tRNA modification reprogramming occurs in ART-R parasites post-drug

To assess whether ART-resistant (ART-R) parasites differentially alter their tRNA modification profiles as compared with ART-sensitive (ART-S) lines in response to a pulse of dihydroartemisinin (DHA), we used a modified, large-scale RSA13, which measures the survival of newly invaded intra-erythrocytic ring-stage parasites exposed to a brief, 6 h pulse of the ART active metabolite, DHA. This assay was combined with previously described workflows to quantify tRNA modifications and link them to proteomic changes and codon use bias (Fig. 1a)62. We selected the Asian, ART-S Dd2 parasite (expressing the wild-type (WT) k13 allele with silent binding-site mutations) and its isogenic, ART-R Dd2R539T line that expresses the K13 R539T variant7,12,23. Initial RSAs confirmed the ART resistance phenotype in Dd2R539T parasites, with a survival level of 25% at 24 h post-DHA treatment, as compared with <1% survival in Dd2 parasites, consistent with earlier reports7,23 (Extended Data Fig. 1). Dd2R539T parasites that survived DHA treatment remained as ring stages after 24 h.

Fig. 1: ART-R parasites differentially alter their tRNA modifications in response to ART stress.

a, The workflow for data generation and integration to assess tRNA modification and proteomic changes as well as codon bias translation. Isogenic, edited Dd2 (ART-S harbouring K13-silent binding-site mutations) and Dd2R539T (ART-R K13 R539T mutant) P. falciparum (Pf) parasites7 were sorbitol synchronized to early ring stages (0–6 hpi) then pulsed with either 700 nM DHA or 0.1% DMSO. For tRNA experiments, samples were collected at 0 and 6 h post-exposure. For proteomics, samples were collected at 0 h and 12 h, with the drug having been removed by wash offs at 6 h. tRNA molecules were purified and modifications analysed by LC–MS/MS. Proteomics was performed using TMT-tagged samples and LC–MS/MS (Methods). Codon bias analysis was run using a codon-counting algorithm and further analysed by principal component analysis. These data were combined to identify particular modification changes that led to codon bias changes. Findings were validated using a cKD of the tRNA 2-thiouridylase PfMnmA. b,c, Changes in the relative quantities of modified ribonucleosides, as quantified by LC–MS/MS in total tRNA extracted from parasites at the timepoints indicated in a. Average fold-change values (range −0.8 to 2.7) were calculated for DHA treatment versus DMSO treatment of the Dd2R539T or Dd2 parasites (relative to t = 0 values) (b) or Dd2R539T parasites versus Dd2 parasites for either DMSO treatment or DHA treatment (c). The results were subjected to hierarchical clustering analysis (log2 transformed data). n = 7 independent biological replicates. Statistics were performed using two-tailed t-tests on data normalized to t = 0, *P < 0.05% (Source data). D, dihydrouridine; Y, pseudouridine. d, A schematic of the tRNA secondary structure with location of key modifications. Wobble positions 34–36 are shown in red, position 37 is shown in purple and position 32 is shown in green.

Source data

To examine tRNA modifications, tRNA from highly synchronized early ring-stage (0–6 h post invasion (hpi)) parasites was prepared and purified at t = 0 and after a 6 h pulse of 700 nM DHA or dimethylsulfoxide (DMSO) vehicle control (Fig. 1a). tRNA modifications were analysed using liquid chromatography-coupled mass spectrometry (LC–MS/MS)62. Lines were cultured simultaneously for each biological replicate to minimize variation in temperature, nutrient supply and other stressors41.

We detected 27 tRNA modifications with high confidence, similar to the 28 detected in earlier P. falciparum profiling62. To standardize analyses, all modification levels were normalized to t = 0 for each line and biological replicate. First, we examined whether tRNA modifications differed between DHA or DMSO treatments, for both Dd2 and Dd2R539T parasites (Fig. 1b). ART-S Dd2 parasites had minimal changes in their tRNA modification levels after DHA exposure relative to DMSO, with only mcm5s2U, mcm5Um and m6,6A showing evidence of a slight increase after DHA treatment compared with DMSO. In marked contrast, Dd2R539T parasites had a global decrease in tRNA modifications after DHA treatment relative to DMSO. Significant decreases (P < 0.05, two-tailed Student’s t-test) were observed with ncm5U, m5C, m5U, mcm5U and m1G. Two other modifications, mcm5s2U and mcm5Um, also decreased in DHA-treated Dd2R539T and, notably, increased in DHA-treated Dd2 parasites. This finding suggested differential tRNA reprogramming between the K13 mutant and WT parasites in response to DHA.

To identify changes specific to ART-R parasites after DHA exposure, we compared tRNA modification levels in Dd2R539T versus Dd2 parasites by comparing the ratio of tRNA modifications for Dd2R539T versus Dd2 for each of DMSO and DHA treatments (Fig. 1c). These two lines showed minimal differences after DMSO exposure, with none reaching significance. Nonetheless, two modifications (mcm5Um and m6,6A) increased in DMSO-treated parasites, although these were also observed in DHA-treated parasites, suggesting a drug-independent effect that probably reflects temporal changes. DHA treatment resulted in 12 other modifications that were decreased in Dd2R539T parasites, to an extent greater than observed post-DMSO exposure. Two of these modifications, mcm5s2U and mCm, attained significance (P < 0.05, two-tailed Student’s t-test), suggesting that their targeted reprogramming may be a specific response to DHA treatment in mutant K13 parasites (Fig. 1c). The mcm5s2U modification combines a 5-carboxymethonylmethyl (mcm5) group and a 2-thio (s2) group on the U34 position, with each part of the modification having its own biosynthetic pathway47.

The mcm5s2U modification was of particular interest as (1) it is located on the wobble position 34 of the Lys, Glu and Gln tRNAs (Fig. 1d) and therefore has the potential to alter translation and (2) an earlier study noted that genes involved in 2-thio (s2) biosynthesis were differentially expressed in K13 mutant versus WT isogenic parasites following DHA exposure23. These data suggest that ART-R parasites differentially alter their tRNA modification profile in response to ART stress, raising the possibility that these changes may have a direct link to translation of proteins important for the stress response and/or emergence from quiescence.

ART-R parasites alter their proteome after DHA exposure

We next evaluated changes in the Dd2 and Dd2R539T proteomes after DHA or DMSO exposure. Samples were collected from synchronized ring-stage parasites (0–6 hpi) at t = 0 (Fig. 1a). These parasites were exposed to either 700 nM DHA or DMSO vehicle control for 6 h, washed and allowed to recover in drug-free media until 12 h post-pulse, when they were collected for proteomic analyses (Extended Data Fig. 1). We identified a total of 1,315 proteins based on 40,955 peptide spectral matches (PSMs) across all samples, using quantitative isobaric tags (tandem mass tag (TMT)) with a labelling efficiency >99%. We represented these proteins as a heat map that depicts relative changes at t = 12 for both DHA and DMSO samples compared with the Dd2 t = 0 proteome. Unsupervised data clustering found that in the 12 h samples, compared with the Dd2 t = 0 samples, DHA-treated Dd2R539T and Dd2 parasites showed very similar proteome profiles. In contrast, substantial differences were observed between Dd2 and Dd2R539T in the DMSO controls (Fig. 2a). We then compared our different experimental conditions to ascertain similarities and differences between each proteome (Extended Data Fig. 2).

Fig. 2: The Dd2R539T parasite proteome is differentially altered after DHA exposure.

TMT-tagged proteomics analysis identified 1,315 proteins with 40,955 PSMs from Dd2 or Dd2R539T parasites at 0 h or 12 h after a 6 h DHA or DMSO pulse. Isogenic, edited Dd2 and Dd2R539T parasites7 were highly sorbitol synchronized to early ring stages (0–6 hpi) then pulsed with either 700 nM DHA or 0.1% DMSO. Samples were collected at 0 h and 12 h, with the drug having been removed by wash offs at 6 h (Fig. 1a). a, A heat map of hierarchical clustering analysis of log2-transformed fold changes in the protein levels of each proteome normalized to the Dd2 t = 0 proteome. b–e, Venn diagrams showing unique and common significant proteins and their GO terms in the Dd2 or Dd2R539T parasite proteomes that were upregulated at 0 h (b), upregulated post-DMSO vehicle control (c) and upregulated (d) or downregulated post DHA (e). PTEX, Plasmodium translocon of exported proteins.

Source data

We compared differentially regulated proteins at t = 0 versus 12 h post-DMSO for Dd2 and Dd2R539T parasites. For the t = 0 Dd2 sample, 25 of the 88 proteins enriched compared with the 12 h timepoint had a Gene Ontology (GO) enrichment category of host cell entry (Fig. 2b, Supplementary Table 1 and Supplementary Results). Proteins involved in response to unfolded proteins were significantly upregulated in Dd2R539T parasites at 12 h post-DMSO, but not in Dd2 parasites (Fig. 2c and Supplementary Table 1). We also compared differentially regulated proteins in Dd2 versus Dd2R539T parasites after ART versus DMSO exposure. Dd2R539T parasites showed a strong downregulation in genes involved in translation (Fig. 2d,e, Supplementary Table 1 and Supplementary Results)

To identify the selective response of DHA-treated mutant parasites, we examined proteins differentially regulated in DHA-treated Dd2R539T parasites that did not significantly change in DHA-treated Dd2 parasites. Forty-four proteins were significantly upregulated and were involved in protein refolding and mitochondrial physiology. Of the 70 downregulated proteins, several were involved in translation, with 14 proteins involved in ribosome biogenesis (Supplementary Table 1).

The ART-R parasite proteome displays codon use bias

We tested whether biased use of synonymous codons occurred in the top up- or downregulated proteins identified in Dd2R539T parasites after DHA exposure, as compared with Dd2R539T parasites sampled at t = 0. We excluded proteins that were similarly up- or downregulated at the translational level in DMSO-treated Dd2R539T and/or DHA-treated Dd2, to identify protein changes unique to DHA-treated Dd2R539T parasites. To analyse these data, we employed a codon-counting algorithm to quantify codon usage patterns in the top 44 upregulated and bottom 70 downregulated proteins (that is, proteins >0.5 or <−0.5 log2 fold change in Dd2R539T DHA versus t = 0 samples and between 0.5 and −0.5 log2 fold change for Dd2 DHA versus t = 0 samples). Principal component analysis revealed a separation in the codon usage patterns of these two groups, mainly in principal component 1 (PC1; Fig. 3a). The corresponding loadings plot demonstrated a strong association of three codons with the upregulated proteins LysAAA, HisCAT and AspGAT, with enrichment of their cognate codons (LysAAG, HisCAC and AspGAC) in the downregulated proteins (Fig. 3b). LysAAA/AAG was the greatest driver amongst codon pairs. Of note, the majority of codons were unchanged between up- and downregulated proteins (Extended Data Fig. 3a, major codon changes are shown in Fig. 3b).

Fig. 3: A subset of proteins, including K13, are regulated by lysine codon bias translation in Dd2R539T parasites.

a, The top 44 upregulated proteins and bottom 70 downregulated proteins in Dd2R539T parasites after DHA exposure were analysed for codon usage patterns (Source data). The codon usage percentages in each gene were used to prepare a data matrix for principal component analysis. The scores plot shows codon use distinction between increased proteins and decreased proteins, with changes greatest in decreased proteins along PC1. b, The corresponding loadings plot for a shows codons contributing most strongly to this separation. For ease of visualization, unchanged codons were removed with the full loadings plot shown in Extended Data Fig. 3a. Cognate codon pairs significantly contributing to this separation are joined by coloured lines (Lys, pink; Asp, blue; His, orange and Asn, green). c, An assessment of differentially regulated proteins for lysine codon usage versus transcriptional direction post-DHA in Dd2R539T parasites. Increased proteins and decreased proteins were evaluated for LysAAA codon usage with z-scores >0.5 or <−0.5 considered significant (y axis). Transcriptomic data from Mok et al.23 were analysed for Dd2R539T parasites after a 6 h DHA pulse and assessed for log2 fold change compared with parasites at timepoint 0 (Source data and Extended Data Fig. 3). Candidate proteins regulated by Lys codon bias translation were considered those that displayed Lys codon bias and had either increased abundance with decreased translation (red-shaded region, Supplementary Table 3) or decreased abundance with increased translation (blue-shaded region, Supplementary Table 4). Proteins that met criteria are numbered and detailed in Table 1. d, Box-and-whisker plot showing Lys codon usage for all differentially translated proteins. The z-score for LysAAA codon usage for increased proteins and decreased proteins as compared with the z-score for LysAAG codon usage for increased and decreased proteins. Data were derived from n = 3 independent biological replicates. Centre line, median; box limits, upper and lower quartiles; and whiskers, minimum and maximum values. e, GO analysis for increased and decreased codon bias proteins with the number of genes per GO slim term on the x axis. The heat map shading represents −log10P values (two-tailed Fisher exact test) (Supplementary Tables 3 and 4).

Source data

We next searched for up- and downregulated proteins enriched for LysAAA or LysAAG as evaluated by z-scores ≥0.5. Among upregulated proteins, 48% were enriched for LysAAA compared with 16% for LysAAG. In contrast, for downregulated proteins, 34% were enriched for LysAAG versus 23% for LysAAA (Supplementary Table 2). Differences in the usage of His and Asp cognate codons in the up- and downregulated proteins were less pronounced (Supplementary Table 2). Interestingly, the differentially regulated mcm5s2U modification (Fig. 1c,d) occurs on the U34 wobble position of LysAAA/AAG codons to regulate translational fidelity44,47, providing a mechanistic link between our tRNA reprogramming changes and the Lys codon bias translation noted above.

Stress-response proteins show Lys codon-biased translation post-ART

To explore whether changes in protein levels were attributable to codon-biased translation rather than to transcriptional regulation, we searched for translationally up- or downregulated proteins that displayed Lys codon bias and whose transcript levels were unchanged or moving in opposite directions to their protein levels. We analysed published transcriptomic data23 that profiled highly synchronized Dd2R539T isogenic parasites before and after a 6 h DHA pulse. Given the lag between transcript and protein level changes, we focused on altered protein levels 6 h after completing the DHA pulse in Dd2R539T parasites. Dd2 parasites were not explored as they are effectively dead after 6 h of DHA exposure. In the Dd2R539T line, 50% of upregulated proteins and 43% of downregulated proteins were found to change transcriptionally in the equal or opposite direction to the protein changes (Supplementary Table 2). By integrating the proteomic and transcriptomic changes and LysAAA codon usage (Fig. 3c), we identified a subset of 12 translationally upregulated proteins that were enriched for LysAAA and transcriptionally downregulated (Fig. 3c, Table 1 and Supplementary Table 3). We also identified a separate set of ten translationally downregulated proteins that were enriched for LysAAG but increased transcriptionally (Fig. 3c, Table 1 and Supplementary Table 4). Within this set of 22 differentially translated proteins in our DHA-treated ART-R parasites, the upregulated proteins showed a clear Lys codon bias (Fig. 3d). Importantly, not all up- or downregulated proteins displayed codon bias, nor did they all have opposing transcription profiles, suggesting that we had identified a unique subset of proteins regulated by Lys codon bias translation in the ART-R parasites. We performed a similar analysis for His and Asp codon pairs (Extended Data Fig. 3b,c) and identified similar, although smaller, sets of codon-biased regulated proteins (Supplementary Tables 5–8).

Table 1 Up- and downregulated lysine codon bias proteins

We analysed the GO slim and PlasmoDB databases for protein functionality and essentiality, respectively. For LysAAA-enriched upregulated proteins, top functional terms included ‘unfolded protein response’ and ‘ATP dependent activity’. For the LysAAG-enriched downregulated proteins, top GO slim terms included RNA binding and ribosome structural components (Fig. 3e, Extended Data Fig. 3d and Supplementary Tables 3 and 4). Three of the downregulated proteins displayed codon bias for Lys, His and Asp, suggesting that these proteins may have a regulatory role in the DHA-induced stress response. This included a 60S ribosomal protein (Pf3D7_1142500), an inner membrane complex subcompartment protein (Pf3D7_1460600) and the conserved translation factor eEF1-α64.

Several upregulated proteins in our Dd2R539T parasites demonstrated bias for at least two codons (Supplementary Table 9). Most striking was K13, which was upregulated translationally, downregulated transcriptionally and had a codon bias for both Lys and Asp. By quantifying protein levels (based on TMT proteomics), we observed decreased PfK13 levels in Dd2R539T parasites compared with Dd2, as previously reported15,65. Interestingly, for Dd2R539T, K13 protein levels increased in DHA-treated parasites while remaining unchanged upon DMSO treatment (Extended Data Fig. 4a). For K13, 52 of 57 lysine codons were LysAAA, which clustered mostly in the first half of the gene (Extended Data Fig. 4b). These data suggest that K13 levels may be modulated by this codon-biased translational mechanism, providing a means for a rapid increase as parasites prepare to exit DHA-induced quiescence.

Pf3D7_1019800 (PfMnmA) is required for parasite development

tRNA s2U modifications are known to regulate protein levels of Lys codon-biased proteins in yeast44, creating a mechanistic correlation between our tRNA and proteomic observations of DHA-treated ART-R parasites. Further support for a role of this pathway in ART resistance came from (1) previous transcriptomic data, which demonstrated that three genes in the s2U biosynthesis pathway were significantly over-represented (P = 0.003) in DHA-treated Dd2R539T parasites, namely a putative tRNA 2-thiouridylase (PF3D7_1019800, PfMnmA), an aminomethyltransferase (PF3D7_134000) and a GTPase (PF3D7_0817100)23; (2) U34 s2U modification changes that are linked to translational fidelity and amino acid homeostasis49,66,67,68 and (3) U34 s2U hypomodification that leads to translational stalling on LysAAA codons, which in yeast causes a substantial growth slowdown44.

To test the potential contribution of the U34 s2U modification, we generated a conditional knockdown (cKD) of PfMnmA (Pf3D7_1019800). This gene was selected as its product catalyses the terminal step in s2U biosynthesis in bacteria and eukaryotic mitochondria (in the eukaryotic cytosol the Ncs6–Urm1 pathway is used)47. In our dataset, PfMnmA was differentially regulated in Dd2R539T, but not Dd2 parasites, after DHA exposure23. To generate this cKD, we used the TetR–DOZI system that uses anhydrotetracycline (aTc) to regulate translation69 (Fig. 4a). Translation occurs in the presence of aTc, whereas removal leads to translation repression. cKD parasites were generated in an NF54 (ART-S) line that constitutively expresses the T7 polymerase and Cas9 (referred to as NF54 below)69. Successful creation of NF54_PfMnmA_cKD parasites (referred to below as PfMnmA_cKD) was confirmed using PCR, Sanger sequencing and western blot analysis (Extended Data Fig. 5a,b and Supplementary Results).

Fig. 4: Knockdown of PfMnmA, the terminal thiouridylase in s2U biosynthesis, leads to increased ART survival.

a, A schematic of donor plasmid PSN054, the endogenous Pf3D7_1019800 (PfmnmA) locus and the recombinant locus of the edited cKD parasite. +aTc, normal translation and −aTc, protein knockdown. Edited parasites were confirmed via PCR and western blot analyses (Extended Data Fig. 5a,b). UTR, untranslated region; BSD, blasticidin S deaminase; LHR, left homology region. b,c, Synchronized, ring-stage PfMnmA_cKD parasites were washed to remove aTc and assayed in parallel with NF54 parasites. Washed parasites were inoculated in high (500 nM), low (3 nM) or no (0 nM) aTc and growth was followed by flow cytometry (b). Data were normalized to high aTc parasitaemias and represented as a percentage of growth. n = 5 independent biological replicates. The error bars represent ±s.e.m. Washed parasites were cultured ±aTc. Thin smears were Giemsa stained and 100 RBCs were counted (c). The y axis shows total parasitaemias (Extended Data Fig. 5d). d, A schematic of the modified RSA. Parasites were cultured with aTc, washed 3× and split into cultures ±aTc for 96 h. Synchronized, early ring-stage parasites (0–6 hpi) were exposed to a 6 h pulse of a range of DHA concentrations, the drug was washed off and then allowed to recover in 30 nM, 3 nM or 0 nM aTc for 72 h. e, RSA survival rates for NF54 and PfMnmA_cKD parasites cultured −aTc for 96 h before DHA exposure and allowed to recover on 30 nM, 3 nM or 0 nM aTc for 72 h. The results demonstrate the percentage of parasites that survived a range of DHA concentrations (≤700 nM aTc) relative to no-drug control parasites assayed in parallel. Percent survival values are shown as means ± s.e.m. f, RSA survival rates for parasites without MnmA knockdown (maintained with aTc) and with MnmA knockdown (maintained without aTc) exposed to 700 nM and 350 nM DHA for 6 h. n = 5 independent biological replicates. Statistical significance was determined via two-tailed Mann–Whitney U-tests as compared with the isogenic line or for the knockdown ±aTc. *P < 0.05 and **P < 0.01 (Source data and Extended Data Fig. 8).

Source data

Growth studies showed that PfMnmA_cKD parasites cultured in low (3 nM) or no aTc displayed a slow onset of death as compared with parasites cultured with high aTc (500 nM). NF54 parasites had no change in growth (Fig. 4b and Extended Data Fig. 5c). cKD parasites grown without aTc had evident defects in schizont morphology (Fig. 4c, Extended Data Figs. 5d and 6, Supplementary Fig. 1 and Supplementary Results). LC–MS/MS evaluation of global mcm5s2U modifications in PfMnmA_cKD parasites ±aTc revealed specific decreases in total levels of mcm5s2U in parasites grown without aTc, as compared with those grown with aTc. There were no changes, however, in m2,2G or m6A (Extended Data Fig. 7 and Supplementary Results). These findings suggest that the PfMnmA knockdown leads to specific decreases in global mcm5s2U modification levels, although this does not abolish the modification fully, probably because of the cytosolic s2U biosynthetic pathway.

Knockdown of MnmA results in increased resistance to ART

We predicted that if s2U hypomodification and its downstream consequences contribute to ART resistance, then a PfMnmA knockdown should have decreased ART sensitivity. To test this, we modified the RSA to incorporate the growth kinetics of our cKD line (Fig. 4d and Methods). At 96 h before drug exposure, parasites were washed and split into media ±aTc. On the day of the assay, highly synchronized early rings (0–6 hpi) were pulsed for 6 h with DHA (concentration range: 700 nM to 1.4 nM), washed and allowed to recover for 72 h in the presence of 30 nM, 3 nM or 0 nM aTc. Parasites maintained on 30 nM aTc before and after DHA comprised the ‘translation on’ control. Parasites cultured without aTc before and after DHA constituted the ‘translation off’ control. Chloroquine (CQ) was used as an unrelated control.

PfMnmA_cKD parasites that underwent protein knockdown (no aTc for 96 h) before DHA exposure demonstrated an aTc concentration-dependent increase in ART survival, as compared with NF54 (Fig. 4e). At both 700 and 350 nM DHA, NF54 parasites did not survive. However, PfMnmA_cKD parasites cultured on 30, 3 or 0 nM aTc post-DHA pulse survived significantly more than NF54 controls (P < 0.05%). At 350 nM DHA, survival differences were more pronounced with an aTc concentration-dependent increase in survival, evident at DHA concentrations as low as 2.7 nM in PfMnmA_cKD parasites (Extended Data Fig. 8a and Supplementary Results).

PfMnmA parasites cultured on 30 nM aTc before the DHA pulse (that is, with translation on) demonstrated no significant differences in survival as compared with DMSO controls (Extended Data Fig. 8b). These data suggest that protein knockdown is essential before DHA exposure to prepare the parasites for this response. There were no differences in survival after CQ exposure for any condition (Supplementary Fig. 2a,b).

To confirm that our phenotype was secondary to PfMnmA knockdown, we compared our ‘translation off’ and ‘translation on’ parasites. At 350 nM ART, the former had significantly more survival than the latter (11% versus 4% survival, P < 0.05), further suggesting that decreased levels of MnmA led to increased ART survival (Fig. 4f). These experiments supported the importance of s2U tRNA hypomodification in the ART-induced stress response.

MnmA knockdown parasites show altered anti-malarial susceptibility

We next addressed whether PfMnmA knockdown would affect parasite susceptibility to other anti-malarials. Parasites were cultured for 96 h (±aTc) and then exposed to twofold serial dilutions of drug for 72 h ±aTc (Extended Data Fig. 9a). We tested three groups of compounds. The first group contained the apicoplast-targeting compounds azithromycin (AZT) and fosmidomycin (FSM), which were selected based on recent data showing that PfMnmA is necessary for apicoplast maintenance70. Knockdown of MnmA led to low-level but significant twofold sensitization to both compounds, as compared with non-knockdown conditions (Fig. 5b,c and Supplementary Results).

Fig. 5: PfMnmA contributes to parasite responses to multiple stressors.

a, A schematic of molecular sites of action for anti-malarials used in this study. Hb, haemoglobin; LUM, lumefantrine; MFQ, mefloquine; PPQ, piperaquine. b–e,g, IC50 data shown as means ± s.e.m. from 72 h dose–response assays of asynchronous NF54 parasites ±aTc, PfMnmA parasites cultured with aTc and PfMnmA parasites cultured without aTc for 96 h before drug pulse (Extended Data Fig. 9a) for FSM (b), AZT (c), DSM265 (d), ATQ (e) and WLL (g). n = 5–7. Statistical significance was determined via two-tailed Mann–Whitney U-tests. *P < 0.05 and **P < 0.01. f, Dose–response curves for ATQ for NF54 parental line with and without aTc, PfMnmA parasites cultured with aTc and PfMnmA parasites cultured without aTc for 96 h before drug pulse, and Dd2 and Dd2_ATQ-R (ATQ resistant, Dd2-CYT1-V259L). The error bars represent s.e.m. n = 6–7 independent biological replicates per parasite line.

Source data

The second group contained the mitochondrial inhibitors DSM265 and atovaquone (ATQ), which were selected because eukaryotic homologues of PfMnmA have been localized to the mitochondria47. While no change was observe