pfexo gene and amino acid prediction
The nucleotide sequence of pfexo from 3D7 was obtained from PlasmoDB with the accession number PF3D7_1362500. The P. falciparum B5 line was obtained from the cloning of a Cambodian P. falciparum isolate [24]. This isolate is ART- and PPQ-sensitive, but CQ-resistant. The full-length pfexo gene of B5 was amplified from P. falciparum B5 genomic DNA using polymerase chain reaction (PCR) with primers PA_exon1 and Screening_3UTR_Rev (Table S1). The encoded amino acid sequences and the nucleotide sequences were aligned using Clustal Omega [30]. The signal peptide region was predicted using Signa-P.5.0 [31] and the InterPro program was used for protein family classification [32].
P. falciparum culture
Asexual blood-stages of P. falciparum were maintained in fresh human erythrocytes (O+) in RPMI1640 (Sigma-Aldrich, USA) supplemented with 5.94 gL-1 HEPES, 2.1 gL-1 sodium bicarbonate, 0.1 gL-1 gentamycin sulphate, 0.5% (w/v) Albumax II, 4 gL-1 dextrose 0.05 gL-1 hypoxanthine, and 10-15% of human serum (complete medium) [33]. Human blood products (erythrocytes and serum) were obtained from the Thai Red Cross. Culture flasks were gassed with 5% CO2, 5% O2, 90% N2 gas and incubated at 37 °C. For synchronisation, mature schizont stage parasites were isolated on cushions of 75% (v/v) isotonic Percoll cushion (GE Healthcare Life Science) as previously described [34, 35] whereas enrichment for ring stages following invasion was performed using 5% (w/v) D-sorbitol [36] The final ring cultures were washed and returned to culture or used as required.
Construction of plasmid constructs to genetically modify P. falciparum
PCR amplicons used in plasmid cloning were generated using Fusion, High Fidelity DNA polymerase (New England Biolabs) and purified using Qiagen PCR purification or Qiagen Gel extraction kits. For diagnostic PCR amplification, GoTaq (Promega) DNA master mix was used. All constructed plasmids were sequenced to verify authenticity (Biobasic, Canada). For parasite genomic DNA extraction, total cell pellets were first treated with 0.15% saponin in PBS for 10 min, then washed with PBS before DNA was extracted using a DNeasy Blood & Tissue Kit (Qiagen).
Construct for donor plasmids
A fragment of exo sequence with E415G mutation (1,441 bp) was commercially synthesised (GenScript, USA), comprising a stretch of native P. falciparum 3D7 exo sequence (covering residues Lys283, exo intron, and Asn408) followed by a stretch of recodonized exo gene sequence encoding the 3D7 amino acid residues Met409 to Glu514 with the point mutation at residue 415 from Glu to Gly but using a different codon usage, and finally a stretch of native 3D7 exo sequence covering amino acid residues 515 to the stop codon. This fragment was cloned into pUC57 by EcoRV generated pUC57-Exo-E415G plasmid. To generate a donor plasmid to introduce Glu415Glu wild-type, pUC57-Exo-E415E plasmid was derived from pUC57-Exo-E415G by Q5 site-directed mutagenesis (New England Biolabs) to alter the codon encoding at amino acid position 415 from GGC to GAA primers Q5SDM_G415E_F and Q5SDM_G415E_R were used (Table S1).
Construct for CRISPR-Cas9 plasmids
pDC2-Cas9-hDHFR-yFCU, containing a Cas9 expression cassette and the drug selection marker human dihydrofolate reductase (hdhfr) [37] was used as a template vector to generate pDC2-Cas9-bsd-yFCU. Since the P. falciparum B5 strain is resistant to pyrimethamine, the hdhfr gene was replaced with the blasticidin gene (bsd). The bsd gene (399 bp) was amplified from PkpSKIP_Pk47 plasmid [38] using primers bsd_NcoI_F and bsd_SacII_R (Table S1) and was cloned into the NcoI/SacII-digested pDC2-Cas9-hDHFRyFCU, giving rise to pDC2-Cas9-bsd-yFCU.
Guide RNA sequences specific for targeting at amino acid position 415 were identified using Benchling (https://www.benchling.com/crispr/) (Fig. S1). A pair of complementary oligonucleotides (sgE415G-1F and sgE415G-1R) corresponding to the 19 nucleotides adjacent to the identified PAM sequences were phosphorylated using T4 polynucleotide kinase, annealed and ligated into pDC2-Cas9-bsd-yFCU predigested with BbsI, resulting in the guide vector pGuide1.4-bsd.
Generation of P. falciparum lines expressing exo-E415G mutant
The donor plasmid pUC57-Exo-E415G was linearized with ScaI prior to electroporation. Percoll-enriched mature schizonts of P. falciparum B5 were electroporated with 20 µg of pGuide1.4-bsd and 60 µg of linearized pUC57-Exo-E415G using either Amaxa P3 primary cell 4D Nucleofector X Kit L (Lonza) or Amaxa Basic Parasite Nucleofector Starter Kit as described [39, 40]. Twenty-four hours post-transfection, the electroporated parasites were treated with 5.45 µM blasticidin-S-hydrochloride (Sigma-Aldrich, USA) to select for transfectants harbouring pGuide1.4-bsd before returning the cultures to medium without drug. Detection of the exo-E415G modified locus was carried out by diagnostic PCR using primer pairs ExonI_K283_F and Screen_WT_Rev_V616, ExonI_K283_F and Recodon_R, and Recodon_F and Screen_3´UTR_R. The wild-type pfexo locus was detected by diagnostic PCR using primers ExonI_K283_F and Screen_WT_Rev_V616, with a PCR product of 766 bp. Transgenic parasite clones were obtained by limiting dilution cloning by plating a calculated 0.3 parasite per well (200 µL and 1% haematocrit) in flat-bottomed 96-well microplate wells as described [41]. Wells containing single plaques were identified after 10-14 days using an inverted microscope and the parasites subsequently expanded into round-bottomed wells for further analysis. Transgenic parasite clones were finally checked by diagnostic PCR for integration and modification of the endogenous pfexoE415G gene. A pfexoE415E transgenic line expressing the wild-type Glu415 codon was generated in the same manner of that for a pfexoE415G transgenic line by transfecting P. falciparum B5 with 20 µg of pGuide1.4-bsd and 60 µg of linearized pUC57-Exo-E415E. Once established, all transgenic clones were maintained in medium without any drug.
pfk13, pfcrt, and pfexo genotyping
Master Cycler Nexus Gradient (Eppendorf) was employed to evaluate the propeller domain of the P. falciparum kelch13 (pfk13) (amino acid residues 442-727) [42, 43], P. falciparum exonuclease (pfexo) SNP at a codon corresponding to the amino acid position 415 [13], and the P. falciparum chloroquine resistant transporter (pfcrt) SNPs at codons corresponding to amino acid positions 93, 97, 145, 218, 343, 350, and 353 [19, 44]. Primers used to identify pfk13, pfexo, and pfcrt SNPs are shown in Table S1. P. falciparum reference DNAs from 3D7 and W2 clones (Malaria Research & Reference Reagent Resource, Manassas, VA) were used as positive controls, and all samples were performed in duplicate.
Plasmepsin (pfpm) 1, 2 and 3 Copy number variation assay
To determine copy numbers of pfpm1 (PF3D7_1407900), pfpm2 (PF3D7_1408000), and pfpm3 (PF3D7_1408100) gene, real-time quantitative PCR (qPCR) was performed on genomic DNA as previously described [29]. The amplification reactions were performed according to Lunaâ Universal qPCR master mix kit (New England Biolabs) with 200 nM of each forward and reverse primer (Table 3) and 2 ng of DNA template using Rotor-Gene Q (QIAGEN, Valencia, CA). For the housekeeping gene, b-tubulin (PF3D7_1008700), b-tubulin forward and reverse primers were designed and used as a reference control for all experiments with the same validated PCR conditions as target primers. P. falciparum 3D7 was used as a reference clone. All samples including the references clones were performed in triplicate. The average copy number values for each gene were calculated using 2−ΔΔCt method where ΔΔCt is [Ct pfpm - Ct pf β-tubulin] sample - [Ct pfpm2 - Ct pf β-tubulin] 3D7. Parasites with copy number greater than 1.6 copies for pfpm2 [15] were interpreted to contain multiple copies.
Plasmepsin 2/3 (pfpm2/3) duplication breakpoint PCR assay
The pfpm2/3 breakpoint PCR assay was performed as previously described [45]. Three pairs of primers (Table 3) were used in this assay. Primers AF_for and AR_rev amplified a 623 bp product surrounding the breakpoint located at the 3′ end of pfpm1. Primers BF_for and BR_rev amplified a 484 bp product surrounding the breakpoint at the 3′ end of pfpm3. Primers BF_for and AR_rev amplified the junction between the breakpoint, producing a 497 bp product in parasite isolates with pfpm2/3 amplifications. A pfpm2/3 single copy isolate is not expected to have the PCR product with these primers. One copy isolate was only noted when the control primer sets amplified a product, and the duplication PCR was negative. Two or more copies were annotated as > 1 copy of pfpm2/3 only when both the control and duplication primer sets generated a product. PCR reactions contained 12.5 µL GoTaqÒ Green Master Mix (Promega), 1 µL of each primer (10 µM stocks), 3 µL of DNA up to 25 µL final volume with water. PCR conditions were as follow: 95°C for 2 min, followed by 30 cycles of 95°C for 45 s, 50°C for 30 s, 72°C for 1 min, followed by a 5-min extension at 72°C.
In vitro drug susceptibility
Drug susceptibility testing used HRP-2 ELISA to measure 50% or 90% inhibitory concentration (IC50 and IC90) performed as previously published [46, 47]. In vitro drug susceptibility testing was carried out for control reference clones (W2, D6, C2B) (Malaria Research & Reference Reagent Resource, Manassas, Vermont, USA) as described previously [48]. IC50s and IC90s were estimated by nonlinear regression analysis using GraphPad Prism version 6.0 program. Samples having poor growth rate, as perceived by obtaining an OD ratio of < 1.7 between the no-drug test wells and the maximum tested drug concentration, were excluded from data analysis.
Bimodal dose response curve
To determine a bimodal-dose response curve, the PPQ concentration (2.44 to 100,000 ng/mL) and the dilution series were increased from 8 to 24 points, according to previously published reports [16, 24]. Culture-adapted clinical isolates or engineered parasites were prepared in the similar manner as in in vitro drug susceptibility testing. The synchronized rings were grown for 72 hours in the presence of different concentrations of PPQ (24-point dilution) in 96-well plates at 1.5% haematocrit, 0.5% starting parasitaemia in 0.5% Albumax RPMI 1640. Growth at 72 hours was measured by HRP-2 ELISA. Assays were carried out in three biological replicates and the control reference clone W2 was tested along with each culture-adapted clinical isolate. The area under the curve (AUC) for the dose response curve at 0.01 - 100 µM was calculated using GraphPad Prism 6.0.
Ring-staged survival assay (RSA)
In vitro RSA0-3 h was performed on 0-3 h post-invasion rings obtained from selected culture-adapted clinical isolates following published methods [12] with slight modifications. Briefly, parasite cultures were tightly synchronized using 5% (w/v) D-sorbitol and 75% Percoll to obtain 0 to 3-h post-invasion rings which were adjusted to 0.5-1% starting parasitaemia with a 2% haematocrit in culture media. (0.5% Albumax RPMI 1640 with 2.5% AB serum) and cultured in a 48-well microplate with 700 nM DHA and 0.1 % DMSO in separate wells for growth control. The culture plate was then incubated for 6 hours at 37 °C in modular incubator chambers and gassed with 5% CO2, 5% O2 and 90% N2 gas. Cells were then washed once, resuspended in drug-free medium, and cultured further for 66 hours. Susceptibility to DHA was assessed microscopically on thin films by estimating the percentage of viable parasites, relative to control (% survival rate). For the controls, the RSA0-3h was also performed on P. falciparum reference clones W2 (ART-sensitive control), IPC-4884 and IPC-5202 (BEI Resources, NIAID, NIH, USA) as ART-resistant control lines. A survival rate > 1% was deemed resistant for RSA.
Piperaquine survival assay (PSA)
PSA0-3h was performed on culture-adapted clinical isolates with 0–3-hour ring-stage parasite cultures following a previously published method [17]. Briefly, parasite cultures were tightly synchronized using 5% (w/v) D-sorbitol and 75% Percoll to obtain 0 to 3-hours post-invasion. Synchronized ring parasites at 0.5-1% starting parasitaemia and 2% haematocrit were incubated with 200 nM PPQ or 0.5% lactic acid in water at 37 °C for 48 hours in a 48-well microplate. The cultures were then washed once, resuspended in drug-free medium, and cultured further for 24 hours. Susceptibility to PPQ was assessed microscopically on thin films by estimating the percentage of viable parasites in the similar manner as RSA. A survival >10% was deemed resistant to PPQ.
Growth Assays
For longer-term replication assays, cultures were synchronized as described and resulting in ring stage cultures. The ring stage parasites were synchronized using 5% (w/v) D-sorbitol and parasitaemia levels were calculated, and cultures adjusted to 0.1% parasitaemia, 2% haematocrit in a final volume of 1 mL per well of a 12-well plate. Samples were then taken at t = 0, 24, 72, 120, and 168 h, fixed in 0.8% glutaraldehyde in PBS and stored at 4°C for flow cytometry analysis. Culture media was replaced at 48, 71, and 120 h. Giemsa-stained thin films were also prepared as required for microscopic analysis.
Flow cytometry for parasite quantification
Parasite samples were fixed in 0.8% glutaraldehyde in PBS and stored at 4°C. Cells were prepared for analysis by staining with 2X SYBR Green I nucleic acid gel stain (Invitrogen, Thermo Fisher Scientific) for 30 min at 37°C. Labelling was stopped with an equal volume of PBS and samples acquired using a CytoFlex (Beckman Coulter, USA) with CyEXpert software. Total RBC numbers were calculated using forward- and side-scatter while fluorescence was detected using the 530/630 blue detection laser. Fluorescence intensity was used to distinguish uninfected from infected RBCs, low fluorescence indicating uninfected cells and gating fixed accordingly. Data were analysed using FlowJo.
Statistical analysis
Statistical analysis was performed using GraphPad Prism version 6.0 (GraphPad Software, Inc., San Diego, CA, USA). The difference of the data between groups was assessed by nonparametric Mann-Whitney test, as appropriate. Statistical significance was defined as a P value of < 0.05.