Genome-wide functional analysis reveals key roles for kinesins in the mammalian and mosquito stages of the malaria parasite life cycle.

Kinesins are microtubule (MT)-based motors important in cell division, motility, polarity, and intracellular transport in many eukaryotes. However, they are poorly studied in the divergent eukaryotic pathogens Plasmodium spp., the causative agents of malaria, which manifest atypical aspects of cell division and plasticity of morphology throughout the life cycle in both mammalian and mosquito hosts. Here, we describe a genome-wide screen of Plasmodium kinesins, revealing diverse subcellular locations and functions in spindle assembly, axoneme formation, and cell morphology. Surprisingly, only kinesin-13 is essential for growth in the mammalian host while the other 8 kinesins are required during the proliferative and invasive stages of parasite transmission through the mosquito vector. In-depth analyses of kinesin-13 and kinesin-20 revealed functions in MT dynamics during apical cell polarity formation, spindle assembly, and axoneme biogenesis. These findings help us to understand the importance of MT motors and may be exploited to discover new therapeutic interventions against malaria.

Molecular characterization of the conoid complex in Toxoplasma reveals its conservation in all apicomplexans, including Plasmodium species.

The apical complex is the instrument of invasion used by apicomplexan parasites, and the conoid is a conspicuous feature of this apparatus found throughout this phylum. The conoid, however, is believed to be heavily reduced or missing from Plasmodium species and other members of the class Aconoidasida. Relatively few conoid proteins have previously been identified, making it difficult to address how conserved this feature is throughout the phylum, and whether it is genuinely missing from some major groups. Moreover, parasites such as Plasmodium species cycle through 3 invasive forms, and there is the possibility of differential presence of the conoid between these stages. We have applied spatial proteomics and high-resolution microscopy to develop a more complete molecular inventory and understanding of the organisation of conoid-associated proteins in the model apicomplexan Toxoplasma gondii. These data revealed molecular conservation of all conoid substructures throughout Apicomplexa, including Plasmodium, and even in allied Myzozoa such as Chromera and dinoflagellates. We reporter-tagged and observed the expression and location of several conoid complex proteins in the malaria model P. berghei and revealed equivalent structures in all of its zoite forms, as well as evidence of molecular differentiation between blood-stage merozoites and the ookinetes and sporozoites of the mosquito vector. Collectively, we show that the conoid is a conserved apicomplexan element at the heart of the invasion mechanisms of these highly successful and often devastating parasites.

Real-time dynamics of Plasmodium NDC80 reveals unusual modes of chromosome segregation during parasite proliferation.

Eukaryotic cell proliferation requires chromosome replication and precise segregation to ensure daughter cells have identical genomic copies. Species of the genus Plasmodium, the causative agents of malaria, display remarkable aspects of nuclear division throughout their life cycle to meet some peculiar and unique challenges to DNA replication and chromosome segregation. The parasite undergoes atypical endomitosis and endoreduplication with an intact nuclear membrane and intranuclear mitotic spindle. To understand these diverse modes of Plasmodium cell division, we have studied the behaviour and composition of the outer kinetochore NDC80 complex, a key part of the mitotic apparatus that attaches the centromere of chromosomes to microtubules of the mitotic spindle. Using NDC80-GFP live-cell imaging in Plasmodium berghei, we observe dynamic spatiotemporal changes during proliferation, including highly unusual kinetochore arrangements during sexual stages. We identify a very divergent candidate for the SPC24 subunit of the NDC80 complex, previously thought to be missing in Plasmodium, which completes a canonical, albeit unusual, NDC80 complex structure. Altogether, our studies reveal the kinetochore to be an ideal tool to investigate the non-canonical modes of chromosome segregation and cell division in Plasmodium.

Comparative 3D genome organization in apicomplexan parasites.

The positioning of chromosomes in the nucleus of a eukaryotic cell is highly organized and has a complex and dynamic relationship with gene expression. In the human malaria parasite Plasmodium falciparum, the clustering of a family of virulence genes correlates with their coordinated silencing and has a strong influence on the overall organization of the genome. To identify conserved and species-specific principles of genome organization, we performed Hi-C experiments and generated 3D genome models for five Plasmodium species and two related apicomplexan parasites. Plasmodium species mainly showed clustering of centromeres, telomeres, and virulence genes. In P. falciparum, the heterochromatic virulence gene cluster had a strong repressive effect on the surrounding nuclear space, while this was less pronounced in Plasmodium vivax and Plasmodium berghei, and absent in Plasmodium yoelii In Plasmodium knowlesi, telomeres and virulence genes were more dispersed throughout the nucleus, but its 3D genome showed a strong correlation with gene expression. The Babesia microti genome showed a classical Rabl organization with colocalization of subtelomeric virulence genes, while the Toxoplasma gondii genome was dominated by clustering of the centromeres and lacked virulence gene clustering. Collectively, our results demonstrate that spatial genome organization in most Plasmodium species is constrained by the colocalization of virulence genes. P. falciparum and P. knowlesi, the only two Plasmodium species with gene families involved in antigenic variation, are unique in the effect of these genes on chromosome folding, indicating a potential link between genome organization and gene expression in more virulent pathogens.

Plasmodium kinesin-8X associates with mitotic spindles and is essential for oocyst development during parasite proliferation and transmission.

Kinesin-8 proteins are microtubule motors that are often involved in regulation of mitotic spindle length and chromosome alignment. They move towards the plus ends of spindle microtubules and regulate the dynamics of these ends due, at least in some species, to their microtubule depolymerization activity. Plasmodium spp. exhibit an atypical endomitotic cell division in which chromosome condensation and spindle dynamics in the different proliferative stages are not well understood. Genome-wide shared orthology analysis of Plasmodium spp. revealed the presence of two kinesin-8 motor proteins, kinesin-8X and kinesin-8B. Here we studied the biochemical properties of kinesin-8X and its role in parasite proliferation. In vitro, kinesin-8X has motility and depolymerization activities like other kinesin-8 motors. To understand the role of Plasmodium kinesin-8X in cell division, we used fluorescence-tagging and live cell imaging to define its location, and gene targeting to analyse its function, during all proliferative stages of the rodent malaria parasite P. berghei life cycle. The results revealed a spatio-temporal involvement of kinesin-8X in spindle dynamics and an association with both mitotic and meiotic spindles and the putative microtubule organising centre (MTOC). Deletion of the kinesin-8X gene revealed a defect in oocyst development, confirmed by ultrastructural studies, suggesting that this protein is required for oocyst development and sporogony. Transcriptome analysis of Δkinesin-8X gametocytes revealed modulated expression of genes involved mainly in microtubule-based processes, chromosome organisation and the regulation of gene expression, supporting a role for kinesin-8X in cell division. Kinesin-8X is thus required for parasite proliferation within the mosquito and for transmission to the vertebrate host.

Systematic analysis of Plasmodium myosins reveals differential expression, localisation, and function in invasive and proliferative parasite stages.

The myosin superfamily comprises of actin-dependent eukaryotic molecular motors important in a variety of cellular functions. Although well studied in many systems, knowledge of their functions in Plasmodium, the causative agent of malaria, is restricted. Previously, six myosins were identified in this genus, including three Class XIV myosins found only in Apicomplexa and some Ciliates. The well characterized MyoA is a Class XIV myosin essential for gliding motility and invasion. Here, we characterize all other Plasmodium myosins throughout the parasite life cycle and show that they have very diverse patterns of expression and cellular location. MyoB and MyoE, the other two Class XIV myosins, are expressed in all invasive stages, with apical and basal locations, respectively. Gene deletion revealed that MyoE is involved in sporozoite traversal, MyoF and MyoK are likely essential in the asexual blood stages, and MyoJ and MyoB are not essential. Both MyoB and its essential light chain (MCL-B) are localised at the apical end of ookinetes but expressed at completely different time points. This work provides a better understanding of the role of actomyosin motors in Apicomplexan parasites, particularly in the motile and invasive stages of Plasmodium during sexual and asexual development within the mosquito.

Compositional and expression analyses of the glideosome during the Plasmodium life cycle reveal an additional myosin light chain required for maximum motility.

Myosin A (MyoA) is a Class XIV myosin implicated in gliding motility and host cell and tissue invasion by malaria parasites. MyoA is part of a membrane-associated protein complex called the glideosome, which is essential for parasite motility and includes the MyoA light chain myosin tail domain-interacting protein (MTIP) and several glideosome-associated proteins (GAPs). However, most studies of MyoA have focused on single stages of the parasite life cycle. We examined MyoA expression throughout the Plasmodium berghei life cycle in both mammalian and insect hosts. In extracellular ookinetes, sporozoites, and merozoites, MyoA was located at the parasite periphery. In the sexual stages, zygote formation and initial ookinete differentiation precede MyoA synthesis and deposition, which occurred only in the developing protuberance. In developing intracellular asexual blood stages, MyoA was synthesized in mature schizonts and was located at the periphery of segmenting merozoites, where it remained throughout maturation, merozoite egress, and host cell invasion. Besides the known GAPs in the malaria parasite, the complex included GAP40, an additional myosin light chain designated essential light chain (ELC), and several other candidate components. This ELC bound the MyoA neck region adjacent to the MTIP-binding site, and both myosin light chains co-located to the glideosome. Co-expression of MyoA with its two light chains revealed that the presence of both light chains enhances MyoA-dependent actin motility. In conclusion, we have established a system to study the interplay and function of the three glideosome components, enabling the assessment of inhibitors that target this motor complex to block host cell invasion.

Plasmodium P-Type Cyclin CYC3 Modulates Endomitotic Growth during Oocyst Development in Mosquitoes.

Cell-cycle progression and cell division in eukaryotes are governed in part by the cyclin family and their regulation of cyclin-dependent kinases (CDKs). Cyclins are very well characterised in model systems such as yeast and human cells, but surprisingly little is known about their number and role in Plasmodium, the unicellular protozoan parasite that causes malaria. Malaria parasite cell division and proliferation differs from that of many eukaryotes. During its life cycle it undergoes two types of mitosis: endomitosis in asexual stages and an extremely rapid mitotic process during male gametogenesis. Both schizogony (producing merozoites) in host liver and red blood cells, and sporogony (producing sporozoites) in the mosquito vector, are endomitotic with repeated nuclear replication, without chromosome condensation, before cell division. The role of specific cyclins during Plasmodium cell proliferation was unknown. We show here that the Plasmodium genome contains only three cyclin genes, representing an unusual repertoire of cyclin classes. Expression and reverse genetic analyses of the single Plant (P)-type cyclin, CYC3, in the rodent malaria parasite, Plasmodium berghei, revealed a cytoplasmic and nuclear location of the GFP-tagged protein throughout the lifecycle. Deletion of cyc3 resulted in defects in size, number and growth of oocysts, with abnormalities in budding and sporozoite formation. Furthermore, global transcript analysis of the cyc3-deleted and wild type parasites at gametocyte and ookinete stages identified differentially expressed genes required for signalling, invasion and oocyst development. Collectively these data suggest that cyc3 modulates oocyst endomitotic development in Plasmodium berghei.

A putative homologue of CDC20/CDH1 in the malaria parasite is essential for male gamete development.

Cell-cycle progression is governed by a series of essential regulatory proteins. Two major regulators are cell-division cycle protein 20 (CDC20) and its homologue, CDC20 homologue 1 (CDH1), which activate the anaphase-promoting complex/cyclosome (APC/C) in mitosis, and facilitate degradation of mitotic APC/C substrates. The malaria parasite, Plasmodium, is a haploid organism which, during its life-cycle undergoes two stages of mitosis; one associated with asexual multiplication and the other with male gametogenesis. Cell-cycle regulation and DNA replication in Plasmodium was recently shown to be dependent on the activity of a number of protein kinases. However, the function of cell division cycle proteins that are also involved in this process, such as CDC20 and CDH1 is totally unknown. Here we examine the role of a putative CDC20/CDH1 in the rodent malaria Plasmodium berghei (Pb) using reverse genetics. Phylogenetic analysis identified a single putative Plasmodium CDC20/CDH1 homologue (termed CDC20 for simplicity) suggesting that Plasmodium APC/C has only one regulator. In our genetic approach to delete the endogenous cdc20 gene of P. berghei, we demonstrate that PbCDC20 plays a vital role in male gametogenesis, but is not essential for mitosis in the asexual blood stage. Furthermore, qRT-PCR analysis in parasite lines with deletions of two kinase genes involved in male sexual development (map2 and cdpk4), showed a significant increase in cdc20 transcription in activated gametocytes. DNA replication and ultra structural analyses of cdc20 and map2 mutants showed similar blockage of nuclear division at the nuclear spindle/kinetochore stage. CDC20 was phosphorylated in asexual and sexual stages, but the level of modification was higher in activated gametocytes and ookinetes. Changes in global protein phosphorylation patterns in the Δcdc20 mutant parasites were largely different from those observed in the Δmap2 mutant. This suggests that CDC20 and MAP2 are both likely to play independent but vital roles in male gametogenesis.

A unique protein phosphatase with kelch-like domains (PPKL) in Plasmodium modulates ookinete differentiation, motility and invasion.

Protein phosphorylation and dephosphorylation (catalysed by kinases and phosphatases, respectively) are post-translational modifications that play key roles in many eukaryotic signalling pathways, and are often deregulated in a number of pathological conditions in humans. In the malaria parasite Plasmodium, functional insights into its kinome have only recently been achieved, with over half being essential for blood stage development and another 14 kinases being essential for sexual development and mosquito transmission. However, functions for any of the plasmodial protein phosphatases are unknown. Here, we use reverse genetics in the rodent malaria model, Plasmodium berghei, to examine the role of a unique protein phosphatase containing kelch-like domains (termed PPKL) from a family related to Arabidopsis BSU1. Phylogenetic analysis confirmed that the family of BSU1-like proteins including PPKL is encoded in the genomes of land plants, green algae and alveolates, but not in other eukaryotic lineages. Furthermore, PPKL was observed in a distinct family, separate to the most closely-related phosphatase family, PP1. In our genetic approach, C-terminal GFP fusion with PPKL showed an active protein phosphatase preferentially expressed in female gametocytes and ookinetes. Deletion of the endogenous ppkl gene caused abnormal ookinete development and differentiation, and dissociated apical microtubules from the inner-membrane complex, generating an immotile phenotype and failure to invade the mosquito mid-gut epithelium. These observations were substantiated by changes in localisation of cytoskeletal tubulin and actin, and the micronemal protein CTRP in the knockout mutant as assessed by indirect immunofluorescence. Finally, increased mRNA expression of dozi, a RNA helicase vital to zygote development was observed in ppkl(-) mutants, with global phosphorylation studies of ookinete differentiation from 1.5-24 h post-fertilisation indicating major changes in the first hours of zygote development. Our work demonstrates a stage-specific essentiality of the unique PPKL enzyme, which modulates parasite differentiation, motility and transmission.

Plasmodium ARK2-EB1 axis drives the unconventional spindle dynamics, scaffold formation and chromosome segregation of sexual transmission stages.

Mechanisms of cell division are remarkably diverse, suggesting the underlying molecular networks among eukaryotes differ extensively. The Aurora family of kinases orchestrates the process of chromosome segregation and cytokinesis during cell division through precise spatiotemporal regulation of their catalytic activities by distinct scaffolds. Plasmodium spp., the causative agents of malaria, are unicellular eukaryotes that have three divergent aurora-related kinases (ARKs) and lack most canonical scaffolds/activators. The parasite uses unconventional modes of chromosome segregation during endomitosis and meiosis in sexual transmission stages within mosquito host. This includes a rapid threefold genome replication from 1N to 8N with successive cycles of closed mitosis, spindle formation and chromosome segregation within eight minutes (termed male gametogony). Kinome studies had previously suggested likely essential functions for all three Plasmodium ARKs during asexual mitotic cycles; however, little is known about their location, function, or their scaffolding molecules during unconventional sexual proliferative stages. Using a combination of super-resolution microscopy, mass spectrometry, and live-cell fluorescence imaging, we set out to investigate the role of the atypical Aurora paralog ARK2 to proliferative sexual stages using rodent malaria model Plasmodium berghei . We find that ARK2 primarily localises to the spindle apparatus in the vicinity of kinetochores during both mitosis and meiosis. Interactomics and co-localisation studies reveal a unique ARK2 scaffold at the spindle including the microtubule plus end-binding protein EB1, lacking conserved Aurora scaffold proteins. Gene function studies indicate complementary functions of ARK2 and EB1 in driving endomitotic divisions and thereby parasite transmission. Our discovery of a novel Aurora kinase spindle scaffold underlines the emerging flexibility of molecular networks to rewire and drive unconventional mechanisms of chromosome segregation in the malaria parasite Plasmodium .

Plasmodium ARK2-EB1 axis drives the unconventional spindle dynamics, scaffold formation and chromosome segregation of sexual transmission stages.

Mechanisms of cell division are remarkably diverse, suggesting the underlying molecular networks among eukaryotes differ extensively. The Aurora family of kinases orchestrates the process of chromosome segregation and cytokinesis during cell division through precise spatiotemporal regulation of their catalytic activities by distinct scaffolds. Plasmodium spp., the causative agents of malaria, are unicellular eukaryotes that have three divergent aurora-related kinases (ARKs) and lack most canonical scaffolds/activators. The parasite uses unconventional modes of chromosome segregation during endomitosis and meiosis in sexual transmission stages within mosquito host. This includes a rapid threefold genome replication from 1N to 8N with successive cycles of closed mitosis, spindle formation and chromosome segregation within eight minutes (termed male gametogony). Kinome studies had previously suggested likely essential functions for all three Plasmodium ARKs during asexual mitotic cycles; however, little is known about their location, function, or their scaffolding molecules during unconventional sexual proliferative stages. Using a combination of super-resolution microscopy, mass spectrometry, omics and live-cell fluorescence imaging, we set out to investigate the contribution of the atypical Aurora paralog ARK2 to proliferative sexual stages using rodent malaria model Plasmodium berghei. We find that ARK2 primarily localises to the spindle apparatus associated with kinetochores during both mitosis and meiosis. Interactomics and co-localisation studies reveal a unique ARK2 scaffold at the spindle including the microtubule plus end-binding protein EB1 and lacking some other conserved molecules. Gene function studies indicate complementary functions of ARK2 and EB1 in driving endomitotic divisions and thereby parasite transmission. Our discovery of a novel Aurora spindle scaffold underlines the emerging flexibility of molecular networks to rewire and drive unconventional mechanisms of chromosome segregation in the malaria parasite Plasmodium.

Plasmodium ARK2 and EB1 drive unconventional spindle dynamics, during chromosome segregation in sexual transmission stages.

The Aurora family of kinases orchestrates chromosome segregation and cytokinesis during cell division, with precise spatiotemporal regulation of its catalytic activities by distinct protein scaffolds. Plasmodium spp., the causative agents of malaria, are unicellular eukaryotes with three unique and highly divergent aurora-related kinases (ARK1-3) that are essential for asexual cellular proliferation but lack most canonical scaffolds/activators. Here we investigate the role of ARK2 during sexual proliferation of the rodent malaria Plasmodium berghei, using a combination of super-resolution microscopy, mass spectrometry, and live-cell fluorescence imaging. We find that ARK2 is primarily located at spindle microtubules in the vicinity of kinetochores during both mitosis and meiosis. Interactomic and co-localisation studies reveal several putative ARK2-associated interactors including the microtubule-interacting protein EB1, together with MISFIT and Myosin-K, but no conserved eukaryotic scaffold proteins. Gene function studies indicate that ARK2 and EB1 are complementary in driving endomitotic division and thereby parasite transmission through the mosquito. This discovery underlines the flexibility of molecular networks to rewire and drive unconventional mechanisms of chromosome segregation in the malaria parasite.

Plasmodium SAS4: basal body component of male cell which is dispensable for parasite transmission.

The centriole/basal body (CBB) is an evolutionarily conserved organelle acting as a microtubule organising centre (MTOC) to nucleate cilia, flagella, and the centrosome. SAS4/CPAP is a conserved component associated with BB biogenesis in many model flagellated cells. Plasmodium, a divergent unicellular eukaryote and causative agent of malaria, displays an atypical, closed mitosis with an MTOC (or centriolar plaque), reminiscent of an acentriolar MTOC, embedded in the nuclear membrane. Mitosis during male gamete formation is accompanied by flagella formation. There are two MTOCs in male gametocytes: the acentriolar nuclear envelope MTOC for the mitotic spindle and an outer centriolar MTOC (the basal body) that organises flagella assembly in the cytoplasm. We show the coordinated location, association and assembly of SAS4 with the BB component, kinesin-8B, but no association with the kinetochore protein, NDC80, indicating that SAS4 is part of the BB and outer centriolar MTOC in the cytoplasm. Deletion of the SAS4 gene produced no phenotype, indicating that it is not essential for either male gamete formation or parasite transmission.

Mechanochemical tuning of a kinesin motor essential for malaria parasite transmission.

Plasmodium species cause malaria and kill hundreds of thousands annually. The microtubule-based motor kinesin-8B is required for development of the flagellated Plasmodium male gamete, and its absence completely blocks parasite transmission. To understand the molecular basis of kinesin-8B's essential role, we characterised the in vitro properties of kinesin-8B motor domains from P. berghei and P. falciparum. Both motors drive ATP-dependent microtubule gliding, but also catalyse ATP-dependent microtubule depolymerisation. We determined these motors' microtubule-bound structures using cryo-electron microscopy, which showed very similar modes of microtubule interaction in which Plasmodium-distinct sequences at the microtubule-kinesin interface influence motor function. Intriguingly however, P. berghei kinesin-8B exhibits a non-canonical structural response to ATP analogue binding such that neck linker docking is not induced. Nevertheless, the neck linker region is required for motility and depolymerisation activities of these motors. These data suggest that the mechanochemistry of Plasmodium kinesin-8Bs is functionally tuned to support flagella formation.

Protein phosphatase 1 regulates atypical mitotic and meiotic division in Plasmodium sexual stages.

PP1 is a conserved eukaryotic serine/threonine phosphatase that regulates many aspects of mitosis and meiosis, often working in concert with other phosphatases, such as CDC14 and CDC25. The proliferative stages of the malaria parasite life cycle include sexual development within the mosquito vector, with male gamete formation characterized by an atypical rapid mitosis, consisting of three rounds of DNA synthesis, successive spindle formation with clustered kinetochores, and a meiotic stage during zygote to ookinete development following fertilization. It is unclear how PP1 is involved in these unusual processes. Using real-time live-cell and ultrastructural imaging, conditional gene knockdown, RNA-seq and proteomic approaches, we show that Plasmodium PP1 is implicated in both mitotic exit and, potentially, establishing cell polarity during zygote development in the mosquito midgut, suggesting that small molecule inhibitors of PP1 should be explored for blocking parasite transmission.

Plasmodium berghei Kinesin-5 Associates With the Spindle Apparatus During Cell Division and Is Important for Efficient Production of Infectious Sporozoites.

Kinesin-5 motors play essential roles in spindle apparatus assembly during cell division, by generating forces to establish and maintain the spindle bipolarity essential for proper chromosome segregation. Kinesin-5 is largely conserved structurally and functionally in model eukaryotes, but its role is unknown in the Plasmodium parasite, an evolutionarily divergent organism with several atypical features of both mitotic and meiotic cell division. We have investigated the function and subcellular location of kinesin-5 during cell division throughout the Plasmodium berghei life cycle. Deletion of kinesin-5 had little visible effect at any proliferative stage except sporozoite production in oocysts, resulting in a significant decrease in the number of motile sporozoites in mosquito salivary glands, which were able to infect a new vertebrate host. Live-cell imaging showed kinesin-5-GFP located on the spindle and at spindle poles during both atypical mitosis and meiosis. Fixed-cell immunofluorescence assays revealed kinesin-5 co-localized with α-tubulin and centrin-2 and a partial overlap with kinetochore marker NDC80 during early blood stage schizogony. Dual-color live-cell imaging showed that kinesin-5 is closely associated with NDC80 during male gametogony, but not with kinesin-8B, a marker of the basal body and axonemes of the forming flagella. Treatment of gametocytes with microtubule-specific inhibitors confirmed kinesin-5 association with nuclear spindles and not cytoplasmic axonemal microtubules. Altogether, our results demonstrate that kinesin-5 is associated with the spindle apparatus, expressed in proliferating parasite stages, and important for efficient production of infectious sporozoites.

Plasmodium DEH is ER-localized and crucial for oocyst mitotic division during malaria transmission.

Cells use fatty acids (FAs) for membrane biosynthesis, energy storage, and the generation of signaling molecules. 3-hydroxyacyl-CoA dehydratase-DEH-is a key component of very long chain fatty acid synthesis. Here, we further characterized in-depth the location and function of DEH, applying in silico analysis, live cell imaging, reverse genetics, and ultrastructure analysis using the mouse malaria model Plasmodium berghei DEH is evolutionarily conserved across eukaryotes, with a single DEH in Plasmodium spp. and up to three orthologs in the other eukaryotes studied. DEH-GFP live-cell imaging showed strong GFP fluorescence throughout the life-cycle, with areas of localized expression in the cytoplasm and a circular ring pattern around the nucleus that colocalized with ER markers. Δdeh mutants showed a small but significant reduction in oocyst size compared with WT controls from day 10 postinfection onwards, and endomitotic cell division and sporogony were completely ablated, blocking parasite transmission from mosquito to vertebrate host. Ultrastructure analysis confirmed degeneration of Δdeh oocysts, and a complete lack of sporozoite budding. Overall, DEH is evolutionarily conserved, localizes to the ER, and plays a crucial role in sporogony.

MRE11 Is Crucial for Malaria Parasite Transmission and Its Absence Affects Expression of Interconnected Networks of Key Genes Essential for Life.

The meiotic recombination 11 protein (MRE11) plays a key role in DNA damage response and maintenance of genome stability. However, little is known about its function during development of the malaria parasite Plasmodium. Here, we present a functional, ultrastructural and transcriptomic analysis of Plasmodium parasites lacking MRE11 during its life cycle in both mammalian and mosquito vector hosts. Genetic disruption of Plasmodium berghei mre11 (PbMRE11) results in significant retardation of oocyst development in the mosquito midgut associated with cytoplasmic and nuclear degeneration, along with concomitant ablation of sporogony and subsequent parasite transmission. Further, absence of PbMRE11 results in significant transcriptional downregulation of genes involved in key interconnected biological processes that are fundamental to all eukaryotic life including ribonucleoprotein biogenesis, spliceosome function and iron-sulfur cluster assembly. Overall, our study provides a comprehensive functional analysis of MRE11's role in Plasmodium development during the mosquito stages and offers a potential target for therapeutic intervention during malaria parasite transmission.

A divergent cyclin/cyclin-dependent kinase complex controls the atypical replication of a malaria parasite during gametogony and transmission.

Cell cycle transitions are generally triggered by variation in the activity of cyclin-dependent kinases (CDKs) bound to cyclins. Malaria-causing parasites have a life cycle with unique cell-division cycles, and a repertoire of divergent CDKs and cyclins of poorly understood function and interdependency. We show that Plasmodium berghei CDK-related kinase 5 (CRK5), is a critical regulator of atypical mitosis in the gametogony and is required for mosquito transmission. It phosphorylates canonical CDK motifs of components in the pre-replicative complex and is essential for DNA replication. During a replicative cycle, CRK5 stably interacts with a single Plasmodium-specific cyclin (SOC2), although we obtained no evidence of SOC2 cycling by transcription, translation or degradation. Our results provide evidence that during Plasmodium male gametogony, this divergent cyclin/CDK pair fills the functional space of other eukaryotic cell-cycle kinases controlling DNA replication.