scholarly journals Inhibition of protein N-myristoylation blocks Plasmodium falciparum intraerythrocytic development, egress and invasion

PLoS Biology ◽  
2021 ◽  
Vol 19 (10) ◽  
pp. e3001408
Author(s):  
Anja C. Schlott ◽  
Ellen Knuepfer ◽  
Judith L. Green ◽  
Philip Hobson ◽  
Aaron J. Borg ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Infected Erythrocyte ◽  
Complex Function ◽  
Pleiotropic Effect ◽  
Substantial Effect ◽  
Parasite Development ◽  
Human Malaria Parasite ◽  
Erythrocyte Invasion ◽  
Erythrocytic Cycle ◽  
Parasite Egress

We have combined chemical biology and genetic modification approaches to investigate the importance of protein myristoylation in the human malaria parasite, Plasmodium falciparum. Parasite treatment during schizogony in the last 10 to 15 hours of the erythrocytic cycle with IMP-1002, an inhibitor of N-myristoyl transferase (NMT), led to a significant blockade in parasite egress from the infected erythrocyte. Two rhoptry proteins were mislocalized in the cell, suggesting that rhoptry function is disrupted. We identified 16 NMT substrates for which myristoylation was significantly reduced by NMT inhibitor (NMTi) treatment, and, of these, 6 proteins were substantially reduced in abundance. In a viability screen, we showed that for 4 of these proteins replacement of the N-terminal glycine with alanine to prevent myristoylation had a substantial effect on parasite fitness. In detailed studies of one NMT substrate, glideosome-associated protein 45 (GAP45), loss of myristoylation had no impact on protein location or glideosome assembly, in contrast to the disruption caused by GAP45 gene deletion, but GAP45 myristoylation was essential for erythrocyte invasion. Therefore, there are at least 3 mechanisms by which inhibition of NMT can disrupt parasite development and growth: early in parasite development, leading to the inhibition of schizogony and formation of “pseudoschizonts,” which has been described previously; at the end of schizogony, with disruption of rhoptry formation, merozoite development and egress from the infected erythrocyte; and at invasion, when impairment of motor complex function prevents invasion of new erythrocytes. These results underline the importance of P. falciparum NMT as a drug target because of the pleiotropic effect of its inhibition.

scholarly journals Inhibition of protein N-myristoylation blocks Plasmodium falciparum intraerythrocytic development, egress, and invasion

2020 ◽  
Author(s):  
Anja C. Schlott ◽  
Ellen Knuepfer ◽  
Judith L. Green ◽  
Philip Hobson ◽  
Aaron J. Borg ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Infected Erythrocyte ◽  
Complex Function ◽  
Pleiotropic Effect ◽  
Substantial Effect ◽  
Parasite Development ◽  
Human Malaria Parasite ◽  
Erythrocyte Invasion ◽  
Erythrocytic Cycle ◽  
Parasite Egress

ABSTRACTWe have combined chemical biology and genetic modification approaches to investigate the importance of protein myristoylation in the human malaria parasite, Plasmodium falciparum. Parasite treatment during schizogony in the last ten to fifteen hours of the erythrocytic cycle with IMP-1002, an inhibitor of N-myristoyl transferase (NMT), led to a significant blockade in parasite egress from the infected erythrocyte. Two rhoptry proteins were mislocalized in the cell, suggesting that rhoptry function is disrupted. We identified sixteen NMT substrates for which myristoylation was significantly reduced by NMT inhibitor treatment, and of these, six proteins were substantially reduced in abundance. In a viability screen, we showed that for four of these proteins replacement of the N-terminal glycine with alanine to prevent myristoylation had a substantial effect on parasite fitness. In detailed studies of one NMT substrate, glideosome associated protein 45 (GAP45), loss of myristoylation had no impact on protein location or glideosome assembly, in contrast to the disruption caused by GAP45 gene deletion, but GAP45 myristoylation was essential for erythrocyte invasion. Therefore, there are at least three mechanisms by which inhibition of NMT can disrupt parasite development and growth: early in parasite development, leading to the inhibition of schizogony and formation of ‘pseudoschizonts’, which has been described previously; at the end of schizogony, with disruption of rhoptry formation, merozoite development and egress from the infected erythrocyte; and at invasion, when impairment of motor complex function prevents invasion of new erythrocytes. These results underline the importance of P. falciparum NMT as a drug target because of the pleiotropic effect of its inhibition.

scholarly journals Exploring the lower thermal limits for development of the human malaria parasite, Plasmodium falciparum

2019 ◽  
Vol 15 (6) ◽  
pp. 20190275 ◽  
Author(s):  
Jessica L. Waite ◽  
Eunho Suh ◽  
Penelope A. Lynch ◽  
Matthew B. Thomas
Keyword(s):  
Plasmodium Falciparum ◽  
Future Climate Change ◽  
Parasite Development ◽  
Temperature Dependent ◽  
Human Malaria Parasite ◽  
Thermal Limits ◽  
Degree Day ◽  
Extrinsic Incubation Period ◽  
Parasite Plasmodium ◽  
Parasite Plasmodium Falciparum

The rate of malaria transmission is strongly determined by parasite development time in the mosquito, known as the extrinsic incubation period (EIP), since the quicker parasites develop, the greater the chance that the vector will survive long enough for the parasite to complete development and be transmitted. EIP is known to be temperature-dependent but this relationship is surprisingly poorly characterized. There is a single degree-day model for EIP of Plasmodium falciparum that derives from a limited number of poorly controlled studies conducted almost a century ago. Here, we show that the established degree-day model greatly underestimates the rate of development of P. falciparum in both Anopheles stephensi and An. gambiae mosquitoes at temperatures in the range of 17–20°C. We also show that realistic daily temperature fluctuation further speeds parasite development. These novel results challenge one of the longest standing models in malaria biology and have potentially important implications for understanding the impacts of future climate change.

scholarly journals Protein ubiquitylation is essential for the schizont to merozoite transition in Plasmodium falciparum blood-stage development

2019 ◽  
Author(s):  
Yang Wu ◽  
Vesela Encheva ◽  
Judith L. Green ◽  
Edwin Lasonder ◽  
Adchara Prommaban ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Homology Model ◽  
Food Vacuole ◽  
Blood Stage ◽  
Parasite Development ◽  
Parasite Line ◽  
Schizont Stage ◽  
Post Translational Modification ◽  
Asexual Blood Stage ◽  
Erythrocyte Invasion

AbstractUbiquitylation is a common post translational modification of eukaryotic proteins and in the human malaria parasite, Plasmodium falciparum (Pf) overall ubiquitylation increases in the transition from intracellular schizont to extracellular merozoite stages in the asexual blood stage cycle. Here, we identify specific ubiquitylation sites of protein substrates in three intracellular parasite stages and extracellular merozoites; a total of 1464 sites in 546 proteins were identified (data available via ProteomeXchange with identifier PXD014998). 469 ubiquitylated proteins were identified in merozoites compared with only 160 in the preceding intracellular schizont stage, indicating a large increase in protein ubiquitylation associated with merozoite maturation. Following merozoite invasion of erythrocytes, few ubiquitylated proteins were detected in the first intracellular ring stage but as parasites matured through trophozoite to schizont stages the extent of ubiquitylation increased. We identified commonly used ubiquitylation motifs and groups of ubiquitylated proteins in specific areas of cellular function, for example merozoite pellicle proteins involved in erythrocyte invasion, exported proteins, and histones. To investigate the importance of ubiquitylation we screened ubiquitin pathway inhibitors in a parasite growth assay and identified the ubiquitin activating enzyme (UBA1 or E1) inhibitor MLN7243 (TAK-243) to be particularly effective. This small molecule was shown to be a potent inhibitor of recombinant PfUBA1, and a structural homology model of MLN7243 bound to the parasite enzyme highlights avenues for the development of P. falciparum specific inhibitors. We created a genetically modified parasite with a rapamycin-inducible functional deletion of uba1; addition of either MLN7243 or rapamycin to the recombinant parasite line resulted in the same phenotype, with parasite development blocked at the late schizont stage. These results indicate that the intracellular target of MLN7243 is UBA1, and this activity is essential for the final differentiation of schizonts to merozoites. The ubiquitylation of many merozoite proteins and their disappearance in ring stages are consistent with the idea that ubiquitylation leads to their destruction via the proteasome once their function is complete following invasion, which would allow amino acid recycling in the period prior to the parasite’s elaboration of a new food vacuole.

scholarly journals Delivery of the Malaria Virulence Protein PfEMP1 to the Erythrocyte Surface Requires Cholesterol-Rich Domains

2006 ◽  
Vol 5 (5) ◽  
pp. 849-860 ◽  
Author(s):  
Sarah Frankland ◽  
Akinola Adisa ◽  
Paul Horrocks ◽  
Theodore F. Taraschi ◽  
Timothy Schneider ◽  
...  
Keyword(s):  
Erythrocyte Membrane ◽  
Infected Erythrocyte ◽  
Membrane Microdomains ◽  
Human Malaria Parasite ◽  
Vesicle Budding ◽  
Erythrocyte Invasion ◽  
Major Virulence Factor ◽  
Erythrocyte Surface ◽  
Virulence Protein ◽  
Maurer's Clefts

ABSTRACT The particular virulence of the human malaria parasite Plasmodium falciparum derives from export of parasite-encoded proteins to the surface of the mature erythrocytes in which it resides. The mechanisms and machinery for the export of proteins to the erythrocyte membrane are largely unknown. In other eukaryotic cells, cholesterol-rich membrane microdomains or “rafts” have been shown to play an important role in the export of proteins to the cell surface. Our data suggest that depletion of cholesterol from the erythrocyte membrane with methyl-β-cyclodextrin significantly inhibits the delivery of the major virulence factor P. falciparum erythrocyte membrane protein 1 (PfEMP1). The trafficking defect appears to lie at the level of transfer of PfEMP1 from parasite-derived membranous structures within the infected erythrocyte cytoplasm, known as the Maurer's clefts, to the erythrocyte membrane. Thus our data suggest that delivery of this key cytoadherence-mediating protein to the host erythrocyte membrane involves insertion of PfEMP1 at cholesterol-rich microdomains. GTP-dependent vesicle budding and fusion events are also involved in many trafficking processes. To determine whether GTP-dependent events are involved in PfEMP1 trafficking, we have incorporated non-membrane-permeating GTP analogs inside resealed erythrocytes. Although these nonhydrolyzable GTP analogs reduced erythrocyte invasion efficiency and partially retarded growth of the intracellular parasite, they appeared to have little direct effect on PfEMP1 trafficking.

scholarly journals Genetic disruption of Plasmodium falciparum Merozoite surface antigen 180 (PfMSA180) suggests an essential role during parasite egress from erythrocytes

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Vanndita Bahl ◽  
Kritika Chaddha ◽  
Syed Yusuf Mian ◽  
Anthony A. Holder ◽  
Ellen Knuepfer ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Surface Antigen ◽  
Essential Role ◽  
Gene Knockout ◽  
Intervention Strategy ◽  
Merozoite Surface Antigen ◽  
Erythrocytic Cycle ◽  
And Function ◽  
Parasite Egress

AbstractPlasmodium falciparum, the parasite responsible for severe malaria, develops within erythrocytes. Merozoite invasion and subsequent egress of intraerythrocytic parasites are essential for this erythrocytic cycle, parasite survival and pathogenesis. In the present study, we report the essential role of a novel protein, P. falciparum Merozoite Surface Antigen 180 (PfMSA180), which is conserved across Plasmodium species and recently shown to be associated with the P. vivax merozoite surface. Here, we studied MSA180 expression, processing, localization and function in P. falciparum blood stages. Initially we examined its role in invasion, a process mediated by multiple ligand-receptor interactions and an attractive step for targeting with inhibitory antibodies through the development of a malaria vaccine. Using antibodies specific for different regions of PfMSA180, together with a parasite containing a conditional pfmsa180-gene knockout generated using CRISPR/Cas9 and DiCre recombinase technology, we demonstrate that this protein is unlikely to play a crucial role in erythrocyte invasion. However, deletion of the pfmsa180 gene resulted in a severe egress defect, preventing schizont rupture and blocking the erythrocytic cycle. Our study highlights an essential role of PfMSA180 in parasite egress, which could be targeted through the development of a novel malaria intervention strategy.

scholarly journals The Transcription Factor PfAP2-O Influences Virulence Gene Transcription and Sexual Development in Plasmodium falciparum

2021 ◽  
Vol 11 ◽  
Author(s):  
Eliana F. G. Cubillos ◽  
Isadora Oliveira Prata ◽  
Wesley Luzetti Fotoran ◽  
Lisa Ranford-Cartwright ◽  
Gerhard Wunderlich
Keyword(s):  
Plasmodium Falciparum ◽  
Transcription Factor ◽  
Severe Malaria ◽  
Infected Erythrocyte ◽  
Virulence Gene ◽  
Transcriptional Regulators ◽  
Parasite Transmission ◽  
Human Malaria Parasite ◽  
Important Target ◽  
Transcriptional Memory

The human malaria parasite Plasmodium falciparum expresses variant PfEMP1 proteins on the infected erythrocyte, which function as ligands for endothelial receptors in capillary vessels, leading to erythrocyte sequestration and severe malaria. The factors that orchestrate the mono-allelic expression of the 45–90 PfEMP1-encoding var genes within each parasite genome are still not fully identified. Here, we show that the transcription factor PfAP2-O influences the transcription of var genes. The temporary knockdown of PfAP2-O leads to a complete loss of var transcriptional memory and a decrease in cytoadherence in CD36 adherent parasites. AP2-O-knocked-down parasites exhibited also significant reductions in transmission through Anopheles mosquitoes. We propose that PfAP2-O is, beside its role in transmission stages, also one of the virulence gene transcriptional regulators and may therefore be exploited as an important target to disrupt severe malaria and block parasite transmission.

scholarly journals Cholesterol dynamics are essential for the growth and development of Plasmodium falciparum within the erythrocyte

2021 ◽  
Author(s):  
Avantika I. Ahiya ◽  
Suyash Bhatnagar ◽  
Joanne Morrisey ◽  
Josh R. Beck ◽  
Akhil B. Vaidya
Keyword(s):  
Infected Erythrocyte ◽  
De Novo ◽  
Cholesterol Content ◽  
Cholesterol Homeostasis ◽  
Parasite Development ◽  
Trophozoite Stage ◽  
Parasite Survival ◽  
Erythrocytic Cycle ◽  
Erythrocyte Plasma Membrane ◽  
Rapid Expulsion

AbstractPlasmodium spp. lack de novo cholesterol synthetic pathways and can only scavenge it from their host erythrocyte. Here we report that depletion of cholesterol from the erythrocyte plasma membrane by methyl-β-cyclodextrin (MBCD) has dramatic consequences. The removal of cholesterol results in invasion defects as well as inhibition of parasite development through the intra-erythrocytic cycle. These defects could be rescued by reconstitution with cholesterol and desmosterol but not with epicholesterol. By using live microscopy of fluorescently tagged trophozoite stage parasites, we detected rapid expulsion of the parasites from erythrocyte when exposed to MBCD for just 30 mins. Strikingly, the parasites transition from being intra-erythrocytic to extracellular within 10 seconds and do so without rupturing the erythrocyte membrane. These extruded parasites were still surrounded by the parasitophorous vacuolar membrane (PVM) and remained tethered to the erythrocyte. Electron microscopy revealed that although extracellular parasites retained their PVM, it was heavily compromised. Treatment with antimalarials that disrupt cholesterol homeostasis prior to MBCD exposure prevented the extrusion of trophozoites. These results reveal importance of cholesterol during the intra-erythrocytic development of P. falciparum and the dramatic consequences resulting from tampering with cholesterol content in the infected erythrocyte. These findings suggest dynamic nature of cholesterol within the infected erythrocyte that is critical for parasite survival.

scholarly journals Plasmodium falciparum-infected erythrocytes utilize a synthetic truncated ceramide precursor for synthesis and secretion of truncated sphingomyelin

1995 ◽  
Vol 308 (1) ◽  
pp. 335-341 ◽  
Author(s):  
I Ansorge ◽  
D Jeckel ◽  
F Wieland ◽  
K Lingelbach
Keyword(s):  
Plasmodium Falciparum ◽  
Mammalian Cells ◽  
Membrane Fraction ◽  
Infected Erythrocyte ◽  
Brefeldin A ◽  
Parasite Development ◽  
Phospholipid Content ◽  
Unicellular Eukaryote ◽  
Fungal Metabolite ◽  
Sphingomyelin Synthase

Plasmodium falciparum is an intracellular parasite of human erythrocytes. Parasite development is accompanied by an increase of the phospholipid content of the infected erythrocyte, but it results in a selective decrease of sphingomyelin. We have studied sphingomyelin biosynthesis in infected erythrocytes using as substrate a synthetic radiolabelled ceramide precursor, truncated in both hydrophobic chains. Lysates of infected, unlike those of non-infected, erythrocytes contained sphingomyelin synthase activity, which therefore is of parasite origin. The enzyme activity was associated with a membrane fraction. In contrast to mammalian cells, the parasite did not synthesize detectable levels of glycosphingolipids. In intact infected erythrocytes the ceramide precursor was converted into a correspondingly truncated soluble sphingomyelin which was released into the medium at 37 degrees C. Release of truncated sphingomyelin was inhibited by low temperature (15 degrees C) but not by the fungal metabolite brefeldin A which, however, arrests protein export from the parasite. While membranes of mammalian cells, including the plasma membrane of non-infected erythrocytes, are impermeable to truncated sphingomyelin, the membrane of infected erythrocytes allowed passage of the molecule in both directions. The results obtained with the unicellular eukaryote used here as an experimental model are discussed in comparison with sphingomyelin synthesis and transport in mammalian cells.

scholarly journals Cell trace far-red is a suitable erythrocyte dye for multi-color Plasmodium falciparum invasion phenotyping assays

2020 ◽  
Vol 245 (1) ◽  
pp. 11-20
Author(s):  
Laty G Thiam ◽  
Yaw Aniweh ◽  
Evelyn B Quansah ◽  
Jacob K Donkor ◽  
Theresa M Gwira ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Fluorescence Intensity ◽  
Parasite Development ◽  
Fluorescent Intensity ◽  
Erythrocyte Invasion ◽  
Low Concentrations ◽  
Donor And Acceptor ◽  
Succinimidyl Ester ◽  
Dna Dyes ◽  
Over Time

Plasmodium falciparum erythrocyte invasion phenotyping assays are a very useful tool for assessing parasite diversity and virulence, and for characterizing the formation of ligand–receptor interactions. However, such assays need to be highly sensitive and reproducible, and the selection of labeling dyes for differentiating donor and acceptor erythrocytes is a critical factor. We investigated the suitability of cell trace far-red (CTFR) as a dye for P. falciparum invasion phenotyping assays. Using the dyes carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) and dichloro dimethyl acridin one succinimidyl ester (DDAO-SE) as comparators, we used a dye-dilution approach to assess the limitations and specific staining procedures for the applicability of CTFR in P. falciparum invasion phenotyping assays. Our data show that CTFR effectively labels acceptor erythrocytes and provides a stable fluorescent intensity at relatively low concentrations. CTFR also yielded a higher fluorescence intensity relative to DDAO-SE and with a more stable fluorescence intensity over time. Furthermore, CTFR did not affect merozoites invasion of erythrocytes and was not toxic to the parasite’s intraerythrocytic development. Additionally, CTFR offers flexibility in the choice of combinations with several other DNA dyes, which broaden its usage for P. falciparum erythrocyte invasion assays, considering a wider range of flow cytometers with various laser settings. Impact statement In recent years, flow cytometry has become a cornerstone in investigating P. falciparum phenotypic diversity using multiple dyes to discriminate between donor and acceptor erythrocytes. To broaden the applicability of such assays, we optimized the staining conditions of a newly developed cytoplasmic dye, cell trace far-red (CTFR), and assessed its suitability for use in P. falciparum invasion phenotyping assays. We showed that CTFR has a very narrow emission peak excited by red lasers. Furthermore, CTFR labeling of target erythrocytes, achieved even at low concentrations, is stable over time and did not impair parasite development. P. falciparum erythrocyte invasion phenotyping assays revealed that CTFR is suitable for use in combination with several DNA dyes in multiplex assays. This will allow for high throughput phenotyping of parasites as well as facilitate the evaluation of preference of erythrocytes by merozoites. Altogether, these make screening for potential invasion-blocking interventions possible.

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