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Malaria remains a heavy public health and socioeconomic burden in tropical and subtropical regions. Increasing resistance against front-line treatments implies that novel targets for antimalarial intervention are urgently required. Protein kinases of both the parasites and their host cells possess strong potential in this respect. We present an overview of the updated kinome of Plasmodium falciparum, the species that is the largest contributor to malaria mortality, and of current knowledge pertaining to the function of parasite-encoded protein kinases during the parasite's life cycle. Furthermore, we detail recent advances in drug initiatives targeting Plasmodium kinases and outline the potential of protein kinases in the context of the growing field of host-directed therapies, which is currently being explored as a novel way to combat parasite drug resistance.

Malaria is a mosquito-borne disease caused by apicomplexan parasites of the genus Plasmodium. Completion of the parasite’s life cycle depends on the transmission of sexual stages, the gametocytes, from an infected human host to the mosquito vector. Sexual commitment occurs in only a small fraction of asexual blood-stage parasites and is initiated by external cues. The gametocyte development protein 1 (GDV1) has been described as a key facilitator to trigger sexual commitment. GDV1 interacts with the silencing factor heterochromatin protein 1 (HP1), leading to its dissociation from heterochromatic DNA at the genomic locus encoding AP2-G, the master transcription factor of gametocytogenesis. How this process is regulated is not known. In this study, we have addressed the role of protein kinases implicated in gametocyte development. From a pool of available protein kinase knockout (KO) lines, we identified two kinase knockout lines which fail to produce gametocytes. However, independent genetic verification revealed that both kinases are not required for gametocytogenesis but that both lines harbor the same mutation that leads to a truncation in the extreme C terminus of GDV1. Introduction of the identified nonsense mutation into the genome of wild-type parasite lines replicates the observed phenotype. Using a GDV1 overexpression line, we show that the truncation in the GDV1 C terminus does not interfere with the nuclear import of GDV1 or its interaction with HP1 in vitro but appears to be important to sustain GDV1 protein levels and thereby sexual commitment. IMPORTANCE Transmission of malaria-causing Plasmodium species by mosquitos requires the parasite to change from a continuously growing asexual parasite form growing in the blood to a sexually differentiated form, the gametocyte. Only a small subset of asexual parasites differentiates into gametocytes that are taken up by the mosquito. Transmission represents a bottleneck in the life cycle of the parasite, so a molecular understanding of the events that lead to stage conversion may identify novel intervention points. Here, we screened a subset of kinases we hypothesized to play a role in this process. While we did not identify kinases required for sexual conversion, we identified a mutation in the C terminus of the gametocyte development 1 protein (GDV1), which abrogates sexual development. The mutation destabilizes the protein but not its interaction with its cognate binding partner HP1. This suggests an important role for the GDV1 C terminus beyond trafficking and protein stability.

It is well established that intracellular pathogens mobilise signalling pathways to manipulate gene expression of their host cell to promote their own survival. Surprisingly, there is evidence that specific host signalling molecules are likewise activated in a-nucleated erythrocytes in response to infection with malaria parasites. In this paper (Adderley et al., Nature Communications 2020), we report the system-wide assessment of host erythrocyte signalling during the course of infection with Plasmodium falciparum. This was achieved through the use of antibody microarrays containing >800 antibodies directed against human signalling proteins, which enabled us to interrogate the status of host erythrocyte signalling pathways at the ring, trophozoite and schizont stages of parasite development. This not only confirmed the pre-existing fragmentary data on the activation of a host erythrocyte PAK-MEK pathway, but also identified dynamic changes to many additional signalling elements, with trophozoite-infected erythrocytes displaying the largest mobilisation of host cell signalling. This study generated a comprehensive dataset on the modulation of host erythrocyte signalling during infection with P. falciparum, and provides the proof of principle that human protein kinases activated by Plasmodium infection represent attractive targets for antimalarial intervention.

Plasmodium species are evolutionarily distant from model eukaryotes, and as a consequence they exhibit many non-canonical cellular processes. In the post-genomic era, functional "omics" disciplines (transcriptomics, proteomics, and metabolomics) have accelerated our understanding of unique aspects of the biology of malaria parasites. Functional "omics" tools, in combination with genetic manipulations, have offered new opportunities to investigate the function of previously uncharacterized genes. Knowledge of basic parasite biology is fundamental to understanding drug modes of action, mechanisms of drug resistance, and relevance of vaccine candidates. This Perspective highlights recent "omics"-based discoveries in basic biology and gene function of the most virulent human malaria parasite, Plasmodium falciparum.

Intracellular pathogens mobilize host signaling pathways of their host cell to promote their own survival. Evidence is emerging that signal transduction elements are activated in a-nucleated erythrocytes in response to infection with malaria parasites, but the extent of this phenomenon remains unknown. Here, we fill this knowledge gap through a comprehensive and dynamic assessment of host erythrocyte signaling during infection with Plasmodium falciparum. We used arrays of 878 antibodies directed against human signaling proteins to interrogate the activation status of host erythrocyte phospho-signaling pathways at three blood stages of parasite asexual development. This analysis reveals a dynamic modulation of many host signalling proteins across parasite development. Here we focus on the hepatocyte growth factor receptor (c-MET) and the MAP kinase pathway component B-Raf, providing a proof of concept that human signaling kinases identified as activated by malaria infection represent attractive targets for antimalarial intervention., Plasmodium infection activates signaling pathways in a-nucleated erythrocytes. Here, Adderley et al. use a comprehensive antibody microarray to show that infection extensively modulates host cell signalling and that the host receptor tyrosine kinase c-MET supports Plasmodium falciparum proliferation.

Summary The search for antimalarial chemotypes with modes of action unrelated to existing drugs has intensified with the recent failure of first-line therapies across Southeast Asia. Here, we show that the trisubstituted imidazole MMV030084 potently inhibits hepatocyte invasion by Plasmodium sporozoites, merozoite egress from asexual blood stage schizonts, and male gamete exflagellation. Metabolomic, phosphoproteomic, and chemoproteomic studies, validated with conditional knockdown parasites, molecular docking, and recombinant kinase assays, identified cGMP-dependent protein kinase (PKG) as the primary target of MMV030084. PKG is known to play essential roles in Plasmodium invasion of and egress from host cells, matching MMV030084's activity profile. Resistance selections and gene editing identified tyrosine kinase-like protein 3 as a low-level resistance mediator for PKG inhibitors, while PKG itself never mutated under pressure. These studies highlight PKG as a resistance-refractory antimalarial target throughout the Plasmodium life cycle and promote MMV030084 as a promising Plasmodium PKG-targeting chemotype., Graphical Abstract, Highlights • MMV030084 inhibits P. falciparum liver and asexual blood stages and male gametes • Proteomic and conditional knockdown studies identified PfPKG as the target • Resistance selection studies identified TKL3 as a low-level resistance mediator • PKG is a promising resistance-refractory target for antimalarial drug development, Vanaerschot et al. report an antimalarial, MMV030084, with potent antiplasmodial activity against all stages of human infection by Plasmodium falciparum. Metabolomic, phosphoproteomic, chemoproteomic, and gene-editing studies identified cGMP-dependent protein kinase (PKG) as the primary target, which did not mutate under selective drug pressure.

Introduction:\ud Despite the positive effects of intervention strategies that include insecticide-impregnated bed nets and artemisinin-based drug therapies, malaria still kills nearly 500,000 people per year and infects more than 200 million individuals globally. This, together with the emerging resistance of the parasite to frontline antimalarials, means that there is an urgent need for novel treatments that not only offer a cure for malaria but also prevent transmission. We show that by inhibiting an essential protein kinase that is a key regulator of RNA processing, we are able to kill the parasite in the blood and liver stages as well as prevent the development of the sexual-stage gametocytes, thereby blocking transmission to the mosquito.\ud \ud Rationale:\ud Our group has previously published a list of 36 protein kinases that are essential for blood-stage survival of the most virulent form of the human malaria parasite, Plasmodium falciparum. Here, we focused on one of these protein kinases from the P. falciparum CLK (cyclin-dependent–like kinase) family, PfCLK3, and reasoned that inhibition of this protein kinase by a small drug-like molecule would be effective at killing blood-stage parasites. We further hypothesized that because PfCLK3 plays a key role in RNA splicing, inhibition of this kinase would be effective at killing the parasite at all stages of the life cycle where RNA splicing is required. This would include blood, liver, and sexual stages.\ud \ud Results:\ud By screening a focused library of nearly 30,000 compounds, we identified a probe molecule that selectively inhibited PfCLK3 and killed blood-stage P. falciparum. Using a combination of evolved resistance and chemogenetics, we established that our probe molecule had parasiticidal activity by inhibition of PfCLK3. We further showed that inhibition of PfCLK3 in parasites resulted in a reduction in more than 400 gene transcripts known to be essential for parasite survival. The finding that the vast majority of the genes down-regulated by PfCLK3 inhibition contained introns supported the notion that inhibition of PfCLK3 killed the malaria parasite by preventing the splicing of essential parasite genes. Because there is a high degree of homology between orthologs of CLK3 in other Plasmodium species, it might be expected that our probe molecule would both inhibit CLK3 contained in other malaria parasite species and have effective parasiticidal activity in these parasites. This was indeed found to be the case, with our molecule showing potent inhibition of CLK3 from P. vivax and P. berghei as well as killing the blood stages of P. berghei and P. knowlesi. Furthermore, we demonstrated that CLK3 inhibition also kills liver-stage P. berghei parasites and prevents P. berghei infection in mice. Finally, we showed that inhibition of PfCLK3 prevents the development of P. falciparum gametocytes, thereby blocking the infection of mosquitoes.\ud \ud Conclusion:\ud We found that inhibition of the essential malaria protein kinase CLK3 can kill multiple species of malaria parasites at the blood stage as well as killing liver-stage parasites and blocking transmission of the parasite to mosquitoes by preventing gametocyte development. In this way, we validate Plasmodium spp. CLK3 as a target that can offer prophylactic, curative, and transmission-blocking potential.

Plasmodium falciparum is the main cause of disease and death from malaria. P. falciparum virulence resides in the ability of infected erythrocytes (IEs) to sequester in various tissues through the interaction between members of the polymorphic P. falciparum erythrocyte membrane protein 1 (PfEMP1) adhesin family to various host receptors. Here, we investigated the effect of phosphorylation of variant surface antigen 2-CSA (VAR2CSA), a member of the PfEMP1 family associated to placental sequestration, on its capacity to adhere to chondroitin sulfate A (CSA) present on the placental syncytium. We showed that phosphatase treatment of IEs impairs cytoadhesion to CSA. MS analysis of recombinant VAR2CSA phosphosites prior to and after phosphatase treatment, as well as of native VAR2CSA expressed on IEs, identified critical phosphoresidues associated with CSA binding. Site-directed mutagenesis on recombinant VAR2CSA of 3 phosphoresidues localised within the CSA-binding region confirmed in vitro their functional importance. Furthermore, using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein-9 nuclease (CRISPR/Cas9), we generated a parasite line in which the phosphoresidue T934 is changed to alanine and showed that this mutation strongly impairs IEs cytoadhesion to CSA. Taken together, these results demonstrate that phosphorylation of the extracellular region of VAR2CSA plays a major role in IEs cytoadhesion to CSA and provide new molecular insights for strategies aiming to reduce the morbidity and mortality of PM., Placental sequestration of malaria-infected erythrocytes is mediated by the interaction between parasite VAR2CSA and the placental receptor chondroitin sulfate A; this study shows that phosphorylation of VAR2CSA plays a major role in this process, with implications for protecting pregnant women and their babies against placental malaria.

International audience; Wolbachia-infected mosquitoes are refractory to super-infection with arthropod-borne pathogens, but the role of host cell signaling proteins in pathogen-blocking mechanisms remains to be elucidated. Here, we use an antibody microarray approach to provide a comprehensive picture of the signaling response of Aedes aegypti-derived cells to Wolbachia. This approach identifies the host cell insulin receptor as being downregulated by the bacterium. Furthermore, siRNA-mediated knockdown and treatment with a small-molecule inhibitor of the insulin receptor kinase concur to assign a crucial role for this enzyme in the replication of dengue and Zika viruses in cultured mosquito cells. Finally, we show that the production of Zika virus in Wolbachia-free live mosquitoes is impaired by treatment with the selective inhibitor mimicking Wolbachia infection. This study identifies Wolbachia-mediated downregulation of insulin receptor kinase activity as a mechanism contributing to the blocking of super-infection by arboviruses.

Signal transduction and kinomics have been rapidly expanding areas of investigation within the malaria research field. Here, we provide an overview of phosphosignalling pathways that operate in all stages of the Plasmodium life cycle. We review signalling pathways in the parasite itself, in the cells it invades, and in other cells of the vertebrate host with which it interacts. We also discuss the potential of these pathways as novel targets for antimalarial intervention.

Aurora kinases are eukaryotic serine/threonine protein kinases that regulate key events associated with chromatin condensation, centrosome and spindle function and cytokinesis. Elucidating the roles of Aurora kinases in apicomplexan parasites is crucial to understand the cell cycle control during Plasmodium schizogony or Toxoplasma endodyogeny. Here, we report on the localization of two previously uncharacterized Toxoplasma Aurora-related kinases (Ark2 and Ark3) in tachyzoites and of the uncharacterized Ark3 orthologue in Plasmodium falciparum erythrocytic stages. In Toxoplasma gondii, we show that TgArk2 and TgArk3 concentrate at specific sub-cellular structures linked to parasite division: the mitotic spindle and intranuclear mitotic structures (TgArk2), and the outer core of the centrosome and the budding daughter cells cytoskeleton (TgArk3). By tagging the endogenous PfArk3 gene with the green fluorescent protein in live parasites, we show that PfArk3 protein expression peaks late in schizogony and localizes at the periphery of budding schizonts. Disruption of the TgArk2 gene reveals no essential function for tachyzoite propagation in vitro, which is surprising giving that the P. falciparum and P. berghei orthologues are essential for erythrocyte schizogony. In contrast, knock-down of TgArk3 protein results in pronounced defects in parasite division and a major growth deficiency. TgArk3-depleted parasites display several defects, such as reduced parasite growth rate, delayed egress and parasite duplication, defect in rosette formation, reduced parasite size and invasion efficiency and lack of virulence in mice. Our study provides new insights into cell cycle control in Toxoplasma and malaria parasites and highlights Aurora kinase 3 as potential drug target.

Malaria is a major global health burden, affecting over 200 million people worldwide. Resistance against all currently available antimalarial drugs is a growing threat, and represents a major and long-standing obstacle to malaria eradication. Like many intracellular pathogens, Plasmodium parasites manipulate host cell signaling pathways, in particular programmed cell death pathways. Interference with apoptotic pathways by malaria parasites is documented in the mosquito and human liver stages of infection, but little is known about this phenomenon in the erythrocytic stages. Although mature erythrocytes have lost all organelles, they display a form of programmed cell death termed eryptosis. Numerous features of eryptosis resemble those of nucleated cell apoptosis, including surface exposure of phosphatidylserine, cell shrinkage and membrane ruffling. Upon invasion, Plasmodium parasites induce significant stress to the host erythrocyte, while delaying the onset of eryptosis. Many eryptotic inducers appear to have a beneficial effect on the course of malaria infection in murine models, but major gaps remain in our understanding of the underlying molecular mechanisms. All currently available antimalarial drugs have parasite-encoded targets, which facilitates the emergence of resistance through selection of mutations that prevent drug-target binding. Identifying host cell factors that play a key role in parasite survival will provide new perspectives for host-directed anti-malarial chemotherapy. This review focuses on the interrelationship between Plasmodium falciparum and the eryptosis of its host erythrocyte. We summarize the current knowledge in this area, highlight the different schools of thoughts and existing gaps in knowledge, and discuss future perspectives for host-directed therapies in the context of antimalarial drug discovery.

Malaria causes every year over half-a-million deaths. The emergence of parasites resistant to available treatments makes the identification of new targets and their inhibitors an urgent task for the development of novel anti-malaria drugs. Protein kinase CK2 is an evolutionary-conserved eukaryotic serine/threonine protein kinase that in Plasmodium falciparum (PfCK2) has been characterized as a promising target for chemotherapeutic intervention against malaria. Here we report a crystallographic structure of the catalytic domain of PfCK2α (D179S inactive single mutant) in complex with ATP at a resolution of 3.0 Å. Compared to the human enzyme, the structure reveals a subtly altered ATP binding pocket comprising five substitutions in the vicinity of the adenine base, that together with potential allosteric sites, could be exploited to design novel inhibitors specifically targeting the Plasmodium enzyme. We provide evidence for the dual autophosphorylation of residues Thr63 and Tyr30 of PfCK2. We also show that CX4945, a human CK2 inhibitor in clinical trials against solid tumor cancers, is effective against PfCK2 with an IC50 of 13.2 nM.

Second messengers such as phosphoinositides and calcium are known to control diverse processes involved in the development of malaria parasites. However, the underlying molecular mechanisms and pathways need to be unraveled, which may be achieved by understanding the regulation of effectors of these second messengers. Calcium-dependent protein kinase (CDPK) family members regulate diverse parasitic processes. Because CDPKs are absent from the host, these kinases are considered as potential drug targets. We have dissected the function of an atypical CDPK from Plasmodium falciparum, PfCDPK7. The domain architecture of PfCDPK7 is very different from that of other CDPKs; it has a pleckstrin homology domain adjacent to the kinase domain and two calcium-binding EF-hands at its N terminus. We demonstrate that PfCDPK7 interacts with PI(4,5)P-2 via its pleckstrin homology domain, which may guide its subcellular localization. Disruption of PfCDPK7 caused a marked reduction in the growth of the blood stage parasites, as maturation of rings to trophozoites was markedly stalled. In addition, parasite proliferation was significantly attenuated. These findings shed light on an important role for PfCDPK7 in the erythrocytic asexual cycle of malaria parasites.

Background. The mechanism of action of artemisinins against malaria is unclear, despite their widespread use in combination therapies and the emergence of resistance. Results. Here, we report expression of PfATP6 (a SERCA pump) in yeast and demonstrate its inhibition by artemisinins. Mutations in PfATP6 identified in field isolates (such as S769N) and in laboratory clones (such as L263E) decrease susceptibility to artemisinins, whereas they increase susceptibility to unrelated inhibitors such as cyclopiazonic acid. As predicted from the yeast model, Plasmodium falciparum with the L263E mutation is also more susceptible to cyclopiazonic acid. An inability to knockout parasite SERCA pumps provides genetic evidence that they are essential in asexual stages of development. Thaperoxides are a new class of potent antimalarial designed to act by inhibiting PfATP6. Results in yeast confirm this inhibition. Conclusions. The identification of inhibitors effective against mutated PfATP6 suggests ways in which artemisinin resistance may be overcome.

Central to the pathogenesis of malaria is the proliferation of Plasmodium falciparum parasites within human erythrocytes. Parasites invade erythrocytes via a coordinated sequence of receptor-ligand interactions between the parasite and host cell. One key ligand, Apical Membrane Antigen 1 (AMA1), is a leading blood-stage vaccine and previous work indicates that phosphorylation of its cytoplasmic domain (CPD) is important to its function during invasion. Here we investigate the significance of each of the six available phospho-sites in the CPD. We confirm that the cyclic AMP/protein kinase A (PKA) signalling pathway elicits a phospho-priming step upon serine 610 (S610), which enables subsequent phosphorylation in vitro of a conserved, downstream threonine residue (T613) by glycogen synthase kinase 3 (GSK3). Both phosphorylation steps are required for AMA1 to function efficiently during invasion. This provides the first evidence that the functions of key invasion ligands of the malaria parasite are regulated by sequential phosphorylation steps.

Protein phosphorylation plays a fundamental role in the biology of apicomplexan parasites. Many apicomplexan protein kinases are substantially different from their mammalian orthologues, and thus constitute a landscape of potential drug targets. Here, we integrate genomic, biochemical, genetic and evolutionary information to provide an integrated and up-to-date analysis of twelve apicomplexan kinomes. All kinome sequences are available through the Kinomer database.

The most severe form of malaria in humans is caused by the protozoan parasite Plasmodium falciparum. The invasive form of malaria parasites is termed a merozoite and it employs an array of parasite proteins that bind to the host cell to mediate invasion. In Plasmodium falciparum, the erythrocyte binding-like (EBL) and reticulocyte binding-like (Rh) protein families are responsible for binding to specific erythrocyte receptors for invasion and mediating signalling events that initiate active entry of the malaria parasite. Here we have addressed the role of the cytoplasmic tails of these proteins in activating merozoite invasion after receptor engagement. We show that the cytoplasmic domains of these type 1 membrane proteins are phosphorylated in vitro. Depletion of PfCK2, a kinase implicated to phosphorylate these cytoplasmic tails, blocks P. falciparum invasion of red blood cells. We identify the crucial residues within the PfRh4 cytoplasmic domain that are required for successful parasite invasion. Live cell imaging of merozoites from these transgenic mutants show they attach but do not penetrate erythrocytes implying the PfRh4 cytoplasmic tail conveys signals important for the successful completion of the invasion process., Author Summary Malaria parasites must invade red blood cells to survive within the human host. Members of the erythrocyte binding-like (EBL) and reticulocyte binding-like (Rh) protein families, which are present at the apical tip of merozoites as single-pass transmembrane proteins, mediate recognition of red blood cells. Although extracellular domains of Plasmodium adhesins are required for binding red blood receptors, only the cytoplasmic region is in contact with the parasite’s cellular machinery to initiate invasion. Therefore any signal that is initiated upon binding must be communicated via the cytoplasmic domain to other targets within the malaria parasites. We investigate the role of adhesin phosphorylation in the P. falciparum invasion process. We show that the majority of adhesin cytoplasmic tails are phosphorylated in vitro. Mutational analyses provide identification of the important residues required for phosphorylation and suggest that PfCK2 may be responsible. Using a conditional knockdown line, we show that PfCK2 is essential for parasite growth and invasion. Using transgenic lines, we show that mutation of the PfRh4 cytoplasmic tail results in defects in parasite invasion. This work describes the critical residues important for signaling events and implicates PfCK2 as the essential kinase involved in P. falciparum invasion.

The lifestyle of intracellular pathogens, such as malaria parasites, is intimately connected to that of their host(s), primarily for nutrient supply. Nutrients act not only as primary sources of energy but also as regulators of gene expression, metabolism and growth, through various signaling networks that confer to cells the ability to sense and adapt to varying environmental conditions1,2. Canonical nutrient-sensing pathways are presumably absent in the causing agent of malaria Plasmodium3–5, thus raising the question of whether these parasites possess the capacity to sense and cope with host nutrient fluctuations. Here, we show that Plasmodium blood-stage parasites actively respond to host dietary calorie alterations through a rearrangement of their transcriptome accompanied by a significant adjustment of their multiplication rate. A kinome analysis combined with chemical and genetic approaches identified KIN as a critical regulator that mediates sensing of nutrients and controls a transcriptional response to the host nutritional status. KIN shares homology to SNF1/AMPKα and yeast complementation studies suggest functional conservation of an ancient cellular energy sensing pathway. Overall, these findings reveal a key parasite nutrient-sensing mechanism that is critical to modulate parasite replication and virulence.

Merozoites of malaria parasites invade red blood cells (RBCs), where they multiply by schizogony, undergoing development through ring, trophozoite and schizont stages that are responsible for malaria pathogenesis. Here, we report that a protein kinase-mediated signalling pathway involving host RBC PAK1 and MEK1, which do not have orthologues in the Plasmodium kinome, is selectively stimulated in Plasmodium falciparum-infected (versus uninfected) RBCs, as determined by the use of phospho-specific antibodies directed against the activated forms of these enzymes. Pharmacological interference with host MEK and PAK function using highly specific allosteric inhibitors in their known cellular IC50 ranges results in parasite death. Furthermore, MEK inhibitors have parasiticidal effects in vitro on hepatocyte and erythrocyte stages of the rodent malaria parasite Plasmodium berghei, indicating conservation of this subversive strategy in malaria parasites. These findings have profound implications for the development of novel strategies for antimalarial chemotherapy.

Aurora kinases compose a family of conserved Ser/Thr protein kinases playing essential roles in eukaryotic cell division. To date, Aurora homologues remain uncharacterized in the protozoan phylum Apicomplexa. In malaria parasites, the characterization of Aurora kinases may help understand the cell cycle control during erythrocytic schizogony where asynchronous nuclear divisions occur. In this study, we revisited the kinome of Plasmodium falciparum and identified three Aurora-related kinases, Pfark-1, -2, -3. Among these, Pfark-1 is highly conserved in malaria parasites and also appears to be conserved across Apicomplexa. By tagging the endogenous Pfark-1 gene with the green fluorescent protein (GFP) in live parasites, we show that the Pfark-1-GFP protein forms paired dots associated with only a subset of nuclei within individual schizonts. Immunofluorescence analysis using an anti-α-tubulin antibody strongly suggests a recruitment of Pfark-1 at duplicated spindle pole bodies at the entry of the M phase of the cell cycle. Unsuccessful attempts at disrupting the Pfark-1 gene with a knockout construct further indicate that Pfark-1 is required for parasite growth in red blood cells. Our study provides new insights into the cell cycle control of malaria parasites and reports the importance of Aurora kinases as potential targets for new antimalarials.

Malaria still remains one of the deadliest infectious diseases, and has a tremendous morbidity and mortality impact in the developing world. The propensity of the parasites to develop drug resistance, and the relative reluctance of the pharmaceutical industry to invest massively in the developments of drugs that would offer only limited marketing prospects, are major issues in antimalarial drug discovery. Protein kinases (PKs) have become a major family of targets for drug discovery research in a number of disease contexts, which has generated considerable resources such as kinase-directed libraries and high throughput kinase inhibition assays. The phylogenetic distance between malaria parasites and their human host translates into important divergences in their respective kinomes, and most Plasmodium kinases display atypical properties (as compared to mammalian PKs) that can be exploited towards selective inhibition. Here, we discuss the taxon-specific kinases possessed by malaria parasites, and give an overview of target PKs that have been validated by reverse genetics, either in the human malaria parasite Plasmodium falciparum or in the rodent model Plasmodium berghei. We also briefly allude to the possibility of attacking Plasmodium through the inhibition of human PKs that are required for survival of this obligatory intracellular parasite, and which are targets for other human diseases.

Summary A Plasmodium falciparum hexose transporter (PfHT) has previously been shown to be a facilitative glucose and fructose transporter. Its expression in Xenopus laevis oocytes and the use of a glucose analogue inhibitor permitted chemical validation of PfHT as a novel drug target. Following recent re-annotations of the P. falciparum genome, other putative sugar transporters have been identified. To investigate further if PfHT is the key supplier of hexose to P. falciparum and to extend studies to different stages of Plasmodium spp., we functionally analysed the hexose transporters of both the human parasite P. falciparum and the rodent parasite Plasmodium berghei using gene targeting strategies. We show here the essential function of pfht for the erythrocytic parasite growth as it was not possible to knockout pfht unless the gene was complemented by an episomal construct. Also, we show that parasites are rescued from the toxic effect of a glucose analogue inhibitor when pfht is overexpressed in these transfectants. We found that the rodent malaria parasite orthologue, P. berghei hexose transporter (PbHT) gene, was similarly refractory to knockout attempts. However, using a single cross-over transfection strategy, we generated transgenic P. berghei parasites expressing a PbHT-GFP fusion protein suggesting that locus is amenable for gene targeting. Analysis of pbht-gfp transgenic parasites showed that PbHT is constitutively expressed through all the stages in the mosquito host in addition to asexual stages. These results provide genetic support for prioritizing PfHT as a target for novel antimalarials that can inhibit glucose uptake and kill parasites, as well as unveiling the expression of this hexose transporter in mosquito stages of the parasite, where it is also likely to be critical for survival.

The molecular control of cell division and development in malaria parasites is far from understood. We previously showed that a Plasmodium gametocyte-specific NIMA-related protein kinase, nek-4, is required for completion of meiosis in the ookinete, the motile form that develops from the zygote in the mosquito vector. Here, we show that another NIMA-related kinase, Pfnek-2, is also predominantly expressed in gametocytes, and that Pfnek-2 is an active enzyme displaying an in vitro substrate preference distinct from that of Pfnek-4. A functional nek-2 gene is required for transmission of both Plasmodium falciparum and the rodent malaria parasite Plasmodium berghei to the mosquito vector, which is explained by the observation that disruption of the nek-2 gene in P. berghei causes dysregulation of DNA replication during meiosis and blocks ookinete development. This has implications (i) in our understanding of sexual development of malaria parasites and (ii) in the context of control strategies aimed at interfering with malaria transmission.

PfPK7 is an orphan protein kinase of Plasmodium falciparum with maximal homology to MEK3/6 and to fungal protein kinase A proteins in its C-terminal and N-terminal regions, respectively. We showed previously that recombinant PfPK7 is active on various substrates but is unable to phosphorylate the Plasmodium falciparum mitogen-activated protein kinase homologues, suggesting that it is not a MEK functional homologue. Using a reverse genetics approach to investigate the function of this enzyme in live parasites, we now show that Pf PK7 − parasite clones display phenotypes at two stages of their life cycle: first, a decrease in the rate of asexual growth in erythrocytes associated with a lower number of daughter merozoites generated per schizont, and second, a dramatic reduction in the ability to produce oocysts in the mosquito vector. A normal asexual growth rate and the ability to produce oocysts are restored if a functional copy of the Pf PK7 gene is reintroduced into the Pf PK7 − parasites. Hence, PfPK7 is involved in a pathway that regulates parasite proliferation and development.

The kinome of the human malaria parasite Plasmodium falciparum includes two genes encoding mitogen-activated protein kinase (MAPK) homologues, pfmap-1 and pfmap-2, but no clear orthologue of the MAPK kinase (MAPKK) family, raising the question of the mode of activation and function of the plasmodial MAPKs. Functional studies in the rodent malaria model Plasmodium berghei recently showed the map-2 gene to be dispensable for asexual growth and gametocytogenesis, but essential for male gametogenesis in the mosquito vector. Here, we demonstrate by using a reverse genetics approach that the map-2 gene is essential for completion of the asexual cycle of P. falciparum, an unexpected result in view of the non-essentiality of the orthologous gene for P. berghei erythrocytic schizogony. This validates Pfmap-2 as a potential target for chemotherapeutic intervention. In contrast, the other P. falciparum MAPK, Pfmap-1, is required neither for in vitro schizogony and gametocytogenesis in erythrocytes, nor for gametogenesis and sporogony in the mosquito vector. However, Pfmap-2 protein levels are elevated in pfmap-1(-) parasites, suggesting that Pfmap-1 fulfils an important function in asexual parasites that necessitates compensatory adaptation in parasites lacking this enzyme.

Protein kinases (PKs) are prime targets for drug discovery in a variety of diseases, including cancer and neurodegenerative pathologies. The characterisation of the kinome of the human malaria parasite Plasmodium falciparum has revealed profound divergences, at several levels, between PKs of the parasite and those of its host. Here, the authors review the major issues and recent advances regarding the development of Plasmodium-selective PK inhibitors, with emphasis on target identification and validation, and on structure-based design. The authors also discuss the possibility of interfering with: i) Plasmodium PKs regulating transmission to the mosquito vector; and ii) host PKs that may be required for parasite survival.

Aurora kinases are eukaryotic serine/threonine protein kinases that regulate key events associated with chromatin condensation, centrosome and spindle function, and cytokinesis. Elucidating the roles of Aurora kinases in apicomplexan parasites is crucial to understand the cell cycle control during Plasmodium schizogony or Toxoplasma endodyogeny. Here, we report on the localization of two previously uncharacterized Toxoplasma Aurora-related kinases (Ark2 and Ark3) in tachyzoites and of the uncharacterized Ark3 orthologue in Plasmodium falciparum erythrocytic stages. In T. gondii, we show that TgArk2 and TgArk3 concentrate at specific sub-cellular structures linked to parasite division: the mitotic spindle and intranuclear mitotic structures (TgArk2), and the outer core of the centrosome and the budding daughter cells cytoskeleton (TgArk3). By tagging the endogenous PfArk3 gene with the green fluorescent protein (GFP) in live parasites, we show that PfArk3 protein expression peaks late in schizogony and localizes at the periphery of budding schizonts. Disruption of the TgArk2 gene reveals no essential function for tachyzoite propagation in vitro, which is surprising giving that the P. falciparum and P. berghei orthologues are essential for erythrocyte schizogony. In contrast, knock-down of TgArk3 protein results in pronounced defects in parasite division and a major growth deficiency. TgArk3-depleted parasites display several defects, such as reduced parasite growth rate, delayed egress and parasite duplication, defect in rosette formation, reduced parasite size and invasion efficiency and lack of virulence in mice. Our study provides new insights into cell cycle control in Toxoplasma and malaria parasites, and highlights Aurora kinase 3 as potential drug target.

With respect to parasite-induced infectious diseases of worldwide importance, members of the genera Plasmodium and Schistosoma are top pathogens. Nearly half a billion people suffer from malaria caused by Plasmodium spp. and schistosomiasis (bilharzia) induced by Schistosoma spp. Resistance against essentially all drugs used for malaria treatment has been reported. For schistosomiasis justified fear of upcoming resistance is discussed against the background of only one widely used drug for treatment. Research of the recent decade has demonstrated that essential steps of the biology of these and other parasites are controlled by kinases, which represent attractive targets for new-generation antiparasitic compounds. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases.

Processes at the surface of Plasmodium falciparum-infected erythrocytes such as antigenic variation and cytoadhesion may be modulated by active signalling between host and parasite. Potential candidates for this role include the putative kinases of the FIKK family. The novel Apicomplexa-specific FIKK gene has expanded in P. falciparum to 20 sequence-related members distributed between 11 chromosomes. Specific antibodies raised against different members indicated that most FIKK proteins locate to punctate foci in the erythrocyte cytoplasm that colocalized with Maurer's clefts proteins. One FIKK member dissociates at the trophozoite stage from the Maurer's clefts and relocates with the erythrocyte cytoskeleton. Another FIKK protein, despite having a PEXEL motif, remains located within the parasite. FIKK proteins possess the essential residues for phosphotransferase activity. We show that protein kinase activity was detected in immunoprecipitates obtained with two anti-FIKK antibodies. Quantitative PCR analysis revealed differential gene transcription of the FIKK paralogues in asexual blood stages parasites. We observed significant changes in the transcription pattern between parasites with different adhesion phenotypes. Our data suggest a role of FIKK proteins in the remodelling of the erythrocyte surface and reveal the existence of an adaptable parasite system able to sense intra- and possibly extracellular changes.

The molecular mechanisms underlying gametocytogenesis in malaria parasites are not understood. Plasmodium falciparum cdc2-related kinase 1 (pfcrk-1), a gene that is expressed predominantly in gametocytes, bears homology to the PITSLRE subfamily of cyclin-dependent kinases and has been hypothesized to function as a negative regulator of the cell cycle. We attempted to knock-out pbcrk-1, the P. berghei orthologue of pfcrk-1, but were unable to recover P. berghei parasites with a disrupted pbcrk-1 locus. In contrast, an integration event at this locus that did not result in a loss-of-function of the pbcrk-1 gene was readily observed. This strongly suggests that a functional pbcrk-1 gene product is essential to intraerythrocytic asexual multiplication.

The transmission of malaria parasites to the mosquito depends critically on the rapid initiation of sexual reproduction in response to triggers from the mosquito midgut environment. We here identify an essential function for an atypical mitogen-activated protein kinase of the rodent malaria parasite Plasmodium berghei, Pbmap-2, in male sexual differentiation and parasite transmission to the mosquito. A deletion mutant no longer expressing the Pbmap-2 protein develops as wild type throughout the asexual erythrocytic phase of the life cycle. Gametocytes, the sexual transmission stages, form normally and respond in vitro to the appropriate environmental cues by rounding up and emerging from their host cells. However, microgametocytes fail to release flagellated microgametes. Female development is not affected, as judged by the ability of macrogametes to become cross-fertilized by microgametes from a donor strain. Cellular differentiation of Pbmap-2 KO microgametocytes is blocked at a late stage of male gamete formation, after replication and mitoses have been completed and axonemes have been assembled. These data demonstrate a function for Pbmap-2 in initiating cytokinesis and axoneme motility, possibly downstream of a cell cycle checkpoint for the completion of replication and/or mitosis, which are extraordinarily rapid in the male gametocyte.

Differentiation of malaria parasites into sexual forms (gametocytes) in the vertebrate host and their subsequent development into gametes in the mosquito vector are crucial steps in the completion of the parasite's life cycle and transmission of the disease. The molecular mechanisms that regulate the sexual cycle are poorly understood. Although several signal transduction pathways have been implicated, a clear understanding of the pathways involved has yet to emerge. Here, we show that a Plasmodium berghei homologue of Plasmodium falciparum mitogen-activated kinase-2 (Pfmap-2), a gametocyte-specific mitogen-activated protein kinase (MAPK), is required for male gamete formation. Parasites lacking Pbmap-2 are competent for gametocytogenesis, but exflagellation of male gametocytes, the process that leads to male gamete formation, is almost entirely abolished in mutant parasites. Consistent with this result, transmission of mutant parasites to mosquitoes is grossly impaired. This finding identifies a crucial role for a MAPK pathway in malaria transmission.

Global production of chickens has trebled in the past two decades and they are now the most important source of dietary animal protein worldwide. Chickens are subject to many infectious diseases that reduce their performance and productivity. Coccidiosis, caused by apicomplexan protozoa of the genus Eimeria, is one of the most important poultry diseases. Understanding the biology of Eimeria parasites underpins development of new drugs and vaccines needed to improve global food security. We have produced annotated genome sequences of all seven species of Eimeria that infect domestic chickens, which reveal the full extent of previously described repeat-rich and repeat-poor regions and show that these parasites possess the most repeat-rich proteomes ever described. Furthermore, while no other apicomplexan has been found to possess retrotransposons, Eimeria is home to a family of chromoviruses. Analysis of Eimeria genes involved in basic biology and host-parasite interaction highlights adaptations to a relatively simple developmental life cycle and a complex array of co-expressed surface proteins involved in host cell binding.

Casein kinase 2 (protein kinase CK2) is a conserved eukaryotic serine/theronine kinase with multiple substrates and roles in the regulation of cellular processes such as cellular stress, cell proliferation and apoptosis. Here we report a detailed analysis of the Plasmodium falciparum CK2, PfCK2, demonstrating that this kinase, like the mammalian orthologue, is a dual specificity kinase able to phosphorylate at both serine and tyrosine. However, unlike the human orthologue that is auto-phosphorylated on tyrosine within the activation loop, PfCK2 shows no activation loop auto-phosphorylation but rather is auto-phosphorylated at threonine 63 within subdomain I. Phosphorylation at this site in PfCK2 is shown here to regulate the intrinsic kinase activity of PfCK2. Furthermore, we generate an homology model of PfCK2 in complex with the known selective protein kinase CK2 inhibitor, quinalizarin, and in so doing identify key co-ordinating residues in the ATP binding pocket that could aid in designing selective inhibitors to PfCK2.

Cyclin-dependent kinase-like kinases (CLKs) are dual specificity protein kinases that phosphorylate Serine/Arginine-rich (SR) proteins involved in pre-mRNA processing. Four CLKs, termed PfCLK-1-4, can be identified in the human malaria parasite Plasmodium falciparum, which show homology with the yeast SR protein kinase Sky1p. The four PfCLKs are present in the nucleus and cytoplasm of the asexual blood stages and of gametocytes, sexual precursor cells crucial for malaria parasite transmission from humans to mosquitoes. We identified three plasmodial SR proteins, PfSRSF12, PfSFRS4 and PfSF-1, which are predominantly present in the nucleus of blood stage trophozoites, PfSRSF12 and PfSF-1 are further detectable in the nucleus of gametocytes. We found that recombinantly expressed SR proteins comprising the Arginine/Serine (RS)-rich domains were phosphorylated by the four PfCLKs in in vitro kinase assays, while a recombinant PfSF-1 peptide lacking the RS-rich domain was not phosphorylated. Since it was hitherto not possible to knock-out the pfclk genes by conventional gene disruption, we aimed at chemical knock-outs for phenotype analysis. We identified five human CLK inhibitors, belonging to the oxo-beta-carbolines and aminopyrimidines, as well as the antiseptic chlorhexidine as PfCLK-targeting compounds. The six inhibitors block P. falciparum blood stage replication in the low micromolar to nanomolar range by preventing the trophozoite-to-schizont transformation. In addition, the inhibitors impair gametocyte maturation and gametogenesis in in vitro assays. The combined data show that the four PfCLKs are involved in phosphorylation of SR proteins with essential functions for the blood and sexual stages of the malaria parasite, thus pointing to the kinases as promising targets for antimalarial and transmission blocking drugs.

In Plasmodium falciparum, the causative agent of human malaria, the catalytic subunit gene of cAMP-dependent protein kinase (Pfpka-c) exists as a single copy. Interestingly, its expression appears developmentally regulated, being at higher levels in the pathogenic asexual stages than in the sexual forms of parasite that are responsible for transmission to the mosquito vector. Within asexual parasites, PfPKA activity can be readily detected in schizonts. Similar to endogenous PKA activity of noninfected red blood cells, the parasite enzyme can be stimulated by cAMP and inhibited by protein kinase inhibitor.Importantly, ex vivo treatment of infected erythrocytes with the classical PKA-C inhibitor H89 leads to a block in parasite growth. This suggests that the PKA activities of infected red blood cells are essential for parasite multiplication. Finally, structural considerations suggest that drugs targeting the parasite, rather than the erythrocyte enzyme, might be developed that could help in the fight against malaria.

The development of novel chemotherapeutic agents has become an urgent task due to the development and rapid spread of drug resistance in Plasmodium falciparum, the protozoan parasite responsible for cerebral malaria. Cyclin-dependent kinases (CDKs) are essential for the regulation of the eukaryotic cell cycle, and several enzymes of this family have been identified in P. falciparum. In recent years, a number of purine-derived kinase inhibitors have been synthesised, some of which display selective activity against CDKs. This report describes a study in which various purine derivatives were screened for in vitro antimalarial activity. The erythrocytic asexual stages of the chloroquine-resistant P. falciparum strain (FCR-3) were cultivated in vitro in the presence of the various purines, and their effect on parasite proliferation was determined by the [3H]hypoxanthine incorporation assay. Our results show considerable variation in the sensitivity of P. falciparum to the different purines, as well as a general independence from their effect on purified starfish CDK1/cyclin B activity, which has been the standard assay used to identify CDK-specific inhibitors. Two subfamilies of purines with moderate to poor activity against CDK1/cyclin B activity showed submicromolar activity against P. falciparum. Structure-activity analysis indicates that certain structural features are associated with increased activity against P. falciparum. These features can be exploited to synthesise compounds with higher activity and specificity towards P. falciparum.

Two studies identify protein kinases of parasitic protists with roles in either their own development or the control of the behavior of their host cell.

Crystal structures of a calcium-dependent protein kinase from Toxoplasma gondii uncover a unique mechanism of activation and potential avenues for selective inhibition.

BACKGROUND: Chemical inhibitors of cyclin-dependent kinases (CDKs) have great therapeutic potential against various proliferative and neurodegenerative disorders. Olomoucine, a 2,6,9-trisubstituted purine, has been optimized for activity against CDK1/cyclin B by combinatorial and medicinal chemistry efforts to yield the purvalanol inhibitors. Although many studies support the action of purvalanols against CDKs, the actual intracellular targets of 2,6, 9-trisubstituted purines remain unverified. RESULTS: To address this issue, purvalanol B (95. ) and an N6-methylated, CDK-inactive derivative (95M. ) were immobilized on an agarose matrix. Extracts from a diverse collection of cell types and organisms were screened for proteins binding purvalanol B. In addition to validating CDKs as intracellular targets, a variety of unexpected protein kinases were recovered from the 95. matrix. Casein kinase 1 (CK1) was identified as a principal 95. matrix binding protein in Plasmodium falciparum, Leishmania mexicana, Toxoplasma gondii and Trypanosoma cruzi. Purvalanol compounds also inhibit the proliferation of these parasites, suggesting that CK1 is a valuable target for further screening with 2,6,9-trisubstituted purine libraries. CONCLUSIONS: That a simple batchwise affinity chromatography approach using two purine derivatives facilitated isolation of a small set of highly purified kinases suggests that this could be a general method for identifying intracellular targets relevant to a particular class of ligands. This method allows a close correlation to be established between the pattern of proteins bound to a small family of related compounds and the pattern of cellular responses to these compounds.

The antifolate proguanil is commonly used in the prophylaxis and treatment of Plasmodium falciparum malaria. A series of point mutations in the dihydrofolate reductase (DHFR) gene has been linked to differential susceptibility of varied P. falciparum clones or isolates to this drug. To survey the efficiency of proguanil prophylaxis in an African endemic region, and to evaluate the level of proguanil resistance in the corresponding parasite population, we performed drug susceptibility assays with P. falciparum isolates from Senegal, Kenya, and Niger. In parallel, we developed a mutation-specific polymerase chain reaction assay that enabled us to characterize mutations in the DHFR gene of the same isolates without in vitro parasite cultivation. We confirm previously available data showing that parasites harboring a point mutation from Ser108 to Asn present a decrease in susceptibility to cycloguanil (the active metabolite of proguanil), and we show that mutations in codons 51 and 59 appear to modulate the level of resistance to cycloguanil. No mutations in codons 16 and 164 were detected in resistant parasites, in contrast with results from some previous studies.

The important role of histone posttranslational modifications, particularly methylation and acetylation, in Plasmodium falciparum gene regulation has been established. However, the role of histone phosphorylation remains understudied. Here, we investigate histone phosphorylation utilizing liquid chromatography and tandem mass spectrometry to analyze histones extracted from asexual blood stages using two improved protocols to enhance preservation of PTMs. Enrichment for phosphopeptides lead to the detection of 14 histone phospho-modifications in P. falciparum. The majority of phosphorylation sites were observed at the N-terminal regions of various histones and were frequently observed adjacent to acetylated lysines. We also report the identification of one novel member of the P. falciparum histone phosphosite binding protein repertoire, Pf14-3-3I. Recombinant Pf14-3-3I protein bound to purified parasite histones. In silico structural analysis of Pf14-3-3 proteins revealed that residues responsible for binding to histone H3 S10ph and/or S28ph are conserved at the primary and the tertiary structure levels. Using a battery of H3 specific phosphopeptides, we demonstrate that Pf14-3-3I preferentially binds to H3S28ph over H3S10ph, independent of modification of neighbouring residues like H3S10phK14ac and H3S28phS32ph. Our data provide key insight into histone phosphorylation sites. The identification of a second member of the histone modification reading machinery suggests a widespread use of histone phosphorylation in the control of various nuclear processes in malaria parasites.

Eukaryotic, prokaryotic and viral pathogens are known to interfere with signaling pathways of their host to promote their own survival and proliferation. Here, we present selected examples of modulation of PAK activity in human cells by both intracellular and extracellular pathogens, focusing on one eukaryotic pathogen, the human malaria parasite Plasmodium falciparum, two Gram-negative bacteria (Helicobacter pylori and Pseudomonas aeruginosa), and two viruses belonging to distinct groups, the lentivirus HIV and the orthomyxovirus Influenza virus A.

The human malaria parasite Plasmodium falciparum is auxotrophic for most amino acids. Its amino acid needs are met largely through the degradation of host erythrocyte hemoglobin; however the parasite must acquire isoleucine exogenously, because this amino acid is not present in adult human hemoglobin. We report that when isoleucine is withdrawn from the culture medium of intraerythrocytic P. falciparum , the parasite slows its metabolism and progresses through its developmental cycle at a reduced rate. Isoleucine-starved parasites remain viable for 72 h and resume rapid growth upon resupplementation. Protein degradation during starvation is important for maintenance of this hibernatory state. Microarray analysis of starved parasites revealed a 60% decrease in the rate of progression through the normal transcriptional program but no other apparent stress response. Plasmodium parasites do not possess a TOR nutrient-sensing pathway and have only a rudimentary amino acid starvation-sensing eukaryotic initiation factor 2α (eIF2α) stress response. Isoleucine deprivation results in GCN2-mediated phosphorylation of eIF2α, but kinase-knockout clones still are able to hibernate and recover, indicating that this pathway does not directly promote survival during isoleucine starvation. We conclude that P. falciparum , in the absence of canonical eukaryotic nutrient stress-response pathways, can cope with an inconsistent bloodstream amino acid supply by hibernating and waiting for more nutrient to be provided.

Background Protein kinase CK2 is a pleiotropic serine/threonine protein kinase with hundreds of reported substrates, and plays an important role in a number of cellular processes. The cellular functions of Plasmodium falciparum CK2 (PfCK2) are unknown. The parasite's genome encodes one catalytic subunit, PfCK2α, which we have previously shown to be essential for completion of the asexual erythrocytic cycle, and two putative regulatory subunits, PfCK2β1 and PfCK2β2. Results We now show that the genes encoding both regulatory PfCK2 subunits (PfCK2β1 and PfCK2β2) cannot be disrupted. Using immunofluorescence and electron microscopy, we examined the intra-erythrocytic stages of transgenic parasite lines expressing hemagglutinin (HA)-tagged catalytic and regulatory subunits (HA-CK2α, HA-PfCK2β1 or HA-PfCK2β2), and localized all three subunits to both cytoplasmic and nuclear compartments of the parasite. The same transgenic parasite lines were used to purify PfCK2β1- and PfCK2β2-containing complexes, which were analyzed by mass spectrometry. The recovered proteins were unevenly distributed between various pathways, with a large proportion of components of the chromatin assembly pathway being present in both PfCK2β1 and PfCK2β2 precipitates, implicating PfCK2 in chromatin dynamics. We also found that chromatin-related substrates such as nucleosome assembly proteins (Naps), histones, and two members of the Alba family are phosphorylated by PfCK2α in vitro. Conclusions Our reverse-genetics data show that each of the two regulatory PfCK2 subunits is required for completion of the asexual erythrocytic cycle. Our interactome study points to an implication of PfCK2 in many cellular pathways, with chromatin dynamics being identified as a major process regulated by PfCK2. This study paves the way for a kinome-wide interactomics-based approach to elucidate protein kinase function in malaria parasites.

Some apicomplexan parasites have evolved distinct protein kinase families to modulate host cell structure and function. Toxoplasma gondii rhoptry protein kinases and pseudokinases are involved in virulence and modulation of host cell signalling. The proteome of Plasmodium falciparum contains a family of putative kinases called FIKKs, some of which are exported to the host red blood cell and might play a role in erythrocyte remodelling. In this review we will discuss kinases known to be critical for host cell invasion, intracellular growth and egress, focusing on (i) calcium-dependent protein kinases and (ii) the secreted kinases that are unique to Toxoplasma (rhoptry protein kinases and pseudokinases) and Plasmodium (FIKKs)., American Heart Association (0835099N), Massachusetts Life Sciences Center (New Investigator Award), Singapore-MIT Alliance for Research and Technology, Pew Charitable Trusts, New England Regional Center of Excellence (Developmental Grant), National Institutes of Health (U.S.) (RO1-AI080621), Merck Research Laboratories (Postdoctoral Fellowship), Massachusetts Institute of Technology. Department of Biology

Merozoites of malaria parasites invade red blood cells (RBCs), where they multiply by schizogony, undergoing development through ring, trophozoite and schizont stages that are responsible for malaria pathogenesis. Here, we report that a protein kinase-mediated signalling pathway involving host RBC PAK1 and MEK1, which do not have orthologues in the Plasmodium kinome, is selectively stimulated in Plasmodium falciparum-infected (versus uninfected) RBCs, as determined by the use of phospho-specific antibodies directed against the activated forms of these enzymes. Pharmacological interference with host MEK and PAK function using highly specific allosteric inhibitors in their known cellular IC50 ranges results in parasite death. Furthermore, MEK inhibitors have parasiticidal effects in vitro on hepatocyte and erythrocyte stages of the rodent malaria parasite Plasmodium berghei, indicating conservation of this subversive strategy in malaria parasites. These findings have profound implications for the development of novel strategies for antimalarial chemotherapy.

Physical contact is important for the interaction between animal cells, but it can represent a major challenge for protists like malaria parasites. Recently, novel filamentous cell-cell contacts have been identified in different types of eukaryotic cells and termed nanotubes due to their morphological appearance. Nanotubes represent small dynamic membranous extensions that consist of F-actin and are considered an ancient feature evolved by eukaryotic cells to establish contact for communication. We here describe similar tubular structures in the malaria pathogen Plasmodium falciparum, which emerge from the surfaces of the forming gametes upon gametocyte activation in the mosquito midgut. The filaments can exhibit a length of > 100 mu m and contain the F-actin isoform actin 2. They actively form within a few minutes after gametocyte activation and persist until the zygote transforms into the ookinete. The filaments originate from the parasite plasma membrane, are close ended and express adhesion proteins on their surfaces that are typically found in gametes, like Pfs230, Pfs48/45 or Pfs25, but not the zygote surface protein Pfs28. We show that these tubular structures represent long-distance cell-to-cell connections between sexual stage parasites and demonstrate that they meet the characteristics of nanotubes. We propose that malaria parasites utilize these adhesive "nanotubes" in order to facilitate intercellular contact between gametes during reproduction in the mosquito midgut.

Four new meroterpenes, alisiaquinones A-C (1-3) and alisiaquinol (4), were isolated from a New Caledonian deep water sponge. Their structures and relative stereochemistry were elucidated by spectroscopic data analysis. They are related to xestoquinone, but showed unusual substitution on a tetrahydrofuran junction. They displayed micromolar range activity on two enzymatic targets of importance for the control of malaria, the plasmodial kinase Pfnek-1 and a protein farnesyl transferase, as well as on different chloroquine-sensitive and -resistant strains of Plasmodium falciparum. Alisiaquinone C displayed a submicromolar activity on P. falciparum and a competitive selectivity index on the different plasmodial strains.