Measuring Solute Transport in Toxoplasma gondii Parasites

Intracellular parasites, such as the apicomplexan Toxoplasma gondii, are adept at scavenging nutrients from their host. However, there is little understanding of how parasites sense and respond to the changing nutrient environments they encounter during an infection. TgApiAT1, a member of the apicomplexan ApiAT family of amino acid transporters, is the major uptake route for the essential amino acid L-arginine (Arg) in T. gondii. Here, we show that the abundance of TgApiAT1, and hence the rate of uptake of Arg, is regulated by the availability of Arg in the parasite’s external environment, increasing in response to decreased [Arg]. Using a luciferase-based ‘biosensor’ strain of T. gondii, we demonstrate that the expression of TgApiAT1 varies between different organs within the host, indicating that parasites are able to modulate TgApiAT1-dependent uptake of Arg as they encounter different nutrient environments in vivo. Finally, we show that Arg-dependent regulation of TgApiAT1 expression is post-transcriptional, mediated by an upstream open reading frame (uORF) in the TgApiAT1 transcript, and we provide evidence that the peptide encoded by this uORF is critical for mediating regulation. Together, our data reveal the mechanism by which an apicomplexan parasite responds to changes in the availability of a key nutrient.

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“…Experiments to measure uptake of [ 14 C]Arg through Tg ApiAT1 were performed as described previously [ 15 , 57 ]. Briefly, extracellular T .…”

Section: Methodsmentioning

confidence: 99%

“…Experiments to measure uptake of [ 14 C]Arg through TgApiAT1 were performed as described previously [15,57]. Briefly, extracellular T. gondii parasites were incubated in PBS containing 10 mM D-glucose, 0.1 μCi/ml [ 14 C]Arg, 50 μM unlabelled Arg, and 80 μM unlabelled Lys for a range of times.…”

Section: Arg Uptake Experimentsmentioning

confidence: 99%

Substrate-mediated regulation of the arginine transporter of Toxoplasma gondii

et al. 2021

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“…Time course radiolabelled uptake assays in extracellular T. gondii parasites were performed as described previously [58]. Briefly, WT and rTgApiAT6-1 parasites were cultured in parasites from unincorporated radiolabel.…”

Section: Radiolabel Uptake Assays In T Gondii Parasitesmentioning

confidence: 99%

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

Fairweather

Rajendran

Blume

et al. 2021

Preprint

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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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“…Radiolabel uptake assays with extracellular T . gondii parasites were performed as described previously [ 58 ]. Briefly, WT and r Tg ApiAT6-1 parasites were cultured in Dulbecco’s Modified Eagle’s medium (containing 400 μM Arg and 800 μM Lys) in the absence or presence of ATc for 2 days.…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2021

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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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Measuring Solute Transport in Toxoplasma gondii Parasites

Cited by 5 publications

References 30 publications

Substrate-mediated regulation of the arginine transporter of Toxoplasma gondii

Substrate-mediated regulation of the arginine transporter of Toxoplasma gondii

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

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

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