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PubMed2001

Anopheles gambiae laminin interacts with the P25 surface protein of Plasmodium berghei ookinetes.

Laminin is a major constituent of the basal lamina surrounding the midgut of the malaria vectors that has been implicated in the development of the Plasmodium oocyst. In this report we describe the cloning of the Anopheles gambiae gene encoding the laminin gamma 1 polypeptide and follow its expression during mosquito development. To further investigate the putative role of laminin in the transmission of the malaria parasite we studied the potential binding of the P25 surface protein of Plasmodium berghei using a yeast two-hybrid system. Heterodimer formation was observed and does not require any additional protein factors since purified fusion proteins can also bind each other in vitro. Laminin gamma 1 also interacts with the paralogue of P25, namely P28, albeit more weakly, possibly explaining why the two parasite proteins can substitute for each other in deletion mutants. This represents the first direct evidence for molecular interactions between a surface protein of the Plasmodium parasite with an Anopheles protein; the strong interplay between laminin gamma 1 and P25 suggests that this pair of proteins may function as a receptor/ligand complex regulating parasite development in the mosquito vector.

Amino Acid Sequence↗
PubMed2001

Gene targeting in the rodent malaria parasite Plasmodium yoelii.

It is anticipated that the sequencing of Plasmodium falciparum genome will soon be completed. Rodent models of malaria infection and stable transformation systems provide powerful means of using this information to study gene function in vivo. To date, gene targeting has only been developed for one rodent malaria species, Plasmodium berghei. Another rodent species, Plasmodium yoelii, however, is favored to study the mechanisms of protective immunity to the pre-erythrocytic stages of infection and vaccine development. In addition, it offers the opportunity to investigate unique aspects of pathogenesis of blood stage infection. Here, we report on the stable transfection and gene targeting of P. yoelii. Purified late blood stage schizonts were used as targets for electroporation with a plasmid that contains a pyrimethamine-resistant form of the P. berghei dihydrofolate reductase-thymidylate synthase (Pbdhfr-ts) fused to green fluorescent protein (gfp) gene. After drug selection, fluorescent parasites contained intact, non-rearranged plasmids that remain stable under drug-pressure. In addition, we used another dhfr-ts/gfp based plasmid to disrupt the P. yoelii trap (thrombospondin-related anonymous protein) locus by site-specific integration. The phenotype of P. yoelii TRAP knockout was identical to that previously reported for the P. berghei TRAP knockout. In the absence of TRAP, the erythrocytic cycle, gametocyte and oocyst development of the mutant parasites were indistinguishable from wild type (WT). Although the sporozoites appeared morphologically normal, they failed to glide and to invade the salivary glands of mosquitoes.

Animals↗
PubMed2001

The 22 kDa component of the protein complex on the surface of Plasmodium falciparum merozoites is derived from a larger precursor, merozoite surface protein 7.

The gene coding for merozoite surface protein 7 has been identified and sequenced in three lines of Plasmodium falciparum. The gene encodes a 351 amino acid polypeptide that is the precursor of a 22-kDa protein (MSP7(22)) on the merozoite surface and non-covalently associated with merozoite surface protein 1 (MSP1) complex shed from the surface at erythrocyte invasion. A second 19-kDa component of the complex (MSP7(19)) was shown to be derived from MSP7(22) and the complete primary structure of this polypeptide was confirmed by mass spectrometry. The protein sequence contains several predicted helical and two beta elements, but has no similarity with sequences outside the Plasmodium databases. Four sites of sequence variation were identified in MSP7, all within the MSP7(22) region. The MSP7 gene is expressed in mature schizonts, at the same time as other merozoite surface protein genes. It is proposed that MSP7(22) is the result of cleavage by a protease that may also cleave MSP1 and MSP6. A related gene was identified and cloned from the rodent malaria parasite, Plasmodium yoelii YM; at the amino acid level this sequence was 23% identical and 50% similar to that of P. falciparum MSP7.

Amino Acid Sequence↗
PubMed2001

Characterization of the ornithine aminotransferase from Plasmodium falciparum.

The ornithine aminotransferase from Plasmodium falciparum 3D7 was cloned, functionally expressed, and characterized. The gene exists as a single copy in the malarial genome and is located on chromosomes 6/7/8. The deduced amino acid sequence was found to be 85% identical to a similar sequence discovered in Plasmodium yoelii, 82% identical to a partial sequence from Plasmodium vivax, and 42-53% identical to ornithine aminotransferases from other eukaryotes. The enzyme had a very narrow substrate specificity, and could only catalyze the transamination of alpha-ketoglutarate with ornithine or N-acetylornithine, and of glutamate-5-semialdehyde with glutamate and alanine. The aminooxy analogue of ornithine, canaline, was found to inhibit the ornithine aminotransferase uncompetatively with a Ki of 492+/-98 nM. As the enzyme effectively catalyzed both ornithine catabolism and formation, its potential role in ornithine biosynthesis from glutamine, via glutamate, glutamate-5-phosphate, and glutamate-5-semialdehyde, was examined. Over the course of a 3.5 h incubation, P. falciparum converted 34% of exogenous, radiolabeled glutamine to glutamate and 0.68% to ornithine. This low level of conversion suggests that the parasite may have alternative mechanisms for obtaining ornithine for polyamine biosynthesis.

Amino Acid Sequence↗
PubMed2002

Antibodies raised against receptor-binding domain of Plasmodium knowlesi Duffy binding protein inhibit erythrocyte invasion.

Erythrocyte invasion by malaria parasites requires specific receptor-ligand interactions. Plasmodium vivax and Plasmodium knowlesi are completely dependent on binding the Duffy blood group antigen to invade human erythrocytes. P. knowlesi invades rhesus erythrocytes by multiple pathways using the Duffy antigen as well as alternative receptors. Plasmodium falciparum binds sialic acid residues on glycophorin A as well as other sialic acid-independent receptors to invade human erythrocytes. Parasite proteins that mediate these interactions belong to a family of erythrocyte binding proteins, which includes the P. vivax Duffy binding protein, 175 kDa P. falciparum erythrocyte binding antigen (EBA-175), P. knowlesi alpha protein, which binds human and rhesus Duffy antigens, and P. knowlesi beta and gamma proteins, which bind Duffy-independent receptors on rhesus erythrocytes. The receptor-binding domains of these proteins lie in conserved, N-terminal, cysteine-rich regions that are referred to as region II. Here, we have examined the feasibility of inhibiting erythrocyte invasion with antibodies directed against receptor-binding domains of erythrocyte binding proteins. Region II of P. knowelsi alpha protein (Pk(alpha)RII), which binds the Duffy antigen, was expressed as a secreted protein in insect cells and purified from culture supernatants. Rabbit antibodies raised against recombinant Pk(alpha)RII were tested for inhibition of erythrocyte binding and invasion. Antibodies raised against Pk(alpha)RII inhibit P. knowlesi invasion of both human and rhesus erythrocytes. These data provide support for the development of recombinant vaccines based on the homologous binding domains of P. vivax Duffy binding protein and P. falciparum EBA-175.

Animals↗
PubMed2002

Plasmodium falciparum MAEBL is a unique member of the ebl family.

Malaria is one of the deadliest human diseases and efforts to control it have been difficult due to the protozoan parasites' complex biology. Malaria merozoite invasion of erythrocytes is an essential part of blood-stage infections. The invasion process is mediated by numerous parasite molecules, such as EBA-175, a member of the ebl family of erythrocyte binding proteins. We have identified maebl, an ebl paralogue, in Plasmodium falciparum and found it highly conserved with its orthologues in P. yoelii and P. berghei, but distinct from other Plasmodium ebl. Importantly, the putative MAEBL ligand domains are highly conserved and are similar to AMA-1, but not the consensus DBL ligand domains present in all other ebl. In mature merozoites, MAEBL localized with rhoptry proteins (RhopH2, RAP-1), including surface localization with RhopH2, but not microneme proteins (EBA-175, BAEBL). MAEBL appears as proteolytically processed fragments in P. falciparum parasites. The amino cysteine-rich ligand domains were present primarily in culture supernatants, while the carboxyl cysteine-rich domain adjacent to the transmembrane domain was preferentially isolated from Triton X-100 extracted fractions. These data indicate that the primary structure of maebl is highly conserved among Plasmodium species, while its characteristics demonstrate a function unique among the ebl proteins.

Amino Acid Sequence↗
PubMed2002

Identification, expression, and functional characterization of MAEBL, a sporozoite and asexual blood stage chimeric erythrocyte-binding protein of Plasmodium falciparum.

MAEBL is a chimeric erythrocyte binding protein reported in rodent malaria parasites Plasmodium yoelii and Plasmodium berghei, that has the gene structure similar to erythrocyte binding proteins, but N-terminal homology to subdomains I and II of Apical membrane antigen-1. We report here the sequence analysis and gene structure of the Plasmodium falciparum maebl gene. We have cloned and expressed a putative red cell binding domain, M2, of this gene in Escherichia coli, purified the recombinant protein (r-PfM2) and studied its in vitro binding specificity to human red cells. Binding of r-PfM2 protein to red cells was abolished by pretreatment with papain, while increased binding was observed to neuraminidase-treated red cells. Polyclonal antibodies to r-PfM2 recognized native MAEBL protein in blood stage schizont extracts of the parasite on Western blots and within the apical complex of free merozoites, by indirect immunofluorescent assay (IFA). MAEBL expression in P. falciparum sporozoites was also detected by reverse transcriptase polymerase chain reaction (RT-PCR) and IFA. High titer antibodies to r-PfM2 were observed in human sera obtained from a malaria endemic region some of which inhibited r-PfM2 binding to red cells. Individuals immunized with irradiated sporozoites tested positive for anti-MAEBL antibodies by ELISA. The dual stage expression of MAEBL makes it an excellent pre-erythrocytic and erythrocytic stage vaccine target antigen.

Amino Acid Sequence↗
PubMed2003

Characterization of a unique aspartate-rich protein of the SET/TAF-family in the human malaria parasite, Plasmodium falciparum, which inhibits protein phosphatase 2A.

A search for physiological inhibitors of protein phosphatases led to the identification of a Plasmodium falciparum (Pf) cDNA that had the potential to code for an aspartate-rich protein and hence named ARP. The PfARP was virtually identical to its Plasmodium berghei counterpart in gene structure and protein sequence. The PfARP coding sequence contained two introns, and the predicted protein contained 269 amino acid residues. Its primary structure showed significant similarity to eukaryotic proteins of the SET and TAF-family that included two inhibitors of mammalian serine/threonine protein phosphatase 2A (PP2A), namely I1(PP2A) and I2(PP2A). Like the SET and TAF proteins, it had an extremely acidic tail. The cDNA was confirmed by recombinant expression in bacteria. Native parasitic ARP was purified and was found to be highly thermostable. PfARP specifically inhibited the parasitic PP2A at nanomolar concentrations, with no effect on PP1, PP2B, PP5, or PPJ. Expression of PfARP in HeLa cells led to elevated phosphorylation of c-Jun, and activation of transcription factors AP1 and NF-kappa B. These functional properties are also characteristic of the SET/TAF-family proteins. The ARP mRNA and protein were detectable in all the erythrocytic asexual stages of the parasite, and the protein was located mainly in the parasitic cytoplasm. Thus, PfARP is a unique cytoplasmic member of the SET/TAF-family and a candidate physiological regulator of the Plasmodium PP2A.

Amino Acid Sequence↗
PubMed1997

Stable transgene expression in Plasmodium falciparum.

Plasmid vectors designed to express transgenes and a selectable marker in Plasmodiumfalciparum were constructed. These consist of a selectable gene cassette comprising the Toxoplasma gondii dihydrofolate reductase-thymidylate synthase (DHFR-TS) gene mutated to confer pyrimethamine resistance flanked by either Plasmodium chabaudi DHFR-TS or P. falciparum calmodulin promoter sequences and the P. falciparum histidine rich protein 2 3' region. Also, each vector includes a different expression cassette driven by various Plasmodium transcriptional control sequences. Initially, the chloramphenicol acetyl transferase (CAT) reporter gene was cloned into the expression site of two vectors, pCC6-CAT and pCC13-CAT, which were identical except for the orientation of the expression cassette with respect to the selectable gene cassette. Approximately 8-fold more CAT activity was detected when the direction of transcription of the expression cassettes was in a head to head, rather than a tail to head, orientation. Importantly, it was found that stable transfection could only be achieved when the gene cassettes were in the head to head direction suggesting that this orientation also has an effect on the level of expression of the selectable marker. All other plasmids were designed with the cassettes in a head to head orientation. With the exception of pCC6-CAT and a second vector pHC4-CAT, stable transfectants were obtained with each vector in which the CAT gene had been inserted into the expression cassette. This is the first time vectors for the stable expression in Plasmodium parasites of transgenes other than a selectable marker have been described.

Animals↗
PubMed1999

Plasmodium vivax merozoite surface protein-3 contains coiled-coil motifs in an alanine-rich central domain.

Plasmodium merozoites are covered with a palisade layer of proteins that are arranged as organized bundles or appear as protruding spikes by electron microscopy. Here we present a third Plasmodium vivax merozoite surface protein, PvMSP-3, which is associated with but not anchored in the merozoite membrane. Serum from a P. vivax immune squirrel monkey was used to screen a lambdagt11 P. vivax genomic DNA (gDNA) library. Plaque-selected antibodies from clone no. 6.1, and rabbit antisera against its encoded protein, produced a pattern in immunofluorescence assays (IFAs) that is consistent with a localization at the surface of mature schizonts and free merozoites. Specific antisera also agglutinated merozoites and recognized a protein of 150 000 Da by SDS-PAGE. The complete msp-3 gene and flanking sequences were cloned from a P. vivax lambda Dash II gDNA library and also partly characterized by RACE (rapid amplification of cDNA ends). The immediate upstream sequence contains non-coding repeats and a putative protein encoding open reading frame (ORF), which are also present on the msp-3 5'RACE gene product. Pvmsp-3 encodes a protein with a calculated mass of 89 573 Da, which has a potential signal peptide and a major central alanine-rich domain (31%) that exhibits largely alpha-helical secondary structure and is flanked by charged regions. The protein does not have a putative transmembrane domain or a consensus sequence for a glycosylphosphatidylinositol (GPI) anchor modification. However, the alanine-rich domain has heptad repeats that are predicted to form coiled-coil tertiary structures, which mediate protein-protein interactions. PvMSP-3 is structurally related to P. falciparum MSP-3 and the 140000 Da MSP of P. knowlesi. Characterization of PvMSP-3, thus, also begins to define a new interspecies family of evolutionarily related Plasmodium merozoite proteins.

Alanine↗
PubMed1987

Modulator effect of Toxoplasma lysate antigen in mice experimentally infected with Plasmodium berghei.

Normal mice were pretreated twice at an interval of 2 weeks with an emulsion of TLA (Toxoplasma lysate antigen), PLA (Plasmodium lysate antigen) or both in LMO (light mineral oil) or with a combination of the emulsion and Obioactin or Tp-LKs (Toxoplasma lymphokines) as an immunopotentiator. They were then given Obioactin or Tp-LKs 3 and 25 days after the first treatment and were further given parasitized erythrocytes with 1 X 10(2)-10(4) P. berghei 2 weeks after the second treatment. Thirty (3/10, number of survival/number of examined) per cent of mice treated with TLA, 50 (5/10)% of those treated with a combination of TLA and Tp-LKs and 60 (6/10)% of those treated with a combination of TLA and Obioactin survived as long as 20 days postinfection while none of untreated controls survived more than 15 days postinfection. Only 18.2 (2/11)% of mice treated with PLA or TLA + PLA survived and 20 (2/10), 18.2 (2/11) and 60 (6/10)% of those treated with TLA + Obioactin, PLA + Obioactin or TLA + PLA + Obioactin survived throughout the experiment, respectively while none of controls survived more than 13 days postinfection. Five mice of each group were killed right before infection, and 5, 10 and 15 days postinfection. In mice treated with TLA + Obioactin, more macrophage phagocytosis and macrophage migration inhibition induced by sensitized T-cells were observed than in those treated otherwise. No appreciable differences were noted according to the method of treatment in blood examination values. Cross immunities between Toxoplasma and Plasmodium antigens were tested by counter-immunoelectrophoresis and indirect fluorescent antibody technique. By using counter-immunoelectrophoresis, a specific precipitin line was observed between TLA and anti-PLA which was absorbed by mouse erythrocytes, leucocytes and liver powder. By the indirect fluorescent antibody technique, anti-Plasmodium IgM and IgG titers were detected in sera from mice treated with TLA or TLA-Obioactin before infection.

Adjuvants, Immunologic↗
PubMed1999

Growth-stimulating influence of human chorionic gonadotropin (hCG) on Plasmodium falciparum in vitro.

In this study, it is reported that human Chorionic Gonadotropin (hCG), being one of the most important hormones of pregnancy, has a growth-stimulating effect on the asexual stages of Plasmodium falciparum in vitro. On the one hand, it is shown that the effect of the hormone is dose-related: The highest growth-rates of Plasmodium falciparum in vitro are achieved, when doses of 8.32 i.u./ml (= 50 i.u. hCG/6 ml) and 16.67 I.U./ml (= 100 i.u. hCG/6 ml) are added to the culture medium. These doses correspond to the physiological peak amounts of hCG between the 9th and 16th week of pregnancy, when parasitaemia also reaches its highest rate. On the other hand, it is shown, that any growth-stimulating effect disappears after inactivation of the hormone by heating at 120 degrees C for 20 minutes. These data support the hypothesis, that hCG does not only possess immunosuppressive properties acting on the response of T-lymphocytes, but also increases the growth of Plasmodium falciparum in vitro. The combination of both effects may explain why malaria still remains one of the most serious complications of pregnancy.

Animals↗
PubMed2002

Crystals of peptide deformylase from Plasmodium falciparum reveal critical characteristics of the active site for drug design.

Peptide deformylase catalyzes the deformylation reaction of the amino terminal fMet residue of newly synthesized proteins in bacteria, and most likely in Plasmodium falciparum, and has therefore been identified as a potential antibacterial and antimalarial drug target. The structure of P. falciparum peptide deformylase, determined at 2.8 A resolution with ten subunits per asymmetric unit, is similar to the bacterial enzyme with the residues involved in catalysis, the position of the bound metal ion, and a catalytically important water structurally conserved between the two enzymes. However, critical differences in the substrate binding region explain the poor affinity of E. coli deformylase inhibitors and substrates toward the Plasmodium enzyme. The Plasmodium structure serves as a guide for designing novel antimalarials.

Amidohydrolases↗
PubMed2001

Plasmodium ookinete-secreted chitinase and parasite penetration of the mosquito peritrophic matrix.

Malaria transmission-blocking strategies aimed at disrupting parasite-mosquito interactions have the potential to make important contributions to global malaria control. It has been suggested that Plasmodium-secreted chitinase plays a crucial role in allowing the ookinete to initiate its invasion of the mosquito midgut, which suggests that this enzyme is a candidate target for blocking malaria transmission. In this review, the authors discuss Plasmodium chitinases from the molecular, biochemical and cell biology viewpoints. Future directions of study could involve developing strategies for interrupting the function of Plasmodium chitinases within the mosquito midgut, including transmission-blocking drugs or vaccines, or the development of chitinase-inhibitor-producing transgenic mosquitoes.

Animals↗
PubMed2002

Phylogenetic analysis of the genus Plasmodium based on the gene encoding adenylosuccinate lyase.

Phylogenetic studies of the genus Plasmodium have been performed using sequences of the nuclear, mitochondrial and plastid genes. Here we have analyzed the adenylosuccinate lyase (ASL) gene, which encodes an enzyme involved in the salvage of host purines needed by malaria parasites for DNA synthesis. The ASL gene is present in several eukaryotic as well as prokaryotic organisms and does not have repeat regions, which facilitates the accuracy of the alignment. Furthermore, it has been shown that ASL is not subject to positive natural selection. We have sequenced the ASL gene of several different Plasmodium species infecting humans, rodents, monkeys and birds and used the obtained sequences along with the previously known P. falciparum ASL sequence, for structural and phylogenetic analysis of the genus Plasmodium. The genetic divergence of ASL is comparable with that observed in other nuclear genes such as cysteine proteinase, although ASL cannot be considered conserved when compared to aldolase or superoxide dismutase, which exhibit a slower rate of evolution. Nevertheless, a protein like ASL has a rate of evolution that provides enough information for elucidating evolutionary relationships. We modeled 3D structures of the ASL protein based on sequences used in the phylogenetic analysis and obtained a consistent structure for four different species despite the divergence observed. Such models would facilitate alignment in further studies with a greater number of plasmodial species or other Apicomplexa.

Adenylosuccinate Lyase↗
PubMed1998

Plasmodium ookinete development in the mosquito midgut: a case of reciprocal manipulation.

The ookinete is one of the most important stages of Plasmodium development in the mosquito. It is morphologically and biochemically distinct from the earlier sexual stages--gametocytes and zygote, and from the later stages--oocyst and sporozoites. Development to ookinete allows the parasite to escape from the tightly packed blood bolus, to cross the sturdy peritrophic matrix (PM), to be protected from the digestive environment of the midgut lumen, and to invade the gut epithelium. The success of each of these activities may depend on the degree of the biochemical and physical barriers in the mosquito (such as density of blood bolus, thickness of peritrophic matrix, proteolytic activities in the gut lumen etc.) and the ability of the ookinete to overcome these barriers. Ookinete motility, secretion of chitinase, resistance to the digestive enzymes, and recognition/invasion of the midgut epithelium all may play crucial roles in the transformation to oocyst. The overall sporogonic development of Plasmodium, therefore, depends on the results of the two-way manipulations between the parasite and the vector mosquito. Study of ookinete development and of the cellular and biochemical complexities of the mosquito gut may therefore lead to the design of novel strategies to block the transmission of malaria. This article reviews the intricate interactions between the parasite and the mosquito midgut in the context of development and transmission of Plasmodium parasites.

Animals↗
PubMed2006

Regulation of host cell survival by intracellular Plasmodium and Theileria parasites.

Plasmodium and Theileria parasites are obligate intracellular protozoa of the phylum Apicomplexa. Theileria infection of bovine leukocytes induces transformation of host cells and infected leukocytes can be kept indefinitely in culture. Theileria-dependent host cell transformation has been the subject of interest for many years and the molecular basis of this unique phenomenon is quite well understood. The equivalent life cycle stage of Plasmodium is the infection of mammalian hepatocytes, where parasites reside for 2-7 days depending on the species. Some of the molecular details of parasite-host interactions in P. berghei-infected hepatocytes have emerged only very recently. Similar to what has been shown for Theileria-infected leukocytes these data suggest that malaria parasites within hepatocytes also protect their host cell from programmed cell death. However, the strategies employed to inhibit host cell apoptotic pathways appear to be different to those used by Theileria. This review discusses similarities and differences at the molecular level of Plasmodium- and Theileria-induced regulation of the host cell survival machinery.

Animals↗
PubMed1999

Blood digestion in the mosquito, Anopheles stephensi: the effects of Plasmodium yoelii nigeriensis on midgut enzyme activities.

Midgut proteases contribute to the success or failure of Plasmodium infection of the mosquito. This paper examines the reciprocal effect of Plasmodium yoelii nigeriensis on midgut trypsin, chymotrypsin, aminopeptidase and carboxypeptidase in the mosquito Anopheles stephensi. The total protein ingested and the rate of protein digestion were unaffected by the parasite, but more protein was ingested at the first than the second bloodmeal. All peptidases were unaffected by the presence of the parasite during the first gonotrophic cycle, when ookinetes were penetrating the midgut. In the second gonotrophic cycle, trypsin and chymotrypsin were unaffected by growing oocysts, but aminopeptidase activity was reduced in the midguts of infected mosquitoes. Chymotrypsin activity was depressed and aminopeptidase activity elevated during the second gonotrophic cycle. Plasmodium infection has a negligible effect on bloodmeal digestion and does not limit the availability of the protein for egg production. The significance of changes in aminopeptidase activity when oocysts are present is discussed.

Aminopeptidases↗