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PubMed2002

Novel antifolate resistant mutations of Plasmodium falciparum dihydrofolate reductase selected in Escherichia coli.

A simple and effective system has been developed from which a number of Plasmodium falciparum dihydrofolate reductase (pfDHFR) mutants conferring resistance to antifolates were randomly generated and characterized. The system exploited error-prone PCR to generate random mutations in the pfDHFR. Using the synthetic gene encoding for wild-type and quadruple mutant (N51I+C59R+S108N+I164L) pfDHFRs as templates, mutants resistant to pyrimethamine (Pyr), m-Cl analogue of Pyr (SO3) and WR99210 were selected by bacterial complementation system in which the endogenous DHFR activity of bacterial host cells, but not of Plasmodium, is selectively inhibited by trimethoprim (Tmp). Mutants conferring resistance to antimalarial antifolates were selected under the condition that inhibited the growth of the wild-type pfDHFR. All obtained Pyr resistant mutants possessed S108 mutation, in combination with common mutations of N51I, C59R and I164L previously found in the field. New Pyr resistant mutants with novel mutations (K27T, N121D, N144K and V213E) not found in the field were also identified. Exposure of the randomly mutated pfDHFR libraries to WR99210 or SO3 resulted in selection of novel single and multiple mutants including D54N, F58L and a combination of C50R, K181R, T219P and K227E, which exhibited 2- to over 2000-fold increase in resistance against antifolates. Kinetic analysis of these mutants suggested that apart from the active site residues that are crucial for DHFR activity, residues remote from the binding pocket also play essential roles in substrate and inhibitor binding.

Animals↗
PubMed2002

PSLAP, a protein with multiple adhesive motifs, is expressed in Plasmodium falciparum gametocytes.

A gene coding for a protein containing two Scavenger Receptor Cysteine-Rich (SRCR) motifs, four Limulus factor C, Coch-5b2 and Lgl1 (LCCL) motifs; and one Polycystin-1, Lipoxygenase and Alpha Toxin (PLAT) motif was cloned from Plasmodium chabaudi and homologues identified in the P. falciparum and P. yoelii genome data bases. At least one of these sequence motifs (SRCR) has adhesive properties in other proteins, therefore, we propose to name this protein PSLAP for Plasmodium SRCR, LCCL Adhesive-like Protein. Southern blotting and chromosome analysis showed that pslap is a single copy gene on chromosome 14 in P. falciparum 3D7. pslap mRNA is strongly expressed in P. falciparum gametocytes, but was undetectable on Northern blots of RNA from the asexual blood stages. Polyclonal antibodies raised to different parts of PSLAP detected a protein expressed in late gametocytes, but not in the early stages of gametocytogenesis or asexual blood stages of P. falciparum. We suggest that PSLAP functions in the mosquito, for example, in modulation of the invertebrate host immune response or in protection against complement factors in the blood meal.

Amino Acid Motifs↗
PubMed2002

Laser capture microdissection of Plasmodium falciparum liver stages for mRNA analysis.

Plasmodium falciparum liver-stages are important targets for vaccine-induced protective immune responses and prophylactic treatment against malaria. Little is known of the gene expression profile of malaria parasites during their development inside hepatocytes. The sequencing of the P. falciparum genome and the development of DNA microarray technology give new opportunities to identify genes expressed during the development of Plasmodium. However, transcriptome analysis cannot currently be applied to the hepatic stages, due to difficulties in obtaining sufficient amounts of parasite material that lie among the large excess of host cell RNA. Here, we describe the isolation of liver-stages by a modified laser capture microdissection approach applied to human hepatocyte cultures infected with P. falciparum. RT-PCR amplification of several P. falciparum transcripts demonstrated the high quality of the RNA recovered after microdissection. This approach should enable analyses of P. falciparum transcriptome during its hepatic development and substantially assist the identification of new therapeutic and vaccine targets.

Animals↗
PubMed2002

Bacterially expressed and refolded receptor binding domain of Plasmodium falciparum EBA-175 elicits invasion inhibitory antibodies.

Malaria parasites make specific receptor-ligand interactions to invade erythrocytes. A 175 kDa Plasmodium falciparum erythrocyte binding antigen (EBA-175) binds sialic acid residues on glycophorin A during invasion of human erythrocytes. The receptor-binding domain of EBA-175 lies in a conserved, amino-terminal, cysteine-rich region, region F2 of EBA-175 (PfF2), that is homologous to the binding domains of other erythrocyte binding proteins such as Plasmodium vivax Duffy binding protein. We have developed methods to produce recombinant PfF2 in its functional form. Recombinant PfF2 was expressed in Escherichia coli, purified from inclusion bodies, renatured by oxidative refolding and purified to homogeneity by ion-exchange and gel filtration chromatography. Refolded PfF2 has been characterized using biochemical and biophysical methods and shown to be pure, homogenous and functional in that it binds human erythrocytes with specificity. Immunization with refolded PfF2 yields high titre antibodies that efficiently inhibit P. falciparum invasion of erythrocytes in vitro. Importantly, antibodies raised against PfF2 block invasion by a P. falciparum field isolate that invades erythrocytes using multiple pathways. These observations support the development of recombinant PfF2 as a vaccine candidate for P. falciparum malaria.

Animals↗
PubMed2003

Plasmodium falciparum histidine-rich protein II binds to actin, phosphatidylinositol 4,5-bisphosphate and erythrocyte ghosts in a pH-dependent manner and undergoes coil-to-helix transitions in anionic micelles.

The recombinant histidine-rich protein II (HRPII) from Plasmodium falciparum was shown to bind actin and phosphatidylinositol 4,5-bisphosphate (PIP(2)) in vitro in a pH-dependent manner, very similar to hisactophilin, an actin-binding protein from ameba. Binding of HRPII to actin and PIP(2) occurred at pH 6.0 and 6.5, but not above pH 7.0. Circular dichroism (CD) spectroscopy confirmed that HRPII interacts with actin at pH below 7.0, as judged by the changes induced in the secondary structure of the HRPII/actin mixture. Further CD analysis demonstrated that HRPII adopts a predominantly alpha-helical conformation with anionic micelles of PIP(2) and SDS, but not with neutral micelles of phosphatidylcholine (PC), a feature that is common to many actin-binding proteins involved in cytoskeleton remodeling. Similarly to hisactophilin, a GFP-HRPII fusion protein shuttled from the cytoplasm to the nucleus of HeLa cells as the cellular pH was lowered from 8.0 to 6.0. HeLa cells transfected with the HRPII gene showed increased levels of histidine-rich proteins (HRPs) in the soluble cell fraction at pH 8.0. At pH 6.0, however, HRPs were detected mainly in the insoluble cell fraction. Interestingly, we found that HRPII binds to human erythrocyte membranes at pH 6.0 and 6.5 but not at pH above 7.0. Our results point to remarkable similarities between HRPII, hisactophilin, and actin-binding proteins. Possible roles of the HRPII during Plasmodium infection are discussed in the light of these findings.

Actins↗
PubMed2003

Linker scanning mutagenesis of the Plasmodium gallinaceum sexual stage specific gene pgs28 reveals a novel downstream cis-control element.

Protozoan parasites undergo complex life cycles that depend on regulated gene expression. However, limited studies on gene regulation in these parasites have repeatedly shown characteristics different from other eukaryotes. Within the Apicomplexa family, little is known about the mechanism of gene expression and regulation in Plasmodium spp. We have been investigating the cis-elements that control basal expression of a sexual stage specific gene in Plasmodium gallinaceum. Previously, we identified by 5' deletion analysis of a reporter construct that the 333bp upstream of the translational start site of pgs28 is sufficient for basal expression, and that the sequence between -333 and 316bp is necessary for such expression. In this report, we identified by linker scanning mutagenesis an 8-bp sequence that is essential for pgs28 transgene expression. This sequence is a target of sequence-specific nuclear factors. Primer extension studies demonstrate that, interestingly, the endogenous pgs28 transcript has two 5' ends, at -65 and +1. We suggest that this 8-bp sequence, CAGACAGC that is situated at +24 to +31 (with respect to the proximal start site), is a novel downstream promoter element in P. gallinaceum that appears to function independently of a TATA box or an Inr element.

Animals↗
PubMed1996

Further characterization of a 58 kDa Plasmodium berghei phosphoprotein as a cochaperone.

Molecular chaperones are important for proper protein folding during protein biogenesis. This report describes a protein from Plasmodium berghei which is 30% identical and 40% similar to a recently described mammalian cochaperone, or heat shock protein 70 interacting protein. The P. berghei cochaperone accumulates throughout the trophozoite stage and decreases during the schizont stage. The stage specific expression is consistent with its presumed role in protein folding or protein-protein interactions. The largest difference between the Plasmodium and mammalian sequences is a more extensive domain of imperfect glycine-glycine-methionine-proline (GGMP) tandem repeats in the parasite's cochaperone sequence. Immunofluorescence studies show that the protein is an abundant cytosolic protein of the parasite. However, antibodies raised against the GGMP repeat domain, which is also found in other parasite chaperones, react with both the parasite and host erythrocyte membrane. The reactivity with the host membrane suggests that the parasite exports molecular chaperones into the infected erythrocyte.

Amino Acid Sequence↗
PubMed1997

The antimalarial drug, chloroquine, interacts with lactate dehydrogenase from Plasmodium falciparum.

We have previously shown that a radioiodinated photoreactive analogue of chloroquine, [125I]N-(4-(4-diethylamino-1-methylbutylamino)quinolin-6-yl) -4-azido-2-hydroxybenzamide ([125I]ASA-Q), specifically labels two proteins in Plasmodium falciparum with apparent molecular weights (Mr) of 42 and 33 kDa (Foley M, Deady LW, Ng K, Cowman AF, Tilley L. J Biol Chem 1994:269:6955-6961). We now report the identification of the 33 kDa protein. The 33 kDa protein was purified from Plasmodium falciparum using photoaffinity labeling with [125I]ASA-Q to monitor the enrichment process. N-terminal sequence analysis of the purified protein revealed exact identity of the first 35 amino acids with P. falciparum lactate dehydrogenase (PfLDH). The plasmodial enzyme was cloned and expressed in E. coli and the recombinant protein used to produce a rabbit antiserum. Immunoprecipitation using affinity-purified anti-PfLDH antibodies confirmed the identity of the 33 kDa CQ-binding protein. The enzyme activity of purified PfLDH was not significantly affected by chloroquine indicating that PfLDH is not a direct target of CQ. PfLDH was, however, shown to be exquisitely sensitive to inhibition by free heme and chloroquine protected against this inhibitory effect.

Affinity Labels↗
PubMed1997

Developmentally regulated expression of pfs16, a marker for sexual differentiation of the human malaria parasite Plasmodium falciparum.

Sexual differentiation is essential for the transmission of Plasmodium to mosquitoes and therefore, for the spread of malaria. The molecular mechanisms underlying sexual differentiation are poorly understood but may be elucidated by a detailed study of the regulation of expression of sexual stage specific genes. In the present work we describe the differential expression of the gene encoding the sexual stage specific protein, Pfs16. We have conducted a comparative analysis of pfs16 promoter activity, RNA levels and the rate of de novo protein synthesis during development of Plasmodium falciparum. Furthermore, we have determined the pattern of expression of pfs16 transcripts at the single cell level by in situ hybridisation. We show that the expression of pfs16 is induced immediately following the invasion of a red blood cell in sexually committed ring stage parasites and continues throughout gametocytogenesis and in macrogametes. The expression of pfs16 is regulated at the level of transcription initiation and modulated by a post-transcriptional process. These results demonstrate that the expression of the pfs16 gene is the earliest event in the sexual differentiation process of P. falciparum described to date.

Animals↗
PubMed1997

Anopheles stephensi salivary glands bear receptors for region I of the circumsporozoite protein of Plasmodium falciparum.

In the mosquito, Plasmodium sporozoites rupture from oocysts found on the midgut wall, circulate in the hemolymph and invade salivary glands where they wait to be injected into a vertebrate host during a bloodmeal. The mechanisms by which sporozoites specifically attach to and invade salivary glands are not known but evidence suggests that it is a receptor-mediated process. Here we show that the major surface protein of sporozoites, the circumsporozoite protein (CS), binds preferentially to salivary glands when compared to other organs exposed to the circulating hemolymph. In addition, we show that a peptide encompassing region I, a highly conserved sequence found in all rodent and primate Plasmodium CS proteins, inhibits binding of CS to mosquito salivary glands.

Amino Acid Sequence↗
PubMed1998

Developmental regulation of a Plasmodium gene involves the generation of stage-specific 5' untranslated sequences.

The B7 gene of Plasmodium berghei, highly conserved within the genus Plasmodium, encodes a nuclear protein most likely involved in chromatin assembly. In this study we describe the transcription pattern of B7 during asexual multiplication and sexual differentiation of the parasites in the blood of the vertebrate host. Two alternative transcripts have been identified: one, 1.4 kb in length is specific for asexual blood stages; the other, 1.8 kb in length is specific for sexually differentiated cells (gametocytes). The processed mRNAs are identical in their coding region and differ only in their 5' untranslated regions (5' UTRs). We show here that the differences in 5' UTRs are the result of two mechanisms: (1) the use of alternative transcription initiation sites mapped at least 1.4 kb apart, which imply the existence of separate, stage-specific promoters; (2) the splicing of a 765 bp gametocyte-specific intron at the 5' UTR of the 1.8 kb transcript.

5' Untranslated Regions↗
PubMed1999

Identification of the transcription initiation site of the asexually expressed rRNA genes of the malaria parasite Plasmodium berghei.

The start site of the A-type ribosomal RNA transcription units of the rodent malaria parasite, Plasmodium berghei, has been identified. The two A-type units cannot be distinguished within the transcription unit, yet exist as single copies on different chromosomes. Gene transcription initiates 820 bp upstream of the A-type small subunit (SSU) ribosomal gene and two major processing sites were mapped 610 and 611 nucleotides upstream of the SSU in the external transcribed spacer region. Surprisingly the nucleotide sequence of the DNA region containing the putative ribosomal promoter lacked repetitive DNA sequences typical of ribosomal promoters. This region was further analysed by computer using programs designed to reveal sequence-dependent structural features. Comparison of DNA curvature, duplex stability and pattern of twist angle variation revealed a striking degree of conservation between the ribosomal promoters from Plasmodium and other eukaryotes.

Animals↗
PubMed1999

Chelation of iron within the erythrocytic Plasmodium falciparum parasite by iron chelators.

To examine the site of action of antimalarial iron chelators, iron ligands were added to control erythrocytes and to erythrocytes parasitized with Plasmodium falciparum, and the concentration of intracellular labile iron was monitored with the fluorescent probe, calcein. The fluorescence of calcein quenches upon binding iron and increases upon releasing iron. The chelators included desferrioxamine B, 2',2'-bipyridyl, and aminophenol II, a compound that is being newly reported as having anti-plasmodial properties. Calcein-loaded parasitized cells displayed fluorescence predominantly within the cytosol of both rings and trophozoites. The addition of chelators to both control and parasitized erythrocytes led to significant increases of fluorescence (P < 0.001). Fluorescence was observed to increase within the parasite itself after addition of iron chelators, indicating that these agents bound labile iron within the plasmodium. The relative increases of fluorescence after addition of chelators were greater in control than parasitized erythrocytes (P < 0.05) as were the estimated labile iron concentrations (P < or = 0.001). These results suggest that (i) the anti-malarial action of iron chelators might result from the ability to reach the infected cell's parasite compartment and bind iron within the parasite cytosol, and (ii) the labile iron pool of the host red cell may be either utilized or stored during plasmodial growth.

2,2'-Dipyridyl↗
PubMed2000

3' UTR elements enhance expression of Pgs28, an ookinete protein of Plasmodium gallinaceum.

In Plasmodium parasites the fusion of gametes to form a fertilized zygote and morphogenesis into the motile ookinete are critical developmental stages in the parasite's complex life cycle. In analogous developmental stages of metazoan organisms 3' gene flanking regions are critical in the regulation of gene expression. To determine whether these mechanisms are conserved in the protozoan parasite we studied the 3' gene flanking elements necessary for the expression of Pgs28, the major surface protein of mature zygotes and ookinetes of the chicken malaria Plasmodium gallinaceum. The DNA sequence of the pgs28 3' gene flanking region contains 7 eukaryotic polyadenylation consensus signals (AATAAA/ATTAAA). An unusual 82% T-rich region is located 55 nucleotides upstream of the fifth polyadenylation signal (ATTAAA). The pgs28 mRNA terminates approximately 20 nucleotides from the polyadenylation signal in a poly (A) tail. To determine whether the T-rich region and polyadenylation signals were necessary for Pgs28 protein expression, sexual stage parasites were transfected with plasmids containing deletions of these elements utilizing firefly luciferase (LUC) and beta-glucuronidase (GUS) as markers of transient gene transfection. The parasites were allowed to develop in vitro to the ookinete stage and assayed for enzymatic activity. Cells transfected with plasmids containing deletions of the T-rich region or fifth eukaryotic polyadenylation consensus signal expressed 89 and 92%, less enzymatic activity respectively than those transfected with the full length pgs28 3' gene flanking region. The U-rich element and fifth eukaryotic polyadenylation consensus sequence within the pgs28 3' UTR are therefore necessary for Pgs28 protein expression.

3' Untranslated Regions↗
PubMed2000

The selectable marker human dihydrofolate reductase enables sequential genetic manipulation of the Plasmodium berghei genome.

Genetic transformation of malaria parasites has been limited by the number of selectable markers available. For the rodent malaria parasite, Plasmodium berghei, only a single selection marker has been at hand, utilising the dihydrofolate reductase-thymidylate synthase gene from either P. berghei or Toxoplasma gondii to confer resistance to the anti-malarial drug pyrimethamine. Here we report the use of the human dihydrofolate reductase (hDHFR) gene as a new selectable marker, which confers resistance to the antifolate inhibitor WR99210 upon both pyrimethamine sensitive and resistant isolates of P. berghei. Transfection with circular constructs containing the hDHFR gene resulted in the generation of highly resistant parasites containing multiple copies of episomally-maintained plasmids. These parasites showed around a 1000-fold increase in resistance to WR99210 compared to the parental parasites. We were also able to generate and select transgenic parasites harbouring only a single copy of hDHFR targeted into their genome, despite the fact that these parasites showed only a fivefold increase in resistance to WR99210 compared to the parental parasites. Importantly, and for the first time with malaria parasites, the hDHFR gene could be used in conjunction with the existing pyrimethamine selectable markers. This was demonstrated by reintroducing the circumsporozoite (CS) gene into transgenic CS-knockout mutant parasites that contained the P. berghei DHFR-TS selectable marker. The development of hDHFR as a second selectable marker will greatly expand the use of transformation technology in Plasmodium, enabling more extensive genetic manipulation and thus facilitating more comprehensive studies on the biology of the malaria parasite.

Animals↗
PubMed2000

Cyclin H activation and drug susceptibility of the Pfmrk cyclin dependent protein kinase from Plasmodium falciparum.

The eukaryotic cell cycle is regulated by a group of highly conserved cyclin dependent protein kinases (CDKs). Several CDKs have been identified in Plasmodium falciparum, however, their regulatory mechanisms as well as their role in parasite growth and differentiation are not understood fully. To further our understanding of Plasmodium CDK regulation, we have characterized Pfmrk kinase activity. Pfmrk was expressed and purified as a 6xHis tagged recombinant protein from Escherichia coli and assayed for histone H1 kinase activity. Pfmrk has significant histone H1 kinase activity and is autophosphorylated in vitro. Human cyclin H forms a stable complex with Pfmrk and stimulates kinase activity. This is the first indication that Plasmodial CDKs are partially regulated by cyclin subunits, as are human CDKs. CDKs are attractive drug targets due to their role in cellular proliferation. Specific CDK inhibitors were selected to evaluate Pfmrk as a potential drug target. Olomoucine and roscovitine failed to inhibit Pfmrk kinase activity which places Pfmrk with a class of CDKs that are insensitive to these compounds. A molecular model of Pfmrk provides a structural explanation for the failure of these compounds to inhibit Pfmrk.

Amino Acid Sequence↗
PubMed2000

cDNA cloning of a novel cysteine protease of Plasmodium falciparum.

The cDNA for a novel Plasmodium cysteine protease (falcipain-2) has been isolated from a Plasmodium falciparum cDNA library. A 602 bp fragment was amplified from P. falciparum by PCR using degenerate oligonucleotide primers. The primers were designed based upon the amino acids flanking the active site cysteine and asparagine residues that are conserved in the eukaryotic cysteine proteases. This fragment was used to screen a P. falciparum cDNA library and isolated a 2.1 kb clone that encoded a novel cysteine protease. The sequence of the 2.1 kb clone predicted a 56 kDa protein containing a typical signal sequence, a prosequence and a 24.7 kDa mature protease with 37% identity to falcipain-1, a hemoglobin-degrading cysteine protease of P. falciparum. Northern blot analysis detected a 2.1 kb message in trophozoites. Taken together, we have isolated a novel cysteine protease of P. falciparum, which may play an important role at the late stages of the erythrocytic cycle of the parasite.

Amino Acid Sequence↗
PubMed2000

Identification of differentially regulated genes of Plasmodium by suppression subtractive hybridization.

Plasmodium, the causative agent of malaria, has many morphologically and functionally distinct developmental stages. In the mosquito host alone, there are five transitions during the development of a gametocyte into a sporozoite. Determining which genes are expressed at the different developmental stages is vital to our understanding of the parasite. There are a growing number of techniques designed to study gene expression, including microarray. Here, Johannes Dessens, Gabrielle Margos, Maria del Carmen Rodriguez and Robert Sinden describe a novel method: suppression subtractive hybridization (SSH) and its successful application in obtaining mosquito midgut stage-specific genes of Plasmodium.

Animals↗