27 research outputs found
Transgenic <it>Plasmodium </it>parasites stably expressing <it>Plasmodium vivax </it>dihydrofolate reductase-thymidylate synthase as <it>in vitro </it>and <it>in vivo </it>models for antifolate screening
No full textAbstract Background Plasmodium vivax is the most prevalent cause of human malaria in tropical regions outside the African continent. The lack of a routine continuous in vitro culture of this parasite makes it difficult to develop specific drugs for this disease. To facilitate the development of anti-P. vivax drugs, bacterial and yeast surrogate models expressing the validated P. vivax target dihydrofolate reductase-thymidylate synthase (DHFR-TS) have been generated; however, they can only be used as primary screening models because of significant differences in enzyme expression level and in vivo drug metabolism between the surrogate models and P. vivax parasites. Methods Plasmodium falciparum and Plasmodium berghei parasites were transfected with DNA constructs bearing P. vivax dhfr-ts pyrimethamine sensitive (wild-type) and pyrimethamine resistant (mutant) alleles. Double crossover homologous recombination was used to replace the endogenous dhfr-ts of P. falciparum and P. berghei parasites with P. vivax homologous genes. The integration of Pvdhfr-ts genes via allelic replacement was verified by Southern analysis and the transgenic parasites lines validated as models by standard drug screening assays. Results Transgenic P. falciparum and P. berghei lines stably expressing PvDHFR-TS replacing the endogenous parasite DHFR-TS were obtained. Anti-malarial drug screening assays showed that transgenic parasites expressing wild-type PvDHFR-TS were pyrimethamine-sensitive, whereas transgenic parasites expressing mutant PvDHFR-TS were pyrimethamine-resistant. The growth and sensitivity to other types of anti-malarial drugs in the transgenic parasites were otherwise indistinguishable from the parental parasites. Conclusion With the permanent integration of Pvdhfr-ts gene in the genome, the transgenic Plasmodium lines expressing PvDHFR-TS are genetically stable and will be useful for screening anti-P. vivax compounds targeting PvDHFR-TS. A similar approach could be used to generate transgenic models specific for other targets of interest, thus facilitating the development of anti-P. vivax drugs in general.</p
Antimalarial target vulnerability of the putative Plasmodium falciparum methionine synthase
No full textBackground Plasmodium falciparum possesses a cobalamin-dependent methionine synthase (MS). MS is putatively encoded by the PF3D7_1233700 gene, which is orthologous and syntenic in Plasmodium. However, its vulnerability as an antimalarial target has not been assessed. Methods We edited the PF3D7_1233700 and PF3D7_0417200 (dihydrofolate reductase-thymidylate synthase, DHFR-TS) genes and obtained transgenic P. falciparum parasites expressing epitope-tagged target proteins under the control of the glmS ribozyme. Conditional loss-of-function mutants were obtained by treating transgenic parasites with glucosamine. Results DHFR-TS, but not MS mutants showed a significant proliferation defect over 96 h, suggesting that P. falciparum MS is not a vulnerable antimalarial target
Deletion of Plasmodium falciparum ubc13 increases parasite sensitivity to the mutagen, methyl methanesulfonate and dihydroartemisinin
Get PDFThe inducible Di-Cre system was used to delete the putative ubiquitin-conjugating enzyme 13 gene (ubc13) of Plasmodium falciparum to study its role in ubiquitylation and the functional consequence during the parasite asexual blood stage. Deletion resulted in a significant reduction of parasite growth in vitro, reduced ubiquitylation of the Lys63 residue of ubiquitin attached to protein substrates, and an increased sensitivity of the parasite to both the mutagen, methyl methanesulfonate and the antimalarial drug dihydroartemisinin (DHA), but not chloroquine. The parasite was also sensitive to the UBC13 inhibitor NSC697923. The data suggest that this gene does code for an ubiquitin conjugating enzyme responsible for K63 ubiquitylation, which is important in DNA repair pathways as was previously demonstrated in other organisms. The increased parasite sensitivity to DHA in the absence of ubc13 function indicates that DHA may act primarily through this pathway and that inhibitors of UBC13 may both enhance the efficacy of this antimalarial drug and directly inhibit parasite growth
Monitoring drug resistance in Plasmodium falciparum clinical isolates collected from Northern Thailand
No full textFunctionalized Boron Nanoparticles as Potential Promising Antimalarial Agents
No full textBoron nanoparticles
(BNPs), functionalized with hydroxyl groups, were synthesized in situ by a cascade process, followed by bromination and
hydrolyzation reactions. These functionalized BNPs, (Bm(OH)n), were characterized
using 1H and 11B NMR spectra, Fourier-transform
infrared (FT-IR) spectroscopy, inductively coupled plasma-optical
emission spectroscopy (ICP-OES), transmission electron microscopy
(TEM), dynamic light scattering (DLS), and X-ray photoelectron spectroscopy
(XPS) methods. These nanoparticles were also evaluated in
vitro for their antimalarial activity against Plasmodium falciparum (3D7 strain) with an IC50 value of 0.0021 μM and showed low toxicity to Uppsala
87 malignant glioma (U87MG) cell lines, malignant melanoma A375 cell
lines, KB human oral cancer cell lines, rat cortical neuron cell lines,
and rat fibroblast-like synoviocyte (FLS) cell lines
Apicoplast ribosomal protein S10-V127M enhances artemisinin resistance of a Kelch13 transgenic Plasmodium falciparum
Get PDFBACKGROUND: The resistance of Plasmodium falciparum to artemisinin-based (ART) drugs, the front-line drug family used in artemisinin-based combination therapy (ACT) for treatment of malaria, is of great concern. Mutations in the kelch13 (k13) gene (for example, those resulting in the Cys580Tyr [C580Y] variant) were identified as genetic markers for ART-resistant parasites, which suggests they are associated with resistance mechanisms. However, not all resistant parasites contain a k13 mutation, and clearly greater understanding of resistance mechanisms is required. A genome-wide association study (GWAS) found single nucleotide polymorphisms associated with ART-resistance in fd (ferredoxin), arps10 (apicoplast ribosomal protein S10), mdr2 (multidrug resistance protein 2), and crt (chloroquine resistance transporter), in addition to k13 gene mutations, suggesting that these alleles contribute to the resistance phenotype. The importance of the FD and ARPS10 variants in ART resistance was then studied since both proteins likely function in the apicoplast, which is a location distinct from that of K13. METHODS: The reported mutations were introduced, together with a mutation to produce the k13-C580Y variant into the ART-sensitive 3D7 parasite line and the effect on ART-susceptibility using the 0−3 h ring survival assay (RSA(0−3 h)) was investigated. RESULTS AND CONCLUSION: Introducing both fd-D193Y and arps10-V127M into a k13-C580Y-containing parasite, but not a wild-type k13 parasite, increased survival of the parasite in the RSA(0−3 h). The results suggest epistasis of arps10 and k13, with arps10-V127M a modifier of ART susceptibility in different k13 allele backgrounds. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12936-022-04330-3
Additional file 1 of Apicoplast ribosomal protein S10-V127M enhances artemisinin resistance of a Kelch13 transgenic Plasmodium falciparum
No full textAdditional file 1: TableS1 Oligonucleotides used to generate guide RNA, primers in PCR analysis ofparasite genetic modification and primers for sequence confirmation
Deletion of Plasmodium falciparum ubc13 increases parasite sensitivity to the mutagen, methyl methanesulfonate and dihydroartemisinin.
No full textThe inducible Di-Cre system was used to delete the putative ubiquitin-conjugating enzyme 13 gene (ubc13) of Plasmodium falciparum to study its role in ubiquitylation and the functional consequence during the parasite asexual blood stage. Deletion resulted in a significant reduction of parasite growth in vitro, reduced ubiquitylation of the Lys63 residue of ubiquitin attached to protein substrates, and an increased sensitivity of the parasite to both the mutagen, methyl methanesulfonate and the antimalarial drug dihydroartemisinin (DHA), but not chloroquine. The parasite was also sensitive to the UBC13 inhibitor NSC697923. The data suggest that this gene does code for an ubiquitin conjugating enzyme responsible for K63 ubiquitylation, which is important in DNA repair pathways as was previously demonstrated in other organisms. The increased parasite sensitivity to DHA in the absence of ubc13 function indicates that DHA may act primarily through this pathway and that inhibitors of UBC13 may both enhance the efficacy of this antimalarial drug and directly inhibit parasite growth
Additional file 4 of Apicoplast ribosomal protein S10-V127M enhances artemisinin resistance of a Kelch13 transgenic Plasmodium falciparum
No full textAdditional file 4: Fig. S2. Sequence analysis demonstratingthe presence of fd-D193Y (upper) and arps10-V127M (lower) alleles in the MRA1240parasite line. The mutant codons are enclosed in thered box, compared with the 3D7 reference line
Additional file 2 of Apicoplast ribosomal protein S10-V127M enhances artemisinin resistance of a Kelch13 transgenic Plasmodium falciparum
No full textAdditional file 2: Table S2. The sequences of designed elements (HR1-Recodonized part-HR2) of therepair plasmids for each mutagenesis
