The Basal Complex Protein PfMORN1 Is Not Required for Asexual Replication of Plasmodium falciparum.

Plasmodium falciparum, the Apicomplexan parasite that causes the most severe form of human malaria, divides via schizogony during the asexual blood stage of its life cycle. In this method of cell division, multiple daughter cells are generated from a single schizont by segmentation. During segmentation, the basal complex forms at the basal end of the nascent daughter parasites and likely facilitates cell shape and cytokinesis. The requirement and function for each of the individual protein components within the basal complex remain largely unknown in P. falciparum. In this work, we demonstrate that the P. falciparum membrane occupation and recognition nexus repeat-containing protein 1 (PfMORN1) is not required for asexual replication. Following inducible knockout of PfMORN1, we find no detectable defect in asexual parasite morphology or replicative fitness. IMPORTANCE Plasmodium falciparum parasites cause the most severe form of human malaria. During the clinically relevant blood stage of its life cycle, the parasites divide via schizogony. In this divergent method of cell division, the components for multiple daughter cells are generated within a common cytoplasm. At the end of schizogony, segmentation partitions the organelles into invasive daughter parasites. The basal complex is a ring-shaped molecular machine that is critical for segmentation. The requirement for individual proteins within the basal complex is incompletely understood. We demonstrate that the PfMORN1 protein is dispensable for blood stage replication of P. falciparum. This result highlights important differences between Plasmodium parasites and Toxoplasma gondii, where the ortholog T. gondii MORN1 (TgMORN1) is required for asexual replication.

Mechanisms of triggering malaria gametocytogenesis by AP2-G.

The transcription factor (TF) AP2-G is essential for gametocytogenesis in the malaria parasite; however, it remains unclear if AP2-G determines commitment to sexual stage development fate in the schizont stage, or whether AP2-G directly initiates sexual stage differentiation and development beginning in the late-trophozoite stage. In this study, we addressed this issue by investigating the expression profile of AP2-G and determining genome-wide target genes in Plasmodium berghei. Fluorescence microscopy showed that AP2-G expression was first observed in the parasite 12 h after erythrocyte invasion and peaked at 18 h when sexual features were first manifested in early gametocytes. Expression of AP2-G decreased with manifestation of sex-specific features. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) was performed at peak AP2-G expression and identified over 1000 binding sites in the genome. The main binding motif of the TF predicted from the binding sites was GTACNY. Predicted targets contained a number of genes related to protein biogenesis, suggesting that AP2-G plays a role in establishing a cellular basis required for sexual differentiation. AP2-G binding sites also existed upstream of gametocyte-specific TFs, namely AP2-G2, AP2-FG, and AP2-G itself. Furthermore, the target contained two AP2 TF-related genes. Disruption of these genes resulted in the arrest of ookinete development. These results suggest another role of AP2-G: activating a transcriptional cascade to promote conversion into early gametocytes. Taken together, AP2-G is involved not in establishing sexual commitment of schizonts, but rather in triggering the initiation of differentiation and the early development of gametocytes in the late trophozoite stage.

Malaria Parasite Schizont Egress Antigen-1 Plays an Essential Role in Nuclear Segregation during Schizogony.

Malaria parasites cause disease through repeated cycles of intraerythrocytic proliferation. Within each cycle, several rounds of DNA replication produce multinucleated forms, called schizonts, that undergo segmentation to form daughter merozoites. Upon rupture of the infected cell, the merozoites egress to invade new erythrocytes and repeat the cycle. In human malarial infections, an antibody response specific for the Plasmodium falciparum protein PF3D7_1021800 was previously associated with protection against malaria, leading to an interest in PF3D7_1021800 as a candidate vaccine antigen. Antibodies to the protein were reported to inhibit egress, resulting in it being named schizont egress antigen-1 (SEA1). A separate study found that SEA1 undergoes phosphorylation in a manner dependent upon the parasite cGMP-dependent protein kinase PKG, which triggers egress. While these findings imply a role for SEA1 in merozoite egress, this protein has also been implicated in kinetochore function during schizont development. Therefore, the function of SEA1 remains unclear. Here, we show that P. falciparum SEA1 localizes in proximity to centromeres within dividing nuclei and that conditional disruption of SEA1 expression severely impacts the distribution of DNA and formation of merozoites during schizont development, with a proportion of SEA1-null merozoites completely lacking nuclei. SEA1-null schizonts rupture, albeit with low efficiency, suggesting that neither SEA1 function nor normal segmentation is a prerequisite for egress. We conclude that SEA1 does not play a direct mechanistic role in egress but instead acts upstream of egress as an essential regulator required to ensure the correct packaging of nuclei within merozoites.IMPORTANCE Malaria is a deadly infectious disease. Rationally designed novel therapeutics will be essential for its control and eradication. The Plasmodium falciparum protein PF3D7_1021800, annotated as SEA1, is under investigation as a prospective component of a malaria vaccine, based on previous indications that antibodies to SEA1 interfere with parasite egress from infected erythrocytes. However, a consensus on the function of SEA1 is lacking. Here, we demonstrate that SEA1 localizes to dividing parasite nuclei and is necessary for the correct segregation of replicated DNA into individual daughter merozoites. In the absence of SEA1, merozoites develop defectively, often completely lacking a nucleus, and, consequently, egress is impaired and/or aberrant. Our findings provide insights into the divergent mechanisms by which intraerythrocytic malaria parasites develop and divide. Our conclusions regarding the localization and function of SEA1 are not consistent with the hypothesis that antibodies against it confer protective immunity to malaria by blocking merozoite egress.

Depletion of the mini-chromosome maintenance complex binding protein allows the progression of cytokinesis despite abnormal karyokinesis during the asexual development of Plasmodium falciparum.

The eukaryotic cell cycle is typically divided into distinct phases with cytokinesis immediately following mitosis. To ensure proper cell division, each phase is tightly coordinated via feedback controls named checkpoints. During its asexual replication cycle, the malaria parasite Plasmodium falciparum undergoes multiple asynchronous rounds of mitosis with segregation of uncondensed chromosomes followed by nuclear division with intact nuclear envelope. The multi-nucleated schizont is then subjected to a single round of cytokinesis that produces dozens of daughter cells called merozoites. To date, no cell cycle checkpoints have been identified that regulate the Plasmodium spp. mode of division. Here, we identify the Plasmodium homologue of the Mini-Chromosome Maintenance Complex Binding Protein (PfMCMBP), which co-purified with the Mini-Chromosome Maintenance (MCM) complex, a replicative helicase required for genomic DNA replication. By conditionally depleting PfMCMBP, we disrupt nuclear morphology and parasite proliferation without causing a block in DNA replication. By immunofluorescence microscopy, we show that PfMCMBP depletion promotes the formation of mitotic spindle microtubules with extensions to more than one DNA focus and abnormal centrin distribution. Strikingly, PfMCMBP-deficient parasites complete cytokinesis and form aneuploid merozoites with variable cellular and nuclear sizes. Our study demonstrates that the parasite lacks a robust checkpoint response to prevent cytokinesis following aberrant karyokinesis.

Transition from Plasmodial Hypnozoite to Schizont Demonstrated.

The progression to schizont formation of individual activated hypnozoites has been observed in vitro for the first time by Voorberg-van der Wel et al. Green-fluorescent protein-positive hypnozoites turned red-fluorescent (mCherry) upon activation. Thus, we now have empirical parasitological proof that supports the 40-year-old hypnozoite theory of relapse in malaria.

The malaria parasite Plasmodium falciparum in red blood cells selectively takes up serum proteins that affect host pathogenicity.

BACKGROUND: The malaria parasite Plasmodium falciparum is a protozoan that develops in red blood cells (RBCs) and requires various host factors. For its development in RBCs, nutrients not only from the RBC cytosol but also from the extracellular milieu must be acquired. Although the utilization of host nutrients by P. falciparum has been extensively analysed, only a few studies have reported its utilization of host serum proteins. Hence, the aim of the current study was to comprehensively identify host serum proteins taken up by P. falciparum parasites and to elucidate their role in pathogenesis. METHODS: Plasmodium falciparum was cultured with human serum in vitro. Uptake of serum proteins by parasites was comprehensively determined via shotgun liquid chromatography-mass spectrometry/mass spectrometry and western blotting. The calcium ion concentration in serum was also evaluated, and coagulation activity of the parasite lysate was assessed. RESULTS: Three proteins, vitamin K-dependent protein S, prothrombin, and vitronectin, were selectively internalized under sufficient Ca2+ levels in the culture medium. The uptake of these proteins was initiated before DNA replication, and increased during the trophozoite and schizont stages, irrespective of the assembly/disassembly of actin filaments. Coagulation assay revealed that prothrombin was activated and thereby induced blood coagulation. CONCLUSIONS: Serum proteins were taken up by parasites under culture conditions with sufficient Ca2+ levels. This uptake phenomenon was associated with their pathogenicity.

Interplay of Plasmodium falciparum and thrombin in brain endothelial barrier disruption.

Recent concepts suggest that both Plasmodium falciparum factors and coagulation contribute to endothelial activation and dysfunction in pediatric cerebral malaria (CM) pathology. However, there is still limited understanding of how these complex inflammatory stimuli are integrated by brain endothelial cells. In this study, we examined how mature-stage P. falciparum infected erythrocytes (IE) interact with tumor necrosis factor α (TNFα) and thrombin in the activation and permeability of primary human brain microvascular endothelial cell (HBMEC) monolayers. Whereas trophozoite-stage P. falciparum-IE have limited effect on the viability of HBMEC or the secretion of pro-inflammatory cytokines or chemokines, except at super physiological parasite-host cell ratios, schizont-stage P. falciparum-IE induced low levels of cell death. Additionally, schizont-stage parasites were more barrier disruptive than trophozoite-stage P. falciparum-IE and prolonged thrombin-induced barrier disruption in both resting and TNFα-activated HBMEC monolayers. These results provide evidence that parasite products and thrombin may interact to increase brain endothelial permeability.

Transitions in morphological forms and rapid development of the asexual schizonts of Eimeria tenella through serial passaging in chicks.

Attenuated strains of avian Eimeria parasites, generated by the selection of precocious lines through serial passaging in chicks, have been used widely as live vaccines. Detailed morphological transitions including their life cycle depending on the passages remain poorly understood. Here, we showed early development and acceleration of transitions in morphological forms of the asexual schizonts of E. tenella that had been attenuated for virulence by serial passaging. Our results may be helpful in understanding parasitism, facilitating further molecular analyses such as comparative genomic or transcriptomic tests.

An essential contractile ring protein controls cell division in Plasmodium falciparum.

During the blood stage of human malaria, Plasmodium falciparum parasites divide by schizogony-a process wherein components for several daughter cells are produced within a common cytoplasm and then segmentation, a synchronized cytokinesis, produces individual invasive daughters. The basal complex is hypothesized to be required for segmentation, acting as a contractile ring to establish daughter cell boundaries. Here we identify an essential component of the basal complex which we name PfCINCH. Using three-dimensional reconstructions of parasites at electron microscopy resolution, we show that while parasite organelles form and divide normally, PfCINCH-deficient parasites develop inviable conjoined daughters that contain components for multiple cells. Through biochemical evaluation of the PfCINCH-containing complex, we discover multiple previously undescribed basal complex proteins. Therefore, this work provides genetic evidence that the basal complex is required for precise segmentation and lays the groundwork for a mechanistic understanding of how the parasite contractile ring drives cell division.

Cutting Edge: FHR-1 Binding Impairs Factor H-Mediated Complement Evasion by the Malaria Parasite Plasmodium falciparum.

Human complement is the first line of defense against invading pathogens, including the malaria parasite Plasmodium falciparum We previously demonstrated that human complement represents a particular threat for the clinically relevant blood stages of the parasite. To evade complement-mediated destruction, the parasites acquire factor H (FH) via specific receptors. We now report that the FH-related protein FHR-1 competes with FH for binding to the parasites. FHR-1, which is composed of five complement control protein domains with variable homology to FH but lacks C3b regulatory activity, accumulates on the surfaces of intraerythrocytic schizonts and free merozoites. Although binding of FH to schizont-infected RBCs and merozoites is increased in FHR-1-deficient human serum, the addition of recombinant FHR-1 decreases FH binding. The presence of FHR-1 consequently impairs C3b inactivation and parasite viability. We conclude that FHR-1 acts as a protective factor in human immunity by counteracting FH-mediated microbial complement evasion.

Modelling dynamic change of malaria transmission in holoendemic setting (Dielmo, Senegal) using longitudinal measures of antibody prevalence to Plasmodium falciparum crude schizonts extract.

BACKGROUND: Evaluation of local Plasmodium falciparum malaria transmission has been investigated previously using the reversible catalytic model based on prevalence of antibody responses to single antigen to estimate seroconversion rates. High correlations were observed between seroconversion rates and entomological inoculation rates (EIR). However, in this model, the effects of malaria control interventions and clinical episodes on serological measurements were not assessed. This study monitors the use of antibody responses to P. falciparum crude extracts for assessing malaria transmission, compares seroconversion rates estimated from longitudinal data to those derived from cross-sectional surveys and investigates the effects of malaria control interventions on these measures in an area of declining malaria transmission. In addition, the validity of this model was evaluated by comparison with the alternative model. METHODS: Five cross-sectional surveys were carried out at the end of the wet season in Dielmo, a malaria-endemic Senegalese rural area in 2000, 2002, 2008, 2010 and 2012. Antibodies against schizonts crude extract of a local P. falciparum strain adapted to culture (Pf 07/03) were measured by ELISA. Age-specific seroprevalence model was used both for cross-sectional surveys and longitudinal data (combined data of all surveys). RESULTS: A total of 1504 plasma samples obtained through several years follow-up of 350 subjects was used in this study. Seroconversion rates based on P. falciparum schizonts crude extract were estimated for each cross-sectional survey and were found strongly correlated with EIR. High variability between SCRs from cross-sectional and longitudinal surveys was observed. In longitudinal studies, the alternative catalytic reversible model adjusted better with serological data than the catalytic model. Clinical malaria attacks and malaria control interventions were found to have significant effect on seroconversion. DISCUSSION: The results of the study suggested that crude extract was a good serological tool that could be used to assess the level of malaria exposure in areas where malaria transmission is declining. However, additional parameters such as clinical malaria and malaria control interventions must be taken into account for determining serological measurements for more accuracy in transmission assessment.

Ned-19 inhibition of parasite growth and multiplication suggests a role for NAADP mediated signalling in the asexual development of Plasmodium falciparum.

BACKGROUND: Although malaria is a preventable and curable human disease, millions of people risk to be infected by the Plasmodium parasites and to develop this illness. Therefore, there is an urgent need to identify new anti-malarial drugs. Ca2+ signalling regulates different processes in the life cycle of Plasmodium falciparum, representing a suitable target for the development of new drugs. RESULTS: This study investigated for the first time the effect of a highly specific inhibitor of nicotinic acid adenine dinucleotide phosphate (NAADP)-induced Ca2+ release (Ned-19) on P. falciparum, revealing the inhibitory effect of this compound on the blood stage development of this parasite. Ned-19 inhibits both the transition of the parasite from the early to the late trophozoite stage and the ability of the late trophozoite to develop to the multinucleated schizont stage. In addition, Ned-19 affects spontaneous intracellular Ca2+ oscillations in ring and trophozoite stage parasites, suggesting that the observed inhibitory effects may be associated to regulation of intracellular Ca2+ levels. CONCLUSIONS: This study highlights the inhibitory effect of Ned-19 on progression of the asexual life cycle of P. falciparum. The observation that Ned-19 inhibits spontaneous Ca2+ oscillations suggests a potential role of NAADP in regulating Ca2+ signalling of P. falciparum.

Generation of transgenic rodent malaria parasites by transfection of cell culture-derived merozoites.

BACKGROUND: Malaria research is greatly dependent on and has drastically advanced with the possibility of genetically modifying Plasmodium parasites. The commonly used transfection protocol by Janse and colleagues utilizes blood stage-derived Plasmodium berghei schizonts that have been purified from a blood culture by density gradient centrifugation. Naturally, this transfection protocol depends on the availability of suitably infected mice, constituting a time-based variable. In this study, the potential of transfecting liver stage-derived merozoites was explored. In cell culture, upon merozoite development, infected cells detach from the neighbouring cells and can be easily harvested from the cell culture supernatant. This protocol offers robust experimental timing and temporal flexibility. METHODS: HeLa cells are infected with P. berghei sporozoites to obtain liver stage-derived merozoites, which are harvested from the cell culture supernatant and are transfected using the Amaxa Nucleofector® electroporation technology. RESULTS: Using this protocol, wild type P. berghei ANKA strain and marker-free PbmCherryHsp70-expressing P. berghei parasites were successfully transfected with DNA constructs designed for integration via single- or double-crossover homologous recombination. CONCLUSION: An alternative protocol for Plasmodium transfection is hereby provided, which uses liver stage-derived P. berghei merozoites for transfection. This protocol has the potential to substantially reduce the number of mice used per transfection, as well as to increase the temporal flexibility and robustness of performing transfections, if mosquitoes are routinely present in the laboratory. Transfection of liver stage-derived P. berghei parasites should enable generation of transgenic parasites within 8-18 days.

Plasmodium falciparum CRK4 directs continuous rounds of DNA replication during schizogony.

Plasmodium parasites, the causative agents of malaria, have evolved a unique cell division cycle in the clinically relevant asexual blood stage of infection1. DNA replication commences approximately halfway through the intracellular development following invasion and parasite growth. The schizont stage is associated with multiple rounds of DNA replication and nuclear division without cytokinesis, resulting in a multinucleated cell. Nuclei divide asynchronously through schizogony, with only the final round of DNA replication and segregation being synchronous and coordinated with daughter cell assembly2,3. However, the control mechanisms for this divergent mode of replication are unknown. Here, we show that the Plasmodium-specific kinase PfCRK4 is a key cell-cycle regulator that orchestrates multiple rounds of DNA replication throughout schizogony in Plasmodium falciparum. PfCRK4 depletion led to a complete block in nuclear division and profoundly inhibited DNA replication. Quantitative phosphoproteomic profiling identified a set of PfCRK4-regulated phosphoproteins with greatest functional similarity to CDK2 substrates, particularly proteins involved in the origin of replication firing. PfCRK4 was required for initial and subsequent rounds of DNA replication during schizogony and, in addition, was essential for development in the mosquito vector. Our results identified an essential S-phase promoting factor of the unconventional P. falciparum cell cycle. PfCRK4 is required for both a prolonged period of the intraerythrocytic stage of Plasmodium infection, as well as for transmission, revealing a broad window for PfCRK4-targeted chemotherapeutics.

Study of Plasmodium falciparum DHHC palmitoyl transferases identifies a role for PfDHHC9 in gametocytogenesis.

Palmitoylation is the post-translational reversible addition of the acyl moiety, palmitate, to cysteine residues of proteins and is involved in regulating protein trafficking, localization, stability and function. The Aspartate-Histidine-Histidine-Cysteine (DHHC) protein family, named for their highly conserved DHHC signature motif, is thought to be responsible for catalysing protein palmitoylation. Palmitoylation is widespread in all eukaryotes, including the malaria parasite, Plasmodium falciparum, where over 400 palmitoylated proteins are present in the asexual intraerythrocytic schizont stage parasites, including proteins involved in key aspects of parasite maturation and development. The P. falciparum genome includes 12 proteins containing the conserved DHHC motif. In this study, we adapted a palmitoyl-transferase activity assay for use with P. falciparum proteins and demonstrated for the first time that P. falciparum DHHC proteins are responsible for the palmitoylation of P. falciparum substrates. This assay also reveals that multiple DHHCs are capable of palmitoylating the same substrate, indicating functional redundancy at least in vitro. To test whether functional redundancy also exists in vivo, we investigated the endogenous localization and essentiality of a subset of schizont-expressed PfDHHC proteins. Individual PfDHHC proteins localized to distinct organelles, including parasite-specific organelles such as the rhoptries and inner membrane complex. Knock-out studies identified individual DHHCs that may be essential for blood-stage growth and others that were functionally redundant in the blood stages but may have functions in other stages of parasite development. Supporting this hypothesis, disruption of PfDHHC9 had no effect on blood-stage growth but reduced the formation of gametocytes, suggesting that this protein could be exploited as a transmission-blocking target. The localization and stage-specific expression of the DHHC proteins may be important for regulating their substrate specificity and thus may provide a path for inhibitor development.

In vivo photoacoustic flow cytometry for early malaria diagnosis.

In vivo photoacoustic (PA) flow cytometry (PAFC) has already demonstrated a great potential for the diagnosis of deadly diseases through ultrasensitive detection of rare disease-associated circulating markers in whole blood volume. Here, we demonstrate the first application of this powerful technique for early diagnosis of malaria through label-free detection of malaria parasite-produced hemozoin in infected red blood cells (iRBCs) as high-contrast PA agent. The existing malaria tests using blood smears can detect the disease at 0.001-0.1% of parasitemia. On the contrary, linear PAFC showed a potential for noninvasive malaria diagnosis at an extremely low level of parasitemia of 0.0000001%, which is ∼10(3) times better than the existing tests. Multicolor time-of-flight PAFC with high-pulse repetition rate lasers at wavelengths of 532, 671, and 820 nm demonstrated rapid spectral and spatial identification and quantitative enumeration of individual iRBCs. Integration of PAFC with fluorescence flow cytometry (FFC) provided real-time simultaneous detection of single iRBCs and parasites expressing green fluorescence proteins, respectively. A combination of linear and nonlinear nanobubble-based multicolor PAFC showed capability to real-time control therapy efficiency by counting of iRBCs before, during, and after treatment. Our results suggest that high-sensitivity, high-resolution ultrafast PAFC-FFC platform represents a powerful research tool to provide the insight on malaria progression through dynamic study of parasite-cell interactions directly in bloodstream, whereas portable hand-worn PAFC device could be broadly used in humans for early malaria diagnosis. © 2016 International Society for Advancement of Cytometry.

Pellicle formation in the malaria parasite.

The intraerythrocytic developmental cycle of Plasmodium falciparum is completed with the release of up to 32 invasive daughter cells, the merozoites, into the blood stream. Before release, the final step of merozoite development is the assembly of the cortical pellicle, a multi-layered membrane structure. This unique apicomplexan feature includes the inner membrane complex (IMC) and the parasite's plasma membrane. A dynamic ring structure, referred to as the basal complex, is part of the IMC and helps to divide organelles and abscises in the maturing daughter cells. Here, we analyze the dynamics of the basal complex of P. falciparum. We report on a novel transmembrane protein of the basal complex termed BTP1, which is specific to the genus Plasmodium. It colocalizes with the known basal complex marker protein MORN1 and shows distinct dynamics as well as localization when compared to other IMC proteins during schizogony. Using a parasite plasma membrane marker cell line, we correlate dynamics of the basal complex with the acquisition of the maternal membrane. We show that plasma membrane invagination and IMC propagation are interlinked during the final steps of cell division.

Effect of Exposure Dose on Ichthyophonus Prevalence and Infection Intensity in Experimentally Infected Rainbow Trout, Oncorhynchus mykiss.

This study describes the effect of increasing exposure dose on Ichthyophonus prevalence and infection intensity in experimentally infected rainbow trout, Oncorhynchus mykiss. Specific-pathogen free trout were exposed per os to increasing numbers of Ichthyophonus schizonts obtained from naturally infected donor fish, then sampled after 30 and 60 days post-exposure. Both in vitro explant culture and histology revealed that as the number of schizonts per dose increased there was a proportionate increase in the number of infected fish, as well as an increase in the number of infected organs; parasite density in individual infected organs also increased with dose. Explant culture revealed that all fish exposed to the highest dose (≥2,080 schizonts) became infected, while only 67% of those exposed to the intermediate dose (1,040-1,153 schizonts) were Ichthyophonus-positive after 60 days; Ichthyophonus was not detected in fish exposed to the 2 lowest doses (≤280 schizonts). Histologic examination of individual infected organs also revealed increasing infection prevalence and parasite density in response to exposure to increasing numbers of Ichthyophonus schizonts.

Phosphoproteomics reveals malaria parasite Protein Kinase G as a signalling hub regulating egress and invasion.

Our understanding of the key phosphorylation-dependent signalling pathways in the human malaria parasite, Plasmodium falciparum, remains rudimentary. Here we address this issue for the essential cGMP-dependent protein kinase, PfPKG. By employing chemical and genetic tools in combination with quantitative global phosphoproteomics, we identify the phosphorylation sites on 69 proteins that are direct or indirect cellular targets for PfPKG. These PfPKG targets include proteins involved in cell signalling, proteolysis, gene regulation, protein export and ion and protein transport, indicating that cGMP/PfPKG acts as a signalling hub that plays a central role in a number of core parasite processes. We also show that PfPKG activity is required for parasite invasion. This correlates with the finding that the calcium-dependent protein kinase, PfCDPK1, is phosphorylated by PfPKG, as are components of the actomyosin complex, providing mechanistic insight into the essential role of PfPKG in parasite egress and invasion.

Regulation of Plasmodium falciparum†Origin Recognition Complex subunit 1 (PfORC1) function through phosphorylation mediated by CDK-like kinase PK5.

Plasmodium falciparum Origin Recognition Complex subunit 1 (PfORC1) has been implicated in DNA replication and var gene regulation. While the C-terminus is involved in DNA replication, the specific role of N-terminus has been suggested in var gene regulation in a Sir2-dependent manner. PfORC1 is localized at the nuclear periphery, where the clustering of chromosomal ends at the early stage of parasite development may be crucial for the regulation of subtelomeric var gene expression. Upon disassembly of telomeric clusters at later stages of parasite development, ORC1 is distributed in the nucleus and parasite cytoplasm where it may be required for its other cellular functions including DNA replication. The level of ORC1 decreases dramatically at the late schizont stage. The mechanisms that mediate regulation of PfORC1 function are largely unknown. Here we show, by the use of recombinant proteins and of transgenic parasites expressing wild type or mutant forms of ORC1, that phosphorylation of the PfORC1-N terminal domain by the cyclin-dependent kinase (CDK) PfPK5 abolishes DNA-binding activity and leads to changes in subcellular localization and proteasome-mediated degradation of the protein in schizonts. These results reveal that PfORC1 phosphorylation by a CDK is central to the regulation of important biological functions like DNA replication and var gene silencing.