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PubMed2001

Serial analysis of gene expression in Plasmodium falciparum reveals the global expression profile of erythrocytic stages and the presence of anti-sense transcripts in the malarial parasite.

Serial analysis of gene expression (SAGE) was applied to the malarial parasite Plasmodium falciparum to characterize the comprehensive transcriptional profile of erythrocytic stages. A SAGE library of approximately 8335 tags representing 4866 different genes was generated from 3D7 strain parasites. Basic local alignment search tool analysis of high abundance SAGE tags revealed that a majority (88%) corresponded to 3D7 sequence, and despite the low complexity of the genome, 70% of these highly abundant tags matched unique loci. Characterization of these suggested the major metabolic pathways that are used by the organism under normal culture conditions. Furthermore several tags expressed at high abundance (30% of tags matching to unique loci of the 3D7 genome) were derived from previously uncharacterized open reading frames, demonstrating the use of SAGE in genome annotation. The open platform "profiling" nature of SAGE also lead to the important discovery of a novel transcriptional phenomenon in the malarial pathogen: a significant number of highly abundant tags that were derived from annotated genes (17%) corresponded to antisense transcripts. These SAGE data were validated by two independent means, strand specific reverse transcription-polymerase chain reaction and Northern analysis, where antisense messages were detected in both asexual and sexual stages. This finding has implications for transcriptional regulation of Plasmodium gene expression.

Animals↗
PubMed2005

A Plasmodium actin-depolymerizing factor that binds exclusively to actin monomers.

ADF/cofilins (AC) are essential F- and G-actin binding proteins that modulate microfilament turnover. The genome of Plasmodium falciparum, the parasite causing malaria, contains two members of the AC family. Interestingly, P. falciparum ADF1 lacks the F-actin binding residues of the AC consensus. Reverse genetics in the rodent malaria model system suggest that ADF1 performs vital functions during the pathogenic red blood cell stages, whereas ADF2 is not present in these stages. We show that recombinant PfADF1 interacts with monomeric actin but does not bind to actin polymers. Although other AC proteins inhibit nucleotide exchange on monomeric actin, the Plasmodium ortholog stimulates nucleotide exchange. Thus, PfADF1 differs in its biochemical properties from previously known AC proteins and seems to promote turnover exclusively by interaction with actin monomers. These findings provide important insights into the low cytosolic abundance and unique turnover characteristics of actin polymers in parasites of the phylum Apicomplexa.

Actins↗
PubMed1987

A microprocessor-based control unit for Plasmodium falciparum culture apparatus.

We describe a microprocessor-based control unit for apparatus which effects changes of culture medium on Plasmodium falciparum parasites maintained in vitro. The unit has required minimal attention during the two years it has been in operation and it has markedly facilitated the production of mature sexual stages of Plasmodium falciparum on a routine basis.

Algorithms↗
PubMed1997

Plasmodium activates the innate immune response of Anopheles gambiae mosquitoes.

Innate immune-related gene expression in the major disease vector mosquito Anopheles gambiae has been analyzed following infection by the malaria parasite, Plasmodium berghei. Substantially increased levels of mRNAs encoding the antibacterial peptide defensin and a putative Gram-negative bacteria-binding protein (GNBP) are observed 20-30 h after ingestion of an infected blood-meal, at a time which indicates that this induction is a response to parasite invasion of the midgut epithelium. The induction is dependent upon the ingestion of infective, sexual-stage parasites, and is not due to opportunistic co-penetration of resident gut micro-organisms into the hemocoel. The response is activated following infection both locally (in the midgut) and systemically (in remaining tissues, presumably fat body and/or hemocytes). The observation that Plasmodium can trigger a molecularly defined immune response in the vector constitutes an important advance in our understanding of parasite-vector interactions that are potentially involved in malaria transmission, and extends knowledge of the innate immune system of insects to encompass responses to protozoan parasites.

Acute-Phase Proteins↗
PubMed1999

Guanylyl cyclases with the topology of mammalian adenylyl cyclases and an N-terminal P-type ATPase-like domain in Paramecium, Tetrahymena and Plasmodium.

We cloned a guanylyl cyclase of 280 kDa from the ciliate Paramecium which has an N-terminus similar to that of a P-type ATPase and a C-terminus with a topology identical to mammalian adenylyl cyclases. Respective signature sequence motifs are conserved in both domains. The cytosolic catalytic C1a and C2a segments of the cyclase are inverted. Genes coding for topologically identical proteins with substantial sequence similarities have been cloned from Tetrahymena and were detected in sequences from Plasmodium deposited by the Malaria Genome Project. After 99 point mutations to convert the Paramecium TAA/TAG-Gln triplets to CAA/CAG, together with partial gene synthesis, the gene from Paramecium was heterologously expressed. In Sf9 cells, the holoenzyme is proteolytically processed into the two domains. Immunocytochemistry demonstrates expression of the protein in Paramecium and localizes it to cell surface membranes. The data provide a novel structural link between class III adenylyl and guanylyl cyclases and imply that the protozoan guanylyl cyclases evolved from an ancestral adenylyl cyclase independently of the mammalian guanylyl cyclase isoforms. Further, signal transmission in Ciliophora (Paramecium, Tetrahymena) and in the most important endoparasitic phylum Apicomplexa (Plasmodium) is, quite unexpectedly, closely related.

Amino Acid Sequence↗
PubMed1999

The A-domain and the thrombospondin-related motif of Plasmodium falciparum TRAP are implicated in the invasion process of mosquito salivary glands.

Sporozoites from all Plasmodium species analysed so far express the thrombospondin-related adhesive protein (TRAP), which contains two distinct adhesive domains. These domains share sequence and structural homology with von Willebrand factor type A-domain and the type I repeat of human thrombospondin (TSP). Increasing experimental evidence indicates that the adhesive domains bind to vertebrate host ligands and that TRAP is involved, through an as yet unknown mechanism, in the process of sporozoite motility and invasion of both mosquito salivary gland and host hepatocytes. We have generated transgenic P.berghei parasites in which the endogenous TRAP gene has been replaced by either P.falciparum TRAP (PfTRAP) or mutated versions of PfTRAP carrying amino acid substitutions or deletions in the adhesive domains. Plasmodium berghei sporozoites carrying the PfTRAP gene develop normally, are motile, invade mosquito salivary glands and infect the vertebrate host. A substitution in a conserved residue of the A-domain or a deletion in the TSP motif of PfTRAP impairs the sporozoites' ability to invade mosquito salivary glands. Notably, midgut sporozoites from these transgenic parasites are still able to infect mice. Midgut sporozoites carrying a mutation in the A-domain of PfTRAP are motile, while no gliding motility could be detected in sporozoites with a TSP motif deletion.

Animals↗
PubMed2002

Levels of circumsporozoite protein in the Plasmodium oocyst determine sporozoite morphology.

The sporozoite stage of the Plasmodium parasite is formed by budding from a multinucleate oocyst in the mosquito midgut. During their life, sporozoites must infect the salivary glands of the mosquito vector and the liver of the mammalian host; both events depend on the major sporozoite surface protein, the circumsporozoite protein (CS). We previously reported that Plasmodium berghei oocysts in which the CS gene is inactivated do not form sporozoites. Here, we analyzed the ultrastructure of P.berghei oocyst differentiation in the wild type, recombinants that do not produce or produce reduced amounts of CS, and corresponding complemented clones. The results indicate that CS is essential for establishing polarity in the oocyst. The amounts of CS protein correlate with the extent of development of the inner membranes and associated microtubules underneath the oocyst outer membrane, which normally demarcate focal budding sites. This is a first example of a protein controlling both morphogenesis and infectivity of a parasite stage.

Animals↗
PubMed2003

Dynamics of molecular markers linked to the resistance loci in a mosquito-Plasmodium system.

Models on the evolution of resistance to parasitism generally assume fitness tradeoffs between the costs of being parasitized and the costs associated with resistance. This study tested this assumption using the yellow fever mosquito Aedes aegypti and malaria parasite Plasmodium gallinaceum system. Experimental mosquito populations were created by mixing susceptible and resistant strains in equal proportions, and then the dynamics of markers linked to loci for Plasmodium resistance and other unlinked neutral markers were determined over 12 generations. We found that when the mixed population was maintained under parasite-free conditions, the frequencies of alleles specific to the susceptible strain at markers closely linked to the loci for resistance (QTL markers) as well as other unlinked markers increased significantly in the first generation and then fluctuated around equilibrium frequencies for all six markers. However, when the mixed population was exposed to an infected blood meal every generation, allele frequencies at the QTL markers for resistance were not significantly changed. Small population size caused significant random fluctuations of allele frequencies at all marker loci. Consistent allele frequency changes in the QTL markers and other unlinked markers suggest that the reduced fitness in the resistant population has a genome-wide effect on the genetic makeup of the mixed population. Continuous exposure to parasites promoted the maintenance of alleles from the resistant Moyo-R strain in the mixed population. The results are discussed in relation to the proposed malaria control strategy through genetic disruption of vector competence.

Aedes↗
PubMed1994

A population-based clinical trial with the SPf66 synthetic Plasmodium falciparum malaria vaccine in Venezuela.

A phase III malaria vaccine trial in 13 villages in an endemic area, South Venezuela, compared incidence rates of Plasmodium falciparum and Plasmodium vivax infections in 1422 vaccinated and 938 nonvaccinated subjects over 18 months. The SPf66 vaccine was given in three doses, on days 0, 20, and 112. Vaccination was complete in 976 subjects (68.7%). Minor side effects requiring no treatment were reported by 123 (12.6%), with an apparent increase in frequency from the first to the third vaccine dose. No autoimmune evidence was observed in a sample of subjects. Antibodies against SPf66 were present at low titers in 24.7% of tested subjects before vaccination, increasing to 53.6% after the second dose and to 73.6% after the third dose; 26.4% of subjects initially seronegative never seroconverted. The SPf66 malaria vaccine showed a protective efficacy of 55% (95% confidence interval, 21%-75%) against P. falciparum and of 41% (19%-57%) against P. vivax malaria.

Adolescent↗
PubMed1997

Evidence for CD4+ T cell responses common to Plasmodium falciparum and recall antigens.

In individual donors which have never been exposed to malaria parasites, the CD4+ T cell precursor frequencies for tetanus toxoid (TT) and Plasmodium falciparum responses are similar (range 1:850-1:4800). Limiting dilution cultures set up in response to P. falciparum trophozoites can be re-stimulated with the same stage of the parasite or TT and respond with similar frequencies. A substantial overlap in the responses to different agents was confirmed in suicide selection experiments where cells responding to malaria parasite, TT or influenza virus antigens were deleted using the cell cycle inhibitor cytosine arabinoside (Ara-C). The responses of the remaining cells to P. falciparum were almost completely abrogated and only weak responses were observed to different recall antigens (0.2-21% of untreated control). Little or no effect was observed on the responses to superantigen or mitogen. Furthermore, in contrast to superantigen, the observed responses to TT and Plasmodium were polyclonal, the blastoid cells generated reacting with a range of anti-TCR Vbeta antibodies with little preferential usage.

Adult↗
PubMed1990

Effect of Plasmodium falciparum on the survival of naturally infected afrotropical Anopheles (Diptera: Culicidae).

The effect of the malarial parasite, Plasmodium falciparum Welch, on the daily survival rates and longevity of Anopheles gambiae Giles sensu lato and Anopheles funestus Giles was determined for wild-caught, naturally infected females from western Kenya. Mosquitoes were collected inside houses and held in cages until death, after which they were assayed for P. falciparum circumsporozoite protein by an enzyme-linked immunosorbent assay (ELISA). Survival rates of field populations determined by parity rates were significantly higher than survival rates estimated by regression for Anopheles dying in cages. Overall, An. gambiae s.l. had a significantly higher daily mortality rate (means = 17.5%) than An. funestrus (means = 13.2%). P. falciparum ELISA infection rates, which were higher for An. gambiae s.l. (means = 19.8%; n = 1,221) than for An. funestus (means = 11.9%; n = 1,128), did not increase as a function of time for caged Anopheles. For An. gambiae s.l., there was a significant negative correlation between holding time and P. falciparum ELISA absorbance, suggesting that detectable circumsporozoite protein and perhaps the number of sporozoites may decrease with time in the vector. In western Kenya, an area where Anopheles populations often have extremely high malaria infection rates. Plasmodium infections did not reduce vector survivorship.

Animals↗
PubMed1991

Plasmodium species identification by ELISA for sporozoites removed from dried dissection slides.

This study tested the feasibility of identifying salavary gland sporozoites to species by Plasmodium falciparum ELISA by drying them on slides or in vials. The glands were dissected from Anopheles gambiae Giles s.l. and An. funestus Giles collected in western Kenya. In 119 gland infections containing a geometric mean of 1,222 sporozoites, a mean of 72.5% of sporozoites were removed in 60 microliters saline from slides at the time of dissection. Each of the 119 samples was divided into three 18 microliters aliquots. Subsamples were stored at -70 degrees C, dried in vials, or dried on a microslide. When tested by Plasmodium falciparum ELISA, positive reactions were observed in 86.6% of frozen samples, 70.6% of samples held dry in vials, and 50.4% of samples held dry on microslides for 1 mo. Of 90 gland infections where coverslips were removed and slides were left to dry for 1 mo before adding blocking buffer, 81.1% were positive for P. falciparum. This was not significantly different from either frozen gland samples (where 85.5% of 392 infections were identified or frozen gland plus corresponding thorax samples where 86.2% of 160 samples were identified). In malaria field studies, where it is not always practical to freeze samples, sporozoites from dissected mosquitoes can be preserved adequately for ELISA identification by simply removing coverslips and drying dissection slides.

Animals↗
PubMed2005

Asymptomatic carriers of Plasmodium spp. as infection source for malaria vector mosquitoes in the Brazilian Amazon.

We have described the existence of asymptomatic carriers of Plasmodium vivax and Plasmodium falciparum infections in native Amazon populations. Most of them had low parasitemias, detected only by polymerase chain reaction (PCR). Because they remain symptomless and untreated, we wanted to determine whether they could infect Anopheles darlingi Root, the main Brazilian vector, and act as disease reservoirs. Fifteen adult asymptomatic patients (PCR positive only) were selected, and experimental infections of mosquitoes were performed by direct feeding and by a membrane-feeding system. Seventeen adult symptomatic patients with high parasitemias were used as controls. We found an infection rate in An. darlingi of 1.2% for the asymptomatic carriers and 22% for the symptomatic carriers. Although the asymptomatic group infected mosquitoes at a much lower rate, these patients remain infective longer than treated, symptomatic patients. Also, the prevalence of asymptomatic infections is 4 to 5 times higher than symptomatic infections among natives. These results have implications for the malaria control program in Brazil, which focuses essentially on the treatment of symptomatic patients.

Animals↗
PubMed1985

Organization of Plasmodium falciparum genome: 1. Evidence for a highly repeated DNA sequence.

Plasmodium falciparum DNA, isolated from the merozoite stage, was cleaved with HindIII and cloned in pBR322 and lambda L47.1 vectors. Plasmid clones containing 13.4, 7.0, 4.3, 4.1 and 1.5 kb inserts were characterized in some detail. The inserts contain several repeating units of smaller size. Nucleic acid hybridization studies showed that the repeat element is present in the Plasmodium DNA at a very high copy number and appears to be distributed widely throughout the genome.

Animals↗
PubMed1992

Extensive turnover of telomeric DNA at a Plasmodium berghei chromosomal extremity marked by a rare recombinational event.

The dynamics of telomere turnover were studied in Plasmodium, whose telomeric structures consist of linear, recognisable sequences of two distinct repeats (TTTAGGG and TTCAGGG). Independent recombinant clones containing a well-defined chromosomal extremity of Plasmodium berghei, both before and after a rare insertion event took place, were obtained from clonal parasite populations and analysed. The insertion, which splits the original telomere and causes a significant reduction in the size of the telomeric structure, is shown to consist of an integer number of subtelomeric repeats typical of P.berghei, flanked on both sides by telomere-derived motifs. Analysis of the telomeric repeat sequence heterogeneity in the otherwise homogeneous populations examined, is compatible with a model in which diversification of a given telomere is driven by the occurrence of breakpoints whose frequency rapidly increases along the telomeric tract when moving in the outward direction. The breakpoints might be due either to terminal deletions followed by random serial addition of the two repeat versions, or to recombination events. The shortening/elongation mechanism is favoured against the recombination hypothesis because of the absence of higher-order patterns in the sequence of telomeric repeats.

Animals↗
PubMed1993

The gene encoding DNA polymerase alpha from Plasmodium falciparum.

The gene encoding DNA polymerase alpha from the human malaria parasite Plasmodium falciparum has been sequenced and characterised. The deduced amino acid sequence possesses the seven sequence motifs which characterise eukaryotic replicative DNA polymerases (I-VII) and four of five motifs (A-E) identified in alpha DNA polymerases. The predicted protein also contains sequences which are reminiscent of Plasmodium proteins but absent from other DNA polymerases. These include four blocks of additional amino acids interspersed with the conserved motifs of the DNA polymerases, four asparagine rich sequences and a novel carboxy-terminal extension. Repetitive sequences similar to those found in other malarial proteins are also present. cDNA-directed PCR was used to establish the presence of these features in the approximately 7kb mRNA. The coding sequence contains a single intron. The gene for DNAPol alpha is located on chromosome 4 and is transcribed in both asexual and sexual erythrocytic stages of the parasite.

Amino Acid Sequence↗
PubMed1996

Molecular cloning of a Plasmodium falciparum gene interrupted by 15 introns encoding a functional primase 53 kDa subunit as demonstrated by expression in a baculovirus system.

The gene encoding the primase small subunit was isolated from genomic DNA of strain K1 of the human malarial parasite Plasmodium falciparum. Isolation of a complete cDNA clone revealed the presence of 15 introns in the genomic sequence. This is unprecedented for Plasmodium genes, which usually contain no or only 1 or 2 introns. The gene is present as a single copy and the cDNA contains an open reading frame of 1356 nt encoding a protein of 452 amino acids. A single mRNA of 2.1 kb was identified by Northern blotting. Comparison of the amino acid sequence with five eukaryotic small primase subunits revealed the presence of eight conserved regions. Sequence alignments allowed the identification of putative motifs A, B and C that are essential features of the catalytic centre of DNA polymerases, RNA polymerases and reverse transcriptases. Also, similarity of a C-terminal region of approximately 100 amino acids to a conserved region in herpes virus primases, alpha-like DNA polymerases and RNA polymerase II was noted. The complete gene was expressed as a fusion product containing an N-terminal polyhistidine tag using a baculovirus expression vector. The protein was overproduced in insect cells and purified. Activity assays demonstrated the ability of the p53 subunit to initiate de novo primer formation.

Amino Acid Sequence↗
PubMed2002

Transcripts of developmentally regulated Plasmodium falciparum genes quantified by real-time RT-PCR.

Plasmodium falciparum intraerythrocytic development is a complex process. Development proceeds rapidly from the trophozoite phase of nutrient acquisition and growth through to the synthetic and reproductive schizont phase, which ends with production of new invasive merozoites. During this process, the malaria parasite must express a series of different gene products, depending on its metabolic and synthetic needs. We are particularly interested in the development of the merozoite's organelles in the apical complex, which form during the later schizont stages. We have used quantitative real-time RT-PCR fluorogenic 5' nuclease assays (TaqMan) for the first time on malaria parasites for analysis of erythrocytic stage-specific gene expression. We analyzed transcripts of the P.falciparum eba-175 and other erythrocyte binding-like (ebl) family genes in temperature-synchronized parasites and found ebl genes have tightly controlled, stage-specific transcription. As expected, eba-175 transcripts were abundant only at the end of schizont development in a pattern most common among ebl, including baebl, pebl and jesebl. The maebl transcript pattern was unique, peaking at mid-late trophozoite stage, but absent in late-stage schizonts. ebl-1 demonstrated another pattern of expression, which peaked during mid-schizont stage and then significantly diminished in late-stage schizonts. Our analysis demonstrates that using real-time RT-PCR fluorogenic 5' nuclease assays is a sensitive, quantitative method for analysis of Plasmodium transcripts.

Animals↗