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PubMed1989

Subtelomeric chromosome deletions in field isolates of Plasmodium falciparum and their relationship to loss of cytoadherence in vitro.

Subtelomeric deletions are responsible for the loss of expression of several Plasmodium falciparum antigens, including the knob-associated histidine-rich protein (KAHRP). Such deletions are detectable by two-dimensional pulsed-field gradient electrophoresis (PFGE) in which the chromosomes separated in dimension 1 are cleaved with Apa I, and the sizes of telomeric fragments are determined in dimension 2. This sensitive technique has enabled us to examine the role of subtelomeric deletions in two aspects of the biology of Plasmodium falciparum. First, we show that similar subtelomeric deletions to those that occur in vitro also occur in field isolates. Second, we demonstrate a correlation between subtelomeric deletions and loss of the phenotype of "cytoadherence" in cultured isolates. Subclones were generated from the cytoadherent cloned isolate ItG2F6, and their phenotypes were examined with respect to cytoadherence, the expression of "knobs," and agglutination of infected erythrocytes with rabbit antiserum. The only chromosomal change detectable by two-dimensional PFGE among subclones that differ from wild type in each of these three characteristics is a deletion of approximately 100 kilobases at one end of chromosome 2. This deletion includes the gene coding for KAHRP and the subtelomeric repeat designated rep20.

Agglutination↗
PubMed1991

Human cytotoxic T lymphocytes against the Plasmodium falciparum circumsporozoite protein.

Cytotoxic T lymphocytes (CTL) against the circumsporozoite (CS) protein of malaria sporozoites protect against malaria in rodents. Although there is interest in developing human vaccines that induce CTL against the Plasmodium falciparum CS protein, humans have never been shown to produce CTL against any Plasmodium species protein or other parasite protein. We report that when peripheral blood mononuclear cells (PBMC) from three of four volunteers immunized with irradiated P. falciparum sporozoites were stimulated in vitro with a recombinant vaccinia virus expressing the P. falciparum CS protein or a peptide including only amino acids 368-390 of the P. falciparum CS protein [CS-(368-390)], the PBMC lysed autologous Epstein-Barr virus-transformed B cells transfected with the P. falciparum CS protein gene or incubated with CS-(368-390) tricosapeptide. Activity was antigen specific, genetically restricted, and dependent on CD8+ T cells. In one volunteer, seven peptides reflecting amino acids 311-400 were tested, and, as in B10.BR mice, CTL activity was only associated with the CS-(368-390) peptide. Development of an assay for studying human CTL against the CS and other malaria proteins and a method for constructing target cells by direct gene transfection provide a foundation for studying the role of CTL in protection against malaria.

Amino Acid Sequence↗
PubMed1992

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.

Amino Acid Sequence↗
PubMed1996

Trafficking of Plasmodium chabaudi adami-infected erythrocytes within the mouse spleen.

Plasmodium chabaudi adami causes a nonlethal infection in mice. We found that crisis, the time of rapidly dropping parasitemia, was abrogated by splenectomy, indicating the role of spleen in parasite killing. The factors that mediate spleen-dependent immunity are not known. An earlier study in Plasmodium berghei-infected rats showed an association between increased clearance of heat-treated erythrocytes and the onset of crisis [Wyler, D. J., Quinn, T. C. & Chen, L.-T. (1982) J. Clin. Invest. 67, 1400-1404]. To determine the potential effects of different vascular beds in parasite killing, we studied the distribution of parasitized erythrocytes and bacteria in the spleens of P. chabaudi adami-infected mice during precrisis (a period of rising parasitemia) and during crisis. After intravenous injection, bacteria were localized predominantly in the marginal zone. In contrast, parasitized erythrocytes were found in the red pulp. We also found that during precrisis, a time of no immunity, the uptake of radiolabeled infected erythrocytes by the spleen was increased, not decreased. These data imply that no change occurs in the flow of parasitized erythrocytes through the spleen during the transition to an immune state (crisis). Our observations suggest that immune effector mechanisms, not circulatory changes, account for spleen-dependent parasite killing during a P. chabaudi adami infection in mice.

Animals↗
PubMed1997

A novel alternate secretory pathway for the export of Plasmodium proteins into the host erythrocyte.

The malarial parasite dramatically alters its host cell by exporting and targeting proteins to specific locations within the erythrocyte. Little is known about the mechanisms by which the parasite is able to carry out this extraparasite transport. The fungal metabolite brefeldin A (BFA) has been used to study the secretory pathway in eukaryotes. BFA treatment of infected erythrocytes inhibits protein export and results in the accumulation of exported Plasmodium proteins into a compartment that is at the parasite periphery. Parasite proteins that are normally localized to the erythrocyte membrane, to nonmembrane bound inclusions in the erythrocyte cytoplasm, or to the parasitophorous vacuolar membrane accumulate in this BFA-induced compartment. A single BFA-induced compartment is detected per parasite and the various exported proteins colocalize to this compartment regardless of their final destinations. Parasite membrane proteins do not accumulate in this novel compartment, but accumulate in the endoplasmic reticulum (ER), suggesting that the parasite has two secretory pathways. This alternate secretory pathway is established immediately after merozoite invasion and at least some dense granule proteins also use the alternate pathway. The BFA-induced compartment exhibits properties that are similar to the ER, but it is clearly distinct from the ER. We propose to call this new organelle the secondary ER of apicomplexa. This ER-like organelle is an early, if not the first, step in the export of Plasmodium proteins into the host erythrocyte.

Animals↗
PubMed1998

Nuclear-encoded proteins target to the plastid in Toxoplasma gondii and Plasmodium falciparum.

A vestigial, nonphotosynthetic plastid has been identified recently in protozoan parasites of the phylum Apicomplexa. The apicomplexan plastid, or "apicoplast," is indispensable, but the complete sequence of both the Plasmodium falciparum and Toxoplasma gondii apicoplast genomes has offered no clue as to what essential metabolic function(s) this organelle might perform in parasites. To investigate possible functions of the apicoplast, we sought to identify nuclear-encoded genes whose products are targeted to the apicoplast in Plasmodium and Toxoplasma. We describe here nuclear genes encoding ribosomal proteins S9 and L28 and the fatty acid biosynthetic enzymes acyl carrier protein (ACP), beta-ketoacyl-ACP synthase III (FabH), and beta-hydroxyacyl-ACP dehydratase (FabZ). These genes show high similarity to plastid homologues, and immunolocalization of S9 and ACP verifies that the proteins accumulate in the plastid. All the putatively apicoplast-targeted proteins bear N-terminal presequences consistent with plastid targeting, and the ACP presequence is shown to be sufficient to target a recombinant green fluorescent protein reporter to the apicoplast in transgenic T. gondii. Localization of ACP, and very probably FabH and FabZ, in the apicoplast implicates fatty acid biosynthesis as a likely function of the apicoplast. Moreover, inhibition of P. falciparum growth by thiolactomycin, an inhibitor of FabH, indicates a vital role for apicoplast fatty acid biosynthesis. Because the fatty acid biosynthesis genes identified here are of a plastid/bacterial type, and distinct from those of the equivalent pathway in animals, fatty acid biosynthesis is potentially an excellent target for therapeutics directed against malaria, toxoplasmosis, and other apicomplexan-mediated diseases.

Animals↗
PubMed1999

A set of independent selectable markers for transfection of the human malaria parasite Plasmodium falciparum.

Genomic information is rapidly accumulating for the human malaria pathogen, Plasmodium falciparum. Our ability to perform genetic manipulations to understand Plasmodium gene function is limited. Dihydrofolate reductase is the only selectable marker presently available for transfection of P. falciparum. Additional markers are needed for complementation and for expression of mutated forms of essential genes. We tested parasite sensitivity to different drugs for which selectable markers are available. Two of these drugs that were very effective as antiplasmodial inhibitors in culture, blasticidin and geneticin (G418), were selected for further study. The genes BSD, encoding blasticidin S deaminase of Aspergillus terreus, and NEO, encoding neomycin phosphotransferase II from transposon Tn 5, were expressed under the histidine-rich protein III (HRPIII) gene promoter and tested for their ability to confer resistance to blasticidin or G418, respectively. After transfection, blasticidin and G418-resistant parasites tested positive for plasmid replication and BSD or NEO expression. Cross-resistance assays indicate that these markers are independent. The plasmid copy number and the enzymatic activity depended directly on the concentration of the drug used for selection. These markers set the stage for new methods of functional analysis of the P. falciparum genome.

Aminohydrolases↗
PubMed1999

Emergence of FY*A(null) in a Plasmodium vivax-endemic region of Papua New Guinea.

In Papua New Guinea (PNG), numerous blood group polymorphisms and hemoglobinopathies characterize the human population. Human genetic polymorphisms of this nature are common in malarious regions, and all four human malaria parasites are holoendemic below 1500 meters in PNG. At this elevation, a prominent condition characterizing Melanesians is alpha(+)-thalassemia. Interestingly, recent epidemiological surveys have demonstrated that alpha(+)-thalassemia is associated with increased susceptibility to uncomplicated malaria among young children. It is further proposed that alpha(+)-thalassemia may facilitate so-called "benign" Plasmodium vivax infection to act later in life as a "natural vaccine" against severe Plasmodium falciparum malaria. Here, in a P. vivax-endemic region of PNG where the resident Abelam-speaking population is characterized by a frequency of alpha(+)-thalassemia >/=0.98, we have discovered the mutation responsible for erythrocyte Duffy antigen-negativity (Fy[a-b-]) on the FY*A allele. In this study population there were 23 heterozygous and no homozygous individuals bearing this new allele (allele frequency, 23/1062 = 0.022). Flow cytometric analysis illustrated a 2-fold difference in erythroid-specific Fy-antigen expression between heterozygous (FY*A/FY*A(null)) and homozygous (FY*A/FY*A) individuals, suggesting a gene-dosage effect. In further comparisons, we observed a higher prevalence of P. vivax infection in FY*A/FY*A (83/508 = 0.163) compared with FY*A/FY*A(null) (2/23 = 0.087) individuals (odds ratio = 2.05, 95% confidence interval = 0.47-8.91). Emergence of FY*A(null) in this population suggests that P. vivax is involved in selection of this erythroid polymorphism. This mutation would ultimately compromise alpha(+)-thalassemia/P. vivax-mediated protection against severe P. falciparum malaria.

Animals↗
PubMed1999

The chitinase PfCHT1 from the human malaria parasite Plasmodium falciparum lacks proenzyme and chitin-binding domains and displays unique substrate preferences.

Within hours after the ingestion of a blood meal, the mosquito midgut epithelium synthesizes a chitinous sac, the peritrophic matrix. Plasmodium ookinetes traverse the peritrophic matrix while escaping the mosquito midgut. Chitinases (EC 3.2.1.14) are critical for parasite invasion of the midgut: the presence of the chitinase inhibitor, allosamidin, in an infectious blood meal prevents oocyst development. A chitinase gene, PgCHT1, recently has been identified in the avian malaria parasite P. gallinaceum. We used the sequence of PgCHT1 to identify a P. falciparum chitinase gene, PfCHT1, in the P. falciparum genome database. PfCHT1 differs from PgCHT1 in that the P. falciparum gene lacks proenzyme and chitin-binding domains. PfCHT1 was expressed as an active recombinant enzyme in Escherichia coli. PfCHT1 shares with PgCHT1 a substrate preference unique to Plasmodium chitinases: the enzymes cleave tri- and tetramers of GlcNAc from penta- and hexameric oligomers and are unable to cleave smaller native chitin oligosaccharides. The pH activity profile of PfCHT1 and its IC(50) (40 nM) to allosamidin are distinct from endochitinase activities secreted by P. gallinaceum ookinetes. Homology modeling predicts that PgCHT1 has a novel pocket in the catalytic active site that PfCHT1 lacks, which may explain the differential sensitivity of PfCHT1 and PgCHT1 to allosamidin. PfCHT1 may be the ortholog of a second, as yet unidentified, chitinase gene of P. gallinaceum. These results may allow us to develop novel strategies of blocking human malaria transmission based on interfering with P. falciparum chitinase.

Acetylglucosamine↗
PubMed1996

The disulfide bond structure of Plasmodium apical membrane antigen-1.

Apical membrane antigen-1 (AMA-1) of Plasmodium falciparum is one of the leading asexual blood stage antigens being considered for inclusion in a malaria vaccine. The ability of this molecule to induce a protective immune response has been shown to be dependent upon a conformation stabilized by disulfide bonds. In this study we have utilized the reversed-phase high performance liquid chromatography of dithiothreitol-reduced and nonreduced tryptic digests of Plasmodium chabaudi AMA-1 secreted from baculovirus-infected insect cells, in conjunction with N-terminal sequencing and electrospray-ionization mass spectrometry, to identify and assign disulfide-linked peptides. All 16 cysteine residues that are conserved in all known sequences of AMA-1 are incorporated into intramolecular disulfide bonds. Six of the eight bonds have been assigned unequivocally, whereas the two unassigned disulfide bonds connect two Cys-Xaa-Cys sequences separated by 14 residues. The eight disulfide bonds fall into three nonoverlapping groups that define three possible subdomains within the AMA-1 ectodomain. Although the pattern of disulfide bonds within subdomain III has not been fully elucidated, one of only two possible linkage patterns closely resembles the cystine knot motif found in growth factors. Sites of amino acid substitutions in AMA-1 that are well separated in the primary sequence are clustered by the disulfide bonds in subdomains II and III. These findings are consistent with the conclusion that these amino acid substitutions are defining conformational disulfide bond-dependent epitopes that are recognized by protective immune responses.

Amino Acid Sequence↗
PubMed1997

Selective cytotoxicity of dermaseptin S3 toward intraerythrocytic Plasmodium falciparum and the underlying molecular basis.

The antimicrobial activity of various naturally occurring microbicidal peptides was reported to result from their interaction with microbial membrane. In this study, we investigated the cytotoxicity of the hemolytic peptide dermaseptin S4 (DS4) and the nonhemolytic peptide dermaseptin S3 (DS3) toward human erythrocytes infected by the malaria parasite Plasmodium falciparum. Both DS4 and DS3 inhibited the parasite's ability to incorporate [3H]hypoxanthine. However, while DS4 was toxic toward both the parasite and the host erythrocyte, DS3 was toxic only toward the intraerythrocytic parasite. To gain insight into the mechanism of this selective cytotoxicity, we labeled the peptides with fluorescent probes and investigated their organization in solution and in membranes. In Plasmodium-infected cells, rhodamine-labeled peptides interacted directly with the intracellular parasite, in contrast to noninfected cells, where the peptides remained bound to the erythrocyte plasma membrane. Binding experiments to phospholipid membranes revealed that DS3 and DS4 had similar binding characteristics. Membrane permeation studies indicated that the peptides were equally potent in permeating phosphatidylserine/phosphatidylcholine vesicles, whereas DS4 was more permeative with phosphatidylcholine vesicles. In aqueous solutions, DS4 was found to be in a higher aggregation state. Nevertheless, both DS3 and DS4 spontaneously dissociated to monomers upon interaction with vesicles, albeit with different kinetics. In light of these results, we propose a mechanism by which dermaseptins permeate cells and affect intraerythrocytic parasites.

Amino Acid Sequence↗
PubMed1997

Species-specific regulation and switching of transcription between stage-specific ribosomal RNA genes in Plasmodium berghei.

Malaria parasites (Plasmodium spp.) differentially express structurally distinct sets of rRNA genes in a stage-specific manner. The four rRNA genes of the rodent malaria parasite, P. berghei, form two classes of 2 units that are genetically unlinked and termed A-type and S-type. Through Northern analysis and in situ hybridization, expression of the units was demonstrated in synchronized parasite preparations covering the developmental pathway from the initiation of the blood-stage asexual cycle to the production of mature ookinetes. A-type units were transcribed in direct response to cell growth in bloodstage asexual parasites yet were differentially regulated during male (inactive) and female (active) gametocytogenesis. S-type expression was not confined solely to the mosquito stages and exhibited a finite period of expression in a subset of bloodstage trophozoites that was significantly elevated in gametocyte-producing parasites. Unlike in the human parasite, P. falciparum, there was no evidence for accumulation of precursor forms of the S-type transcripts in gametocytes. No significant rRNA transcription was observed in cultured, fertilized ookinetes until approximately 20 h of development when S-type transcription was initiated. The results further demonstrate that in Plasmodium the expression of the different rRNA units is linked to developmental progression but in a species-specific manner.

Animals↗
PubMed1998

Transport and metabolism of the essential vitamin pantothenic acid in human erythrocytes infected with the malaria parasite Plasmodium falciparum.

The growth of the human malaria parasite, Plasmodium falciparum, within its host erythrocyte is reliant on the uptake of a number of essential nutrients from the extracellular medium. One of these is pantothenic acid, a water-soluble vitamin that is a precursor of coenzyme A. In this study we show that normal uninfected erythrocytes are impermeable to pantothenate but that the vitamin is taken up rapidly into malaria-infected cells via a transport pathway that has the characteristics (furosemide sensitivity, nonsaturability) of previously characterized, broad specificity permeation pathways induced by the intracellular parasite in the host cell membrane. The transport of pantothenate therefore constitutes a critical physiological role for these pathways. Inside the parasitized cell pantothenate undergoes phosphorylation, the first step in its conversion to coenzyme A. Parasites within saponin-permeabilized erythrocytes were shown to take up and phosphorylate pantothenate, consistent with the intracellular parasite having both a pantothenate transporter and a pantothenate kinase. Comparisons of the rate of phosphorylation of pantothenate by lysates prepared from uninfected and infected erythrocytes revealed that the pantothenate kinase activity of the P. falciparum trophozoite is some 10-fold higher than that of its host cell and that most, if not all, of the phosphorylation of pantothenate within the malaria-infected cell occurs within the intracellular parasite. These results contrast with those of previous studies in which it was proposed that the avian malaria parasite Plasmodium lophurae lacks pantothenate kinase (as well as the other enzymes for the synthesis of coenzyme A) and is reliant upon the uptake of preformed coenzyme A from the host cell cytosol.

Animals↗
PubMed2000

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.

Amino Acid Sequence↗
PubMed2000

Chitinases of the avian malaria parasite Plasmodium gallinaceum, a class of enzymes necessary for parasite invasion of the mosquito midgut.

The Plasmodium ookinete produces chitinolytic activity that allows the parasite to penetrate the chitin-containing peritrophic matrix surrounding the blood meal in the mosquito midgut. Since the peritrophic matrix is a physical barrier that the parasite must cross to invade the mosquito, and the presence of allosamidin, a chitinase inhibitor, in a blood meal prevents the parasite from invading the midgut epithelium, chitinases (3.2.1.14) are potential targets of malaria parasite transmission-blocking interventions. We have purified a chitinase of the avian malaria parasite Plasmodium gallinaceum and cloned the gene, PgCHT1, encoding it. PgCHT1 encodes catalytic and substrate-binding sites characteristic of family 18 glycohydrolases. Expressed in Escherichia coli strain AD494 (DE3), recombinant PgCHT1 was found to hydrolyze polymeric chitin, native chitin oligosaccharides, and 4-methylumbelliferone derivatives of chitin oligosaccharides. Allosamidin inhibited recombinant PgCHT1 with an IC(50) of 7 microM and differentially inhibited two chromatographically separable P. gallinaceum ookinete-produced chitinase activities with IC(50) values of 7 and 12 microM, respectively. These two chitinase activities also had different pH activity profiles. These data suggest that the P. gallinaceum ookinete uses products of more than one chitinase gene to initiate mosquito midgut invasion.

Amino Acid Sequence↗
PubMed2001

Transition state analogue inhibitors of purine nucleoside phosphorylase from Plasmodium falciparum.

Immucillins are logically designed transition-state analogue inhibitors of mammalian purine nucleoside phosphorylase (PNP) that induce purine-less death of Plasmodium falciparum in cultured erythrocytes (Kicska, G. A., Tyler, P. C., Evans, G. B., Furneaux, R. H., Schramm, V. L., and Kim, K. (2002) J. Biol. Chem. 277, 3226-3231). PNP is present at high levels in human erythrocytes and in P. falciparum, but the Plasmodium enzyme has not been characterized. A search of the P. falciparum genome data base yielded an open reading frame similar to the PNP from Escherichia coli. PNP from P. falciparum (P. falciparum PNP) was cloned, overexpressed in E. coli, purified, and characterized. The primary amino acid sequence has 26% identity with E. coli PNP, has 20% identity with human PNP, and is phylogenetically unique among known PNPs with equal genetic distance between PNPs and uridine phosphorylases. Recombinant P. falciparum PNP is catalytically active for inosine and guanosine but is less active for uridine. The immucillins are powerful inhibitors of P. falciparum PNP. Immucillin-H is a slow onset tight binding inhibitor with a K(i)* value of 0.6 nm. Eight related immucillins are also powerful inhibitors with dissociation constants from 0.9 to 20 nm. The K(m)/K(i)* value for immucillin-H is 9000, making this inhibitor the most powerful yet reported for P. falciparum PNP. The PNP from P. falciparum differs from the human enzyme by a lower K(m) for inosine, decreased preference for deoxyguanosine, and reduced affinity for the immucillins, with the exception of 5'-deoxy-immucillin-H. These properties of P. falciparum PNP are consistent with a metabolic role in purine salvage and provide an explanation for the antibiotic effect of the immucillins on P. falciparum cultured in human erythrocytes.

Amino Acid Sequence↗
PubMed2001

Binding and invasion of liver cells by Plasmodium falciparum sporozoites. Essential involvement of the amino terminus of circumsporozoite protein.

Plasmodium sporozoites display circumsporozoite (CS) protein on their surface, which is involved in the attachment of sporozoites to liver cells. CS protein is a member of the thrombospondin type I repeat (TSR) domain family and possess a single copy of TSR domain toward its carboxyl terminus. We show by a direct measurement the correlation between the binding activity of various segments of the CS protein and their ability to inhibit the invasion of liver cells by the sporozoites. We made eight truncated versions of Plasmodium falciparum CS protein to elucidate the role of various regions in the binding and invasion process. Deletion of the TSR domain actually enhanced binding activity by 2-3-fold without the loss of receptor specificity, indicating that TSR may not be the only domain in defining the specificity of binding. These same deletions blocked invasion of live sporozoites more efficiently than proteins that include the TSR domain. Deletion of as little as six amino acids from amino terminus of the protein, however, renders it incapable of binding to liver cells and as an inhibitor of sporozoite invasion. Hence, the binding of CS protein to liver cells and its ability to inhibit the invasion process are affected in a parallel manner, both positively and negatively, by sequence changes in the encoded CS gene. This indicates that both assays are measuring interrelated phenomenon and points to the essential involvement for the amino-terminal portion of the CS protein in these processes.

Amino Acids↗
PubMed2002

Plasmodium falciparum possesses a cell cycle-regulated short type replication protein A large subunit encoded by an unusual transcript.

DNA replication in Plasmodium parasites takes place at multiple distinct points during their complex life cycle in the mosquito and vertebrate hosts. Although several parasite proteins involved in DNA replication have been described, the various mechanisms engaged in DNA metabolism of this major pathogen remain largely unexplored. As a step toward understanding this complex network, we describe the identification of Plasmodium falciparum replication protein A large subunit (pfRPA1) through affinity purification and mass spectral analysis of a purified 55-kDa factor. Gel retardation experiments revealed that pfRPA is the major single-stranded DNA binding activity in parasite protein extracts. The activity was expressed in a cell cycle-dependent manner with peak activities in late trophozoites and schizonts, thus correlating with the beginning of chromosomal DNA replication. Accordingly, the pfrpa1 message was detected in parasites 20-24 h post-invasion which is in agreement with the expression of other P. falciparum DNA replication genes. Our results show that pfRPA1 is encoded by an unusual 6.5-kb transcript containing a single open reading frame of which only the C-terminal 42% of the deduced protein sequence shows homologies to other reported RPA1s. Like the orthologues of other protozoan parasites, pfRPA1 lacks the N-terminal protein interaction domain and is thus remarkably smaller than the RPA1s of higher eukaryotes.

Amino Acid Sequence↗