Anti-PfGARP activates programmed cell death of parasites and reduces severe malaria.

Malaria caused by Plasmodium falciparum remains the leading single-agent cause of mortality in children1, yet the promise of an effective vaccine has not been fulfilled. Here, using our previously described differential screening method to analyse the proteome of blood-stage P. falciparum parasites2, we identify P. falciparum glutamic-acid-rich protein (PfGARP) as a parasite antigen that is recognized by antibodies in the plasma of children who are relatively resistant-but not those who are susceptible-to malaria caused by P. falciparum. PfGARP is a parasite antigen of 80 kDa that is expressed on the exofacial surface of erythrocytes infected by early-to-late-trophozoite-stage parasites. We demonstrate that antibodies against PfGARP kill trophozoite-infected erythrocytes in culture by inducing programmed cell death in the parasites, and that vaccinating non-human primates with PfGARP partially protects against a challenge with P. falciparum. Furthermore, our longitudinal cohort studies showed that, compared to individuals who had naturally occurring anti-PfGARP antibodies, Tanzanian children without anti-PfGARP antibodies had a 2.5-fold-higher risk of severe malaria and Kenyan adolescents and adults without these antibodies had a twofold-higher parasite density. By killing trophozoite-infected erythrocytes, PfGARP could synergize with other vaccines that target parasite invasion of hepatocytes or the invasion of and egress from erythrocytes.

Three-dimensional ultrastructure of Plasmodium falciparum throughout cytokinesis.

New techniques for obtaining electron microscopy data through the cell volume are being increasingly utilized to answer cell biologic questions. Here, we present a three-dimensional atlas of Plasmodium falciparum ultrastructure throughout parasite cell division. Multiple wild type schizonts at different stages of segmentation, or budding, were imaged and rendered, and the 3D structure of their organelles and daughter cells are shown. Our high-resolution volume electron microscopy both confirms previously described features in 3D and adds new layers to our understanding of Plasmodium nuclear division. Interestingly, we demonstrate asynchrony of the final nuclear division, a process that had previously been reported as synchronous. Use of volume electron microscopy techniques for biological imaging is gaining prominence, and there is much we can learn from applying them to answer questions about Plasmodium cell biology. We provide this resource to encourage readers to consider adding these techniques to their cell biology toolbox.

A PPP-type pseudophosphatase is required for the maintenance of basal complex integrity in Plasmodium falciparum.

During its asexual blood stage, P. falciparum replicates via schizogony, wherein dozens of daughter cells are formed within a single parent. The basal complex, a contractile ring that separates daughter cells, is critical for schizogony. In this study, we identify a Plasmodium basal complex protein essential for basal complex maintenance. Using multiple microscopy techniques, we demonstrate that PfPPP8 is required for uniform basal complex expansion and maintenance of its integrity. We characterize PfPPP8 as the founding member of a novel family of pseudophosphatases with homologs in other Apicomplexan parasites. By co-immunoprecipitation, we identify two additional new basal complex proteins. We characterize the unique temporal localizations of these new basal complex proteins (late-arriving) and of PfPPP8 (early-departing). In this work, we identify a novel basal complex protein, determine its specific role in segmentation, identify a new pseudophosphatase family, and establish that the P. falciparum basal complex is a dynamic structure.

Essential function of alveolin PfIMC1g in the Plasmodium falciparum asexual blood stage.

IMPORTANCE: Infection by the Plasmodium falciparum parasite is responsible for the most severe form of human malaria. The asexual blood stage of the parasite, which occurs inside human red blood cells, is responsible for the symptoms of malaria and is the target of most antimalarial drugs. Plasmodium spp. rely on their highly divergent cytoskeletal structures to scaffold their cell division, sustain the mechanical stress of invasion, and survive in both the human bloodstream and the mosquito. We investigate the function of a class of divergent intermediate filament-like proteins called alveolins in the clinically important blood stage. The functional role of individual alveolins in Plasmodium remains poorly understood due to pleiotropic effects of gene knockouts and redundancy among alveolins. We evaluate the localization and essentiality of the four asexual-stage alveolins and find that PfIMC1g and PfIMC1c are essential. Furthermore, we demonstrate that PfIMC1g is critical for survival of the parasite post-invasion.

Novel CRISPR-Associated Gene-Editing Systems Discovered in Metagenomic Samples Enable Efficient and Specific Genome Engineering.

Development of medicines using gene editing has been hampered by enzymological and immunological impediments. We described previously the discovery and characterization of improved, novel gene-editing systems from metagenomic data. In this study, we substantially advance this work with three such gene-editing systems, demonstrating their utility for cell therapy development. All three systems are capable of reproducible, high-frequency gene editing in primary immune cells. In human T cells, disruption of the T cell receptor (TCR) alpha-chain was induced in >95% of cells, both paralogs of the TCR beta-chain in >90% of cells, and >90% knockout of ß2-microglobulin, TIGIT, FAS, and PDCD1. Simultaneous double knockout of TRAC and TRBC was obtained at a frequency equal to that of the single edits. Gene editing with our systems had minimal effect on T cell viability. Furthermore, we integrate a chimeric antigen receptor (CAR) construct into TRAC (up to ∼60% of T cells), and demonstrate CAR expression and cytotoxicity. We next applied our novel gene-editing tools to natural killer (NK) cells, B cells, hematopoietic stem cells, and induced pluripotent stem cells, generating similarly efficient cell-engineering outcomes including the creation of active CAR-NK cells. Interrogation of our gene-editing systems' specificity reveals a profile comparable with or better than Cas9. Finally, our nucleases lack preexisting humoral and T cell-based immunity, consistent with their sourcing from nonhuman pathogens. In all, we show these new gene-editing systems have the activity, specificity, and translatability necessary for use in cell therapy development.

Versatile Labeling and Detection of Endogenous Proteins Using Tag-Assisted Split Enzyme Complementation.

Recent advances in genome engineering have expanded our capabilities to study proteins in their natural states. In particular, the ease and scalability of knocking-in small peptide tags has enabled high throughput tagging and analysis of endogenous proteins. To improve enrichment capacities and expand the functionality of knock-ins using short tags, we developed the tag-assisted split enzyme complementation (TASEC) approach, which uses two orthogonal small peptide tags and their cognate binders to conditionally drive complementation of a split enzyme upon labeled protein expression. Using this approach, we have engineered and optimized the tag-assisted split HaloTag complementation system (TA-splitHalo) and demonstrated its versatile applications in improving the efficiency of knock-in cell enrichment, detection of protein-protein interaction, and isolation of biallelic gene edited cells through multiplexing.

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.

An essential contractile ring protein controls cell division in Plasmodium falciparum.

During the blood stage of human malaria, Plasmodium falciparum parasites divide by schizogony-a process wherein components for several daughter cells are produced within a common cytoplasm and then segmentation, a synchronized cytokinesis, produces individual invasive daughters. The basal complex is hypothesized to be required for segmentation, acting as a contractile ring to establish daughter cell boundaries. Here we identify an essential component of the basal complex which we name PfCINCH. Using three-dimensional reconstructions of parasites at electron microscopy resolution, we show that while parasite organelles form and divide normally, PfCINCH-deficient parasites develop inviable conjoined daughters that contain components for multiple cells. Through biochemical evaluation of the PfCINCH-containing complex, we discover multiple previously undescribed basal complex proteins. Therefore, this work provides genetic evidence that the basal complex is required for precise segmentation and lays the groundwork for a mechanistic understanding of how the parasite contractile ring drives cell division.

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.

Anti-Self Phosphatidylserine Antibodies Recognize Uninfected Erythrocytes Promoting Malarial Anemia.

Plasmodium species, the parasitic agents of malaria, invade erythrocytes to reproduce, resulting in erythrocyte loss. However, a greater loss is caused by the elimination of uninfected erythrocytes, sometimes long after infection has been cleared. Using a mouse model, we found that Plasmodium infection induces the generation of anti-self antibodies that bind to the surface of uninfected erythrocytes from infected, but not uninfected, mice. These antibodies recognize phosphatidylserine, which is exposed on the surface of a fraction of uninfected erythrocytes during malaria. We find that phosphatidylserine-exposing erythrocytes are reticulocytes expressing high levels of CD47, a "do-not-eat-me" signal, but the binding of anti-phosphatidylserine antibodies mediates their phagocytosis, contributing to anemia. In human patients with late postmalarial anemia, we found a strong inverse correlation between the levels of anti-phosphatidylserine antibodies and plasma hemoglobin, suggesting a similar role in humans. Inhibition of this pathway may be exploited for treating malarial anemia.