Novel insights from the Plasmodium falciparum sporozoite-specific proteome by probabilistic integration of 26 studies.

Plasmodium species, the causative agent of malaria, have a complex life cycle involving two hosts. The sporozoite life stage is characterized by an extended phase in the mosquito salivary glands followed by free movement and rapid invasion of hepatocytes in the human host. This transmission stage has been the subject of many transcriptomics and proteomics studies and is also targeted by the most advanced malaria vaccine. We applied Bayesian data integration to determine which proteins are not only present in sporozoites but are also specific to that stage. Transcriptomic and proteomic Plasmodium data sets from 26 studies were weighted for how representative they are for sporozoites, based on a carefully assembled gold standard for Plasmodium falciparum (Pf) proteins known to be present or absent during the sporozoite life stage. Of 5418 Pf genes for which expression data were available at the RNA level or at the protein level, 975 were identified as enriched in sporozoites and 90 specific to them. We show that Pf sporozoites are enriched for proteins involved in type II fatty acid synthesis in the apicoplast and GPI anchor synthesis, but otherwise appear metabolically relatively inactive in the salivary glands of mosquitos. Newly annotated hypothetical sporozoite-specific and sporozoite-enriched proteins highlight sporozoite-specific functions. They include PF3D7_0104100 that we identified to be homologous to the prominin family, which in human has been related to a quiescent state of cancer cells. We document high levels of genetic variability for sporozoite proteins, specifically for sporozoite-specific proteins that elicit antibodies in the human host. Nevertheless, we can identify nine relatively well-conserved sporozoite proteins that elicit antibodies and that together can serve as markers for previous exposure. Our understanding of sporozoite biology benefits from identifying key pathways that are enriched during this life stage. This work can guide studies of molecular mechanisms underlying sporozoite biology and potential well-conserved targets for marker and drug development.

The PHIST protein GEXP02 targets the host cytoskeleton during sexual development of Plasmodium falciparum.

A hallmark of the biology of Plasmodium falciparum blood stage parasites is their extensive host cell remodelling, facilitated by parasite proteins that are exported into the erythrocyte. Although this area has received extensive attention, only a few exported parasite proteins have been analysed in detail, and much of this remodelling process remains unknown, particularly for gametocyte development. Recent advances to induce high rates of sexual commitment enable the production of large numbers of gametocytes. We used this approach to study the Plasmodium helical interspersed subtelomeric (PHIST) protein GEXP02, which is expressed during sexual development. We show by immunofluorescence that GEXP02 is exported to the gametocyte-infected host cell periphery. Co-immunoprecipitation revealed potential interactions between GEXP02 and components of the erythrocyte cytoskeleton as well as other exported parasite proteins. This indicates that GEXP02 targets the erythrocyte cytoskeleton and is likely involved in its remodelling. GEXP02 knock-out parasites show no obvious phenotype during gametocyte maturation, transmission through mosquitoes, and hepatocyte infection, suggesting auxiliary or redundant functions for this protein. In summary, we performed a detailed cellular and biochemical analysis of a sexual stage-specific exported parasite protein using a novel experimental approach that is broadly applicable to study the biology of P. falciparum gametocytes.

Identification of essential exported Plasmodium falciparum protein kinases in malaria-infected red blood cells.

FIKK kinases in the human malaria parasite Plasmodium falciparum are attractive targets for new anti-malaria drugs, as they have no orthologues in humans and have been linked to disease severity. Six FIKKs are known to be exported into red blood cells (RBCs) where they mediate dramatic structural and functional changes to RBCs that are central to pathogenesis. Eleven members of this family, which are predicted to be exported into infected RBCs (iRBCs), remain uncharacterised. Using a targeted gene-knockout approach, we have characterised these FIKKs and discovered that five are essential for parasite survival. Three of these five FIKKs (FIKK9.1, FIKK10.1, FIKK10.2) were exported from the parasite into iRBCs and for two of these (FIKK9.1 and FIKK10.1), export was via Maurer's clefts (parasite-derived structures involved in protein trafficking and pathognomonic of falciparum malaria). Of the remaining two essential kinases, FIKK3 was associated with rhoptries (specialised protein secretory organelles in the parasite) and FIKK9.5 was localised in the parasite nucleus. The diverse localisation and essentiality of these FIKKs demonstrate that they play different but essential roles in the survival of P. falciparum in RBCs and therefore are attractive new drug targets for the prevention or treatment of falciparum malaria.

AlbuMAX supplemented media induces the formation of transmission-competent P. falciparum gametocytes.

Asexual blood stage culture of Plasmodium falciparum is routinely performed but reproducibly inducing commitment to and maturation of viable gametocytes remains difficult. Culture media can be supplemented with human serum substitutes to induce commitment but these generally only allow for long-term culture of asexual parasites and not transmission-competent gametocytes due to their different lipid composition. Recent insights demonstrated the important roles lipids play in sexual commitment; elaborating on this we exposed ring stage parasites (20-24 hours hpi) for one day to AlbuMAX supplemented media to trigger induction to gametocytogenesis. We observed a significant increase in gametocytes after AlbuMAX induction compared to serum. We also tested the transmission potential of AlbuMAX inducted gametocytes and found a significant higher oocyst intensity compared to serum. We conclude that AlbuMAX supplemented media induces commitment, allows a more stable and predictable production of transmittable gametocytes than serum alone.

Quantifying the deformability of malaria-infected red blood cells using deep learning trained on synthetic cells.

Several hematologic diseases, including malaria, diabetes, and sickle cell anemia, result in a reduced red blood cell deformability. This deformability can be measured using a microfluidic device with channels of varying width. Nevertheless, it is challenging to algorithmically recognize large numbers of red blood cells and quantify their deformability from image data. Deep learning has become the method of choice to handle noisy and complex image data. However, it requires a significant amount of labeled data to train the neural networks. By creating images of cells and mimicking noise and plasticity in those images, we generate synthetic data to train a network to detect and segment red blood cells from video-recordings, without the need for manually annotated labels. Using this new method, we uncover significant differences between the deformability of RBCs infected with different strains of Plasmodium falciparum, providing clues to the variation in virulence of these strains.

Pfs230 Domain 7 is targeted by a potent malaria transmission-blocking monoclonal antibody.

Malaria transmission-blocking vaccines (TBVs) aim to induce antibodies that block Plasmodium parasite development in the mosquito midgut, thus preventing mosquitoes from becoming infectious. While the Pro-domain and first of fourteen 6-Cysteine domains (Pro-D1) of the Plasmodium gamete surface protein Pfs230 are known targets of transmission-blocking antibodies, no studies to date have discovered other Pfs230 domains that are functional targets. Here, we show that a murine monoclonal antibody (mAb), 18F25.1, targets Pfs230 Domain 7. We generated a subclass-switched complement-fixing variant, mAb 18F25.2a, using a CRISPR/Cas9-based hybridoma engineering method. This subclass-switched mAb 18F25.2a induced lysis of female gametes in vitro. Importantly, mAb 18F25.2a potently reduced P. falciparum infection of Anopheles stephensi mosquitoes in a complement-dependent manner, as assessed by standard membrane feeding assays. Together, our data identify Pfs230 Domain 7 as target for transmission-blocking antibodies and provide a strong incentive to study domains outside Pfs230Pro-D1 as TBV candidates.

CRISPR/Cas9-engineered inducible gametocyte producer lines as a valuable tool for Plasmodium falciparum malaria transmission research.

The malaria parasite Plasmodium falciparum replicates inside erythrocytes in the blood of infected humans. During each replication cycle, a small proportion of parasites commits to sexual development and differentiates into gametocytes, which are essential for parasite transmission via the mosquito vector. Detailed molecular investigation of gametocyte biology and transmission has been hampered by difficulties in generating large numbers of these highly specialised cells. Here, we engineer P. falciparum NF54 inducible gametocyte producer (iGP) lines for the routine mass production of synchronous gametocytes via conditional overexpression of the sexual commitment factor GDV1. NF54/iGP lines consistently achieve sexual commitment rates of 75% and produce viable gametocytes that are transmissible by mosquitoes. We also demonstrate that further genetic engineering of NF54/iGP parasites is a valuable tool for the targeted exploration of gametocyte biology. In summary, we believe the iGP approach developed here will greatly expedite basic and applied malaria transmission stage research.

A Prioritized and Validated Resource of Mitochondrial Proteins in Plasmodium Identifies Unique Biology.

Plasmodium species have a single mitochondrion that is essential for their survival and has been successfully targeted by antimalarial drugs. Most mitochondrial proteins are imported into this organelle, and our picture of the Plasmodium mitochondrial proteome remains incomplete. Many data sources contain information about mitochondrial localization, including proteome and gene expression profiles, orthology to mitochondrial proteins from other species, coevolutionary relationships, and amino acid sequences, each with different coverage and reliability. To obtain a comprehensive, prioritized list of Plasmodium falciparum mitochondrial proteins, we rigorously analyzed and integrated eight data sets using Bayesian statistics into a predictive score per protein for mitochondrial localization. At a corrected false discovery rate of 25%, we identified 445 proteins with a sensitivity of 87% and a specificity of 97%. They include proteins that have not been identified as mitochondrial in other eukaryotes but have characterized homologs in bacteria that are involved in metabolism or translation. Mitochondrial localization of seven Plasmodium berghei orthologs was confirmed by epitope labeling and colocalization with a mitochondrial marker protein. One of these belongs to a newly identified apicomplexan mitochondrial protein family that in P. falciparum has four members. With the experimentally validated mitochondrial proteins and the complete ranked P. falciparum proteome, which we have named PlasmoMitoCarta, we present a resource to study unique proteins of Plasmodium mitochondria. IMPORTANCE The unique biology and medical relevance of the mitochondrion of the malaria parasite Plasmodium falciparum have made it the subject of many studies. However, we actually do not have a comprehensive assessment of which proteins reside in this organelle. Many omics data are available that are predictive of mitochondrial localization, such as proteomics data and expression data. Individual data sets are, however, rarely complete and can provide conflicting evidence. We integrated a wide variety of available omics data in a manner that exploits the relative strengths of the data sets. Our analysis gave a predictive score for the mitochondrial localization to each nuclear encoded P. falciparum protein and identified 445 likely mitochondrial proteins. We experimentally validated the mitochondrial localization of seven of the new mitochondrial proteins, confirming the quality of the complete list. These include proteins that have not been observed mitochondria before, adding unique mitochondrial functions to P. falciparum.