Nucleotide sequence and deduced amino acid sequence of a Plasmodium falciparum actin gene

The nucleotide sequence of a Plasmodium falciparum actin gene has been established. The gene codes for a protein of 376 amino acids and is not interrupted by introns. The nucleotide sequence reveals an extreme bias in codon usage. Not less than 85% of the codons possess an A or T at the third position. As has been found for the actins in other unicellular eukaryotes, P. falciparum actin is related both to vertebrate cytoplasmic and vertebrate muscle specific actins. However, the malarial actin is one of the most alpha-like actins hitherto found in lower eukaryotes.     

Immunosuppression in malaria: effect of hemozoin produced by Plasmodium berghei and Plasmodium falciparum

To a considerable degree, malaria-induced immunosuppression has been attributed to an inhibition of macrophage accessory cell function. In this study hemozoin, a plasmodium hemoglobin degradation product which readily accumulates in phagocytic cells and tissues during infection, was examined for its influence on immune responses. Hemozoin-laden liver and splenic macrophages from Plasmodium berghei-infected mice, displayed accessory cell dysfunction which was likely due to hemozoin loading by these phagocytic cells. This indicated by the observation that hemozoin obtained from livers and spleens of infected mice as well as from Plasmodium falciparum cultures greatly inhibited splenic plaque-forming cell responses to sheep red blood cells. The results of the present study strongly suggest that the inhibition of macrophage accessory cell activity is due, at least in part, to the uptake and accumulation of hemozoin in their cytoplasms.     

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.     

Purification and characterization of 37-kilodalton proteases from Plasmodium falciparum and Plasmodium berghei which cleave erythrocyte cytoskeletal components

Cytosoluble 100,000 X g extracts from Plasmodium berghei or Plasmodium falciparum infected red blood cells were shown to hydrolyze erythrocyte spectrin. By Fast Protein Liquid Chromatography (FPLC), these enzymes were purified and exhibited a pI of 4.5 and Mr of 37,000 using SDS-PAGE under reducing conditions. An immunochemical enzyme assay using anti-spectrin antibodies was developed. The optimal activity using spectrin as substrate was at pH 5.0, and the enzymes were strongly inhibited by HgCl2, ZnCl2, chymostatin, leupeptin and aprotinin, and moderately by pepstatin. These properties of the Pf37 and Pb37 proteases differ from the Plasmodium lophurae and P. falciparum 'cathepsin D-like' enzymes and from the serine or cysteine neutral proteases previously described in P. falciparum and P. berghei infected red blood cells. While the Pf37 and Pb37 enzymes cleaved spectrin preferentially, degradation of band 4.1 was also observed with high concentration of enzyme. The parasite origin of the Pf37 protease was clearly demonstrated, since purified radiolabeled enzyme was active on spectrin. A high-molecular-weight polymer (greater than 240 kDa) was often observed on incubating purified spectrin and Pf37 protease. The breakdown of erythrocyte cytoskeletal components could be of interest in the release of merozoites from segmented schizonts or during the process of invasion of erythrocytes by merozoites.     

Bioassay-guided isolation and characterization of active antiplasmodial compounds from Murraya koenigii extracts against Plasmodium falciparum and Plasmodium berghei

Malaria is an overwhelming impact in the poorest countries in the world due to their prevalence, virulence and drug resistance ability. Currently, there is inadequate armoury of drugs for the treatment of malaria. This underscores the continuing need for the discovery and development of new effective and safe antimalarial drugs. To evaluate the in vitro and in vivo antimalarial activity of the leaf ethyl acetate extract of Murraya koenigii, bioassay-guided chromatographic fractionation was employed for the isolation and purification of antimalarial compounds. The in vitro antimalarial activity was assayed by the erythrocytic stages of chloroquine-sensitive strain of Plasmodium falciparum (3D7) in culture using the fluorescence-based SYBR Green I assay. The in vivo assay was done by administering mice infected with Plasmodium berghei (NK65) four consecutive daily doses of the extracts through oral route following Peter’s 4-day curative standard test. The percentage suppression of parasitaemia was calculated for each dose level by comparing the parasitaemia in untreated control with those of treated mice. Cytotoxicity was determined against HeLa cells using MTT assay. Histopathology was studied in kidney, liver and spleen of isolated compound-treated Swiss albino mice. The leaf crude ethyl acetate extract of M. koenigii showed good in vitro antiplasmodial activity against P. falciparum. The in vivo test of the leaf crude ethyl acetate extract (600 mg/kg) showed reduced malaria parasitaemia by 86.6 % against P. berghei in mice. Bioassay-guided fractionation of the leaf ethyl acetate extract of M. koenigii led to the isolation of two purified fractions C3B2 (2.84 g) and C3B4 (1.97 g). The purified fractions C3B2 and C3B4 were found to be active with IC₅₀ values of 10.5?±?0.8 and 8.25?±?0.2 μg/mL against P. falciparum, and in vivo activity significantly reduced parasitaemia by 82.6 and 88.2 % at 100 mg/kg/body weight on day 4 against P. berghei, respectively. The isolated fractions C3B2 and C3B4 were monitored by thin-layer chromatography until a single spot was obtained with R _f values of 0.36 and 0.52, respectively. The pure compounds obtained in the present investigation were subjected to UV–visible spectroscopy, Fourier transformer infrared spectroscopy, 1D and 2D ¹H-Nuclear magnetic resonance (NMR), ¹³C NMR, DEPT, COSY and Mass spectral analysis. Based on the spectral analysis, it is concluded that the isolated compounds were myristic acid (C3B2) and β-caryophyllene (C3B4). The cytotoxic effect of myristic acid and β-caryophyllene showed the TC₅₀ values of >100 and 80.5 μg/mL, respectively against HeLa cell line. The histopathology study showed that protection against nephrotoxicity of kidney, hepatic damage of liver and splenocytes protection in spleen was achieved with the highest dose tested at 100 mg/kg/body weight. The present study provides evidence of antiplasmodial compounds from M. koenigii and is reported for the first time.     

Inhibition of the growth of Plasmodium falciparum and Plasmodium berghei by the DNA polymerase inhibitor HPMPA.

The acyclic adenosine analogue (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine [HPMPA] belongs to a class of nucleoside analogues originally described as having potent activity against a broad spectrum of DNA viruses. We examined the effects of this class of drugs on the growth of cultured Plasmodium falciparum. Strong inhibition was observed by HPMPA (ID50 = 47 nM) at concentrations more than 1000-fold less than the cytotoxic dose for human cells. 3-deaza-HPMPA was even more strongly inhibitory (ID50 = 8 nM), whereas several other acyclic nucleosides were not effective. In mice infected with Plasmodium berghei, increase of parasitaemia can be blocked for 4-6 days by a single injection of HPMPA. Repeated drug administration blocks parasite growth for prolonged periods at doses that are clinically feasible. We also determined the inhibition of several purified Plasmodium DNA polymerases by diphosphorylated HPMPA (HPMPApp). DNA polymerase alpha-like enzymes of P. falciparum and P. berghei are inhibited with an IC50 = 40 microM and a gamma-like DNA polymerase from P. falciparum is even 40-fold more sensitive to the drug. The inhibition by HPMPApp is competitive with dATP, strongly suggesting that Plasmodium DNA polymerases are targets for this class of nucleotide analogue.     

Characterisation and sequence of a protective rhoptry antigen from Plasmodium falciparum

We have recently demonstrated that a non-polymorphic rhoptry antigen, RAP-1 (rhoptry associated protein-1), which is recognised by human immune serum, can successfully protect Saimiri monkeys from a lethal infection of Plasmodium falciparum malaria. In this report we further characterise the antigen, which consists of four major proteins of 80, 65, 42 and 40 kDa and two minor proteins of 77 and 70 kDa, and present the antigen's gene sequence. Monoclonal antibody evidence, autocatalytic processing and immunological cross-reactivity suggest that all components of this antigen are derived from the same precursor protein. The antigen is lipophilic, and disulphide bonding plays an important role in its structure. We discuss the structure and function of RAP-1 in the light of its deduced amino acid sequence and consider the relationship of this antigen to other rhoptry antigens of similar subunit size and composition.     

A conserved peptide sequence of the Plasmodium falciparum circumsporozoite protein and antipeptide antibodies inhibit Plasmodium berghei sporozoite invasion of Hep-G2 cells and protect immunized mice against P. berghei sporozoite challenge.

Minutes after injection into the circulation, malaria sporozoites enter hepatocytes. The speed and specificity of the invasion process suggest that it is receptor mediated. The region II sequence of Plasmodium falciparum circumsporozoite (CS) protein includes a nonapeptide (WSPCSVTCG) which is highly conserved in all of the CS proteins sequenced to data, including the one from Plasmodium berghei. We have found that two peptides based on the P. falciparum region II sequence, P18 (EWSPCSVTCGNGIQVRIK) and P32 (IEQYLKKIKNS ISTEWSPCSVTCGNGIQVRIK), significantly inhibited P. berghei sporozoite invasion into Hep-G2 cells in vitro. This inhibition was enhanced if either peptide was preincubated with Hep-G2 cells prior to sporozoite invasion. We confirm that region II is a sporozoite ligand for the hepatocyte receptor; moreover, despite the few differences between P. falciparum and P. berghei region II sequences around the nonapeptide sequence (66% homology), the functional characteristics of the motif sequences are not affected. Since the conserved motifs represent a crucial sequence involved in Plasmodium sporozoite invasion of hepatocytes, antibodies to region II should inhibit sporozite invasion into hepatocytes. Indeed, we found that polyclonal antibodies generated to the P. falciparum-based peptide P32 inhibited P. berghei sporozoite invasion of Hep-G2 cells. Furthermore, inbred mice (C57BL/6) immunized with P32 were protected against a lethal challenge of P. berghei sporozoites. Our results suggest that the conserved region II of the CS protein contains crucial B- and T-cell epitopes, that such peptide sequences from the human malaria parasite P. falciparum can be screened in the P. berghei rodent model, and, finally, that region II can be considered useful as one of the components of a malaria vaccine.     

Strain variation in the circumsporozoite protein gene of Plasmodium falciparum

The complete nucleotide sequence of the cloned circumsporozoite protein gene of the Plasmodium falciparum Wellcome (West African) isolate has been determined. The sequence shows two striking differences from that of the published Brazilian strain; the total number of tandem 12 base pair repeats is 46 compared to 41, and the 5' coding region contains an additional 30 nucleotides. From Southern blot experiments, two out of four cloned Thai lines also have a similar, higher number of repeats. Heterogeneity in the CSP gene repeat region and in the length of the 5' coding region allows the strains to be classed into three groups, with the Wellcome strain being indistinguishable from the Thai line T9-94.     

Possible roles of Ca2+ and cGMP as mediators of the exflagellation of Plasmodium berghei and Plasmodium falciparum

The roles of Ca2+ and cyclic nucleotides as secondary, intracellular messengers for exflagellation of Plasmodium berghei and Plasmodium falciparum were investigated. Treatment with Ca2+ antagonists such as TMB-8 (an inhibitor of intracellular Ca2+ release) or W-7 (a calmodulin inhibitor) strongly inhibited exflagellation induced by alkaline medium at pH 8.0 whereas EGTA (a Ca2+ chelator) or nicardipine and nifedipine (Ca2+ channel inhibitors) had no effect. These results may indicate that mobilization of parasites' internal resources of Ca2+ is a prerequisite for exflagellation. Agents which increase cAMP levels did not induce exflagellation at the non-permissive pH of 7.3, and had no significant inhibitory effect at the permissive pH of 8.0. IBMX (cAMP/cGMP-phosphodiesterase inhibitor), however, enhanced exflagellation at pH 7.3, indicating the possibility that cGMP, but not cAMP, may be involved in the induction of exflagellation. Furthermore, cGMP or agents which increase cGMP levels such as nitroprusside (a potent activator of guanylate cyclase), enhanced exflagellation at pH 7.3, whereas N-methyl-hydroxylamine (guanylate cyclase inhibitor) inhibited the exflagellation at pH 8.0. From these results, it may be concluded that the induction of exflagellation requires both Ca2+ mobilization and an increase in cGMP levels.     

Heterologous expression of proteins from Plasmodium falciparum: results from 1000 genes

As part of a structural genomics initiative, 1000 open reading frames from Plasmodium falciparum, the causative agent of the most deadly form of malaria, were tested in an E. coli protein expression system. Three hundred and thirty-seven of these targets were observed to express, although typically the protein was insoluble. Sixty-three of the targets provided soluble protein in yields ranging from 0.9 to 406.6 mg from one liter of rich media. Higher molecular weight, greater protein disorder (segmental analysis, SEG), more basic isoelectric point (pI), and a lack of homology to E. coli proteins were all highly and independently correlated with difficulties in expression. Surprisingly, codon usage and the percentage of adenosines and thymidines (%AT) did not appear to play a significant role. Of those proteins which expressed, high pI and a hypothetical annotation were both strongly and independently correlated with insolubility. The overwhelmingly important role of pI in both expression and solubility appears to be a surprising and fundamental issue in the heterologous expression of P. falciparum proteins in E. coli. Twelve targets which did not express in E. coli from the native gene sequence were codon-optimized through whole gene synthesis, resulting in the (insoluble) expression of three of these proteins. Seventeen targets which were expressed insolubly in E. coli were moved into a baculovirus/Sf-21 system, resulting in the soluble expression of one protein at a high level and six others at a low level. A variety of factors conspire to make the heterologous expression of P. falciparum proteins challenging, and these observations lay the groundwork for a rational approach to prioritizing and, ultimately, eliminating these impediments.     

Characterisation of a repetitive DNA sequence from the malaria parasite,Plasmodium falciparum

A repetitive DNA fragment cloned from the malaria parasite, Plasmodium falciparum, has been analysed. It contains a 21 base pair sequence which occurs in multiple tandem repeats. Two clusters of the same repeat are found in opposite orientations on the same DNA fragment. The repetitive DNA provides an additional way to distinguish between different strains of parasite by hybridisation to genomic blots and may serve as a species-specific probe for diagnosis.     

An S antigen gene from Plasmodium falciparum contains a novel repetitive sequence

The complete sequence of the gene coding for the S antigen from the Wellcome (West African) strain of Plasmodium falciparum has been obtained. It contains a central repetitive region consisting of 65 copies of a partially degenerate 24 bp sequence, coding for a conserved 8 amino acid repeat (Gly Pro Asn Ser Asp Gly Asp Lys). The repeat sequence is different from those of S antigens characterised in other strains and thus represents a new S antigen serotype.