Human p38 mitogen-activated protein kinase inhibitor drugs inhibit Plasmodium falciparum replication

We recently demonstrated that human p38 mitogen-activated protein kinase (MAPK) inhibitors reduced in vitro and in vivo replication of the protozoan parasites Toxoplasma gondii and Encephalitozoon cuniculi. In this study, we assessed the efficacy of five p38 MAPK inhibitors to block the replication of Plasmodium falciparum in human erythrocytes cultured ex vivo and demonstrate that the pyridinylimidazole RWJ67657 and the pyrrolobenzimidazole RWJ68198 reduced P. falciparum replication, yielded trophozoites that were greatly diminished in size at 24 h, and that these two agents interfered with stage differentiation. Interestingly, the chloroquine-resistant strain W2 was significantly more sensitive to these drugs than was the chloroquine-sensitive strain HB3. These results suggest that pyridinylimidazoles and pyrrolobenzimidazoles designed to inhibit human p38 MAPK activation can be developed to treat malaria.     

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.     

Crystal structure of Plasmodium falciparum phosphoglycerate kinase: evidence for anion binding in the basic patch

3-Phosphoglycerate kinase (EC 2.7.2.3) is a key enzyme in the glycolytic pathway and catalyzes an important phosphorylation step leading to the production of ATP. The crystal structure of Plasmodium falciparum phosphoglycerate kinase (PfPGK) in the open conformation is presented in two different groups, namely I222 and P6₁22. The structure in I222 space group is solved using MAD and refined at 3 Å whereas that in P6₁22A is solved using MR and refined at 2.7 Å. I222 form has three monomers in asymmetric unit whereas P6₁22 form has two monomers in the asymmetric unit. In both crystal forms a sulphate ion is located at the active site where ATP binds, but no Mg²⁺ ion is observed. For the first time another sulphate ion is found at the basic patch where the 3-phosphate of 1,3-biphosphoglycerate normally binds. This was found in both chains of P6₁22 form but only in chain A of I222 form.     

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.     

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.     

Immunogenicity of synthetic peptides derived from Plasmodium falciparum proteins

To obtain antibodies suitable to be used in an antigen-capture assay, we have identified, synthesized, and evaluated a series of peptides from different Plasmodium falciparum excretory-secretory proteins: glutamate-rich protein (GLURP); histidine-rich protein 2; histidine-rich protein 3; Falciparum interspersed repeat antigen and, serine-rich antigen homologous. Conformational as well as antigenic predictions were performed using the ANTHEPROT package. Chemical synthesis was carried out by the multiple manual synthesis using the t-boc strategy. The peptides were used as antigens for the preparation of polyclonal antibodies in rabbits. Out of the 14 peptide constructs, eight by ELISA and, six by MABA elicited antibodies that showed correspondence between the predictive study and the immunogenicity obtained in rabbits. All antipeptide (GLURP, HRP2, and FIRA) antisera were found to bind to the corresponding synthetic sequence in an ELISA assay. The binding activity and specificity of antibodies were determined by Western blot with supernatant culture from P. falciparum. Anti-GLURP (IMT-94 and IMT-200) antisera bound to five molecules present in supernatant with molecular weight of 73, 82, 116, 124, and 128 kDa. Anti-HRP2 (IMT-192) antisera recognized a band of 58 kDa. In both cases, the specific molecules were inhibited by preincubation with the homologous peptide. Anti-HRP3, anti-FIRA neither anti-SERPH antisera showed reactivity. Anti-peptides HRP2 antibodies recognized the recombinant protein present in Parasight-F test. The same way, synthetic peptides from HRPII molecule were recognized by monoclonal antibody present in the Parasight-F assay. Our results confirm the potential value of synthetic peptides when inducing monospecific polyclonal antibodies for the development of diagnostic tests based on the capture of antigens.     

Conflicting immune responses can prolong the length of infection in Plasmodium falciparum malaria

We have recently proposed a new model for antigenic variation in Plasmodium falciparum that relies on a network of partially cross-protective immune responses to orchestrate this complex immune evasion process. In addition to exhibiting prolonged oscillations of single variants that resemble the sequential dominance of immunologically distinct antigenic types, the model implies that a higher efficacy of cross-reactive immunity actually increases the length of infection while reducing severity of disease. Here, we analyse the behaviour of a reduced system under conditions of perfect synchrony between variants to demonstrate that these features of this system can be attributed to the antagonism between cross-reactive and variant-specific responses.     

Plasmodium falciparum expresses a multidrug resistance-associated protein

Plasmodium falciparum proteins that efflux toxic metabolic products such as oxidised glutathione (GSSG) are possible targets for anti-malarial drug development. Proteins capable of transporting GSSG and glutathione conjugates include the multidrug resistance-associated transporters (MRPs). A gene, PFA0590w, encoding a MRP homologue, has been identified in P. falciparum. Here we show the presence of full-length mRNA (5.5 kb) of this PfMRP in trophozoites by RT-PCR and Northern blotting. A polyclonal anti-PfMRP antibody generated against two unique, hydrophilic peptides in the predicted sequence produced a strong immunoreactive protein band of 210-215 kDa on Western blots of schizonts of chloroquine-sensitive and chloroquine-resistant strains, confirming expression of PfMRP protein. Using confocal microscopy the protein was seen to be localised at the edge of the schizonts with no obvious staining of the food vacuole. We suggest that PfMRP may act as the GSSG transporter in the parasite plasma membrane.     

Chloroquine mediates specific proteome oxidative damage across the erythrocytic cycle of resistant Plasmodium falciparum

Resistance of Plasmodium falciparum to chloroquine hinders malaria control in endemic areas. Current hypotheses on the action mechanism of chloroquine evoke its ultimate interference with the parasite's oxidative defence systems. Through carbonyl derivatization by 2,4-dinitrophenylhydrazine and proteomics, we compared oxidatively modified proteins across the parasite's intraerythrocytic stages in untreated and transiently IC(50) chloroquine-treated cultures of the chloroquine-resistant P. falciparum strain Dd2. Functional plasmodial protein groups found to be most oxidatively damaged were among those central to the parasite's physiological processes, including protein folding, proteolysis, energy metabolism, signal transduction, and pathogenesis. While an almost constant number of oxidized proteins was detected across the P. falciparum life cycle, chloroquine treatment led to increases in both the extent of protein oxidation and the number of proteins oxidized as the intraerythrocytic cycle progressed to mature stages. Our data provide new insights into early molecular effects produced by chloroquine in the parasite, as well as into the normal protein-oxidation modifications along the parasite cycle. Oxidized proteins involved in the particular parasite drug-response suggest that chloroquine causes specific oxidative stress, sharing common features with eukaryotic cells. Targeting these processes might provide ways of combating chloroquine-resistance and developing new antimalarial drugs.     

Intraerythrocytic stages of Plasmodium falciparum biosynthesize vitamin E

The 2-C-methyl-d-erythritol-4-phosphate and shikimate pathways were found to be active in Plasmodium falciparum and both can result in vitamin E biosynthesis in plants and algae. This study biochemically confirmed vitamin E biosynthesis in the malaria parasite, which can be inhibited by usnic acid. Furthermore, we found evidence pointing to a role of this vitamin in infected erythrocytes. These findings not only contribute to current understanding of P. falciparum biology but also reveal a pathway that could serve as a chemotherapeutic target.     

Dynamic changes in white blood cell counts in uncomplicated Plasmodium falciparum and P. vivax malaria

Total and differential white blood cell (WBC) counts are basic and essential indicators in any type of illness resulting from infection. In malaria, WBC counts are generally characterized as low to normal during treatment. WBC-counts data, before and during treatment with artemisinin derivatives, was gathered for patients with either Plasmodium falciparum or Plasmodium vivax infection (at 28-day follow-up), to investigate dynamic changes in WBC count. We analyzed and compared the WBC counts of 1310 inpatients presenting with uncomplicated P. falciparum and P. vivax malaria at the Hospital for Tropical Diseases, in Bangkok, Thailand. Before-treatment, a statistically significant negative correlation was found between initial WBC count and highest temperature on admission. Before and during treatment, WBC counts were significantly lower in P. falciparum than P. vivax infection on days 0 and 7, but the numerical difference was small. We also found clinically significantly low WBC counts during the acute stages of both types of malaria, which subsequently normalized by day 28 follow-up. This finding has important clinical implications for the conventional method of estimating parasitemia using an assumed WBC count of 8000 cells/μL. The most significant finding in our analysis is that WBC counts in acute P. falciparum and P. vivax malaria are significantly lower than previously assumed for estimating malaria-parasite density. However, these abnormalities returned to normal within several weeks after artemisinin-derivative-based treatment.     

Genetic polymorphisms in the glutamate-rich protein of Plasmodium falciparum field isolates from a malaria-endemic area of Brazil

The genetic diversity displayed by Plasmodium falciparum, the most deadly Plasmodium species, is a significant obstacle for effective malaria vaccine development. In this study, we identified genetic polymorphisms in P. falciparum glutamate-rich protein (GLURP), which is currently being tested in clinical trials as a malaria vaccine candidate, from isolates found circulating in the Brazilian Amazon at variable transmission levels. The study was performed using samples collected in 1993 and 2008 from rural villages situated near Porto Velho, in the state of Rondônia. DNA was extracted from 126 P. falciparum-positive thick blood smears using the phenol-chloroform method and subjected to a nested polymerase chain reaction protocol with specific primers against two immunodominant regions of GLURP, R0 and R2. Only one R0 fragment and four variants of the R2 fragment were detected. No differences were observed between the two time points with regard to the frequencies of the fragment variants. Mixed infections were uncommon. Our results demonstrate conservation of GLURP-R0 and limited polymorphic variation of GLURP-R2 in P. falciparum isolates from individuals living in Porto Velho. This is an important finding, as genetic polymorphisms in B and T-cell epitopes could have implications for the immunological properties of the antigen.     

The role of ICAM-1 in Plasmodium falciparum cytoadherence

Parasite sequestration at microvascular sites is a fundamental phenomenon in the manifestation of the symptoms of malaria and the progression to severe disease. Here, we review the endothelial cell-expressed intercellular adhesion molecule-1 (ICAM-1) and its role in mediating the interaction between the parasitised red blood cell (PRBC) and the vascular endothelium. We highlight the nature of the interaction between ICAM-1 and the parasite-expressed PfEMP-1 molecule at the molecular level. The review also discusses the complexity of the PRBC-endothelial cell interaction and the mechanisms that underlie parasite cytoadherence.     

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.     

Over-production of lactate dehydrogenase from Plasmodium falciparum opens a route to new antimalarials

Over-production of lactate dehydrogenase (PfLDH) from Plasmodium falciparum from E. coli TG2 cells transformed with a pKK223-3 plasmid containing the wild type gene isolated by Bzik DJ, Fox BA, and Gonyer K (1993) Mol. Biochem. Parasit. 59, 155–166, gave mostly an inactive protein after isolation. Sequencing the N-terminus of the over-produced protein showed that the major product commenced at an internal methionine. Truncation of the protein occurred due to the inappropriate priming from a Shine–Dalgarno (SD) sequence upstream of Met 35. Silent mutations of this SD sequence to remove the purine-rich region allowed over-production of the full length PfLDH up to 15 mg protein l^–1 broth. The purified protein exhibited biochemical properties of an authentic LDH enzyme. However, high activity with 3-acetylpyridine adenine dinucleotide as well as with the natural cofactor, NAD, was also observed. The high-resolution X-ray structure obtained from the recombinant enzyme has provided the opportunity for the development of inhibitors specific to PfLDH.     

Fine structure of the malaria parasite Plasmodium falciparum in human hepatocytes in vitro

Summary Recent advances in the ability to culture the hepatic forms of mammalian malaria parasites, particularly of the important human pathogen Plasmodium falciparum have provided novel opportunities to study the ultrastrucural organisation of the parasite in its natural host cell the human hepatocyte. In this electron-microscopic and immunofluorescence study we have found the morphology of both parasite and host cell to be well preserved. The exoerythrocytic forms, which may be found at densities of up to 100/cm², grow at rates comparable to that in vivo in the chimpanzee. In the multiplying 5- and 7-day schizogonic forms the ultrastructural organisation of the parasite bears striking resemblances to other mammalian parasites, e.g., the secretory activity and distribution of the peripheral vacuole system, but also homology with avian parasites, e.g., in nuclear and nucleolar structure and mitochondrial form. The latter homologies support earlier suggestions of the close phylogenetic relationship of P. falciparum with the avian parasites. Evidence is also presented showing the persistence of the cytoskeleton of the invasive sporozoite within the cytoplasm of the ensuing rapidly growing vegetative parasites.     

15-Deoxyspergualin modulates Plasmodium falciparum heat shock protein function

Heat shock proteins are essential for the survival of all cells. The C-terminal EEVD motif of Hsp70 has previously been implicated in binding 15-deoxyspergualin (DSG), an immunosuppressant with antimalarial activity whose mechanism of action is uncertain. We report the cloning, overexpression, and characterization of three members of the heat shock family, PfHsp70-1 (an Hsp70 protein with a C-terminal EEVD motif), PfHsp70-2 (an Hsp70 protein without the EEVD motif), and PfHsp70 interacting protein. The chaperone activity of PfHsp70-1, and PfHsp70-2 was enhanced by ATP and by PfHip. Interestingly, while binding of protein substrates to PfHsp70-1, PfHsp70-2 and PfHip was unaffected in the presence of DSG, the ATP enhanced chaperone activity of PfHsp70-1 but not PfHsp70-2 was stimulated further by DSG. Our finding suggests that the binding partner of DSG in the parasite cellular milieu is PfHsp70-1 and paves the way for the elucidation of the mechanism of antimalarial action of DSG.