Project description:Proteomic characterization of complexity and dynamics of detergent resistant membranes of sexual and asexual stages of the rodent malaria parasite Plasmodium berghei
Project description:Malaria sporozoites, the form transmitted by mosquitoes, are quiescent while in the insect salivary glands. It is only after the sporozoites are deposited in the host’s skin, migrate to the liver and infect hepatocytes that the parasites continue the life cycle. We show that the sporozoite latency is an active process that requires phosphorylation of the eukaryotic initiation factor-2α (eIF2α) by a sporozoite-specific kinase. Inactivation of the kinase gene leads to an overall enhancement of protein synthesis including of silenced liver stage proteins, and inhibits transmission of malaria. Specific inhibition of the eIF2α phosphatase by salubrinal has the opposite effect. Thus, to prevent premature transformation into liver stages, Plasmodium sporozoites exploit the same mechanism that regulates stress responses in mammalian cells.
Project description:PP1 is a conserved serine/threonine phosphatase that regulates many aspects of mitosis and meiosis, often working in concert with other phosphatases, such as CDC14 and CDC25, in model eukaryotes. However, the malaria parasite, Plasmodium spp. lacks the CDC14 and CDC25 genes. The proliferative stages of the parasite life cycle include sexual development within the mosquito vector, with male gamete formation characterized by an atypical rapid mitosis, with three rounds of DNA synthesis, successive spindle formation with clustered kinetochore, and a meiotic stage during zygote to ookinete development following fertilization. It is unclear how PP1 is involved these unusual processes. Using real-time live-cell imaging, conditional gene knockdown, RNAseq and proteomic approaches, we show that Plasmodium PP1 is involved in both chromosome segregation during mitotic exit, and establishment of cell polarity during these sexual stages, suggesting that PP1 inhibitors may block parasite transmission.
Project description:Malaria sporozoites, the form transmitted by mosquitoes, are quiescent while in the insect salivary glands. It is only after the sporozoites are deposited in the hostâs skin, migrate to the liver and infect hepatocytes that the parasites continue the life cycle. We show that the sporozoite latency is an active process that requires phosphorylation of the eukaryotic initiation factor-2α (eIF2α) by a sporozoite-specific kinase. Inactivation of the kinase gene leads to an overall enhancement of protein synthesis including of silenced liver stage proteins, and inhibits transmission of malaria. Specific inhibition of the eIF2α phosphatase by salubrinal has the opposite effect. Thus, to prevent premature transformation into liver stages, Plasmodium sporozoites exploit the same mechanism that regulates stress responses in mammalian cells. 4 samples overall, 2 wt and 2 KO (PbeIK2 knockout) samples
Project description:During the malaria infection, Plasmodium parasites invade the host’s red blood cells where they can differentiate into two different life forms. The majority will replicate asexually and infect new erythrocytes. A small percentage, however, will transform into gametocytes – a specialized sexual stage able to survive and develop when taken up by Anopheles mosquito. As the gametocytes ensure the parasite’s transmission to a new host, their generation is an attractive target for new antimalarial interventions. The molecular mechanisms controlling gametocytogenesis, however, remain largely unknown due to the technical challenges: the early gametocytes are morphologically indistinguishable from asexual parasites and present in very low numbers during the infection. Recently, AP2-G - a transcription factor from an apicomplexa-specific apiAP2 family – was described as indispensable for gametocyte commitment in both human malaria parasite Plasmodium falciparum and rodent malaria model Plasmodium berghei. Therefore, we have decided to test whether the overexpression of this factor alone could increase gametocyte production and enable the investigation of uncharacterised, earliest stages of gametocyte development. To this end, we have engineered PBGAMi - a Plasmodium berghei line, in which all parasites were ap2-g deficient by default but able to overexpress it when induced with rapamycin. While the control parasites (PBGAMi R-), as expected, differentiated into asexual forms (schizonts) only, almost all rapamycin-treated parasites (PBGAMi R+) transformed into gametocytes. We used the generated line to perform RNA-seq analysis of the R- and R+ populations at different time points of their development and identify the changes arising between them, mapping the sequence of events leading to the formation of gametocytes.
Project description:During the malaria infection, Plasmodium parasites invade the host’s red blood cells where they can differentiate into two different life forms. The majority will replicate asexually and infect new erythrocytes. A small percentage, however, will transform into gametocytes – a specialized sexual stage able to survive and develop when taken up by Anopheles mosquito. As the gametocytes ensure the parasite’s transmission to a new host, their generation is an attractive target for new antimalarial interventions. The molecular mechanisms controlling gametocytogenesis, however, remain largely unknown due to the technical challenges: the early gametocytes are morphologically indistinguishable from asexual parasites and present in very low numbers during the infection. Recently, AP2-G - a transcription factor from an apicomplexa-specific apiAP2 family – was described as indispensable for gametocyte commitment in both human malaria parasite Plasmodium falciparum and rodent malaria model Plasmodium berghei. Therefore, we have decided to test whether the overexpression of this factor alone could increase gametocyte production and enable the investigation of uncharacterised, earliest stages of gametocyte development. To this end, we have engineered PBGAMi - a Plasmodium berghei line, in which all parasites were ap2-g deficient by default but able to overexpress it when induced with rapamycin. While the control parasites (PBGAMi R-), as expected, differentiated into asexual forms (schizonts) only, almost all rapamycin-treated parasites (PBGAMi R+) transformed into gametocytes. We used the generated line to perform RNA-seq analysis of the R- and R+ populations at different time points of their development and identify the changes arising between them, mapping the sequence of events leading to the formation of gametocytes. At the same time we have generated purified transcriptomes of male and female gametocytes for the reference
Project description:The purpose of this research is to identify and evaluate the global gene expression of the rodent malaria parasites Plasmodium yoelii, Plasmodium berghei and Plasmodium chabaudi blood-stage parasites and specifically compare the blood stage gene expression profiles of samples derived from previous studies on Plasmodium falciparum, Plasmodium vivax and Plasmodium knowlesi
Project description:Gametogenesis is essential for malaria parasite transmission, but the molecular mechanism of this process remains to be refined. Here, we identified a G-protein-coupled receptor 180 (GPR180) that plays a critical role in signal transduction during gametogenesis in Plasmodium. P. berghei GPR180 was predominantly expressed in gametocytes and ookinetes and associated with the plasma membrane in female gametes and ookinetes. Knockout of pbgpr180 (Δpbgpr180) had no noticeable effect on blood-stage development but impaired gamete formation and reduced transmission of the parasites to mosquitoes. Transcriptome analysis revealed that a large proportion of the dysregulated genes in the Δpbgpr180 gametocytes had assigned functions in cyclic nucleotide signaling transduction. In the Δpbgpr180 gametocytes, the intracellular cGMP level was significantly reduced, and the cytosolic Ca2+ mobilization showed a delay and a reduction in the magnitude during gametocyte activation. These results suggest that PbGPR180 functions upstream of the cGMP-protein kinase G-Ca2+ signaling pathway. In line with this functional prediction, the PbGPR180 protein was found to interact with several transmembrane transporter proteins and the small GTPase Rab6 in activated gametocytes. Allele replacement of pbgpr180 with the P. vivax ortholog pvgpr180 showed equal competence of the transgenic parasite in sexual development, suggesting functional conservation of this gene in Plasmodium spp. Furthermore, an anti-PbGPR180 monoclonal antibody and the anti-PvGPR180 serum possessed robust transmission-blocking activities. These results indicate that GPR180 is involved in signal transduction during gametogenesis in malaria parasites and is a promising target for blocking parasite transmission.
Project description:Investigation of whole genome gene expression level changes in Plasmodium falciparum 3D7 delta-PfPuf2 mutant, compared to the wild-type strain 3D7. The mutation engineered into this strain render tanslational control. The mutants analyzed in this study are further described in Miao J, Li J, Fan Q, Li X, Li X, Cui L.2010. The Puf-family RNA-binding protein PfPuf2 regulates sexual development and sex differentiation in the malaria parasite Plasmodium falciparum. J Cell Sci. 123(7):1039-49 (PMID 20197405).