Falciparum Homologue Research Articles

PfADA2, a Plasmodium falciparum homologue of the transcriptional coactivator ADA2 and its in vivo association with the histone acetyltransferase PfGCN5

The transcriptional coactivator ADA2 is an evolutionarily conserved component of histone acetyltransferase (HAT) complexes involved in chromatin remodeling and transcriptional regulation in eukaryotes. The Plasmodium falciparum homologue, PfADA2, has a 7737 bp open reading frame, encoding a protein of 2578 amino acids with an ADA2-like domain located near the C-terminus. The annotated PfADA2 in the parasite genome is Pf10_143, located on chromosome 10. Sequence analysis demonstrated the presence of an ADA2 homologue in each Plasmodium species selected for genome sequencing. Mapping of the 5′ transcriptional initiation sites suggested that PfADA2 transcription was initiated from multiple sites. Northern analysis detected a major transcript of ∼8.5 kb in erythrocytic stage parasites. An antiserum raised against the internal ADA2-like domain detected multiple proteins from mixed blood stages, suggesting that PfADA2 may be proteolytically processed. In comparison, affinity-purified anti-GCN5 antibodies reacted with a major protein of ∼200 kDa and immunoprecipitated proteins from the parasite lysate with HAT activity similar to that of the recombinant GCN5. Moreover, this GCN5-like HAT activity could also be precipitated with anti-PfADA2 antibodies, indicating that PfADA2 is associated with PfGCN5 in vivo. To illustrate whether PfADA2 could functionally replace the yADA2, complementation experiments were performed. However, the ADA2-like domain of PfADA2 failed to rescue the yeast ada2 − mutant, probably due to significant divergence between the two genes. Taken together, these results indicate the presence of PfADA2-PfGCN5 complex(es) in the malaria parasite, which may have conserved functions in chromatin remodeling and gene regulation.

PfPKB, a novel protein kinase B-like enzyme from Plasmodium falciparum: I. Identification, characterization, and possible role in parasite development.

Extracellular signals control various important functions of a eukaryotic cell, which is often achieved by regulating a battery of protein kinases and phosphatases. Protein Kinase B (PKB) is an important member of the phosphatidylinositol 3-kinase-dependent signaling pathways in several eukaryotes, but the role of PKB in protozoan parasites is not known. We have identified a protein kinase B homologue in Plasmodium falciparum (PfPKB) that is expressed mainly in the schizonts and merozoites. Even though PfPKB shares high sequence homology with PKB catalytic domain, it lacks a pleckstrin homology domain typically found at the N terminus of the mammalian enzyme. Biochemical studies performed to understand the mechanism of PfPKB catalytic activation suggested (i) its activation is dependent on autophosphorylation of a serine residue (Ser-271) in its activation loop region and (ii) PfPKB has an unusual N-terminal region that was found to negatively regulate its catalytic activity. We also identified an inhibitor of PfPKB activity that also inhibits P. falciparum growth, suggesting that this enzyme may be important for the development of the parasite.

A Plasmodium falciparum homologue of the Ran binding protein 1, a protein involved in nucleocytoplasmic transport

Keywords: Sequence Homology, Amino Acid Reference EPFL-ARTICLE-148279doi:10.1016/S0166-6851(02)00119-6 Record created on 2010-04-14, modified on 2017-01-24

Evidence for a role for a Plasmodium falciparum homologue of Sec31p in the export of proteins to the surface of malaria parasite-infected erythrocytes.

The malaria parasite, Plasmodium falciparum, spends part of its life cycle inside the enucleated erythrocytes of its human host. The parasite modifies the cytoplasm and plasma membrane of its host cell by exporting proteins beyond the confines of its own plasma membrane. We have previously provided evidence that a plasmodial homologue of the COPII protein, Sar1p, is involved in the trafficking of proteins across the erythrocyte cytoplasm. We have now characterised an additional plasmodial COPII protein homologue, namely Sec31p. Recombinant proteins corresponding to the WD-40 and the intervening domains of the PfSec31p sequence were used to raise antibodies. The affinity-purified antisera recognised a protein with an apparent relative molecular mass of 1.6 x 10(5) on western blots of malaria parasite-infected erythrocytes but not on blots of uninfected erythrocytes. PfSec31p was shown to be largely insoluble in nonionic detergent, suggesting cytoskeletal attachment. Confocal immunofluorescence microscopy of malaria parasite-infected erythrocytes was used to show that PfSec31p is partly located within the parasite and partly exported to structures outside the parasite in the erythrocyte cytoplasm. We have also shown that PfSec31p and PfSar1p occupy overlapping locations. Furthermore, the location of PfSec31p overlaps that of the cytoadherence-mediating protein PfEMP1. These data support the suggestion that the malaria parasite establishes a vesicle-mediated trafficking pathway outside the boundaries of its own plasma membrane - a novel paradigm in eukaryotic biology.

Conserved regions of the Plasmodium yoelii rhoptry protein RhopH3 revealed by comparison with the P. falciparum homologue

Conserved regions of the Plasmodium yoelii rhoptry protein RhopH3 revealed by comparison with the P. falciparum homologue

Activation of a Plasmodium falciparum cdc2-related Kinase by Heterologous p25 and Cyclin H: FUNCTIONAL CHARACTERIZATION OF A P. FALCIPARUM CYCLIN HOMOLOGUE

Several Plasmodium falciparum genes encoding cdc2-related protein kinases have been identified, but the modalities of their regulation remains largely unexplored. In the present study, we investigated the regulation in vitro of PfPK5, a putative homologue of Cdk1 (cdc2) in P. falciparum. We show that (i) PfPK5 is efficiently activated by heterologous (human) cyclin H and p25, a cyclin-like molecule that specifically activates human Cdk5; (ii) the activated enzyme can be inhibited by chemical Cdk inhibitors; (iii) Pfmrk, a putative P. falciparum homologue of the Cdk-activating kinase, does neither activate nor phosphorylate PfPK5; and (iv) PfPK5 is able to autophosphorylate in the presence of a cyclin. Taken together, these results suggest that the regulation of Plasmodium Cdks may differ in important aspects from that of their human counterparts. Furthermore, we cloned an open reading frame encoding a novel P. falciparum protein possessing maximal homology to cyclin H from various organisms, and we show that this protein, called Pfcyc-1, is able to activate recombinant PfPK5 in vitro with an efficiency similar to that of human cyclin H and p25. This work opens the way to the development of screening procedures aimed at identifying compounds that specifically target the parasite Cdks.

Stage-specific expression of 14-3-3 in asexual blood-stage Plasmodium

This paper reports the identification of 14-3-3 in Plasmodium. 14-3-3 is an evolutionarily conserved protein that is most noted as a mediator in signal transduction events and cell cycle regulation. The complete cDNA (∼2.6 kb) and gDNA (∼3.4 kb) of a Plasmodium knowlesi 14-3-3 ( Pk14-3-3) is reported. The gene has three introns; two near the beginning and one close to the end of the coding sequence. Also reported, is the gDNA of the Plasmodium falciparum homologue ( Pf14-3-3). Unlike in many other organisms, where multiple gene copies and different functional isoforms exist, Plasmodium 14-3-3 is encoded as a single-copy gene. Northern blot analyses show that the Pk14-3-3 transcript in asexual blood stages begins to be expressed in the ring-stage, predominates in young trophozoites, and thereafter declines. An antiserum produced against recombinant Pk14-3-3 reacts via immunoblot and immunoprecipitation with the ∼30 kDa and the ∼32 kDa Pk14-3-3 and Pf14-3-3 proteins, respectively. Protein expression in P. knowlesi closely mimics the pattern of the transcript.

A Plasmodium falciparum novel gene encoding a coronin-like protein which associates with actin filaments

Plasmodium falciparum, the major causative agent of human malaria, is an Apicomplexa protozoan parasite which invades in its intermediate host hepatocytes and erythrocytes. The driving force underlying internalization into the host cell is thought to involve both polymerization of parasite actin, as entry is inhibited by the cytochalasins, and an actin motor-associated protein. In the related Apicomplexa parasite, Toxoplasma gondii, the involvement of parasite actin during both processes of motility and host cell entry has been genetically established. In a search for molecules that can regulate actin dynamics within Apicomplexa parasites, we have identified a P. falciparum homologue of the actin associated protein called coronin originally described in the amoeba Dictyostelium discoideum. The single copy gene displays a strong homology with the amoeba sequence and with the bovine and human coronin homologues recently cloned. This homology lies not only within the N-terminus containing the five WD repeats that characterize coronin but also extends in the C-terminal part. Furthermore, using an affinity-purified mouse monoclonal antibody against D. discoideum coronin, we have detected in extracts of P. falciparum young and mature schizonts a 42-kDa polypeptide which binds this antibody and is present in a Triton insoluble fraction that also contains parasite actin filaments. In addition, the recombinant protein encoded by the homologue nucleotidic sequence of P. falciparum coronin is indeed recognized by the antibody against D. discoideum coronin. Finally, the cross-reactive polypeptide displays the ability to cosediment with exogenous F-actin, a property which fits with its involvement in actin dynamics.

A Plasmodium falciparum homologue of the ATPase subunit of a multi-protein complex involved in chromatin remodelling for transcription

A Plasmodium falciparum homologue of one of the components of a chromatin-remodelling complex which controls binding of transcription factors to nucleosome core particles has been cloned and characterised. The gene encodes 1422 amino acids with an estimated molecular mass of 167 kDa. The protein, SNF2L, shares 60% amino acid identity in its conserved DNA-dependent ATPase domain with yeast transcription factors originally identified by characterising mating type switch mutants. It also contains sequences related to the so-called SWI3, ADA2, N-CoR and TFIIIB B″ or SANT DNA binding domains which are characteristic of these transcriptional activation factors. The SNF2L gene has two short introns in the 3′ region of the coding sequence of the gene and is transcribed into a single ∼6.5 kb messenger RNA species which is present throughout the asexual stages of the cell cycle. Southern blotting and pulsed field gel electrophoresis experiments show that SNF2L is a single copy gene, located on P. falciparum chromosome 11.

Characterization of P0, a Ribosomal Phosphoprotein ofPlasmodium falciparum: ANTIBODY AGAINST AMINO-TERMINAL DOMAIN INHIBITS PARASITE GROWTH

A cDNA expression clone of the human malarial parasite Plasmodium falciparum, lambdaPf4, which was reactive only to the immune sera and not to the patient sera, has recently been found to be the P. falciparum homologue of the P0 ribosomal phosphoprotein gene. A Northern analysis of the P0 gene revealed the presence of two transcripts, both present in all the different intraerythrocytic stages of the parasite life cycle. A 138-base pair amino-terminal domain of this gene was expressed as a fusion protein with glutathione S-transferase in Escherichia coli. Polyclonal antibodies raised against this domain immunoprecipitated the expected 38-kDa P0 protein from the 35S-labeled as well as 32P-labeled P. falciparum cultures. Monospecific human immune sera affinity-purified using the expression clone lambdaPf4 also immunoprecipitated the same size protein from [35S]methionine-labeled P. falciparum protein extract. Purified IgG from polyclonal antibodies raised against the amino-terminal domain of P0 protein completely inhibited the growth of P. falciparum in vitro. This inhibition appears to be mainly at the step of erythrocyte invasion by the parasites.

Assessment of pfmdr 1 gene copy number by tandem competitive polymerase chain reaction

The pfmdr 1 gene encodes a Plasmodium falciparum homologue of the human P-glycoprotein expressed on the surface of the parasite food vacuole. Variation in copy number and specific codon mutations of pfmdr 1 have been implicated in the development of parasite resistance to antimalarial drugs. We describe here the technique of Tandem-Competitive Polymerase Chain Reaction (TC-PCR), which allows accurate measurement of pfmdr 1 copy number in parasite DNA obtained directly from small quantities (100 μl) of red blood cells. We reliably quantified pfmdr 1 in previously well characterised strains of Plasmodium falciparum with differing pfmdr 1 gene copy numbers using starting amounts of between 3000 and 40 000 gene copies. We then used TC-PCR to determine pfmdr 1 gene copy number in field specimens of venous blood taken from 10 patients with malaria contracted along the Thai—Burmese border. In this region of high grade parasite resistance to mefloquine greater than 70% of samples had a copy number greater than 1 of pfmdr 1 determined with a repeatability coefficient of 0.58.

Identification and Characterization of the Protective Hepatocyte Erythrocyte Protein 17 kDa Gene of Plasmodium yoelii, homolog of Plasmodium falciparum Exported Protein 1

We recently reported the discovery of a 17-kDa Plasmodium yoelii protein expressed in infected hepatocytes and erythrocytes, P. yoelii hepatocyte erythrocyte protein 17 (PyHEP17), and have demonstrated that this protein is a target of protective antibodies and T cells. Here, we report the identification and characterization of the gene encoding this protein and reveal that it is composed of two exons. Immunization of mice with PyHEP17 plasmid DNA induces antibodies, cytotoxic T lymphocytes, and protective immunity directed against the infected hepatocyte. Based on extensive sequence homology, expression pattern, and antigenic cross-reactivity, the Plasmodium falciparum homolog of PyHEP17 is identified as the protein known as exported protein-1 (PfExp-1), also called antigen 5.1, circumsporozoite related antigen, or QF116. Identity between PyHEP17 and PfExp-1 is 37% at the amino acid level (60/161 residues), mapping primarily to two regions within the second exon of 73% (16/22 residues) and 71% (25/35 residues) identity. On this basis, PfExp-1 is proposed as an important component of pre-erythrocytic human malaria vaccines.

Circumventing genetic restriction of protection against malaria with multigene DNA immunization: CD8+ cell-, interferon gamma-, and nitric oxide-dependent immunity.

Despite efforts to develop vaccines that protect against malaria by inducing CD8+ T cells that kill infected hepatocytes, no subunit vaccine has been shown to circumvent the genetic restriction inherent in this approach, and little is known about the interaction of subunit vaccine-induced immune effectors and infected hepatocytes. We now report that immunization with plasmid DNA encoding the plasmodium yoelii circumsporozoite protein protected one of five strains of mice against malaria (H-2d, 75%); a PyHEP17 DNA vaccine protected three of the five strains (H-2a, 71%; H-2k, 54%; H-2d, 26%); and the combination protected 82% of H-2a, 90% of H-2k, and 88% of H-2d mice. Protection was absolutely dependent on CD8+ T cells, INF-gamma, or nitric oxide. These data introduce a new target of protective preerythrocytic immune responses, PyHEP 17 and its P. falciparum homologue, and provide a realistic perspective on the opportunities and challenges inherent in developing malaria vaccines that target the infected hepatocyte.

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.

Cloning and characterization of the vacuolar ATPase B subunit from Plasmodium falciparum

The transvacuolar pH gradient determines, to a significant extent, the distribution of the antimalarial drug chloroquine in Plasmodium falciparum. A proton pump, similar to the vacuolar ATPase found in many cell types, appears to regulate a pH gradient across the membranes of acidic compartments of the parasite. In order to understand and define the components involved in the maintenance of the vacuolar pH gradient, we have cloned and characterized a gene, designated VAP B, encoding a P. falciparum homologue of the B subunit of the vacuolar ATPase. The VAP B gene encodes a protein of 494 amino acids which has between 69% and 74% amino acid identity with the sequences of vacuolar ATPase B subunits of other organisms. The VAP B gene exists as a single copy gene on chromosome 4 that gives rise to a RNA transcript of 2.4 kb. Antibodies raised to the VAP B protein react specifically with a protein of 56-kDa, consistent with the size predicted from the gene sequence and with the homologous protein from other organisms. The 56-kDa protein is expressed throughout the asexual life cycle and subcellular localization by indirect immunofluorescence shows that the protein has a heterogenous distribution over most of the parasite. This suggests that the function of the vacuolar proton ATPase is not confined to the regulation of the pH of the digestive vacuole.

Cloning and characterization of a vacuolar ATPase A subunit homologue from Plasmodium falciparum

The distribution of the antimalarial drug chloroquine is determined to a significant extent by a transvacuolar pH gradient in Plasmodium falciparum. A proton pump similar to the vacuolar ATPase found in many cell types has been suggested to maintain a pH gradient across the membranes of acidic compartments in the parasite. In order to understand and define the components involved in the mechanism of acidification of parasite vesicles, we have cloned and characterized a gene, designated VAP-A, encoding a P. falciparum homologue of the catalytic A subunit of the vacuolar ATPase. The VAP-A gene encodes a polypeptide of 611 amino acids which shows between 56 to 61% amino acid identity over its entire length with the sequences of vacuolar ATPase A subunits from several species. The VAP-A gene exists as a single copy gene on P. falciparum chromosome 13 and gives rise to a transcript of 3.7 kb. Antibodies raised against a VAP-A gene segment expressed in Escherichia coli react specifically with a 67-kDa polypeptide, consistent with the size predicted from the sequence and with the size of the corresponding polypeptide in other organisms. The 67-kDa protein is present throughout the asexual erythrocytic cycle and is expressed at similar levels in 5 P. falciparum isolates of differing chloroquine sensitivity. Sequence analysis of the coding region of the VAP-A gene from 2-chloroquine-sensitive and 3 chloroquine-resistant isolates has shown no changes that are linked to chloroquine resistance. Therefore, a proposed chloroquine resistance-linked vacuolar acidification defect does not involve mutations in the VAP-A gene in the isolates we have studied.

Characterization of Plasmodium falciparum sporozoite surface protein 2.

Immunization of mice with Plasmodium yoelii sporozoite surface protein 2 (PySSP2) and circumsporozoite protein protects completely against P. yoelii. The amino acid sequence of PySSP2 suggested that the thrombospondin-related anonymous protein (TRAP) [Robson, K. J. H., Hall, J. R. S., Jennings, M. W., Harris, T. J. R., Marsh, K., Newbold, C. I., Tate, V. E. & Weatherall, D. J. (1988) Nature (London) 335, 79-82] is the Plasmodium falciparum homolog of PySSP2. We report data confirming that TRAP is P. falciparum SSP2 (PfSSP2). Murine antibodies against recombinant PfSSP2 identify a 90-kDa protein in extracts of P. falciparum sporozoites, recognize sporozoites and infected hepatocytes by immunofluorescence, localize PfSSP2 to the sporozoite micronemes by immunoelectron microscopy and to the surface membrane by live immunofluorescence, and inhibit sporozoite invasion and development in hepatocytes in vitro. Human volunteers immunized with irradiated sporozoites and protected against malaria develop antibody and proliferative T-cell responses to PfSSP2, suggesting that, like PySSP2, PfSSP2 is a target of protective immunity, and supporting inclusion of PfSSP2 in a multicomponent malaria vaccine.

A P-glycoprotein homologue of Plasmodium falciparum is localized on the digestive vacuole.

Resistance to chloroquine in Plasmodium falciparum bears a striking similarity to the multi-drug resistance (MDR) phenotype of mammalian tumor cells which is mediated by overexpression of P-glycoprotein. We show here that the P. falciparum homologue of the P-glycoprotein (Pgh1) is a 160,000-D protein that is expressed throughout the asexual erythrocytic life cycle of the parasite. Quantitative immunoblotting analysis has shown that the protein is expressed at approximately equal levels in chloroquine resistant and sensitive isolates suggesting that overexpression of Pgh1 is not essential for chloroquine resistance. The chloroquine-resistant cloned line FAC8 however, does express approximately threefold more Pgh1 protein than other isolates which is most likely because of the increased pfmdr1 gene copy number present in this isolate. Immunofluorescence and immunoelectron microscopy has demonstrated that Pgh1 is localized on the membrane of the digestive vacuole of mature parasites. This subcellular localization suggests that Pgh1 may modulate intracellular chloroquine concentrations and has important implications for the normal physiological function of this protein.

Amplification of the multidrug resistance gene in some chloroquine-resistant isolates of P. falciparum

Resistance of Plasmodium falciparum to chloroquine shares features with the multidrug resistance (MDR) phenotype of mammalian tumor cells. We report here the sequence of pfmdr, the P. falciparum homolog of mdr. We show that pfmdr is amplified in some chloroquine-resistant parasites but not in any of the sensitive isolates examined and that pfmdr transcript levels are increased. The gene is located on chromosome 5, and in one chloroquine-resistant line with an amplified pfmdr gene, chromosome 5 is greatly enlarged. The chromosome heterogeneity is due to varying copy numbers of different-sized pfmdr-containing amplicons. The existence of an mdr gene in P. falciparum and its amplification in some chloroquine-resistant lines greatly adds to the circumstantial evidence that pfmdr mediates chloroquine resistance in these lines.