Berghei Life Cycle Research Articles

An ApiAp2 Transcription Factor with a Dispensable Role in Plasmodium berghei Life Cycle.

Malaria parasites have a complex life cycle and undergo replication and population expansion within vertebrate hosts and mosquito vectors. These developmental transitions rely on changes in gene expression and chromatin reorganization that result in the activation and silencing of stage-specific genes. The ApiAp2 family of DNA-binding proteins plays an important role in regulating gene expression in malaria parasites. Here, we characterized the ApiAp2 protein in Plasmodium berghei, which we termed Ap2-D. In silico analysis revealed that Ap2-D has three beta-sheets followed by a helix at the C-terminus for DNA binding. Using gene tagging with 3XHA-mCherry, we found that Ap2-D is expressed in Plasmodium blood stages and is present in the parasite cytoplasm and nucleus. Surprisingly, our gene deletion study revealed a completely dispensable role for Ap2-D in the entirety of the P. berghei life cycle. Ap2-D KO parasites were found to grow in the blood successfully and progress through the mosquito midgut and salivary glands. Sporozoites isolated from mosquito salivary glands were infective for hepatocytes and achieved similar patency as WT in mice. We emphasize the importance of genetic validation of antimalarial drug targets before progressing them to drug discovery.

Protein kinase 9 is not required for completion of the Plasmodium berghei life cycle

Protein kinases uniquely expressed in Plasmodium represent attractive drug targets. Previous studies have reported that Plasmodium falciparum Protein kinase 9 (Pk9) phosphorylates regulatory serine 106 of the ubiquitin-conjugating enzyme (Ubc13) thereby negatively regulating its activity. We investigated the effect of Pk9 depletion and Ubc13 mutation at S106 on the progression of rodent malaria model P. berghei life cycle. Our studies demonstrate that while phosphorylation of the regulatory serine 106 of Ubc13 is essential in blood stages, the lack of Pk9 expression neither altered Ubc13 phosphorylation nor parasite viability at all life cycle stages, though Ubc13 and Pk9 showed co-localization in the cytosol of erythrocytic and liver stages. Further, phosphorylation of Ubc13 in the absence of Pk9 reiterated the redundancy of its regulation in P. berghei. These results highlight the indispensable role of Ubc13 in P. berghei life cycle and redundancy in its phosphorylation by protein kinase and reiterate the need to validate novel gene function through genetic approaches for drug development strategies.

Gene editing in Plasmodium berghei made easy: Development of a CRISPR/Cas9 protocol using linear donor template and ribozymes for sgRNA generation

Efficient reverse genetics approaches are critical for the study of many organisms. The CRISPR/Cas9 gene editing system has led to a plethora of new tools for geneticists. Here, we successfully established a simplified CRISPR/Cas9 system for the malaria model parasite Plasmodium berghei. The homologous directed repair (HDR) template is provided as a linear template with homologous arms of 600−700bp while the CRISPR elements sgRNA and Cas9 are encoded from a single plasmid utilizing the Ribozyme-Guide-Ribozyme (RGR) expression strategy. Our approach eliminates the need for negative selection markers since the plasmid cannot be incorporated into the genome. As a test case we inserted the FLAG encoding sequence into the ACT2 locus using this new approach. We showed that the genetic modification of this locus had no adverse effects on the completion of the P. berghei life cycle, including transmission through the mosquito.

Mitochondrial apurinic/apyrimidinic endonuclease Apn1 is not critical for the completion of the Plasmodium berghei life cycle

Mitochondrion is an essential organelle in malaria parasite and its DNA must be maintained for optimal function during its complex life cycle. Base excision repair is one of the major pathways by which this is achieved. Apurinic/apyrimidinic (AP) endonucleases are important components of this pathway as they create a nick at the 5′-phosphodiester bond in the AP site and generate free 5′-phosphate and 3′-hydroxyl groups. Two class II AP endonucleases (Apn1 and Ape1) have been annotated in the Plasmodium berghei genome. Using reverse genetic approaches, we provide direct evidence that Apn1 is exclusively localized to the mitochondria of P. berghei. Surprisingly, our gene deletion study revealed a completely dispensable role of Apn1 for the entirety of the P. berghei life cycle. Apn1− parasites were found to successfully grow in the blood. They were transmitted normally to the mosquito midguts and salivary glands. Sporozoites obtained from the salivary glands were infective and achieved similar patency as WT. Our results help emphasize the non-availability of this enzyme as a plausible drug target. We also emphasize the importance of genetic validation of antimalarial drug targets before furthering them down the drug discovery pipeline.

Identification and Assessment of Plasmodium berghei Merozoites and Cell Cycle by Flow Cytometry.

The asexual blood stages of the Plasmodium berghei life cycle including merozoites are attractive targets for transmission blocking vaccines and drugs. Improved understanding of P. berghei life cycle stage growth and development would provide new opportunities to evaluate antimalarial vaccines and drugs. Blood stage samples from C57BL/6 albino mice infected with P. berghei sporozoites were singly stained with a high binding affinity deoxyribonucleic acid dye, YOYO-1, and measured by flow cytometry (FCM). Duplicate slides were made from samples and stained with diluted Giemsa's and YOYO-1, respectively. Correlated results were compared by FCM, light microscopy, and fluorescent microscopy. Complete life cycle stage determination and analysis by FCM is reported to include merozoites, ring forms, trophozoites, immature, and mature schizonts. FCM demonstrated a clear separation between each stage using their unique fluorescence distribution. When compared to light microscopy, a strong correlation (r 2 = 0.925 to 0.974) was observed in determining the ring forms, trophozoites, and schizonts phases, but only a moderate correlation (r 2 = 0.684 to 0.778) was observed for merozoites. The identification and measurement of merozoites suggest that FCM is a useful technique to monitor the entire life stage of the parasite. Initial stage-specific data demonstrated that mefloquine has a mode of action on mature parasite forms, and artesunic acid was rapidly effective against merozoites and other immature and mature parasite forms with higher killing. Blood stage parasites in each individual life stage, including merozoites, are reliably identified and quantified quickly by FCM, making this technique an ideal alternative to microscopy. This integrated whole life stage model, particularly with confirmed determination of merozoite population, could widely be used for drug and vaccine research in malaria therapy and prophylaxis.

Plasmodium berghei Kinesin-5 Associates With the Spindle Apparatus During Cell Division and Is Important for Efficient Production of Infectious Sporozoites.

Kinesin-5 motors play essential roles in spindle apparatus assembly during cell division, by generating forces to establish and maintain the spindle bipolarity essential for proper chromosome segregation. Kinesin-5 is largely conserved structurally and functionally in model eukaryotes, but its role is unknown in the Plasmodium parasite, an evolutionarily divergent organism with several atypical features of both mitotic and meiotic cell division. We have investigated the function and subcellular location of kinesin-5 during cell division throughout the Plasmodium berghei life cycle. Deletion of kinesin-5 had little visible effect at any proliferative stage except sporozoite production in oocysts, resulting in a significant decrease in the number of motile sporozoites in mosquito salivary glands, which were able to infect a new vertebrate host. Live-cell imaging showed kinesin-5-GFP located on the spindle and at spindle poles during both atypical mitosis and meiosis. Fixed-cell immunofluorescence assays revealed kinesin-5 co-localized with α-tubulin and centrin-2 and a partial overlap with kinetochore marker NDC80 during early blood stage schizogony. Dual-color live-cell imaging showed that kinesin-5 is closely associated with NDC80 during male gametogony, but not with kinesin-8B, a marker of the basal body and axonemes of the forming flagella. Treatment of gametocytes with microtubule-specific inhibitors confirmed kinesin-5 association with nuclear spindles and not cytoplasmic axonemal microtubules. Altogether, our results demonstrate that kinesin-5 is associated with the spindle apparatus, expressed in proliferating parasite stages, and important for efficient production of infectious sporozoites.

Characterization of a novel Plasmodium Virulence Factor

Abstract Plasmodium is a deadly parasite that claims lives of nearly half a million individuals each year. Identification of the mechanisms the parasite uses to evade the immune response is a critical goal to develop the much needed vaccine against this pathogen. However, we know very little about how individual parasite genes function. Here, by using CRISPR/Cas9 strategy, we altered the DNA binding domain of a Transcription Factor (TF) (PBANKA_011210) that is essential for the completion of Plasmodium Berghei life cycle. In mice, compared to the WT parasite, the mutant one showed differential expression of 46 genes, most of which are thought to be involved in generating parasite surface antigen diversity. Furthermore, unlike WT parasite which successfully hides from immune system, mutant parasite triggered high levels of IFN-g which translated into formation of prolonged germinal centers and development of higher titers of parasite specific IgG2b and IgG2c type antibodies. At low inoculum levels this resulted in successful clearance of infection and development of protective immunological memory in mutant parasite infected animals in contrast to a deadly outcome in WT parasite infected group. The differential development of the immune response was dependent on the host’s ability to produce IFNg as the phenotype was lost in IFNg KO mice. We therefore conclude that (PBANKA_011210) TF is a novel virulence factor acts as an immune modulator and dampens IFNg mediated immune response against parasite

Nuclear Pore Complex Components in the Malaria Parasite Plasmodium berghei

The nuclear pore complex (NPC) is a large macromolecular assembly of around 30 different proteins, so-called nucleoporins (Nups). Embedded in the nuclear envelope the NPC mediates bi-directional exchange between the cytoplasm and the nucleus and plays a role in transcriptional regulation that is poorly understood. NPCs display modular arrangements with an overall structure that is generally conserved among many eukaryotic phyla. However, Nups of yeast or human origin show little primary sequence conservation with those from early-branching protozoans leaving those of the malaria parasite unrecognized. Here we have combined bioinformatic and genetic methods to identify and spatially characterize Nup components in the rodent infecting parasite Plasmodium berghei and identified orthologs from the human malaria parasite P. falciparum, as well as the related apicomplexan parasite Toxoplasma gondii. For the first time we show the localization of selected Nups throughout the P. berghei life cycle. Largely restricted to apicomplexans we identify an extended C-terminal poly-proline extension in SEC13 that is essential for parasite survival and provide high-resolution images of Plasmodium NPCs obtained by cryo electron tomography. Our data provide the basis for full characterization of NPCs in malaria parasites, early branching unicellular eukaryotes with significant impact on human health.

Compositional and expression analyses of the glideosome during the Plasmodium life cycle reveal an additional myosin light chain required for maximum motility

Myosin A (MyoA) is a Class XIV myosin implicated in gliding motility and host cell and tissue invasion by malaria parasites. MyoA is part of a membrane-associated protein complex called the glideosome, which is essential for parasite motility and includes the MyoA light chain myosin tail domain–interacting protein (MTIP) and several glideosome-associated proteins (GAPs). However, most studies of MyoA have focused on single stages of the parasite life cycle. We examined MyoA expression throughout the Plasmodium berghei life cycle in both mammalian and insect hosts. In extracellular ookinetes, sporozoites, and merozoites, MyoA was located at the parasite periphery. In the sexual stages, zygote formation and initial ookinete differentiation precede MyoA synthesis and deposition, which occurred only in the developing protuberance. In developing intracellular asexual blood stages, MyoA was synthesized in mature schizonts and was located at the periphery of segmenting merozoites, where it remained throughout maturation, merozoite egress, and host cell invasion. Besides the known GAPs in the malaria parasite, the complex included GAP40, an additional myosin light chain designated essential light chain (ELC), and several other candidate components. This ELC bound the MyoA neck region adjacent to the MTIP-binding site, and both myosin light chains co-located to the glideosome. Co-expression of MyoA with its two light chains revealed that the presence of both light chains enhances MyoA-dependent actin motility. In conclusion, we have established a system to study the interplay and function of the three glideosome components, enabling the assessment of inhibitors that target this motor complex to block host cell invasion.

Functional characterization of Plasmodium berghei PSOP25 during ookinete development and as a malaria transmission-blocking vaccine candidate

BackgroundPlasmodium ookinete surface proteins as post-fertilization target antigens are potential malaria transmission-blocking vaccine (TBV) candidates. Putative secreted ookinete protein 25 (PSOP25) is a highly conserved ookinete surface protein, and has been shown to be a promising novel TBV target. Here, we further investigated the TBV activities of the full-length recombinant PSOP25 (rPSOP25) protein in Plasmodium berghei, and characterized the potential functions of PSOP25 during the P. berghei life-cycle.MethodsWe expressed the full-length P. berghei PSOP25 protein in a prokaryotic expression system, and developed polyclonal mouse antisera and a monoclonal antibody (mAb) against the recombinant protein. Indirect immunofluorescence assay (IFA) and Western blot were used to test the specificity of antibodies. The transmission-blocking (TB) activities of antibodies were evaluated by the in vitro ookinete conversion assay and by direct mosquito feeding assay (DFA). Finally, the function of PSOP25 during Plasmodium development was studied by deleting the psop25 gene.ResultsBoth polyclonal mouse antisera and anti-rPSOP25 mAb recognized the PSOP25 proteins in the parasites, and IFA showed the preferential expression of PSOP25 on the surface of zygotes, retorts and mature ookinetes. In vitro, these antibodies significantly inhibited ookinetes formation in an antibody concentration-dependent manner. In DFA, mice immunized with the rPSOP25 and those receiving passive transfer of the anti-rPSOP25 mAb reduced the prevalence of mosquito infection by 31.2 and 26.1%, and oocyst density by 66.3 and 63.3%, respectively. Genetic knockout of the psop25 gene did not have a detectable impact on the asexual growth of P. berghei, but significantly affected the maturation of ookinetes and the formation of midgut oocysts.ConclusionsThe full-length rPSOP25 could elicit strong antibody response in mice. Polyclonal and monoclonal antibodies against PSOP25 could effectively block the formation of ookinetes in vitro and transmission of the parasites to mosquitoes. Genetic manipulation study indicated that PSOP25 is required for ookinete maturation in P. berghei. These results support further testing of the PSOP25 orthologs in human malaria parasites as promising TBV candidates.

Gene disruption reveals a dispensable role for Plasmepsin VII in the Plasmodium berghei life cycle

Plasmepsins (PM), aspartic proteases of Plasmodium, comprises a family of ten proteins that perform critical functions in Plasmodium life cycle. Except VII and VIII, functions of the remaining plasmepsin members have been well characterized. Here, we have generated a mutant parasite lacking PM VII in Plasmodium berghei using reverse genetics approach. Systematic comparison of growth kinetics and infection in both mosquito and vertebrate host revealed that PM VII depleted mutants exhibited no defects in development and progressed normally throughout the parasite life cycle. These studies suggest a dispensable role for PM VII in Plasmodium berghei life cycle.

Predicting the parasite killing effect of artemisinin combination therapy in a murine malaria model.

To develop a mechanism-based model that describes the time course of the malaria parasite in infected mice receiving a combination therapy regimen of dihydroartemisinin and piperaquine. Total parasite density-time data from Swiss mice inoculated with Plasmodium berghei were used for the development of population models in S-ADAPT. The mice were administered a single intraperitoneal dose of 30 mg/kg dihydroartemisinin, 10 mg/kg piperaquine phosphate or a combination of both antimalarials at 64 h post-inoculation. In a separate study, mice received multiple dihydroartemisinin doses (5 × 10 mg/kg or 30 mg/kg dihydroartemisinin followed by two 10 mg/kg doses). Parasite recrudescence after treatment was defined using a model that incorporated each erythrocytic stage of the P. berghei life cycle. The disposition of dihydroartemisinin and piperaquine was described by a one-compartment and two-compartment model, respectively. The estimated clearance was 1.95 L/h for dihydroartemisinin and 0.109 L/h for piperaquine. A turnover model described the parasite killing curve after single-agent dosing, with an estimated mean IC50 of 0.747 μg/L for dihydroartemisinin and 16.8 μg/L for piperaquine. In addition, the rate of parasite killing by dihydroartemisinin was almost 50-fold faster than for piperaquine. Parameters from the monotherapy models adequately described the parasite density-time curve following dihydroartemisinin/piperaquine combination therapy or multiple-dose regimens of dihydroartemisinin. This study has developed mechanistic models that describe the parasite-time curve after single, multiple or combination dosing of antimalarials to mice. These structural models have potential application for pre-clinical investigations to design and refine artemisinin-based combination therapy dosage regimens.

The ETRAMP Family Member SEP2 Is Expressed throughout Plasmodium berghei Life Cycle and Is Released during Sporozoite Gliding Motility

The early transcribed membrane proteins ETRAMPs belong to a family of small, transmembrane molecules unique to Plasmodium parasite, which share a signal peptide followed by a short lysine-rich stretch, a transmembrane domain and a variable, highly charged C-terminal region. ETRAMPs are usually expressed in a stage-specific manner. In the blood stages they localize to the parasitophorous vacuole membrane and, in described cases, to vesicle-like structures exported to the host erythrocyte cytosol. Two family members of the rodent parasite Plasmodium berghei, uis3 and uis4, localize to secretory organelles of sporozoites and to the parasitophorous membrane vacuole of the liver stages. By the use of specific antibodies and the generation of transgenic lines, we showed that the P. berghei ETRAMP family member SEP2 is abundantly expressed in gametocytes as well as in mosquito and liver stages. In intracellular parasite stages, SEP2 is routed to the parasitophorous vacuole membrane while, in invasive ookinete and sporozoite stages, it localizes to the parasite surface. To date SEP2 is the only ETRAMP protein detected throughout the parasite life cycle. Furthermore, SEP2 is also released during gliding motility of salivary gland sporozoites. A limited number of proteins are known to be involved in this key function and the best characterized, the CSP and TRAP, are both promising transmission-blocking candidates. Our results suggest that ETRAMP members may be viewed as new potential candidates for malaria control.

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

Murine 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) for Plasmodium berghei and 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 with Plasmodium berghei were 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-hour P. berghei life 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(-1) and 0.851 liter, respectively. Parasite killing was described by a turnover model, with DHA inhibiting the production of physiological intermediates (IC(50), 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.

Arrested Oocyst Maturation in Plasmodium Parasites Lacking Type II NADH:Ubiquinone Dehydrogenase

The Plasmodium mitochondrial electron transport chain has received considerable attention as a potential target for new antimalarial drugs. Atovaquone, a potent inhibitor of Plasmodium cytochrome bc(1), in combination with proguanil is recommended for chemoprophylaxis and treatment of malaria. The type II NADH:ubiquinone oxidoreductase (NDH2) is considered an attractive drug target, as its inhibition is thought to lead to the arrest of the mitochondrial electron transport chain and, as a consequence, pyrimidine biosynthesis, an essential pathway for the parasite. Using the rodent malaria parasite Plasmodium berghei as an in vivo infection model, we studied the role of NDH2 during Plasmodium life cycle progression. NDH2 can be deleted by targeted gene disruption and, thus, is dispensable for the pathogenic asexual blood stages, disproving the candidacy for an anti-malarial drug target. After transmission to the insect vector, NDH2-deficient ookinetes display an intact mitochondrial membrane potential. However, ndh2(-) parasites fail to develop into mature oocysts in the mosquito midgut. We propose that Plasmodium blood stage parasites rely on glycolysis as the main ATP generating process, whereas in the invertebrate vector, a glucose-deprived environment, the malaria parasite is dependent on an intact mitochondrial respiratory chain.

The Ribosomal Genes of Plasmodium

Publisher Summary The chapter describes the Plasmodium rDNA unit. The chapter discusses briefly a feature of the rDNA units of other eukaryotic organisms and then compares and contrasts this with what has been found about the Plasmodium rDNA unit. There are three main areas of interest: (1) the number of ribosomal genes in a haploid genome, (2) the structural variations among individual rDNA units, and (3) the arrangement of these genes with regard to each other. The most striking feature of eukaryotic rDNA units on an evolutionary scale is their similarity. In contrast, perhaps the most interesting features of the rDNA units of the Plasmodium species, and the subject of this review, are the sharp digressions made from what was thought to be standard features of the eukaryotic ribosomal gene. The chapter illustrates a generalized life cycle of mammalian malarias, discussing only the Plasmodium berghei life cycle. The exoerythrocytic forms of the parasite compose the first developmental stage in the mammalian host. This stage initiates with inoculation of sporozoites into the bloodstream and invasion of the liver. The parasites replicate asexually in the liver and are released as exoerythrocytic merozoites into the bloodstream. The organism goes through at least 11 distinct stages in its life cycle, both haploid and diploid. Protein synthesis varies in rate and occurs at different temperatures throughout the cycle.