Effect of thioredoxin peroxidase-1 gene disruption on the liver stages of the rodent malaria parasite Plasmodium berghei.

Phenotypic observation of thioredoxin peroxidase-1 (TPx-1) gene-disrupted Plasmodium berghei (TPx-1 KO) in the liver-stage was performed with an in vitro infection system in order to investigate defective liver-stage development in a mouse infection model. Indirect immunofluorescence microscopy assay with anti-circumsporozoite protein antibody revealed that in the liver schizont stage, TPx-1 KO parasite cells were significantly smaller than cells of the wild-type parent strain (WT). Indirect immunofluorescence microscopy assay with anti-merozoite surface protein-1 antibody, which was used to evaluate late schizont-stage development, indicated that TPx-1 KO schizont development was similar to WT strain development towards the merozoite-forming stage (mature schizont). However, fewer merozoites were produced in the mature TPx-1 KO schizont than in the mature WT schizont. Taken together, the results suggest that TPx-1 may be involved in merozoite formation during liver schizont development.

Magnetic isolation of Plasmodium falciparum schizonts iRBCs to generate a high parasitaemia and synchronized in vitro culture.

BACKGROUND: The establishment of methods for an in vitro continuous culture of Plasmodium falciparum is essential for gaining knowledge into its biology and for the development of new treatments. Previously, several techniques have been used to synchronize, enrich and concentrate P. falciparum, although obtaining cultures with high parasitaemia continues being a challenging process. Current methods produce high parasitaemia levels of synchronized P. falciparum cultures by frequent changes of culture medium or reducing the haematocrit. However, these methods are time consuming and sometimes lead to the loss of synchrony. METHODS: A procedure that combines Percoll and sorbitol treatments, the use of magnetic columns, and the optimization of the in vitro culture conditions to reach high parasitaemia levels for synchronized Plasmodium falciparum cultures is described. RESULTS: A new procedure has been established using P. falciparum 3D7, combining previous reported methodologies to achieve in vitro parasite cultures that reach parasitaemia up to 40% at any intra-erythrocytic stage. High parasitaemia levels are obtained only one day after magnetic column purification without compromising the parasite viability and synchrony. CONCLUSIONS: The described procedure allows obtaining a large scale synchronized parasite culture at a high parasitaemia with less manipulations than other methods previously described.

Targeting Plasmodium PI(4)K to eliminate malaria.

Achieving the goal of malaria elimination will depend on targeting Plasmodium pathways essential across all life stages. Here we identify a lipid kinase, phosphatidylinositol-4-OH kinase (PI(4)K), as the target of imidazopyrazines, a new antimalarial compound class that inhibits the intracellular development of multiple Plasmodium species at each stage of infection in the vertebrate host. Imidazopyrazines demonstrate potent preventive, therapeutic, and transmission-blocking activity in rodent malaria models, are active against blood-stage field isolates of the major human pathogens P. falciparum and P. vivax, and inhibit liver-stage hypnozoites in the simian parasite P. cynomolgi. We show that imidazopyrazines exert their effect through inhibitory interaction with the ATP-binding pocket of PI(4)K, altering the intracellular distribution of phosphatidylinositol-4-phosphate. Collectively, our data define PI(4)K as a key Plasmodium vulnerability, opening up new avenues of target-based discovery to identify drugs with an ideal activity profile for the prevention, treatment and elimination of malaria.

Plasmodium berghei MAPK1 displays differential and dynamic subcellular localizations during liver stage development.

Mitogen-activated protein kinases (MAPKs) regulate key signaling events in eukaryotic cells. In the genomes of protozoan Plasmodium parasites, the causative agents of malaria, two genes encoding kinases with significant homology to other eukaryotic MAPKs have been identified (mapk1, mapk2). In this work, we show that both genes are transcribed during Plasmodium berghei liver stage development, and analyze expression and subcellular localization of the PbMAPK1 protein in liver stage parasites. Live cell imaging of transgenic parasites expressing GFP-tagged PbMAPK1 revealed a nuclear localization of PbMAPK1 in the early schizont stage mediated by nuclear localization signals in the C-terminal domain. In contrast, a distinct localization of PbMAPK1 in comma/ring-shaped structures in proximity to the parasite's nuclei and the invaginating parasite membrane was observed during the cytomere stage of parasite development as well as in immature blood stage schizonts. The PbMAPK1 localization was found to be independent of integrity of a motif putatively involved in ATP binding, integrity of the putative activation motif and the presence of a predicted coiled-coil domain in the C-terminal domain. Although PbMAPK1 knock out parasites showed normal liver stage development, the kinase may still fulfill a dual function in both schizogony and merogony of liver stage parasites regulated by its dynamic and stage-dependent subcellular localization.

Features of autophagic cell death in Plasmodium liver-stage parasites.

Analyzing molecular determinants of Plasmodium parasite cell death is a promising approach for exploring new avenues in the fight against malaria. Three major forms of cell death (apoptosis, necrosis and autophagic cell death) have been described in multicellular organisms but which cell death processes exist in protozoa is still a matter of debate. Here we suggest that all three types of cell death occur in Plasmodium liver-stage parasites. Whereas typical molecular markers for apoptosis and necrosis have not been found in the genome of Plasmodium parasites, we identified genes coding for putative autophagy-marker proteins and thus concentrated on autophagic cell death. We characterized the Plasmodium berghei homolog of the prominent autophagy marker protein Atg8/LC3 and found that it localized to the apicoplast. A relocalization of PbAtg8 to autophagosome-like vesicles or vacuoles that appear in dying parasites was not, however, observed. This strongly suggests that the function of this protein in liver-stage parasites is restricted to apicoplast biology.

Novel RuvB nuclear ATPase is specific to intraerythrocytic mitosis during schizogony of Plasmodium falciparum.

RuvB protein belongs to AAA+ family of enzymes involved in diverse cellular activities. In addition to the annotated two RuvB proteins in Plasmodium falciparum database, we report that a third RuvB protein is also present. The amino acid sequence analysis has revealed that P. falciparum RuvB3 (PfRuvB3) possesses Walker motif A, Walker motif B, sensor I and sensor II conserved motifs similar to yeast and human RuvB like proteins. The phylogenetic analysis revealed that PfRuvB3 is closely related to yeast RuvB like proteins which are essential for the survival of yeast. The biochemical characterization of recombinant PfRuvB3 confirms its ssDNA dependent ATPase activity. Using the truncated derivatives we show that Walker motif A is essential for the enzymatic activity of PfRuvB3. Using the immunodepletion assays we further show that the ATPase activity is attributable to PfRuvB3 protein. The endogenous P. falciparum RuvB3 contains the characteristic ATPase and some DNA helicase activities. The confocal microscopy analysis showed that this protein is mainly expressed during intraerythrocytic schizont stages of the parasite and is localized to the nuclear region. Once merozoite comes out from schizont, PfRuvB3 protein distinctly relocalized to the subnuclear region. The co-localization studies with a nucleolar marker PfNop1 further suggest that in P. falciparum RuvB3 localizes into a discrete nuclear compartment. On the basis of these studies it can be speculated that P. falciparum RuvB3 is most likely required for intraerythrocytic schizogony.

Novel putative glycosylphosphatidylinositol-anchored micronemal antigen of Plasmodium falciparum that binds to erythrocytes.

We have identified a new Plasmodium falciparum erythrocyte binding protein that appears to be located in the micronemes of the merozoite stage of the parasite and membrane linked through a glycosylphosphatidylinositol (GPI) anchor. The protein is designated GPI-anchored micronemal antigen (GAMA) and was identified by applying a set of selection criteria to identify previously uncharacterized merozoite proteins that may have a role in cell invasion. The protein is also present in the proteomes of the sporozoite and ookinete micronemes and is conserved throughout the genus. GAMA contains a novel domain that may be constrained by disulfide bonds and a predicted C-terminal hydrophobic sequence that is presumably replaced by the GPI. The protein is synthesized late during schizogony, processed into two fragments that are linked by a disulfide bond, and translocated to an apical location, which is probably the micronemes. In a proportion of free merozoites GAMA can also be detected on the parasite surface. Following erythrocyte invasion the bulk of the protein is shed in a soluble form, although a short C-terminal fragment may be carried into the newly invaded red blood cell. The protein was shown to bind reversibly to erythrocytes and therefore represents a new example of a host cell binding protein.