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

mBio ◽  
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.

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

2020 ◽  
Author(s):  
Stephanie D. Nofal ◽  
Avnish Patel ◽  
Michael J. Blackman ◽  
Christian Flueck ◽  
David A. Baker
Keyword(s):  
Plasmodium Falciparum ◽  
Guanylyl Cyclase ◽  
Blood Stage ◽  
Primary Role ◽  
Atpase Domain ◽  
Asexual Blood Stage ◽  
Dependent Protein ◽  
Point Mutagenesis ◽  
Cgmp Synthesis

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.

scholarly journals The Structure of the Cysteine-Rich Domain of Plasmodium falciparum P113 Identifies the Location of the RH5 Binding Site

mBio ◽  
2020 ◽  
Vol 11 (5) ◽  
Author(s):  
Ivan Campeotto ◽  
Francis Galaway ◽  
Shahid Mehmood ◽  
Lea K. Barfod ◽  
Doris Quinkert ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Binding Site ◽  
Structural Information ◽  
Blood Stage ◽  
Site Directed Mutagenesis ◽  
Effective Vaccine ◽  
Binding Region ◽  
Fab Fragment ◽  
Content Type ◽  
Blood Stage Malaria

ABSTRACT Plasmodium falciparum RH5 is a secreted parasite ligand that is essential for erythrocyte invasion through direct interaction with the host erythrocyte receptor basigin. RH5 forms a tripartite complex with two other secreted parasite proteins, CyRPA and RIPR, and is tethered to the surface of the parasite through membrane-anchored P113. Antibodies against RH5, CyRPA, and RIPR can inhibit parasite invasion, suggesting that vaccines containing these three components have the potential to prevent blood-stage malaria. To further explore the role of the P113-RH5 interaction, we selected monoclonal antibodies against P113 that were either inhibitory or noninhibitory for RH5 binding. Using a Fab fragment as a crystallization chaperone, we determined the crystal structure of the RH5 binding region of P113 and showed that it is composed of two domains with structural similarities to rhamnose-binding lectins. We identified the RH5 binding site on P113 by using a combination of hydrogen-deuterium exchange mass spectrometry and site-directed mutagenesis. We found that a monoclonal antibody to P113 that bound to this interface and inhibited the RH5-P113 interaction did not inhibit parasite blood-stage growth. These findings provide further structural information on the protein interactions of RH5 and will be helpful in guiding the development of blood-stage malaria vaccines that target RH5. IMPORTANCE Malaria is a deadly infectious disease primarily caused by the parasite Plasmodium falciparum. It remains a major global health problem, and there is no highly effective vaccine. A parasite protein called RH5 is centrally involved in the invasion of host red blood cells, making it—and the other parasite proteins it interacts with—promising vaccine targets. We recently identified a protein called P113 that binds RH5, suggesting that it anchors RH5 to the parasite surface. In this paper, we use structural biology to locate and characterize the RH5 binding region on P113. These findings will be important to guide the development of new antimalarial vaccines to ultimately prevent this disease, which affects some of the poorest people on the planet.

scholarly journals Bacterially Expressed Full-Length Recombinant Plasmodium falciparum RH5 Protein Binds Erythrocytes and Elicits Potent Strain-Transcending Parasite-Neutralizing Antibodies

2013 ◽  
Vol 82 (1) ◽  
pp. 152-164 ◽  
Author(s):  
K. Sony Reddy ◽  
Alok K. Pandey ◽  
Hina Singh ◽  
Tajali Sahar ◽  
Amlabu Emmanuel ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Neutralizing Antibodies ◽  
Malaria Vaccine ◽  
Expression System ◽  
Full Length ◽  
Blood Stage ◽  
Parasite Protein ◽  
Content Type ◽  
Erythrocyte Binding ◽  
Blood Stage Malaria

ABSTRACTPlasmodium falciparumreticulocyte binding-like homologous protein 5 (PfRH5) is an essential merozoite ligand that binds with its erythrocyte receptor, basigin. PfRH5 is an attractive malaria vaccine candidate, as it is expressed by a wide number ofP. falciparumstrains, cannot be genetically disrupted, and exhibits limited sequence polymorphisms. Viral vector-induced PfRH5 antibodies potently inhibited erythrocyte invasion. However, it has been a challenge to generate full-length recombinant PfRH5 in a bacterial-cell-based expression system. In this study, we have produced full-length recombinant PfRH5 inEscherichia colithat exhibits specific erythrocyte binding similar to that of the native PfRH5 parasite protein and also, importantly, elicits potent invasion-inhibitory antibodies against a number ofP. falciparumstrains. Antibasigin antibodies blocked the erythrocyte binding of both native and recombinant PfRH5, further confirming that they bind with basigin. We have thus successfully produced full-length PfRH5 as a functionally active erythrocyte binding recombinant protein with a conformational integrity that mimics that of the native parasite protein and elicits potent strain-transcending parasite-neutralizing antibodies.P. falciparumhas the capability to develop immune escape mechanisms, and thus, blood-stage malaria vaccines that target multiple antigens or pathways may prove to be highly efficacious. In this regard, antibody combinations targeting PfRH5 and other key merozoite antigens produced potent additive inhibition against multiple worldwideP. falciparumstrains. PfRH5 was immunogenic when immunized with other antigens, eliciting potent invasion-inhibitory antibody responses with no immune interference. Our results strongly support the development of PfRH5 as a component of a combination blood-stage malaria vaccine.

scholarly journals Shedding Light on the Role of the Skin in Vaccine-Induced Protection against the Malaria Sporozoite

mBio ◽  
2018 ◽  
Vol 9 (6) ◽  
Author(s):  
Johanna Patricia Daily
Keyword(s):  
Plasmodium Falciparum ◽  
Blood Vessels ◽  
Blood Stream ◽  
Mosquito Vectors ◽  
Protective Mechanisms ◽  
Partial Protection ◽  
Content Type ◽  
Shed Light ◽  
In Infants And Children

ABSTRACT The most advanced vaccine against Plasmodium falciparum malaria, RTS,S/AS01, provides partial protection in infants and children living in areas of malaria endemicity. Further understanding its mechanisms of protection may allow the development of improved second-generation vaccines. The RTS,S/AS01 vaccine targets the sporozoites injected by mosquito vectors into the dermis which then travel into the blood stream to establish infection in the liver. Flores-Garcia et al. (Y. Flores-Garcia, G. Nasir, C. S. Hopp, C. Munoz, et al., mBio 9:e02194-18, 2018, https://doi.org/10.1128/mBio.02194-18) shed light on early protective responses occurring in the dermis in immunized animals. They demonstrated that immunization impairs sporozoite motility and entry into blood vessels. Furthermore, they established that challenge experiments performed using a dermal route conferred greater protection than intravenous challenge in immunized mice. Thus, the dermal challenge approach captures the additional protective mechanisms occurring in the dermis that reflect the natural physiology of infection. Those studies highlighted the fascinating biology of skin-stage sporozoites and provided additional insights into vaccine-induced protection.

scholarly journals Combined Transcriptome and Proteome Profiling for Role of pfEMP1 in Antimalarial Mechanism of Action of Dihydroartemisinin

2021 ◽  
Author(s):  
Lina Chen ◽  
Zhongyuan Zheng ◽  
Hui Liu ◽  
Xi Wang ◽  
Shuiqing Qu ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Mechanism Of Action ◽  
Blood Cells ◽  
Artemisinin Derivative ◽  
Combination Therapies ◽  
Proteomic Profiling ◽  
First Line ◽  
Content Type ◽  
Artemisinin Combination Therapies

Malaria parasites induce morphological and biochemical changes in the membranes of parasite-infected red blood cells (iRBCs) for propagation, with artemisinin combination therapies as the first-line treatments. To understand whether artemisinin targets or interacts with iRBC membrane proteins, this study investigated the molecular changes caused by dihydroartemisinin (DHA), an artemisinin derivative, in Plasmodium falciparum 3D7 using a combined transcriptomic and membrane proteomic profiling approach.

C-type natriuretic peptide inhibits ANP secretion and atrial dynamics in perfused atria: NPR-B-cGMP signaling

2000 ◽  
Vol 278 (1) ◽  
pp. H208-H221 ◽  
Author(s):  
Sook Jeong Lee ◽  
Sung Zoo Kim ◽  
Xun Cui ◽  
Suhn Hee Kim ◽  
Kyung Sun Lee ◽  
...  
Keyword(s):  
Stroke Volume ◽  
Natriuretic Peptide ◽  
Rank Order ◽  
Extracellular Fluid ◽  
Atrial Pacing ◽  
Atrial Myocytes ◽  
Cgmp Production ◽  
Cgmp Signaling ◽  
Intracellular Ca

The purpose of the present experiments was to define the role of C-type natriuretic peptide (CNP) in the regulation of atrial secretion of atrial natriuretic peptide (ANP) and atrial stroke volume. Experiments were performed in perfused beating and nonbeating quiescent atria, single atrial myocytes, and atrial membranes. CNP suppressed in a dose-related fashion the increase in atrial stroke volume and ANP secretion induced by atrial pacing. CNP caused a right shift in the positive relationships between changes in the secretion of ANP and atrial stroke volume or translocation of the extracellular fluid (ECF), which indicates the suppression of atrial myocytic release of ANP into the paracellular space. The effects of CNP on the secretion and contraction were mimicked by 8-bromoguanosine 3′,5′-cyclic monophosphate (8-BrcGMP). CNP increased cGMP production in the perfused atria, and the effects of CNP on the secretion of ANP and atrial dynamics were accentuated by pretreatment with an inhibitor of cGMP phosphodiesterase, zaprinast. An inhibitor of the biological natriuretic peptide receptor (NPR), HS-142-1, attenuated the effects of CNP. The suppression of ANP secretion by CNP and 8-BrcGMP was abolished by a depletion of extracellular Ca2+ in nonbeating atria. Natriuretic peptides increased cGMP production in atrial membranes with a rank order of potency of CNP > BNP > ANP, and the effect was inhibited by HS-142-1. CNP and 8-BrcGMP increased intracellular Ca2+ concentration transients in single atrial myocytes, and mRNAs for CNP and NPR-B were expressed in the rabbit atrium. From these results we conclude that atrial ANP release and stroke volume are controlled by CNP via NPR-B-cGMP mediated signaling, which may in turn act via regulation of intracellular Ca2+.

scholarly journals A Single-Dose Combination Study with the Experimental Antimalarials Artefenomel and DSM265 To Determine Safety and Antimalarial Activity against Blood-Stage Plasmodium falciparum in Healthy Volunteers

2019 ◽  
Vol 64 (1) ◽  
Author(s):  
James S. McCarthy ◽  
Thomas Rückle ◽  
Suzanne L. Elliott ◽  
Emma Ballard ◽  
Katharine A. Collins ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Antimalarial Activity ◽  
Plasma Concentrations ◽  
Parasite Clearance ◽  
Blood Stage ◽  
Content Type ◽  
Dose Combination ◽  
Combination Study ◽  
Study Support ◽  
New Compounds

ABSTRACT Artefenomel and DSM265 are two new compounds that have been shown to be well tolerated and effective when administered as monotherapy malaria treatment. This study aimed to determine the safety, pharmacokinetics, and pharmacodynamics of artefenomel and DSM265 administered in combination to healthy subjects in a volunteer infection study using the Plasmodium falciparum-induced blood-stage malaria model. Thirteen subjects were inoculated with parasite-infected erythrocytes on day 0 and received a single oral dose of artefenomel and DSM265 on day 7. Cohort 1 (n = 8) received 200 mg artefenomel plus 100 mg DSM265, and cohort 2 (n = 5) received 200 mg artefenomel plus 50 mg DSM265. Blood samples were collected to measure parasitemia, gametocytemia, and artefenomel-DSM265 plasma concentrations. There were no treatment-related adverse events. The pharmacokinetic profiles of artefenomel and DSM265 were similar to those of the compounds when administered as monotherapy, suggesting no pharmacokinetic interactions. A reduction in parasitemia occurred in all subjects following treatment (log10 parasite reduction ratios over 48 h [PRR48] of 2.80 for cohort 1 and 2.71 for cohort 2; parasite clearance half-lives of 5.17 h for cohort 1 and 5.33 h for cohort 2). Recrudescence occurred in 5/8 subjects in cohort 1 between days 19 and 28 and in 5/5 subjects in cohort 2 between days 15 and 22. Low-level gametocytemia (1 to 330 female gametocytes/ml) was detected in all subjects from day 14. The results of this single-dosing combination study support the further clinical development of the use of artefenomel and DSM265 in combination as a treatment for falciparum malaria. (This study has been registered at ClinicalTrials.gov under identifier NCT02389348.)

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

Nature ◽  
2007 ◽  
Vol 446 (7131) ◽  
pp. 88-91 ◽  
Author(s):  
Heather J. Painter ◽  
Joanne M. Morrisey ◽  
Michael W. Mather ◽  
Akhil B. Vaidya
Keyword(s):  
Plasmodium Falciparum ◽  
Electron Transport ◽  
Blood Stage ◽  
Specific Role

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

2013 ◽  
Vol 57 (12) ◽  
pp. 6050-6062 ◽  
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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