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PubMed1992

Polymorphism in the circumsporozoite protein of the human malaria parasite Plasmodium vivax.

The circumsporozoite (CS) protein that covers the surface of infectious sporozoites is a candidate antigen in malaria vaccine development. To determine the extent of B- and T-epitope polymorphism and to understand the mechanisms of antigenic variability, we have characterized the CS protein gene of Plasmodium vivax from field isolates representing geographically distant regions of Papua New Guinea (PNG) and Brazil. In the central repeat region of the CS protein, in addition to variation in the number of repeats, an array of mutations was observed which suggests that point mutations have led to the emergence of the variant CS repeat sequence ANGA(G/D)(N/D)QPG from GDRA(D/A)GQPA. Outside the repeat region of the protein, the nonsilent nucleotide substitutions of independent origin are localized in three domains of the protein that either harbor known T-cell determinants or are analogous to the Plasmodium falciparum immunodominant determinants, Th2R and Th3R. We have found that, with the exception of one CS clone sequence that was shared by one P. vivax isolate each from PNG and Brazil, the P. vivax CS protein types can be grouped into Papuan and Brazilian types. These results suggest that an in-depth study of parasite population dynamics is required before field trials for vaccine formulation based on polymorphic immunodominant determinants are conducted.

Amino Acid Sequence↗
PubMed1993

The gamma-tubulin gene of the malaria parasite Plasmodium falciparum.

By screening of cDNA and genomic libraries of Plasmodium falciparum with a DNA probe derived from the cognate beta-tubulin gene, gene Pf gamma tub has been identified that codes for gamma-tubulin, a newly discovered member of the tubulin superfamily that is indispensible for nuclear division and microtubule assembly [12]. Gene Pf gamma tub is not interrupted by introns and only present as a single-copy in the parasite genome. Its encoded amino acid sequence (452 amino acids; M(r) 50,560) has a 63% similarity to the gamma-tubulins encoded by Aspergillus nidulans, Schizosaccharomyces pombe, Drosophila melanogaster, Xenopus laevis and Homo sapiens. This figure is significantly (approx. 8%) lower than the average identity between the gamma-tubulins of the latter five species suggesting that during evolution the genes have been exposed to different selection pressures. The identity of gamma-tubulin to the Plasmodium falciparum encoded alpha- and beta-tubulins is 30 and 33%, respectively.

Amino Acid Sequence↗
PubMed1993

Selective inhibition of Plasmodium falciparum aldolase by a tubulin derived peptide and identification of the binding site.

Aldolase of the human malaria parasite Plasmodium falciparum (PfAldo) may be a potential target for the development of novel antimalarial drugs. Using in vitro mutagenesis we analyzed the function of the carboxy-terminus of the recombinant enzyme. Deletion of the carboxy-terminus of PfAldo confirmed its critical role in catalysis; exchange of conserved residues minimally affected enzyme activity. We exchanged a pair of parasite specific lysine residues with corresponding amino acids of the host. These mutant enzymes exhibited an increased catalytic activity and reduced binding to erythrocyte band 3 protein. Homologous peptides of human band 3 protein and P. falciparum alpha-tubulin were competitive inhibitors of PfAldo. Selective inhibition of PfAldo by the alpha-tubulin peptide depends on the presence of tandem lysine residues and the fine structure of the inhibitor peptide. Our data support the concept of a matrix organisation of glycolytic enzymes in Plasmodium falciparum.

Amino Acid Sequence↗
PubMed1993

Molecular characterization of the largest subunit of Plasmodium falciparum RNA polymerase I.

Plasmodium species possess developmentally regulated ribosomal RNA (rRNA) genes. This report describes the expression and gene structure of the largest subunit of P. falciparum RNA polymerase I (RNAPI), which is responsible for the synthesis of rRNA. The RNAPI largest subunit gene was present as a single copy gene on chromosome 9. Three exons encode the 2910-amino acid RNAPI polypeptide (340 140 Da). A comparison of Plasmodium, Trypanosoma, and Saccharomyces cerevisiae nuclear RNAP largest subunits identified conserved amino acid positions and class-specific amino acid positions. Novel amino acid insertions were found between RNAPI conserved regions A and B (region A'), D and DE1 (region D'), DE2 and E (region DE2'), and F and G (region F'). Leucine zipper domains were found within regions D', DE2, and DE2'. A novel serine-rich repeat domain, a domain with homology to the C-terminal domain of eukaryotic upstream binding factor (UBF), and 4 highly conserved casein kinase II (CKII) Ser/Thr phosphorylation motifs were found within a 127-amino acid sub-region of enlarged region F'. The novel RNAPI serine-rich repeat contained a conserved motif, Ser-X3-Ser, which was also identified in the serine-rich repeat domains of the P. falciparum RNAPII and RNAPIII largest subunits, as well as within a highly homologous serine-rich repeat from trophozoite antigen R45. The results of this molecular analysis indicate that phosphorylation and dephosphorylation mechanisms regulate the activity of P. falciparum RNAPI.

Amino Acid Sequence↗
PubMed1994

Cloning of the glucose 6-phosphate dehydrogenase gene from Plasmodium falciparum.

Glucose 6-phosphate dehydrogenase (G6PD) deficiency is one of the human genetic traits that confer relative resistance against malaria caused by Plasmodium falciparum. It has been previously shown that this organism, during its intraerythrocytic development, produces its own G6PD, which has properties different from those of human G6PD. In order to investigate the role of this enzyme in parasite-host cell interactions, we have isolated the G6PD gene from Plasmodium falciparum as a set of overlapping lambda gt11 clones. By sequence analysis we have found a single open reading frame, uninterrupted by introns, coding for a protein of 910 amino acids, almost twice as long as any previously sequenced G6PD molecule. The P. falciparum G6PD mRNA is 5.1 kb in size and has an exceptionally long 5' untranslated region of some 1000 nucleotides. We have mapped the G6PD gene to chromosome 14. The C-terminal portion of the predicted protein, from amino acid 310-910 (except for an 'insert' of 62 amino acids), has 39% homology to human G6PD, with a number of characteristic, fully conserved peptides. The N-terminal portion of the predicted protein has no homology to G6PD, but it contains a peptide in which 7 out of 12 amino acids are identical to the putative glutathione binding site of human glutathione S-transferase.

Amino Acid Sequence↗
PubMed1994

Analysis of stage-specific transcripts of the Plasmodium falciparum serine repeat antigen (SERA) gene and transcription from the SERA locus.

We evaluated the stage-specific transcription and processing of serine repeat antigen (SERA) messenger RNA to further examine mechanisms regulating gene expression in Plasmodium falciparum. SERA mRNA was expressed exclusively in trophozoite and schizont stages. Transcription from the SERA gene was first detected between 24 and 29 h following erythrocyte invasion. The transcript mapping data revealed heterogeneity of the SERA mRNA 5' and 3' ends. RNA sequencing revealed that SERA transcripts were not generated by a trans-splicing mechanism. A new SERA gene, SERA3, was identified 1.8 kb upstream of SERA. The direction of transcription of the SERA locus genes, SERA3, SERA, and SERA2, was mapped relative to the location of other chromosome 2 genetic markers. The SERA locus and the closely linked MSA2 locus were found to be transcriptionally regulated in a coordinate fashion. Collectively, the results of these experiments show that parallel and coordinately controlled transcription units reside on chromosome 2. These results implicate a novel mechanism of transcriptional control in Plasmodium.

Amino Acid Sequence↗
PubMed1995

A genetic locus on Plasmodium falciparum chromosome 12 linked to a defect in mosquito-infectivity and male gametogenesis.

Infection of mosquitoes by Plasmodium spp. requires sexual differentiation of the malarial parasite in the vertebrate host and mating of the heterogametes in the vector midgut. A Plasmodium falciparum clone, Dd2, differentiates into normal-appearing gametocytes, yet poorly infects mosquitoes. The Dd2 clone, however, effectively cross-fertilized HB3, a Central American P. falciparum clone, and yielded several independent recombinant progeny. We have examined 11 HB3 x Dd2 progeny for their ability to infect mosquitoes and to differentiate into male gametes. Our analyses indicate that the poor mosquito-infectivity of the Dd2 clone results from a defect in male gametogenesis. This defect was inherited as a single locus in the independent recombinant progeny of HB3 x Dd2. Comparison with a restriction fragment length polymorphism map of the HB3 x Dd2 cross indicates that the defective phenotype of Dd2 maps to a locus on P. falciparum chromosome 12. This genetic locus may contain determinants that play a crucial role in male gametogenesis by P. falciparum.

Animals↗
PubMed1994

Allelic variants of the Plasmodium falciparum merozoite surface antigen 2 (MSA-2) in a geographically restricted area of Irian Jaya.

Blood samples were collected from 12 residents of 4 villages in the Oksibil area of Irian Jaya. Eleven patients were positive for Plasmodium falciparum infection as evidenced by successful amplification of the MSA-2 gene by the polymerase chain reaction. Two patients showed evidence of infection by 2 strains of Plasmodium falciparum. All MSA-2 genes were completely sequenced and all could be assigned to one of the two major allelic families of MSA-2, however all MSA-2 gene sequences differed from previously described alleles. Five new allelic forms were identified, one of which was present in 8 of the 11 patients. Within small natural populations of P. falciparum, it appears that variation in MSA-2 approximates that seen world-wide. All samples were also analysed by hybridisation of amplified DNA to family specific probes and all samples hybridised to known probes. Our results demonstrate that there is a degree of microheterogeneity of MSA-2 that is undetectable by hybridisation studies alone.

Alleles↗
PubMed1994

Analysis of expressed sequence tags from Plasmodium falciparum.

An initiative was undertaken to sequence all genes of the human malaria parasite Plasmodium falciparum in an effort to gain a better understanding at the molecular level of the parasite that inflicts much suffering in the developing world. 550 random complimentary DNA clones were partially sequenced from the intraerythrocytic form of the parasite as one of the approaches to analyze the transcribed sequences of its genome. The sequences, after editing, generated 389 expressed sequence tag sites and over 105 kb of DNA sequences. About 32% of these clones showed significant homology with other genes in the database. These clones represent 340 new Plasmodium falciparum expressed sequence tags.

Animals↗
PubMed1994

Regular initiation of translation of Plasmodium berghei aldolase-2 after pre-mRNA splicing.

In Plasmodium falciparum aldolase a UAG or a regular AUG codon has been proposed for the initiation of ribosomal protein synthesis. A UAG codon present at the beginning of the coding sequence of the aldolase 2 gene (aldo-2) of Plasmodium berghei is not recognised in vitro as an initiation codon, which suggests addition of a regular AUG codon by mRNA splicing. Sequence analysis of cDNA amplified by the reversed polymerase chain reaction reveals addition of an ATG codon with a splice donor consensus sequence to the aldo-2 exon. By the same technique and northern blot analysis, substantial amounts of partially spliced P. berghei aldo-2 precursor mRNA are detected which could explain the isolation of immature P. falciparum aldolase cDNA clones starting with a stop codon.

Animals↗
PubMed1994

Cloning and characterisation of a Plasmodium falciparum homologue of the Ran/TC4 signal transducing GTPase involved in cell cycle control.

On the basis of conserved sequences characteristic of the Ran/TC4 subfamily of the GTPase superfamily, a fragment of the gene encoding a Plasmodium falciparum Ran/TC4 homologue was amplified in the polymerase chain reaction. The fragment was used to screen a cDNA library to obtain clones which allowed determination of the complete gene sequence. The gene, designated pfran (Plasmodium falciparum ras-like nuclear protein), has around 70% amino acid identity with previously characterised Ran/TC4 proteins. Like other malarial mRNAs, the pfran mRNA contains a long (at least 679 bp) 5' untranslated region. Southern blotting experiments show that pfran is a single copy gene located on chromosome 11. RNA hybridisation experiments indicate that pfran mRNA is abundant in late trophozoite and schizont stages, but present at very low levels in gametocytes and early asexual stages.

Amino Acid Sequence↗
PubMed1994

A Plasmodium falciparum isolate with a chromosome 9 deletion expresses a trypsin-resistant cytoadherence molecule.

Sequestration of Plasmodium falciparum infected erythrocytes in the cerebral circulation is strongly implicated in the pathogenesis of cerebral malaria. From previous studies it was postulated that genes essential for cytoadherence were located on the right arm of chromosome 9 as P. falciparum isolates with a deletion in this region lost the capacity to cytoadhere in vitro and no longer expressed Plasmodium falciparum erythrocyte membrane protein-1 (PfEMP-1) on the surface of the infected cells. We have selected a P. falciparum isolate from Papua New Guinea for high levels of cytoadherence to human umbilical vein endothelial cells (HUVECs) and have shown that the cloned parasite has several novel properties related to cytoadherence. The cloned parasite adheres to HUVECs, does not bind to melanoma cells, and expresses a surface molecule with most of the properties of PfEMP-1, despite a deletion in the right arm of chromosome 9. Interestingly, the surface expressed PfEMP-1 in this strain is resistant to trypsin treatment and infected cells continue to cytoadhere after trypsin digestion at a concentration of 100 micrograms ml-1. The receptor on HUVECs for the cloned parasite lines is a molecule different from any previously described, as parasitized cells do not adhere to soluble intercellular adhesion molecule 1, thrombospondin, vascular cell adhesion molecule 1, E-selectin or P-selectin, nor to CD36. Our work, taken together with the results from previous studies, suggest that the ability of parasites to cytoadhere is encoded in at least two distinct genomic locations in the parasite, and the diversity of receptor-ligand interaction is greater than previously described.

Animals↗
PubMed1995

Natural selection on Plasmodium surface proteins.

Numbers of synonymous (ds) and nonsynonymous (dN) nucleotide substitutions per site were analysed in eight polymorphic Plasmodium genes: circumsporozoite protein gene (CSP), sporozoite surface protein 2 (thrombospondin related anonymous protein, TRAP), merozoite surface antigen 2 (MSA-2), apical membrane antigen 1 (PF83), liver-stage antigen-1 (LSA-1), knob-associated histidine-rich protein (KAHRP), ring-infected erythrocyte surface antigen (RESA) and S-antigen. In certain regions of genes coding for proteins of the sporozoite and merozoite surface (CSP, TRAP, MSA-2 and PF83), dN was significantly greater than dS. This unusual pattern of nucleotide substitution is indicative of positive Darwinian selection acting to promote diversity at the amino-acid level; thus our results suggest that the sporozoite and merozoite surface proteins are under positive selection, presumably exerted by the host immune system. No such pattern of substitution was observed on LSA-1, KAHRP, RESA, or S-antigen. Observed patterns of nucleotide substitution were not explicable by nucleotide content bias. G + C content in the 5' nonrepeat region of CSP in nine Plasmodium species was positively correlated with that in the 3' nonrepeat region; however, there was no relationship between G + C content and the ratio dS/dN in either CSP or a larger sample of all regions of all genes analysed.

Animals↗
PubMed1995

Comparison of introns in a cdc2-homologous gene within a number of Plasmodium species.

The first three introns of CRK2, a cdc2-homologous gene, have been compared in a total of seven Plasmodium species. The introns were located at conserved sites, suggesting an ancestral origin. Interspecific comparison of intron sequences agreed with the previously inferred evolutionary relationships of the malaria parasites. Unlike the introns in the rodent malaria species, the similarity of the CRK2 introns was regionalized between the human parasite P. vivax and the simian parasite P. knowlesi: the central region of all three introns showed markedly less interspecific similarity than the 5' and 3' regions. This was also in contrast with the organisation and composition of homologous intron pairs from other genes of the same two species. No conservation at the level of secondary structure could be detected, even between highly similar introns. A database search for intron-containing Plasmodium genes was performed. All introns obtained in this way plus the additional CRK2 introns were scanned for the presence of putative branching site consensus sequences. For P. falciparum, we present an update of the 5' and 3' splice-site consensus.

Amino Acid Sequence↗
PubMed1995

Cloning and characterization of a Plasmodium falciparum cyclophilin gene that is stage-specifically expressed.

An immunosuppressive agent, cyclosporin A (CsA), has antimalarial activity in several Plasmodium species. Cyclophilins of several species including Plasmodium falciparum exhibit peptidyl-prolyl cis-trans isomerase activity which is inhibited by CsA. A gene encoding P. falciparum cyclophilin (PFCyP) was cloned and characterized. This gene has the entire coding sequence for the mature protein plus a 39-amino-acid-long N-terminal extension. Most of the amino acids predicted to be involved in the peptidyl-prolyl cis-trans isomerase activity and CsA binding are present in the cloned gene. The PFCyP also has the single highly conserved tryptophan residue that is a major determinant in the inhibition of PPIase activity by CsA. The PFCyP coding sequence with or without the N-terminal amino-acid extension was used to construct recombinant expression vectors which were transformed into E. coli. Both vectors produced enzymatically active mature PFCyP proteins that were sensitive to CsA. Northern blot analysis of RNA isolated from the synchronized parasite cultures verified the expression of PFCyP in all erythrocytic stages of the parasite, but at variable levels. The highest level of expression was observed in ring-stage parasites, a stage shown to be more susceptible to CsA. Inhibition of P. falciparum growth in vitro by CsA was re-evaluated for chloroquine-sensitive and chloroquine-resistant strains of the parasite. Essentially, there was no difference between the two strains for the concentration of CsA required to yield 50% inhibition in 48 h of exposure (0.25-0.4 microM).

Amino Acid Isomerases↗
PubMed1995

Molecular cloning of a gene from Plasmodium falciparum that codes for a protein sharing motifs found in adhesive molecules from mammals and plasmodia.

Adhesion of Plasmodium to host cells is an important phenomenon in parasite invasion and in malaria-associated pathology. We report here the molecular cloning of a putative adhesive molecule from P. falciparum that shares both sequence and structural similarities with a sporozoite surface molecule from Plasmodium termed the thrombospondin-related anonymous protein (TRAP) and, to a lesser extent, with the circumsporozoite (CS) protein. The gene, which is present on chromosome 3 as a single copy, was termed CTRP for CS protein-TRAP-related protein. The full-length CTRP encodes a protein containing a putative signal sequence followed by a long extracellular region of 1990 amino acids, a transmembrane domain, and a short cytoplasmic segment. The putative extracellular region of CTRP is defined by two separated adhesive domains. The first domain contains six 210-amino acid-long homologous repeats, the sequence of which is related to the A-type domain found in adhesive molecules including the alpha subunits of several integrins and a number of extracellular matrix glycoproteins. The second domain contains seven repeats of 87-60 amino acids in length, which share similarities with the thrombospondin type 1 domain found in a variety of adhesive molecules. Finally, CTRP also contains consensus motifs found in the superfamily of haematopoietin receptors. Interstrain analysis of eight different parasite isolates revealed that CTRP does not show size polymorphism except in repetitive regions flanking potential adhesive domains.

Amino Acid Sequence↗
PubMedDate not supplied

Characterization of iron-dependent endogenous superoxide dismutase of Plasmodium falciparum.

Two main superoxide dismutase activities at isoelectric points (pI) 6.2 and 6.8 and two minor at pI 5.6 and 6.4 were found in crude extracts of Plasmodium falciparum. These activities were cyanide-resistant and hydrogen peroxide-sensitive and represented 20-30% of the total SOD activity found in the crude extract. A fragment of 424 bp, amplified from genomic DNA from P. falciparum, was cloned and sequenced. The deduced amino acid sequence identified this fragment as a coding region of an SOD gene. A cDNA corresponding to SOD was then isolated from a P. falciparum cDNA library and sequenced. The deduced amino acid sequence of SOD (197 aa) was compared with 32 known Feor Mn-SODs by the 'DARWIN' system. This analysis showed that the parasitic enzyme was related to typical Fe-SODs. The SOD subunit was purified and the N-terminal sequence, determined up to 29 residues, corresponded to that of cDNA isolated. The iron-dependent SOD activity found in Plasmodium falciparum represents the first level of the antioxidant defence system of the parasite. It is also the first SOD characterized in the parasitic Apicomplexa phylum whose sequence can be compared to equivalent iron-dependent enzymes known in other protozoa and bacteria.

Amino Acid Sequence↗
PubMed1996

Amodiaquine accumulation in Plasmodium falciparum as a possible explanation for its superior antimalarial activity over chloroquine.

Amodiaquine is a 4-aminoquinoline antimalarial whose structure is similar to chloroquine. In contrast to the wealth of information available about chloroquine accumulation and its relationship to activity, little is known about the uptake characteristics of amodiaquine, a drug that is inherently more active against malaria parasites. In this study we have investigated the accumulation of amodiaquine in Plasmodium falciparum in vitro, in order to gain an insight into the mechanisms responsible for its superior activity over chloroquine. The driving force for parasite accumulation of the 4-aminoquinolines is proposed to be a transmembrane proton gradient maintained by a vacuolar ATPase. In the present study, amodiaquine accumulation was greatly reduced, at steady state, in the absence of glucose and at 0 degrees C indicating a clear energy dependence of uptake. Amodiaquine accumulation in Plasmodium falciparum was shown to be 2- to 3-fold greater than chloroquine accumulation. This observation probably accounts for amodiaquine's greater inherent activity but is surprising given that amodiaquine is a weaker base than chloroquine. With this in mind we present evidence for an intraparasitic binding component in the accumulation of the 4-aminoquinolines. Differences in binding affinity of this 'receptor' for amodiaquine and chloroquine may partially explain the greater accumulation and in vitro potency of amodiaquine compared to chloroquine.

Adenosine Triphosphate↗