scholarly journals Assessment of biological role and insight into druggability of the Plasmodium falciparum protease plasmepsin V

2018 ◽  
Author(s):  
Alexander J. Polino ◽  
S. Nasamu Armiyaw ◽  
Jacquin C. Niles ◽  
Daniel E. Goldberg
Keyword(s):  
Plasmodium Falciparum ◽  
Host Cell ◽  
Drug Targets ◽  
Parasite Growth ◽  
Aspartic Protease ◽  
Protein Export ◽  
New Drugs ◽  
Functional Studies ◽  
Parasite Survival ◽  
Post Transcriptional Regulation

AbstractUpon infection of a red blood cell (RBC), the malaria parasite Plasmodium falciparum drastically remodels its host by exporting hundreds of proteins into the RBC cytosol. This program of protein export is essential for parasite survival, hence there is interest in export-related proteins as potential drug targets. One proposed target is plasmepsin V (PMV), an aspartic protease that cleaves export-destined proteins in the parasite ER at a motif called the Plasmodium export element (PEXEL). This cleavage is essential for effector export across the vacuolar membrane. Despite long-standing interest in PMV, functional studies have been hindered by the failure of current technologies to produce a regulatable lethal depletion of PMV. To overcome this technical barrier, we designed a facile system for stringent post-transcriptional regulation, allowing a tightly controlled, tunable knockdown of PMV. Under maximal knockdown conditions, parasite growth was arrested, validating PMV as essential for parasite survival in RBCs. We found that PMV levels had to be dramatically depleted to affect parasite growth, suggesting that the parasite maintains this enzyme in substantial excess. This has important implications for antimalarial development. Additionally, we found that PMV-depleted parasites arrest immediately after invasion of the host cell, suggesting that PMV has an unappreciated role in early development that is distinct from its previously reported role in protein export in later-stage parasites.ImportanceMalaria is endemic to large swaths of the developing world, causing nearly 500,000 deaths each year. While infection can be treated with antimalarial drugs, resistance continues to emerge to frontline antimalarials, spurring calls for new drugs and targets to feed the drug development pipeline. One proposed target is the aspartic protease plasmepsin V (PMV) that processes exported proteins, enabling the export program that remodels the host cell. This work uses facile genetic tools to produce lethal depletion of PMV, validating it as a drug target and showing that PMV is made in substantial excess in blood-stage parasites. Unexpectedly, PMV depletion leads to parasite death immediately after invasion of RBCs, distinct from other disruptions of the export pathway. This suggests that PMV inhibitors could lead to relatively rapid parasite death, and that PMV has additional unexplored role(s) during RBC infection.

scholarly journals The Exported Chaperone PfHsp70x Is Dispensable for the Plasmodium falciparum Intraerythrocytic Life Cycle

mSphere ◽  
2017 ◽  
Vol 2 (5) ◽  
Author(s):  
David W. Cobb ◽  
Anat Florentin ◽  
Manuel A. Fierro ◽  
Michelle Krakowiak ◽  
Julie M. Moore ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Life Cycle ◽  
Host Cell ◽  
Protein Trafficking ◽  
Human Disease ◽  
Clinical Manifestations ◽  
Parasite Protein ◽  
Content Type ◽  
Parasite Survival ◽  
Outstanding Question

ABSTRACT Half of the world’s population lives at risk for malaria. The intraerythrocytic life cycle of Plasmodium spp. is responsible for clinical manifestations of malaria; therefore, knowledge of the parasite’s ability to survive within the erythrocyte is needed to combat the deadliest agent of malaria, P. falciparum. An outstanding question in the field is how P. falciparum undertakes the essential process of trafficking its proteins within the host cell. In most organisms, chaperones such as Hsp70 are employed in protein trafficking. Of the Plasmodium species causing human disease, the chaperone PfHsp70x is unique to P. falciparum, and it is the only parasite protein of its kind exported to the host (S. Külzer et al., Cell Microbiol 14:1784–1795, 2012). This has placed PfHsp70x as an ideal target to inhibit protein trafficking and kill the parasite. However, we show that PfHsp70x is not required for export of parasite effectors and it is not essential for parasite survival inside the RBC. Export of parasite proteins into the host erythrocyte is essential for survival of Plasmodium falciparum during its asexual life cycle. While several studies described key factors within the parasite that are involved in protein export, the mechanisms employed to traffic exported proteins within the host cell are currently unknown. Members of the Hsp70 family of chaperones, together with their Hsp40 cochaperones, facilitate protein trafficking in other organisms, and are thus likely used by P. falciparum in the trafficking of its exported proteins. A large group of Hsp40 proteins is encoded by the parasite and exported to the host cell, but only one Hsp70, P. falciparum Hsp70x (PfHsp70x), is exported with them. PfHsp70x is absent in most Plasmodium species and is found only in P. falciparum and closely related species that infect apes. Herein, we have utilized clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 genome editing in P. falciparum to investigate the essentiality of PfHsp70x. We show that parasitic growth was unaffected by knockdown of PfHsp70x using both the dihydrofolate reductase (DHFR)-based destabilization domain and the glmS ribozyme system. Similarly, a complete gene knockout of PfHsp70x did not affect the ability of P. falciparum to proceed through its intraerythrocytic life cycle. The effect of PfHsp70x knockdown/knockout on the export of proteins to the host red blood cell (RBC), including the critical virulence factor P. falciparum erythrocyte membrane protein 1 (PfEMP1), was tested, and we found that this process was unaffected. These data show that although PfHsp70x is the sole exported Hsp70, it is not essential for the asexual development of P. falciparum. IMPORTANCE Half of the world’s population lives at risk for malaria. The intraerythrocytic life cycle of Plasmodium spp. is responsible for clinical manifestations of malaria; therefore, knowledge of the parasite’s ability to survive within the erythrocyte is needed to combat the deadliest agent of malaria, P. falciparum. An outstanding question in the field is how P. falciparum undertakes the essential process of trafficking its proteins within the host cell. In most organisms, chaperones such as Hsp70 are employed in protein trafficking. Of the Plasmodium species causing human disease, the chaperone PfHsp70x is unique to P. falciparum, and it is the only parasite protein of its kind exported to the host (S. Külzer et al., Cell Microbiol 14:1784–1795, 2012). This has placed PfHsp70x as an ideal target to inhibit protein trafficking and kill the parasite. However, we show that PfHsp70x is not required for export of parasite effectors and it is not essential for parasite survival inside the RBC.

scholarly journals The Effect of Aqueous Extract of Cinnamon on the Metabolome ofPlasmodium falciparumUsing1HNMR Spectroscopy

2016 ◽  
Vol 2016 ◽  
pp. 1-5 ◽  
Author(s):  
Shirin Parvazi ◽  
Sedigheh Sadeghi ◽  
Mehri Azadi ◽  
Maryam Mohammadi ◽  
Mohammad Arjmand ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Aqueous Extract ◽  
Drug Targets ◽  
Inhibitory Concentration ◽  
Biological Fluids ◽  
Medicinal Herbs ◽  
New Drugs ◽  
Lysine Biosynthesis ◽  
Glutathione Metabolism

Malaria is responsible for estimated 584,000 deaths in 2013. Researchers are working on new drugs and medicinal herbs due to drug resistance that is a major problem facing them; the search is on for new medicinal herbs. Cinnamon is the bark of a tree with reported antiparasitic effects. Metabonomics is the simultaneous study of all the metabolites in biological fluids, cells, and tissues detected by high throughput technology. It was decided to determine the mechanism of the effect of aqueous extract of cinnamon on the metabolome ofPlasmodium falciparum in vitrousing1HNMR spectroscopy. Prepared aqueous extract of cinnamon was added to a culture ofPlasmodium falciparum3D7 and its 50% inhibitory concentration determined, and, after collection, their metabolites were extracted and1HNMR spectroscopy by NOESY method was done. The spectra were analyzed by chemometric methods. The differentiating metabolites were identified using Human Metabolome Database and the metabolic cycles identified by Metaboanalyst. 50% inhibitory concentration of cinnamon onPlasmodium falciparumwas 1.25 mg/mL withp<0.001. The metabolites were identified as succinic acid, glutathione, L-aspartic acid, beta-alanine, and 2-methylbutyryl glycine. The main metabolic cycles detected were alanine and aspartame and glutamate pathway and pantothenate and coenzyme A biosynthesis and lysine biosynthesis and glutathione metabolism, which are all important as drug targets.

scholarly journals Artemisinin activity-based probes identify multiple molecular targets within the asexual stage of the malaria parasites Plasmodium falciparum 3D7

2016 ◽  
Vol 113 (8) ◽  
pp. 2080-2085 ◽  
Author(s):  
Hanafy M. Ismail ◽  
Victoria Barton ◽  
Matthew Phanchana ◽  
Sitthivut Charoensutthivarakul ◽  
Michael H. L. Wong ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Drug Targets ◽  
Antioxidant Defense ◽  
Protein Profiling ◽  
Parasite Protein ◽  
Know How ◽  
Parasite Survival ◽  
Hemoglobin Degradation ◽  
Insight Into

The artemisinin (ART)-based antimalarials have contributed significantly to reducing global malaria deaths over the past decade, but we still do not know how they kill parasites. To gain greater insight into the potential mechanisms of ART drug action, we developed a suite of ART activity-based protein profiling probes to identify parasite protein drug targets in situ. Probes were designed to retain biological activity and alkylate the molecular target(s) of Plasmodium falciparum 3D7 parasites in situ. Proteins tagged with the ART probe can then be isolated using click chemistry before identification by liquid chromatography–MS/MS. Using these probes, we define an ART proteome that shows alkylated targets in the glycolytic, hemoglobin degradation, antioxidant defense, and protein synthesis pathways, processes essential for parasite survival. This work reveals the pleiotropic nature of the biological functions targeted by this important class of antimalarial drugs.

scholarly journals Knockdown of the translocon protein EXP2, reduces growth and protein export in malaria parasites

2018 ◽  
Author(s):  
Sarah C. Charnaud ◽  
Rasika Kumarasingha ◽  
Hayley E. Bullen ◽  
Brendan S. Crabb ◽  
Paul R. Gilson
Keyword(s):  
Parasite Growth ◽  
Protein Export ◽  
Cell Protein ◽  
Host Cell Protein ◽  
Growth Reduction ◽  
Malaria Parasites ◽  
Asexual Blood Stage ◽  
Parasite Survival ◽  
Core Components ◽  
Exported Protein

AbstractMalaria parasites remodel their host erythrocytes to gain nutrients and avoid the immune system. Host erythrocytes are modified by hundreds of effectors proteins exported from the parasites into the host cell. Protein export is mediated by the PTEX translocon comprising five core components of which EXP2 is considered to form the putative pore that spans the vacuole membrane enveloping the parasite within its erythrocyte. To explore the function and importance of EXP2 for parasite survival in the asexual blood stage of Plasmodium falciparum we inducibly knocked down the expression of EXP2. Reduction in EXP2 expression strongly reduced parasite growth proportional to the degree of protein knockdown and tended to stall development about half way through the asexual cell cycle. Once the knockdown inducer was removed and EXP2 expression restored, parasite growth recovered dependent upon the length and degree of knockdown. To establish EXP2 function and hence the basis for growth reduction, the trafficking of an exported protein was monitored following EXP2 knockdown. This resulted in severe attenuation of protein export and is consistent with EXP2, and PTEX in general, being the conduit for export of proteins into the host compartment.

scholarly journals Plasmodium falciparum parasites deploy RhopH2 into the host erythrocyte to obtain nutrients, grow and replicate

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Natalie A Counihan ◽  
Scott A Chisholm ◽  
Hayley E Bullen ◽  
Anubhav Srivastava ◽  
Paul R Sanders ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
De Novo ◽  
Parasite Growth ◽  
Toxic Waste ◽  
De Novo Synthesis ◽  
Parasite Survival ◽  
Cell Remodeling ◽  
Causative Agents ◽  
New Permeability Pathways ◽  
Rhoptry Protein

Plasmodium falciparum parasites, the causative agents of malaria, modify their host erythrocyte to render them permeable to supplementary nutrient uptake from the plasma and for removal of toxic waste. Here we investigate the contribution of the rhoptry protein RhopH2, in the formation of new permeability pathways (NPPs) in Plasmodium-infected erythrocytes. We show RhopH2 interacts with RhopH1, RhopH3, the erythrocyte cytoskeleton and exported proteins involved in host cell remodeling. Knockdown of RhopH2 expression in cycle one leads to a depletion of essential vitamins and cofactors and decreased de novo synthesis of pyrimidines in cycle two. There is also a significant impact on parasite growth, replication and transition into cycle three. The uptake of solutes that use NPPs to enter erythrocytes is also reduced upon RhopH2 knockdown. These findings provide direct genetic support for the contribution of the RhopH complex in NPP activity and highlight the importance of NPPs to parasite survival.

scholarly journals Using Lipoamidase as a Novel Probe To Interrogate the Importance of Lipoylation in Plasmodium falciparum

mBio ◽  
2018 ◽  
Vol 9 (6) ◽  
Author(s):  
Hugo Jhun ◽  
Maroya S. Walters ◽  
Sean T. Prigge
Keyword(s):  
Plasmodium Falciparum ◽  
Parasite Growth ◽  
Malaria Parasites ◽  
Subcellular Compartment ◽  
Content Type ◽  
Growth And Survival ◽  
Bacterial Enzyme ◽  
Parasite Survival ◽  
Key Aspects ◽  
Insight Into

ABSTRACT Lipoate is a redox active cofactor that is covalently bound to key enzymes of oxidative metabolism. Plasmodium falciparum is auxotrophic for lipoate during the intraerythrocytic stages, but it is not known whether lipoate attachment to protein is required or whether attachment is required in a specific subcellular compartment of the parasite. To address these questions, we used an enzyme called lipoamidase (Lpa) as a probe of lipoate metabolism. Lpa was first described in Enterococcus faecalis, and it specifically cleaves protein-bound lipoate, inactivating enzymes requiring this cofactor. Enzymatically active Lpa could be expressed in the cytosol of P. falciparum without any effect on protein lipoylation or parasite growth. Similarly, Lpa could be expressed in the apicoplast, and although protein lipoylation was reduced, parasite growth was not inhibited. By contrast, while an inactive mutant of Lpa could be expressed in the mitochondrion, the active enzyme could not. We designed an attenuated mutant of Lpa and found that this enzyme could be expressed in the parasite mitochondrion, but only in conjunction with a chemical bypass system. These studies suggest that acetyl-CoA production and a cryptic function of the H protein are both required for parasite survival. Our study validates Lpa as a novel probe of metabolism that can be used in other systems and provides new insight into key aspects of mitochondrial metabolism that are responsible for lipoate auxotrophy in malaria parasites. IMPORTANCE Lipoate is an essential cofactor for a small number of enzymes that are important for central metabolism. Malaria parasites require lipoate scavenged from the human host for growth and survival; however, it is not known why this cofactor is so important. To address this question, we designed a probe of lipoate activity based on the bacterial enzyme lipoamidase (Lpa). Expression of this probe in different subcellular locations allowed us to define the mitochondrion as the compartment housing essential lipoate metabolism. To gain further insight into the specific uses of lipoate in the mitochondrion, we designed a series of catalytically attenuated probes and employed the probes in conjunction with a chemical bypass system. These studies suggest that two lipoylated proteins are required for parasite survival. We were able to express Lpa with different catalytic abilities in different subcellular compartments and driven by different promoters, demonstrating the versatility of this tool and suggesting that it can be used as a probe of lipoate metabolism in other organisms.

scholarly journals An essential ER-resident N-acetyltransferase in Plasmodium falciparum

2021 ◽  
Author(s):  
Alexander J Polino ◽  
Katherine Floyd ◽  
Yolotzin Avila-Cruz ◽  
Yujuan Yang ◽  
Daniel E Goldberg
Keyword(s):  
Plasmodium Falciparum ◽  
Malaria Parasite ◽  
Protein Modification ◽  
Essential Role ◽  
Parasite Growth ◽  
Protein Export ◽  
Development Cycle ◽  
Parasite Plasmodium ◽  
Parasite Plasmodium Falciparum ◽  
Acetyl Group

N-terminal acetylation is a common eukaryotic protein modification that involves the addition of an acetyl group to the N-terminus of a polypeptide. This modification is largely performed by cytosolic N-terminal acetyltransferases (NATs). Most associate with the ribosome, acetylating nascent polypeptides co-translationally. In the malaria parasite Plasmodium falciparum, exported effectors are translated into the ER, processed by the aspartic protease plasmepsin V and then N-acetylated, despite having no clear access to cytosolic NATs. Here, we used post-transcriptional knockdown to investigate the most obvious candidate, Pf3D7_1437000. We found that it co-localizes with the ER-resident plasmepsin V and is required for parasite growth. However, depletion of Pf3D7_1437000 had no effect on protein export or acetylation of the exported proteins HRP2 and HRP3. Pf3D7_1437000-depleted parasites arrested later in their development cycle than export-blocked parasites, suggesting the protein's essential role is distinct from protein export.

scholarly journals Involvement of δ-aminolaevulinate synthase encoded by the parasite gene in de novo haem synthesis by Plasmodium falciparum

2002 ◽  
Vol 367 (2) ◽  
pp. 321-327 ◽  
Author(s):  
S. VARADHARAJAN ◽  
S. DHANASEKARAN ◽  
Z.Q. BONDAY ◽  
P.N. RANGARAJAN ◽  
G. PADMANABAN
Keyword(s):  
Plasmodium Falciparum ◽  
Enzyme Activity ◽  
Drug Target ◽  
Malaria Parasite ◽  
De Novo ◽  
Parasite Growth ◽  
Pcr Analysis ◽  
Trophozoite Stage ◽  
Parasite Survival ◽  
A Cell

The malaria parasite can synthesize haem de novo. In the present study, the expression of the parasite gene for Δ-aminolaevulinate synthase (PfALAS) has been studied by reverse transcriptase PCR analysis of the mRNA, protein expression using antibodies to the recombinant protein expressed in Escherichia coli and assay of ALAS enzyme activity in Plasmodium falciparum in culture. The gene is expressed through all stages of intra-erythrocytic parasite growth, with a small increase during the trophozoite stage. Antibodies to the erythrocyte ALAS do not cross-react with the parasite enzyme and vice versa. The recombinant enzyme activity is inhibited by ethanolamine and the latter inhibits haem synthesis in P. falciparum and growth in culture. The parasite ALAS is localized in the mitochondrion and its import into mitochondria in a cell-free import assay has been demonstrated. The import is blocked by haemin. On the basis of these results, the following conclusions are arrived at: PfALAS has distinct immunological identity and inhibitor specificity and is therefore a drug target. The malaria parasite synthesizes haem through the mitochondrion/cytosol partnership, and this assumes significance in light of the presence of apicoplasts in the parasite that may be capable of independent haem synthesis. The PfALAS gene is functional and vital for parasite haem synthesis and parasite survival.

scholarly journals In Silico Knockout Screening of Plasmodium falciparum Reactions and Prediction of Novel Essential Reactions by Analysing the Metabolic Network

2018 ◽  
Vol 2018 ◽  
pp. 1-11 ◽  
Author(s):  
Jelili Oyelade ◽  
Itunuoluwa Isewon ◽  
Efosa Uwoghiren ◽  
Olufemi Aromolaran ◽  
Olufunke Oladipupo
Keyword(s):  
Infectious Disease ◽  
Plasmodium Falciparum ◽  
Metabolic Network ◽  
Antimalarial Drug ◽  
Drug Target ◽  
Drug Targets ◽  
New Drugs ◽  
Biological Evidence ◽  
Essential Reactions ◽  
Extensive List

Malaria is an infectious disease that affects close to half a million individuals every year and Plasmodium falciparum is a major cause of malaria. The treatment of this disease could be done effectively if the essential enzymes of this parasite are specifically targeted. Nevertheless, the development of the parasite in resisting existing drugs now makes discovering new drugs a core responsibility. In this study, a novel computational model that makes the prediction of new and validated antimalarial drug target cheaper, easier, and faster has been developed. We have identified new essential reactions as potential targets for drugs in the metabolic network of the parasite. Among the top seven (7) predicted essential reactions, four (4) have been previously identified in earlier studies with biological evidence and one (1) has been with computational evidence. The results from our study were compared with an extensive list of seventy-seven (77) essential reactions with biological evidence from a previous study. We present a list of thirty-one (31) potential candidates for drug targets in Plasmodium falciparum which includes twenty-four (24) new potential candidates for drug targets.

scholarly journals Characterization of the erythrocyte GTPase Rac1 in relation to Plasmodium falciparum invasion

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Silvio Paone ◽  
Sarah D’Alessandro ◽  
Silvia Parapini ◽  
Francesco Celani ◽  
Valentina Tirelli ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Host Cell ◽  
Drug Targets ◽  
Parasitophorous Vacuole ◽  
Erythrocyte Invasion ◽  
Inhibitory Compounds ◽  
Novel Drug ◽  
Novel Drug Targets

AbstractMalaria is still a devastating disease with 228 million cases globally and 405,000 lethal outcomes in 2018, mainly in children under five years of age. The threat of emerging malaria strains resistant to currently available drugs has made the search for novel drug targets compelling. The process by which Plasmodium falciparum parasites invade the host cell has been widely studied, but only a few erythrocyte proteins involved in this process have been identified so far. The erythrocyte protein Rac1 is a GTPase that plays an important role in host cell invasion by many intracellular pathogens. Here we show that Rac1 is recruited in proximity to the site of parasite entry during P. falciparum invasion process and that subsequently localizes to the parasitophorous vacuole membrane. We also suggest that this GTPase may be involved in erythrocyte invasion by P. falciparum, by testing the effect of specific Rac1 inhibitory compounds. Finally, we suggest a secondary role of the erythrocyte GTPase also in parasite intracellular development. We here characterize a new erythrocyte protein potentially involved in P. falciparum invasion of the host cell and propose the human GTPase Rac1 as a novel and promising antimalarial drug target.

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