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Scientific Reports Jul 2017It is important to understand intrinsic variation in asexual blood stage multiplication rates of the most virulent human malaria parasite, Plasmodium falciparum. Here,...
It is important to understand intrinsic variation in asexual blood stage multiplication rates of the most virulent human malaria parasite, Plasmodium falciparum. Here, multiplication rates of long-term laboratory adapted parasite clones and new clinical isolates were measured, using a newly standardised assay of growth from low starting density in replicate parallel cultures with erythrocytes from multiple different donors, across multiple cycles. Multiplication rates of long-term established clones were between 7.6 and 10.5 fold per 48 hours, with clone Dd2 having a higher rate than others (clones 3D7, HB3 and D10). Parasite clone-specific growth was then analysed in co-culture assays with all possible heterologous pairwise combinations. This showed that co-culture of different parasites did not affect their replication rates, indicating that there were no suppressive interactions operating between parasites. Multiplication rates of eleven new clinical isolates were measured after a few weeks of culture, and showed a spectrum of replication rates between 2.3 and 6.0 fold per 48 hours, the entire range being lower than for the long-term laboratory adapted clones. Multiplication rate estimates remained stable over time for several isolates tested repeatedly up to three months after culture initiation, indicating considerable persistence of this important trait variation.
Topics: Coculture Techniques; Erythrocytes; Humans; Malaria, Falciparum; Microsatellite Repeats; Plasmodium falciparum
PubMed: 28743888
DOI: 10.1038/s41598-017-06295-9 -
Transactions of the Royal Society of... 1997
Plasmodium falciparum: adaptation in vitro of isolates from symptomatic individuals in Gabon: polymerase chain reaction typing and evaluation of chloroquine susceptibility.
Topics: Animals; Antimalarials; Chloroquine; Drug Resistance; Gabon; Humans; Malaria, Falciparum; Plasmodium falciparum; Polymerase Chain Reaction; Polymorphism, Genetic
PubMed: 9196771
DOI: 10.1016/s0035-9203(97)90226-1 -
Malaria Journal Apr 2016Transmission of the malaria parasite Plasmodium falciparum from humans to the mosquito vector requires differentiation of a sub-population of asexual forms replicating...
BACKGROUND
Transmission of the malaria parasite Plasmodium falciparum from humans to the mosquito vector requires differentiation of a sub-population of asexual forms replicating within red blood cells into non-dividing male and female gametocytes. The nature of the molecular mechanism underlying this key differentiation event required for malaria transmission is not fully understood.
METHODS
Whole genome sequencing was used to examine the genomic diversity of the gametocyte non-producing 3D7-derived lines F12 and A4. These lines were used in the recent detection of the PF3D7_1222600 locus (encoding PfAP2-G), which acts as a genetic master switch that triggers gametocyte development.
RESULTS
The evolutionary changes from the 3D7 parental strain through its derivatives F12 (culture-passage derived cloned line) and A4 (transgenic cloned line) were identified. The genetic differences including the formation of chimeric var genes are presented.
CONCLUSION
A genomics resource is provided for the further study of gametocytogenesis or other phenotypes using these parasite lines.
Topics: Gametogenesis; Genome, Protozoan; Plasmodium falciparum; Polymorphism, Genetic; Sequence Analysis, DNA
PubMed: 27098483
DOI: 10.1186/s12936-016-1254-1 -
Plasmodium falciparum: differentiation of isolates with DNA hybridization using antigen gene probes.Experimental Parasitology Aug 1985Chromosomal DNA was prepared from seven Plasmodium falciparum isolates that had been cultured in vitro and from a cloned P. falciparum line. The DNA was cleaved with...
Chromosomal DNA was prepared from seven Plasmodium falciparum isolates that had been cultured in vitro and from a cloned P. falciparum line. The DNA was cleaved with restriction endonucleases, fractionated by agarose gel electrophoresis, blotted to nitrocellulose, and hybridized with a series of radioactively labeled DNA probes. The probes had been derived from cDNA clones encoding portions of P. falciparum antigens. Simple, reproducible band patterns that differed for many of the isolates were obtained. Parasite isolates collected from different continents could be readily distinguished, as could some but not all isolates collected from one restricted region of Papua New Guinea. Application of this technique for the identification and differentiation of parasite strains was explored. The patterns of hybridization observed were consistent with the proposition that blood stages of P. falciparum have a haploid genome.
Topics: Animals; Antigens, Protozoan; Autoradiography; Collodion; DNA; DNA Restriction Enzymes; Deoxyribonuclease EcoRI; Deoxyribonuclease HindIII; Electrophoresis, Agar Gel; Nucleic Acid Hybridization; Plasmodium falciparum; Polymorphism, Genetic
PubMed: 2990991
DOI: 10.1016/s0014-4894(85)80025-4 -
Antimicrobial Agents and Chemotherapy Dec 2019Management of uncomplicated malaria worldwide is threatened by the emergence in Asia of carrying variants of the locus and exhibiting reduced susceptibility to...
Management of uncomplicated malaria worldwide is threatened by the emergence in Asia of carrying variants of the locus and exhibiting reduced susceptibility to artemisinin. Mutations in two other genes, and , are associated with artemisinin resistance in rodent malaria and with clinical failure of combination therapy in African malaria patients. Transgenic clones, each carrying orthologues of mutations in and associated with artemisinin resistance in , were derived by Cas9 gene editing. Susceptibility to artemisinin and other antimalarial drugs was determined. Following exposure to 700 nM dihydroartemisinin in the ring-stage survival assay, we found strong evidence that transgenic parasites expressing the I592T variant (11% survival), but not the S160N variant (1% survival), of the AP2μ adaptin subunit were significantly less susceptible than the parental wild-type parasite population. The V3275F variant of UBP1, but not the V3306F variant, also displayed reduced susceptibility to dihydroartemisinin (8.5% survival versus 0.5% survival). AP2μ and UBP1 variants did not elicit reduced susceptibility to 48 h of exposure to artemisinin or to other antimalarial drugs. Therefore, variants of the AP2 adaptor complex μ-subunit and of the ubiquitin hydrolase UBP1 reduce artemisinin susceptibility at the early ring stage in These findings confirm the existence of multiple pathways to perturbation of either the mode of action of artemisinin, the parasite's adaptive mechanisms of resistance, or both. The cellular role of UBP1 and AP2μ in parasites should now be elucidated.
Topics: Antimalarials; Artemisinins; Gene Editing; Plasmodium falciparum; Protozoan Proteins
PubMed: 31636063
DOI: 10.1128/AAC.01542-19 -
Antimicrobial Agents and Chemotherapy Apr 2009
Topics: Animals; Mutation; Plasmodium falciparum; Tetrahydrofolate Dehydrogenase
PubMed: 19299520
DOI: 10.1128/AAC.01427-08 -
The Journal of Antimicrobial... Sep 2013Plasmodium gametocytes, responsible for malaria parasite transmission from humans to mosquitoes, represent a crucial target for new antimalarial drugs to achieve malaria...
OBJECTIVES
Plasmodium gametocytes, responsible for malaria parasite transmission from humans to mosquitoes, represent a crucial target for new antimalarial drugs to achieve malaria elimination/eradication. We developed a novel colorimetric screening method for anti-gametocyte compounds based on the parasite lactate dehydrogenase (pLDH) assay, already standardized for asexual stages, to measure gametocyte viability and drug susceptibility.
METHODS
Gametocytogenesis of 3D7 and NF54 Plasmodium falciparum strains was induced in vitro and asexual parasites were depleted with N-acetylglucosamine. Gametocytes were treated with dihydroartemisinin, epoxomicin, methylene blue, primaquine, puromycin or chloroquine in 96-well plates and the pLDH activity was evaluated using a modified Makler protocol. Mosquito infectivity was measured by the standard membrane feeding assay (SMFA).
RESULTS
A linear correlation was found between gametocytaemia determined by Giemsa staining and pLDH activity. A concentration-dependent reduction in pLDH activity was observed after 72 h of drug treatment, whereas an additional 72 h of incubation without drugs was required to obtain complete inhibition of gametocyte viability. SMFA on treated and control gametocytes confirmed that a reduction in pLDH activity translates into reduced oocyst development in the mosquito vector.
CONCLUSIONS
The gametocyte pLDH assay is fast, easy to perform, cheap and reproducible and is suitable for screening novel transmission-blocking compounds, which does not require parasite transgenic lines.
Topics: Animals; Antimalarials; Cell Survival; Colorimetry; Drug Evaluation, Preclinical; Humans; L-Lactate Dehydrogenase; Plasmodium falciparum
PubMed: 23645588
DOI: 10.1093/jac/dkt165 -
Cell Reports Aug 2020Southeast Asia has been the hotbed for the development of drug-resistant malaria parasites, including those with resistance to artemisinin combination therapy. While...
Southeast Asia has been the hotbed for the development of drug-resistant malaria parasites, including those with resistance to artemisinin combination therapy. While mutations in the kelch propeller domain (K13 mutations) are associated with artemisinin resistance, a range of evidence suggests that other factors are critical for the establishment and subsequent transmission of resistance in the field. Here, we perform a quantitative analysis of DNA damage and repair in the malaria parasite Plasmodium falciparum and find a strong link between enhanced DNA damage repair and artemisinin resistance. This experimental observation is further supported when variations in seven known DNA repair genes are found in resistant parasites, with six of these mutations being associated with K13 mutations. Our data provide important insights on confounding factors that are important for the establishment and spread of artemisinin resistance and may explain why resistance has not yet arisen in Africa.
Topics: Africa; Artemisinins; Asia, Southeastern; DNA Damage; DNA Repair; Drug Resistance; Genotype; Geography; Humans; Life Cycle Stages; Phenotype; Plasmodium falciparum; Protein Domains; Protozoan Proteins
PubMed: 32755588
DOI: 10.1016/j.celrep.2020.107996 -
Scientific Reports Dec 2018Glucose is an essential nutrient for Plasmodium falciparum and robust glycolytic activity is indicative of viable parasites. Using NMR spectroscopy, we show that P....
Glucose is an essential nutrient for Plasmodium falciparum and robust glycolytic activity is indicative of viable parasites. Using NMR spectroscopy, we show that P. falciparum infected erythrocytes consume ~20 times more glucose, and trophozoites metabolize ~6 times more glucose than ring stage parasites. The glycolytic activity, and hence parasite viability, can be measured within a period of 2 h to 5 h, using this method. This facilitates antimalarial bioactivity screening on ring and trophozoite stage parasites, exclusively. We demonstrate this using potent and mechanistically distinct antimalarial compounds such as chloroquine, atovaquone, cladosporin, DDD107498 and artemisinin. Our findings indicate that ring stage parasites are inherently more tolerant to antimalarial inhibitors, a feature which may facilitate emergence of drug resistance. Thus, there is a need to discover novel antimalarial compounds, which are potent and fast acting against ring stage parasites. The NMR method reported here can facilitate the identification of such molecules.
Topics: Antimalarials; Cells, Cultured; Glycolysis; Humans; Life Cycle Stages; Magnetic Resonance Spectroscopy; Plasmodium falciparum
PubMed: 30584241
DOI: 10.1038/s41598-018-36197-3 -
Trends in Parasitology Jul 2008In a recent study published in Nature, Daily et al. profiled parasite gene expression in Plasmodium falciparum infections and identified three in vivo 'states' based on...
In a recent study published in Nature, Daily et al. profiled parasite gene expression in Plasmodium falciparum infections and identified three in vivo 'states' based on parasite transcription patterns. Despite similar host clinical features, two states displayed highly divergent gene expression, whereas the third was found in individuals with increased inflammatory markers. These findings suggest that parasites exist in different physiological states in vivo, providing an important foundation for future studies investigating how these states might contribute to malaria pathogenesis and outcome.
Topics: Animals; Gene Expression Profiling; Gene Expression Regulation; Genetic Variation; Humans; Malaria, Falciparum; Plasmodium falciparum; Transcription, Genetic
PubMed: 18538633
DOI: 10.1016/j.pt.2008.04.004