A single point mutation in the Plasmodium falciparum 3′–5′ exonuclease does not alter piperaquine susceptibility

Background The rise in Plasmodium falciparum resistance to dihydroartemisinin–piperaquine (DHA–PPQ) treatment has been documented in the Greater Mekong Subregion with associations with mutations in the P. falciparum chloroquine resistance transporter (pfcrt) and plasmepsin 2 (pfpm2) genes. However, it is unclear whether other genes also play a role with PPQ resistance, such as the E415G mutation in the exonuclease (pfexo) gene. The aim of this study was to investigate the role of this mutation in PPQ resistance by generating transgenic parasites expressing the pfexo-E415G mutant allele. Methods Transgenic parasite clones carrying the E415G mutation in PfEXO of the B5 isolate were derived by CRISPR-Cas9 gene editing and verified using PCR and gene sequencing. Polymorphisms of pfkelch-13, pfcrt, and pfexo were examined by PCR while the copy number variations of pfpm2 were examined by both relative quantitative real-time PCR and the duplication breakpoint assay. Drug sensitivity against a panel of antimalarials, the ring-stage survival assay (RSA), the PPQ survival assay (PSA), and bimodal dose-response curves were used to evaluate antimalarial susceptibility. Results The transgenic line, B5-rexo-E415G-B8, was successfully generated. The PPQ-IC90, %PPQ survival, and the bimodal dose-response clearly showed that E415G mutation in PfEXO of B5 isolate remained fully susceptible to PPQ. Furthermore, growth assays demonstrated that the engineered parasites grew slightly faster than the unmodified parental isolates whereas P. falciparum isolates harbouring pfkelch-13, pfcrt, and pfexo mutations with multiple copies of pfpm2 grew much more slowly. Conclusions Insertion of the E415G mutation in PfEXO did not lead to increased PPQ-IC90 and %PPQ survival, suggesting that this mutation alone may not be associated with PPQ resistance, but could still be an important marker if used in conjunction with other markers for monitoring PPQ-resistant parasites. The results also highlight the importance of monitoring and evaluating suspected genetic mutations with regard to parasite fitness and resistance. Supplementary information The online version contains supplementary material available at 10.1186/s12936-022-04148-z.

While the implication of multiple copies of pfpm2 and novel PfCRT mutations on PPQ resistance have been elucidated [19,20,29], and the PPQ-resistance phenotype can be observed in the presence of novel PfCRT mutations with the PfEXO-E415G mutation [24], there is still a lack of confirmation as to whether the PfEXO-E415G mutation alone causes PPQ-resistance. To investigate whether the PfEXO-E415G is directly associated with PPQ resistance, in this study, CRISPR-Cas9 genome editing was used to generate P. falciparum Cambodian parasites harbouring PfEXO-E415G. The in vitro susceptibility to piperaquine and other frontline antimalarials of these gene-edited parasites was determined and compared to those of validated PPQ-resistant P. falciparum clinical isolates of Cambodian origin.

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 CQresistant. 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_3'UTR_Rev (Additional file 1: 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].

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 (1441 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 (Additional file 1: Table S1).

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]. Twentyfour 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 pfexo E415G gene. A pfexo E415E transgenic line expressing the wild-type Glu415 codon was generated in the same manner of that for a pfexo E415G transgenic line by transfecting P. falciparum B5 with 20 µg of pGuide1.4bsd and 60 µg of linearized pUC57-Exo-E415E. Once established, all transgenic clones were maintained in medium without any drug.

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 Luna ® Universal qPCR master mix kit (New England Biolabs) with 200 nM of each forward and reverse primer (Additional file 1: Table S1) and 2 ng of DNA template using Rotor-Gene Q (QIAGEN, Valencia, CA). For the housekeeping gene, β-tubulin (PF3D7_1008700), β-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 Parasites with copy number greater than 1.6 copies for pfpm2 and pfpm2 [15,16] 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 (Additional file 1: Table S1) 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 GoTaq ® 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 (IC 50 and IC 90 ) 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]. IC 50 s and IC 90 s 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 h in the presence of different concentrations of PPQ (24point dilution) in 96-well plates at 1.5% haematocrit, 0.5% starting parasitaemia in 0.5% Albumax RPMI 1640. Growth at 72 h 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-stage survival assay (RSA)
In vitro RSA 0-3 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 h at 37 °C in modular incubator chambers and gassed with 5% CO 2 , 5% O 2 and 90% N 2 gas. Cells were then washed once, resuspended in drug-free medium, and cultured further for 66 h. 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 RSA 0-3 h 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)
PSA 0-3 h was performed on culture-adapted clinical isolates with 0-3-h 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-h 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 h in a 48-well microplate. The cultures were then washed once, resuspended in drug-free medium, and cultured further for 24 h. 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. Giemsastained 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 2× 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 U test, as appropriate. Statistical significance was defined as a P value of < 0.05.

CRISPR-Cas9-mediated editing to introduce pfexo E415G into P. falciparum parasites
The pfexo gene from P. falciparum 3D7 (PF3D7_1362500) consists of 2274 bp with 2 exons and 1 intron. Exon 1 comprises nucleotides 1-949, while the exon 2 starts from nucleotides 1082-2274 (Additional file 1: Fig. S1). PfEXO consists of 713 amino acids with a molecular mass of 86.7 kDa. The enzyme contains a peptide signal region (residues 1-70) and a 3′ to 5′ exonuclease domain (residues 445-618). The DNA sequence of the pfexo gene from P. falciparum B5 [24], the parental line for genome editing in this study, showed a single non-synonymous mutation, leading to a missense mutation from Lys to Asn at residue 614 (Additional file 1: Fig. S2).
To evaluate the impact of the PfEXO-E415G mutation on parasite susceptibility to piperaquine and other frontline chemotherapies, CRISPR-Cas9 editing was used to introduce the E415G mutation into the native pfexo gene (Fig. 1A). A pfexo E415G was efficiently installed onto the P. falciparum B5 background [24]. In parallel, a pfexo E415E transgenic line expressing wild-type E415 codon in the context of recodonization abating the Cas9 protospacer adjacent motif (PAM) site was also attempted to rule out a role for silent mutations in drug susceptibility. Eleven transfections were performed to introduce pfexo E415G mutation and 3 transfections for pfexo E415E mutation but only three transfections provided revived parasites. Two of the three transfections gave rise to the modified pfexo gene, and one transfection did not provide the modified parasites. Successful modification of the pfexo gene in the transfected population following the introduction of the targeting vector was observed around 21 days after transfection and confirmed by diagnostic PCR (Fig. 1B, pre-cloning). Limiting dilution cloning of the modified parasites resulted in the isolation of parasite clone, B5-rexo-E415G-B8. It is noted that several clones obtained from the cloning did not contain the modified region of the pfexo gene. Modification of the native pfexo locus was then confirmed in the transgenic line by diagnostic PCR (Fig. 1B, B5-rexo-E415G-B8) and genomic sequences of transgenic lines (Fig. 1C). It is noted that the band of 828 bp appearing on lane 2 (Fig. 1B) arose from the non-specificity of primer 3 which can anneal to both parental and transgenic parasite lines. Unfortunately, the pfexo E415E transgenic line could not be obtained from this study.

Molecular genotyping of modified pfexo transgenic lines and clinical isolates with PfEXO-E415G mutation
The genotypic profiling for piperaquine and artemisinin molecular markers is outlined in Table 1. In addition to the reference clones and engineered parasite lines, two Cambodian isolates from a previous P. falciparum clinical efficacy study (ASAP-21 and ASAP-168) [49] were included as they harboured PfEXO-E415G with and without novel PfCRT mutations. As expected, the transgenic B5-rexo-E415G-B8 line carried a similar genotypic profile as the parental B5 line except for the E415G mutation in the pfexo gene. Regarding the two clinical isolates, both carried the PfK13-C580Y and PfEXO-E415G mutations. The ASAP-21 isolate harboured the novel PfCRT -F145I mutation while ASAP-168 did not. Genetic studies have identified copy number variations in the pfpm2 and pfpm3 genes that associate with clinical and in vitro PPQ resistance [13,15]. For pfpm2/3 amplification, the putative breakpoints lie near the 3′ end of both pfpm1 and pfpm3 so each amplification produces an intact extra copy of pfpm2 together with a new chimeric pfpm3 with its 3′ end replaced by the 3′ end of pfpm1 (Additional file 1: Fig. S3). This result showed that the copy number variation determined by qPCR assay showed that all parasite lines harboured a single copy of pfpm1. Two methods were then carried out to determine the copy number variation of the pfpm2 and pfpm3 gene: the SYBR-green qPCR and breakpoint assays [45]. By using the qPCR assay, ASAP-21 was found to contain multiple copies of pfpm2 and ASAP-168 carried a single copy of pfpm2, whereas B5 parental line and transgenic B5-rexo-E415G-B8 were found to have multiple copies of pfpm3. On the contrary, both ASAP-21 and ASAP-168 isolates were positive for pfpm2/3 amplification (>1 copy) while B5 and B5-rexo-E415G-B8 lines showed no pfpm2/3 amplification as detected by the breakpoint assay (Additional file 1: Fig. S3). The non-concordant detection between the two methods were also reported by Ansbro et al. [45] and it was suggested that the breakpoint PCR assay is more sensitive than the qPCR assay for detecting minor clones containing the duplication in field isolates. Therefore, in this study, ASAP-21 and ASAP-168 isolates were treated as having multiple copies of pfpm2/3 amplification, while B5 and B5-rexo-E415G-B8 lines contained a single copy of pfpm2 and pfpm3.

Drug sensitivity and survival assay of modified pfexo transgenic lines and clinical isolates
The transgenic parasites expressing E415G mutant of PfEXO and the clinical isolates were tested for their PPQ susceptibility (      To evaluate if the modified pfexo transgenic parasites and clinical isolates with PfEXO-E415G mutation have cross-resistance, parasite drug sensitivity was also assessed against a panel of antimalarial drugs (Table 3). No significant difference in IC 50 values between B5 and B5-rexo-E415G-B8 parasites were observed for most of antimalarial drugs except for atovaquone (ATG) and proguanil (PG), where higher IC 50 values were detected in comparison with the B5 parental line. The ASAP-21 parasite had significantly lower IC 50 values for quinine (QN), chloroquine (CQ), atovaquone (ATQ) and doxycycline (DOX) but higher IC 50 value for pyronaridine (PND) compared to the B5 parental line, while the ASAP-168 parasite had increased drug sensitivity against dihydroartemisinin (DHA), mefloquine (MQ), QN, and ATQ. The reduced IC 50 values of QN and ATQ were observed in both ASAP-21 and ASAP-168 parasites compared to the B5 parental line.
To gain a better understanding of the ART and PPQ resistance phenotypes, the survival assay (RSA 0-3 h and PSA 0-3 h ) and PPQ-bimodal dose response curve were performed (Fig. 2). For RSA 0-3 h (Fig. 2A), P. falciparum W2 was used as a control of ART-sensitive parasites, while IPC-4884 and IPC-5202 parasites were controls for ART-resistant parasites. The ASAP-21 and ASAP-168 parasites, containing the PfK13-C580Y mutation, exhibited % RSA survival rate of greater than 1, while B5 and B5-rexo-E415G-B8 parasites, carrying wild-type PfK13, exhibited % RSA survival rate of less than 1, clearly validating the correlation between PfK13-C580Y mutation and ART resistance. The % PSA survival rate of the modified pfexo transgenic lines and clinical isolates were next examined (Fig. 2B). PPQ-sensitive parasites P. falciparum W2, IPC-4884 and IPC-5202 were used as controls. The transgenic B5-rexo-E415G-B8 parasites harbouring merely PfEXO-E415G mutation had a % PSA survival rate of less than 10 (a cut-off for PPQ resistance), similar to that of the parental B5 parasites. This suggested that the presence of PfEXO-E415G mutation alone could not confer the PPQ resistance phenotype. The ASAP-21 and ASAP-168 parasites, containing the PfEXO-E415G mutation in combination with either PfCRT mutation or multiple copies of pfpm2, exhibited a % PSA survival rate higher than 10, indicative of PPQ-resistance.
In addition to PSA 0-3 h , PPQ-resistant parasites have been reported to exhibit a bimodal dose-response curve with a second peak between 0.01 and 100 µM and that the AUC correlates with the degree of PPQ resistance [16]. B5 and B5-rexo-E415G-B8 parasites did not exhibit the bimodal dose-response curve, confirming the PPQsensitive phenotype, whereas both ASAP-21 and ASAP-168 parasites clearly showed the bimodal dose-response curve with the AUC of 1029 and 596, respectively ( Fig. 3C-F). The AUC of these parasites was in good agreement with both IC 50 and IC 90 values in that ASAP-21 parasites are more resistant to PPQ than ASAP-168 parasites.

Discussion
Validated molecular markers, such as pfk13 for ART resistance, have been widely employed as identification and prediction tools for the emergence of drug-resistant were synchronized (initiated at 0.1% parasitaemia ring stage, 2% haematocrit) and followed for 168 hours. Nucleic acids were stained with SYBR Green I and parasitaemia assessed by flow cytometry. Raw values were corrected, and data represent mean ± S.D. from 3 independent experiments P. falciparum [50]. While a molecular marker of PPQ resistant P. falciparum malaria has been reported [13,15,16,19,20,29], there is still a pressing need for validating potential new or additive molecular markers to determine effects on PPQ as a partner drug in ACT. This study found that the E415G mutation in the gene pfexo cannot alone reduce PPQ susceptibility neither improve parasite survival rates that are characteristic of PPQ resistant isolates circulating in the Greater Mekong Subregion [17].
Exonucleases are essential to genome stability, catalysing the removal of a single nucleotide monophosphate (dNMP) from the end of one strand of DNA and acting as a proof-reader during DNA replication [51]. Exonucleases are highly conserved and can be classified into families based on both sequence and functional homology, including the 5′-3′ exo C-terminal domain (CTD) superfamily, the RNAaseH domain superfamily i.e., DnaQ-like family and other 3′-5′ exonucleases. The pfexo gene from P. falciparum (PF3D7_1362500) encodes a protein with a mass of 86.7 kDa with a 3′-5′ exonuclease domain at the C-terminus. In this study, the P. falciparum B5 line used for genome editing had one mutation (K614N) in exonuclease, located in the 3′-5′ exonuclease domain. Since no in vivo relevance of this mutation has yet been proved in this study, it is suggested that the prevalence of this mutation should be determined. Nucleases may be partially or fully redundant, depending on the pathway, and such redundancy might complement functional losses. Zhang et al. [52] used bioinformatics to predict eight putative RNA exosome-associated proteins in the P. falciparum genome, including exoribonuclease functional domain-containing proteins Dis3 and Rrp6, the latter of which was found in PfEXO of this study.
CRISPR/Cas9 approaches have transformed the speed and scale with which Plasmodium genome editing can be achieved, and the approach has been employed to introduce point mutations into several P. falciparum genes [53]. In this study, instead of using P. falciparum 3D7 or Dd2 lines, the Cambodia adapted-B5 line [24] was employed with the hypothesis that the B5 line has a close genetic background to the currently circulating P. falciparum in Cambodia. It has been previously shown that different phenotypes were observed when using different parasite backgrounds. Targeted gene disruption of either pfpm2 or pfpm3 in the 3D7 genetic background caused a only slight decrease in PPQ susceptibility [54] and pfpm2 and pfpm3 overexpression in 3D7 did not alter the sensitivity of P. falciparum to PPQ [55]. However, when P. falciparum Dd2 parasites with copy number variation in pfpm2 were generated [29], pfpm2 amplification contributed to PPQ resistance, with a bimodal dose-response observed. In addition to a qPCR assay to detect pfpm2 copy number variation, the breakpoint assay, as described by Imwong et al. [56], was carried out. It was found that in parasites with a pfpm2/3 copy number above the cut-off of 1.52, 88% were confirmed to have pfpm2/3 amplification by the breakpoint assay, while for those < 1.14 (the cutoff for a single copy number) and for an intermediate value between 1.14 and 1.52, the proportion of pfpm2/3 amplified parasites using the breakpoint SNP were 4% and 38%, respectively. In this study, the ASAP-168 parasite was found to have pfpm2 copy number variation of 1.11, lower than the cut-off for a single copy number, but the breakpoint assay confirmed the gene duplication of pfpm2. As for pfpm3 copy number variation, the detection by qPCR was not 100% concordant with and pfpm2/3 breakpoint assay for B5 and B5-rexo-E415G-B8 lines as qPCR assay showed the pfpm3 multiple copy for Cambodia isolates, which was different from the previous report by Ansbro et al. [45]. It is not lost on the authors that different primers were used to identify the copy number of pfpm3 and further investigation could be done. Therefore, it is important to also apply the breakpoint assay to isolates with copy number values < 1.52 for pfpm2, to capture all isolates with pfpm2/3 amplification.
IC 90 values, PPQ survival rates, and bimodal doseresponse curves were used for assessing PPQ resistance in vitro [16,17,48]. All three assays confirmed that the engineered parasites harbouring the E415G-PfEXO mutation (B5-rexo-E415G-B8) did not show a PPQ resistant phenotype. On the contrary, parasites with the combination of PfEXO-E415G mutation with either novel PfCRT mutations or pfpm2 multiple copies demonstrated reduced IC 90 susceptibility, high PPQ survival rates, and a second peak of bimodal curve (Fig. 3). If the PfEXO-E415G mutation does not alter the PPQ susceptibility, this finding also recapitulates the work of Silva et al. [29] that parasites with the pfpm2 multiple copy alone (i.e. ASAP-168) show reduced PPQ susceptibility and involved in PPQ resistance. Nonetheless, when the novel PfCRT mutation was added on top of the pfpm2 multiple copies, the level of PPQ resistance increased tremendously as judged by the IC 50 and IC 90 values as well as AUC between 0.01 and 100 µM of PPQ in the bimodal dose-response curve. Bopp et al. [16] showed that when exposed up to 10 µM PPQ for 12 h, PPQ resistance parasites could survive and complete their lifecycle. It was also previously evident that the introduction of novel PfCRT mutations resulted in a fitness cost for the mutations [19,20]. Similar trends were observed in this study; parasites holding either novel PfCRT mutations or pfpm2 multiple copies grew much slower than those without mutations. It is likely that introducing PfEXO-E415G mutations may account for better fitness compared to parasites with the same genetic background. Even though the scope of this work could not address the function of PfEXO, it is evidently showed that the E415G mutation in PfEXO does not alter PPQ susceptivity, but rather affects parasite fitness.

Conclusions
In summary, this study suggests that the E415G mutation in PfEXO could still be an important marker if used in conjunction with other markers. The insertion of the PfEXO-E415G mutation did not lead to an increased PPQ-IC 90 or improve %PPQ survival, suggesting that this mutation is not associated with PPQ resistance. Additionally, this specific mutation resulted in parasites that grew better than those with the same background, highlighting the importance of genetic mutations toward parasite fitness and resistance.