Fig. 3: Pyrimethamine susceptibility measured via growth inhibition assays of parasite lines.

Pkdhfr^WT (Parental parasite line, yellow dot), Pkdhfr^OR (recodonised P. knowlesi DHFR, transfection control line, green dot), Pfdhfr^OR_PfS (P. falciparum sensitive DHFR genotype: N51, C59, S108, purple dot), Pmdhfr^OR_PmS (P. malariae hypothetical sensitive sequence with two mutations: 58S and 114S, navy dot), Pmdhfr^OR_PmUG01 (P. malariae reference sequence, with suspected pyrimethamine resistance: R58 and N114, red dot), Pmdhfr^OR_PmM5 (P. malariae reference sequence: R58 and N114, with additional mutation F57L, pink dot), Pfdhfr^OR_PfR (P. falciparum resistant IRN sequence: 51I, 59 R and 108 N, aqua dot). All data points in both plots represent an independent biological replicate, with the overall mean EC50 in nM given in the summary table. For all lines, a minimum of 3 independent biological replicates were performed. For each independent biological replicate, 3 technical replicates were performed, and the mean EC₅₀ value across the technical replicates is visualised as an individual data point in each graph. In the table, the mean EC50 value across all the biological replicates is shown (Mean (nM)), with the standard deviation (SD) given. A Susceptibility to pyrimethamine using serial dilutions. A log₁₀ scale is used on the plot, with mean EC₅₀ values in nM in the table (standard deviations, SD). Regression analysis was used to investigate the differences in pyrimethamine susceptibility and asterisks used to demonstrate levels of significance (p = 0 − 0.001, ***; p = 0.001 − 0.01, **; p = 0.01-0-.1, *, p values). Regression analysis is a two-sided statistical test, and there is no adjustment for multiple comparisons as the testing is nested within the same model. Pkdhfr^WT (n = 6 biological replicates, minima 0.629, maxima 0.851, median 0.673, Q1 0.653, Q3 0.734), Pkdhfr^OR (n = 4, 0.691, maxima 0.836, median 0.828, Q1 0.792, Q3 0.831), Pfdhfr^OR_PfS (n = 3, minima 0.905, maxima 1.49, median 1.33, Q1 1.12, Q3 1.41), Pmdhfr^OR_PmS (n = 3, minima 1.06, maxima 1.23, median 1.18, Q1 1.12, Q3 1.20), Pmdhfr^OR_PmUG01 (n = 5, minima 3.44, maxima 3.89, median 3.57,Q1 3.51, Q3 3.87), Pmdhfr^OR_PmM5 (n = 3, minima 4.39, maxima 4.56, median 4.39, Q1 4.39, Q3 4.47), Pfdhfr^OR_PfR (n = 3, minima 4.66, maxima 4.92, median 4.80, Q1 4.73, Q3 4.86). P-values not included in the table include pfdhfr^OR_PfS 0.000768, pmdhfr^OR_PmUG01 2e-16, pmdhfr^OR_PmM5 2e-16, pfdhfr^OR_PfR 2e-16. B Dihydroartemisinin (DHA) was used as a control drug, with EC₅₀ values below 7 nM for all parasite lines. Pkdhfr^WT (n = 4 biological replicates, minima 0.231, maxima 0.466, median 0.319, Q1 0.313, Q3 0.466), Pkdhfr^OR (n = 3, minima 0.485, maxima 0.555, median 0.512, Q1 0.499, Q3 0. 533), Pfdhfr^OR_PfS (n = 4, minima 0.424, maxima 0.742, median 0.697, Q1 610, Q3 727), Pmdhfr^OR_PmS (n = 5, minima 0.071, maxima 0.569, median 0.214, Q1 0.180, Q3 0.254), Pmdhfr^OR_PmUG01 (n = 4, minima 0.437, maxima 0.872, median 0.557, Q1 0.522, Q3 0.641), Pmdhfr^OR_PmM5 (n = 3, minima 0.372, maxima 0.820, median 0.805, Q1 0.589, Q3 0.812), Pfdhfr^OR_PfR (n = 5, minima 0.474, maxima 0.668, median 0.638, Q1 0.489, Q3 0.638). In both assays (for pyrimethamine and DHA), pkdhfr^OR is first compared to the parental parasite line (pkdhfr^WT) to confirm that the transfection process itself has no impact on drug susceptibility. Following this, all other parasite lines are compared to pkdhfr^OR to determine the change in drug susceptibility.