A male gametocyte osmiophilic body and microgamete surface protein of the rodent malaria parasite Plasmodium yoelii (PyMiGS) plays a critical role in male osmiophilic body formation and exflagellation

Anopheles mosquitoes transmit Plasmodium parasites of mammals, including the species that cause malaria in humans. Malaria pathology is caused by rapid multiplication of parasites in asexual intraerythrocytic cycles. Sexual stage parasites are also produced during the intraerythrocytic cycle and are ingested by the mosquito, initiating gametogenesis and subsequent sporogonic stage development. Here, we present a Plasmodium protein, termed microgamete surface protein (MiGS), which has an important role in male gametocyte osmiophilic body (MOB) formation and microgamete function. MiGS is expressed exclusively in male gametocytes and microgametes, in which MiGS localises to the MOB and microgamete surface. Targeted gene disruption of MiGS in a rodent malaria parasite Plasmodium yoelii 17XNL generated knockout parasites (ΔPyMiGS) that proliferate normally in erythrocytes and form male and female gametocytes. The number of MOB in male gametocyte cytoplasm is markedly reduced and the exflagellation of microgametes is impaired in ΔPyMiGS. In addition, anti‐PyMiGS antibody severely blocked the parasite development in the Anopheles stephensi mosquito. MiGS might thus be a potential novel transmission‐blocking vaccine target candidate.     

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.     

Calcium and a calcium-dependent protein kinase regulate gamete formation and mosquito transmission in a malaria parasite

Transmission of malaria parasites to mosquitoes is initiated by the obligatory sexual reproduction of the parasite within the mosquito bloodmeal. Differentiation of specialized transmission stages, the gametocytes, into male and female gametes is induced by a small mosquito molecule, xanthurenic acid (XA). Using a Plasmodium berghei strain expressing a bioluminescent calcium sensor, we show that XA triggers a rapid rise in cytosolic calcium specifically in gametocytes that is essential for their differentiation into gametes. A member of a family of plant-like calcium dependent protein kinases, CDPK4, is identified as the molecular switch that translates the XA-induced calcium signal into a cellular response by regulating cell cycle progression in the male gametocyte. CDPK4 is shown to be essential for the sexual reproduction and mosquito transmission of P. berghei. This study reveals an unexpected function for a plant-like signaling pathway in cell cycle regulation and life cycle progression of a malaria parasite.     

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.     

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.     

PfCCp proteins of Plasmodium falciparum: gametocyte-specific expression and role in complement-mediated inhibition of exflagellation

The sexual phase of the malaria parasite Plasmodium falciparum is essential for transmission of the disease and is accompanied by the co-ordinated expression of sexual stage proteins. Six of these proteins belong to a highly conserved apicomplexan family of multi-domain adhesion proteins, termed PfCCps. PfCCp1, PfCCp2 and PfCCp3 are co-dependently expressed in the parasitophorous vacuole associated with the gametocyte plasma membrane. PfCCp2 and PfCCp3 also play an essential role for parasite development in the mosquito. We show that the six PfCCp proteins are expressed in stages II-V of gametocytogenesis as well as during early gamete formation. The proteins are expressed in association with the surface of both male and female gametocytes and macrogametes, but are not present in exflagellating microgametes. Further, the newly described protein PfCCp4 co-localizes with the transmission blocking candidate Pfs230, with which it forms a protein complex. In contrast to the phenotypes that are observed following targeted gene disruption of PfCCp2, PfCCp3 or Pfs230, the lack of PfCCp4 expression does not inhibit parasite development in the mosquito vector. This indicates a non-essential role for this protein during parasite transmission. Exflagellation assays revealed that antibodies directed against distinct domains of PfCCp1 through PfCCp4 and PfFNPA support a complement-mediated decrease in gametocyte emergence. We conclude that the six PfCCp proteins are specifically expressed during gametocytogenesis and gamete formation, and that select members may represent prospective candidates for transmission blocking vaccines.     

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.     

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.     

The effects of mosquito transmission and population bottlenecking on virulence, multiplication rate and rosetting in rodent malaria

Malaria parasites vary in virulence, but the effects of mosquito transmission on virulence phenotypes have not been systematically analysed. Using six lines of malaria parasite that varied widely in virulence, three of which had been serially blood-stage passaged many times, we found that mosquito transmission led to a general reduction in malaria virulence. Despite that, the between-line variation in virulence remained. Forcing serially passaged lines through extreme population bottlenecks (<5 parasites) reduced virulence in only one of two lines. That reduction was to a level intermediate between that of the virulent parental and avirulent ancestral line. Mosquito transmission did not reverse the increased parasite replication rates that had accrued during serial passage, but it did increase rosetting frequencies. Re-setting of asexual stage genes during the sexual stages of the life cycle, coupled with stochastic sampling of parasites with variable virulence during population bottlenecks, could account for the virulence reductions and increased rosetting induced by mosquito transmission.     

Live-cell fluorescence imaging of microgametogenesis in the human malaria parasite Plasmodium falciparum

Formation of gametes in the malaria parasite occurs in the midgut of the mosquito and is critical to onward parasite transmission. Transformation of the male gametocyte into microgametes, called microgametogenesis, is an explosive cellular event and one of the fastest eukaryotic DNA replication events known. The transformation of one microgametocyte into eight flagellated microgametes requires reorganisation of the parasite cytoskeleton, replication of the 22.9 Mb genome, axoneme formation and host erythrocyte egress, all of which occur simultaneously in <20 minutes. Whilst high-resolution imaging has been a powerful tool for defining stages of microgametogenesis, it has largely been limited to fixed parasite samples, given the speed of the process and parasite photosensitivity. Here, we have developed a live-cell fluorescence imaging workflow that captures the entirety of microgametogenesis. Using the most virulent human malaria parasite, Plasmodium falciparum, our live-cell approach captured early microgametogenesis with three-dimensional imaging through time (4D imaging) and microgamete release with two-dimensional (2D) fluorescence microscopy. To minimise the phototoxic impact to parasites, acquisition was alternated between 4D fluorescence, brightfield and 2D fluorescence microscopy. Combining live-cell dyes specific for DNA, tubulin and the host erythrocyte membrane, 4D and 2D imaging together enables definition of the positioning of newly replicated and segregated DNA. This combined approach also shows the microtubular cytoskeleton, location of newly formed basal bodies, elongation of axonemes and morphological changes to the erythrocyte membrane, the latter including potential echinocytosis of the erythrocyte membrane prior to microgamete egress. Extending the utility of this approach, the phenotypic effects of known transmission-blocking inhibitors on microgametogenesis were confirmed. Additionally, the effects of bortezomib, an untested proteasomal inhibitor, revealed a clear block of DNA replication, full axoneme nucleation and elongation. Thus, as well as defining a framework for broadly investigating microgametogenesis, these data demonstrate the utility of using live imaging to validate potential targets for transmission-blocking antimalarial drug development.     

Copper‐transporting ATPase is important for malaria parasite fertility

Homeostasis of the trace element copper is essential to all eukaryotic life. Copper serves as a cofactor in metalloenzymes and catalyses electron transfer reactions as well as the generation of potentially toxic reactive oxygen species. Here, we describe the functional characterization of an evolutionarily highly conserved, predicted copper‐transporting P‐type ATPase (CuTP) in the murine malaria model parasite Plasmodium berghei. Live imaging of a parasite line expressing a fluorescently tagged CuTP demonstrated that CuTP is predominantly located in vesicular bodies of the parasite. A P. berghei loss‐of‐function mutant line was readily obtained and showed no apparent defect in in vivo blood stage growth. Parasite transmission through the mosquito vector was severely affected, but not entirely abolished. We show that male and female gametocytes are abundant in cutp^? parasites, but activation of male microgametes and exflagellation were strongly impaired. This specific defect could be mimicked by addition of the copper chelator neocuproine to wild‐type gametocytes. A cross‐fertilization assay demonstrated that female fertility was also severely abrogated. In conclusion, we provide experimental genetic and pharmacological evidence that a healthy copper homeostasis is critical to malaria parasite fertility of both genders of gametocyte and, hence, to transmission to the mosquito vector.     

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.     

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.     

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.     

The developmental migration of Plasmodium in mosquitoes

Migration of the protozoan parasite Plasmodium through the mosquito is a complex and delicate process, the outcome of which determines the success of malaria transmission. The mosquito is not simply the vector of Plasmodium but, in terms of the life cycle, its definitive host: there, the parasite undergoes its sexual development, which results in colonization of the mosquito salivary glands. Two of the parasite's developmental stages in the mosquito, the ookinete and the sporozoite, are invasive and depend on gliding motility to access, penetrate and traverse their host cells. Recent advances in the field have included the identification of numerous Plasmodium molecules that are essential for parasite migration in the mosquito vector.     

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.