Fairweather SJ et al. (2021), Coordinated action of multiple transporters in ...

XB-ART-58396

PLoS Pathog 2021 Aug 25;178:e1009835. doi: 10.1371/journal.ppat.1009835.

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Coordinated action of multiple transporters in the acquisition of essential cationic amino acids by the intracellular parasite Toxoplasma gondii.

Fairweather SJ , Rajendran E , Blume M , Javed K , Steinhöfel B , McConville MJ , Kirk K , Bröer S , van Dooren GG .

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Intracellular parasites of the phylum Apicomplexa are dependent on the scavenging of essential amino acids from their hosts. We previously identified a large family of apicomplexan-specific plasma membrane-localized amino acid transporters, the ApiATs, and showed that the Toxoplasma gondii transporter TgApiAT1 functions in the selective uptake of arginine. TgApiAT1 is essential for parasite virulence, but dispensable for parasite growth in medium containing high concentrations of arginine, indicating the presence of at least one other arginine transporter. Here we identify TgApiAT6-1 as the second arginine transporter. Using a combination of parasite assays and heterologous characterisation of TgApiAT6-1 in Xenopus laevis oocytes, we demonstrate that TgApiAT6-1 is a general cationic amino acid transporter that mediates both the high-affinity uptake of lysine and the low-affinity uptake of arginine. TgApiAT6-1 is the primary lysine transporter in the disease-causing tachyzoite stage of T. gondii and is essential for parasite proliferation. We demonstrate that the uptake of cationic amino acids by TgApiAT6-1 is 'trans-stimulated' by cationic and neutral amino acids and is likely promoted by an inwardly negative membrane potential. These findings demonstrate that T. gondii has evolved overlapping transport mechanisms for the uptake of essential cationic amino acids, and we draw together our findings into a comprehensive model that highlights the finely-tuned, regulated processes that mediate cationic amino acid scavenging by these intracellular parasites.

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Species referenced: Xenopus laevis
Genes referenced: cat1 cat2 rps3a
GO keywords: arginine transmembrane transporter activity [+]

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	Fig 1. TgApiAT6-1 is important for parasite proliferation.A. Western blot analysis of proteins extracted from rTgApiAT6-1-HA parasites cultured in the presence of ATc for 0–2 days, and detected with antibodies against HA or TgTom40 (as a loading control). B-D. Fluorescence growth assays measuring the proliferation of rTgApiAT6-1 parasites (B), WT parasites (C), or rTgApiAT6-1 parasites complemented with a constitutively-expressed copy of TgApiAT6-1 (rTgApiAT6-1/cTgApiAT6-1; D). Parasites were cultured for 6 or 7 days in the absence (black) or presence (red) of ATc. Parasite proliferation is expressed as a percentage of parasite proliferation in the -ATc condition on the final day of the experiment for each strain. Data are averaged from 3 technical replicates (± S.D.) and are representative of three independent experiments.
	Fig 2. TgApiAT6-1 is a cationic and neutral amino acid transporter with high affinity for Lysine.A. Analysis of [13C] amino acid uptake in parasites expressing or lacking TgApiAT6-1. rTgApiAT6-1 parasites were cultured for 2 days in the absence (black) or presence (red) of ATc until natural egress, then incubated in medium containing [13C]-L-amino acids for 15 min. Metabolites were extracted and the fractions of [13C]-L-amino acids determined by GC-MS. Amino acids are represented by single letter codes; OxoP, 5-oxoproline. The data represent the mean ± S.D of 3 replicate experiments (, P < 0.001, Student’s t test. Where significance values are not shown, differences were not significant; P > 0.05). B. Uptake of a range of amino acids into oocytes expressing TgApiAT6-1. Uptake was measured in the presence of 100 μM unlabelled substrate and 1.0 μCi/ml [3H] or [14C] substrate. Amino acid substrates are represented by single letter codes, while for other metabolites: Pu, putrescine; Sp, spermidine; and GA, γ-amino butyric acid (GABA). Each bar represents the mean ± S.D. uptake of 10 oocytes for a single experiment, and each is representative of three independent experiments. The uptake into uninjected oocytes (shown in S3A Fig) was subtracted for all substrates tested. Statistical analysis compares injected oocyte uptake to uninjected oocyte uptake for the same substrate (P < 0.05, one-way ANOVA, Dunnett’s post-hoc test). C-D. Inhibition of Arg uptake into TgApiAT6-1-expressing oocytes by a range of amino acids. Uptake of 100 μM unlabelled Arg and 1.0 μCi/ml [14C]Arg was measured in the presence of 1 mM (C) or 10 mM (D) of the competing amino acid. Amino acid substrates are represented by single letter codes. Each bar represents the mean ± S.D. uptake of 10 oocytes for a single experiment, and are representative of three independent experiments. The first bar in each graph represents the Arg-only uptake control. The uptake in uninjected oocytes (shown in S3B and S3C Fig for the 1 mM and 10 mM competition experiments, respectively) has been subtracted for all conditions. Statistical analysis compares all bars to the Arg uptake control (*, P < 0.05, one-way ANOVA, Dunnett’s post-hoc test). E-F. Steady-state kinetic analysis of Lys (E) and Arg (F) uptake into TgApiAT6-1-expressing oocytes. Uptake was measured at a range of concentrations of unlabelled Lys (E) or Arg (F) as indicated on the x-axis and 1.0 μCi/ml [14C]Arg or [14C]Lys. Each data point represents the mean ± S.D. uptake of 10 oocytes for a single experiment, and are representative of three independent experiments. The uptake into uninjected oocytes has been subtracted for all substrate concentrations tested.
	Fig 3. Arg and Lys compete for the same binding site in TgApiAT6-1.Electrophysiology measurements in TgApiAT6-1 expressing oocytes. All currents were recorded in two-voltage clamp configuration to record membrane current. All oocytes were voltage clamped at −50 mV and the application of substrate (Arg or Lys) are indicated by the dashed horizontal lines above the tracings. Representative current tracings were normalised to 0 nA to remove background (non-substrate induced) current. A. Representative current tracing of TgApiAT6-1 expressing oocytes upon the addition and subsequent washout of 1 mM extracellular Arg ([Arg]o) with no pre-injection of substrate. The perfusion buffer used was ND96 (pH 7.3). B. Representative current tracing of a TgApiAT6-1 expressing oocyte repeatedly pulsed with 1 mM Arg for 1 min, 2 min, and 10 min with 5 min gaps in between pulses. The perfusion buffer used was ND96 (pH 7.3). C. Representative current tracing of a TgApiAT6-1 expressing oocyte pre-injected with 1 mM Arg ([Arg]i = 1 mM) upon the addition and subsequent washout of 1 mM extracellular Arg ([Arg]o). The perfusion buffer used was ND96 (pH 7.3). D. Representative current tracings of TgApiAT6-1 expressing oocytes upon the addition and subsequent washout of either 1 mM extracellular Arg ([Arg]o) or Lys ([Lys]o) with no pre-injection of substrate. The perfusion buffer used was ND96 (pH 7.3). E-F. Inhibition kinetics of increasing [Lys] on Arg-induced inward currents in TgApiAT6-1 expressing oocytes. All oocytes were voltage-clamped at −50 mV and exposed to a range of Arg concentrations at one of 3 concentrations of Lys (0, 50, 500 μM) until a new steady-state current baseline was achieved. Each data point represents the mean ± S.D. steady-state inward current (n = 14 oocytes). All 3 inhibitory [Lys] plots were fitted to either the Michaelis-Menten equation (E) with R2 values of 0.97 (0 mM Lys), 0.84 (50 μM), 0.63 (500 μM), or a Lineweaver-Burke linear regression (F) with R2 values of 0.94 (0 mM Lys), 0.93 (50 μM), and 0.67 (500 μM).
	Fig 4. TgApiAT6-1 mediates uptake of Lys and Arg in T. gondii parasites.A-B. Initial rate of Lys (A) and Arg (B) uptake in WT and rTgApiAT6-1 parasites cultured in DME in the absence (black) or presence (red) of ATc for 2 days. Uptake was measured in 50 μM unlabelled Lys and 0.1 μCi/ml [14C]Lys (A) or 80 μM unlabelled Arg and 0.1 μCi/ml [14C]Arg (B). Initial rates were calculated from fitted curves obtained in time-course uptake experiments (S5 Fig). Data represent the mean initial uptake rate ± S.D. from three independent experiments.(**, P = 0.01; n.s. not significant; ANOVA with Sidak’s multiple comparisons test).
	Fig 5. Trans-simulation of AA+ transport in TgApiAT6-1- and TgApiAT1-expressing oocytes.A-B.TgApiAT6-1-injected (A) and TgApiAT1-injected (B) oocytes were pre-loaded with either 1 mM unlabelled Lys and 1.0 μCi/ml of [14C]Lys (TgApiAT6-1) or 1 mM unlabelled Arg and 1.0 μCi/ml of [14C]Arg (TgApiAT1) for 3–6 hr. The retention of substrates in TgApiAT6-1- or TgApiAT1-expressing oocytes were measured in the presence of 1 mM external substrate (closed symbols) or in the absence of an external substrate (open symbols). Data points represent the mean ± S.D. from 3 batches of 5 oocytes from one experiment, and are representative of 3 independent experiments. C-D. Arg efflux and retention in TgApiAT6-1 expressing (C) and TgApiAT1 expressing (D) oocytes in the presence of candidate trans-stimulating substrates. Oocytes were pre-loaded (PL) with Arg by first microinjecting Arg to a final concentration of ~5 mM, then incubated oocytes in a solution containing 1 mM unlabelled Arg and 1.0 μCi/ml of [14C]Arg for 1 hr (TgApiAT6-1) or 2 hr (TgApiAT1). This pre-loading was followed by addition of 1 mM unlabelled amino acids or amino acid derivatives to the outside of the oocyte. Efflux of pre-loaded Arg (Argo; black bars) and retention of pre-loaded Arg (Argi; white bars) were measured after 1 hr (TgApiAT6-1) or 2 hr (TgApiAT1) in the presence of 1 mM of the metabolites or with ND96 buffer in the extracellular medium (−). The horizontal dashed line across both figures indicates the amount of Arg pre-loaded (PL) into oocytes (left-most bar). Amino acid substrates are represented by single letter codes, while for other metabolites: Cr, creatine; Ag, agmatine; Sp, spermidine; Pu, putrescine; Ci, citrulline; Ur, urea; and Or, ornithine. Each bar represents the mean ± S.D. efflux or retention in from 3 batches of 5 oocytes from one experiment, and are representative of three independent experiments. Statistical analysis compares all bars to oocyte efflux in the presence of ND96 (−) (pH 7.3) (* P < 0.05, one-way ANOVA, Dunnett’s post-hoc test). E-F. Lys (E) or Arg (F) uptake into TgApiAT6-1 expressing oocytes pre-loaded with a range of candidate trans-stimulating substrates. TgApiAT6-1-injected oocytes were pre-loaded by microinjecting the indicated substrates to a final concentration of ~5 mM (or with the same volume of H2O as a control), and the uptake of 15 μM Lys and 1.0 μCi/ml of [14C]Lys (E) or 1 mM Arg and 1.0 μCi/ml of [14C]Arg (F) was determined. Uptake of Arg and Lys into control oocytes not expressing TgApiAT6-1 using the same trans-stimulation conditions (shown in S3D and S3E Fig for Arg and Lys uptake, respectively) were subtracted for all conditions. Amino acid substrates are represented by single letter codes, while for other metabolites: Cr, creatine; Ag, agmatine; Sp, spermidine; Pu, putrescine; Ci, citrulline; and Or, ornithine. Each bar represents the mean ± S.D. uptake of 10 oocytes for a single experiment, and are representative of three independent experiments. Statistical analysis compares all bars to Lys or Arg uptake in control H2O ‘pre-loaded’ oocytes (* P < 0.05, one-way ANOVA, Dunnett’s post-hoc test).
	Fig 6. TgApiAT6-1 and TgApiAT1 are net accumulators of cationic amino acids.A-C. Time-course measuring the Lys (A) or Arg (B-C) concentration in TgApiAT6-1 expressing oocytes (A-B; squares), TgApiAT1 expressing oocytes (C; squares), or H2O-injected oocytes (A-C; circles) incubated in 1 mM Lys (A) or 1 mM Arg (B-C) as quantified by LC-MS/MS. Following 32–34 hr of incubation measuring accumulation inside oocytes, samples were split into two groups, one continuing with substrate incubation (closed symbols), the other with substrate replaced by substrate-free incubation media (open symbols). Each data point represents the mean intracellular amino acid concentration ± S.D. of 12 individual oocytes (substrate-incubated) or 3 batches containing 5 oocytes each (substrate-free), and are representative of 3 independent experiments.
	Fig 7. Model for AA+ uptake into intracellular T. gondii parasites.The proliferation of T. gondii parasites (light blue) inside infected host cells (yellow) causes a depletion of host cell Arg, leading to an upregulation of the host cell AA+ transporters CAT1 and, possibly, CAT2 (dark blue), and a concomitant increase in the uptake of Lys and Arg into host cells. In organs with high Arg catabolism (e.g. liver, right), the intracellular ratio of [Arg]:[Lys] is low, and Lys uptake through TgApiAT6-1 (green) outcompetes Arg uptake through this transporter. The parasite responds by upregulating the abundance of its selective Arg transporter, TgApiAT1 (red), enabling Arg uptake through this transporter. In organs in which Arg is synthesised (e.g. kidneys, left), the intracellular ratio of [Arg]:[Lys] is high, resulting in increased uptake of Arg through TgApiAT6-1. Parasites respond by downregulating TgApiAT1 abundance. The transport activity of TgApiAT6-1 is increased by the exchange of Lys and Arg with cationic (AA+) and neutral (AA0) amino acids. The activity of both TgApiAT1 and TgApiAT6-1 may be increased by an inwardly negative membrane potential (Em) at the parasite plasma membrane.

References [+] :

Augusto, Regulation of arginine transport by GCN2 eIF2 kinase is important for replication of the intracellular parasite Toxoplasma gondii. 2019, Pubmed