A mitogen-activated protein kinase regulates male gametogenesis and transmission of the malaria parasite Plasmodium berghei (original) (raw)
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Molecular Microbiology, 2005
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.
Proceedings of the National Academy of Sciences, 2005
A male gametocyte defect in the Plasmodium falciparum Dd2 parasite was previously discovered through the observation that all progeny clones in a Dd2 ؋ HB3 genetic cross were the result of fertilization events between Dd2 female and HB3 male gametes. A determinant linked to the defect in Dd2 was subsequently mapped to an 800-kb segment on chromosome 12. Here, we report further mapping of the determinant to an 82-kb region and the identification of a candidate gene, P. falciparum male development gene 1 (pfmdv-1), that is expressed at a lower level in Dd2 compared with the wild-type normal male gametocyte-producing ancestor W2. Pfmdv-1 protein is sexual-stage specific and is located on the gametocyte plasma membrane, parasitophorous vacuole membrane, and the membranes of cleft-like structures within the erythrocyte. Disruption of pfmdv-1 results in a dramatic reduction in mature gametocytes, especially functional male gametocytes, with the majority of sexually committed parasites developmentally arrested at stage I. The pfmdv-1-knockout parasites show disturbed membrane structures, particularly multimembrane vesi-cles͞tubes that likely derive from deformed cleft-like structures. Mosquito infectivity of the knockout parasites was also greatly reduced but not completely lost. The results suggest that pfmdv-1 plays a key role in gametocyte membrane formation and integrity.
From Genes to Biomarkers: Understanding the Biology of Malaria Gametocytes and Their Detection
Genetic Polymorphisms - New Insights [Working Title], 2021
Each year, approximately 230 million malaria cases and 400,00 malaria deaths are reported worldwide. Malaria is a life-threatening disease caused by Plasmodium parasites that are transmitted from one individual to another through the bites of infected female Anopheles mosquitoes. Malaria parasites replicate asexually in the human host, and, in each replication cycle, a portion of the asexual stages develops into sexual gametocytes that permit transmission. The proportion of infections that carries gametocytes and the infectivity of gametocytes are indicators of human-to-mosquito transmission potential. In P. falciparum, gametocytes appear 10–14 days after infection, whereas in P. vivax gametocytes appear simultaneously with asexual schizonts. Such difference in development not only increases the length of time that an individual is infectious, but also increases the likelihood of transmission before treatment. The conversion from asexual parasites to gametocytes is also highly varia...
PLoS pathogens, 2012
Malaria transmission requires the production of male and female gametocytes in the human host followed by fertilization and sporogonic development in the mosquito midgut. Although essential for the spread of malaria through the population, little is known about the initiation of gametocytogenesis in vitro or in vivo. Using a gametocyte-defective parasite line and genetic complementation, we show that Plasmodium falciparumgametocyte development 1 gene (Pfgdv1), encoding a peri-nuclear protein, is critical for early sexual differentiation. Transcriptional analysis of Pfgdv1 negative and positive parasite lines identified a set of gametocytogenesis early genes (Pfge) that were significantly down-regulated (>10 fold) in the absence of Pfgdv1 and expression was restored after Pfgdv1 complementation. Progressive accumulation of Pfge transcripts during successive rounds of asexual replication in synchronized cultures suggests that gametocytes are induced continuously during asexual grow...
Gametocytes: insights gained during a decade of molecular monitoring
Trends in Parasitology, 2008
In vertebrate hosts, malaria parasites produce specialized male and female sexual stages (gametocytes). Soon after being taken up by a mosquito, gametocytes rapidly produce gametes and, once mated, they infect their vector and can be transmitted to new hosts. Despite being the parasite stages that were first identified (over a century ago), gametocytes have remained elusive, and basic questions remain concerning their biology. However, the postgenomic era has substantiated information on the specialized molecular machinery of gametocytogenesis and expedited the development of molecular tools to detect and quantify gametocytes. The application of such highly sensitive and specific tools has opened up novel approaches and provided new insights into gametocyte biology. Here, we review the discoveries made during the past decade, highlight unanswered questions and suggest new directions.
Molecular microbiology, 2008
Osmiophilic bodies are membrane-bound vesicles, found predominantly in Plasmodium female gametocytes, that become progressively more abundant as the gametocyte reaches full maturity. These vesicles lie beneath the subpellicular membrane of the gametocyte, and the release of their contents into the parasitophorous vacuole has been postulated to aid in the escape of gametocytes from the erythrocyte after ingestion by the mosquito. Currently, the only protein known to be associated with osmiophilic bodies in Plasmodium falciparum is Pfg377, a gametocyte-specific protein expressed at the onset of osmiophilic body development. Here we show by targeted gene disruption that Pfg377 plays a fundamental role in the formation of these organelles, and that female gametocytes lacking the full complement of osmiophilic bodies are significantly less efficient both in vitro and in vivo in their emergence from the erythrocytes upon induction of gametogenesis, a process whose timing is critical for fertilization with the short-lived male gamete. This reduced efficiency of emergence explains the significant defect in oocyst formation in mosquitoes fed blood meals containing Pfg377-negative gametocytes, resulting in an almost complete blockade of infection.
Molecular basis of gametocytogenesis in malaria parasites
2014
Malaria, a parasitic disease caused by five species of the protozoan parasite Plasmodium, still kills an estimated 0.6 million people each year, almost all in the third world African countries. With renewed emphasis on global eradication of malaria, genome-based discovery of novel anti-transmission candidates has been identified as one of the priority research areas for the immediate future. The aim of this study was to exploit the benefits of a combination of classical forward/reverse genetics approaches, flow cytometry and high throughput whole genome sequencing to examine the molecular basis of gametocytogenesis in the rodent malaria parasite, P. berghei. Plasmodium is known to spontaneously generate gametocyte non-producing (GNP) mutants if asexually maintained for a long time. Using a sex-specific fluorescently labelled P. berghei parental line, ten parallel isogenic lineages were asexually maintained in mice by repeated mechanical passage for a year. Three, out of the potential ten lines, developed the GNP phenotype at the end of the study. The three GNP and their isogenic parental lines were sequenced using the Illumina platform and the sequence variations analysed. It was discovered that one single gene, a transcription factor with an AP2 domain (PBANKA_143750), was uniquely mutated in all the three de novo GNP mutants and also in the two pre-existing GNP mutants. The gene, called AP2-G, was thus implicated in regulating a switch associated with commitment to gametocytogenesis. Further conclusive evidence was generated using targeted AP2-G knockout studies (producing the GNP phenotype) and complementation studies in the AP2-G mutants (restoring the WT phenotype). AP2-G was also shown to recognize and bind to a conserved DNA motif in the selected gene promoters in a sequencespecific manner. Inhibition of this interaction by a synthetic customized polyamide compound, ISS-15, was also demonstrated in vitro. Collectively, the work done in the thesis (together with simultaneous independent evidence of involvement of the P. falciparum orthologue of AP2-G in gametocytogenesis) established AP2-G as the critical regulator of the commitment to gametocytogenesis in the form of a molecular switch.
SummarySexual development is an essential phase in the Plasmodium life cycle, where male gametogenesis is an original and extraordinarily rapid process. It produces 8 haploid motile microgametes, from a microgametocyte within 15 minutes. Its unique achievement lies in linking the assembly of 8 axonemes in the cytoplasm to the three rounds of intranuclear genome replication, forming motile microgametes, which are in a process called exflagellation. Surprisingly little is known about the actors involved in this process. We were interested in kinesins, molecular motors that could play potential roles in male gametogenesis. We have undertaken a functional characterization in Plasmodium berghei of a kinesin identified in male gametocytes and gametes, kinesin-8B (PbKIN8B). By generating Pbkin8B-gfp parasites, we show that PbKIN8B is specifically expressed during male gametogenesis and is associated with the axoneme. We created a ΔPbkin8B knockout cell line and analysed the consequences of...
MBio, 2021
Gametocytes of the malaria parasite Plasmodium are taken up by the mosquito vector with an infectious blood meal, representing a critical stage for parasite transmission. Calcium-independent protein kinases (CDPKs) play key roles in calcium-mediated signaling across the complex life cycle of the parasite. We sought to understand their role in human parasite transmission from the host to the mosquito vector and thus investigated the role of the human-infective parasite Plasmodium falciparum CDPK4 in the parasite life cycle. P. falciparum cdpk4 2 parasites created by targeted gene deletion showed no effect in blood stage development or gametocyte development. However, cdpk4 2 parasites showed a severe defect in male gametogenesis and the emergence of flagellated male gametes. To understand the molecular underpinnings of this defect, we performed mass spectrometry-based phosphoproteomic analyses of wild-type and Plasmodium falciparum cdpk4 2 late gametocyte stages to identify key CDPK4-mediated phosphorylation events that may be important for the regulation of male gametogenesis. We further employed in vitro assays to identify these putative substrates of Plasmodium falciparum CDPK4. This indicated that CDPK4 regulates male gametogenesis by directly or indirectly controlling key essential events, such as DNA replication, mRNA translation, and cell motility. Taken together, our work demonstrates that PfCDPK4 is a central kinase that regulates exflagellation and thereby is critical for parasite transmission to the mosquito vector. IMPORTANCE Transmission of the malaria parasite to the mosquito vector is critical for the completion of the sexual stage of the parasite life cycle and is dependent on the release of male gametes from the gametocyte body inside the mosquito midgut. In the present study, we demonstrate that PfCDPK4 is critical for male gametogenesis and is involved in phosphorylation of proteins essential for male gamete emergence. Targeting PfCDPK4 and its substrates may provide insights into achieving effective malaria transmission-blocking strategies.