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 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 The Plasmodium falciparum var gene switching rate, switching mechanism and patterns of parasite recrudescence described by mathematical modelling

Parasitology ◽  
2002 ◽  
Vol 124 (3) ◽  
pp. 225-235 ◽  
Author(s):  
S. PAGET-MCNICOL ◽  
M. GATTON ◽  
I. HASTINGS ◽  
A. SAUL
Keyword(s):  
Plasmodium Falciparum ◽  
Chronic Infection ◽  
Malaria Parasite ◽  
Gene Repertoire ◽  
Switching Rate ◽  
Specific Immunity ◽  
Parasite Survival ◽  
Gene Switching ◽  
Variant Antigen ◽  
Var Gene

Recrudescing Plasmodium falciparum parasitaemia is attributed to the switching of PfEMP1, a variant antigen family encoded by the var gene repertoire, and the host's immune response. We have developed a mathematical model which incorporates var gene switching, and variant specific, non-variant specific and non-specific immunity. By conducting a sensitivity analysis of the model we have defined the parameter limits which produce chronic and recrudescing infections. We explore 3 switching mechanisms: ordered, random and uncoupled switching. We show that if var genes switch on and off independently at variable rates through the repertoire a chronic clinical infection is predicted. The fastest switching-on rate that produces a chronic infection is 0·03% per generation. The model predicts that non-variant specific immunity plays an important role in reducing disease severity. This work illustrates the complex relationship between the malaria parasite and its host and shows that var gene switching at rates substantially slower than 2% are essential for parasite survival.

scholarly journals A Novel Case of Multiple Endocrine Neoplasia Type 2A Associated with Two de Novo Mutations of the RETProtooncogene*

1999 ◽  
Vol 84 (10) ◽  
pp. 3522-3527 ◽  
Author(s):  
Alessandra Tessitore ◽  
Antonio A. Sinisi ◽  
Daniela Pasquali ◽  
Monica Cardone ◽  
Domenico Vitale ◽  
...  
Keyword(s):  
Multiple Endocrine Neoplasia ◽  
De Novo ◽  
Multiple Endocrine Neoplasia Type ◽  
Pcr Analysis ◽  
De Novo Mutations ◽  
Endocrine Neoplasia ◽  
Endocrine Neoplasia Type ◽  
A Cell ◽  
Glycine Substitution ◽  
Men 2A

Abstract We report a novel case of multiple endocrine neoplasia type 2A (MEN 2A) associated with two mutations of the protooncogene RET. One affects codon 634 and causes a cysteine to arginine substitution; the second at codon 640 causes an alanine to glycine substitution in the transmembrane region. The two mutations were present on the same RET allele and were detected in germline and tumor DNA. Both mutations were de novo, i.e. they were not found in the DNA of the parents or relatives. Immunohistochemical and RT-PCR analysis showed that the pheochromocytoma expressed calcitonin as well as both RET alleles. A cell line established from the tumor and propagated in culture sustained the expression of RET and calcitonin, as did the original pheochromocytoma. Because the patient presented with medullary thyroid carcinoma and pheochromocytoma without parathyroid gland involvement, we speculate that this clinical picture could be correlated with the two RET mutations and to the unusual calcitonin production. This is the first report of a MEN 2A case due to two mutations of the RET gene and associated with a calcitonin-producing pheochromocytoma.

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 Disruption of Plasmodium falciparum histidine-rich protein II may affect haem metabolism in the blood stage

2020 ◽  
Author(s):  
Yingchao Yang ◽  
Tongke Tang ◽  
Bo Feng ◽  
Shanshan Li ◽  
Nan Hou ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Malaria Parasite ◽  
De Novo ◽  
Transcript Level ◽  
Blood Stage ◽  
Gene Transcript ◽  
Parasite Line ◽  
Genes Encoding ◽  
Comparison Groups ◽  
Transgenic Parasites

Abstract Background: Haem is a key metabolic factor in the life cycle of the malaria parasite. In the blood stage, the parasite acquires host haemoglobin to generate amino acids for protein synthesis and the by-product haem for metabolic use. The malaria parasite can also synthesize haem de novo by itself. Plasmodium falciparum-specific histidine-rich protein 2 (PfHRP2) has a haem-binding site to mediate the formation of haemozoin, a biocrystallized form of haem aggregates. Notably, the gene regulates the mechanism of haemoglobin-derived haem metabolism and the de novo haem biosynthetic pathway in the Pfhrp2-disrupted parasite line during the intraerythrocytic stages. Methods: The CRISPR/Cas9 system was used to disrupt the gene locus of Pfhrp2. DNA was extracted from the transgenic parasite, and polymerase chain reaction (PCR), Southern blotting and Western blotting were used to confirm the establishment of transgenic parasites. RNA-Seq and comparative transcriptome analysis were performed to identify differences in gene expression between 3D7 and Pfhrp2- 3D7 parasites.Results: Pfhrp2- transgenic parasites were successfully established by the CRISPR/Cas9 system. A total of 964, 1261, 3138, 1064, 2512, and 1778 differentially expressed genes (DEGs) were identified in the six comparison groups, and a total of 373, 520, 1499, 353, 1253, and 742 of the DEGs were upregulated, and 591, 741, 1639, 711, 1259, and 1036 of the DEGs were downregulated, respectively. Five DEGs related to haem metabolism and synthesis were identified in the comparison groups of six time points (0, 8, 16, 24, 32, and 40 h after merozoite invasion). The genes encoding ALAS and FC, related to haem biosynthesis, were found to be significantly upregulated in the comparison groups, and the HO, SPP, and PBGD genes were found to be significantly downregulated. No GO terms were significantly enriched in haem-related processes (Q value=1).Conclusion: Our data revealed changes in the transcriptome expression profile of the Pfhrp2-3D7 parasite during the intraerythrocytic stages. The above findings provide insight at the gene transcript level for further research and development of anti-malaria drugs.

scholarly journals Disruption of Plasmodium falciparum histidine-rich protein 2 may affect haem metabolism in the blood stage

2020 ◽  
Vol 13 (1) ◽  
Author(s):  
Yingchao Yang ◽  
Tongke Tang ◽  
Bo Feng ◽  
Shanshan Li ◽  
Nan Hou ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Malaria Parasite ◽  
De Novo ◽  
Aminolevulinic Acid ◽  
Transcript Level ◽  
Blood Stage ◽  
Gene Transcript ◽  
Parasite Line ◽  
Comparison Groups ◽  
Transgenic Parasites

Abstract Background Haem is a key metabolic factor in the life cycle of the malaria parasite. In the blood stage, the parasite acquires host haemoglobin to generate amino acids for protein synthesis and the by-product haem for metabolic use. The malaria parasite can also synthesize haem de novo on its own. Plasmodium falciparum-specific histidine-rich protein 2 (PfHRP2) has a haem-binding site to mediate the formation of haemozoin, a biocrystallized form of haem aggregates. Notably, the gene regulates the mechanism of haemoglobin-derived haem metabolism and the de novo haem biosynthetic pathway in the Pfhrp2-disrupted parasite line during the intraerythrocytic stages. Methods The CRISPR/Cas9 system was used to disrupt the gene locus of Pfhrp2. DNA was extracted from the transgenic parasite, and PCR, Southern blotting and Western blotting were used to confirm the establishment of transgenic parasites. RNA-sequencing and comparative transcriptome analysis were performed to identify differences in gene expression between 3D7 and Pfhrp2--3D7 parasites. Results Pfhrp2- transgenic parasites were successfully established by the CRISPR/Cas9 system. A total of 964, 1261, 3138, 1064, 2512 and 1778 differentially expressed genes (DEGs) were identified in the six comparison groups, respectively, with 373, 520, 1499, 353, 1253 and 742 of these DEGs upregulated and 591, 741, 1639, 711, 1259 and 1036 of them downregulated, respectively. Five DEGs related to haem metabolism and synthesis were identified in the comparison groups at six time points (0, 8, 16, 24, 32, and 40 h after merozoite invasion). The genes encoding delta-aminolevulinic acid synthetase and ferrochelatase, both related to haem biosynthesis, were found to be significantly upregulated in the comparison groups, and those encoding haem oxygenase, stromal-processing peptidase and porphobilinogen deaminase were found to be significantly downregulated. No GO terms were significantly enriched in haem-related processes (Q value = 1). Conclusion Our data revealed changes in the transcriptome expression profile of the Pfhrp2--3D7 parasite during the intraerythrocytic stages. The findings provide insight at the gene transcript level that will facilitate further research on and development of anti-malaria drugs.

scholarly journals Inhibition of PfMYST Histone Acetyltransferase Activity Blocks Plasmodium falciparum Growth and Survival

2020 ◽  
Vol 65 (1) ◽  
pp. e00953-20
Author(s):  
Utsav Sen ◽  
Akshaykumar Nayak ◽  
Juhi Khurana ◽  
Deepu Sharma ◽  
Ashish Gupta
Keyword(s):  
Catalytic Activity ◽  
Drug Target ◽  
Cell Cycle Progression ◽  
Histone Acetyltransferase ◽  
Antigenic Variation ◽  
Therapeutic Potential ◽  
Parasite Growth ◽  
Trophozoite Stage ◽  
Content Type ◽  
Growth And Survival

ABSTRACTOne of the major barriers in the prevention and control of malaria programs worldwide is the growing emergence of multidrug resistance in Plasmodium parasites, and this necessitates continued efforts to discover and develop effective drug molecules targeting novel proteins essential for parasite survival. In recent years, epigenetic regulators have evolved as an attractive drug target option owing to their crucial role in survival and development of Plasmodium at different stages of its life cycle. PfMYST, a histone acetyltransferase protein, is known to regulate key cellular processes, such as cell cycle progression, DNA damage repair, and antigenic variation, that facilitate parasite growth, adaptation, and survival inside its host. With the aim of assessing the therapeutic potential of PfMYST as a novel drug target, we examined the effect of NU9056 (an HsTIP60 inhibitor) on the rate of parasite growth and survival. In the present study, by using a yeast complementation assay, we established that PfMYST is a true homolog of TIP60 and showed that NU9056 can inhibit PfMYST catalytic activity and kill P. falciparum parasites in culture. Inhibiting the catalytic activity of PfMYST arrests the parasite in the trophozoite stage and inhibits its further transition to the schizont stage, eventually leading to its death. Overall, our study provides proof of concept that PfMYST catalytic activity is essential for parasite growth and survival and that PfMYST can be a potential target for antimalarial therapy.

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