Figure 3.

Parasites harboring the PfCRT L272F mutation require methionine for growth. (a) Growth rates of Dd2^Dd2 (left panel) and Dd2^{Dd2 L272F} (right panel) in amino acid-rich media (RPMI-VLA) and minimal media (lacking all amino acids save for supplementation with the ones denoted by single letter codes, e.g. IM: isoleucine plus methionine). Note that for Cysteine (C), this was supplemented as the oxidized dimer cystine that is presumably reduced in the host RBC. Unlike Dd2^Dd2, Dd2^{Dd2 L272F} cannot grow in medium containing only isoleucine, but can be rescued with the addition of methionine (IM and IMC). Cumulative parasitemia expansions, calculated as Ln-transformed parasitemias, were calculated from three independent experiments for each line and media condition. Data are presented as the natural log of parasitemia adjusted for dilution factor over 240 hours. Student t tests performed at the last time point showed significant differences only for the Dd2^{Dd2 L272F} line cultured in +I versus +IM or +IC versus +IMC. For this parasite line, no significant differences were observed between RPMI-VLA and any other media. All comparisons for different media were non-significant with Dd2^Dd2 parasites. (b) Average DV volumes (represented as μm³/cell) of Dd2^GC03, Dd2^Dd2 and Dd2^{Dd2 L272F} lines post amino acid-starvation for either 1 hour (left panel) or 6 hours (right panel). DV volumes are represented as mean ± SEM calculated from 30 independent experiments. Statistical comparisons used Mann-Whitney U tests, which for each line compared volumes in amino-acid depleted media vs. those in rich RPMI-VLA media. *p < 0.05.