Research into the aetiological agent of the most widespread form of severe malaria, Plasmodium falciparum, has benefitted enormously from the ability to culture and genetically manipulate blood-stage forms of the parasite in vitro. However, most malaria outside Africa is caused by a distinct Plasmodium species, Plasmodium vivax, and it has become increasingly apparent that zoonotic infection by the closely related simian parasite Plasmodium knowlesi is a frequent cause of life-threatening malaria in regions of southeast Asia. Neither of these important malarial species can be cultured in human cells in vitro, requiring access to primates with the associated ethical and practical constraints. We report the successful adaptation of P. knowlesi to continuous culture in human erythrocytes. Humanadapted P. knowlesi clones maintain their capacity to replicate in monkey erythrocytes and can be genetically modified with unprecedented efficiency, providing an important and unique model for studying conserved aspects of malarial biology as well as speciesspecific features of an emerging pathogen.invasion | transfection T he development of a continuous culture system for asexual blood stages of the most deadly human malaria parasite, Plasmodium falciparum (1, 2), proved a milestone in malaria research, enabling genetic modification of the parasite (3), high-throughput drug screening (4), and other fundamental advances in parasite biology. Adaptation of other human malaria parasite species to in vitro culture has proved more challenging, and none of the additional four parasite species that cause human malaria can be continuously maintained in human RBC. This difficulty is a significant obstacle to studying these pathogens, which differ from P. falciparum in important aspects of biology and the pathology they cause. Furthermore, although considerable progress has been made in the development of transgenic technologies for Plasmodium, P. falciparum remains poorly amenable to genetic manipulation, with a typical transfection efficiency of only ∼10 −6 (5). Additional in vitro human malaria parasite models that are genetically tractable and that complement the P. falciparum system have tremendous potential.Much of the early work on the mechanics of RBC invasion by the malaria parasite used the simian parasite Plasmodium knowlesi. This species has a 24-h erythrocytic life cycle and large, long-lived invasive merozoites, facilitating the use of electron and video microscopy to dissect the dynamics of erythrocyte invasion (6-8). P. knowlesi can be cultured in vitro in rhesus monkey (Macaca mulata) RBC with rhesus or human serum (9, 10). Importantly, P. knowlesi is amenable to genetic manipulation, with reported transfection efficiencies similar to those achieved with the rodent malaria model Plasmodium berghei and far surpassing those attained in P. falciparum (10, 11). P. knowlesi is phylogenetically closely related to Plasmodium vivax, the most important cause of malaria outside of Africa (12), so its study can provide insights ...

SummaryHeterochromatin-dependent gene silencing is central to the adaptation and survival of Plasmodium falciparum malaria parasites, allowing clonally variant gene expression during blood infection in humans. By assessing genome-wide heterochromatin protein 1 (HP1) occupancy, we present a comprehensive analysis of heterochromatin landscapes across different Plasmodium species, strains, and life cycle stages. Common targets of epigenetic silencing include fast-evolving multi-gene families encoding surface antigens and a small set of conserved HP1-associated genes with regulatory potential. Many P. falciparum heterochromatic genes are marked in a strain-specific manner, increasing the parasite's adaptive capacity. Whereas heterochromatin is strictly maintained during mitotic proliferation of asexual blood stage parasites, substantial heterochromatin reorganization occurs in differentiating gametocytes and appears crucial for the activation of key gametocyte-specific genes and adaptation of erythrocyte remodeling machinery. Collectively, these findings provide a catalog of heterochromatic genes and reveal conserved and specialized features of epigenetic control across the genus Plasmodium.

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

The ookinete is a motile stage in the malaria life cycle which forms in the mosquito blood meal from the zygote. Ookinetes use an acto-myosin motor to glide towards and penetrate the midgut wall to establish infection in the vector. The regulation of gliding motility is poorly understood. Through genetic interaction studies we here describe a signalling module that identifies guanosine 3′, 5′-cyclic monophosphate (cGMP) as an important second messenger regulating ookinete differentiation and motility. In ookinetes lacking the cyclic nucleotide degrading phosphodiesterase δ (PDEδ), unregulated signalling through cGMP results in rounding up of the normally banana-shaped cells. This phenotype is suppressed in a double mutant additionally lacking guanylyl cyclase β (GCβ), showing that in ookinetes GCβ is an important source for cGMP, and that PDEδ is the relevant cGMP degrading enzyme. Inhibition of the cGMP-dependent protein kinase, PKG, blocks gliding, whereas enhanced signalling through cGMP restores normal gliding speed in a mutant lacking calcium dependent protein kinase 3, suggesting at least a partial overlap between calcium and cGMP dependent pathways. These data demonstrate an important function for signalling through cGMP, and most likely PKG, in dynamically regulating ookinete gliding during the transmission of malaria to the mosquito.