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

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

Antimalarial target vulnerability of the putative Plasmodium falciparum methionine synthase

Background 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

Inducible Knockdown of <i>Plasmodium</i> Gene Expression Using the <i>glmS</i> Ribozyme

<div><p>Conventional reverse genetic approaches for study of <i>Plasmodium</i> malaria parasite gene function are limited, or not applicable. Hence, new inducible systems are needed. Here we describe a method to control <i>P. falciparum</i> gene expression in which target genes bearing a <i>glmS</i> ribozyme in the 3′ untranslated region are efficiently knocked down in transgenic <i>P. falciparum</i> parasites in response to glucosamine inducer. Using reporter genes, we show that the <i>glmS</i> ribozyme cleaves reporter mRNA <i>in vivo</i> leading to reduction in mRNA expression following glucosamine treatment. Glucosamine-induced ribozyme activation led to efficient reduction of reporter protein, which could be rapidly reversed by removing the inducer. The <i>glmS</i> ribozyme was validated as a reverse-genetic tool by integration into the essential gene and antifolate drug target dihydrofolate reductase-thymidylate synthase (<i>Pf</i>DHFR-TS). Glucosamine treatment of transgenic parasites led to rapid and efficient knockdown of <i>Pf</i>DHFR-TS mRNA and protein. <i>Pf</i>DHFR-TS knockdown led to a growth/arrest mutant phenotype and hypersensitivity to pyrimethamine. The <i>glmS</i> ribozyme may thus be a tool for study of essential genes in <i>P. falciparum</i> and other parasite species amenable to transfection.</p></div

<i>glmS</i> ribozyme cleavage and control of <i>P. falciparum</i> mRNA expression.

<p>(A) Schematic diagram of the DHFR-TS-GFP reporter gene with <i>glmS</i> ribozyme in the 3′-UTR position (reporter_<i>glmS</i>). The reporter gene is flanked by 5′-hsp86 and PbDT-3′ <i>Plasmodium</i> transcriptional regulatory sequences. The sequence regions analyzed in parts B and C are marked: FL probe, antisense RNA probe for RNase protection assay; 5′-P and 3′-P, 5′ and 3′ ribozyme cleavage products, respectively; RT-qPCR, amplicon for RT-qPCR analysis of reporter mRNA levels. (B) RNase protection assay revealed <i>glmS</i> ribozyme cleavage products (arrowed as 5′-P and 3′-P respectively) in <i>P. falciparum</i> expressing reporter_<i>glmS</i>. 10% ring-stage synchronized parasites were treated for 24 h in the presence of 10 mM GlcN prior to harvesting and extraction of total parasite RNA. The 3D7 wild-type parasite was used as a control to test for probe specificity. Control hybridizations and gel analysis without RNase (lanes 1 and 2 marked as -) were also performed to demonstrate integrity of the RNA probe. The migration of the full-length RNA probe complementary to the <i>glmS</i> RNA (FL probe) and small RNA ladder (New England Biolabs) bands are marked. (C) Analysis of reporter mRNA expression in response to treatment with 10 mM GlcN and Fru. The expression levels of reporter_<i>glmS</i> mRNA or reporter_M9 mRNA (bearing inactivating mutations in the <i>glmS</i> ribozyme cleavage site) in treated cultures relative to untreated were determined from RT-qPCR using the ΔΔCq method normalized to BSD mRNA. Starting with 10% ring-stage synchronized cultures, parasites were treated with 10 mM GlcN or Fru for 24 h prior to harvesting and RNA extraction. Error bars represent S.E.M. (<i>n</i> = 7 for GlcN experiments, <i>n</i> = 3 for Fru experiments). One sample two-tailed <i>t</i>-tests were performed to determine if change in mRNA expression was significant; NS denotes not significant, *** denotes highly significant. The calculated <i>P</i>-values comparing sample means against hypothetical mean = 1 were: reporter_<i>glmS</i> GlcN treatment, <0.0001; reporter_M9 GlcN treatment, 0.5136; reporter_<i>glmS</i> Fru treatment, 0.3986; reporter_M9 Fru treatment, 0.0619.</p

Ribozyme-mediated control of endogenous <i>Pf</i>DHFR-TS expression.

<p>(A) Knockdown of <i>Pf</i>DHFR-TS expression in DHFR-TS-GFP_<i>glmS</i> integrant parasite clonal lines in response to GlcN. Parasitized erythrocytes expressing <i>Pf</i>DHFR-TS-GFP were enumerated by flow cytometry based on the level of GFP fusion partner. Extra sum-of-squares F- test comparing individual curve fits with the null hypothesis that slope and EC₅₀ are the same for both clone #1 and #2, <i>P</i> = 0.19. (B) Ribozyme-mediated knockdown of DHFR-TS-GFP protein is reversible. DHFR-TS-GFP_<i>glmS</i> integrant parasite clone #1 was cultured and treated with GlcN, and western immunoblotting to quantify DHFR-TS-GFP protein was performed as described in Fig. 3. Data are the mean from triplicate experiments and error bars represent S.E.M. (C) Representative images from Ponceau S staining of parasite lysates following electrophoresis and transfer to membrane, and chemiluminescent detection of GFP using specific antibodies. The pre-stained marker lane is marked M above the Ponceau S panel, and the sizes of two marker proteins are indicated.</p

Ribozyme-mediated knockdown of reporter protein expression in response to sugar treatment.

<p>Ring-stage synchronized parasites from reporter_<i>glmS</i> (parts A, C) and reporter_M9 (parts B, D) transfected lines were treated for 24 h with varying concentrations of different sugars (GlcN, ManN, GalN, Fru and Man) prior to flow cytometric analysis. The numbers of GFP positive and viable cells were normalized to untreated controls, which were set as 100%. Data are the mean from triplicate experiments and error bars represent S.E.M. Part E shows that ribozyme-mediated knockdown of reporter protein is reversible. 10% ring-stage parasites were treated with 10 mM GlcN for 24 h and samples taken after 12 and 24 h of treatment. The parasite culture medium was changed and the culture was continued, with samples taken after 12 and 24 h in the new GlcN-free medium. The experiment was performed in triplicate with independent cultures for each replicate (5 sampled time points from each culture). Western immunoblot analysis of DHFR-TS-GFP reporter protein expression was performed using an anti-GFP polyclonal antibody. The normalized intensity of the ∼100 kDa band corresponding to DHFR-TS-GFP protein was measured relative to the normalized intensity at 0 h, which was taken as 100%. Data are the mean and error bars represent S.E.M. Representative images from Ponceau S staining of parasite lysates following electrophoresis and transfer to membrane, and the corresponding chemiluminescent detection of GFP with specific antibodies are shown in part F. The pre-stained marker lane is marked M above the Ponceau S panel, and the sizes of two marker proteins are indicated.</p

Integration of <i>glmS</i> ribozyme sequence into the essential <i>Pf</i>DHFR-TS gene.

<p>(A) Schematic showing the strategy used to incorporate the GFP and <i>glmS</i> ribozyme sequences at the 3′ end of the <i>Pf</i>DHFR-TS gene by single crossover homologous recombination. The expected sizes of PCR products shown in part B are marked. (B) PCR testing of plasmid integration from two cloned transgenic lines, clone #1 and #2. PCR primer combinations used are indicated above the lanes and the migrations of 1 kb+ DNA marker (Invitrogen) bands are indicated on the left.</p