The molecular mechanisms regulating the sexual development of malaria parasites from gametocytes to oocysts in their mosquito vector are still largely unexplored. In other eukaryotes, NIMA-related kinases (Neks) regulate cell cycle progression and have been implicated in the regulation of meiosis. Here, we demonstrate that Nek-4, a new Plasmodium member of the Nek family, is essential for completion of the sexual cycle of the parasite. Recombinant Plasmodium falciparum Nek-4 possesses protein kinase activity and displays substrate preferences similar to those of other Neks. Nek-4 is highly expressed in gametocytes, yet disruption of the nek-4 gene in the rodent malaria parasite P. berghei has no effect on gamete formation and subsequent fertilization. However, further differentiation of zygotes into ookinetes is abolished. Measurements of nuclear DNA content indicate that zygotes lacking Nek-4 fail to undergo the genome replication to the tetraploid level that precedes meiosis. Cell cycle progression in the zygote is identified as a likely precondition for its morphological transition to the ookinete and for the successful establishment of a malaria infection in the mosquito.Malaria remains a devastating disease in most tropical and subtropical regions. The problem has been exacerbated over the last decades of the twentieth century by the emergence and spread of resistance of the causative agents, parasitic protists of the genus Plasmodium, to available antimalarials (Plasmodium falciparum is the species responsible for the vast majority of lethal cases) (1). Infection of the human host is initiated by the bite of an infected Anopheles mosquito, which delivers sporozoites into the bloodstream. The sporozoites rapidly gain the liver, where they invade hepatocytes and undergo a first round of schizogony. The merozoites produced in this process are released into the bloodstream and invade erythrocytes, where they undergo recurrent and synchronized schizogony. This is the phase of the parasite's life cycle that is responsible for malaria pathogenesis. A proportion of merozoites, upon invasion of a new red blood cell, do not enter schizogony, but arrest their cell cycle and develop into male or female gametocytes, the only forms capable of infecting the mosquito vector. Ingestion of gametocytes during a blood meal triggers their further development into gametes, a process, which for the male gametocyte, involves three rounds of genome replication and generation of eight flagellated male gametes. Fertilization is followed by nuclear fusion, one round of genome replication, and meiosis, which occurs within 3 h (2, 3). The nuclear envelope remains intact throughout this process, and meiosis is not followed by nuclear division. As a consequence the ookinete, a motile form that develops from the zygote and exits the mosquito midgut lumen, is tetraploid. The ookinete establishes an oocyst at the basal lamina, which produces several thousand sporozoites. These accumulate in the insect salivary glands and render the mosquito...

The cDNA encoding Pfmap-2, an enzyme of the human malaria parasite Plasmodium falciparum, was cloned, sequenced, and expressed in Escherichia coli. The open reading frame carried by the Pfmap-2 cDNA encodes a 508-amino acid polypeptide of 59.2 kDa with maximal homology to mitogen-activated protein kinases (MAPKs) from various organisms. The purified recombinant enzyme displayed functional characteristics of MAPKs such as (i) ability to undergo autophosphorylation, (ii) ability to phosphorylate myelin basic protein, a classical MAPK substrate, (iii) regulation of kinase activity by a MAPK-specific phosphatase, and (iv) ability to be activated by component(s) present in cell extracts. Mutational analysis of the recombinant protein allowed the identification of residues that are important for enzymatic activity. Northern blot analysis and immunofluorescence assays indicated that Pfmap-2 is expressed specifically in gametocytes, the form that is responsible for transmission of the parasite to the mosquito vector. Gametocyte extracts activated recombinant Pfmap-2 more efficiently than extracts from asexual parasites, which is consistent with this stage specificity. Despite its overall high level of homology to MAPKs, Pfmap-2 presents the peculiarity of not possessing the conserved threonine-X-tyrosine activation motif usually found in enzymes of this family; instead, it has a threonine-serine-histidine at the same location. This atypical feature formed the basis for a detailed analysis of the primary structure of MAPKs, allowing us to define an operational MAPK signature, which is shared by Pfmap-2. The fact that no MAPK from vertebrates diverge in the activation motif suggests that the fine mechanisms of Pfmap-2 regulation may offer an opportunity for antimalarial drug targeting.The spread of drug resistance in Plasmodium falciparum, the parasite responsible for the lethal form of human malaria, represents one of the most pressing public health problems in many parts of the world (1, 2). Parasites that are resistant to anti-malarials are selected under drug pressure in treated patients, develop into male and female gametocytes that are infective to the mosquito vector, and hence can be transmitted to new human hosts. One possible way to limit the spread of P. falciparum resistance might consist in interfering with sexual development of the parasite, thereby preventing transmission. A rational approach to this goal requires a detailed knowledge of the molecular mechanisms of Plasmodium sexual development.After invasion of a red blood cell, a merozoite can either embark on a new cycle of asexual multiplication leading to the formation of a schizont ultimately releasing 8 -32 new merozoites or undergo sexual differentiation (gametocytogenesis), a process characterized by cell cycle arrest, a shift in the transcriptional repertoire, and morphological changes (reviewed in Refs. 3-4). Mature gametocytes maintain their cell cycle arrested while in the blood of the human host, but this block is relieved immediately after th...

SummaryThe kinome of the human malaria parasite Plasmodium falciparum includes two genes encoding mitogen-activated protein kinase (MAPK) homologues, pfmap-1 and pfmap-2, but no clear orthologue of the MAPK kinase (MAPKK) family, raising the question of the mode of activation and function of the plasmodial MAPKs. Functional studies in the rodent malaria model Plasmodium berghei recently showed the map-2 gene to be dispensable for asexual growth and gametocytogenesis, but essential for male gametogenesis in the mosquito vector. Here, we demonstrate by using a reverse genetics approach that the map-2 gene is essential for completion of the asexual cycle of P. falciparum, an unexpected result in view of the non-essentiality of the orthologous gene for P. berghei erythrocytic schizogony. This validates Pfmap-2 as a potential target for chemotherapeutic intervention. In contrast, the other P. falciparum MAPK, Pfmap-1, is required neither for in vitro schizogony and gametocytogenesis in erythrocytes, nor for gametogenesis and sporogony in the mosquito vector. However, Pfmap-2 protein levels are elevated in pfmap-1 -parasites, suggesting that Pfmap-1 fulfils an important function in asexual parasites that necessitates compensatory adaptation in parasites lacking this enzyme.

Merozoites of malaria parasites invade red blood cells (RBCs), where they multiply by schizogony, undergoing development through ring, trophozoite and schizont stages that are responsible for malaria pathogenesis. Here, we report that a protein kinase-mediated signalling pathway involving host RBC PAK1 and MEK1, which do not have orthologues in the Plasmodium kinome, is selectively stimulated in Plasmodium falciparum-infected (versus uninfected) RBCs, as determined by the use of phospho-specific antibodies directed against the activated forms of these enzymes. Pharmacological interference with host MEK and PAK function using highly specific allosteric inhibitors in their known cellular IC50 ranges results in parasite death. Furthermore, MEK inhibitors have parasiticidal effects in vitro on hepatocyte and erythrocyte stages of the rodent malaria parasite Plasmodium berghei, indicating conservation of this subversive strategy in malaria parasites. These findings have profound implications for the development of novel strategies for antimalarial chemotherapy.