scholarly journals PfClpC Is An Essential Clp Chaperone Required For Plastid Integrity And Clp Protease Stability In Plasmodium falciparum

2016 ◽  
Author(s):  
Anat Florentin ◽  
David W Cobb ◽  
Jillian D Fishburn ◽  
Michael J Cipriano ◽  
Paul S Kim ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Growth Arrest ◽  
Parasite Line ◽  
Functional Interactions ◽  
Conditional Mutant ◽  
Caseinolytic Protease ◽  
Active Protease ◽  
Protease Stability ◽  
Essential Function ◽  
Conditional Inhibition

SummaryThe deadly malaria parasite, Plasmodium falciparum, contains a non-photosynthetic plastid known as the apicoplast, that functions to produce essential metabolites. Little is known about its biology or regulation, but drugs that target the apicoplast are clinically effective. Using phylogenetic analysis, we identified a putative complex of clp (caseinolytic protease) genes. We genetically targeted members of this complex and generated conditional mutants of the PfClpC chaperone and PfClpP protease and found that they co-localize in the apicoplast. Conditional inhibition of the PfClpC chaperone resulted in growth arrest and apicoplast loss, and was rescued by addition of the essential apicoplast-derived metabolite, IPP. Using a double conditional-mutant parasite line, we discovered that the chaperone activity is required to stabilize the active protease, revealing functional interactions. These data demonstrate the essential function of PfClpC in maintaining apicoplast integrity and its role in regulating the proteolytic activity of the Clp complex.

scholarly journals PfClpC Is an Essential Clp Chaperone Required for Plastid Integrity and Clp Protease Stability in Plasmodium falciparum

Cell Reports ◽  
2017 ◽  
Vol 21 (7) ◽  
pp. 1746-1756 ◽  
Author(s):  
Anat Florentin ◽  
David W. Cobb ◽  
Jillian D. Fishburn ◽  
Michael J. Cipriano ◽  
Paul S. Kim ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Clp Protease ◽  
Protease Stability

Ability of Plasmodium falciparum to invade Southeast Asian ovalocytes varies between parasite lines

Blood ◽  
2004 ◽  
Vol 104 (9) ◽  
pp. 2961-2966 ◽  
Author(s):  
Alfred Cortés ◽  
Ariadna Benet ◽  
Brian M. Cooke ◽  
John W. Barnwell ◽  
John C. Reeder
Keyword(s):  
Plasmodium Falciparum ◽  
Red Blood Cells ◽  
Blood Cells ◽  
Clinical Observation ◽  
Southeast Asian ◽  
Parasite Line ◽  
Invasion Pathways ◽  
Receptor Interactions ◽  
Multiple Ligand

Abstract Plasmodium falciparum, the causative agent of the most lethal form of human malaria, uses multiple ligand-receptor interactions to invade host red blood cells (RBCs). We studied the invasion of P falciparum into abnormal RBCs from humans carrying the Southeast Asian ovalocytosis (SAO) trait. One particular parasite line, 3D7-A, invaded these cells efficiently, whereas all other lines studied invaded SAO RBCs to only about 20% of the extent of normal (non-SAO) cells. This result is consistent with the clinical observation that SAO individuals can experience high-density P falciparum infections and provides an explanation for previous discrepant results on invasion of SAO RBCs. Characterization of the invasion phenotype of 3D7-A revealed that efficient invasion of SAO RBCs was paralleled by relatively efficient invasion of normal RBCs treated with either neuraminidase, trypsin, or chymotrypsin and a novel capacity to invade normal RBCs treated sequentially with both neuraminidase and trypsin. Our results suggest that only parasites able to use some particular invasion pathways can invade SAO RBCs efficiently in culture. A similar situation might occur in the field.

scholarly journals A Novel Tool for the Generation of Conditional Knockouts To Study Gene Function across the Plasmodium falciparum Life Cycle

mBio ◽  
2019 ◽  
Vol 10 (5) ◽  
Author(s):  
Marta Tibúrcio ◽  
Annie S. P. Yang ◽  
Kazuhide Yahata ◽  
Pablo Suárez-Cortés ◽  
Hugo Belda ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Life Cycle ◽  
Genetic Modification ◽  
Gene Deletion ◽  
Genetically Modify ◽  
Complex Life Cycle ◽  
Mosquito Vector ◽  
Parasite Line ◽  
Content Type ◽  
First Time

ABSTRACT Plasmodium falciparum has a complex life cycle that involves interaction with multiple tissues inside the human and mosquito hosts. Identification of essential genes at all different stages of the P. falciparum life cycle is urgently required for clinical development of tools for malaria control and eradication. However, the study of P. falciparum is limited by the inability to genetically modify the parasite throughout its life cycle with the currently available genetic tools. Here, we describe the detailed characterization of a new marker-free P. falciparum parasite line that expresses rapamycin-inducible Cre recombinase across the full life cycle. Using this parasite line, we were able to conditionally delete the essential invasion ligand AMA1 in three different developmental stages for the first time. We further confirm efficient gene deletion by targeting the nonessential kinase FIKK7.1. IMPORTANCE One of the major limitations in studying P. falciparum is that so far only asexual stages are amenable to rapid conditional genetic modification. The most promising drug targets and vaccine candidates, however, have been refractory to genetic modification because they are essential during the blood stage or for transmission in the mosquito vector. This leaves a major gap in our understanding of parasite proteins in most life cycle stages and hinders genetic validation of drug and vaccine targets. Here, we describe a method that supports conditional gene deletion across the P. falciparum life cycle for the first time. We demonstrate its potential by deleting essential and nonessential genes at different parasite stages, which opens up completely new avenues for the study of malaria and drug development. It may also allow the realization of novel vaccination strategies using attenuated parasites.

scholarly journals The single CCA-adding enzyme of T. brucei has distinct functions in the cytosol and in mitochondria

2020 ◽  
Vol 295 (18) ◽  
pp. 6138-6150 ◽  
Author(s):  
Shikha Shikha ◽  
André Schneider
Keyword(s):  
Amino Acids ◽  
Growth Arrest ◽  
Trna Genes ◽  
Potential Candidate ◽  
Mitochondrial Localization ◽  
Putative Protein ◽  
Blast Search ◽  
Genes Encoding ◽  
Essential Function ◽  
Nucleotide Triplet

tRNAs universally carry a CCA nucleotide triplet at their 3′-ends. In eukaryotes, the CCA is added post-transcriptionally by the CCA-adding enzyme (CAE). The mitochondrion of the parasitic protozoan Trypanosoma brucei lacks tRNA genes and therefore imports all of its tRNAs from the cytosol. This has generated interest in the tRNA modifications and their distribution in this organism, including how CCA is added to tRNAs. Here, using a BLAST search for genes encoding putative CAE proteins in T. brucei, we identified a single ORF, Tb927.9.8780, as a potential candidate. Knockdown of this putative protein, termed TbCAE, resulted in the accumulation of truncated tRNAs, abolished translation, and inhibited both total and mitochondrial CCA-adding activities, indicating that TbCAE is located both in the cytosol and mitochondrion. However, mitochondrially localized tRNAs were much less affected by the TbCAE ablation than the other tRNAs. Complementation assays revealed that the N-terminal 10 amino acids of TbCAE are dispensable for its activity and mitochondrial localization and that deletion of 10 further amino acids abolishes both. A growth arrest caused by the TbCAE knockdown was rescued by the expression of the cytosolic isoform of yeast CAE, even though it was not imported into mitochondria. This finding indicated that the yeast enzyme complements the essential function of TbCAE by adding CCA to the primary tRNA transcripts. Of note, ablation of the mitochondrial TbCAE activity, which likely has a repair function, only marginally affected growth.

scholarly journals A clonal Plasmodium falciparum population in an isolated outbreak of malaria in the Republic of Cabo Verde

Parasitology ◽  
1999 ◽  
Vol 118 (4) ◽  
pp. 347-355 ◽  
Author(s):  
A. P. AREZ ◽  
G. SNOUNOU ◽  
J. PINTO ◽  
C. A. SOUSA ◽  
D. MODIANO ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Genetic Analysis ◽  
Epidemiological Data ◽  
Parasite Line ◽  
Cross Sectional ◽  
Blood Samples ◽  
Cabo Verde ◽  
The Republic ◽  
Santiago Island ◽  
Falciparum Population

We present the first parasitological, molecular and longitudinal analysis of an isolated outbreak of malaria. This outbreak occurred on Santiago Island (Republic of Cabo Verde), a region where malaria is hypoendemic and controlled, and thus the population is considered non-immune. Blood samples were collected from the inhabitants over 1 month and during cross-sectional surveys in the following year. The presence and nature of the parasites was determined by PCR. Plasmodium falciparum was the only species detected. Genetic analysis revealed that the circulating parasites were genetically homogeneous, and probably clonal. Gametocytes were found throughout this period. Our data suggest that this represented a focal outbreak, resulting in the infection of at least 40% of the villagers with a clonal parasite line. Thus, P. falciparum infections can persist for at least 1 year in a substantial proportion (10%) of the hosts. Implications for malaria control and the interpretation of epidemiological data are discussed.

scholarly journals Protein ubiquitylation is essential for the schizont to merozoite transition in Plasmodium falciparum blood-stage development

2019 ◽  
Author(s):  
Yang Wu ◽  
Vesela Encheva ◽  
Judith L. Green ◽  
Edwin Lasonder ◽  
Adchara Prommaban ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Homology Model ◽  
Food Vacuole ◽  
Blood Stage ◽  
Parasite Development ◽  
Parasite Line ◽  
Schizont Stage ◽  
Post Translational Modification ◽  
Asexual Blood Stage ◽  
Erythrocyte Invasion

AbstractUbiquitylation is a common post translational modification of eukaryotic proteins and in the human malaria parasite, Plasmodium falciparum (Pf) overall ubiquitylation increases in the transition from intracellular schizont to extracellular merozoite stages in the asexual blood stage cycle. Here, we identify specific ubiquitylation sites of protein substrates in three intracellular parasite stages and extracellular merozoites; a total of 1464 sites in 546 proteins were identified (data available via ProteomeXchange with identifier PXD014998). 469 ubiquitylated proteins were identified in merozoites compared with only 160 in the preceding intracellular schizont stage, indicating a large increase in protein ubiquitylation associated with merozoite maturation. Following merozoite invasion of erythrocytes, few ubiquitylated proteins were detected in the first intracellular ring stage but as parasites matured through trophozoite to schizont stages the extent of ubiquitylation increased. We identified commonly used ubiquitylation motifs and groups of ubiquitylated proteins in specific areas of cellular function, for example merozoite pellicle proteins involved in erythrocyte invasion, exported proteins, and histones. To investigate the importance of ubiquitylation we screened ubiquitin pathway inhibitors in a parasite growth assay and identified the ubiquitin activating enzyme (UBA1 or E1) inhibitor MLN7243 (TAK-243) to be particularly effective. This small molecule was shown to be a potent inhibitor of recombinant PfUBA1, and a structural homology model of MLN7243 bound to the parasite enzyme highlights avenues for the development of P. falciparum specific inhibitors. We created a genetically modified parasite with a rapamycin-inducible functional deletion of uba1; addition of either MLN7243 or rapamycin to the recombinant parasite line resulted in the same phenotype, with parasite development blocked at the late schizont stage. These results indicate that the intracellular target of MLN7243 is UBA1, and this activity is essential for the final differentiation of schizonts to merozoites. The ubiquitylation of many merozoite proteins and their disappearance in ring stages are consistent with the idea that ubiquitylation leads to their destruction via the proteasome once their function is complete following invasion, which would allow amino acid recycling in the period prior to the parasite’s elaboration of a new food vacuole.

scholarly journals Identification of Three Novel Plasmodium Factors Involved in Ookinete to Oocyst Developmental Transition

2021 ◽  
Vol 11 ◽  
Author(s):  
Chiamaka V. Ukegbu ◽  
George K. Christophides ◽  
Dina Vlachou
Keyword(s):  
Plasmodium Falciparum ◽  
Plasmodium Berghei ◽  
Disease Transmission ◽  
Sub Saharan Africa ◽  
Parasite Transmission ◽  
Targeted Disruption ◽  
Transmission Blocking ◽  
Essential Function ◽  
Sub Saharan ◽  
Transmission Biology

Plasmodium falciparum malaria remains a major cause of global morbidity and mortality, mainly in sub-Saharan Africa. The numbers of new malaria cases and deaths have been stable in the last years despite intense efforts for disease elimination, highlighting the need for new approaches to stop disease transmission. Further understanding of the parasite transmission biology could provide a framework for the development of such approaches. We phenotypically and functionally characterized three novel genes, PIMMS01, PIMMS57, and PIMMS22, using targeted disruption of their orthologs in the rodent parasite Plasmodium berghei. PIMMS01 and PIMMS57 are specifically and highly expressed in ookinetes, while PIMMS22 transcription starts already in gametocytes and peaks in sporozoites. All three genes show strong phenotypes associated with the ookinete to oocyst transition, as their disruption leads to very low numbers of oocysts and complete abolishment of transmission. PIMMS22 has a secondary essential function in the oocyst. Our results enrich the molecular understanding of the parasite-vector interactions and identify PIMMS01, PIMMS57, and PIMMS22 as new targets of transmission blocking interventions.

scholarly journals The Clp System in Malaria Parasites Degrades Essential Substrates to Regulate Plastid Biogenesis

2019 ◽  
Author(s):  
A. Florentin ◽  
D.R. Stephens ◽  
C.F. Brooks ◽  
R.P. Baptista ◽  
V Muralidharan
Keyword(s):  
Plasmodium Falciparum ◽  
Malaria Parasite ◽  
Affinity Purification ◽  
Nuclear Genome ◽  
Clp Protease ◽  
Complex Composition ◽  
Malaria Parasites ◽  
Human Malaria Parasite ◽  
Essential Function ◽  
Comprehensive Study

AbstractThe human malaria parasite, Plasmodium falciparum, contains an essential plastid called the apicoplast. Most of apicoplast proteins are encoded by the nuclear genome and it is unclear how the plastid proteome is regulated. Here, we study an apicoplast-localized caseinolytic-protease (Clp) system and how it regulates organelle proteostasis. Using null and conditional mutants, we demonstrated that the Clp protease (PfClpP) has robust enzymatic activity that is essential for apicoplast biogenesis. We developed a CRISPR/Cas9 based system to express catalytically-dead PfClpP, which showed that PfClpP oligomerizes as a zymogen and matured via trans-autocatalysis. The expression of a Clp chaperone (PfClpC) mutant led to the discovery of a functional chaperone-protease interaction essential for plastid function. Conditional mutants of the substrate-adaptor (PfClpS) demonstrated its essential function in plastid biogenesis. A combination of multiple affinity purification screens identified the Clp complex composition as well as putative Clp substrates. This comprehensive study reveals the molecular composition and interactions influencing the proteolytic function of the apicoplast Clp system and demonstrates its central role in the biogenesis of the plastid in malaria parasites.

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.

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