Cyclic AMP signalling controls key components of malaria parasite host cell invasion machinery

Patel, Avnish; Perrin, Abigail J.; Flynn, Helen; Bisson, C.; Withers‐Martinez, Chrislaine; Treeck, Moritz; Flueck, Christian; Nicastro, Giuseppe; Martin, Stephen R.; Ramos, Andrés; Gilberger, Tim‐Wolf; Snijders, Ambrosius P.; Blackman, Michael J.; Baker, David A.

doi:10.1371/journal.pbio.3000264

Cited by 79 publications

(144 citation statements)

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“…Invasion is an orchestrated process, comprising several steps including merozoite attachment, deformation of the RBC membrane, merozoite reorientation, formation of a high affinity interaction between the apical zone of the merozoite and the RBC surface, active entry, and finally sealing of the RBC membrane behind the intracellular parasite [1][2][3]. Invasion is generally immediately followed by a period of transient RBC echinocytosis, a morphological transformation of the RBC surface into an undulated or 'spiky' appearance, although this can also be induced under certain conditions even in the absence of successful invasion [3][4][5]. Entry into the host cell occurs concomitantly with formation of a membrane-bound parasitophorous vacuole (PV) within which the invading parasite comes to rest.…”

Section: Introductionmentioning

confidence: 99%

The Plasmodium falciparum rhoptry bulb protein RAMA plays an essential role in rhoptry neck morphogenesis and host red blood cell invasion

et al. 2019

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The malaria parasite Plasmodium falciparum invades, replicates within and destroys red blood cells in an asexual blood stage life cycle that is responsible for clinical disease and crucial for parasite propagation. Invasive malaria merozoites possess a characteristic apical complex of secretory organelles that are discharged in a tightly controlled and highly regulated order during merozoite egress and host cell invasion. The most prominent of these organelles, the rhoptries, are twinned, club-shaped structures with a body or bulb region that tapers to a narrow neck as it meets the apical prominence of the merozoite. Different protein populations localise to the rhoptry bulb and neck, but the function of many of these proteins and how they are spatially segregated within the rhoptries is unknown. Using conditional disruption of the gene encoding the only known glycolipid-anchored malarial rhoptry bulb protein, rhoptry-associated membrane antigen (RAMA), we demonstrate that RAMA is indispensable for blood stage parasite survival. Contrary to previous suggestions, RAMA is not required for trafficking of all rhoptry bulb proteins. Instead, RAMA-null parasites display selective mislocalisation of a subset of rhoptry bulb and neck proteins (RONs) and produce dysmorphic rhoptries that lack a distinct neck region. The mutant parasites undergo normal intracellular development and egress but display a fatal defect in invasion and do not induce echinocytosis in target red blood cells. Our results indicate that distinct pathways regulate biogenesis of the two main rhoptry sub-compartments in the malaria parasite. Author summaryDespite improved control measures over recent decades, malaria is still a considerable health burden across much of the globe. The disease is caused by a single-celled parasite PLOS Pathogens | https://doi.

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Section: Introductionmentioning

confidence: 99%

The Plasmodium falciparum rhoptry bulb protein RAMA plays an essential role in rhoptry neck morphogenesis and host red blood cell invasion

et al. 2019

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“…Of the PKG inhibitors used for synchronization of parasite cultures, Compound 2 has been the most commonly used [14, 38, 42, 44, 45, 54, 55, 61]. However, the results shown here indicate that ML10 has advantages over Compound 2.…”

Section: Discussionmentioning

confidence: 81%

Use of a highly specific kinase inhibitor for rapid, simple and precise synchronization ofPlasmodium falciparumandPlasmodium knowlesiasexual stage parasites

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Thomas

Nofal

et al. 2020

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During the course of the asexual erythrocytic stage of development, Plasmodium spp. parasites undergo a series of morphological changes and induce alterations in the host cell. At the end of this stage, the parasites exit the host cell, after which the progeny invade a new host cell. These processes are rapid and occur in a time-dependent manner. Of particular importance, egress and invasion of erythrocytes by the parasite are difficult to capture in an unsynchronized culture, or even a culture that has been synchronized to within hours. Therefore, precise synchronization of parasite cultures is of paramount importance for the investigation of these processes. Here we describe a method for synchronizing Plasmodium falciparum and Plasmodium knowlesi asexual blood stage parasites with ML10, a highly specific inhibitor of the cGMP-dependent protein kinase (PKG) that arrests parasite growth approximately 15 minutes prior to egress. This inhibitor allows parasite cultures to be synchronized to within minutes, with a simple wash step. Furthermore, we show that parasites remain viable for several hours after becoming arrested by the compound and that ML10 has advantages over the previously used PKG inhibitor Compound 2. Here, we demonstrate that ML10 is an invaluable tool for the study of Plasmodium spp. asexual blood stage biology and for the routine synchronization of P. falciparum and P. knowlesi cultures.

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“…These 500 protrusions may therefore be merozoites that have differentiated into ring-stage parasites on 501 the outside of the RBC as newly invaded merozoites often produce mobile, pseudopodial 502 extensions within the iRBC prior to ring differentiation which can be observed in a normal 503 invasion event in Supplementary Video 3. Live cell microscopy of failed invasion events 504 have not been previously described to form protrusions as recently observed with PKA and 505 adenylate cyclase beta gene disruptions (69). Further support that MMV020291 treatment 506 causes unsuccessful merozoites to differentiate into rings is that there was no significant 507 difference observed between the time taken for pseudopod formation to become visible on 508 the surface of MMV020291 treated RBCs compared to normally invaded RBCs.…”

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confidence: 78%

Screening the Medicines for Malaria Venture Pathogen Box for invasion and egress inhibitors of the blood stage of Plasmodium falciparum reveals several inhibitory compounds

Dans

Weiss

Wilson

et al. 2019

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To identify potential inhibitors of egress and invasion in the asexual blood stage of Plasmodium falciparum, we screened the Medicines for Malaria Venture (MMV) Pathogen Box. This compound library comprises of 400 drugs against neglected tropical diseases, including 125 with antimalarial activity. For this screen, we utilised transgenic parasites expressing a bioluminescent reporter, Nanoluciferase (Nluc), to measure inhibition of parasite egress and invasion in the presence of the Pathogen Box compounds. At a concentration of 2 µM, we found 15 compounds that inhibited parasite egress by >40% and 24 invasion-specific compounds that inhibited invasion by >90%. We further characterised 11 of these inhibitors through cell-based assays and live cell microscopy and found two compounds that inhibited merozoite maturation in schizonts, one compound that inhibited merozoite egress, one compound that directly inhibited parasite invasion and one compound that slowed down invasion and arrested ring formation. The remaining compounds were general growth inhibitors that acted during the egress and invasion phase of the cell cycle. We found the sulfonylpiperazine, MMV020291, to be the most invasion-specific inhibitor, blocking successful merozoite internalisation within human RBCs and having no substantial effect on other stages of the cell cycle. This has greater implications for the possible development of an invasion-specific inhibitor as an antimalarial in a combination based therapy, in addition to being a useful tool for studying the biology of the invading parasite.ImportancePlasmodium falciparum causes the most severe form of malaria and with emerging resistance to frontline treatments, there is the need to identify new drug targets in the parasite. One of the most critical processes during the asexual blood stage in the parasite’s lifecycle is the egress from old red blood cells (RBCs) and subsequent invasion of new RBCs. Many unique parasite ligands, receptors and enzymes are employed during egress and invasion that are essential for parasite proliferation and survival, therefore making these processes druggable targets. Identifying novel compounds that inhibit these essential processes would further their development into possible antimalarials that would be highly effective at killing asexual RBC stage parasites when used in combination with drugs that target the intraerythrocytic growth phase. These compounds potentially may also be used as novel tools to study the complex biology of parasites to gain further insight into the mechanisms behind egress and invasion.

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Cyclic AMP signalling controls key components of malaria parasite host cell invasion machinery

Cited by 79 publications

References 69 publications

The Plasmodium falciparum rhoptry bulb protein RAMA plays an essential role in rhoptry neck morphogenesis and host red blood cell invasion

The Plasmodium falciparum rhoptry bulb protein RAMA plays an essential role in rhoptry neck morphogenesis and host red blood cell invasion

Use of a highly specific kinase inhibitor for rapid, simple and precise synchronization ofPlasmodium falciparumandPlasmodium knowlesiasexual stage parasites

Screening the Medicines for Malaria Venture Pathogen Box for invasion and egress inhibitors of the blood stage of Plasmodium falciparum reveals several inhibitory compounds

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