Disruption of a Plasmodium falciparum patatin-like phospholipase delays male gametocyte exflagellation.

An essential process in transmission of the malaria parasite to the Anopheles vector is the conversion of mature gametocytes into gametes within the mosquito gut, where they egress from the red blood cell (RBC). During egress, male gametocytes undergo exflagellation, leading to the formation of eight haploid motile microgametes, while female gametes retain their spherical shape. Gametocyte egress depends on sequential disruption of the parasitophorous vacuole membrane and the host cell membrane. In other life cycle stages of the malaria parasite, phospholipases have been implicated in membrane disruption processes during egress, however their importance for gametocyte egress is relatively unknown. Here, we performed comprehensive functional analyses of six putative phospholipases for their role during development and egress of Plasmodium falciparum gametocytes. We localize two of them, the prodrug activation and resistance esterase (PF3D7_0709700) and the lysophospholipase 1 (PF3D7_1476700), to the parasite plasma membrane. Subsequently, we show that disruption of most of the studied phospholipase genes does neither affect gametocyte development nor egress. The exception is the putative patatin-like phospholipase 3 (PF3D7_0924000), whose gene deletion leads to a delay in male gametocyte exflagellation, indicating an important, albeit not essential, role of this enzyme in male gametogenesis.

Atlas of Plasmodium falciparum intraerythrocytic development using expansion microscopy.

Apicomplexan parasites exhibit tremendous diversity in much of their fundamental cell biology, but study of these organisms using light microscopy is often hindered by their small size. Ultrastructural expansion microscopy (U-ExM) is a microscopy preparation method that physically expands the sample by ~4.5×. Here, we apply U-ExM to the human malaria parasite Plasmodium falciparum during the asexual blood stage of its lifecycle to understand how this parasite is organized in three dimensions. Using a combination of dye-conjugated reagents and immunostaining, we have cataloged 13 different P. falciparum structures or organelles across the intraerythrocytic development of this parasite and made multiple observations about fundamental parasite cell biology. We describe that the outer centriolar plaque and its associated proteins anchor the nucleus to the parasite plasma membrane during mitosis. Furthermore, the rhoptries, Golgi, basal complex, and inner membrane complex, which form around this anchoring site while nuclei are still dividing, are concurrently segregated and maintain an association to the outer centriolar plaque until the start of segmentation. We also show that the mitochondrion and apicoplast undergo sequential fission events while maintaining an association with the outer centriolar plaque during cytokinesis. Collectively, this study represents the most detailed ultrastructural analysis of P. falciparum during its intraerythrocytic development to date and sheds light on multiple poorly understood aspects of its organelle biogenesis and fundamental cell biology.

Shigella flexneri remodeling and consumption of host lipids during infection.

IMPORTANCE: Bacterial pathogens have vastly distinct sites that they inhabit during infection. This requires adaptation due to changes in nutrient availability and antimicrobial stress. The bacterial surface is a primary barrier, and here, we show that the bacterial pathogen Shigella flexneri increases its surface decorations when it transitions to an intracellular lifestyle. We also observed changes in bacterial and host cell fatty acid homeostasis. Specifically, intracellular S. flexneri increased the expression of their fatty acid degradation pathway, while the host cell lipid pool was significantly depleted. Importantly, bacterial proliferation could be inhibited by fatty acid supplementation of host cells, thereby providing novel insights into the possible link between human malnutrition and susceptibility to S. flexneri.

Bottoms up! Malaria parasite invasion the right way around.

A critical part of the malaria parasite's life cycle is invasion of red blood cells (RBCs) by merozoites. Inside RBCs, the parasite forms a schizont, which undergoes segmentation to produce daughter merozoites. These cells are released, establishing cycles of invasion. Traditionally, merozoites are represented as nonmotile, egg-shaped cells that invade RBCs 'narrower end' first and pack within schizonts with this narrower end facing outwards. Here, we discuss recent evidence and re-evaluate previous data which suggest that merozoites are capable of motility and have spherical or elongated-teardrop shapes. Furthermore, merozoites invade RBCs 'wider end' first and pack within schizonts with this wider end facing outwards. We encourage the field to review this revised model and consider its implications for future studies.

Sulfonylpiperazine compounds prevent Plasmodium falciparum invasion of red blood cells through interference with actin-1/profilin dynamics.

With emerging resistance to frontline treatments, it is vital that new antimalarial drugs are identified to target Plasmodium falciparum. We have recently described a compound, MMV020291, as a specific inhibitor of red blood cell (RBC) invasion, and have generated analogues with improved potency. Here, we generated resistance to MMV020291 and performed whole genome sequencing of 3 MMV020291-resistant populations. This revealed 3 nonsynonymous single nucleotide polymorphisms in 2 genes; 2 in profilin (N154Y, K124N) and a third one in actin-1 (M356L). Using CRISPR-Cas9, we engineered these mutations into wild-type parasites, which rendered them resistant to MMV020291. We demonstrate that MMV020291 reduces actin polymerisation that is required by the merozoite stage parasites to invade RBCs. Additionally, the series inhibits the actin-1-dependent process of apicoplast segregation, leading to a delayed death phenotype. In vitro cosedimentation experiments using recombinant P. falciparum proteins indicate that potent MMV020291 analogues disrupt the formation of filamentous actin in the presence of profilin. Altogether, this study identifies the first compound series interfering with the actin-1/profilin interaction in P. falciparum and paves the way for future antimalarial development against the highly dynamic process of actin polymerisation.

Atlas of Plasmodium falciparum intraerythrocytic development using expansion microscopy.

Apicomplexan parasites exhibit tremendous diversity in much of their fundamental cell biology, but study of these organisms using light microscopy is often hindered by their small size. Ultrastructural expansion microscopy (U-ExM) is a microscopy preparation method that physically expands the sample ~4.5x. Here, we apply U-ExM to the human malaria parasite Plasmodium falciparum during the asexual blood stage of its lifecycle to understand how this parasite is organized in three-dimensions. Using a combination of dye-conjugated reagents and immunostaining, we have catalogued 13 different P. falciparum structures or organelles across the intraerythrocytic development of this parasite and made multiple observations about fundamental parasite cell biology. We describe that the outer centriolar plaque and its associated proteins anchor the nucleus to the parasite plasma membrane during mitosis. Furthermore, the rhoptries, Golgi, basal complex, and inner membrane complex, which form around this anchoring site while nuclei are still dividing, are concurrently segregated and maintain an association to the outer centriolar plaque until the start of segmentation. We also show that the mitochondrion and apicoplast undergo sequential fission events while maintaining an association with the outer centriolar plaque during cytokinesis. Collectively, this study represents the most detailed ultrastructural analysis of P. falciparum during its intraerythrocytic development to date, and sheds light on multiple poorly understood aspects of its organelle biogenesis and fundamental cell biology.

Defining species-specific and conserved interactions of apical membrane protein 1 during erythrocyte invasion in malaria to inform multi-species vaccines.

Plasmodium falciparum and P. vivax are the major causes of human malaria, and P. knowlesi is an important additional cause in SE Asia. Binding of apical membrane antigen 1 (AMA1) to rhoptry neck protein 2 (RON2) was thought to be essential for merozoite invasion of erythrocytes by Plasmodium spp. Our findings reveal that P. falciparum and P. vivax have diverged and show species-specific binding of AMA1 to RON2, determined by a ß-hairpin loop in RON2 and specific residues in AMA1 Loop1E. In contrast, cross-species binding of AMA1 to RON2 is retained between P. vivax and P. knowlesi. Mutation of specific amino acids in AMA1 Loop1E in P. falciparum or P. vivax ablated RON2 binding without impacting erythrocyte invasion. This indicates that the AMA1-RON2-loop interaction is not essential for invasion and additional AMA1 interactions are involved. Mutations in AMA1 that disrupt RON2 binding also enable escape of invasion inhibitory antibodies. Therefore, vaccines and therapeutics will need to be broader than targeting only the AMA1-RON2 interaction. Antibodies targeting AMA1 domain 3 had greater invasion-inhibitory activity when RON2-loop binding was ablated, suggesting this domain is a promising additional target for vaccine development. Targeting multiple AMA1 interactions involved in invasion may enable vaccines that generate more potent inhibitory antibodies and address the capacity for immune evasion. Findings on specific residues for invasion function and species divergence and conservation can inform novel vaccines and therapeutics against malaria caused by three species, including the potential for cross-species vaccines.

Characterisation of PfCZIF1 and PfCZIF2 in Plasmodium falciparum asexual stages.

Plasmodium falciparum exerts strong temporal control of gene expression across its lifecycle. Proteins expressed exclusively during late schizogony of blood stages, for example, often have a role in facilitating merozoite invasion of the host red blood cell (RBC), through merozoite development, egress, invasion or early establishment of infection in the RBC. Here, we characterise P. falciparum C3H1 zinc finger 1 (PfCZIF1, Pf3D7_1468400) and P. falciparum C3H1 zinc finger 2 (PfCZIF2, Pf3D7_0818100) which we identified as the only C3H1-type zinc finger proteins with peak expression at schizogony. Previous studies reported that antibodies against PfCZIF1 inhibit merozoite invasion, suggesting this protein may have a potential role during RBC invasion. We show using C-terminal truncations and gene knockouts of each of Pfczif1 and Pfczif2 that neither are essential for blood stage growth. However, they could not both be knocked out simultaneously, suggesting that at least one is needed for parasite growth in vitro. Immunofluorescence localisation of PfCZIF1 and PfCZIF2 indicated that both proteins occur in discrete foci on the periphery of the parasite's cytosol and biochemical assays suggest they are peripherally associated to a membrane. Transcriptomic analyses for the C-terminal truncation mutants reveal no significant expression perturbations with PfCZIF1 truncation. However, modification of PfCZIF2 appears to modify the expression for some exported proteins including PfKAHRP. This study does not support a role for PfCZIF1 or PfCZIF2 in merozoite invasion of the RBC and suggests that these proteins may help regulate the expression of proteins exported into the RBC cytosol after merozoite invasion.

Targeting malaria parasites with novel derivatives of azithromycin.

Introduction: The spread of artemisinin resistant Plasmodium falciparum parasites is of global concern and highlights the need to identify new antimalarials for future treatments. Azithromycin, a macrolide antibiotic used clinically against malaria, kills parasites via two mechanisms: 'delayed death' by inhibiting the bacterium-like ribosomes of the apicoplast, and 'quick-killing' that kills rapidly across the entire blood stage development. Methods: Here, 22 azithromycin analogues were explored for delayed death and quick-killing activities against P. falciparum (the most virulent human malaria) and P. knowlesi (a monkey parasite that frequently infects humans). Results: Seventeen analogues showed improved quick-killing against both Plasmodium species, with up to 38 to 20-fold higher potency over azithromycin after less than 48 or 28 hours of treatment for P. falciparum and P. knowlesi, respectively. Quick-killing analogues maintained activity throughout the blood stage lifecycle, including ring stages of P. falciparum parasites (<12 hrs treatment) and were >5-fold more selective against P. falciparum than human cells. Isopentenyl pyrophosphate supplemented parasites that lacked an apicoplast were equally sensitive to quick-killing analogues, confirming that the quick killing activity of these drugs was not directed at the apicoplast. Further, activity against the related apicoplast containing parasite Toxoplasma gondii and the gram-positive bacterium Streptococcus pneumoniae did not show improvement over azithromycin, highlighting the specific improvement in antimalarial quick-killing activity. Metabolomic profiling of parasites subjected to the most potent compound showed a build-up of non-haemoglobin derived peptides that was similar to chloroquine, while also exhibiting accumulation of haemoglobin-derived peptides that was absent for chloroquine treatment. Discussion: The azithromycin analogues characterised in this study expand the structural diversity over previously reported quick-killing compounds and provide new starting points to develop azithromycin analogues with quick-killing antimalarial activity.

Cell biological analysis reveals an essential role for Pfcerli2 in erythrocyte invasion by malaria parasites.

Merozoite invasion of host red blood cells (RBCs) is essential for survival of the human malaria parasite Plasmodium falciparum. Proteins involved with RBC binding and invasion are secreted from dual-club shaped organelles at the apical tip of the merozoite called the rhoptries. Here we characterise P. falciparum Cytosolically Exposed Rhoptry Leaflet Interacting protein 2 (PfCERLI2), as a rhoptry bulb protein that is essential for merozoite invasion. Phylogenetic analyses show that cerli2 arose through an ancestral gene duplication of cerli1. We show that PfCERLI2 is essential for blood-stage growth and localises to the cytosolic face of the rhoptry bulb. Inducible knockdown of PfCERLI2 led to a proportion of merozoites failing to invade and was associated with elongation of the rhoptry organelle during merozoite development and inhibition of rhoptry antigen processing. These findings identify PfCERLI2 as a protein that has key roles in rhoptry biology during merozoite invasion.

Host-directed therapy, an untapped opportunity for antimalarial intervention.

Host-directed therapy (HDT) is gaining traction as a strategy to combat infectious diseases caused by viruses and intracellular bacteria, but its implementation in the context of parasitic diseases has received less attention. Here, we provide a brief overview of this field and advocate HDT as a promising strategy for antimalarial intervention based on untapped targets. HDT provides a basis from which repurposed drugs could be rapidly deployed and is likely to strongly limit the emergence of resistance. This strategy can be applied to any intracellular pathogen and is particularly well placed in situations in which rapid identification of treatments is needed, such as emerging infections and pandemics, as starkly illustrated by the current COVID-19 crisis.

Property activity refinement of 2-anilino 4-amino substituted quinazolines as antimalarials with fast acting asexual parasite activity.

Malaria is a devastating disease caused by Plasmodium parasites. Emerging resistance against current antimalarial therapeutics has engendered the need to develop antimalarials with novel structural classes. We recently described the identification and initial optimization of the 2-anilino quinazoline antimalarial class. Here, we refine the physicochemical properties of this antimalarial class with the aim to improve aqueous solubility and metabolism and to reduce adverse promiscuity. We show the physicochemical properties of this class are intricately balanced with asexual parasite activity and human cell cytotoxicity. Structural modifications we have implemented improved LipE, aqueous solubility and in vitro metabolism while preserving fast acting P. falciparum asexual stage activity. The lead compounds demonstrated equipotent activity against P. knowlesi parasites and were not predisposed to resistance mechanisms of clinically used antimalarials. The optimized compounds exhibited modest activity against early-stage gametocytes, but no activity against pre-erythrocytic liver parasites. Confoundingly, the refined physicochemical properties installed in the compounds did not engender improved oral efficacy in a P. berghei mouse model of malaria compared to earlier studies on the 2-anilino quinazoline class. This study provides the framework for further development of this antimalarial class.

Optimisation of 2-(N-phenyl carboxamide) triazolopyrimidine antimalarials with moderate to slow acting erythrocytic stage activity.

Malaria is a devastating parasitic disease caused by parasites from the genus Plasmodium. Therapeutic resistance has been reported against all clinically available antimalarials, threatening our ability to control the disease and therefore there is an ongoing need for the development of novel antimalarials. Towards this goal, we identified the 2-(N-phenyl carboxamide) triazolopyrimidine class from a high throughput screen of the Janssen Jumpstarter library against the asexual stages of the P. falciparum parasite. Here we describe the structure activity relationship of the identified class and the optimisation of asexual stage activity while maintaining selectivity against the human HepG2 cell line. The most potent analogues from this study were shown to exhibit equipotent activity against P. falciparum multidrug resistant strains and P. knowlesi asexual parasites. Asexual stage phenotyping studies determined the triazolopyrimidine class arrests parasites at the trophozoite stage, but it is likely these parasites are still metabolically active until the second asexual cycle, and thus have a moderate to slow onset of action. Non-NADPH dependent degradation of the central carboxamide and low aqueous solubility was observed in in vitro ADME profiling. A significant challenge remains to correct these liabilities for further advancement of the 2-(N-phenyl carboxamide) triazolopyrimidine scaffold as a potential moderate to slow acting partner in a curative or prophylactic antimalarial treatment.

The Ins and Outs of Plasmodium Rhoptries, Focusing on the Cytosolic Side.

Parasites of the genus Plasmodium cause human and animal malaria, leading to significant health and economic impacts. A key aspect of the complex life cycle of Plasmodium parasites is the invasion of the parasite into its host cell, which is mediated by secretory organelles. The largest of these organelles, the rhoptry, undergoes rapid and profound physiological changes when it secretes its contents during merozoite and sporozoite invasion of the host erythrocyte and hepatocyte, respectively. Here we discuss recent advancements in our understanding of the dynamic rhoptry biology during the parasite's invasive stages, with a focus on the roles of cytosolically exposed rhoptry-interacting proteins (C-RIPs). We explore potential similarities between the molecular mechanisms driving merozoite and sporozoite rhoptry function.

Structure activity refinement of phenylsulfonyl piperazines as antimalarials that block erythrocytic invasion.

The emerging resistance to combination therapies comprised of artemisinin derivatives has driven a need to identify new antimalarials with novel mechanisms of action. Central to the survival and proliferation of the malaria parasite is the invasion of red blood cells by Plasmodium merozoites, providing an attractive target for novel therapeutics. A screen of the Medicines for Malaria Venture Pathogen Box employing transgenic P. falciparum parasites expressing the nanoluciferase bioluminescent reporter identified the phenylsulfonyl piperazine class as a specific inhibitor of erythrocyte invasion. Here, we describe the optimization and further characterization of the phenylsulfonyl piperazine class. During the optimization process we defined the functionality required for P. falciparum asexual stage activity and determined the alpha-carbonyl S-methyl isomer was important for antimalarial potency. The optimized compounds also possessed comparable activity against multidrug resistant strains of P. falciparum and displayed weak activity against sexual stage gametocytes. We determined that the optimized compounds blocked erythrocyte invasion consistent with the asexual activity observed and therefore the phenylsulfonyl piperazine analogues described could serve as useful tools for studying Plasmodium erythrocyte invasion.

Traditional Chinese herbal medicine Astragalus Radix and its effects on intestinal absorption of aconite alkaloids in rats.

Objective: Astragalus Radix (AR, Huangqi in Chinese) has been widely used as a qi (energy) restoring herb that is thought to act through reinvigorating the spleen and lung. Aconite is used to rebalance the body temperature during illness and played an irreplaceable role in disease control since ancient times, but it is limited by its strong neuro and cardiotoxicity. Since the Song Dynasty (1227), the two herbs have been commonly used as herbal pairs including in the famous Qifu Decotion, from the "Wei's Family Prescription". However, many ancient texts also record that they are not compatible using together, suggesting they can have negative outcomes when mixed. This study investigated whether Astragali Radix had either positive or negative effects on absorption of six different active alkaloids derived from aconite. Methods: Single intestinal perfusion model was used to study the effects of Astragali Radix on aconite alkaloids absorption. Response of ABC transporters and distribution of three tight junction proteins on the surface of intestinal enothelium were assessed by Reverse Transcription-Polymerase Chain Reaction (RT-PCR), Western blot and immunofluorescence microscopy, respectively. Results: The results showed that aconite alkaloids absorption could be inhibited, and different concentrations of Astragali Radix considerably increased the expression levels of the ABC transporters and tight junction proteins with Astragali Radix treatment. Conclusion: These results suggest that Astragali Radix can block absorption of aconite alkaloids through the upregulation expression of ATP-binding cassette transporters (ABC transporters) and tight junction proteins. It demonstrates that co-administration of Astragali Radix with other drugs might change the absorption profile of the second drug which is important to know in clinic therapy.

Automated detection and staging of malaria parasites from cytological smears using convolutional neural networks.

Microscopic examination of blood smears remains the gold standard for laboratory inspection and diagnosis of malaria. Smear inspection is, however, time-consuming and dependent on trained microscopists with results varying in accuracy. We sought to develop an automated image analysis method to improve accuracy and standardization of smear inspection that retains capacity for expert confirmation and image archiving. Here, we present a machine learning method that achieves red blood cell (RBC) detection, differentiation between infected/uninfected cells, and parasite life stage categorization from unprocessed, heterogeneous smear images. Based on a pretrained Faster Region-Based Convolutional Neural Networks (R-CNN) model for RBC detection, our model performs accurately, with an average precision of 0.99 at an intersection-over-union threshold of 0.5. Application of a residual neural network-50 model to infected cells also performs accurately, with an area under the receiver operating characteristic curve of 0.98. Finally, combining our method with a regression model successfully recapitulates intraerythrocytic developmental cycle with accurate lifecycle stage categorization. Combined with a mobile-friendly web-based interface, called PlasmoCount, our method permits rapid navigation through and review of results for quality assurance. By standardizing assessment of Giemsa smears, our method markedly improves inspection reproducibility and presents a realistic route to both routine lab and future field-based automated malaria diagnosis.

Development and application of a high-throughput screening assay for identification of small molecule inhibitors of the P. falciparum reticulocyte binding-like homologue 5 protein.

The P. falciparum parasite, responsible for the disease in humans known as malaria, must invade erythrocytes to provide an environment for self-replication and survival. For invasion to occur, the parasite must engage several ligands on the host erythrocyte surface to enable adhesion, tight junction formation and entry. Critical interactions include binding of erythrocyte binding-like ligands and reticulocyte binding-like homologues (Rhs) to the surface of the host erythrocyte. The reticulocyte binding-like homologue 5 (Rh5) is the only member of this family that is essential for invasion and it binds to the basigin host receptor. The essential nature of Rh5 makes it an important vaccine target, however to date, Rh5 has not been targeted by small molecule intervention. Here, we describe the development of a high-throughput screening assay to identify small molecules which interfere with the Rh5-basigin interaction. To validate the utility of this assay we screened a known drug library and the Medicines for Malaria Box and demonstrated the reproducibility and robustness of the assay for high-throughput screening purposes. The screen of the known drug library identified the known leukotriene antagonist, pranlukast. We used pranlukast as a model inhibitor in a post screening evaluation cascade. We procured and synthesised analogues of pranlukast to assist in the hit confirmation process and show which structural moieties of pranlukast attenuate the Rh5 - basigin interaction. Evaluation of pranlukast analogues against P. falciparum in a viability assay and a schizont rupture assay show the parasite activity was not consistent with the biochemical inhibition of Rh5, questioning the developability of pranlukast as an antimalarial. The high-throughput assay developed from this work has the capacity to screen large collections of small molecules to discover inhibitors of P. falciparum Rh5 for future development of invasion inhibitory antimalarials.

Retargeting azithromycin analogues to have dual-modality antimalarial activity.

BACKGROUND: Resistance to front-line antimalarials (artemisinin combination therapies) is spreading, and development of new drug treatment strategies to rapidly kill Plasmodium spp. malaria parasites is urgently needed. Azithromycin is a clinically used macrolide antibiotic proposed as a partner drug for combination therapy in malaria, which has also been tested as monotherapy. However, its slow-killing 'delayed-death' activity against the parasite's apicoplast organelle and suboptimal activity as monotherapy limit its application as a potential malaria treatment. Here, we explore a panel of azithromycin analogues and demonstrate that chemical modifications can be used to greatly improve the speed and potency of antimalarial action. RESULTS: Investigation of 84 azithromycin analogues revealed nanomolar quick-killing potency directed against the very earliest stage of parasite development within red blood cells. Indeed, the best analogue exhibited 1600-fold higher potency than azithromycin with less than 48 hrs treatment in vitro. Analogues were effective against zoonotic Plasmodium knowlesi malaria parasites and against both multi-drug and artemisinin-resistant Plasmodium falciparum lines. Metabolomic profiles of azithromycin analogue-treated parasites suggested activity in the parasite food vacuole and mitochondria were disrupted. Moreover, unlike the food vacuole-targeting drug chloroquine, azithromycin and analogues were active across blood-stage development, including merozoite invasion, suggesting that these macrolides have a multi-factorial mechanism of quick-killing activity. The positioning of functional groups added to azithromycin and its quick-killing analogues altered their activity against bacterial-like ribosomes but had minimal change on 'quick-killing' activity. Apicoplast minus parasites remained susceptible to both azithromycin and its analogues, further demonstrating that quick-killing is independent of apicoplast-targeting, delayed-death activity. CONCLUSION: We show that azithromycin and analogues can rapidly kill malaria parasite asexual blood stages via a fast action mechanism. Development of azithromycin and analogues as antimalarials offers the possibility of targeting parasites through both a quick-killing and delayed-death mechanism of action in a single, multifactorial chemotype.

Analysis of erythrocyte signalling pathways during Plasmodium falciparum infection identifies targets for host-directed antimalarial intervention.

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.