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Malaria Journal Dec 2017Plasmodium falciparum malaria is one of the most widespread parasitic infections in humans and remains a leading global health concern. Malaria elimination efforts are...
BACKGROUND
Plasmodium falciparum malaria is one of the most widespread parasitic infections in humans and remains a leading global health concern. Malaria elimination efforts are threatened by the emergence and spread of resistance to artemisinin-based combination therapy, the first-line treatment of malaria. Promising molecular markers and pathways associated with artemisinin drug resistance have been identified, but the underlying molecular mechanisms of resistance remains unknown. The genomic data from early period of emergence of artemisinin resistance (2008-2011) was evaluated, with aim to define k13 associated genetic background in Cambodia, the country identified as epicentre of anti-malarial drug resistance, through characterization of 167 parasite isolates using a panel of 21,257 SNPs.
RESULTS
Eight subpopulations were identified suggesting a process of acquisition of artemisinin resistance consistent with an emergence-selection-diffusion model, supported by the shifting balance theory. Identification of population specific mutations facilitated the characterization of a core set of 57 background genes associated with artemisinin resistance and associated pathways. The analysis indicates that the background of artemisinin resistance was not acquired after drug pressure, rather is the result of fixation followed by selection on the daughter subpopulations derived from the ancestral population.
CONCLUSIONS
Functional analysis of artemisinin resistance subpopulations illustrates the strong interplay between ubiquitination and cell division or differentiation in artemisinin resistant parasites. The relationship of these pathways with the P. falciparum resistant subpopulation and presence of drug resistance markers in addition to k13, highlights the major role of admixed parasite population in the diffusion of artemisinin resistant background. The diffusion of resistant genes in the Cambodian admixed population after selection resulted from mating of gametocytes of sensitive and resistant parasite populations.
Topics: Antimalarials; Artemisinins; Cambodia; Drug Resistance; Genotype; Humans; Malaria, Falciparum; Mutation; Plasmodium falciparum; Polymorphism, Single Nucleotide; Protozoan Proteins
PubMed: 29258508
DOI: 10.1186/s12936-017-2140-1 -
Methods in Molecular Biology (Clifton,... 2013The in vitro cultivation of Plasmodium falciparum is absolutely essential for the molecular dissection of parasite biology and still poses several challenges. The...
The in vitro cultivation of Plasmodium falciparum is absolutely essential for the molecular dissection of parasite biology and still poses several challenges. The dependence on, and interaction with host red blood cells, the tightly regulated stage-specific expression of proteins, and the parasite peculiar demands on nutrients and gaseous environments are only a few aspects that need to be addressed to successfully cultivate P. falciparum in vitro. In this chapter, we present techniques for normal maintenance of the erythrocytic stages of P. falciparum cultures, their synchronization and the generation of clonal cell lines.
Topics: Cell Culture Techniques; Erythrocytes; Humans; Plasmodium falciparum; Sorbitol
PubMed: 22990767
DOI: 10.1007/978-1-62703-026-7_1 -
Parasitology Jun 1992An in vitro culture of Plasmodium falciparum (isolate FCUP-1/RSA) was arrested in the trophozoite stage with alpha-difluoromethylornithine (DFMO), followed by sorbitol...
An in vitro culture of Plasmodium falciparum (isolate FCUP-1/RSA) was arrested in the trophozoite stage with alpha-difluoromethylornithine (DFMO), followed by sorbitol treatment to liberate intracellular parasites from infected erythrocytes. Most of the unlysed erythrocytes and ghost membranes were removed by filtration, after agglutination with anti-erythrocyte monoclonal antibodies or wheat-germ agglutinin. A highly purified parasite preparation was obtained after chromatography on an immuno-affinity column consisting of polystyrene particles to which anti-erythrocyte monoclonal antibodies had been adsorbed. Isolated parasites were free of surrounding erythrocyte membranes and structurally intact as assessed by transmission electron microscopy, SDS-PAGE and ELISA.
Topics: Agglutination; Animals; Chromatography, Affinity; Electrophoresis, Polyacrylamide Gel; Enzyme-Linked Immunosorbent Assay; Erythrocytes; Filtration; Humans; Mice; Microscopy, Electron; Plasmodium falciparum; Sorbitol
PubMed: 1641237
DOI: 10.1017/s0031182000063629 -
Current Opinion in Microbiology Aug 2001New data on the organization of plasmodial telomeres has recently become available. Telomeres form clusters of four to seven heterologous chromosome ends at the nuclear... (Review)
Review
New data on the organization of plasmodial telomeres has recently become available. Telomeres form clusters of four to seven heterologous chromosome ends at the nuclear periphery in asexual and sexual parasite stages. This subnuclear compartment promotes gene conversion between members of subtelomeric virulence factor genes in heterologous chromosomes resulting in diversity of antigenic and adhesive phenotypes. This has important implications for parasite survival.
Topics: Animals; Gene Expression Regulation; Humans; Malaria, Falciparum; Plasmodium falciparum; Telomere; Virulence
PubMed: 11495803
DOI: 10.1016/s1369-5274(00)00227-7 -
Parasitology Today (Personal Ed.) Oct 2000Cytoadherence is believed to be fundamental for the survival of Plasmodium falciparum in vivo and, uniquely, is a major determinant of the virulence of this parasite.... (Review)
Review
Cytoadherence is believed to be fundamental for the survival of Plasmodium falciparum in vivo and, uniquely, is a major determinant of the virulence of this parasite. Despite the widely professed importance of cytoadhesion in the development of severe disease, there are a number of aspects of this highly complex process that remain poorly understood. Recent progress in the understanding of cytoadhesive phenomena was discussed extensively at the Molecular Approaches to Malaria conference, Lorne, Australia, 2-5 February 2000. Here, Brian Cooke, Mats Wahlgren and Ross Coppel consider just how far we have progressed during the past 30 years and highlight what is still missing in our understanding of the mechanisms and clinical relevance of this apparently vital process.
Topics: Animals; Cell Adhesion; Erythrocytes; Humans; Malaria, Falciparum; Plasmodium falciparum; Virulence
PubMed: 11006472
DOI: 10.1016/s0169-4758(00)01753-1 -
Scientific Reports Jan 2023Artemisinin combination therapies (ACTs) have led to a significant decrease in Plasmodium falciparum malaria mortality. This progress is now threatened by emerging...
Artemisinin combination therapies (ACTs) have led to a significant decrease in Plasmodium falciparum malaria mortality. This progress is now threatened by emerging artemisinin resistance (ART-R) linked originally in SE Asia to polymorphisms in the Kelch propeller protein (K13) and more recently to several other seemingly unrelated genetic mutations. To better understand the parasite response to ART, we are characterizing a P. falciparum mutant with altered sensitivity to ART that was created via piggyBac transposon mutagenesis. The transposon inserted near the putative transcription start site of a gene defined as a "Plasmodium-conserved gene of unknown function," now functionally linked to K13 as the Kelch13 Interacting Candidate 5 protein (KIC5). Phenotype analysis of the KIC5 mutant during intraerythrocytic asexual development identified transcriptional changes associated with DNA stress response and altered mitochondrial metabolism, linking dysregulation of the KIC5 gene to the parasite's ability to respond to ART exposure. Through characterization of the KIC5 transcriptome, we hypothesize that this gene may be essential under ART exposure to manage gene expression of the wild-type stress response at early ring stage, thereby providing a better understanding of the parasite's processes that can alter ART sensitivity.
Topics: Antimalarials; Artemisinins; Drug Resistance; Mutation; Plasmodium falciparum; Protozoan Proteins
PubMed: 36624300
DOI: 10.1038/s41598-023-27417-6 -
Journal of Toxicology and Environmental... Nov 2021Chloroquine (CQ) is an important drug used therapeutically for treatment of malaria. However, due to limited number of studies on metabolic targets of chloroquine (CQ),...
Chloroquine (CQ) is an important drug used therapeutically for treatment of malaria. However, due to limited number of studies on metabolic targets of chloroquine (CQ), it is difficult to attribute mechanisms underlying resistance associated with usage of this drug. The present study aimed to investigate the metabolic signatures of CQ-resistant (PfDd2) compared to CQ-sensitive (Pf3D7). Both Pf3D7 and PfDd2 were treated with CQ at 200 nM for 48 hr; thereafter, the harvested red blood cells (RBCs) and media were subjected to microscopy and high-resolution metabolomics (HRM). Glutathione, γ-L-glutamyl-L-cysteine, spermidine, inosine monophosphate, alanine, and fructose-1,6-bisphosphate were markedly altered in PfDd2 of RBC. In the media, cysteine, cysteic acid, spermidine, phenylacetaldehyde, and phenylacetic acid were significantly altered in PfDd2. These differential metabolic signatures related signaling pathways of PfDd2, such as oxidative stress pathway and glycolysis may provide evidence for understanding the resistance mechanism and pathogenesis of the CQ-resistant parasite.
Topics: Antimalarials; Chloroquine; Drug Resistance; Metabolome; Plasmodium falciparum
PubMed: 34338159
DOI: 10.1080/15287394.2021.1944945 -
BMC Genomics Apr 2010Plasmodium falciparum, the causative agent of severe human malaria, has evolved to become resistant to previously successful antimalarial chemotherapies, most notably...
BACKGROUND
Plasmodium falciparum, the causative agent of severe human malaria, has evolved to become resistant to previously successful antimalarial chemotherapies, most notably chloroquine and the antifolates. The prevalence of resistant strains has necessitated the discovery and development of new chemical entities with novel modes-of-action. Although much effort has been invested in the creation of analogues based on existing drugs and the screening of chemical and natural compound libraries, a crucial shortcoming in current Plasmodial drug discovery efforts remains the lack of an extensive set of novel, validated drug targets. A requirement of these targets (or the pathways in which they function) is that they prove essential for parasite survival. The polyamine biosynthetic pathway, responsible for the metabolism of highly abundant amines crucial for parasite growth, proliferation and differentiation, is currently under investigation as an antimalarial target. Chemotherapeutic strategies targeting this pathway have been successfully utilized for the treatment of Trypanosomes causing West African sleeping sickness. In order to further evaluate polyamine depletion as possible antimalarial intervention, the consequences of inhibiting P. falciparum spermidine synthase (PfSpdSyn) were examined on a morphological, transcriptomic, proteomic and metabolic level.
RESULTS
Morphological analysis of P. falciparum 3D7 following application of the PfSpdSyn inhibitor cyclohexylamine confirmed that parasite development was completely arrested at the early trophozoite stage. This is in contrast to untreated parasites which progressed to late trophozoites at comparable time points. Global gene expression analyses confirmed a transcriptional arrest in the parasite. Several of the differentially expressed genes mapped to the polyamine biosynthetic and associated metabolic pathways. Differential expression of corresponding parasite proteins involved in polyamine biosynthesis was also observed. Most notably, uridine phosphorylase, adenosine deaminase, lysine decarboxylase (LDC) and S-adenosylmethionine synthetase were differentially expressed at the transcript and/or protein level. Several genes in associated metabolic pathways (purine metabolism and various methyltransferases) were also affected. The specific nature of the perturbation was additionally reflected by changes in polyamine metabolite levels.
CONCLUSIONS
This study details the malaria parasite's response to PfSpdSyn inhibition on the transcriptomic, proteomic and metabolic levels. The results corroborate and significantly expand previous functional genomics studies relating to polyamine depletion in this parasite. Moreover, they confirm the role of transcriptional regulation in P. falciparum, particularly in this pathway. The findings promote this essential pathway as a target for antimalarial chemotherapeutic intervention strategies.
Topics: Cyclohexylamines; Gene Expression Profiling; Gene Expression Regulation; Metabolic Networks and Pathways; Plasmodium falciparum; Polyamines; Protozoan Proteins; Spermidine Synthase
PubMed: 20385001
DOI: 10.1186/1471-2164-11-235 -
Infection, Genetics and Evolution :... Jun 2011Malaria is one of the world's most common and serious diseases causing death of about 3 million people each year. Its most severe occurrence is caused by the protozoan...
Malaria is one of the world's most common and serious diseases causing death of about 3 million people each year. Its most severe occurrence is caused by the protozoan Plasmodium falciparum. Reports have shown that the resistance of the parasite to existing drugs is increasing. Therefore, there is a huge and urgent need to discover and validate new drug or vaccine targets to enable the development of new treatments for malaria. The ability to discover these drug or vaccine targets can only be enhanced from our deep understanding of the detailed biology of the parasite, for example how cells function and how proteins organize into modules such as metabolic, regulatory and signal transduction pathways. It has been noted that the knowledge of signalling transduction pathways in Plasmodium is fundamental to aid the design of new strategies against malaria. This work uses a linear-time algorithm for finding paths in a network under modified biologically motivated constraints. We predicted several important signalling transduction pathways in Plasmodium falciparum. We have predicted a viable signalling pathway characterized in terms of the genes responsible that may be the PfPKB pathway recently elucidated in Plasmodium falciparum. We obtained from the FIKK family, a signal transduction pathway that ends up on a chloroquine resistance marker protein, which indicates that interference with FIKK proteins might reverse Plasmodium falciparum from resistant to sensitive phenotype. We also proposed a hypothesis that showed the FIKK proteins in this pathway as enabling the resistance parasite to have a mechanism for releasing chloroquine (via an efflux process). Furthermore, we also predicted a signalling pathway that may have been responsible for signalling the start of the invasion process of Red Blood Cell (RBC) by the merozoites. It has been noted that the understanding of this pathway will give insight into the parasite virulence and will facilitate rational vaccine design against merozoites invasion. And we have a host of other predicted pathways, some of which have been used in this work to predict the functionality of some proteins.
Topics: Algorithms; Computational Biology; Gene Expression Profiling; Gene Expression Regulation; Plasmodium falciparum; Protein Interaction Mapping; Protozoan Proteins; Signal Transduction
PubMed: 21112415
DOI: 10.1016/j.meegid.2010.11.006 -
Molecular Microbiology Jun 2008Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is a potentially important family of immune targets, encoded by an extremely diverse gene family called... (Comparative Study)
Comparative Study
Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is a potentially important family of immune targets, encoded by an extremely diverse gene family called var. Understanding of the genetic organization of var genes is hampered by sequence mosaicism that results from a long history of non-homologous recombination. Here we have used software designed to analyse social networks to visualize the relationships between large collections of short var sequences tags sampled from clinical parasite isolates. In this approach, two sequences are connected if they share one or more highly polymorphic sequence blocks. The results show that the majority of analysed sequences including several var-like sequences from the chimpanzee parasite Plasmodium reichenowi can be either directly or indirectly linked together in a single unbroken network. However, the network is highly structured and contains putative subgroups of recombining sequences. The major subgroup contains the previously described group A var genes, previously proposed to be genetically distinct. Another subgroup contains sequences found to be associated with rosetting, a parasite virulence phenotype. The mosaic structure of the sequences and their division into subgroups may reflect the conflicting problems of maximizing antigenic diversity and minimizing epitope sharing between variants while maintaining their host cell binding functions.
Topics: Amino Acid Sequence; Animals; Antigenic Variation; Antigens, Protozoan; Child; Conserved Sequence; Humans; Malaria, Falciparum; Plasmodium falciparum; Polymorphism, Genetic; Protozoan Proteins; Recombination, Genetic; Sequence Alignment
PubMed: 18433451
DOI: 10.1111/j.1365-2958.2008.06248.x