third ‘echinocytosis’ phase commences when about 36 s after every successful invasion a dramatic dehydration-type morphology was adopted by the infected RBC. During this phase, the echinocytotic effect reached a peak over the next 23.4 s, after which the infected RBC recovered over a 5-11 min period. By then the merozoite had assumed an amoeboid-like state and was apparently free in the cytoplasm. A comparison of our data with that of an earlier study of the distantly related primate parasite Plasmodium knowlesi indicated remarkable similarities, suggesting that the kinetics of invasion are conserved across the Plasmodium genus. This study provides a morphological and kinetic framework onto which the invasion-associated physiological and molecular events can be overlaid.     

Isolation and functional characterization of a dynamin-like gene from Plasmodium falciparum

A novel dynamin-like GTPase gene, Pfdyn1, was cloned from an asexual stage cDNA library of Plasmodium falciparum Dd2 strain. Pfdyn1 contains a highly conserved N-terminal tripartite GTPase domain, a coiled-coil region, and a C-terminal 129 aa unknown function domain. Like yeast Vps1p, it lacks pleckstrin homology domain and proline-rich region. Western blot analysis showed that Pfdyn1 is a Triton X-100 insoluble protein expressed only in the mature sub-stage. Morphological studies indicated that Pfdyn1 is partly co-localized with PfGRP, a known ER-resident protein, and localizes diffusely with several membrane structures and a 60-100 nm vesicle both inside and on surface of the parasites and also in the cytoplasm of infected erythrocytes. The dsRNA originated by C-terminus fragment of Pfdyn1 inhibits markedly the growth of P. falciparum parasite at the erythrocyte stage. Those data showed that Pfdyn1 is a conservative, membrane related protein and plays an essential role for the survival of Plasmodium parasite.     

Plasmodium falciparum: growth response to potassium channel blocking compounds

Potassium channels are essential for cell survival and regulate the cell membrane potential and electrochemical gradient. During its lifecycle, Plasmodium falciparum parasites must rapidly adapt to dramatically variant ionic conditions within the mosquito mid-gut, the hepatocyte and red blood cell (RBC) cytosols, and the human circulatory system. To probe the participation of K⁺ channels in parasite viability, growth response assays were performed in which asexual stage P. falciparum parasites were cultured in the presence of various Ca²⁺-activated K⁺ channel blocking compounds. These data describe the novel anti-malarial effects of bicuculline methiodide and tubocurarine chloride and the novel lack of effect of apamine and verruculogen. Taken together, the data herein imply the presence of K⁺ channels, or other parasite-specific targets, in P. falciparum-infected RBCs that are sensitive to blockade with Ca²⁺-activated K⁺ channel blocking compounds.     

Inhibition of Plasmodium falciparum proliferation in vitro by antisense oligodeoxynucleotides against malarial topoisomerase II

The development of new effective antimalarial agents is urgently needed due to the ineffectiveness of current drug regimes on the most virulent human malaria parasite Plasmodium falciparum. Antisense (AS) oligodeoxynucleotides (ODNs) have shown promise as chemotherapeutic agents. Phosphorothioate AS ODNs against different regions of P. falciparum topoisomerase II gene were investigated. Chloroquine- and pyrimethamine-resistant P. falciparum K1 strain was exposed to phosphorothioate AS ODNs for 48 h and growth was determined by flow cytometric assay or by microscopic assay. Exogenous delivery of phosphorothioate AS ODNs between 0.01 and 0.5 microM significantly inhibited parasite growth compared with sense sequence controls suggesting sequence specific inhibition. This inhibition was shown to occur during maturation stages, with optimal inhibition being detected after 36 h. These results should prove useful in future designs of novel antimalarial agents.     

Epigallocatechin gallate is a slow-tight binding inhibitor of enoyl-ACP reductase from Plasmodium falciparum

Epigallocatechin gallate (EGCG) is known to have numerous pharmacological properties. In the present study, we have shown that EGCG inhibits enoyl-acyl carrier protein reductase of Plasmodium falciparum (PfENR) by following a two-step, slow, tight-binding inhibition mechanism. The association/isomerization rate constant (k₅) of the reversible and loose PfENR-EGCG binary complex to a tight [PfENR-EGCG]^∗ or EI^∗ complex was calculated to be 4.0 × 10⁻² s⁻¹. The low dissociation rate constant (k₆) of the [PfENR-EGCG]^∗ complex confirms the tight-binding nature of EGCG. EGCG inhibited PfENR with the overall inhibition constant (K_i^∗) of 7.0 ± 0.8 nM. Further, we also studied the effect of triclosan on the inhibitory activity of EGCG. Triclosan lowered the k₆ of the EI∗ complex by 100 times, lowering the overall K_i^∗ of EGCG to 97.5 ± 12.5 pM. The results support EGCG as a promising candidate for the development of tea catechin based antimalarial drugs.     

Mitochondrial NADH dehydrogenase from Plasmodium falciparum and Plasmodium berghei

The mitochondrial electron transport system is necessary for growth and survival of malarial parasites in mammalian host cells. NADH dehydrogenase of respiratory complex I was demonstrated in isolated mitochondrial organelles of the human parasite Plasmodium falciparum and the mouse parasite Plasmodium berghei by using the specific inhibitor rotenone on oxygen consumption and enzyme activity. It was partially purified by two sequential steps of fast protein liquid chromatographic techniques from n-octyl glucoside solubilization of the isolated mitochondria of both parasites. In addition, physical and kinetic properties of the malarial enzymes were compared to the host mouse liver mitochondrial respiratory complex I either as intact or as partially purified forms. The malarial enzyme required both NADH and ubiquinone for maximal catalysis. Furthermore, rotenone and plumbagin (ubiquinone analog) showed strong inhibitory effect against the purified malarial enzymes and had antimalarial activity against in vitro growth of P. falciparum. Some unique properties suggest that the enzyme could be exploited as chemotherapeutic target for drug development, and it may have physiological significance in the mitochondrial metabolism of the parasite.     

The role of tryptophan-48 in catalysis and binding of inhibitors of Plasmodium falciparum dihydrofolate reductase

Dihydrofolate reductases (DHFRs) from Plasmodium falciparum (Pf) and various species of both prokaryotic and eukaryotic organisms have a conserved tryptophan (Trp) at position 48 in the active site. The role in catalysis and binding of inhibitors of the conserved Trp48 of PfDHFR has been analysed by site-specific mutagenesis, enzyme kinetics and use of a bacterial surrogate system. All 19 mutant enzymes showed undetectable or very low specific activities, with the highest value of k(cat)/K(m) from the Tyr48 (W48Y) mutant (0.12 versus 11.94M(-1)s(-1)), of about 1% of the wild-type enzyme. The inhibition constants for pyrimethamine, cycloguanil and WR99210 of the W48Y mutants are 2.5-5.3 times those of the wild-type enzyme. All mutants, except W48Y, failed to support the growth of Escherichia coli transformed with the parasite gene in the presence of trimethoprim, indicating the loss of functional activity of the parasite enzyme. Hence, Trp48 plays a crucial role in catalysis and inhibitor binding of PfDHFR. Interestingly, W48Y with an additional mutation at Asn188Tyr (N188Y) was found to promote bacterial growth and yielded a higher amount of purified enzyme. However, the kinetic parameters of the purified W48Y+N188Y enzyme were comparable with W48Y and the binding affinities for DHFR inhibitors were also similar to the wild-type enzyme. Due to its conserved nature, Trp48 of PfDHFR is a potential site for interaction with antimalarial inhibitors which would not be compromised by its mutations.     

Plasmodium falciparum TryThrA antigen synthetic peptides block in vitro merozoite invasion to erythrocytes

Tryptophan-threonine-rich antigen (TryThrA) is a Plasmodium falciparum homologue of Plasmodium yoelii-infected erythrocyte membrane pypAg-1 antigen. pypAg-1 binds to the surface of uninfected mouse erythrocytes and has been used successfully in vaccine studies. The two antigens are characterized by an unusual tryptophan-rich domain, suggesting similar biological properties. Using synthetic peptides spanning the TryThrA sequence and human erythrocyte we have done binding assays to identify possible TryThrA functional regions. We describe four peptides outside the tryptophan-rich domain having high activity binding to normal human erythrocytes. The peptides termed HABPs (high activity binding peptides) are 30884 ((61)LKEKKKKVLEFFENLVLNKKY(80)) located at the N-terminal and 30901 ((401)RKSLEQQFGDNMDKMNKLKKY(420)), 30902 ((421)KKILKFFPLFNYKSDLESIM(440)) and 30913 ((641)DLESTAEQKAEKKGGKAKAKY(660)) located at the C-terminal. Studies with polyclonal goat antiserum against synthetic peptides chosen to represent the whole length of the protein showed that TryThrA has fluorescence pattern similar to PypAg-1 of P. yoelii. All HABPs inhibited merozoite in vitro invasion, suggesting that TryThrA protein may be participating in merozoite-erythrocyte interaction during invasion.     

The human placental derived BeWo cell line: a useful model for selecting Plasmodium falciparum CSA-binding parasites

Chondroitin sulfate A (CSA) present in the placental intervillous blood spaces has been described as the main receptor involved in the massive sequestration of Plasmodium falciparum parasitized erythrocytes to the placenta. Placental parasite isolates are functionally distinct from isolates that sequester in other organs, because they do not cytoadhere to CD36 but instead bind to CSA. To investigate for the parasites molecules associated with the CSA adhesion phenotype, different methodologies have been developed to select for CSA-binding lines in vitro mainly using non-placental sources of CSA that differ in their sulfation pattern. In this study, we show that the human trophoblastic BeWo cell line is a very efficient alternative to select for the CSA-binding phenotype in parasitized erythrocytes.     

Study on the biochemical basis of mefloquine resistant Plasmodium falciparum

Increase in drug detoxification and alteration of drug uptake and efflux of Plasmodium falciparum were investigated for their possible association with mefloquine (MQ) resistance in five different clones of P. falciparum from Thailand (T994b₃, K1CB₂, PR70CB₁, PR71CB₂ and TM₄CB8-2.2.3). Fifty percent inhibitory concentration (IC₅₀) values from these five clones varied between 30- and 50-fold. Regarding the detoxification mechanism, the ability of P. falciparum clones to biotransform MQ was shown in vitro by parasite microsomal protein prepared from parasite infected red blood cells protein (30 μg), NADPH (1 nM) and phosphate buffer pH 7.4, carried out at 37 °C with agitation. Radiolabelled unmetabolized MQ and possible metabolite(s) generated from the reaction was extracted into ethylacetate and separated by radiometric-HPLC after 1 h. All clones were capable of converting MQ into carboxymefloquine (CMQ), which is the main metabolite in human plasma. In addition, another unidentified metabolite eluted at 4.2 min on the chromatograph could be detected from the incubation reaction. This metabolite has never been detected in human liver microsomes before. There was no significant difference in the percentages of CMQ formed in the resistant (T994_b3, PR₇₀CB₁, PR₇₁CB₂) and sensitive (TM₄CB8-2.2.3, K1CB₂) clones. Another possible mechanism, i.e., alteration in the accumulation of MQ in the parasites was investigated in vitro using [¹⁴C]MQ as a tracer. The time courses of [¹⁴C]MQ uptake and efflux were generally characterized by two phases. A trend of increased efflux of [¹⁴C]MQ was observed in the resistant compared with sensitive clones.     

Structural analysis of chorismate synthase from Plasmodium falciparum: a novel target for antimalaria drug discovery

The shikimate pathway in Plasmodium falciparum provides several targets for designing novel antiparasitic agents for the treatment of malaria. Chorismate synthase (CS) is a key enzyme in the shikimate pathway which catalyzes the seventh and final step of the pathway. P. falciparum chorismate synthase (PfCS) is unique in terms of enzymatic behavior, cellular localization and in having two additional amino acid inserts compared to any other CS. The structure of PfCS along with cofactor FMN was predicted by homology modeling using crystal structure of Helicobacter pylori chorismate synthase (HpCS). The quality of the model was validated using structure analysis servers and molecular dynamics. Dimeric form of PfCS was generated and the FMN binding mechanism involving movement of loop near active site has been proposed. Active site pocket has been identified and substrate 5-enolpyruvylshikimate 3-phosphate (EPSP) along with screened potent inhibitors has been docked. The study resulted in identification of putative inhibitors of PfCS with binding efficiency in nanomolar range. The selected putative inhibitors could lead to the development of anti-malarial drugs.     

Comparison of microsatellite and antigen-coding loci for differentiating recrudescing Plasmodium falciparum infections from reinfections in Kenya

We have compared the ability of five Plasmodium falciparum microsatellites and three antigen-coding loci to differentiate recrudescence from reinfection. We used 133 pairs of P. falciparum-infected blood samples collected during in vivo drug efficacy trials from three sites in Kenya with different malaria endemicities. There were no significant differences between the marker subsets in their ability to discriminate recrudescences from new infections across the three sites. Overall, microsatellite loci revealed significantly higher expected heterozygosity and multiplicity of infection levels than antigen-coding loci. The mean expected heterozygosity across all loci in the three populations was significantly higher with microsatellites (0.70, 0.78 and 0.79) than antigen-coding loci (0.53, 0.60 and 0.62) for Mwea, Tiwi and Bondo areas, respectively. These observations can be explained by three non-exclusive hypotheses: (i) microsatellites are more polymorphic than antigenic loci; (ii) partially immune hosts remove certain parasites from infections on the basis of their antigenic alleles; and/or (iii) recombination occurs in vitro or in vivo with microsatellites.     

Plasmodium falciparum: effect of radiation on levels of gene transcripts in sporozoites

Humans immunized by the bites of irradiated Plasmodium falciparum (Pf) sporozoite-infected mosquitoes are protected against malaria. Radiation attenuates the sporozoites preventing them from fully developing and replicating in hepatocytes, but the effects of radiation on gene expression in sporozoites are unknown. We used RT-PCR (35 cycles of PCR followed by densitometry) to assess the expression of ten genes in Pf sporozoites, and in sporozoites irradiated with 15,000cGy. Irradiation reduced expression substantially (>60%) of two DNA repair genes; moderately (30-60%) of PfUIS3, the Pf orthologue of PbUIS3, a gene up-regulated in Plasmodium berghei sporozoites and of a third DNA repair gene; and minimally (<30%) of the Pf18S ribosomal RNA, PfCSP, PfSSP2/TRAP, and PfCELTOS genes. Irradiation increased expression of PfSPATR minimally. PfLSA1 RNA was not detectable in sporozoites. These results establish that radiation of sporozoites affects gene expression levels and provide the foundation for studies to identify specific genes involved in attenuation and protective immunity.     

Plasmodium falciparum infection and exoerythrocytic development in mice with chimeric human livers

The exoerythrocytic stage of Plasmodium falciparum has remained a difficult phase of the parasite life-cycle to study. The host and tissue specificity of the parasite requires the experimental infection of humans or non-human primates and subsequent surgical recovery of parasite-infected liver tissue to analyze this stage of the parasites development. This type of study is impossible in humans due to obvious ethical considerations and the cost and complexity in working with primate models has precluded their use for extensive studies of the exoerythrocytic stage. In this study we assessed, for the first time, the use of transgenic, chimeric mice containing functioning human hepatocytes as an alternative for modeling the in vivo interaction of P. falciparum parasites and human hepatocytes. Infection of these mice with P. falciparum sporozoites produced morphologically and antigenically mature liver stage schizonts containing merozoites capable of invading human red blood cells. Additionally, using microdissection, highly enriched P. falciparum liver stage parasites essentially free of hepatocyte contamination, were recovered for molecular studies. Our results establish a stable murine model for P. falciparum that will have a wide utility for assessing the biology of the parasite, potential anti-malarial chemotherapeutic agents and vaccine design.