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Nature Communications Mar 2023Malaria-causing parasites of the Plasmodium genus undergo multiple developmental phases in the human and the mosquito hosts, regulated by various post-translational...
Malaria-causing parasites of the Plasmodium genus undergo multiple developmental phases in the human and the mosquito hosts, regulated by various post-translational modifications. While ubiquitination by multi-component E3 ligases is key to regulate a wide range of cellular processes in eukaryotes, little is known about its role in Plasmodium. Here we show that Plasmodium berghei expresses a conserved SKP1/Cullin1/FBXO1 (SCF) complex showing tightly regulated expression and localisation across multiple developmental stages. It is key to cell division for nuclear segregation during schizogony and centrosome partitioning during microgametogenesis. It is additionally required for parasite-specific processes including gamete egress from the host erythrocyte, as well as integrity of the apical and the inner membrane complexes (IMC) in merozoite and ookinete, two structures essential for the dissemination of these motile stages. Ubiquitinomic surveys reveal a large set of proteins ubiquitinated in a FBXO1-dependent manner including proteins important for egress and IMC organisation. We additionally demonstrate an interplay between FBXO1-dependent ubiquitination and phosphorylation via calcium-dependent protein kinase 1. Altogether we show that Plasmodium SCF plays conserved roles in cell division and is also important for parasite-specific processes in the mammalian and mosquito hosts.
Topics: Humans; Erythrocytes; Plasmodium berghei; Protein Binding; S-Phase Kinase-Associated Proteins; Ubiquitination
PubMed: 36898988
DOI: 10.1038/s41467-023-36999-8 -
Scientific Reports Jul 2015The erythrocyte is an extraordinary host cell for intracellular pathogens and requires extensive remodelling to become permissive for infection. Malaria parasites modify...
The erythrocyte is an extraordinary host cell for intracellular pathogens and requires extensive remodelling to become permissive for infection. Malaria parasites modify their host red blood cells through protein export to acquire nutrients and evade immune responses. Endogenous fluorescent tagging of three signature proteins of the Plasmodium berghei translocon of exported proteins (PTEX), heat shock protein 101, exported protein 2 (EXP2), and PTEX88, revealed motile, tubular extensions of the parasitophorous vacuole that protrude from the parasite far into the red blood cell. EXP2 displays a more prominent presence at the periphery of the parasite, consistent with its proposed role in pore formation. The tubular compartment is most prominent during trophozoite growth. Distinct spatiotemporal expression of individual PTEX components during sporogony and liver-stage development indicates additional functions and tight regulation of the PTEX translocon during parasite life cycle progression. Together, live cell imaging and correlative light and electron microscopy permitted previously unrecognized spatiotemporal and subcellular resolution of PTEX-containing tubules in murine malaria parasites. These findings further refine current models for Plasmodium-induced erythrocyte makeover.
Topics: Erythrocytes; Heat-Shock Proteins; Life Cycle Stages; Plasmodium berghei; Protein Transport; Protozoan Proteins; Vacuoles
PubMed: 26219962
DOI: 10.1038/srep12532 -
Experimental Parasitology Feb 1973
Topics: Animals; Anopheles; Antibodies; Antibody Formation; Antigens; Female; Immunity, Active; Immunization; Injections, Intravenous; Malaria; Mice; Mice, Inbred Strains; Plasmodium berghei; Radiation Effects; Rats; Rats, Inbred Strains; Time Factors
PubMed: 4570978
DOI: 10.1016/0014-4894(73)90022-2 -
Experimental Parasitology Dec 1978
Topics: Amino Acids; Animals; Anopheles; Carbohydrate Metabolism; Culture Media; Energy Metabolism; Movement; Plasmodium berghei; Salivary Glands
PubMed: 365562
DOI: 10.1016/0014-4894(78)90144-3 -
Malaria Journal Jun 2019Reverse genetics approaches have become powerful tools to dissect the biology of malaria parasites. In a previous study, development of an in vitro drug selection method...
BACKGROUND
Reverse genetics approaches have become powerful tools to dissect the biology of malaria parasites. In a previous study, development of an in vitro drug selection method for generating transgenic parasite of Plasmodium berghei was reported. Using this method, two novel and independent selection markers using the P. berghei heat shock protein 70 promoter was previously established. While the approach permits the easy and flexible genetic manipulation of P. berghei, shortcomings include a low variety in promoter options to drive marker gene expression and increased complexity of the selection procedure. In this study, addressing these issues was attempted.
METHODS
To secure a variety of promoters, the use of a P. berghei elongation factor-1α promoter for marker gene expression was attempted. To simplify the procedure of in vitro selection, the establishment of a two cell-cycle culture method and its application for drug selection were attempted.
RESULTS
The P. berghei elongation factor-1α (pbef-1α) promoter, which is commonly used to drive marker gene expression, was successfully applied as an alternative promoter model for marker gene expression, using the parasite's codon-optimized marker sequence. To simplify the in vitro selection method, a two cell-cycle culture method in which the merozoite was released by filtration of the culture containing matured schizont-infected erythrocytes was also developed and successfully applied for drug selection.
CONCLUSION
The pbef-1α promoter was successfully applied in an in vitro selection system. The in vitro selection procedure also could be simplified for practical use using a two cell-cycle culture method. These improvements provide a more versatile platform for the genetic manipulation of P. berghei.
Topics: Animals; Antimalarials; Cell Culture Techniques; Female; Malaria; Mice; Mice, Inbred BALB C; Mice, Inbred ICR; Microorganisms, Genetically-Modified; Plasmodium berghei
PubMed: 31238932
DOI: 10.1186/s12936-019-2851-6 -
MBio Apr 2024Neddylation is a type of posttranslational modification known to regulate a wide range of cellular processes by covalently conjugating the ubiquitin-like protein Nedd8...
Neddylation is a type of posttranslational modification known to regulate a wide range of cellular processes by covalently conjugating the ubiquitin-like protein Nedd8 to target proteins at lysine residues. However, the role of neddylation in malaria parasites has not been determined. Here, for the first time, we showed that neddylation plays an essential role in malaria transmission in . We found that disruption of Nedd8 did not affect blood-stage propagation, gametocyte development, gamete formation, or zygote formation while abolishing the formation of ookinetes and further transmission of the parasites in mosquitoes. These phenotypic defects in Nedd8 knockout parasites were complemented by reintroducing the gene that restored mosquito transmission to wild-type levels. Our data establish the role of Nedd8 in malaria parasite transmission.IMPORTANCENeddylation is a process by which Nedd8 is covalently attached to target proteins through three-step enzymatic cascades. The attachment of Nedd8 residues results in a range of diverse functions, such as cell cycle regulation, metabolism, immunity, and tumorigenesis. The potential neddylation substrates are cullin (CUL) family members, which are implicated in controlling the cell cycle. Cullin neddylation leads to the activation of cullin-RING ubiquitin ligases, which regulate a myriad of biological processes through target-specific ubiquitylation. Neddylation possibly regulates meiosis in zygotes, which subsequently develop into ookinetes. Our findings point to an essential function of this neddylation pathway and highlight its possible importance in designing novel intervention strategies.
Topics: Animals; Ubiquitins; Plasmodium berghei; Cullin Proteins; Ubiquitin; Ubiquitin-Protein Ligases
PubMed: 38411954
DOI: 10.1128/mbio.00232-24 -
Molecular and Biochemical Parasitology Nov 2021Plasmodium the causative agent of malaria is a member of the phylum Apicomplexa, where all invasive forms have a substrate-dependent motility called gliding, key to...
Plasmodium the causative agent of malaria is a member of the phylum Apicomplexa, where all invasive forms have a substrate-dependent motility called gliding, key to malaria transmission. Gliding allows parasite host-cell recognition, binding, cell entry and trespassing the cytoplasm. In this process Plasmodium releases molecules from micronemes and the cell surface that are deposited on trails left behind on the substratum as the parasite progresses. Previously we identified the heat shock protein 70-1 (HSP 70-1) on the surface and micronemes of P. berghei ookinetes, the parasite form that invades the mosquito midgut. To investigate if this protein is shed of from the parasite during invasion, we searched HSP 70-1 in gliding trails deposited on a solid surface by P. berghei ookinetes.
Topics: Animals; Culicidae; HSP70 Heat-Shock Proteins; Malaria; Plasmodium berghei; Protozoan Proteins
PubMed: 34756988
DOI: 10.1016/j.molbiopara.2021.111428 -
Parasitology International Jun 2020G-strand binding protein 2 (GBP2) is a Ser/Arg-rich (SR) protein involved in mRNA surveillance and nuclear mRNA quality control in yeast. However, the roles of GBP2 in... (Comparative Study)
Comparative Study
G-strand binding protein 2 (GBP2) is a Ser/Arg-rich (SR) protein involved in mRNA surveillance and nuclear mRNA quality control in yeast. However, the roles of GBP2 in virulence and sexual development in Plasmodium parasites are unclear, although GBP2 is involved in the asexual development of Plasmodium berghei, the rodent malaria parasite. In this study, we investigated the role of GBP2 in virulence and sexual development of P. berghei using gbp2-deleted P. berghei (Δgbp2 parasites). Then, to identify factors affected by gbp2 deletion, we performed a comparative proteomic analysis of the Δgbp2 parasites. We found that GBP2 was not associated with the development of experimental cerebral malaria during infection with P. berghei, but asexual development of the parasite was delayed with deletion of gbp2. However, the development of P. berghei gametocytes was significantly reduced with deletion of gbp2. Comparative proteomic analysis revealed that the levels of adenosine deaminase (ADA), purine nucleoside phosphorylase (PNP), and hypoxanthine-guanine phosphoribosyltransferase (HGPRT) in Δgbp2 parasites were significantly higher than those in wild-type (WT) parasites, suggesting that biosynthesis of purine nucleotides may be involved in function of GBP2. Therefore, we investigated the effect of purine starvation on the sexual development and proteome. In nt1-deleted P. berghei (Δnt1 parasites), the production of male and female gametocytes was significantly reduced compared to that in WT parasites. Moreover, we found that protein levels of GBP2 in Δnt1 parasites were markedly lower than in WT parasites. These findings suggest that GBP2 is primarily involved in the sexual development of malaria parasites, and its function may be suppressed by purine starvation.
Topics: Animals; Erythrocytes; Female; Gene Deletion; Malaria, Cerebral; Mice; Mice, Inbred C57BL; Plasmodium berghei; Proteomics; Protozoan Proteins; Purine Nucleotides; Specific Pathogen-Free Organisms
PubMed: 31958569
DOI: 10.1016/j.parint.2020.102059 -
Cellular Microbiology Mar 2021Gametogenesis, the formation of gametes from gametocytes, an essential step for malaria parasite transmission, is targeted by transmission-blocking drugs and vaccines....
Gametogenesis, the formation of gametes from gametocytes, an essential step for malaria parasite transmission, is targeted by transmission-blocking drugs and vaccines. We identified a conserved protein (PBANKA_0305900) in Plasmodium berghei, which encodes a protein of 22 kDa (thus named Pb22) and is expressed in both asexual stages and gametocytes. Its homologues are present in all Plasmodium species and its closely related, Hepatocystis, but not in other apicomplexans. Pb22 protein was localised in the cytosols of schizonts, as well as male and female gametocytes. During gamete-to-ookinete development, Pb22 became localised on the plasma membranes of gametes and ookinetes. Compared to the wild-type (WT) parasites, P. berghei with pb22 knockout (KO) showed a significant reduction in exflagellation (~89%) of male gametocytes and ookinete number (~97%) during in vitro ookinete culture. Mosquito feeding assays showed that ookinete and oocyst formation of the pb22-KO line in mosquito midguts was almost completely abolished. These defects were rescued in parasites where pb22 was restored. Cross-fertilisation experiments with parasite lines defective in either male or female gametes confirmed that the defects in the pb22-KO line were restricted to the male gametes, whereas female gametes in the pb22-KO line were fertile at the WT level. Detailed analysis of male gametogenesis showed that 30% of the male gametocytes in the pb22-KO line failed to assemble the axonemes, whereas ~48.9% of the male gametocytes formed flagella but failed to egress from the host erythrocyte. To explore its transmission-blocking potential, recombinant Pb22 (rPb22) was expressed and used to immunise mice. in vitro assays showed that the rPb22-antisera significantly inhibited exflagellation by ~64.8% and ookinete formation by ~93.4%. Mosquitoes after feeding on rPb22-immunised mice also showed significant decreases in infection prevalence (83.3-93.3%) and oocyst density (93.5-99.6%). Further studies of the Pb22 orthologues in human malaria parasites are warranted.
Topics: Animals; Anopheles; Antibodies, Protozoan; Antigens, Protozoan; Apicomplexa; Cell Membrane; Gene Knockout Techniques; Malaria; Malaria Vaccines; Mice; Mice, Inbred BALB C; Plasmodium berghei; Protozoan Proteins
PubMed: 33222390
DOI: 10.1111/cmi.13294 -
Molecular Microbiology Jun 2006Apicomplexan parasites critically depend on a unique form of gliding motility to colonize their hosts and to invade cells. Gliding requires different stage and...
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.
Topics: Animals; Anopheles; Cell Movement; Epithelium; Gene Deletion; Insect Vectors; Intestines; Mice; Plasmodium berghei; Protein Kinases; Protozoan Proteins
PubMed: 16796674
DOI: 10.1111/j.1365-2958.2006.05189.x