Plasmodium falciparum guanylyl cyclase-alpha and the activity of its appended P4-ATPase domain are essential for cGMP synthesis and blood stage egress

AbstractIn malaria parasites, guanylyl cyclases (GCs), which synthesise cyclic GMP (cGMP), are associated with a P4-ATPase-like domain in a unique bifunctional configuration. P4-ATPases generate membrane bilayer lipid asymmetry by translocating phospholipids from the outer to the inner leaflet. Here we investigate the role of Plasmodium falciparum guanylyl cyclase alpha (GCα) and its associated P4-ATPase module, showing that asexual blood stage parasites lacking both the cyclase and P4-ATPase domains are unable to egress from host erythrocytes. GCα-null parasites cannot synthesise cGMP, or mobilise calcium, a cGMP-dependent protein kinase (PKG)-driven requirement for egress. Using chemical complementation with a cGMP analogue and point mutagenesis of a crucial conserved residue within the P4-ATPase domain, we show that ATPase activity is up stream of and linked to cGMP synthesis. Collectively, our results demonstrate that GCα is a critical regulator of PKG and that its associated P4-ATPase domain plays a primary role in generating cGMP for merozoite egress.

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Plasmodium falciparum Guanylyl Cyclase-Alpha and the Activity of Its Appended P4-ATPase Domain Are Essential for cGMP Synthesis and Blood-Stage Egress

mBio ◽

10.1128/mbio.02694-20 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Stephanie D. Nofal ◽

Avnish Patel ◽

Michael J. Blackman ◽

Christian Flueck ◽

David A. Baker

Keyword(s):

Plasmodium Falciparum ◽

Cyclic Gmp ◽

Guanylyl Cyclase ◽

Blood Stage ◽

Atpase Domain ◽

Content Type ◽

Cgmp Production ◽

Cgmp Signaling ◽

Cgmp Synthesis

ABSTRACT Guanylyl cyclases (GCs) synthesize cyclic GMP (cGMP) and, together with cyclic nucleotide phosphodiesterases, are responsible for regulating levels of this intracellular messenger which mediates myriad functions across eukaryotes. In malaria parasites (Plasmodium spp), as well as their apicomplexan and ciliate relatives, GCs are associated with a P4-ATPase-like domain in a unique bifunctional configuration. P4-ATPases generate membrane bilayer lipid asymmetry by translocating phospholipids from the outer to the inner leaflet. Here, we investigate the role of Plasmodium falciparum guanylyl cyclase alpha (GCα) and its associated P4-ATPase module, showing that asexual blood-stage parasites lacking both the cyclase and P4-ATPase domains are unable to egress from host erythrocytes. GCα-null parasites cannot synthesize cGMP or mobilize calcium, a cGMP-dependent protein kinase (PKG)-driven requirement for egress. Using chemical complementation with a cGMP analogue and point mutagenesis of a crucial conserved residue within the P4-ATPase domain, we show that P4-ATPase activity is upstream of and linked to cGMP synthesis. Collectively, our results demonstrate that GCα is a critical regulator of PKG and that its associated P4-ATPase domain plays a primary role in generating cGMP for merozoite egress. IMPORTANCE The clinical manifestations of malaria arise due to successive rounds of replication of Plasmodium parasites within red blood cells. Once mature, daughter merozoites are released from infected erythrocytes to invade new cells in a tightly regulated process termed egress. Previous studies have shown that the activation of cyclic GMP (cGMP) signaling is critical for initiating egress. Here, we demonstrate that GCα, a unique bifunctional enzyme, is the sole enzyme responsible for cGMP production during the asexual blood stages of Plasmodium falciparum and is required for the cellular events leading up to merozoite egress. We further demonstrate that in addition to the GC domain, the appended ATPase-like domain of GCα is also involved in cGMP production. Our results highlight the critical role of GCα in cGMP signaling required for orchestrating malaria parasite egress.

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A patatin-like phospholipase is crucial for gametocyte induction in the malaria parasite Plasmodium falciparum

10.1101/699363 ◽

2019 ◽

Author(s):

Ansgar Flammersfeld ◽

Atscharah Panyot ◽

Yoshiki Yamaryo ◽

Philipp Auraß ◽

Jude M. Pryborski ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Malaria Parasite ◽

Blood Stage ◽

Gene Encoding ◽

Asexual Blood Stage ◽

Parasite Plasmodium ◽

Parasite Plasmodium Falciparum ◽

Eukaryotic Organisms ◽

Phospholipid Degradation

AbstractPatatin-like phospholipases (PNPLAs) are highly conserved enzymes of prokaryotic and eukaryotic organisms with major roles in lipid homeostasis. The genome of the malaria parasite Plasmodium falciparum encodes four putative PNPLAs with predicted functions during phospholipid degradation. We here investigated the role of one of the plasmodial PNPLAs, a putative PLA2 termed PNPLA1, during blood stage replication and gametocyte development. PNPLA1 is present in the asexual and sexual blood stages and here localizes to the cytoplasm. PNPLA1-deficiency due to gene disruption or conditional gene-knockdown had no effect on erythrocytic replication, gametocyte maturation and gametogenesis. However, blood stage parasites lacking PNPLA1 were severely impaired in gametocyte induction, while PNPLA1 overexpression promotes gametocyte formation. The loss of PNPLA1 further leads to transcriptional down-regulation of genes related to gametocytogenesis, including the gene encoding the sexual commitment regulator AP2-G. Additionally, lipidomics of PNPLA1-deficient asexual blood stage parasites revealed overall increased levels of major phospholipids, including phosphatidylcholine (PC), which is a substrate of PLA2. Because PC synthesis is pivotal for erythrocytic replication, while the reduced availability of PC precursors drives the parasite into gametocytogenesis, we hypothesize that the high PC levels due to PNPLA1-deficiency prevent the blood stage parasites from entering the sexual pathway.

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Faculty Opinions recommendation of Plasmodium falciparum SERA5 plays a non-enzymatic role in the malarial asexual blood-stage lifecycle.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.725319331.793503750 ◽

2015 ◽

Author(s):

Toshihiro Horii ◽

Nirianne Palacpac

Keyword(s):

Plasmodium Falciparum ◽

Blood Stage ◽

Asexual Blood Stage

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Progress towards a broadly neutralizing vaccine against the asexual blood-stage of Plasmodium falciparum

Frontiers in Immunology ◽

10.3389/conf.fimmu.2013.02.00574 ◽

2013 ◽

Vol 4 ◽

Author(s):

Douglas Alexander ◽

Williams Andrew ◽

Illingworth Joseph ◽

Hjerrild Kathryn ◽

Draper Simon

Keyword(s):

Plasmodium Falciparum ◽

Blood Stage ◽

Asexual Blood Stage

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The Malaria Parasite Cyclic GMP-Dependent Protein Kinase Plays a Central Role in Blood-Stage Schizogony

Eukaryotic Cell ◽

10.1128/ec.00186-09 ◽

2009 ◽

Vol 9 (1) ◽

pp. 37-45 ◽

Cited By ~ 123

Author(s):

Helen M. Taylor ◽

Louisa McRobert ◽

Munira Grainger ◽

Audrey Sicard ◽

Anton R. Dluzewski ◽

...

Keyword(s):

Life Cycle ◽

Protein Kinase ◽

Cyclic Gmp ◽

Malaria Parasite ◽

Kinase Inhibitor ◽

Blood Stage ◽

Parasite Life Cycle ◽

Dependent Protein Kinase ◽

Asexual Blood Stage ◽

Dependent Protein

ABSTRACT A role for the Plasmodium falciparum cyclic GMP (cGMP)-dependent protein kinase (PfPKG) in gametogenesis in the malaria parasite was elucidated previously. In the present study we examined the role of PfPKG in the asexual blood-stage of the parasite life cycle, the stage that causes malaria pathology. A specific PKG inhibitor (compound 1, a trisubstituted pyrrole) prevented the progression of P. falciparum schizonts through to ring stages in erythrocyte invasion assays. Addition of compound 1 to ring-stage parasites allowed normal development up to 30 h postinvasion, and segmented schizonts were able to form. However, synchronized schizonts treated with compound 1 for ≥6 h became large and dysmorphic and were unable to rupture or liberate merozoites. To conclusively demonstrate that the effect of compound 1 on schizogony was due to its selective action on PfPKG, we utilized genetically manipulated P. falciparum parasites expressing a compound 1-insensitive PfPKG. The mutant parasites were able to complete schizogony in the presence of compound 1 but not in the presence of the broad-spectrum protein kinase inhibitor staurosporine. This shows that PfPKG is the primary target of compound 1 during schizogony and provides direct evidence of a role for PfPKG in this process. Discovery of essential roles for the P. falciparum PKG in both asexual and sexual development demonstrates that cGMP signaling is a key regulator of both of these crucial life cycle phases and defines this molecule as an exciting potential drug target for both therapeutic and transmission blocking action against malaria.

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Use of a highly specific kinase inhibitor for rapid, simple and precise synchronization of Plasmodium falciparum and Plasmodium knowlesi asexual blood-stage parasites

PLoS ONE ◽

10.1371/journal.pone.0235798 ◽

2020 ◽

Vol 15 (7) ◽

pp. e0235798

Author(s):

Margarida Ressurreição ◽

James A. Thomas ◽

Stephanie D. Nofal ◽

Christian Flueck ◽

Robert W. Moon ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Kinase Inhibitor ◽

Plasmodium Knowlesi ◽

Blood Stage ◽

Asexual Blood Stage ◽

Specific Kinase Inhibitor

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Specific role of mitochondrial electron transport in blood-stage Plasmodium falciparum

Nature ◽

10.1038/nature05572 ◽

2007 ◽

Vol 446 (7131) ◽

pp. 88-91 ◽

Cited By ~ 303

Author(s):

Heather J. Painter ◽

Joanne M. Morrisey ◽

Michael W. Mather ◽

Akhil B. Vaidya

Keyword(s):

Plasmodium Falciparum ◽

Electron Transport ◽

Blood Stage ◽

Specific Role

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Identification of MMV Malaria Box Inhibitors of Plasmodium falciparum Early-Stage Gametocytes Using a Luciferase-Based High-Throughput Assay

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00870-13 ◽

2013 ◽

Vol 57 (12) ◽

pp. 6050-6062 ◽

Cited By ~ 72

Author(s):

Leonardo Lucantoni ◽

Sandra Duffy ◽

Sophie H. Adjalley ◽

David A. Fidock ◽

Vicky M. Avery

Keyword(s):

Plasmodium Falciparum ◽

High Throughput ◽

Early Stage ◽

Blood Stage ◽

Stage I ◽

Mosquito Vector ◽

Asexual Blood Stage ◽

Content Type ◽

Malaria Box ◽

Gametocytocidal Activity

ABSTRACTThe design of new antimalarial combinations to treatPlasmodium falciparuminfections requires drugs that, in addition to resolving disease symptoms caused by asexual blood stage parasites, can also interrupt transmission to the mosquito vector. Gametocytes, which are essential for transmission, develop as sexual blood stage parasites in the human host over 8 to 12 days and are the most accessible developmental stage for transmission-blocking drugs. Considerable effort is currently being devoted to identifying compounds active against mature gametocytes. However, investigations on the drug sensitivity of developing gametocytes, as well as screening methods for identifying inhibitors of early gametocytogenesis, remain scarce. We have developed a luciferase-based high-throughput screening (HTS) assay using tightly synchronous stage I to III gametocytes from a recombinantP. falciparumline expressing green fluorescent protein (GFP)-luciferase. The assay has been used to evaluate the early-stage gametocytocidal activity of the MMV Malaria Box, a collection of 400 compounds with known antimalarial (asexual blood stage) activity. Screening this collection against early-stage (I to III) gametocytes yielded 64 gametocytocidal compounds with 50% inhibitory concentrations (IC50s) below 2.5 μM. This assay is reproducible and suitable for the screening of large compound libraries, with an average percent coefficient of variance (%CV) of ≤5%, an average signal-to-noise ratio (S:N) of >30, and a Z′ of ∼0.8. Our findings highlight the need for screening efforts directed specifically against early gametocytogenesis and indicate the importance of experimental verification of early-stage gametocytocidal activity in the development of new antimalarial candidates for combination therapy.

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