"Be sustainable": EOSC-Life recommendations for implementation of FAIR principles in life science data handling.

The main goals and challenges for the life science communities in the Open Science framework are to increase reuse and sustainability of data resources, software tools, and workflows, especially in large-scale data-driven research and computational analyses. Here, we present key findings, procedures, effective measures and recommendations for generating and establishing sustainable life science resources based on the collaborative, cross-disciplinary work done within the EOSC-Life (European Open Science Cloud for Life Sciences) consortium. Bringing together 13 European life science research infrastructures, it has laid the foundation for an open, digital space to support biological and medical research. Using lessons learned from 27 selected projects, we describe the organisational, technical, financial and legal/ethical challenges that represent the main barriers to sustainability in the life sciences. We show how EOSC-Life provides a model for sustainable data management according to FAIR (findability, accessibility, interoperability, and reusability) principles, including solutions for sensitive- and industry-related resources, by means of cross-disciplinary training and best practices sharing. Finally, we illustrate how data harmonisation and collaborative work facilitate interoperability of tools, data, solutions and lead to a better understanding of concepts, semantics and functionalities in the life sciences.

A Knockout Screen of ApiAP2 Genes Reveals Networks of Interacting Transcriptional Regulators Controlling the Plasmodium Life Cycle.

A family of apicomplexa-specific proteins containing AP2 DNA-binding domains (ApiAP2s) was identified in malaria parasites. This family includes sequence-specific transcription factors that are key regulators of development. However, functions for the majority of ApiAP2 genes remain unknown. Here, a systematic knockout screen in Plasmodium berghei identified ten ApiAP2 genes that were essential for mosquito transmission: four were critical for the formation of infectious ookinetes, and three were required for sporogony. We describe non-essential functions for AP2-O and AP2-SP proteins in blood stages, and identify AP2-G2 as a repressor active in both asexual and sexual stages. Comparative transcriptomics across mutants and developmental stages revealed clusters of co-regulated genes with shared cis promoter elements, whose expression can be controlled positively or negatively by different ApiAP2 factors. We propose that stage-specific interactions between ApiAP2 proteins on partly overlapping sets of target genes generate the complex transcriptional network that controls the Plasmodium life cycle.

A genome-scale vector resource enables high-throughput reverse genetic screening in a malaria parasite.

The genome-wide identification of gene functions in malaria parasites is hampered by a lack of reverse genetic screening methods. We present a large-scale resource of barcoded vectors with long homology arms for effective modification of the Plasmodium berghei genome. Cotransfecting dozens of vectors into the haploid blood stages creates complex pools of barcoded mutants, whose competitive fitness can be measured during infection of a single mouse using barcode sequencing (barseq). To validate the utility of this resource, we rescreen the P. berghei kinome, using published kinome screens for comparison. We find that several protein kinases function redundantly in asexual blood stages and confirm the targetability of kinases cdpk1, gsk3, tkl3, and PBANKA_082960 by genotyping cloned mutants. Thus, parallel phenotyping of barcoded mutants unlocks the power of reverse genetic screening for a malaria parasite and will enable the systematic identification of genes essential for in vivo parasite growth and transmission.

A cascade of DNA-binding proteins for sexual commitment and development in Plasmodium.

Commitment to and completion of sexual development are essential for malaria parasites (protists of the genus Plasmodium) to be transmitted through mosquitoes. The molecular mechanism(s) responsible for commitment have been hitherto unknown. Here we show that PbAP2-G, a conserved member of the apicomplexan AP2 (ApiAP2) family of DNA-binding proteins, is essential for the commitment of asexually replicating forms to sexual development in Plasmodium berghei, a malaria parasite of rodents. PbAP2-G was identified from mutations in its encoding gene, PBANKA_143750, which account for the loss of sexual development frequently observed in parasites transmitted artificially by blood passage. Systematic gene deletion of conserved ApiAP2 genes in Plasmodium confirmed the role of PbAP2-G and revealed a second ApiAP2 member (PBANKA_103430, here termed PbAP2-G2) that significantly modulates but does not abolish gametocytogenesis, indicating that a cascade of ApiAP2 proteins are involved in commitment to the production and maturation of gametocytes. The data suggest a mechanism of commitment to gametocytogenesis in Plasmodium consistent with a positive feedback loop involving PbAP2-G that could be exploited to prevent the transmission of this pernicious parasite.

Efficacy of a Plasmodium vivax malaria vaccine using ChAd63 and modified vaccinia Ankara expressing thrombospondin-related anonymous protein as assessed with transgenic Plasmodium berghei parasites.

Plasmodium vivax is the world's most widely distributed malaria parasite and a potential cause of morbidity and mortality for approximately 2.85 billion people living mainly in Southeast Asia and Latin America. Despite this dramatic burden, very few vaccines have been assessed in humans. The clinically relevant vectors modified vaccinia virus Ankara (MVA) and the chimpanzee adenovirus ChAd63 are promising delivery systems for malaria vaccines due to their safety profiles and proven ability to induce protective immune responses against Plasmodium falciparum thrombospondin-related anonymous protein (TRAP) in clinical trials. Here, we describe the development of new recombinant ChAd63 and MVA vectors expressing P. vivax TRAP (PvTRAP) and show their ability to induce high antibody titers and T cell responses in mice. In addition, we report a novel way of assessing the efficacy of new candidate vaccines against P. vivax using a fully infectious transgenic Plasmodium berghei parasite expressing P. vivax TRAP to allow studies of vaccine efficacy and protective mechanisms in rodents. Using this model, we found that both CD8+ T cells and antibodies mediated protection against malaria using virus-vectored vaccines. Our data indicate that ChAd63 and MVA expressing PvTRAP are good preerythrocytic-stage vaccine candidates with potential for future clinical application.

Comparative genomics in Chlamydomonas and Plasmodium identifies an ancient nuclear envelope protein family essential for sexual reproduction in protists, fungi, plants, and vertebrates.

Fertilization is a crucial yet poorly characterized event in eukaryotes. Our previous discovery that the broadly conserved protein HAP2 (GCS1) functioned in gamete membrane fusion in the unicellular green alga Chlamydomonas and the malaria pathogen Plasmodium led us to exploit the rare biological phenomenon of isogamy in Chlamydomonas in a comparative transcriptomics strategy to uncover additional conserved sexual reproduction genes. All previously identified Chlamydomonas fertilization-essential genes fell into related clusters based on their expression patterns. Out of several conserved genes in a minus gamete cluster, we focused on Cre06.g280600, an ortholog of the fertilization-related Arabidopsis GEX1. Gene disruption, cell biological, and immunolocalization studies show that CrGEX1 functions in nuclear fusion in Chlamydomonas. Moreover, CrGEX1 and its Plasmodium ortholog, PBANKA_113980, are essential for production of viable meiotic progeny in both organisms and thus for mosquito transmission of malaria. Remarkably, we discovered that the genes are members of a large, previously unrecognized family whose first-characterized member, KAR5, is essential for nuclear fusion during yeast sexual reproduction. Our comparative transcriptomics approach provides a new resource for studying sexual development and demonstrates that exploiting the data can lead to the discovery of novel biology that is conserved across distant taxa.

Recombination-mediated genetic engineering of Plasmodium berghei DNA.

DNA of Plasmodium berghei is difficult to manipulate in Escherichia coli by conventional restriction and ligation methods due to its high content of adenine and thymine (AT) nucleotides. This limits our ability to clone large genes and to generate complex vectors for modifying the parasite genome. We here describe a protocol for using lambda Red recombinase to modify inserts of a P. berghei genomic DNA library constructed in a linear, low-copy, phage-derived vector. The method uses primer extensions of 50 bp, which provide sufficient homology for an antibiotic resistance marker to recombine efficiently with a P. berghei genomic DNA insert in E. coli. In a subsequent in vitro Gateway reaction the bacterial marker is replaced with a cassette for selection in P. berghei. The insert is then released and used for transfection. The basic techniques we describe here can be adapted to generate highly efficient vectors for gene deletion, tagging, targeted mutagenesis, or genetic complementation with larger genomic regions.

The alveolin IMC1h is required for normal ookinete and sporozoite motility behaviour and host colonisation in Plasmodium berghei.

Alveolins, or inner membrane complex (IMC) proteins, are components of the subpellicular network that forms a structural part of the pellicle of malaria parasites. In Plasmodium berghei, deletions of three alveolins, IMC1a, b, and h, each resulted in reduced mechanical strength and gliding velocity of ookinetes or sporozoites. Using time lapse imaging, we show here that deletion of IMC1h (PBANKA_143660) also has an impact on the directionality and motility behaviour of both ookinetes and sporozoites. Despite their marked motility defects, sporozoites lacking IMC1h were able to invade mosquito salivary glands, allowing us to investigate the role of IMC1h in colonisation of the mammalian host. We show that IMC1h is essential for sporozoites to progress through the dermis in vivo but does not play a significant role in hepatoma cell transmigration and invasion in vitro. Colocalisation of IMC1h with the residual IMC in liver stages was detected up to 30 hours after infection and parasites lacking IMC1h showed developmental defects in vitro and a delayed onset of blood stage infection in vivo. Together, these results suggest that IMC1h is involved in maintaining the cellular architecture which supports normal motility behaviour, access of the sporozoites to the blood stream, and further colonisation of the mammalian host.

Transmission of malaria to mosquitoes blocked by bumped kinase inhibitors.

Effective control and eradication of malaria will require new tools to prevent transmission. Current antimalarial therapies targeting the asexual stage of Plasmodium do not prevent transmission of circulating gametocytes from infected humans to mosquitoes. Here, we describe a new class of transmission-blocking compounds, bumped kinase inhibitors (BKIs), which inhibit microgametocyte exflagellation. Oocyst formation and sporozoite production, necessary for transmission to mammals, were inhibited in mosquitoes fed on either BKI-1-treated human blood or mice treated with BKI-1. BKIs are hypothesized to act via inhibition of Plasmodium calcium-dependent protein kinase 4 and predicted to have little activity against mammalian kinases. Our data show that BKIs do not inhibit proliferation of mammalian cell lines and are well tolerated in mice. Used in combination with drugs active against asexual stages of Plasmodium, BKIs could prove an important tool for malaria control and eradication.

A scalable pipeline for highly effective genetic modification of a malaria parasite.

In malaria parasites, the systematic experimental validation of drug and vaccine targets by reverse genetics is constrained by the inefficiency of homologous recombination and by the difficulty of manipulating adenine and thymine (A+T)-rich DNA of most Plasmodium species in Escherichia coli. We overcame these roadblocks by creating a high-integrity library of Plasmodium berghei genomic DNA (>77% A+T content) in a bacteriophage N15-based vector that can be modified efficiently using the lambda Red method of recombineering. We built a pipeline for generating P. berghei genetic modification vectors at genome scale in serial liquid cultures on 96-well plates. Vectors have long homology arms, which increase recombination frequency up to tenfold over conventional designs. The feasibility of efficient genetic modification at scale will stimulate collaborative, genome-wide knockout and tagging programs for P. berghei.