Merozoite Proteins Discovered by qRT-PCR-Based Transcriptome Screening of Plasmodium falciparum

The development of malaria vaccines and medicines depends on the discovery of novel malaria protein targets, but the functions of more than 40% of P. falciparum genes remain unknown. Asexual parasites are the critical stage that leads to serious clinical symptoms and that can be modulated by malaria treatments and vaccines. To identify critical genes involved in the development of Plasmodium parasites within erythrocytes, the expression profile of more than 5,000 genes distributed across the 14 chromosomes of the PF3D7 strain during its six critical developmental stages (merozoite, early-ring, late-ring, early trophozoite, late-trophozoite, and middle-schizont) was evaluated. Hence, a qRT-PCR-based transcriptome of the erythrocytic developmental process of P. falciparum was revealed. Weighted gene coexpression network analyses revealed that a large number of genes are upregulated during the merozoite release process. Further gene ontology analysis revealed that a cluster of genes is associated with merozoite and may be apical complex components. Among these genes, 135 were comprised within chromosome 14, and 80% of them were previously unknown in functions. Western blot and immunofluorescence assays using newly developed corresponding antibodies showed that some of these newly discovered proteins are highly expressed in merozoites. Further invasion inhibition assays revealed that specific antibodies against several novel merozoite proteins can interfere with parasite invasion. Taken together, our study provides a developmental transcriptome of the asexual parasites of P. falciparum and identifies a group of previously unknown merozoite proteins that may play important roles in the process of merozoite invasion.

Analysis of Spleen Histopathology, Splenocyte composition and Hematological Parameters in Mice Infected with Plasmodium berghei K173

ABSTRACTMalaria is a fatal disease that presents clinically as a continuum of symptoms and severity, which are determined by complex host-parasite interactions. Clearance of infection is believed to be accomplished by the spleen and mononuclear phagocytic system (MPS), both in the presence and absence of artemisinin treatment. The spleen filters infected RBCs from circulation through immune-mediated recognition of the infected RBCs followed by phagocytosis. Using different strains of mice infected with P. berghei K173 (PbK173), the mechanisms leading to splenomegaly, histopathology, splenocyte activation and proliferation, and their relationship to control of parasitemia and host mortality were examined. Survival time of mice infected with PbK173 varied, although the infection was uniformly lethal. Mice of the C57BL/6 strain were the most resistant, while mice of the strain ICR were the most susceptible. BALB/c and KM mice were intermediate. In the course of PbK173 infection, both strains of mice experienced significant splenomegaly. Parasites were observed in the red pulp at 3 days post infection in all animals. All spleens retained late trophozoite stages as well as a fraction of earlier ring-stage parasites. The percentages of macrophages in infected C57BL/6 and KM mice were higher than uninfected mice on 8 dpi. Spleens of infected ICR and KM mice exhibited structural disorganization and remodeling. Furthermore, parasitemia was significantly higher in KM versus C57BL/6 mice at 8 dpi. The percentages of macrophages in ICR infected mice were lower than uninfected mice, and the parasitemia was higher than other strains. The results presented here demonstrate the rate of splenic mechanical filtration and the splenic macrophages likely contribute to an individual’s total parasite burden. This in turn can influence the pathogenesis of malaria. Finally, different genetic backgrounds of mice have different splenic mechanisms for controlling malaria infection.

The pseudogene SURFIN 4.1 is vital for merozoite formation in blood stage P. falciparum

The surf gene family of the human malaria parasite Plasmodium falciparum encodes for antigens with largely unknown functions. Three of the ten surf genes found in the P. falciparum 3D7 genome are annotated as pseudogenes, and one of these – surf4.1 (PF3D7_0402200) - was continuously transcribed in P. falciparum 3D7 blood stage forms. GFP-tagging revealed that despite several stop codons a full-length protein was expressed, which localized to developing merozoites. Analysis of cDNAs showed that no specific editing occurred pointing to readthrough of stop codons during translation. Intriguingly, attempts to generate parasite lines containing an additional artificial stop codon failed. Transcript knockdown revealed that surf4.1 is essential for merozoite formation in late trophozoite/schizont stages while DNA replication seemed not to be influenced. SURFIN4.1 is the first example of a plasmodial multigene family member of which a knockout is deleterious and may pose as a novel target for anti-malarial therapy.

Mechanism-Based Model of Parasite Growth and Dihydroartemisinin Pharmacodynamics in Murine Malaria

ABSTRACTMurine models are used to study erythrocytic stages of malaria infection, because parasite morphology and development are comparable to those in human malaria infections. Mechanism-based pharmacokinetic-pharmacodynamic (PK-PD) models for antimalarials are scarce, despite their potential to optimize antimalarial combination therapy. The aim of this study was to develop a mechanism-based growth model (MBGM) forPlasmodium bergheiand then characterize the parasiticidal effect of dihydroartemisinin (DHA) in murine malaria (MBGM-PK-PD). Stage-specific (ring, early trophozoite, late trophozoite, and schizont) parasite density data from Swiss mice inoculated withPlasmodium bergheiwere used for model development in S-ADAPT. A single dose of intraperitoneal DHA (10 to 100 mg/kg) or vehicle was administered 56 h postinoculation. The MBGM explicitly reflected all four erythrocytic stages of the 24-hourP. bergheilife cycle. Merozoite invasion of erythrocytes was described by a first-order process that declined with increasing parasitemia. An efflux pathway with subsequent return was additionally required to describe the schizont data, thus representing parasite sequestration or trapping in the microvasculature, with a return to circulation. A 1-compartment model with zero-order absorption described the PK of DHA, with an estimated clearance and distribution volume of 1.95 liters h−1and 0.851 liter, respectively. Parasite killing was described by a turnover model, with DHA inhibiting the production of physiological intermediates (IC50, 1.46 ng/ml). Overall, the MBGM-PK-PD described the rise in parasitemia, the nadir following DHA dosing, and subsequent parasite resurgence. This novel model is a promising tool for studying malaria infections, identifying the stage specificity of antimalarials, and providing insight into antimalarial treatment strategies.

MK-4815, a Potential New Oral Agent for Treatment of Malaria

ABSTRACTMalaria continues to have a significant impact on the health of the developing world. Efforts to combat this disease now focus on combination therapy in order to stem the emergence of resistant parasites. Continued efforts are needed to discover and develop new agents for use in combination antimalarial regimens. MK-4815 is a small molecule with antimalarial activity that was identified from a large pharmaceutical compound collection using a semiautomated version of a well-establishedin vitroassay for the erythrocytic stages ofPlasmodium falciparum. In vitrostudies indicate that the compound selectively accumulates in infected red blood cells and is most effective against the metabolically active late trophozoite/early schizont stages. A variety of drug-resistant field isolates ofP. falciparumwere found to be as sensitive to MK-4815 as the wild-type lines. MK-4815 is orally active in aP. bergheimouse model of acute malaria. In this model, where untreated animals succumb to infection 10 to 12 days postinfection, MK-4815 was completely curative when given as a single dose of 50 mg/kg, 2 doses of 25 mg/kg, or 4.5 doses of 12.5 mg/kg. In pharmacokinetic studies with mice and rhesus monkeys, MK-4815 demonstrated oral bioavailability and low clearance. In addition, MK-4815 is inexpensive to synthesize, an important characteristic for providing affordable antimalaria therapy to the developing world. The attractive biological and pharmaceutical profile of MK-4815 demonstrates its potential for use in combination with other agents in the fight against malaria.

Febrile temperature leads to significant stiffening ofPlasmodium falciparumparasitized erythrocytes

Parasitic infection with Plasmodium falciparum is responsible for the most severe form of human malaria in which patients suffer from periodic fever. It is well established that during intra-erythrocytic maturation of the parasite in the red blood cell (RBC), the RBC becomes significantly more cytoadhesive and less deformable; these and other biochemical factors together with human host factors such as compromised immune status are important contributors to the disease pathology. There is currently substantial interest in understanding the loss of RBC deformability due to P. falciparum infection, but few results are available concerning effects of febrile conditions or parasitization on RBC membrane rheology. Here, for the first time, we report rheology of the single, isolated RBC with and without P. falciparum merozoite invasion, spanning a range from room temperature to febrile conditions (41°C), over all the stages of parasite maturation. As expected, stiffness increased with parasite maturation. Surprisingly, however, stiffness increased acutely with temperature on a scale of minutes, particularly in late trophozoite and schizont stages. This acute stiffening in late falciparum stages may contribute to fever-dependent pathological consequences in the microcirculation.

Potent Plasmodicidal Activity of a Heat-Induced Reformulation of Deoxycholate-Amphotericin B (Fungizone) against Plasmodium falciparum

ABSTRACT The emergence and spread of drug-resistant Plasmodium falciparum continue to pose problems in malaria chemotherapy. Therefore, it is necessary to identify new antimalarial drugs and therapeutic strategies. In the present study, the activity of a heat-treated form of amphotericin B (HT-AMB) against P. falciparum was evaluated. The efficacy and toxicity of HT-AMB were also compared with those of the standard formulation (AMB). HT-AMB showed significant activity against a chloroquine-resistant strain (strain K-1) and a chloroquine-susceptible strain (strain FCR-3) in vitro. The 50% inhibitory concentrations of HT-AMB were 0.32 ± 0.03 μg/ml for strain K-1 and 0.33 ± 0.03 μg/ml for strain FCR-3. In the presence of 1.0 μg of HT-AMB per ml, only pyknotic parasites were observed after 24 h of incubation of early trophozoites (ring forms). However, when late trophozoites and schizonts were cultured with 1.0 μg of HT-AMB per ml, those forms multiplied to ring forms but the number of infected erythrocytes did not increase. These results indicate that HT-AMB possesses potent antiplasmodial activity and that the drug is more effective against the ring-form stage than against the late trophozoite and schizont stages. HT-AMB was observed to have little cytotoxic effect against a human liver cell line (Chang liver cells). In conclusion, the results suggest that HT-AMB has promising properties and merits further in vivo investigations as a treatment for falciparum malaria.

Plasmodium falciparum cysteine protease falcipain-2 cleaves erythrocyte membrane skeletal proteins at late stages of parasite development

Plasmodium falciparum–derived cysteine protease falcipain-2 cleaves host erythrocyte hemoglobin at acidic pH and specific components of the membrane skeleton at neutral pH. Analysis of stage-specific expression of these 2 proteolytic activities of falcipain-2 shows that hemoglobin-hydrolyzing activity is maximum in early trophozoites and declines rapidly at late stages, whereas the membrane skeletal protein hydrolyzing activity is markedly increased at the late trophozoite and schizont stages. Among the erythrocyte membrane skeletal proteins, ankyrin and protein 4.1 are cleaved by native and recombinant falcipain-2 near their C-termini. To identify the precise peptide sequence at the hydrolysis site of protein 4.1, we used a recombinant construct of protein 4.1 as substrate followed by MALDI-MS analysis of the cleaved product. We show that falcipain-2–mediated cleavage of protein 4.1 occurs immediately after lysine 437, which lies within a region of the spectrin–actin-binding domain critical for erythrocyte membrane stability. A 16-mer peptide containing the cleavage site completely inhibited the enzyme activity and blocked falcipain-2–induced fragmentation of erythrocyte ghosts. Based on these results, we propose that falcipain-2 cleaves hemoglobin in the acidic food vacuole at the early trophozoite stage, whereas it cleaves specific components of the red cell skeleton at the late trophozoite and schizont stages. It is the proteolysis of skeletal proteins that causes membrane instability, which, in turn, facilitates parasite release in vivo.

Ribonucleic acid synthesis inPlasmodium knowlesimaintained bothin vivoandin vitro

RNA extracted from purified parasites ofPlasmodium knowlesiwas fractionated using agarose gel electrophoresis. Preparations from parasites grown bothin vivoandin vitrocontained species of RNA with sedimentation coefficients of 4·0S, 5·0S, 16·6S, 24·2S, 31·4S, 38·0S and 48·3S. There was less RNA present in parasites grownin vitrothan the equivalent stage parasites grownin vivobut the proportional amounts of the various species of RNA was similar in both cases. It is suggested that the 24·2S and 16·6S species of RNA are ribosomal and that the high molecular weight 31·4S, 38·0S and 48·0S species are ribosomal precursors. Ribosomal RNA synthesis occurs throughout the cell cycle during growth from the ring to the schizont stage; maximum incorporation of [H3]-adenosine occurs at the late trophozoite stage before nuclear division.

A rational approach to the serial culture of malaria parasites: evidence for a deficiency in RNA synthesis during the first cyclein vitro

When ring-stage parasites are grown to the late trophozoite stagein vitro, the incorporation of [3H]adenosine into parasite DNA and RNA is in the ratio of ~ 1:1·5 and not 1:4 as expected from direct biochemical analysis of the parasite. A method is described by which large quantities of infected blood can be grownin vitrofrom the ring to the trophozoite stage thus allowing direct biochemical analysis of parasites grownin vitro. The results of direct biochemical analysis indicated that parasites grownin vitrohave similar DNA and protein contents to those grownin vitrobut that RNA contents are much lessin vitro. Quantitative histochemical studies also indicated a deficiency in RNA in parasites grownin vitro. It is concluded from the evidence of three distinct methods that RNA synthesis is defective during the first asexual cyclein vitro. In the second cycle all three macromolecular biosyntheses studied are reduced when compared to the first so that the actual rate of RNA synthesis is further reduced when compared to thatin vitro. It is suggested that the successive decrease in RNA synthesis parallels the reduction in multiplication obtained on subculture of the parasite.