An essential malaria protein defines the architecture of blood-stage and transmission-stage parasites

Absalon, Sabrina; Robbins, Jonathan A.; Dvorin, Jeffrey D.

doi:10.1038/ncomms11449

Cited by 37 publications

(54 citation statements)

References 58 publications

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“…Rhoptry and microneme secretion assays. Apical organelle secretion assays were modified from a previous protocol 70 . Briefly, synchronous ring-stage cultures were obtained using sorbitol and either treated with 2.5 mM GLCN or left untreated and incubated in standard culture conditions for 24 h until early trophozoite stage.…”

Section: Assessment Of In Vitro Blood-stage Growth and Invasionmentioning

confidence: 99%

PfCERLI1 is a conserved rhoptry associated protein essential for Plasmodium falciparum merozoite invasion of erythrocytes

et al. 2020

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The disease-causing blood-stage of the Plasmodium falciparum lifecycle begins with invasion of human erythrocytes by merozoites. Many vaccine candidates with key roles in binding to the erythrocyte surface and entry are secreted from the large bulb-like rhoptry organelles at the apical tip of the merozoite. Here we identify an essential role for the conserved protein P. falciparum Cytosolically Exposed Rhoptry Leaflet Interacting protein 1 (PfCERLI1) in rhoptry function. We show that PfCERLI1 localises to the cytosolic face of the rhoptry bulb membrane and knockdown of PfCERLI1 inhibits merozoite invasion. While schizogony and merozoite organelle biogenesis appear normal, biochemical techniques and semi-quantitative superresolution microscopy show that PfCERLI1 knockdown prevents secretion of key rhoptry antigens that coordinate merozoite invasion. PfCERLI1 is a rhoptry associated protein identified to have a direct role in function of this essential merozoite invasion organelle, which has broader implications for understanding apicomplexan invasion biology.

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Section: Assessment Of In Vitro Blood-stage Growth and Invasionmentioning

confidence: 99%

PfCERLI1 is a conserved rhoptry associated protein essential for Plasmodium falciparum merozoite invasion of erythrocytes

et al. 2020

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“…Immunofluorescence microscopy was carried out as previously described (8,25). Briefly, 3D7-PfCDPK5 3HA-DD -PfNPT1 sm-V5 parasites were maintained with or without Shld and E64 was added at 42 h p.i.…”

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confidence: 99%

Influence of Plasmodium falciparum Calcium-Dependent Protein Kinase 5 (PfCDPK5) on the Late Schizont Stage Phosphoproteome

Blomqvist

Helmel

Wang

et al. 2020

mSphere

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Protein kinases are important mediators of signal transduction in cellular pathways, and calcium-dependent protein kinases (CDPKs) compose a unique class of calcium-dependent kinases present in plants and apicomplexans, including Plasmodium parasites, the causative agents of malaria. During the asexual stage of infection, the human malaria parasite Plasmodium falciparum grows inside red blood cells, and P. falciparum calcium-dependent protein kinase 5 (PfCDPK5) is required for egress from the host cell. In this paper, we characterize the late-schizont-stage P. falciparum phosphoproteome by performing large-scale phosphoproteomic profiling on tightly synchronized parasites just prior to egress, identifying 2,704 phosphorylation sites on 919 proteins. Using a conditional knockdown of PfCDPK5, we identify 58 phosphorylation sites on 50 proteins with significant reduction in levels of PfCDPK5-deficient parasites. Furthermore, gene ontology analysis of the identified proteins reveals enrichment in transmembrane- and membrane-associated proteins and in proteins associated with transport activity. Among the identified proteins is PfNPT1, a member of the apicomplexan-specific novel putative transporter (NPT) family of proteins. We show that PfNPT1 is a potential substrate of PfCDPK5 and that PfNPT1 localizes to the parasite plasma membrane. Importantly, P. falciparum egress relies on many proteins unique to Apicomplexa that are therefore attractive targets for antimalarial therapeutics. IMPORTANCE The malaria parasite Plasmodium falciparum is a major cause of morbidity and mortality globally. The P. falciparum parasite proliferates inside red blood cells during the blood stage of infection, and egress from the red blood cell is critical for parasite survival. P. falciparum calcium-dependent protein kinase 5 (PfCDPK5) is essential for egress; parasites deficient in PfCDPK5 remain trapped inside their host cells. We have used a label-free quantitative mass spectrometry approach to identify the phosphoproteome of schizont-stage parasites just prior to egress and identify 50 proteins that display a significant reduction in phosphorylation in PfCDPK5-deficient parasites. We show that a member of the Apicomplexan-specific transport protein family, PfNPT1 is a potential substrate of PfCDPK5 and is localized to the parasite plasma membrane. P. falciparum egress requires several proteins not present in human cells, thus making this pathway an ideal target for new therapeutics.

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“…The use of DD fusion protein has recently validated essentiality of the P. falciparum Merozoite Organizing Protein (PfMOP). Induction of PfMOP degradation resulted in inner membrane complex formation defect, causing maturation arrest with aberrant morphology and parasite death [90]. But although powerful, the use of destabilizing domains is limited by protein location, since proteins that are secreted are not targeted by the protein degradation machinery of the parasite and thus cannot be targeted via this approach [91].…”

Section: Dd/dddmentioning

confidence: 99%

New directions in antimalarial target validation

Batista

Gyau

Vilacha

et al. 2020

Expert Opinion on Drug Discovery

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Introduction: Malaria is one of the most prevalent human infections worldwide with over 40% of the world's population living in malaria-endemic areas. In the absence of an effective vaccine, emergence of drug-resistant strains requires urgent drug development. Current methods applied to drug target validation, a crucial step in drug discovery, possess limitations in malaria. These constraints require the development of techniques capable of simplifying the validation of Plasmodial targets. Areas covered: The authors review the current state of the art in techniques used to validate drug targets in malaria, including our contributionthe protein interference assay (PIA)as an additional tool in rapid in vivo target validation. Expert opinion: Each technique in this review has advantages and disadvantages, implying that future validation efforts should not focus on a single approach, but integrate multiple approaches. PIA is a significant addition to the current toolset of antimalarial validation. Validation of aspartate metabolism as a druggable pathway provided proof of concept of how oligomeric interfaces can be exploited to control specific activity in vivo. PIA has the potential to be applied not only to other enzymes/ pathways of the malaria parasite but could, in principle, be extrapolated to other infectious diseases.

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An essential malaria protein defines the architecture of blood-stage and transmission-stage parasites

Cited by 37 publications

References 58 publications

PfCERLI1 is a conserved rhoptry associated protein essential for Plasmodium falciparum merozoite invasion of erythrocytes

PfCERLI1 is a conserved rhoptry associated protein essential for Plasmodium falciparum merozoite invasion of erythrocytes

Influence of Plasmodium falciparum Calcium-Dependent Protein Kinase 5 (PfCDPK5) on the Late Schizont Stage Phosphoproteome

New directions in antimalarial target validation

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