FLP/FRT-mediated conditional mutagenesis in pre-erythrocytic stages of Plasmodium berghei

We describe here a highly efficient procedure for conditional mutagenesis in Plasmodium. The procedure uses the site-specific recombination FLP-FRT system of yeast and targets the pre-erythrocytic stages of the rodent Plasmodium parasite P. berghei, including the sporozoite stage and the subsequent liver stage. The technique consists of replacing the gene under study by an FRTed copy (i.e., flanked by FRT sites) in the erythrocytic stages of a parasite clone that expresses the flip (FLP) recombinase stage-specifically--called the 'deleter' clone. We present the available deleter clones, which express FLP at different times of the parasite life cycle, as well as the schemes and tools for constructing new deleter parasites. We also outline and discuss the various strategies for exchanging a wild-type gene with an FRTed copy and for generating conditional gene knockout or knockdown parasite clones. Finally, we detail the protocol for obtaining sporozoites that lack a protein of interest and for monitoring sporozoite-specific DNA excision and depletion of the target protein. The protocol should allow the functional analysis of any essential protein in the sporozoite, liver stage or hepatic merozoite stages of rodent Plasmodium parasites.     

FRET peptides reveal differential proteolytic activation in intraerythrocytic stages of the malaria parasites Plasmodium berghei and Plasmodium yoelii

Malaria is still a major health problem in developing countries. It is caused by the protist parasite Plasmodium, in which proteases are activated during the cell cycle. Ca(2+) is a ubiquitous signalling ion that appears to regulate protease activity through changes in its intracellular concentration. Proteases are crucial to Plasmodium development, but the role of Ca(2+) in their activity is not fully understood. Here we investigated the role of Ca(2+) in protease modulation among rodent Plasmodium spp. Using fluorescence resonance energy transfer (FRET) peptides, we verified protease activity elicited by Ca(2+) from the endoplasmatic reticulum (ER) after stimulation with thapsigargin (a sarco/endoplasmatic reticulum Ca(2+)-ATPase (SERCA) inhibitor) and from acidic compartments by stimulation with nigericin (a K(+)/H(+) exchanger) or monensin (a Na(+)/H(+) exchanger). Intracellular (BAPTA/AM) and extracellular (EGTA) Ca(2+) chelators were used to investigate the role played by Ca(2+) in protease activation. In Plasmodium berghei both EGTA and BAPTA blocked protease activation, whilst in Plasmodium yoelii these compounds caused protease activation. The effects of protease inhibitors on thapsigargin-induced proteolysis also differed between the species. Pepstatin A and phenylmethylsulphonyl fluoride (PMSF) increased thapsigargin-induced proteolysis in P. berghei but decreased it in P. yoelii. Conversely, E64 reduced proteolysis in P. berghei but stimulated it in P. yoelii. The data point out key differences in proteolytic responses to Ca(2+) between species of Plasmodium.     

Dendritic cells subsets mediated immune response during Plasmodium berghei ANKA and Plasmodium yoelii infection

The roles of dendritic cells (DCs) in mediating immunity against Plasmodium infection have been extensively investigated, but immune response during pathogenesis of malaria is still poorly understood. In the present study, we compared the splenic DCs phenotype and function during P. berghei ANKA (PbA) or P. yoelii (P. yoelii) infection in Swiss mice. We observed that PbA-infected mice developed more myeloid and mature DCs capable of secreting IL-12, while P. yoelii-infected mice had more plasmacytoid and immature DCs secreting higher levels of IL-10. Expression of FoxP3, IL-17, TGF-β and IL-6 were also different between these two infections. Thus, these results suggest that the phenotypic and functional subsets of splenic DCs are key factors for regulating immune responses to PbA and P. yoelii infections.     

Aminopeptidases from Plasmodium falciparum, Plasmodium chabaudi chabaudi and Plasmodium berghei

ABSTRACT. Using fluorogenic substrates and polyacrylamide gels we detected in cell-free extracts of Plasmodium falciparum, Plasmodium chabaudi chabaudi and Plasmodium berghei only a single aminopeptidase. A comparative study of the aminopeptidase activity in each extract revealed that the enzymes have similar specificities and kinetics, a near-neutral pH optima of 7.2 and are moderately thermophilic. Each has an apparent molecular weight of 80,000 ± 10,000, determined by high performance liquid chromatography on a calibrated SW500 column. Whilst the P. c. chabaudi and P. berghei activity co-migrate in native polyacrylamide gels, that of P. falciparum migrates more slowly. The three enzymes can be selectively inhibited by ortho -phenanthroline and are thus metallo-aminopeptidases; however, in contrast to other aminopeptidases the metal co-factor does not appear to be Zn²⁺.     

Mitochondrial NADH dehydrogenase from Plasmodium falciparum and Plasmodium berghei

The mitochondrial electron transport system is necessary for growth and survival of malarial parasites in mammalian host cells. NADH dehydrogenase of respiratory complex I was demonstrated in isolated mitochondrial organelles of the human parasite Plasmodium falciparum and the mouse parasite Plasmodium berghei by using the specific inhibitor rotenone on oxygen consumption and enzyme activity. It was partially purified by two sequential steps of fast protein liquid chromatographic techniques from n-octyl glucoside solubilization of the isolated mitochondria of both parasites. In addition, physical and kinetic properties of the malarial enzymes were compared to the host mouse liver mitochondrial respiratory complex I either as intact or as partially purified forms. The malarial enzyme required both NADH and ubiquinone for maximal catalysis. Furthermore, rotenone and plumbagin (ubiquinone analog) showed strong inhibitory effect against the purified malarial enzymes and had antimalarial activity against in vitro growth of P. falciparum. Some unique properties suggest that the enzyme could be exploited as chemotherapeutic target for drug development, and it may have physiological significance in the mitochondrial metabolism of the parasite.     

Fine structure of exoerythrocytic merozoite formation of Plasmodium berghei in rat liver

The fine structure of exoerythrocytic merogony of Plasmodium berghei was studied after perfusion-fixation of rat livers from 51 h post-inoculation onwards. Meroblast formation was effected by clefts originating from the parasite plasmalemma and by fusion of vacuoles with each other. Invaginations at the periphery resulted in labyrinthine structures providing the parasites with an enormous increase in surface area, which might facilitate exchange of metabolites. When the parasitophorous vacuole membrane collapsed, the newly formed merozoites were lying free in the hepatocytic cytoplasm, which degenerated until the merozoites were sticking together by a stroma, obviously a remnant of the host hepatocyte. Groups of merozoites, still kept together by the spongy stroma, were subsequently released in the bloodstream. At 53 h most of the developmental stages leading to the release of merozoites could be found and thereafter parasite numbers decreased while large granulomas became apparent.     

Enhanced detection of Plasmodium vivax liver stages by cytocentrifugation

Malaria parasites circulating in the blood or developing in the mosquito host can easily be seen and studied. We know much less about malaria parasites in the liver not only because of their location, but also because there are so few of them. Hepatic parasites are most often grown within hepatoma cells in culture, stained and visualized on slides under direct microscopy. In this report, Chitraporn Karnasuta and George Watt investigate the potential of cytocentrifugation as a tool for improving the detection of liver-stage malaria parasites.     

IFN-gamma inhibits development of Plasmodium berghei exoerythrocytic stages in hepatocytes by an L-arginine-dependent effector mechanism

Primary cultures of BALB/cJ hepatocytes treated with 10(3) U/ml rIFN-gamma consistently inhibited intracellular Plasmodium berghei liver schizont development by 50 to 70%. Monomethyl-L-arginine (NGMMLA), the competitive inhibitor of L-arginine as substrate for production of nitric oxides by hepatocytes, reversed the activity of IFN-gamma on these malaria-infected cells. Reversal of IFN-gamma activity by NGMMLA was dose dependent and was maximal at 0.5 mM NGMMLA. Depletion of L-arginine by addition of arginase to the culture medium blocked the capacity of IFN-gamma to inhibit parasite development in hepatocytes; addition of excess L-arginine to cultures treated with IFN-gamma in the presence of NGMMLA competitively restored IFN-gamma capacity to activate hepatocyte anti-parasite activity. TNF-alpha was neither required for IFN-gamma activity, nor effective at any concentration tested as an inhibitor of schizont development by itself in primary hepatocytes. These data strongly suggest that the action of IFN-gamma on P. berghei-infected hepatocytes is to induce the production of L-arginine-derived nitrogen oxides that are toxic for the intracellular parasite.     

Mitochondrial lipoic acid scavenging is essential for Plasmodium berghei liver stage development

Lipoic acid is an essential cofactor for enzymes that participate in key metabolic pathways in most organisms. While in mammalian cells lipoylated proteins reside exclusively in the mitochondria, apicomplexan parasites of the genus Plasmodium harbour two independent lipoylation pathways in the mitochondrion and the apicoplast, a second organelle of endosymbiotic origin. Protein lipoylation in the apicoplast relies on de novo lipoic acid synthesis while lipoylation of proteins in the mitochondrion depends on scavenging of lipoic acid from the host cell. Here, we analyse the impact of lipoic acid scavenging on the development of Plasmodium berghei liver stage parasites. Treatment of P. berghei-infected HepG2 cells with the lipoic acid analogue 8-bromo-octanoic acid (8-BOA) abolished lipoylation of mitochondrial enzyme complexes in the parasite while lipoylation of apicoplast proteins was not affected. Parasite growth as well as the ability of the parasites to successfully complete liver stage development by merosome formation were severely impaired but not completely blocked by 8-BOA. Liver stage parasites were most sensitive to 8-BOA treatment during schizogony, the phase of development when the parasite grows and undergoes extensive nuclear division to form a multinucleated syncytium. Live cell imaging as well as immunofluorescence analysis and electronmicroscopy studies revealed a close association of both host cell and parasite mitochondria with the parasitophorous vacuole membrane suggesting that host cell mitochondria might be involved in lipoic acid uptake by the parasite from the host cell.