Pyrimethamine

Parasites were grown in hTERT-BJ1 (clontech) cells in supplemented Dulbecco's modified Eagle's medium [67] ). Parasite cloning and plaque assays were performed in human foreskin fibroblasts (HFF). For the selection of stable transgenic lines, drugs were added as follow: 1 µM pyrimethamine added one day after transfection for one week, 20 µM chloramphenicol added the day of transfection for three weeks, 5 µM FUDR added two days after transfection for one week. To repress the regulated promoter, parasites were grown in the presence of 0.5 µM anhydrotetracycline (ATc).
Thalassiosira pseudonana (Hustedt) Hasle et Heimdal CCMP1335 was grown in an artificial seawater medium (EASW) according to the North East Pacific Culture Collection protocol (http://www3.botany.ubc.ca/cccm/NEPCC/esaw.html) at 18°C under constant light. Where indicated, NaNO₃ was omitted from the medium (nitrogen-free medium) or replaced by 0.55 mM NH₄Cl (ammonium medium).

All primer sequences can be found in Table S1. Plasmid pX330 encoding N-terminally FLAG tagged Cas9 with nuclear localization sequences, and the guide RNA under mammalian promoters, as previously published [3] (link)–[5] (link),was generously provided by Tim Wang. The TgTUB1 promoter was cloned into the KpnI and NcoI sites upstream of Cas9 using primers P1 and P2. Constructs with protospacers against SAG1 (pU6-SAG1) and PKG (pU6-PKG), as well as a universal plasmid encoding BsaI sites in place of a protospacer, were synthesized by IDT (Figure S1). These constructs were amplified using P3 and P4, digested with NcoI and XbaI, and cloned into the PciI and XbaI sites of the Cas9-encoding plasmid. The construct targeting the CDPK3 locus was generated by annealing oligos P25 and P26, phosphorylating the duplex, and cloning it into the BsaI-digested universal plasmid. The universal plasmid has been deposited with Addgene (ID no. 52694).
Oligos used to facilitate homologous recombination in PKG and CDPK3 were generated by heating complementary 90- or 119- nucleotide oligomers ordered from IDT to 99 degrees for 2 minutes in a heat block, then removing the block from the heating apparatus and allowing the DNA to cool to room temperature over the course of a few hours. The sequences for these oligomers are listed in Table S1 as P5 and P6 for the PKG oligo and P7 and p8 for the CDPK3 oligo.
The plasmid used to generate the allelic replacements of PKG was made by amplifying the 5′ end of the locus with P17 and P18 from RH genomic DNA and the 3′ end of the gene with P19 and P20 from RH cDNA. The two fragments were spliced together with AvrII and cloned into the PacI/AscI sites of pLIC-YFP-HXGPRT (provided by V. Carruthers, University of Michigan, USA), thus removing the YFP and introducing the two fragments. The gatekeeper residue in this construct was changed to code for T⁷⁶¹M using site-directed mutagenesis.
Control plasmid 1 used in viability experiments was constructed by PCR-amplifying the pyrimethamine resistance cassette from pDHFR-TS [6] (link) using primers P23 and P24, and cloned into the NsiI and SbfI sites of the universal plasmid in place of Cas9. Control plasmid 2 is the universal plasmid. Sequences for both control plasmids are provided in File S1.

Growth inhibition of P. falciparum malaria parasites by S. occidentalis root extract was assessed as described [44 (link)], with minor modification. A ten-fold dilution was prepared from the stock solutions using complete RPMI 1640 and serially diluted 7-fold across a 96-well cell culture plate. This provided a dose-titration range of 250 μg/ml to 1.95 μg/ml for the extracts and 25 μg/ml to 0.20 μg/ml for pyrimethamine. Sorbitol synchronized ring stage parasitized RBCs (1% parasitemia) suspended in complete RPMI at 3% hematocrit was then added to respective wells. Non-infected RBCs as well as DMSO were also included in the assay as controls. The plates were incubated at 37 °C. After 72 hours, thin smears were prepared and parasites quantified by light microscopy. Percentage parasitemia suppression was computed and 50% inhibitory concentration (IC₅₀) values determined for each extract [45 (link), 46 (link)].

A modification of the method by Ryley and Peters (1970) was used to evaluate the curative potency of the extract against P. berghei in mice [53 , 54 (link)]. Mice were inoculated intraperitoneally with 1 × 10⁶P. berghei infected erythrocytes and assigned randomly to 5 experimental groups; A, B, C, D and E (5 mice per group). At approximately 5% parasitemia (day 5 post-infection), treatment was started. Groups A and B were treated with 200 mg/kg and 100 mg/kg of the methanolic extract, respectively. Group C was treated with 200 mg/kg of the aqueous extract. Group D was treated with 1 mg/kg pyrimethamine (Sigma – Aldrich Chemie, Steinheim, Germany). Group E (the placebo group) was administered with 1% DMSO in phosphate buffered saline (vehicle). The treatment was administered orally for 4 consecutive days. The oral route was chosen based on documented ethnomedical usage of the plant [28 (link)]. The mice were monitored for survival till day 30 post infection. Meanwhile, parasitemia suppression was determined. The amount of extract administered was informed by dose recommendations for in vivo administration of crude extracts [55 (link)] as well as previous antimalarial studies involving Senna family [5 (link), 56 (link)].

Senna occidentalis root extracts were tested in vitro for antiplasmodial potency using P. falciparum, strain 3D7 obtained from Kenya Medical Research Institute (KEMRI) repository. A modified version of a previously used method [41 (link)] was utilised to establish a continuous culture of these malaria parasites. In summary, the parasites were propagated at 37 °C in blood group O+ red blood cells (RBCs) maintained in RPMI 1640 growth medium (Life technologies Ltd., Paisley, UK) supplemented with 1 M 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES), (Gibco, Life technologies Ltd., Paisley, UK), 1 M Sodium hydroxide (EMD Millipore corporation, Darmstadt, Germany), 20% D-glucose (PANREAC QUIMICA SA Barcelona, Spain), 200 mM L-glutamine (Gibco, Life technologies Ltd., Paisley, UK), 10% human serum (group O+) and a gas mixture of 90% N₂, 5% O₂ and 5% CO₂. The culture was refreshed every 48 hours at 3% haematocrit.
Dilutions of the extract and standard drug were carried out in the same manner as described [42 (link)], with minor modifications. To make a stock solution (5 mg/ml), each extract was first dissolved in dimethyl sulfoxide (DMSO), vortexed and RPMI 1640 (incomplete medium) added to the required volume. For extract combinations, the extracts were weighed and blended in a 1:1 ratio [43 (link)]. A 0.5 mg/ml stock solution of pyrimethamine was also prepared.