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Nature Communications Jun 2024Differentiation of male gametocytes into flagellated fertile male gametes relies on the assembly of axoneme, a major component of male development for mosquito...
Differentiation of male gametocytes into flagellated fertile male gametes relies on the assembly of axoneme, a major component of male development for mosquito transmission of the malaria parasite. RNA-binding protein (RBP)-mediated post-transcriptional regulation of mRNA plays important roles in eukaryotic sexual development, including the development of female Plasmodium. However, the role of RBP in defining the Plasmodium male transcriptome and its function in male gametogenesis remains incompletely understood. Here, we performed genome-wide screening for gender-specific RBPs and identified an undescribed male-specific RBP gene Rbpm1 in the Plasmodium. RBPm1 is localized in the nucleus of male gametocytes. RBPm1-deficient parasites fail to assemble the axoneme for male gametogenesis and thus mosquito transmission. RBPm1 interacts with the spliceosome E complex and regulates the splicing initiation of certain introns in a group of 26 axonemal genes. RBPm1 deficiency results in intron retention and protein loss of these axonemal genes. Intron deletion restores axonemal protein expression and partially rectifies axonemal defects in RBPm1-null gametocytes. Further splicing assays in both reporter and endogenous genes exhibit stringent recognition of the axonemal introns by RBPm1. The splicing activator RBPm1 and its target introns constitute an axonemal intron splicing program in the post-transcriptional regulation essential for Plasmodium male development.
Topics: Introns; RNA Splicing; RNA-Binding Proteins; Animals; Protozoan Proteins; Male; Axoneme; Female; Gametogenesis; Spliceosomes; Plasmodium berghei; Malaria; Plasmodium
PubMed: 38824128
DOI: 10.1038/s41467-024-49002-9 -
Proceedings of the National Academy of... Feb 2017The first, obligatory replication phase of malaria parasite infections is characterized by rapid expansion and differentiation of single parasites in liver cells,...
The first, obligatory replication phase of malaria parasite infections is characterized by rapid expansion and differentiation of single parasites in liver cells, resulting in the formation and release of thousands of invasive merozoites into the bloodstream. Hepatic development occurs inside a specialized membranous compartment termed the parasitophorous vacuole (PV). Here, we show that, during the parasite's hepatic replication, the C-terminal region of the parasitic PV membrane protein exported protein 1 (EXP-1) binds to host Apolipoprotein H (ApoH) and that this molecular interaction plays a pivotal role for successful liver-stage development. Expression of a truncated EXP-1 protein, missing the specific ApoH interaction site, or down-regulation of ApoH expression in either hepatic cells or mouse livers by RNA interference resulted in impaired intrahepatic development. Furthermore, infection of mice with sporozoites expressing a truncated version of EXP-1 resulted in both a significant reduction of liver burden and delayed blood-stage patency, leading to a disease outcome different from that generally induced by infection with wild-type parasites. This study identifies a host-parasite protein interaction during the hepatic stage of infection by parasites. The identification of such vital interactions may hold potential toward the development of novel malaria prevention strategies.
Topics: Animals; Animals, Genetically Modified; Binding Sites; Down-Regulation; Genes, Protozoan; HEK293 Cells; Hepatocytes; Humans; Liver; Malaria; Membrane Proteins; Mice; Mice, Inbred C57BL; Microscopy, Confocal; Plasmodium berghei; Protein Binding; Protozoan Proteins; RNA Interference; RNA, Small Interfering; Sequence Deletion; Sporozoites; Vacuoles; beta 2-Glycoprotein I
PubMed: 28137845
DOI: 10.1073/pnas.1606419114 -
Single-cell RNA sequencing reveals developmental heterogeneity among Plasmodium berghei sporozoites.Scientific Reports Feb 2021In the malaria-causing parasite's life cycle, Plasmodium sporozoites must travel from the midgut of a mosquito to the salivary glands before they can infect a mammalian...
In the malaria-causing parasite's life cycle, Plasmodium sporozoites must travel from the midgut of a mosquito to the salivary glands before they can infect a mammalian host. However, only a fraction of sporozoites complete the journey. Since salivary gland invasion is required for transmission of sporozoites, insights at the molecular level can contribute to strategies for malaria prevention. Recent advances in single-cell RNA sequencing provide an opportunity to assess sporozoite heterogeneity at a resolution unattainable by bulk RNA sequencing methods. In this study, we use a droplet-based single-cell RNA sequencing workflow to analyze the transcriptomes of over 8000 Plasmodium berghei sporozoites derived from the midguts and salivary glands of Anopheles stephensi mosquitoes. The detection of known marker genes confirms the successful capture and sequencing of samples composed of a mixed population of sporozoites. Using data integration, clustering, and trajectory analyses, we reveal differences in gene expression profiles of individual sporozoites, and identify both annotated and unannotated markers associated with sporozoite development. Our work highlights the utility of a high-throughput workflow for the transcriptomic profiling of Plasmodium sporozoites, and provides new insights into gene usage during the parasite's development in the mosquito.
Topics: Computational Biology; Gene Expression Profiling; Gene Expression Regulation; Gene Ontology; Genetic Heterogeneity; High-Throughput Nucleotide Sequencing; Malaria; Organ Specificity; Plasmodium berghei; Single-Cell Analysis; Sporozoites; Transcriptome
PubMed: 33619283
DOI: 10.1038/s41598-021-82914-w -
PloS One 2018Retroviral protease inhibitors (RPIs) such as lopinavir (LP) and saquinavir (SQ) are active against Plasmodium parasites. However, the exact molecular target(s) for...
Retroviral protease inhibitors (RPIs) such as lopinavir (LP) and saquinavir (SQ) are active against Plasmodium parasites. However, the exact molecular target(s) for these RPIs in the Plasmodium parasites remains poorly understood. We hypothesised that LP and SQ suppress parasite growth through inhibition of aspartyl proteases. Using reverse genetics approach, we embarked on separately generating knockout (KO) parasite lines lacking Plasmepsin 4 (PM4), PM7, PM8, or DNA damage-inducible protein 1 (Ddi1) in the rodent malaria parasite Plasmodium berghei ANKA. We then tested the suppressive profiles of the LP/Ritonavir (LP/RT) and SQ/RT as well as antimalarials; Amodiaquine (AQ) and Piperaquine (PQ) against the KO parasites in the standard 4-day suppressive test. The Ddi1 gene proved refractory to deletion suggesting that the gene is essential for the growth of the asexual blood stage parasites. Our results revealed that deletion of PM4 significantly reduces normal parasite growth rate phenotype (P = 0.003). Unlike PM4_KO parasites which were less susceptible to LP and SQ (P = 0.036, P = 0.030), the suppressive profiles for PM7_KO and PM8_KO parasites were comparable to those for the WT parasites. This finding suggests a potential role of PM4 in the LP and SQ action. On further analysis, modelling and molecular docking studies revealed that both LP and SQ displayed high binding affinities (-6.3 kcal/mol to -10.3 kcal/mol) towards the Plasmodium aspartyl proteases. We concluded that PM4 plays a vital role in assuring asexual stage parasite fitness and might be mediating LP and SQ action. The essential nature of the Ddi1 gene warrants further studies to evaluate its role in the parasite asexual blood stage growth as well as a possible target for the RPIs.
Topics: Animals; Anti-Retroviral Agents; Antimalarials; Aspartic Acid Endopeptidases; Aspartic Acid Proteases; Lopinavir; Mice; Models, Molecular; Molecular Docking Simulation; Plasmodium berghei; Protease Inhibitors; Protozoan Proteins; Reverse Genetics; Saquinavir
PubMed: 30067811
DOI: 10.1371/journal.pone.0201556 -
Proceedings. Biological Sciences May 2015Sexual reproduction is an obligate step in the life cycle of many parasites, including the causative agents of malaria (Plasmodium). Mixed-species infections are common...
Sexual reproduction is an obligate step in the life cycle of many parasites, including the causative agents of malaria (Plasmodium). Mixed-species infections are common in nature and consequently, interactions between heterospecific gametes occur. Given the importance of managing gene flow across parasite populations, remarkably little is understood about how reproductive isolation between species is maintained. We use the rodent malaria parasites P. berghei and P. yoelii to investigate the ecology of mixed-species mating groups, identify proteins involved in pre-zygotic barriers, and examine their evolution. Specifically, we show that (i) hybridization occurs, but at low frequency; (ii) hybridization reaches high levels when female gametes lack the surface proteins P230 or P48/45, demonstrating that these proteins are key for pre-zygotic reproductive isolation; (iii) asymmetric reproductive interference occurs, where the fertility of P. berghei gametes is reduced in the presence of P. yoelii and (iv) as expected for gamete recognition proteins, strong positive selection acts on a region of P230 and P47 (P48/45 paralogue). P230 and P48/45 are leading candidates for interventions to block malaria transmission. Our results suggest that depending on the viability of hybrids, applying such interventions to populations where mixed-species infections occur could either facilitate or hinder malaria control.
Topics: Evolution, Molecular; Gene Flow; Hybridization, Genetic; Molecular Sequence Data; Plasmodium berghei; Plasmodium yoelii; Protozoan Proteins; Reproduction; Sequence Analysis, DNA
PubMed: 25854886
DOI: 10.1098/rspb.2014.3027 -
PloS One 2020The circumsporozoite protein (CSP) and thrombospondin-related adhesion protein (TRAP) are major targets for pre-erythrocytic malaria vaccine development. However, the...
The circumsporozoite protein (CSP) and thrombospondin-related adhesion protein (TRAP) are major targets for pre-erythrocytic malaria vaccine development. However, the CSP-based vaccine RTS,S provides only marginal protection, highlighting the need for innovative vaccine design and development. Here we design and characterize expression and folding of P. berghei (Pb) and P. falciparum (Pf) TRAP-CSP fusion proteins, and evaluate immunogenicity and sterilizing immunity in mice. TRAP N-terminal domains were fused to the CSP C-terminal αTSR domain with or without the CSP repeat region, expressed in mammalian cells, and evaluated with or without N-glycan shaving. Pb and Pf fusions were each expressed substantially better than the TRAP or CSP components alone; furthermore, the fusions but not the CSP component could be purified to homogeneity and were well folded and monomeric. As yields of TRAP and CSP fragments were insufficient, we immunized BALB/c mice with Pb TRAP-CSP fusions in AddaVax adjuvant and tested the effects of absence or presence of the CSP repeats and absence or presence of high mannose N-glycans on total antibody titer and protection from infection by mosquito bite both 2.5 months and 6 months after the last immunization. Fusions containing the repeats were completely protective against challenge and re-challenge, while those lacking repeats were significantly less effective. These results correlated with higher total antibody titers when repeats were present. Our results show that TRAP-CSP fusions increase protein antigen production, have the potential to yield effective vaccines, and also guide design of effective proteins that can be encoded by nucleic acid-based and virally vectored vaccines.
Topics: Animals; Antibodies; Antigens; Disease Models, Animal; Gene Expression Regulation; Humans; Immunization; Malaria; Malaria Vaccines; Mice; Plasmodium berghei; Plasmodium falciparum; Polysaccharides; Protein Folding; Protozoan Proteins; Recombinant Fusion Proteins
PubMed: 31967991
DOI: 10.1371/journal.pone.0216260 -
Malaria Journal Sep 2016Zanthoxylum heitzii (Rutaceae) (olon) is used in traditional medicine in Central and West Africa to treat malaria. To identify novel compounds with anti-parasitic...
BACKGROUND
Zanthoxylum heitzii (Rutaceae) (olon) is used in traditional medicine in Central and West Africa to treat malaria. To identify novel compounds with anti-parasitic activity and validate medicinal usage, extracts and compounds isolated from this tree were tested against the erythrocytic stages of the human malaria parasite Plasmodium falciparum and for inhibition of transmission in rodent malaria parasite Plasmodium berghei.
RESULTS
Hexane bark extract showed activity against P. falciparum (IC50 0.050 μg/ml), while leaf and seed extracts were inactive. Fractionation of the hexane bark extract led to the identification of three active constituents; dihydronitidine, pellitorine and heitziquinone. Dihydronitidine was the most active compound with an IC50 value of 0.0089 µg/ml (25 nM). This compound was slow acting, requiring 50 % longer exposure time than standard anti-malarials to reach full efficacy. Heitziquinone and pellitorine were less potent, with IC50 values of 3.55 μg/ml and 1.96 µg/ml, but were fast-acting. Plasmodium berghei ookinete conversion was also inhibited by the hexane extract (IC50 1.75 µg/ml), dihydronitidine (0.59 µg/ml) and heitziquinone (6.2 µg/ml). Water extracts of Z. heitzii bark contain only low levels of dihydronitidine and show modest anti-parasitic activity.
CONCLUSIONS
Three compounds with anti-parasitic activity were identified in Z. heitzii bark extract. The alkaloid dihydronitidine is the most effective of these, accounting for the bulk of activity in both erythrocytic and transmission-blocking assays. These compounds may present good leads for development of novel anti-malarials and add to the understanding of the chemical basis of the anti-parasitic activity in these classes of natural product.
Topics: Antimalarials; Biological Products; Inhibitory Concentration 50; Plasmodium berghei; Plasmodium falciparum; Zanthoxylum
PubMed: 27649682
DOI: 10.1186/s12936-016-1533-x -
Cellular Microbiology Jul 2019The Plasmodium subtilisin-like serine protease SUB1 is expressed in hepatic and both asexual and sexual blood parasite stages. SUB1 is required for egress of invasive...
The Plasmodium subtilisin-like serine protease SUB1 is expressed in hepatic and both asexual and sexual blood parasite stages. SUB1 is required for egress of invasive forms of the parasite from both erythrocytes and hepatocytes, but its subcellular localisation, function, and potential substrates in the sexual stages are unknown. Here, we have characterised the expression profile and subcellular localisation of SUB1 in Plasmodium berghei sexual stages. We show that the protease is selectively expressed in mature male gametocytes and localises to secretory organelles known to be involved in gamete egress, called male osmiophilic bodies. We have investigated PbSUB1 function in the sexual stages by generating P. berghei transgenic lines deficient in PbSUB1 expression or enzyme activity in gametocytes. Our results demonstrate that PbSUB1 plays a role in male gamete egress. We also show for the first time that the PbSUB1 substrate PbSERA3 is expressed in gametocytes and processed by PbSUB1 upon gametocyte activation. Taken together, our results strongly suggest that PbSUB1 is not only a promising drug target for asexual stages but could also be an attractive malaria transmission-blocking target.
Topics: Animals; Erythrocytes; Germ Cells; Hepatocytes; Malaria; Male; Organelles; Plasmodium berghei; Plasmodium falciparum; Serine Endopeptidases; Subtilisins
PubMed: 30941868
DOI: 10.1111/cmi.13028 -
Cellular Microbiology Oct 2019Intracellular Plasmodium parasites develop inside a parasitophorous vacuole (PV), a specialised compartment enclosed by a membrane (PVM) that contains proteins of both...
Intracellular Plasmodium parasites develop inside a parasitophorous vacuole (PV), a specialised compartment enclosed by a membrane (PVM) that contains proteins of both host and parasite origin. Although exported protein 1 (EXP1) is one of the earliest described parasitic PVM proteins, its function throughout the Plasmodium life cycle remains insufficiently understood. Here, we show that whereas the N-terminus of Plasmodium berghei EXP1 (PbEXP1) is essential for parasite survival in the blood, parasites lacking PbEXP1's entire C-terminal (CT) domain replicate normally in the blood but cause less severe pathology than their wild-type counterparts. Moreover, truncation of PbEXP1's CT domain not only impairs parasite development in the mosquito but also abrogates PbEXP1 localization to the PVM of intrahepatic parasites, severely limiting their replication and preventing their egress into the blood. Our findings highlight the importance of EXP1 during the Plasmodium life cycle and identify this protein as a promising target for antiplasmodial intervention.
Topics: Animals; Cell Line, Tumor; Culicidae; Erythrocytes; Female; Humans; Intracellular Membranes; Life Cycle Stages; Liver; Mice; Mice, Inbred C57BL; Plasmodium berghei; Protein Domains; Protozoan Proteins; Vacuoles
PubMed: 31364224
DOI: 10.1111/cmi.13088 -
Malaria Journal May 2019The protective efficacy of the most promising malaria whole-parasite based vaccine candidates critically depends on the parasite's potential to migrate in the human...
BACKGROUND
The protective efficacy of the most promising malaria whole-parasite based vaccine candidates critically depends on the parasite's potential to migrate in the human host. Key components of the parasite motility machinery (e.g. adhesive proteins, actin/myosin-based motor, geometrical properties) have been identified, however the regulation of this machinery is an unknown process.
METHODS
In vitro microscopic live imaging of parasites in different formulations was performed and analysed, with the quantitative analysis software SMOOT, their motility; their adherence capacity, movement pattern and velocity during forward locomotion.
RESULTS
SMOOT enabled the detailed analysis of the regulation of the motility machinery of Plasmodium berghei in response to specific (macro)molecules in the formulation. Albumin acted as an essential supplement to induce parasite attachment and movement. Glucose, salts and other whole serum components further increased the attachment rate and regulated the velocity of the movement.
CONCLUSIONS
Based on the findings can be concluded that a complex interplay of albumin, glucose and certain salts and amino acids regulates parasite motility. Insights in parasite motility regulation by supplements in solution potentially provide a way to optimize the whole-parasite malaria vaccine formulation.
Topics: Albumins; Animals; Culicidae; Culture Media; Female; Glucose; Intravital Microscopy; Locomotion; Malaria; Mice; Plasmodium berghei; Software; Sporozoites
PubMed: 31046772
DOI: 10.1186/s12936-019-2794-y