Selection of drug resistant mutants from random library of Plasmodium falciparum dihydrofolate reductase in Plasmodium berghei model

<p>Abstract</p> <p>Background</p> <p>The prevalence of drug resistance amongst the human malaria <it>Plasmodium </it>species has most commonly been associated with genomic mutation within the parasites. This phenomenon necessitates evolutionary predictive studies of possible resistance mutations, which may occur when a new drug is introduced. Therefore, identification of possible new <it>Plasmodium falciparum </it>dihydrofolate reductase (<it>Pf</it>DHFR) mutants that confer resistance to antifolate drugs is essential in the process of antifolate anti-malarial drug development.</p> <p>Methods</p> <p>A system to identify mutations in <it>Pfdhfr </it>gene that confer antifolate drug resistance using an animal <it>Plasmodium </it>parasite model was developed. By using error-prone PCR and <it>Plasmodium </it>transfection technologies, libraries of <it>Pfdhfr </it>mutant were generated and then episomally transfected to <it>Plasmodium berghei </it>parasites, from which pyrimethamine-resistant <it>Pf</it>DHFR mutants were selected.</p> <p>Results</p> <p>The principal mutation found from this experiment was S108N, coincident with the first pyrimethamine-resistance mutation isolated from the field. A transgenic <it>P. berghei</it>, in which endogenous <it>Pbdhfr </it>allele was replaced with the mutant <it>Pfdhfr^S108N</it>, was generated and confirmed to have normal growth rate comparing to parental non-transgenic parasite and also confer resistance to pyrimethamine.</p> <p>Conclusion</p> <p>This study demonstrated the power of the transgenic <it>P. berghei </it>system to predict drug-resistant <it>Pfdhfr </it>mutations in an <it>in vivo </it>parasite/host setting. The system could be utilized for identification of possible novel drug-resistant mutants that could arise against new antifolate compounds and for prediction the evolution of resistance mutations.</p

Transgenic Plasmodium parasites stably expressing Plasmodium vivax dihydrofolate reductase-thymidylate synthase as in vitro and in vivo models for antifolate screening

<p>Abstract</p> <p>Background</p> <p><it>Plasmodium vivax </it>is the most prevalent cause of human malaria in tropical regions outside the African continent. The lack of a routine continuous <it>in vitro </it>culture of this parasite makes it difficult to develop specific drugs for this disease. To facilitate the development of anti-<it>P. vivax </it>drugs, bacterial and yeast surrogate models expressing the validated <it>P. vivax </it>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 <it>in vivo </it>drug metabolism between the surrogate models and <it>P. vivax </it>parasites.</p> <p>Methods</p> <p><it>Plasmodium falciparum </it>and <it>Plasmodium berghei </it>parasites were transfected with DNA constructs bearing <it>P. vivax dhfr-ts </it>pyrimethamine sensitive (wild-type) and pyrimethamine resistant (mutant) alleles. Double crossover homologous recombination was used to replace the endogenous <it>dhfr-ts </it>of <it>P. falciparum </it>and <it>P. berghei </it>parasites with <it>P. vivax </it>homologous genes. The integration of <it>Pvdhfr-ts </it>genes via allelic replacement was verified by Southern analysis and the transgenic parasites lines validated as models by standard drug screening assays.</p> <p>Results</p> <p>Transgenic <it>P. falciparum </it>and <it>P. berghei </it>lines stably expressing <it>Pv</it>DHFR-TS replacing the endogenous parasite DHFR-TS were obtained. Anti-malarial drug screening assays showed that transgenic parasites expressing wild-type <it>Pv</it>DHFR-TS were pyrimethamine-sensitive, whereas transgenic parasites expressing mutant <it>Pv</it>DHFR-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.</p> <p>Conclusion</p> <p>With the permanent integration of <it>Pvdhfr-ts </it>gene in the genome, the transgenic <it>Plasmodium </it>lines expressing <it>Pv</it>DHFR-TS are genetically stable and will be useful for screening anti-<it>P. vivax </it>compounds targeting <it>Pv</it>DHFR-TS. A similar approach could be used to generate transgenic models specific for other targets of interest, thus facilitating the development of anti-<it>P. vivax </it>drugs in general.</p

A nuclear targeting system in Plasmodium falciparum

<p>Abstract</p> <p>Background</p> <p>The distinct differences in gene control mechanisms acting in the nucleus between <it>Plasmodium falciparum </it>and the human host could lead to new potential drug targets for anti-malarial development. New molecular toolkits are required for dissecting molecular machineries in the <it>P. falciparum </it>nucleus. One valuable tool commonly used in model organisms is protein targeting to specific sub-cellular locations. Targeting proteins to specified locations allows labeling of organelles for microscopy, or testing of how the protein of interest modulates organelle function. In recent years, this approach has been developed for various malaria organelles, such as the mitochondrion and the apicoplast. A tool for targeting a protein of choice to the <it>P. falciparum </it>nucleus using an exogenous nuclear localization sequence is reported here.</p> <p>Methods</p> <p>To develop a nuclear targeting system, a putative nuclear localization sequence was fused with green fluorescent protein (GFP). The nuclear localization sequence from the yeast transcription factor Gal4 was chosen because of its well-defined nuclear localization signal. A series of truncated Gal4 constructs was also created to narrow down the nuclear localization sequence necessary for <it>P. falciparum </it>nuclear import. Transfected parasites were analysed by fluorescent and laser-scanning confocal microscopy.</p> <p>Results</p> <p>The nuclear localization sequence of Gal4 is functional in <it>P. falciparum</it>. It effectively transported GFP into the nucleus, and the first 74 amino acid residues were sufficient for nuclear localization.</p> <p>Conclusions</p> <p>The Gal4 fusion technique enables specific transport of a protein of choice into the <it>P. falciparum </it>nucleus, and thus provides a tool for labeling nuclei without using DNA-staining dyes. The finding also indicates similarities between the nuclear transport mechanisms of yeast and <it>P. falciparum</it>. Since the nuclear transport system has been thoroughly studied in yeast, this could give clues to research on the same mechanism in <it>P. falciparum</it>.</p

Plasmodium parasites mount an arrest response to dihydroartemisinin, as revealed by whole transcriptome shotgun sequencing (RNA-seq) and microarray study

RNA-seq data analysis from DHA treatment of P. falciparum Limma results from 1 h treatments with 500 nM DHA in P. falciparum K1 rings, trophozoites and schizonts. (XLS 2040 kb

Validation of Plasmodium falciparum deoxyhypusine synthase as an antimalarial target

Background Hypusination is an essential post-translational modification in eukaryotes. The two enzymes required for this modification, namely deoxyhypusine synthase (DHS) and deoxyhypusine hydrolase are also conserved. Plasmodium falciparum human malaria parasites possess genes for both hypusination enzymes, which are hypothesized to be targets of antimalarial drugs. Methods Transgenic P. falciparum parasites with modification of the PF3D7_1412600 gene encoding PfDHS enzyme were created by insertion of the glmS riboswitch or the M9 inactive variant. The PfDHS protein was studied in transgenic parasites by confocal microscopy and Western immunoblotting. The biochemical function of PfDHS enzyme in parasites was assessed by hypusination and nascent protein synthesis assays. Gene essentiality was assessed by competitive growth assays and chemogenomic profiling. Results Clonal transgenic parasites with integration of glmS riboswitch downstream of the PfDHS gene were established. PfDHS protein was present in the cytoplasm of transgenic parasites in asexual stages. The PfDHS protein could be attenuated fivefold in transgenic parasites with an active riboswitch, whereas PfDHS protein expression was unaffected in control transgenic parasites with insertion of the riboswitch-inactive sequence. Attenuation of PfDHS expression for 72 h led to a significant reduction of hypusinated protein; however, global protein synthesis was unaffected. Parasites with attenuated PfDHS expression showed a significant growth defect, although their decline was not as rapid as parasites with attenuated dihydrofolate reductase-thymidylate synthase (PfDHFR-TS) expression. PfDHS-attenuated parasites showed increased sensitivity to N1-guanyl-1,7-diaminoheptane, a structural analog of spermidine, and a known inhibitor of DHS enzymes. Discussion Loss of PfDHS function leads to reduced hypusination, which may be important for synthesis of some essential proteins. The growth defect in parasites with attenuated Pf DHS expression suggests that this gene is essential. However, the slower decline of PfDHS mutants compared with PfDHFR-TS mutants in competitive growth assays suggests that PfDHS is less vulnerable as an antimalarial target. Nevertheless, the data validate PfDHS as an antimalarial target which can be inhibited by spermidine-like compounds

Ubiquitin activation is essential for schizont maturation in Plasmodium falciparum blood-stage development

Ubiquitylation 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 intraerythrocytic 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, suggesting 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 apparent 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 schizont stage. Nuclear division and formation of intracellular structures was interrupted. These results indicate that the intracellular target of MLN7243 is UBA1, and this activity is essential for the final differentiation of schizonts to merozoites

Automated detection and staging of malaria parasites from cytological smears using convolutional neural networks

Microscopic examination of blood smears remains the gold standard for laboratory inspection and diagnosis of malaria. Smear inspection is, however, time-consuming and dependent on trained microscopists with results varying in accuracy. We sought to develop an automated image analysis method to improve accuracy and standardization of smear inspection that retains capacity for expert confirmation and image archiving. Here, we present a machine learning method that achieves red blood cell (RBC) detection, differentiation between infected/uninfected cells, and parasite life stage categorization from unprocessed, heterogeneous smear images. Based on a pretrained Faster Region-Based Convolutional Neural Networks (R-CNN) model for RBC detection, our model performs accurately, with an average precision of 0.99 at an intersection-over-union threshold of 0.5. Application of a residual neural network-50 model to infected cells also performs accurately, with an area under the receiver operating characteristic curve of 0.98. Finally, combining our method with a regression model successfully recapitulates intraerythrocytic developmental cycle with accurate lifecycle stage categorization. Combined with a mobile-friendly web-based interface, called PlasmoCount, our method permits rapid navigation through and review of results for quality assurance. By standardizing assessment of Giemsa smears, our method markedly improves inspection reproducibility and presents a realistic route to both routine lab and future field-based automated malaria diagnosis

Cellular responses to modified Plasmodium falciparum MSP119 antigens in individuals previously exposed to natural malaria infection

<p>Abstract</p> <p>Background</p> <p>MSP1 processing-inhibitory antibodies bind to epitopes on the 19 kDa C-terminal region of the <it>Plasmodium falciparum </it>merozoite surface protein 1 (MSP1₁₉), inhibiting erythrocyte invasion. Blocking antibodies also bind to this antigen but prevent inhibitory antibodies binding, allowing invasion to proceed. Recombinant MSP1₁₉had been modified previously to allow inhibitory but not blocking antibodies to continue to bind. Immunization with these modified proteins, therefore, has the potential to induce more effective protective antibodies. However, it was unclear whether the modification of MSP1₁₉would affect critical T-cell responses to epitopes in this antigen.</p> <p>Methods</p> <p>The cellular responses to wild-type MSP1₁₉and a panel of modified MSP1₁₉antigens were measured using an <it>in-vitro </it>assay for two groups of individuals: the first were malaria-naïve and the second had been naturally exposed to <it>Plasmodium falciparum </it>infection. The cellular responses to the modified proteins were examined using cells from malaria-exposed infants and adults.</p> <p>Results</p> <p>Interestingly, stimulation indices (SI) for responses induced by some of the modified proteins were at least two-fold higher than those elicited by the wild-type MSP1₁₉. A protein with four amino acid substitutions (Glu27→Tyr, Leu31→Arg, Tyr34→Ser and Glu43→Leu) had the highest stimulation index (SI up to 360) and induced large responses in 64% of the samples that had significant cellular responses to the modified proteins.</p> <p>Conclusion</p> <p>This study suggests that specific MSP1₁₉variants that have been engineered to improve their antigenicity for inhibitory antibodies, retain T-cell epitopes and the ability to induce cellular responses. These proteins are candidates for the development of MSP1-based malaria vaccines.</p