An apicoplast-resident folate transporter is essential for sporogony of malaria parasites.

Malaria parasites are fast replicating unicellular organisms and require substantial amounts of folate for DNA synthesis. Despite the central role of this critical co-factor for parasite survival, only little is known about intraparasitic folate trafficking in Plasmodium. Here, we report on the expression, subcellular localisation and function of the parasite's folate transporter 2 (FT2) during life cycle progression in the murine malaria parasite Plasmodium berghei. Using live fluorescence microscopy of genetically engineered parasites, we demonstrate that FT2 localises to the apicoplast. In invasive P. berghei stages, a fraction of FT2 is also observed at the apical end. Upon genetic disruption of FT2, blood and liver infection, gametocyte production and mosquito colonisation remain unaltered. But in the Anopheles vector, FT2-deficient parasites develop inflated oocysts with unusual pulp formation consisting of numerous single-membrane vesicles, which ultimately fuse to form large cavities. Ultrastructural analysis suggests that this defect reflects aberrant sporoblast formation caused by abnormal vesicular traffic. Complete sporogony in FT2-deficient oocysts is very rare, and mutant sporozoites fail to establish hepatocyte infection, resulting in a complete block of parasite transmission. Our findings reveal a previously unrecognised organellar folate transporter that exerts critical roles for pathogen maturation in the arthropod vector.

A conserved malaria parasite antigen Pb22 plays a critical role in male gametogenesis in Plasmodium berghei.

Gametogenesis, the formation of gametes from gametocytes, an essential step for malaria parasite transmission, is targeted by transmission-blocking drugs and vaccines. We identified a conserved protein (PBANKA_0305900) in Plasmodium berghei, which encodes a protein of 22 kDa (thus named Pb22) and is expressed in both asexual stages and gametocytes. Its homologues are present in all Plasmodium species and its closely related, Hepatocystis, but not in other apicomplexans. Pb22 protein was localised in the cytosols of schizonts, as well as male and female gametocytes. During gamete-to-ookinete development, Pb22 became localised on the plasma membranes of gametes and ookinetes. Compared to the wild-type (WT) parasites, P. berghei with pb22 knockout (KO) showed a significant reduction in exflagellation (~89%) of male gametocytes and ookinete number (~97%) during in vitro ookinete culture. Mosquito feeding assays showed that ookinete and oocyst formation of the pb22-KO line in mosquito midguts was almost completely abolished. These defects were rescued in parasites where pb22 was restored. Cross-fertilisation experiments with parasite lines defective in either male or female gametes confirmed that the defects in the pb22-KO line were restricted to the male gametes, whereas female gametes in the pb22-KO line were fertile at the WT level. Detailed analysis of male gametogenesis showed that 30% of the male gametocytes in the pb22-KO line failed to assemble the axonemes, whereas ~48.9% of the male gametocytes formed flagella but failed to egress from the host erythrocyte. To explore its transmission-blocking potential, recombinant Pb22 (rPb22) was expressed and used to immunise mice. in vitro assays showed that the rPb22-antisera significantly inhibited exflagellation by ~64.8% and ookinete formation by ~93.4%. Mosquitoes after feeding on rPb22-immunised mice also showed significant decreases in infection prevalence (83.3-93.3%) and oocyst density (93.5-99.6%). Further studies of the Pb22 orthologues in human malaria parasites are warranted.

Distinct Functional Contributions by the Conserved Domains of the Malaria Parasite Alveolin IMC1h.

Invasive, motile life cycle stages (zoites) of apicomplexan parasites possess a cortical membrane skeleton composed of intermediate filaments with roles in zoite morphogenesis, tensile strength and motility. Its building blocks include a family of proteins called alveolins that are characterized by conserved "alveolin" domains composed of tandem repeat sequences. A subset of alveolins possess additional conserved domains that are structurally unrelated and the roles of which remain unclear. In this structure-function analysis we investigated the functional contributions of the "alveolin" vs. "non-alveolin" domains of IMC1h, a protein expressed in the ookinete and sporozoite life cycle stages of malaria parasites and essential for parasite transmission. Using allelic replacement in Plasmodium berghei, we show that the alveolin domain is responsible for targeting IMC1h to the membrane skeleton and, consequently, its deletion from the protein results in loss of function manifested by abnormally-shaped ookinetes and sporozoites with reduced tensile strength, motility and infectivity. Conversely, IMC1h lacking its non-alveolin conserved domain is correctly targeted and can facilitate tensile strength but not motility. Our findings support the concept that the alveolin module contains the properties for filament formation, and show for the first time that tensile strength makes an important contribution to zoite infectivity. The data furthermore provide new insight into the underlying molecular mechanisms of motility, indicating that tensile strength is mechanistically uncoupled from locomotion, and pointing to a role of the non-alveolin domain in the motility-enhancing properties of IMC1h possibly by engaging with the locomotion apparatus.

A unique profilin-actin interface is important for malaria parasite motility.

Profilin is an actin monomer binding protein that provides ATP-actin for incorporation into actin filaments. In contrast to higher eukaryotic cells with their large filamentous actin structures, apicomplexan parasites typically contain only short and highly dynamic microfilaments. In apicomplexans, profilin appears to be the main monomer-sequestering protein. Compared to classical profilins, apicomplexan profilins contain an additional arm-like ß-hairpin motif, which we show here to be critically involved in actin binding. Through comparative analysis using two profilin mutants, we reveal this motif to be implicated in gliding motility of Plasmodium berghei sporozoites, the rapidly migrating forms of a rodent malaria parasite transmitted by mosquitoes. Force measurements on migrating sporozoites and molecular dynamics simulations indicate that the interaction between actin and profilin fine-tunes gliding motility. Our data suggest that evolutionary pressure to achieve efficient high-speed gliding has resulted in a unique profilin-actin interface in these parasites.

Modulation of host cell SUMOylation facilitates efficient development of Plasmodium berghei and Toxoplasma gondii.

SUMOylation is a reversible post translational modification of proteins that regulates protein stabilization, nucleocytoplasmic transport, and protein-protein interactions. Several viruses and bacteria modulate host SUMOylation machinery for efficient infection. Plasmodium sporozoites are infective forms of malaria parasite that invade mammalian hepatocytes and transforms into exoerythrocytic forms (EEFs). Here, we show that during EEF development, the distribution of SUMOylated proteins in host cell nuclei was significantly reduced and expression of the SUMOylation enzymes was downregulated. Plasmodium EEFs destabilized the host cytoplasmic protein SMAD4 by inhibiting its SUMOylation. SUMO1 overexpression was detrimental to EEF growth, and insufficiency of the only conjugating enzyme Ubc9/E2 promoted EEF growth. The expression of genes involved in suppression of host cell defense pathways during infection was reversed during SUMO1 overexpression, as revealed by transcriptomic analysis. The inhibition of host cell SUMOylation was also observed during Toxoplasma infection. We provide a hitherto unknown mechanism of regulating host gene expression by Apicomplexan parasites through altering host SUMOylation.

Microstructured Blood Vessel Surrogates Reveal Structural Tropism of Motile Malaria Parasites.

Plasmodium sporozoites, the highly motile forms of the malaria parasite, are transmitted naturally by mosquitoes and traverse the skin to find, associate with, and enter blood capillaries. Research aimed at understanding how sporozoites select blood vessels is hampered by the lack of a suitable experimental system. Arrays of uniform cylindrical pillars can be used to study small cells moving in controlled environments. Here, an array system displaying a variety of pillars with different diameters and shapes is developed in order to investigate how Plasmodium sporozoites associate to the pillars as blood vessel surrogates. Investigating the association of sporozoites to pillars in arrays displaying pillars of different diameters reveals that the crescent-shaped parasites prefer to associate with and migrate around pillars with a similar curvature. This suggests that after transmission by a mosquito, malaria parasites may use a structural tropism to recognize blood capillaries in the dermis in order to gain access to the blood stream.

Overexpression of Plasmodium berghei ATG8 by Liver Forms Leads to Cumulative Defects in Organelle Dynamics and to Generation of Noninfectious Merozoites.

UNLABELLED: Plasmodium parasites undergo continuous cellular renovation to adapt to various environments in the vertebrate host and insect vector. In hepatocytes, Plasmodium berghei discards unneeded organelles for replication, such as micronemes involved in invasion. Concomitantly, intrahepatic parasites expand organelles such as the apicoplast that produce essential metabolites. We previously showed that the ATG8 conjugation system is upregulated in P. berghei liver forms and that P. berghei ATG8 (PbATG8) localizes to the membranes of the apicoplast and cytoplasmic vesicles. Here, we focus on the contribution of PbATG8 to the organellar changes that occur in intrahepatic parasites. We illustrated that micronemes colocalize with PbATG8-containing structures before expulsion from the parasite. Interference with PbATG8 function by overexpression results in poor development into late liver stages and production of small merosomes that contain immature merozoites unable to initiate a blood infection. At the cellular level, PbATG8-overexpressing P. berghei exhibits a delay in microneme compartmentalization into PbATG8-containing autophagosomes and elimination compared to parasites from the parental strain. The apicoplast, identifiable by immunostaining of the acyl carrier protein (ACP), undergoes an abnormally fast proliferation in mutant parasites. Over time, the ACP staining becomes diffuse in merosomes, indicating a collapse of the apicoplast. PbATG8 is not incorporated into the progeny of mutant parasites, in contrast to parental merozoites in which PbATG8 and ACP localize to the apicoplast. These observations reveal that Plasmodium ATG8 is a key effector in the development of merozoites by controlling microneme clearance and apicoplast proliferation and that dysregulation in ATG8 levels is detrimental for malaria infectivity. IMPORTANCE: Malaria is responsible for more mortality than any other parasitic disease. Resistance to antimalarial medicines is a recurring problem; new drugs are urgently needed. A key to the parasite's successful intracellular development in the liver is the metabolic changes necessary to convert the parasite from a sporozoite to a replication-competent, metabolically active trophozoite form. Our study reinforces the burgeoning concept that organellar changes during parasite differentiation are mediated by an autophagy-like process. We have identified ATG8 in Plasmodium liver forms as an important effector that controls the development and fate of organelles, e.g., the clearance of micronemes that are required for hepatocyte invasion and the expansion of the apicoplast that produces many metabolites indispensable for parasite replication. Given the unconventional properties and the importance of ATG8 for parasite development in hepatocytes, targeting the parasite's autophagic pathway may represent a novel approach to control malarial infections.

Replication of Plasmodium in reticulocytes can occur without hemozoin formation, resulting in chloroquine resistance.

Most studies on malaria-parasite digestion of hemoglobin (Hb) have been performed using P. falciparum maintained in mature erythrocytes, in vitro. In this study, we examine Plasmodium Hb degradation in vivo in mice, using the parasite P. berghei, and show that it is possible to create mutant parasites lacking enzymes involved in the initial steps of Hb proteolysis. These mutants only complete development in reticulocytes and mature into both schizonts and gametocytes. Hb degradation is severely impaired and large amounts of undigested Hb remains in the reticulocyte cytoplasm and in vesicles in the parasite. The mutants produce little or no hemozoin (Hz), the detoxification by-product of Hb degradation. Further, they are resistant to chloroquine, an antimalarial drug that interferes with Hz formation, but their sensitivity to artesunate, also thought to be dependent on Hb degradation, is retained. Survival in reticulocytes with reduced or absent Hb digestion may imply a novel mechanism of drug resistance. These findings have implications for drug development against human-malaria parasites, such as P. vivax and P. ovale, which develop inside reticulocytes.

P. berghei telomerase subunit TERT is essential for parasite survival.

Telomeres define the ends of chromosomes protecting eukaryotic cells from chromosome instability and eventual cell death. The complex regulation of telomeres involves various proteins including telomerase, which is a specialized ribonucleoprotein responsible for telomere maintenance. Telomeres of chromosomes of malaria parasites are kept at a constant length during blood stage proliferation. The 7-bp telomere repeat sequence is universal across different Plasmodium species (GGGTTT/CA), though the average telomere length varies. The catalytic subunit of telomerase, telomerase reverse transcriptase (TERT), is present in all sequenced Plasmodium species and is approximately three times larger than other eukaryotic TERTs. The Plasmodium RNA component of TERT has recently been identified in silico. A strategy to delete the gene encoding TERT via double cross-over (DXO) homologous recombination was undertaken to study the telomerase function in P. berghei. Expression of both TERT and the RNA component (TR) in P. berghei blood stages was analysed by Western blotting and Northern analysis. Average telomere length was measured in several Plasmodium species using Telomere Restriction Fragment (TRF) analysis. TERT and TR were detected in blood stages and an average telomere length of ∼ 950 bp established. Deletion of the tert gene was performed using standard transfection methodologies and we show the presence of tert- mutants in the transfected parasite populations. Cloning of tert- mutants has been attempted multiple times without success. Thorough analysis of the transfected parasite populations and the parasite obtained from extensive parasite cloning from these populations provide evidence for a so called delayed death phenotype as observed in different organisms lacking TERT. The findings indicate that TERT is essential for P. berghei cell survival. The study extends our current knowledge on telomere biology in malaria parasites and validates further investigations to identify telomerase inhibitors to induce parasite cell death.

The Plasmodium protein P113 supports efficient sporozoite to liver stage conversion in vivo.

Invasive stages of Plasmodium parasites possess distinct integral and peripheral membrane proteins that mediate host cell attachment and invasion. P113 is an abundant protein in detergent-resistant high molecular weight complexes in Plasmodium schizonts, but is unusual since expression extends to gametocytes and sporozoites. In this study, we tested whether P113 performs important functions for parasite propagation in Plasmodium berghei. We show that pre-erythrocytic expression of P113 displays key signatures of upregulated in infectious sporozoites (UIS) genes, including control by the liver stage master regulator SLARP. Targeted gene deletion resulted in viable blood stage parasites that displayed no signs of blood stage growth defects. p113(-) parasites propagated normally through the life cycle until mature sporozoites, but displayed defects during natural sporozoite transmission, leading to a delay to patency in infected animals. By comparative in vitro and in vivo analysis of pre-erythrocytic development and using a xeno-diagnostic test we show that ablation of P113 results in lower sporozoite to liver stage conversion and, as a consequence, reduced merozoite output in vivo, without delaying liver stage development. We conclude that p113 is dispensable for Plasmodium life cycle progression and plays auxiliary roles during pre-erythrocytic development.

A cascade of DNA-binding proteins for sexual commitment and development in Plasmodium.

Commitment to and completion of sexual development are essential for malaria parasites (protists of the genus Plasmodium) to be transmitted through mosquitoes. The molecular mechanism(s) responsible for commitment have been hitherto unknown. Here we show that PbAP2-G, a conserved member of the apicomplexan AP2 (ApiAP2) family of DNA-binding proteins, is essential for the commitment of asexually replicating forms to sexual development in Plasmodium berghei, a malaria parasite of rodents. PbAP2-G was identified from mutations in its encoding gene, PBANKA_143750, which account for the loss of sexual development frequently observed in parasites transmitted artificially by blood passage. Systematic gene deletion of conserved ApiAP2 genes in Plasmodium confirmed the role of PbAP2-G and revealed a second ApiAP2 member (PBANKA_103430, here termed PbAP2-G2) that significantly modulates but does not abolish gametocytogenesis, indicating that a cascade of ApiAP2 proteins are involved in commitment to the production and maturation of gametocytes. The data suggest a mechanism of commitment to gametocytogenesis in Plasmodium consistent with a positive feedback loop involving PbAP2-G that could be exploited to prevent the transmission of this pernicious parasite.

Genetic crosses and complementation reveal essential functions for the Plasmodium stage-specific actin2 in sporogonic development.

Malaria parasites have two actin isoforms, ubiquitous actin1 and specialized actin2. Actin2 is essential for late male gametogenesis, prior to egress from the host erythrocyte. Here, we examined whether the two actins fulfil overlapping functions in Plasmodium berghei. Replacement of actin2 with actin1 resulted in partial complementation of the defects in male gametogenesis and, thus, viable ookinetes were formed, able to invade the midgut epithelium and develop into oocysts. However, these remained small and their DNA was undetectable at day 8 after infection. As a consequence sporogony did not occur, resulting in a complete block of parasite transmission. Furthermore, we show that expression of actin2 is tightly controlled in female stages. The actin2 transcript is translationally repressed in female gametocytes, but translated in female gametes. The protein persists until mature ookinetes; this expression is strictly dependent on the maternally derived expression. Genetic crosses revealed that actin2 functions at an early stage of ookinete formation and that parasites lacking actin2 are unable to undergo sporogony in the mosquito midgut. Our results provide insights into the specialized role of actin2 in Plasmodium development in the mosquito and suggest that the two actin isoforms have distinct biological functions.

High-speed holographic microscopy of malaria parasites reveals ambidextrous flagellar waveforms.

Axonemes form the core of eukaryotic flagella and cilia, performing tasks ranging from transporting fluid in developing embryos to the propulsion of sperm. Despite their abundance across the eukaryotic domain, the mechanisms that regulate the beating action of axonemes remain unknown. The flagellar waveforms are 3D in general, but current understanding of how axoneme components interact stems from 2D data; comprehensive measurements of flagellar shape are beyond conventional microscopy. Moreover, current flagellar model systems (e.g., sea urchin, human sperm) contain accessory structures that impose mechanical constraints on movement, obscuring the "native" axoneme behavior. We address both problems by developing a high-speed holographic imaging scheme and applying it to the (male) microgametes of malaria (Plasmodium) parasites. These isolated flagella are a unique, mathematically tractable model system for the physics of microswimmers. We reveal the 3D flagellar waveforms of these microorganisms and map the differential shear between microtubules in their axonemes. Furthermore, we overturn claims that chirality in the structure of the axoneme governs the beat pattern [Hirokawa N, et al. (2009) Ann Rev Fluid Mech 41:53-72], because microgametes display a left- or right-handed character on alternate beats. This breaks the link between structural chirality in the axoneme and larger scale symmetry breaking (e.g., in developing embryos), leading us to conclude that accessory structures play a critical role in shaping the flagellar beat.

Metabolite extract of Streptomyces hygroscopicus Hygroscopicus inhibit the growth of Plasmodium berghei through inhibition of ubiquitin - proteasome system.

Streptomyces hygroscopicus Hygroscopicus, a member of family of Actinomycetes produces eponemycin a proteasome inhibitor that can inhibit Ubiquitin-Proteasome System (UPS) function in eukaryotic cell. Previous study showed that coronamycin, an active substrate isolated from Streptomyces sp. can act as anti-plasmodial, antibacterial, and antifungal, however the research did not show the mechanism of coronamycin in inhibiting the growth of Plasmodium. This research was done to reveal if eponemycin that is contained in metabolite extract of S. hygroscopicus can inhibit UPS function of Plasmodium berghei. This study was an experimental study using P. berghei infected Balb/C mice as malaria model. Samples were divided into 1 control group (group infected with P. berghei without treatment) and 3 treatment groups (mice infected with P. berghei and treated intra-peritoneal with metabolite extract of S. hygroscopicus dose 130 µg/kgBW, 580 µg/kgBW, and 2600 µg/kgBW for 5 days). The degree of parasitemia and morphology of the parasite were measured from the first day of malaria induction until the last treatment. The accumulation level of polyubiquitin was measured using Western blot and ELISA method. The degree of parasitemia on day 6 showed significant differences among treatment groups and control (p=0,000). Percentage of inhibition showed significant differences between control and group treated with metabolite extract of S. hygroscopicus 2600 µg/kgBW. An increasing dose of extract of S. hygroscopicus followed by an increasing of inhibition in parasite growth (r=0,850). Probit analysis showed that ED50 was 9.418 µg/kgBW. There was a change in morphology of the parasite after treatment. Parasite morphology became crisis form. There was an accumulation of polyubiquitinated protein in the group treated with metabolite extract of S. hygroscopicus 2600 µg/kgBW. It can be concluded that analog eponemycin in metabolite of S. hygroscopicus is a potential candidate for new malarial drug by inhibiting UPS function of the parasite and cause stress and dead of the parasite.

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.

Morphogenesis of Plasmodium zoites is uncoupled from tensile strength.

A shared feature of the motile stages (zoites) of malaria parasites is a cortical cytoskeletal structure termed subpellicular network (SPN), thought to define and maintain cell shape. Plasmodium alveolins comprise structural components of the SPN, and alveolin gene knockout causes morphological abnormalities that coincide with markedly reduced tensile strength of the affected zoites, indicating the alveolins are prime cell shape determinants. Here, we characterize a novel SPN protein of Plasmodium berghei ookinetes and sporozoites named G2 (glycine at position 2), which is structurally unrelated to alveolins. G2 knockout abolishes parasite transmission and causes zoite malformations and motility defects similar to those observed in alveolin null mutants. Unlike alveolins, however, G2 contributes little to tensile strength, arguing against a cause-effect relationship between tensile strength and cell shape. We also show that G2 null mutant sporozoites display an abnormal arrangement of their subpellicular microtubules. These results provide important new understanding of the factors that determine zoite morphogenesis, as well as the potential roles of the cortical cytoskeleton in gliding motility.

Phenotypic dissection of a Plasmodium-refractory strain of malaria vector Anopheles stephensi: the reduced susceptibility to P. berghei and P. yoelii.

Anopheline mosquitoes are the major vectors of human malaria. Parasite-mosquito interactions are a critical aspect of disease transmission and a potential target for malaria control. Current investigations into parasite-mosquito interactions frequently assume that genetically resistant and susceptible mosquitoes exist in nature. Therefore, comparisons between the Plasmodium susceptibility profiles of different mosquito species may contribute to a better understanding of vectorial capacity. Anopheles stephensi is an important malaria vector in central and southern Asia and is widely used as a laboratory model of parasite transmission due to its high susceptibility to Plasmodium infection. In the present study, we identified a rodent malaria-refractory strain of A. stephensi mysorensis (Ehime) by comparative study of infection susceptibility. A very low number of oocysts develop in Ehime mosquitoes infected with P. berghei and P. yoelii, as determined by evaluation of developed oocysts on the basal lamina. A stage-specific study revealed that this reduced susceptibility was due to the impaired formation of ookinetes of both Plasmodium species in the midgut lumen and incomplete crossing of the midgut epithelium. There were no apparent abnormalities in the exflagellation of male parasites in the ingested blood or the maturation of oocysts after the rounding up of the ookinetes. Overall, these results suggest that invasive-stage parasites are eliminated in both the midgut lumen and epithelium in Ehime mosquitoes by strain-specific factors that remain unknown. The refractory strain newly identified in this report would be an excellent study system for investigations into novel parasite-mosquito interactions in the mosquito midgut.

Role of host cell traversal by the malaria sporozoite during liver infection.

Malaria infection starts when the sporozoite stage of the Plasmodium parasite is injected into the skin by a mosquito. Sporozoites are known to traverse host cells before finally invading a hepatocyte and multiplying into erythrocyte-infecting forms, but how sporozoites reach hepatocytes in the liver and the role of host cell traversal (CT) remain unclear. We report the first quantitative imaging study of sporozoite liver infection in rodents. We show that sporozoites can cross the liver sinusoidal barrier by multiple mechanisms, targeting Kupffer cells (KC) or endothelial cells and associated or not with the parasite CT activity. We also show that the primary role of CT is to inhibit sporozoite clearance by KC during locomotion inside the sinusoid lumen, before crossing the barrier. By being involved in multiple steps of the sporozoite journey from the skin to the final hepatocyte, the parasite proteins mediating host CT emerge as ideal antibody targets for vaccination against the parasite.

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.