Global Release of Translational Repression Across Plasmodium's Host-to-Vector Transmission Event.

Malaria parasites must be able to respond quickly to changes in their environment, including during their transmission between mammalian hosts and mosquito vectors. Therefore, before transmission, female gametocytes proactively produce and translationally repress mRNAs that encode essential proteins that the zygote requires to establish a new infection. This essential regulatory control requires the orthologues of DDX6 (DOZI), LSM14a (CITH), and ALBA proteins to form a translationally repressive complex in female gametocytes that associates with many of the affected mRNAs. However, while the release of translational repression of individual mRNAs has been documented, the details of the global release of translational repression have not. Moreover, the changes in spatial arrangement and composition of the DOZI/CITH/ALBA complex that contribute to translational control are also not known. Therefore, we have conducted the first quantitative, comparative transcriptomics and DIA-MS proteomics of Plasmodium parasites across the host-to-vector transmission event to document the global release of translational repression. Using female gametocytes and zygotes of P. yoelii, we found that nearly 200 transcripts are released for translation soon after fertilization, including those with essential functions for the zygote. However, we also observed that some transcripts remain repressed beyond this point. In addition, we have used TurboID-based proximity proteomics to interrogate the spatial and compositional changes in the DOZI/CITH/ALBA complex across this transmission event. Consistent with recent models of translational control, proteins that associate with either the 5' or 3' end of mRNAs are in close proximity to one another during translational repression in female gametocytes and then dissociate upon release of repression in zygotes. This observation is cross-validated for several protein colocalizations in female gametocytes via ultrastructure expansion microscopy and structured illumination microscopy. Moreover, DOZI exchanges its interaction from NOT1-G in female gametocytes to the canonical NOT1 in zygotes, providing a model for a trigger for the release of mRNAs from DOZI. Finally, unenriched phosphoproteomics revealed the modification of key translational control proteins in the zygote. Together, these data provide a model for the essential translational control mechanisms used by malaria parasites to promote their efficient transmission from their mammalian host to their mosquito vector.

Plasmodium RON11 triggers biogenesis of the merozoite rhoptry pair and is essential for erythrocyte invasion.

Malaria is a global and deadly human disease caused by the apicomplexan parasites of the genus Plasmodium. Parasite proliferation within human red blood cells (RBC) is associated with the clinical manifestations of the disease. This asexual expansion within human RBCs, begins with the invasion of RBCs by P. falciparum, which is mediated by the secretion of effectors from two specialized club-shaped secretory organelles in merozoite-stage parasites known as rhoptries. We investigated the function of the Rhoptry Neck Protein 11 (RON11), which contains seven transmembrane domains and calcium-binding EF-hand domains. We generated conditional mutants of the P. falciparum RON11. Knockdown of RON11 inhibits parasite growth by preventing merozoite invasion. The loss of RON11 did not lead to any defects in processing of rhoptry proteins but instead led to a decrease in the amount of rhoptry proteins. We utilized ultrastructure expansion microscopy (U-ExM) to determine the effect of RON11 knockdown on rhoptry biogenesis. Surprisingly, in the absence of RON11, fully developed merozoites had only one rhoptry each. The single rhoptry in RON11 deficient merozoites were morphologically typical with a bulb and a neck oriented into the apical polar ring. Moreover, rhoptry proteins are trafficked accurately to the single rhoptry in RON11 deficient parasites. These data show that in the absence of RON11, the first rhoptry is generated during schizogony but upon the start of cytokinesis, the second rhoptry never forms. Interestingly, these single-rhoptry merozoites were able to attach to host RBCs but are unable to invade RBCs. Instead, RON11 deficient merozoites continue to engage with RBC for prolonged periods eventually resulting in echinocytosis, a result of secreting the contents from the single rhoptry into the RBC. Together, our data show that RON11 triggers the de novo biogenesis of the second rhoptry and functions in RBC invasion.

Nuclear pore complexes undergo Nup221 exchange during blood stage asexual replication of Plasmodium parasites.

Plasmodium parasites, which are the causative agents of malaria, undergo closed mitosis without breakdown of the nuclear envelope. Unlike the closed mitosis in yeast, P. berghei parasites undergo multiple rounds of asynchronous nuclear divisions in a shared cytoplasm result in a multinucleated (8-24) organism prior to formation of daughter cells within an infected red blood cell. During this replication process, intact nuclear pore complexes (NPCs) and their component nucleoporins are likely to play critical roles in parasite growth, facilitating selective bi-directional nucleocytoplasmic transport and genome organization. Here we utilize ultrastructure expansion microscopy (U-ExM) to investigate P. berghei Nup138, Nup221, and Nup313 at the single nucleus level throughout the 24 hour blood-stage replication cycle. Our findings reveal that these Nups are evenly distributed around the nuclei and organized in a rosette structure previously undescribed around the centriolar plaque, which is responsible for intranuclear microtubule nucleation during mitosis. We also detect an increased number of NPCs compared with previously reported, highlighting the power of U-ExM. By adapting the recombination-induced tag exchange (RITE) system to P. berghei, we provide evidence of NPC maintenance, demonstrating Nup221 turnover during parasite asexual replication. Our data shed light on the distribution of NPCs and their homeostasis during the blood-stage replication of P. berghei parasites. Further studies into the nuclear surface of these parasites will allow for a better understanding of parasites nuclear mechanics and organization.

Artemisinin resistance mutations in Pfcoronin impede hemoglobin uptake.

Artemisinin (ART) combination therapies have been critical in reducing malaria morbidity and mortality, but these important drugs are threatened by growing resistance associated with mutations in Pfcoronin and Pfkelch13 . Here, we describe the mechanism of Pfcoronin -mediated ART resistance. Pf Coronin interacts with Pf Actin and localizes to the parasite plasma membrane (PPM), the digestive vacuole (DV) membrane, and membrane of a newly identified preDV compartment-all structures involved in the trafficking of hemoglobin from the RBC for degradation in the DV. Pfcoronin mutations alter Pf Actin homeostasis and impair the development and morphology of the preDV. Ultimately, these changes are associated with decreased uptake of red blood cell cytosolic contents by ring-stage Plasmodium falciparum . Previous work has identified decreased hemoglobin uptake as the mechanism of Pfkelch 13-mediated ART resistance. This work demonstrates that Pf Coronin appears to act via a parallel pathway. For both Pfkelch13 -mediated and Pfcoronin -mediated ART resistance, we hypothesize that the decreased hemoglobin uptake in ring stage parasites results in less heme-based activation of the artemisinin endoperoxide ring and reduced cytocidal activity. This study deepens our understanding of ART resistance, as well as hemoglobin uptake and development of the DV in early-stage parasites.

Expansion microscopy of apicomplexan parasites.

Apicomplexan parasites comprise significant pathogens of humans, livestock and wildlife, but also represent a diverse group of eukaryotes with interesting and unique cell biology. The study of cell biology in apicomplexan parasites is complicated by their small size, and historically this has required the application of cutting-edge microscopy techniques to investigate fundamental processes like mitosis or cell division in these organisms. Recently, a technique called expansion microscopy has been developed, which rather than increasing instrument resolution like most imaging modalities, physically expands a biological sample. In only a few years since its development, a derivative of expansion microscopy known as ultrastructure-expansion microscopy (U-ExM) has been widely adopted and proven extremely useful for studying cell biology of Apicomplexa. Here, we review the insights into apicomplexan cell biology that have been enabled through the use of U-ExM, with a specific focus on Plasmodium, Toxoplasma and Cryptosporidium. Further, we summarize emerging expansion microscopy modifications and modalities and forecast how these may influence the field of parasite cell biology in future.

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.

PEERing forward in parasitology.

PEERs in Parasitology (PiP) is a global scientific grassroots organization founded in 2021 to promote equity and inclusion for persons (currently and) historically excluded from science because of ethnicity and/or race. The article details systemic obstacles PEER parasitologists face and current and future strategies of PiP to overcome them.

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.

Apicoplast Dynamics During Plasmodium Cell Cycle.

The deadly malaria parasite, Plasmodium falciparum, contains a unique subcellular organelle termed the apicoplast, which is a clinically-proven antimalarial drug target. The apicoplast is a plastid with essential metabolic functions that evolved via secondary endosymbiosis. As an ancient endosymbiont, the apicoplast retained its own genome and it must be inherited by daughter cells during cell division. During the asexual replication of P. falciparum inside human red blood cells, both the parasite, and the apicoplast inside it, undergo massive morphological changes, including DNA replication and division. The apicoplast is an integral part of the cell and thus its development is tightly synchronized with the cell cycle. At the same time, certain aspects of its dynamics are independent of nuclear division, representing a degree of autonomy in organelle biogenesis. Here, we review the different aspects of organelle dynamics during P. falciparum intraerythrocytic replication, summarize our current understanding of these processes, and describe the many open questions in this area of parasite basic cell biology.

Hand-in-hand advances in microscopy and Plasmodium nuclear biology.

For 140 years, microscopy has repeatedly revolutionized the study of nucleus biology, but despite this our understanding of the evolutionarily divergent nucleus biology of Plasmodium remains limited. Here, we discuss how microscopy advances have enabled two groundbreaking studies by Simon et al. and Klaus et al. into Plasmodium nucleus biology.

Expansion Microscopy Reveals Plasmodium falciparum Blood-Stage Parasites Undergo Anaphase with A Chromatin Bridge in the Absence of Mini-Chromosome Maintenance Complex Binding Protein.

The malaria parasite Plasmodium falciparum undergoes closed mitosis, which occurs within an intact nuclear envelope, and differs significantly from its human host. Mitosis is underpinned by the dynamics of microtubules and the nuclear envelope. To date, our ability to study P. falciparum mitosis by microscopy has been hindered by the small size of the P. falciparum nuclei. Ultrastructure expansion microscopy (U-ExM) has recently been developed for P. falciparum, allowing the visualization of mitosis at the individual nucleus level. Using U-ExM, three intranuclear microtubule structures are observed: hemispindles, mitotic spindles, and interpolar spindles. A previous study demonstrated that the mini-chromosome maintenance complex binding-protein (MCMBP) depletion caused abnormal nuclear morphology and microtubule defects. To investigate the role of microtubules following MCMBP depletion and study the nuclear envelope in these parasites, we developed the first nuclear stain enabled by U-ExM in P. falciparum. MCMBP-deficient parasites show aberrant hemispindles and mitotic spindles. Moreover, anaphase chromatin bridges and individual nuclei containing multiple microtubule structures were observed following MCMBP knockdown. Collectively, this study refines our understanding of MCMBP-deficient parasites and highlights the utility of U-ExM coupled with a nuclear envelope stain for studying mitosis in P. falciparum.

γδ T cells suppress Plasmodium falciparum blood-stage infection by direct killing and phagocytosis.

Activated Vγ9Vδ2 (γδ2) T lymphocytes that sense parasite-produced phosphoantigens are expanded in Plasmodium falciparum-infected patients. Although previous studies suggested that γδ2 T cells help control erythrocytic malaria, whether γδ2 T cells recognize infected red blood cells (iRBCs) was uncertain. Here we show that iRBCs stained for the phosphoantigen sensor butyrophilin 3A1 (BTN3A1). γδ2 T cells formed immune synapses and lysed iRBCs in a contact, phosphoantigen, BTN3A1 and degranulation-dependent manner, killing intracellular parasites. Granulysin released into the synapse lysed iRBCs and delivered death-inducing granzymes to the parasite. All intra-erythrocytic parasites were susceptible, but schizonts were most sensitive. A second protective γδ2 T cell mechanism was identified. In the presence of patient serum, γδ2 T cells phagocytosed and degraded opsonized iRBCs in a CD16-dependent manner, decreasing parasite multiplication. Thus, γδ2 T cells have two ways to control blood-stage malaria-γδ T cell antigen receptor (TCR)-mediated degranulation and phagocytosis of antibody-coated iRBCs.

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

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

mSphere of Influence: the Dynamic Nature of the Nuclear Envelope during Mitosis of Malaria Parasites.

Sabrina Absalon works in the field of cellular and molecular biology of Plasmodium falciparum, the most virulent parasite causing malaria in humans. In this mSphere of Influence article, she reflects on how the paper "3D nuclear architecture reveals coupled cell cycle dynamics of chromatin and nuclear pores in the malaria parasite Plasmodium falciparum" by Allon Weiner et al. (A. Weiner, N. Dahan-Pasternak, E. Shimoni, V. Shinder, et al., Cell Microbiol 13:967-977, 2011, https://doi.org/10.1111/j.1462-5822.2011.01592.x) triggered her aspiration to study the molecular mechanisms governing nuclear envelope assembly and integrity of P. falciparum throughout the intraerythrocytic development cycle.

Influence of Plasmodium falciparum Calcium-Dependent Protein Kinase 5 (PfCDPK5) on the Late Schizont Stage Phosphoproteome.

Protein kinases are important mediators of signal transduction in cellular pathways, and calcium-dependent protein kinases (CDPKs) compose a unique class of calcium-dependent kinases present in plants and apicomplexans, including Plasmodium parasites, the causative agents of malaria. During the asexual stage of infection, the human malaria parasite Plasmodium falciparum grows inside red blood cells, and P. falciparum calcium-dependent protein kinase 5 (PfCDPK5) is required for egress from the host cell. In this paper, we characterize the late-schizont-stage P. falciparum phosphoproteome by performing large-scale phosphoproteomic profiling on tightly synchronized parasites just prior to egress, identifying 2,704 phosphorylation sites on 919 proteins. Using a conditional knockdown of PfCDPK5, we identify 58 phosphorylation sites on 50 proteins with significant reduction in levels of PfCDPK5-deficient parasites. Furthermore, gene ontology analysis of the identified proteins reveals enrichment in transmembrane- and membrane-associated proteins and in proteins associated with transport activity. Among the identified proteins is PfNPT1, a member of the apicomplexan-specific novel putative transporter (NPT) family of proteins. We show that PfNPT1 is a potential substrate of PfCDPK5 and that PfNPT1 localizes to the parasite plasma membrane. Importantly, P. falciparum egress relies on many proteins unique to Apicomplexa that are therefore attractive targets for antimalarial therapeutics.IMPORTANCE The malaria parasite Plasmodium falciparum is a major cause of morbidity and mortality globally. The P. falciparum parasite proliferates inside red blood cells during the blood stage of infection, and egress from the red blood cell is critical for parasite survival. P. falciparum calcium-dependent protein kinase 5 (PfCDPK5) is essential for egress; parasites deficient in PfCDPK5 remain trapped inside their host cells. We have used a label-free quantitative mass spectrometry approach to identify the phosphoproteome of schizont-stage parasites just prior to egress and identify 50 proteins that display a significant reduction in phosphorylation in PfCDPK5-deficient parasites. We show that a member of the Apicomplexan-specific transport protein family, PfNPT1 is a potential substrate of PfCDPK5 and is localized to the parasite plasma membrane. P. falciparum egress requires several proteins not present in human cells, thus making this pathway an ideal target for new therapeutics.

Calcium-Dependent Protein Kinase 5 Is Required for Release of Egress-Specific Organelles in Plasmodium falciparum.

The human malaria parasite Plasmodium falciparum requires efficient egress out of an infected red blood cell for pathogenesis. This egress event is highly coordinated and is mediated by several signaling proteins, including the plant-like Pfalciparum calcium-dependent protein kinase 5 (PfCDPK5). Knockdown of PfCDPK5 results in an egress block where parasites are trapped inside their host cells. The mechanism of this PfCDPK5-dependent block, however, remains unknown. Here, we show that PfCDPK5 colocalizes with a specialized set of parasite organelles known as micronemes and is required for their discharge, implicating failure of this step as the cause of the egress defect in PfCDPK5-deficient parasites. Furthermore, we show that PfCDPK5 cooperates with the Pfalciparum cGMP-dependent kinase (PfPKG) to fully activate the protease cascade critical for parasite egress. The PfCDPK5-dependent arrest can be overcome by hyperactivation of PfPKG or by physical disruption of the arrested parasite, and we show that both treatments facilitate the release of the micronemes required for egress. Our results define the molecular mechanism of PfCDPK5 function and elucidate the complex signaling pathway of parasite egress.IMPORTANCE The signs and symptoms of clinical malaria result from the replication of parasites in human blood. Efficient egress of the malaria parasite Plasmodium falciparum out of an infected red blood cell is critical for pathogenesis. The Pfalciparum calcium-dependent protein kinase 5 (PfCDPK5) is essential for parasite egress. Following PfCDPK5 knockdown, parasites remain trapped inside their host cell and do not egress, but the mechanism for this block remains unknown. We show that PfCDPK5 colocalizes with parasite organelles known as micronemes. We demonstrate that PfCDPK5 is critical for the discharge of these micronemes and that failure of this step is the molecular mechanism of the parasite egress arrest. We also show that hyperactivation of the cGMP-dependent kinase PKG can overcome this arrest. Our data suggest that small molecules that inhibit the egress signaling pathway could be effective antimalarial therapeutics.

The Malaria Parasite Cyclin H Homolog PfCyc1 Is Required for Efficient Cytokinesis in Blood-Stage Plasmodium falciparum.

All well-studied eukaryotic cell cycles are driven by cyclins, which activate cyclin-dependent kinases (CDKs), and these protein kinase complexes are viable drug targets. The regulatory control of the Plasmodium falciparum cell division cycle remains poorly understood, and the roles of the various CDKs and cyclins remain unclear. The P. falciparum genome contains multiple CDKs, but surprisingly, it does not contain any sequence-identifiable G1-, S-, or M-phase cyclins. We demonstrate that P. falciparum Cyc1 (PfCyc1) complements a G1 cyclin-depleted Saccharomyces cerevisiae strain and confirm that other identified malaria parasite cyclins do not complement this strain. PfCyc1, which has the highest sequence similarity to the conserved cyclin H, cannot complement a temperature-sensitive yeast cyclin H mutant. Coimmunoprecipitation of PfCyc1 from P. falciparum parasites identifies PfMAT1 and PfMRK as specific interaction partners and does not identify PfPK5 or other CDKs. We then generate an endogenous conditional allele of PfCyc1 in blood-stage P. falciparum using a destabilization domain (DD) approach and find that PfCyc1 is essential for blood-stage proliferation. PfCyc1 knockdown does not impede nuclear division, but it prevents proper cytokinesis. Thus, we demonstrate that PfCyc1 has a functional divergence from bioinformatic predictions, suggesting that the malaria parasite cell division cycle has evolved to use evolutionarily conserved proteins in functionally novel ways.IMPORTANCE Human infection by the eukaryotic parasite Plasmodium falciparum causes malaria. Most well-studied eukaryotic cell cycles are driven by cyclins, which activate cyclin-dependent kinases (CDKs) to promote essential cell division processes. Remarkably, there are no identifiable cyclins that are predicted to control the cell cycle in the malaria parasite genome. Thus, our knowledge regarding the basic mechanisms of the malaria parasite cell cycle remains unsatisfactory. We demonstrate that P. falciparum Cyc1 (PfCyc1), a transcriptional cyclin homolog, complements a cell cycle cyclin-deficient yeast strain but not a transcriptional cyclin-deficient strain. We show that PfCyc1 forms a complex in the parasite with PfMRK and the P. falciparum MAT1 homolog. PfCyc1 is essential and nonredundant in blood-stage P. falciparum PfCyc1 knockdown causes a stage-specific arrest after nuclear division, demonstrating morphologically aberrant cytokinesis. This work demonstrates a conserved PfCyc1/PfMAT1/PfMRK complex in malaria and suggests that it functions as a schizont stage-specific regulator of the P. falciparum life cycle.

The disruption of GDP-fucose de novo biosynthesis suggests the presence of a novel fucose-containing glycoconjugate in Plasmodium asexual blood stages.

Glycosylation is an important posttranslational protein modification in all eukaryotes. Besides glycosylphosphatidylinositol (GPI) anchors and N-glycosylation, O-fucosylation has been recently reported in key sporozoite proteins of the malaria parasite. Previous analyses showed the presence of GDP-fucose (GDP-Fuc), the precursor for all fucosylation reactions, in the blood stages of Plasmodium falciparum. The GDP-Fuc de novo pathway, which requires the action of GDP-mannose 4,6-dehydratase (GMD) and GDP-L-fucose synthase (FS), is conserved in the parasite genome, but the importance of fucose metabolism for the parasite is unknown. To functionally characterize the pathway we generated a PfGMD mutant and analyzed its phenotype. Although the labelling by the fucose-binding Ulex europaeus agglutinin I (UEA-I) was completely abrogated, GDP-Fuc was still detected in the mutant. This unexpected result suggests the presence of an alternative mechanism for maintaining GDP-Fuc in the parasite. Furthermore, PfGMD null mutant exhibited normal growth and invasion rates, revealing that the GDP-Fuc de novo metabolic pathway is not essential for the development in culture of the malaria parasite during the asexual blood stages. Nonetheless, the function of this metabolic route and the GDP-Fuc pool that is generated during this stage may be important for gametocytogenesis and sporogonic development in the mosquito.

An essential malaria protein defines the architecture of blood-stage and transmission-stage parasites.

Blood-stage replication of the human malaria parasite Plasmodium falciparum occurs via schizogony, wherein daughter parasites are formed by a specialized cytokinesis known as segmentation. Here we identify a parasite protein, which we name P. falciparum Merozoite Organizing Protein (PfMOP), as essential for cytokinesis of blood-stage parasites. We show that, following PfMOP knockdown, parasites undergo incomplete segmentation resulting in a residual agglomerate of partially divided cells. While organelles develop normally, the structural scaffold of daughter parasites, the inner membrane complex (IMC), fails to form in this agglomerate causing flawed segmentation. In PfMOP-deficient gametocytes, the IMC formation defect causes maturation arrest with aberrant morphology and death. Our results provide insight into the mechanisms of replication and maturation of malaria parasites.