Figure 5: Arginine uptake into T. gondii is mediated by TgNPT1 and by a TgNPT1-independent cationic amino acid uptake pathway.

(a) [¹⁴C]Arg uptake in WT, Δnpt1 and Δnpt1/tubNPT1 parasites in the absence (black) and presence (grey) of 80?μM unlabelled lysine, expressed as a percentage of the initial rate of [¹⁴C]Arg uptake in WT parasite measured in the absence of lysine. Uptake was measured in parasites suspended in PBS containing 10?mM glucose, 40?μM unlabelled arginine and 0.1?μCi?ml⁻¹ (289?nM) [¹⁴C]Arg. The initial rates of [¹⁴C]Arg uptake were derived from the initial slopes of the time courses shown in Supplementary Fig. 7a. The mean initial rate of [¹⁴C]Arg uptake in WT parasite measured in the absence of lysine was 900±61?pmol 10⁷?cells⁻¹?min⁻¹ (mean±s.e.m.; n=3). The data shown represent the mean±s.e.m. from three independent experiments (*P<0.05; **P<0.01; ***P<0.001; ****P<0.0001; Student’s t test). (b) [¹⁴C]Lys uptake in T. gondii. The uptake of 0.1?μCi?ml⁻¹ (307?nM) [¹⁴C]Lys (in the presence of 50?μM unlabelled lysine) was measured over 3?min (within the initial linear phase of uptake; Supplementary Fig. 7b) in WT (black) and Δnpt1 (grey) parasites, suspended either in the presence or absence of a 1?mM concentration of the cationic amino acids lysine (Lys), arginine (Arg) or ornithine (Orn), the anionic amino acid glutamate (Glu), or the small neutral amino acid alanine (Ala). The results are averaged from those obtained in three separate experiments±s.e.m. (*P<0.05; **P<0.01; ****P<0.0001; n.s.=not significant; ANOVA). (c) Fluorescence growth assay for WT (black) and Δnpt1 (red) parasites cultured for 4 days in media having a range of lysine concentrations and a constant 400?μM arginine. Growth is expressed as a percentage of that measured at 50?μM lysine for each parasite strain. The data shown are averaged from three technical replicates (shown±s.d.) and are representative of those obtained in three biological replicates.