The catalytic subunit of Plasmodium falciparum casein kinase 2 is essential for gametocytogenesis.
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ABSTRACT:
SUBMITTER: Hitz E
PROVIDER: S-EPMC7954856 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Gametocytes are essential for Plasmodium transmission, but little is known about the mechanisms that lead to their formation. Using piggyBac transposon-mediated insertional mutagenesis, we screened for parasites that no longer form mature gametocytes, which led to the isolation of 29 clones (insertional gametocyte-deficient mutants) that fail to form mature gametocytes. Additional analysis revealed 16 genes putatively responsible for the loss of gametocytogenesis, none of which has been previously implicated in gametocytogenesis. Transcriptional profiling and detection of an early stage gametocyte antigen determined that a subset of these mutants arrests development at stage I or in early stage II gametocytes, likely representing genes involved in gametocyte maturation. The remaining mutants seem to arrest before formation of stage I gametocytes and may represent genes involved in commitment to the gametocyte lineage.
| S-EPMC3645567 | biostudies-literature
Project description:The molecular triggers of sexual differentiation into gametocytes by blood stage Plasmodium falciparum, the most malignant human malaria parasites, are subject of much investigation for potential transmission-blocking strategies. The parasites are readily grown in vitro with culture media supplemented by the addition of human serum (10%) or by a commercially available substitute (0.5% AlbuMAX). We found better gametocytemia with serum than AlbuMAX, suggesting suboptimal concentrations of some components in the commercial product; consistent with this hypothesis, substantial concentration differences of multiple fatty acids were detected between serum- and AlbuMAX-supplemented media. Mass spectroscopy analysis distinguished the lipid profiles of gametocyte- and asexual stage-parasite membranes. Delivery of various combinations of unsaturated fatty-acid-containing phospholipids to AlbuMAX-supported gametocyte cultures improved gametocyte production to the levels achieved with human-serum-supplemented media. Maturing gametocytes readily incorporated externally supplied d5-labeled glycerol with fatty acids into unsaturated phospholipids. Phospholipids identified in this work thus may be taken up from extracellular sources or generated internally for important steps of gametocyte development. Further study of polyunsaturated fatty-acid metabolism and phospholipid profiles will improve understanding of gametocyte development and malaria parasite transmission.
| S-EPMC6679406 | biostudies-literature
Project description:Live cell imaging of human malaria parasites Plasmodium falciparum during gametocytogenesis revealed that the apicoplast does not grow, whereas the mitochondrion undergoes remarkable morphological development. A close connection of the two organelles is consistently maintained. The apicoplast and mitochondrion are not components of the male gametes, suggesting maternal inheritance.
| S-EPMC2620748 | biostudies-literature
Project description:The malaria parasite harbors a relict plastid called the apicoplast and its discovery opened a new avenue for drug discovery and development due to its unusual, nonmammalian metabolism. The apicoplast is essential during the asexual intraerythrocytic and hepatic stages of the parasite, and there is strong evidence supporting its essential metabolic role during the mosquito stages of the parasite. Supply of the isoprenoid building blocks isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) is the essential metabolic function of the apicoplast during the asexual intraerythrocytic stages. However, the metabolic role of the apicoplast during gametocyte development, the malaria stages transmitted to the mosquito, remains unknown. In this study, we showed that production of IPP for isoprenoid biosynthesis is the essential metabolic function of the apicoplast during gametocytogenesis, by obtaining normal gametocytes lacking the apicoplast when supplemented with IPP. When IPP supplementation was removed early in gametocytogenesis, developmental defects were observed, supporting the essential role of isoprenoids for normal gametocytogenesis. Furthermore, mosquitoes infected with gametocytes lacking the apicoplast developed fewer and smaller oocysts that failed to produce sporozoites. This finding further supports the essential role of the apicoplast in establishing a successful infection in the mosquito vector. Our study supports isoprenoid biosynthesis as a valid drug target for development of malaria transmission-blocking inhibitors.
| S-EPMC4311923 | biostudies-literature
Project description:Casein kinase CK2 is an essential enzyme in higher organisms, catalyzing the transfer of the ? phosphate from ATP to serine and threonine residues on protein substrates. In a number of animal tumors, CK2 activity has been shown to escape normal cellular control, making it a potential target for cancer therapy. Several crystal structures of human CK2 have been published with different conformations for the CK2? catalytic subunit. This variability reflects a high flexibility for two regions of CK2?: the interdomain hinge region, and the glycine-rich loop (p-loop). Here, we present a computational study simulating the equilibrium between three conformations involving these regions. Simulations were performed using well-tempered metadynamics combined with a path collective variables approach. This provides a reference pathway describing the conformational changes being studied, based on analysis of free energy surfaces. The free energies of the three conformations were found to be close and the paths proposed had low activation barriers. Our results indicate that these conformations can exist in water. This information should be useful when designing inhibitors specific to one conformation.
| S-EPMC4026775 | biostudies-literature
Project description:The recent focus on the elimination of malaria has led to an increased interest in the role of sexual stages in its transmission. We introduce Plasmodium falciparum gametocyte exported protein-5 (PfGEXP5) transcript analysis as an important tool for evaluating the earliest (ring) stage sexual gametocytes in the blood of infected individuals. We show that gametocyte rings are detected in the peripheral blood immediately following establishment of asexual infections-without the need for triggers such as high-density asexual parasitemia or drug treatment. Committed gametocytes are refractory to the commonly used drug piperaquine, and mature gametocytes reappear in the bloodstream 10 days after the initial appearance of gametocyte rings. A further wave of commitment is observed following recrudescent asexual parasitemia, and these gametocytes are again refractory to piperaquine treatment. This work has implications for monitoring gametocyte and transmission dynamics and responses to drug treatment.
| S-EPMC5426372 | biostudies-literature
Project description:BackgroundThe maturation of Plasmodium falciparum gametocytes in the human host takes several days, during which the parasites need to efficiently evade the host immune system. Like asexual stage parasites, immature gametocytes can sequester at various sites in the human body, and only mature sexual stages are found in the circulation. Although the fundamental mechanisms of gametocyte immune evasion are still largely unknown, candidate molecules that may be involved include variant antigens encoded by multigene families in the P. falciparum genome, such as the PfEMP1, STEVOR and RIFIN proteins. While expression of the former two families in sexual stages has been investigated earlier, we report here RIFIN expression during gametocytogenesis.Methodology/principal findingsVariants of two previously characterized RIFIN subfamilies (A- and B-type RIFINs) were found to be synthesized in gametocytes. Immunofluorescence experiments showed A-type RIFINs to be accumulated in a crescent-shaped pattern of discrete punctate structures at the infected erythrocyte membrane, while members of the B-type family were associated with the parasite. Transcription analysis demonstrated the existence of diverse transcriptional regulation patterns during sexual differentiation and indicated variant-specific regulation of B-type RIFINs, in contrast to group-specific regulation for A-type RIFINs. Phylogenetic analysis of 5'-upstream regions showed that the rif-gene family falls into five defined clusters, designated rups (rifupstream) A1, A2, AB, B and C. In trophozoites and early gametocytes, rif variants of the rupsA2-type were preferentially expressed.Conclusions/significanceIn this work we demonstrate the expression dynamics of the rif-gene family during sexual differentiation and present indications for subgroup specific regulation patterns. Therefore, our data provide a first foundation and point to new directions for future investigations of the potential role of RIFINs in gametocyte immune evasion.
| S-EPMC2582490 | biostudies-literature
Project description:Palmitoylation is the post-translational reversible addition of the acyl moiety, palmitate, to cysteine residues of proteins and is involved in regulating protein trafficking, localization, stability and function. The Aspartate-Histidine-Histidine-Cysteine (DHHC) protein family, named for their highly conserved DHHC signature motif, is thought to be responsible for catalysing protein palmitoylation. Palmitoylation is widespread in all eukaryotes, including the malaria parasite, Plasmodium falciparum, where over 400 palmitoylated proteins are present in the asexual intraerythrocytic schizont stage parasites, including proteins involved in key aspects of parasite maturation and development. The P. falciparum genome includes 12 proteins containing the conserved DHHC motif. In this study, we adapted a palmitoyl-transferase activity assay for use with P. falciparum proteins and demonstrated for the first time that P. falciparum DHHC proteins are responsible for the palmitoylation of P. falciparum substrates. This assay also reveals that multiple DHHCs are capable of palmitoylating the same substrate, indicating functional redundancy at least in vitro. To test whether functional redundancy also exists in vivo, we investigated the endogenous localization and essentiality of a subset of schizont-expressed PfDHHC proteins. Individual PfDHHC proteins localized to distinct organelles, including parasite-specific organelles such as the rhoptries and inner membrane complex. Knock-out studies identified individual DHHCs that may be essential for blood-stage growth and others that were functionally redundant in the blood stages but may have functions in other stages of parasite development. Supporting this hypothesis, disruption of PfDHHC9 had no effect on blood-stage growth but reduced the formation of gametocytes, suggesting that this protein could be exploited as a transmission-blocking target. The localization and stage-specific expression of the DHHC proteins may be important for regulating their substrate specificity and thus may provide a path for inhibitor development.
| S-EPMC5091645 | biostudies-literature
Project description:BackgroundThe malaria Eradication Research Agenda (malERA) has identified human-to-mosquito transmission of Plasmodium falciparum as a major target for eradication. The cornerstone for identifying and evaluating transmission in the laboratory is standard membrane feeding assays (SMFAs) where mature gametocytes of P. falciparum generated in vitro are offered to mosquitoes as part of a blood-meal. However, propagation of "infectious" gametocytes requires 10-12 days with considerable physico-chemical demands imposed on host RBCs and thus, "fresh" RBCs that are ≤ 1-week old post-collection are generally recommended. However, in addition to the costs, physico-chemical characteristics unique to RBC donors may confound reproducibility and interpretation of SMFAs. Cryogenic storage of RBCs ("cryo-preserved RBCs") is accepted by European and US FDAs as an alternative to refrigeration (4 °C) for preserving RBC "quality" and while cryo-preserved RBCs have been used for in vitro cultures of other Plasmodia and the asexual stages of P. falciparum, none of the studies required RBCs to support parasite development for > 4 days.ResultsUsing the standard laboratory strain, P. falciparum NF54, 11 SMFAs were performed with RBCs from four separate donors to demonstrate that RBCs cryo-preserved in the gaseous phase of liquid nitrogen (- 196 °C) supported gametocytogenesis in vitro and subsequent gametogenesis in Anopheles stephensi mosquitoes. Overall levels of sporogony in the mosquito, as measured by oocyst and sporozoite prevalence, as well as oocyst burden, from each of the four donors thawed after varying intervals of cryopreservation (1, 4, 8, and 12 weeks) were comparable to using ≤ 1-week old refrigerated RBCs. Lastly, the potential for cryo-preserved RBCs to serve as a suitable alternative substrate is demonstrated for a Cambodian isolate of P. falciparum across two independent SMFAs.ConclusionsBasic guidelines are presented for integrating cryo-preserved RBCs into an existing laboratory/insectary framework for P. falciparum SMFAs with significant potential for reducing running costs while achieving greater reliability. Lastly, scenarios are discussed where cryo-preserved RBCs may be especially useful in enhancing the understanding and/or providing novel insights into the patterns and processes underlying human-to-mosquito transmission.
| S-EPMC6282341 | biostudies-literature
Project description:We have previously described the occurrence in Paramecium of a casein kinase (CK) activity (EC 2.7.1.37) with some unusual properties, including inhibition by Ca(2+) (R. Kissmehl, T. Treptau, K. Hauser, and H. Plattner, FEBS Lett. 402:227-235, 1995). We now have cloned four genes, PtCK2alpha1 to PtCK2alpha4, all of which encode the catalytic alpha subunit of type 2 CK (CK2) with calculated molecular masses ranging from 38.9 to 39.4 kDa and pI values ranging from 8.8 to 9.0. They can be classified into two groups, which differ from each other by 28% on the nucleotide level and by 18% on the derived amino acid level. One of them, PtCK2alpha3, has been expressed in Escherichia coli and characterized in vitro. As we also have observed with the isolated CK, the recombinant protein preferentially phosphorylates casein but also phosphorylates some Paramecium-specific substrates, including the exocytosis-sensitive phosphoprotein pp63/parafusin. Characteristically, Ca(2+) inhibits the phosphorylation at elevated concentrations occurring during stimulation of a cell. Reconstitution with a recombinant form of the regulatory subunit from Xenopus laevis, XlCK2beta, confirms Ca(2+) sensitivity also under conditions of autophosphorylation. This is unusual for CK2 but correlates with the presence of two EF-hand calcium-binding motifs, one of which is located in the N-terminal segment essential for constitutive activity, as well as with an aberrant composition of normally basic domains recognizing acidic substrate domains. Immunogold localization reveals a considerable enrichment in the outermost cell cortex layers, excluding cilia. We discuss a potential role of this Ca(2+)-inhibited PtCK2alpha species in a late step of signal transduction.
| S-EPMC326640 | biostudies-literature