Commitment to and completion of sexual development are essential for malaria parasites (protists of the genus Plasmodium) to be transmitted through mosquitoes1. The molecular mechanism(s) responsible for commitment have been hitherto unknown. Here we show that PBAP2-G, a conserved member of the ApiAP2 family of transcription factors, is essential for the commitment of asexually replicating forms to sexual development in P. 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, 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 PBAP2G which might be exploited to prevent the transmission of this pernicious parasite.

SummaryA 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.

powers erythrocyte invasion (6), while PfCDPK5 was shown to be critical for egress of P. falciparum merozoites from erythrocytes (7). In this report, we describe bumped kinase inhibitors (BKIs) that block infection of mosquitoes by malaria parasites. These compounds selectively and potently inhibit CDPK4, which is required for exflagellation of Plasmodium berghei microgametes (8) and has recently been shown to be connected with induction of exflagellation in P. falciparum microgametes (9), before fusion with the macrogamete, to form a zygote. The zygote undergoes transitional ookinete and oocyst stages to mature into infective sporozoites that are injected into a mammalian host during the female mosquito blood meal. Blocking exflagellation through the selective inhibition of Plasmodium CDPK4 would be expected to interrupt malaria transmission without being toxic to humans (10). Results and DiscussionWe have previously demonstrated that the ATP-binding pockets of Toxoplasma gondii and Cryptosporidium parvum CDPK1 can be selectively targeted by BKIs with large aromatic moieties displayed from the 3 position of the pyrazolopyrimidine scaffold due to the anomalously small gatekeeper residues (glycine) present in these kinases. Selective inhibition of Tg/CpCDPK1 with BKIs leads to blockage of mammalian-host cell invasion (11,12). PfCDPK4 has a serine at the gatekeeper position (Figure 1), smaller than the gatekeeper in almost all mammalian kinases, and an overall binding pocket that is very similar to those of TgCDPK1 and CpCDPK1. A number of compounds in our Tg/CpCDPK1 BKI library were found to inhibit recombinant PfCDPK4 (rPfCDPK4), the most potent being BKI-1 with an IC 50 (concentration to inhibit 50% of enzyme activity) of 4 nM (Table 1). However, not all compounds that are potent inhibitors of Tg/CpCDPK1 have comparable activity against rPfCDPK4. Despite the overall structural similarities in the ATP-binding pockets of CDPKs, small differences in the size of the gatekeeper residue may have a large effect on inhibitor potency.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.

SummaryThe 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.

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.

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 (AT) rich Plasmodium DNA in E. coli. We overcome these roadblocks by demonstrating that a high integrity library of P. berghei genomic DNA (>77% AT) in a bacteriophage N15-based vector can be modified efficiently using the lambda Red method of recombineering. We built a pipeline for generating Plasmodium 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 10-fold over conventional designs. The feasibility of efficient genetic modification at scale will stimulate collaborative, genome-wide knockout and tagging programs for P. 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.

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.