Identifying the major lactate transporter of Toxoplasma gondii tachyzoites

Abstract

Toxoplasma gondii and Plasmodium falciparum parasites both extrude L-lactate, a byproduct of glycolysis. The P. falciparum Formate Nitrite Transporter, PfFNT, mediates L-lactate transport across the plasma membrane of P. falciparum parasites and has been validated as a drug target. The T. gondii genome encodes three FNTs that have been shown to transport L-lactate, and which are proposed to be the targets of several inhibitors of T. gondii proliferation. Here, we show that each of the TgFNTs localize to the T. gondii plasma membrane and are capable of transporting L-lactate across it, with TgFNT1 making the primary contribution to L-lactate transport during the disease-causing lytic cycle of the parasite. We use the Xenopus oocyte expression system to provide direct measurements of L-lactate transport via TgFNT1. We undertake a genetic analysis of the importance of the tgfnt genes for parasite proliferation, and demonstrate that all three tgfnt genes can be disrupted individually and together without affecting the lytic cycle under in vitro culture conditions. Together, our experiments identify the major lactate transporter in the disease causing stage of T. gondii, and reveal that this transporter is not required for parasite proliferation, indicating that TgFNTs are unlikely to be targets for anti-Toxoplasma drugs.

Figure 1

TgFNT1 and ectopically-expressed TgFNT2 and TgFNT3 localize to the plasma membrane of T. gondii. (a) Western blot of TgFNT1-HA-expressing parasites performed using an anti-HA antibody. The uncropped image is shown in Supplementary Fig. S3. (b–d) Immunofluorescence assay reveals co-localization of TgFNT1-HA (b; green), TgFNT2-HA₃ (c; green) and TgFNT3-HA₃ (d; green) with the plasma membrane marker P30 (red). The scale bars represent 2 µm.

Figure 2

TgFNTs are not required for tachyzoite proliferation in vitro. (a) Data from a single representative experiment showing the growth of the different parasite lines (wild-type, blue; Δtgfnt1, white; Δtgfnt2, grey; Δtgfnt3, red; Δtgfnt1/2/3, dark red). Parasite growth was normalized to the maximum level reached for each parasite line after subtraction of the background fluorescence. The mean and SD from triplicate measurements shown. Where not shown, error bars fall within the symbols. For clarity, only positive or negative error bars are shown for the data for certain parasite lines. (b) The maximum growth rate for each parasite line. The bars show the mean ± SD obtained from three independent experiments for each line, and the symbols show the results obtained in individual experiments. There was no significant difference in the maximum growth rate between any of the parasite lines (P = 0.8; one-way ANOVA).

Figure 3

Measurements of the effect of l-lactate on cytosolic pH (pH_cyt) reveal a role for TgFNT1 in lactate transport across the parasite plasma membrane. Extracellular tachyzoites were loaded with BCECF and suspended in Saline Solution at 4 °C. The grey arrows denote the addition of 10 mM l-lactate. The red arrow denotes the addition of the protonophore CCCP (10 µM). The top panels show data for Δtgfnt1 (b), Δtgfnt2 (c), Δtgfnt3 (d) parasites and their wild-type parental parasites (a). The bottom panels show data for Δtgfnt1 parasites complemented with tgfnt1 (e), tgfnt2 (f) or tgfnt3 (g). The traces are from a single experiment for each parasite line, and are representative of those obtained in at least three independent experiments. The time taken to reach a minimum pH_cyt value after the addition of l-lactate was > 400 s in all experiments for Δtgfnt1 parasites complemented with tgfnt2, compared to < 150 s in all experiments for WT parasites, Δtgfnt2 parasites, Δtgfnt3 parasites, and Δtgfnt1 parasites complemented with tgfnt1 or tgfnt3.

Figure 4

TgFNT1 transports l-lactate in Xenopus oocytes. l-[¹⁴C]lactate uptake into non-injected oocytes and oocytes expressing PfFNT or TgFNT1-HA was measured in the absence of compound (0.008% v/v DMSO; solvent control; white bars) and in the presence of the PfFNT inhibitor MMV007839 (4 µM; grey bars). The uptake of l-[¹⁴C]lactate is expressed as a percentage of that determined for TgFNT1-HA in the absence of MMV007839. The data are averaged from four independent experiments (using oocytes from different frogs), within which measurements were made from 7 to 10 oocytes per treatment. The symbols show the data from each experiment and the bars show the mean ± SD. Where not shown, error bars fall within the symbols. The data were tested for statistical difference from the non-injected control (black asterisks). The data obtained in the presence or absence of MMV007839 were also compared for each oocyte type (grey asterisks). ***P < 0.001 (one-way ANOVA with post hoc Tukey test); other comparisons did not show significant differences.

Figure 5

TgFNT1 is critical for l-lactate transport across the parasite plasma membrane. (a,c,e) Time courses for the uptake of l-lactate (a), 2-DOG (c) and L-arginine (e) by wild-type parasites (blue symbols), Δtgfnt1 parasites (white symbols) and Δtgfnt1/2/3 parasites (dark red symbols). The data shown are the mean ± SD from three to four independent experiments for each line. For clarity, only positive or negative error bars are shown for the data for certain parasite lines. Where not shown, error bars fall within the symbols. (b,d,f) The initial rates of uptake for l-lactate (b), 2-DOG (d), and L-arginine (f) estimated from the same set of experiments. The bars show the mean ± SD obtained from three or four independent experiments for each line, and the symbols show the results obtained in individual experiments. For panels b,d and f, the data for each parasite line were compared with the data for every other parasite line (one-way ANOVA, followed by post hoc Tukey tests where significant differences were present). ***P < 0.001; other comparisons (those in d and f) did not reveal significant differences.