Coordinated Action of Multiple Transporters in the Acquisition of Essential Cationic Amino Acids by the Intracellular Parasite Toxoplasma gondii

Abstract

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.

Untargeted Metabolomics Uncovers the Essential Lysine Transporter in Toxoplasma gondii

“…In contrast to NPT1, a lack of AT6-1 could not be rescued by providing high levels of exogenous lysine, indicating that AT6-1 is the sole lysine transporter of T. gondii. We further observed a modest but significant decrease in arginine uptake upon the down-regulation of AT6-1, consistent with its role as a low affinity arginine transporter [26,27]. To the best of our knowledge, this is the first study employing untargeted metabolomics to characterize an apicomplexan solute carrier.…”

“…TgApiAT1 and TgApiAT5-3 were identified as arginine and tyrosine transporters, respectively [11,12]. TgApiAT6-1 was recently putatively characterized as a lysine transporter [26,27]. (b) Cartoon scheme of AT6-1 locus modification by in-Figure 1.…”

“…TgApiAT1 and TgApiAT5-3 were identified as arginine and tyrosine transporters, respectively [11,12]. TgApiAT6-1 was recently putatively characterized as a lysine transporter [26,27]. (b) Cartoon scheme of AT6-1 locus modification by introducing a 3-Ty-tag, loxP sites and a U1 recognition site, enabling localization of AT6-1 and its conditional downregulation.…”

“…These findings suggested that NPT1 functions as an arginine transporter but that an additional arginine transporter exists, which facilitates the transport of arginine with a lower affinity. Recently, AT6-1 (TGME49_240810) was proposed as the lysine transporter and low affinity arginine transporter [26,27]. These amino acid transporters were identified based on targeted or semi-targeted analyses using stable isotope labeled amino acids.…”

Characterization of apicomplexan amino acid transporters (ApiATs) in the malaria parasite Plasmodium falciparum

“…P. falciparum possesses five of the eleven ApiAT subfamilies: Pf ApiAT2 ( Pf ApiAT2/MFR4: PF3D7_0914700), Pf ApiAT4 (MFR5: PF3D7_1129900), Pf ApiAT8 (NPT1: PF3D7_0104800), Pf ApiAT9 (MFR2: PF3D7_0104700) and Pf ApiAT10 (MFR3: PF3D7_0312500). Previous work in the rodent malaria species Plasmodium berghei ( Pb ) and the related Apicomplexan parasite Toxoplasma gondii ( Tg ) showed that Pb ApiAT8 (or Pb NPT1) and several other Tg ApiATs are localized at the PPM 29–32 and possess amino acid transport activity 25,26,29–34 . To date, only Pb ApiAT4 has been shown to play an important role in parasite proliferation within erythrocytes 35 .…”