Differential gene expression in the ookinete stage of the malaria parasite Plasmodium berghei

Plasmodium, the malaria parasite, undergoes a complex developmental program in its mosquito vector. The ookinete is the parasite form which invades the mosquito midgut and is an important stage for genetic mixing. To identify genes expressed during ookinete development and mosquito midgut invasion, purified zygotes and ookinetes of the rodent parasite Plasmodium berghei were used to construct a suppression subtractive hybridization cDNA library, enriched in sequences expressed in the ookinete stage. In addition to four genes coding for previously described major ookinete-secreted proteins, we isolated ookinete-expressed sequences representing 18 predicted genes. Their gene products include proteins involved in signal transduction and regulatory processes. For six of these genes our analysis provides the first evidence for expression in the ookinete stage. A majority of the genes are not expressed in the zygote, the preceding developmental stage. Furthermore, four of the genes are also transcribed in sporozoites, and one of these in merozoites, suggesting that they code for proteins with a function common to Plasmodium invasive stages.     

Gene targeting in the rodent malaria parasite Plasmodium yoelii

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

Cytokines inhibit the development of liver schizonts of the malaria parasite Plasmodium berghei in vivo.

The effect of induction of an acute-phase response and its mediators on the development of liver schizonts of the rodent malaria parasite Plasmodium berghei was investigated in Brown Norway rats. Subcutaneous injection of turpentine oil 24 h or 5 min before inoculation of sporozoites resulted in 80% and 35% reduction of schizont development, respectively. Turpentine oil induced high plasma levels of interleukin-6 (IL-6). Intraperitoneal administration of IL-1, IL-6 or both, significantly reduced liver schizont development. This reduction was also present if IL-6 had been administered 24 h after sporozoite inoculation. Inhibition induced by IL-1 could be prevented by simultaneous administration of polyclonal anti-IL-6. Administration of polyclonal anti-IL-6 without IL-1 resulted in a 40% increase of liver schizonts compared to control animals. We conclude that induction of an acute-phase response during experimental Plasmodium berghei infections in Brown Norway rats, strongly inhibits liver schizont development and that IL-6 is a key mediator in this process.     

Cytosolic protein kinase activity associated with the maturation of the malaria parasite Plasmodium berghei

Seven cytosolic phosphoproteins with relative molecular masses of 110, 58, 52, 46, 38, 36 and 34kDa and isoelectric points between 4.2 and 5.0 are identified from the rodent malaria parasite Plasmodium berghei. Similar patterns of phosphorylated proteins are obtained from parasite cytosol after incubation of intact infected erythrocytes with [32P]orthophosphate, or from parasite cytosol incubated with [gamma-32P]ATP. The characteristics of the phosphorylation reaction are similar to the previously described Plasmodium protein kinase [Wiser, M.F., Eaton, J.W. and Sheppard, J.R. (1983) J. Cell. Biochem. 21, 305-314], suggesting that the same protein kinase is involved. More protein phosphorylation activity is associated with the mature parasites than the immature forms, suggesting that these phosphoproteins may play some role in the parasite's erythrocytic stage cycle.     

Impairment of hepatic cytochrome P-450-dependent monooxygenases by the malaria parasite Plasmodium berghei

The effect of Plasmodium berghei infection on hepatic monooxygenase activities and cytochrome P-450 contents was investigated in mice. NIH/NMRI or A/J mice infected with active P. berghei showed 30-40% decreases in hepatic cytochrome P-450 contents and the ability to metabolize the test substrates, ethylmorphine and benzo(a)pyrene. These decreases were observed during the erythrocytic stage of the infection, but not during the initial exoerythrocytic stage, or after heat-inactivated sporozoites were injected. These results strongly suggest that malaria infections may significantly impair the capacity of the liver to metabolize drugs, carcinogens, and other foreign compounds.     

Improved transfection and new selectable markers for the rodent malaria parasite Plasmodium yoelii

The rodent malaria Plasmodium yoelii is a useful model to study protective immunity to pre-erythrocytic stages of infection, pathogenesis of erythrocytic stages, and vaccine development. However, the utility of the P. yoelii model system has not been fully realized because transfection and genetic manipulation methodologies for this rodent species are less developed than that of another rodent species Plasmodium berghei. Here we report improved transfection efficiency using the AMAXA nucleofector system compared to conventional transfection methodologies. We also show that heterologous promoters from P. berghei can be used to drive expression of a green fluorescent protein (GFP) reporter protein in P. yoelii. In an effort to develop additional selectable markers for this parasite, we also tested positive selectable markers that have been used successfully in P. falciparum and P. berghei. Human dihydrofolate reductase (hdhfr) and Toxoplasma gondii dihydrofolate reductase-thymidylate synthase (Tgdhfr-ts) conferred drug resistance to WR99210 and pyrimethamine, respectively, when introduced as episomes. These improvements should make genetic manipulation of P. yoelii more amenable and facilitate further studies of host-parasite interactions using this attractive rodent model.     

Chronic, patent Plasmodium berghei malaria in splenectomized mice.

It was shown that splenectomized mice could develop a certain resistance to Plasmodium berghei but usually no real immunity, since the infection became chronic, often with high parasitemias. A patent infection lasting at least 2 weeks was necessary for the development of this degree of protection. Prolonged suppression to subpatent levels (sulfonamide treatment), rather than radical cure (chloroquine), after 2 weeks of patency yielded a higher proportion of mice resistant to superinfection. An increasing proportion of B10LP, but not C57BL/Rij or BALB/c, mice cleared their chronic infection spontaneously in time. Chronic patent infections were accompanied by anemia, elevated serum glutamate oxaloacetate transaminase levels, indicating liver pathology, and decreased immune reactivity, but the magnitude to these pathological changes was limited compared with changes in primary, lethal infections in intact controls. Parasitemia and pathology did not always develop synchronously.     

Molecular karyotyping of the rodent malarias Plasmodium chabaudi, Plasmodium berghei and Plasmodium vinckei

The molecular karyotypes of four isolates of Plasmodium chabaudi, three of the subspecies P. chabaudi adami and one P. chabaudi chabaudi, as well as P. berghei and P. vinckei were studied by means of pulsed field gradient (PFG) gel electrophoresis. Each species appears to have 14 chromosomes, ranging in size from approximately 730 kb to greater than 2000 kb. The three P. chabaudi adami isolates did not appear any more similar to each other than to the P. c. chabaudi isolate. The chromosome locations of genes for a heat shock protein (hsp) 70, ribosomal RNA (rRNA), the precursor to the major merozoite surface proteins, dihydrofolate reductase and P. falciparum antigen 352 as well as four cloned DNA markers and a telomere probe were determined. However, a number of probes representing cloned P. falciparum antigens failed to hybridize to P. chabaudi DNA. Hence genes for malaria antigens appear to be much more divergent than genes for housekeeping functions.     

TNF and Plasmodium berghei ANKA-induced cerebral malaria

The cerebral pathology observed in Plasmodium berghei ANKA-infected CBA mice has been attributed to overproduction of TNF, the mice in which this syndrome is seen being those with the highest serum TNF levels. To investigate this further, we injected recombinant human TNF into malaria-primed mice to see if we could reproduce the cerebral changes observed in P. berghei ANKA infections. A range of doses, administered as a single or repeated injections, or via osmotic pumps, failed to reproduce these changes, but did induce hypoglycaemia, midzonal liver necrosis and neutrophil adhesion in pulmonary vessels. This pathology is seen in terminal Plasmodium vinckei infections, but absent in terminal P. berghei ANKA. In addition, the permeability of the blood-brain barrier to Evan's blue, which is present in P. berghei ANKA but not in normal or P. vinckei-infected mice, was not induced by exogenous TNF. Serum levels of TNF were measured in an ELISA assay, and found to be consistently higher in P. vinckei rather than P. berghei ANKA terminal infections. This is consistent with the pathological changes we could reproduce by injecting TNF. For these reasons we suggest that the cerebral pathology seen in mice infected with P. berghei ANKA may be governed by TNF produced locally by monocytes sequestered within the cerebral blood vessels, not simply by systemic levels of this cytokine.     

In vitro inhibition of liver forms of the rodent malaria parasite Plasmodium berghei by naphthylisoquinoline alkaloids – structure-activity relationships of dioncophyllines A and C and ancistrocladine

Naphthylisoquinoline alkaloids are derived from Dioncophyllaceae and Ancistrocladaceae species and comprise a new class of promising antimalarials with a demonstrated potential against asexual erythrocytic Plasmodium falciparum and P. berghei stages in vitro. We report herein the pronounced activity of pure naphthylisoquinoline alkaloids against exoerythrocytic malaria parasites. P. berghei-infected human hepatoma cells (Hep G2) were incubated with culture medium containing selected alkaloids at 10 μg/ml. The most active compounds, showing inhibitory activity of more than 40%, were dioncophylline A (compound 1), dioncophyllacine A (compound 6), and ancistrobarterine A (compound 12). For structure-activity investigations of dioncophyllines A (compound 1) and C (compound 3) and ancistrocladine (compound 7) a selection of their analogs from natural or synthetic sources was examined. Dioncophylleine A (compound 16), 5′-O-demethyl-8-O-methyl-7-epi-dioncophylline A (compound 17), N-formyl-8-O-methyl-dioncophylline C (compound 21), and N-formyl-8-O-benzoyldioncophylline C (compound 24) were found to display high levels of activity as well, although the former two compounds caused damage to the host-cell monolayers. As naphthylisoquinoline alkaloids are also highly active against blood forms of Plasmodium spp., they should be regarded as lead compounds for further development as drugs against erythrocytic and exoerythrocytic stages of Plasmodium spp. Received: 28 January 1997 / Accepted: 17 March 1997     

Mixed strain infections and strain-specific protective immunity in the rodent malaria parasite Plasmodium chabaudi chabaudi in mice

Important to malaria vaccine design is the phenomenon of "strain-specific" immunity. Using an accurate and sensitive assay of parasite genotype, real-time quantitative PCR, we have investigated protective immunity against mixed infections of genetically distinct cloned "strains" of the rodent malaria parasite Plasmodium chabaudi chabaudi in mice. Four strains of P. c. chabaudi, AS, AJ, AQ, and CB, were studied. One round of blood infection and drug cure with a single strain resulted in a partial reduction in parasitemia, compared with levels for na?ve mice, in challenge infections with mixed inocula of the immunizing (homologous) strain and a heterologous strain. In all cases, the numbers of blood-stage parasites of each genotype were reduced to similar degrees. After a second, homologous round of infection and drug cure followed by challenge with homologous and heterologous strains, the parasitemias were reduced even further. In these circumstances, moreover, the homologous strain was reduced much faster than the heterologous strain in all of the combinations tested. That the immunity induced by a single infection did not show "strain specificity," while the immunity following a second, homologous infection did, suggests that the "strain-specific" component of protective immunity in malaria may be dependent upon immune memory. The results show that strong, protective immunity induced by and effective against malaria parasites from a single parasite species has a significant "strain-specific" component and that this immunity operates differentially against genetically distinct parasites within the same infection.     

Gametocytogenesis and ribosomal rRNA gene organisation in the rodent malarias Plasmodium chabaudi and Plasmodium berghei

A cloned Plasmodium berghei (ANKA) isolate was syringe passaged repeatedly to generate a line that was non-infective to Anopheles stephensi. Ribosomal gene organisation of this non-infective line was then compared to its infective ancestor. DNA was also prepared from asexual parasites and gametocytes of P. chabaudi and the arrangement of the rRNA genes of this species was studied. Although macrogametocytes have many more ribosomes than microgametocytes, this increase does not appear to stem from an amplification of the rRNA genes, as no differences either in the quantity or the arrangement of the rDNA could be detected. Furthermore, the loss of infectivity of the P. berghei gametocytes does not seem to be due to a reduction or rearrangement of sequences coding for the rRNA genes. P. chabaudi and P. berghei DNA failed to show any homology to a repetitive DNA sequence cloned from P. falciparum. We conclude that this probe, PFH8rep20, is specific for P. falciparum.     

Calcium regulation in the intraerythrocytic malaria parasite Plasmodium falciparum

The regulation of intracellular Ca(2+) in the intraerythrocytic form of the human malaria parasite, Plasmodium falciparum, was investigated using parasites 'isolated' from their host cells by saponin-permeabilisation of the erythrocyte membrane. The isolated parasites maintained tight control over their resting cytosolic Ca(2+) concentration which ranged from approximately 100 nM in the absence of extracellular Ca(2+) to approximately 700 nM in the presence of 1 mM extracellular Ca(2+). The parasite has two functionally discrete intracellular Ca(2+) stores. One is an 'endoplasmic reticulum (ER)-like' store, the other an 'acidic store'. The ER-like store was discharged by cyclopiazonic acid (CPA), an inhibitor of sarco/endoplasmic reticulum Ca(2+)-ATPases (SERCAs) of animal and plant cells, but not by thapsigargin (TG), a more specific inhibitor of SERCAs of animal cells. The acidic store was discharged by nigericin and by NH(4)(+). The amount of Ca(2+) in the ER-like store increased with increasing extracellular Ca(2+) concentration, whereas the amount of Ca(2+) in the acidic store did not. Ca(2+) released from the ER-like store by CPA was cleared from the parasite cytosol by uptake into the acidic store (over a range of extracellular Ca(2+) concentrations), consistent with the acidic store serving as a Ca(2+) reservoir within the intracellular parasite.     

Polymorphism in the circumsporozoite protein of the human malaria parasite Plasmodium vivax.

The circumsporozoite (CS) protein that covers the surface of infectious sporozoites is a candidate antigen in malaria vaccine development. To determine the extent of B- and T-epitope polymorphism and to understand the mechanisms of antigenic variability, we have characterized the CS protein gene of Plasmodium vivax from field isolates representing geographically distant regions of Papua New Guinea (PNG) and Brazil. In the central repeat region of the CS protein, in addition to variation in the number of repeats, an array of mutations was observed which suggests that point mutations have led to the emergence of the variant CS repeat sequence ANGA(G/D)(N/D)QPG from GDRA(D/A)GQPA. Outside the repeat region of the protein, the nonsilent nucleotide substitutions of independent origin are localized in three domains of the protein that either harbor known T-cell determinants or are analogous to the Plasmodium falciparum immunodominant determinants, Th2R and Th3R. We have found that, with the exception of one CS clone sequence that was shared by one P. vivax isolate each from PNG and Brazil, the P. vivax CS protein types can be grouped into Papuan and Brazilian types. These results suggest that an in-depth study of parasite population dynamics is required before field trials for vaccine formulation based on polymorphic immunodominant determinants are conducted.