A mitogen-activated protein kinase regulates male gametogenesis and transmission of the malaria parasite Plasmodium berghei

Differentiation of malaria parasites into sexual forms (gametocytes) in the vertebrate host and their subsequent development into gametes in the mosquito vector are crucial steps in the completion of the parasite's life cycle and transmission of the disease. The molecular mechanisms that regulate the sexual cycle are poorly understood. Although several signal transduction pathways have been implicated, a clear understanding of the pathways involved has yet to emerge. Here, we show that a Plasmodium berghei homologue of Plasmodium falciparum mitogen-activated kinase-2 (Pfmap-2), a gametocyte-specific mitogen-activated protein kinase (MAPK), is required for male gamete formation. Parasites lacking Pbmap-2 are competent for gametocytogenesis, but exflagellation of male gametocytes, the process that leads to male gamete formation, is almost entirely abolished in mutant parasites. Consistent with this result, transmission of mutant parasites to mosquitoes is grossly impaired. This finding identifies a crucial role for a MAPK pathway in malaria transmission.     

A NIMA-related protein kinase is essential for completion of the sexual cycle of malaria parasites

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.     

Gametogenesis in Malaria Parasites Is Mediated by the cGMP-Dependent Protein Kinase

Malaria parasite transmission requires differentiation of male and female gametocytes into gametes within a mosquito following a blood meal. A mosquito-derived molecule, xanthurenic acid (XA), can trigger gametogenesis, but the signalling events controlling this process in the human malaria parasite Plasmodium falciparum remain unknown. A role for cGMP was revealed by our observation that zaprinast (an inhibitor of phosphodiesterases that hydrolyse cGMP) stimulates gametogenesis in the absence of XA. Using cGMP-dependent protein kinase (PKG) inhibitors in conjunction with transgenic parasites expressing an inhibitor-insensitive mutant PKG enzyme, we demonstrate that PKG is essential for XA- and zaprinast-induced gametogenesis. Furthermore, we show that intracellular calcium (Ca²⁺) is required for differentiation and acts downstream of or in parallel with PKG activation. This work defines a key role for PKG in gametogenesis, elucidates the hierarchy of signalling events governing this process in P. falciparum, and demonstrates the feasibility of selective inhibition of a crucial regulator of the malaria parasite life cycle.     

An atypical mitogen-activated protein kinase controls cytokinesis and flagellar motility during male gamete formation in a malaria parasite

The transmission of malaria parasites to the mosquito depends critically on the rapid initiation of sexual reproduction in response to triggers from the mosquito midgut environment. We here identify an essential function for an atypical mitogen-activated protein kinase of the rodent malaria parasite Plasmodium berghei, Pbmap-2, in male sexual differentiation and parasite transmission to the mosquito. A deletion mutant no longer expressing the Pbmap-2 protein develops as wild type throughout the asexual erythrocytic phase of the life cycle. Gametocytes, the sexual transmission stages, form normally and respond in vitro to the appropriate environmental cues by rounding up and emerging from their host cells. However, microgametocytes fail to release flagellated microgametes. Female development is not affected, as judged by the ability of macrogametes to become cross-fertilized by microgametes from a donor strain. Cellular differentiation of Pbmap-2 KO microgametocytes is blocked at a late stage of male gamete formation, after replication and mitoses have been completed and axonemes have been assembled. These data demonstrate a function for Pbmap-2 in initiating cytokinesis and axoneme motility, possibly downstream of a cell cycle checkpoint for the completion of replication and/or mitosis, which are extraordinarily rapid in the male gametocyte.     

Gametogenesis in malaria parasites is mediated by the cGMP-dependent protein kinase

Malaria parasite transmission requires differentiation of male and female gametocytes into gametes within a mosquito following a blood meal. A mosquito-derived molecule, xanthurenic acid (XA), can trigger gametogenesis, but the signalling events controlling this process in the human malaria parasite Plasmodium falciparum remain unknown. A role for cGMP was revealed by our observation that zaprinast (an inhibitor of phosphodiesterases that hydrolyse cGMP) stimulates gametogenesis in the absence of XA. Using cGMP-dependent protein kinase (PKG) inhibitors in conjunction with transgenic parasites expressing an inhibitor-insensitive mutant PKG enzyme, we demonstrate that PKG is essential for XA- and zaprinast-induced gametogenesis. Furthermore, we show that intracellular calcium (Ca²⁺) is required for differentiation and acts downstream of or in parallel with PKG activation. This work defines a key role for PKG in gametogenesis, elucidates the hierarchy of signalling events governing this process in P. falciparum, and demonstrates the feasibility of selective inhibition of a crucial regulator of the malaria parasite life cycle.     

Male fertility of malaria parasites is determined by GCS1, a plant-type reproduction factor

Malaria, which is caused by Plasmodium parasites, is transmitted by anopheline mosquitoes. When gametocytes, the precursor cells of Plasmodium gametes, are transferred to a mosquito, they fertilize and proliferate, which render the mosquito infectious to the next vertebrate host. Although the fertilization of malaria parasites has been considered as a rational target for transmission-blocking vaccines, the underlying mechanism is poorly understood. Here, we show that the rodent malaria parasite gene Plasmodium berghei GENERATIVE CELL SPECIFIC 1 (PbGCS1) plays a central role in its gametic interaction. PbGCS1 knockout parasites show male sterility, resulting in unsuccessful fertilization. Because such a male-specific function of GCS1 has been observed in angiosperms, this indicates, for the first time, that parasite sexual reproduction is controlled by a machinery common to flowering plants. Our present findings provide a new viewpoint for understanding the parasitic fertilization system and important clues for novel strategies to attack life-threatening parasites.     

Plasmodium sex determination and transmission to mosquitoes

In order to be transmitted by their mosquito vector, malaria parasites undergo sexual reproduction, which occurs between specialized male and female parasites (gametes) within the blood meal in the mosquito. Nothing was known about how Plasmodium determines the sex of its gametocytes (gamete precursors), which are produced in the vertebrate host. Recently, erythropoietin, the vertebrate hormone controlling erythropoiesis in response to anaemia, was implicated in Plasmodium sex determination in animal models of malaria. This review examines the available information and addresses the relevance of such a sex determining mechanism for Plasmodium falciparum transmission to mosquitoes, with special reference to low gametocytaemias.     

The novel putative transporter NPT1 plays a critical role in early stages of Plasmodium berghei sexual development

Transmission of Plasmodium species from a mammalian host to the mosquito vector requires the uptake, during an infected blood meal, of gametocytes, the precursor cells of the gametes. Relatively little is known about the molecular mechanisms involved in the developmental switch from asexual development to sexual differentiation or the maturation and survival of gametocytes. Here, we show that a gene coding for a novel putative transporter, NPT1, plays a crucial role in the development of Plasmodium berghei gametocytes. Parasites lacking NPT1 are severely compromised in the production of gametocytes and the rare gametocytes produced are unable to differentiate into fertile gametes. This is the earliest block in gametocytogenesis obtained by reverse genetics and the first to demonstrate the role of a protein with a putative transport function in sexual development. These results and the high degree of conservation of NPT1 in Plasmodium species suggest that this protein could be an attractive target for the development of novel drugs to block the spread of malaria.     

Fit for fertilization: Mating in malaria parasites

The human malaria parasite Plasmodium falciparum has an obligate sexual phase in its life cycle. Male and female gametes must mate in the mosquito midgut for transmission to occur. When mosquitoes ingest a mixture of two parasite clones, the inheritance of nuclear genes suggests that mating between gametes is random. Both cross-fertilization (between unlike male and female gametes) and selfing occur. However, it has been suggested that the inheritance of mitochondrial markers indicates non-random mating. An alternative hypothesis, which is presented here by Lisa Ranford-Cartwright, is that mating is random, but differences in the relative fitnesses of the gametocytes can explain the inheritance patterns observed.     

PbGEST mediates malaria transmission to both mosquito and vertebrate host

The malaria life cycle relies on the successful transfer of the parasite between its human and mosquito hosts. We identified a Plasmodium berghei secreted protein (PBANKA_131270) that plays distinct roles in both the mammal-to-mosquito and the mosquito-to-mammal transitions. This protein, here named gamete egress and sporozoite traversal (GEST), plays an important role in the egress of male and female gametes from the vertebrate red blood cell. Interestingly, GEST is also required following the bite of the infected mosquito, for sporozoite progression through the skin. We found PbGEST to be secreted shortly after activation of the intraerythrocytic gametocyte, and during sporozoite migration. These findings indicate that a single malaria protein may have pleiotropic roles in different parasites stages mediating transmission between its insect and mammalian hosts.     

Commitment to gametocytogenesis in Plasmodium falciparum

To achieve transmission, a subpopulation of asexually dividing bloodstream forms of the human malaria parasite Plasmodium falciparum withdraws from the cell cycle to develop into gametocytes - cells specialized for sexual reproduction and invasion of the mosquito vector. For natural selection to maximize transmission to new hosts, a balance must have evolved between asexual replication and sexual differentiation. Here, Mike Dyer and Karen Day consider observations on the process of commitment to gametocytogenesis and use this information as the framework for a model that begins to explain the control of the dynamics between asexual and sexual development.     

Azadirachtin disrupts formation of organised microtubule arrays during microgametogenesis of Plasmodium berghei

Transmission of malaria parasites from vertebrate blood to the mosquito vector depends critically on the differentiation of the gametocytes into gametes. This occurs in response to environmental stimuli encountered by the parasite in the mosquito bloodmeal. Male gametogenesis involves three rounds of DNA replication and endomitosis, and the assembly de novo of 8 motile axonemes. Azadirachtin, a plant limnoid and insecticide with an unkown mode of action, specifically inhibits the release of motile gametes from activated microgametocytes but does not inhibit growth and replication of a sexual blood stages. We have combined confocal laser scanning microscopy and transmission electron microscopy to examine the effect of azadirachtin on the complex reorganisation of the microtubule cytoskeleton during gametogenesis in Plasmodium berghei. Neither the replication of the genome nor the ability of tubulin monomers to assemble into microtubules upon gametocyte activation were prevented by azadirachtin. However, the drug interfered with the formation of mitotic spindles and with the assembly of microtubules into typical axonemes. Our observations suggest that azadarachtin specifically disrupts the patterning of microtubules into more complex structures, such as mitotic spindles and axonemes.     

Plasmodium falciparum gametocytes: still many secrets of a hidden life

Sexual differentiation and parasite transmission are intimately linked in the life cycle of malaria parasites. The specialized cells providing this crucial link are the Plasmodium gametocytes. These are formed in the vertebrate host and are programmed to mature into gametes emerging from the erythrocytes in the midgut of a blood-feeding mosquito. The ensuing fusion into a zygote establishes parasite infection in the insect vector. Although key mechanisms of gametogenesis and fertilization are becoming progressively clear, the fundamental biology of gametocyte formation still presents open questions, some of which are specific to the human malaria parasite Plasmodium falciparum. Developmental commitment to sexual differentiation, regulation of stage-specific gene expression, the profound molecular and cellular changes accompanying gametocyte specialization, the requirement for tissue-specific sequestration in P. falciparum gametocytogenesis are proposed here as areas for future investigation. The epidemiological relevance of parasite transmission from humans to mosquito in the spread of malaria and of Plasmodium drug resistance genes indicates that understanding molecular mechanisms of gametocyte formation is highly relevant to design strategies able to interfere with the transmission of this disease.     

Plasmodium berghei calcium-dependent protein kinase 3 is required for ookinete gliding motility and mosquito midgut invasion

Apicomplexan parasites critically depend on a unique form of gliding motility to colonize their hosts and to invade cells. Gliding requires different stage and species-specific transmembrane adhesins, which interact with an intracellular motor complex shared across parasite stages and species. How gliding is regulated by extracellular factors and intracellular signalling mechanisms is largely unknown, but current evidence suggests an important role for cytosolic calcium as a second messenger. Studying a Plasmodium berghei gene deletion mutant, we here provide evidence that a calcium-dependent protein kinase, CDPK3, has an important function in regulating motility of the ookinete in the mosquito midgut. We show that a cdpk3- parasite clone produces morphologically normal ookinetes, which fail to engage the midgut epithelium, due to a marked reduction in their ability to glide productively, resulting in marked reduction in malaria transmission to the mosquito. The mutant was successfully complemented with an episomally maintained cdpk3 gene, restoring mosquito transmission to wild-type level. cdpk3- ookinetes maintain their full genetic differentiation potential when microinjected into the mosquito haemocoel and cdpk3- sporozoites produced in this way are motile and infectious, suggesting an ookinete-limited essential function for CDPK3.     

Disruption of a Plasmodium falciparum cyclic nucleotide phosphodiesterase gene causes aberrant gametogenesis

Phosphodiesterase (PDE) and guanylyl cyclase (GC) enzymes are key components of the cGMP signalling pathway and are encoded in the genome of Plasmodium falciparum . Here we investigate the role of specific GC and PDE isoforms in gamete formation – a process that is essential for malaria transmission and occurs in the Anopheles mosquito midgut following feeding on an infected individual. Details of the intracellular signalling events controlling development of the male and female gametes from their precursors (gametocytes) remain sparse in P. falciparum . Previous work involving the addition of pharmacological agents to gametocytes implicated cGMP in exflagellation – the emergence of highly motile, flagellated male gametes from the host red blood cell. In this study we show that decreased GC activity in parasites having undergone disruption of the PfGCβ gene had no significant effect on gametogenesis. By contrast, decreased cGMP-PDE activity during gametocyte development owing to disruption of the PfPDEδ gene, led to a severely reduced ability to undergo gametogenesis. This suggests that the concentration of cGMP must be maintained below a threshold in the developing gametocyte to allow subsequent differentiation to proceed normally. The data indicate that PfPDEδ plays a crucial role in regulating cGMP levels during sexual development.     

Malaria transmission-blocking antigen, Pfs230, mediates human red blood cell binding to exflagellating male parasites and oocyst production

Malaria transmission requires that the parasites differentiate into gametocytes prior to ingestion by a mosquito during a blood meal. Once in the mosquito midgut the gametocytes emerge from red blood cells (RBCs), fertilize, develop into ookinetes and finally infectious sporozoites. Gamete surface antigen, Pfs230, is an important malaria transmission-blocking vaccine candidate, but its function has remained unclear. Two clones with distinct Pfs230 gene disruptions (Delta1.356 and Delta2.560) and a clone with a disruption of Pfs48/45 were used to evaluate the role of Pfs230 in the mosquito midgut. Pfs230 disruptants successfully emerge from RBCs and male gametes exflagellate producing microgametes. However, exflagellating Pfs230-minus males, in the presence or absence of Pfs48/45, are unable to interact with RBCs and form exflagellation centres. Oocyst production and mosquito infectivity is also significantly reduced, 96-92% and 76-71% respectively. In contrast, in the Pfs230 disruptants the expression and localization of other known sexual stage-specific antigens, including Pfs48/45, Pfs47, the Pfs230 paralogue (PfsMR5), Pfs16 or Pfs25, were not altered and the Pfs230-minus gametes retained resistance to the alternative pathway of human complement. These results suggest that Pfs230 is the surface molecule on males that mediates RBC binding and plays an important role in oocyst development, a critical step in malaria transmission.