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TgApiAT6-1 is the primary lysine transporter in the disease-causing tachyzoite stage of T. These findings demonstrate that T. The causative agent of toxoplasmosis, Fluoroplex (Fluorouracil Topical Cream)- Multum gondii, is a versatile intracellular parasite that can proliferate within nucleated cells of warm-blooded organisms.

In order to survive, T. In a previous study, we demonstrated that a plasma membrane-localized protein called TgApiAT1 facilitates the uptake of arginine into the parasite. We found that parasites lacking TgApiAT1 could proliferate when cultured in medium containing high concentrations of arginine, suggesting the existence of an additional uptake pathway for arginine.

In the present study, we demonstrate that this second uptake pathway is advanced powder technology by TgApiAT6-1, a protein belonging to the same solute transporter family as TgApiAT1. We show that TgApiAT6-1 is the major lysine transporter of the parasite, and that it is critical for parasite proliferation.

Furthermore, we demonstrate that TgApiAT6-1 can transport arginine into parasites under conditions in which Fluoroplex (Fluorouracil Topical Cream)- Multum concentrations are high and lysine concentrations are comparatively lower. These data support a model for the finely-tuned acquisition of essential cationic amino acids that involves multiple transporters, and which likely contributes to these parasites being able to survive and proliferate within a wide variety of host cell types.

PLoS Pathog 17(8): e1009835. This is Fluoroplex (Fluorouracil Topical Cream)- Multum open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: The authors confirm that all data underlying the findings are fully available without restriction.

Funding: This work was supported by Discovery Grants from the Australian Research Council to KK, GGvD and SB (DP150102883) and to GGvD and KK (DP200100483). MJM is a NHMRC Principal Research Fellow (APP1154540). The Fluoroplex (Fluorouracil Topical Cream)- Multum had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist. Intracellular parasites of the phylum Apicomplexa are the causative agents Fluoroplex (Fluorouracil Topical Cream)- Multum a diverse range of diseases in humans and domestic livestock, imposing major health and economic burdens in many countries.

The apicomplexan parasite Toxoplasma gondii infects up to one-third of the human population, and is the causative agent of the disease toxoplasmosis. Although usually asymptomatic in healthy adults, toxoplasmosis can cause lethal encephalitis in immunocompromised patients.

Despite initial indications that T. As a result of these auxotrophies, T. To date, three ApiAT proteins have been characterized and shown to transport essential amino acids across the plasma membrane. All three ApiATs have been shown to be important Fluoroplex (Fluorouracil Topical Cream)- Multum particular parasite life-cycle stages: the T. All ApiATs characterized to date have been shown Fluoroplex (Fluorouracil Topical Cream)- Multum be equilibrative transporters, facilitating the transmembrane passage of their amino acid substrates without the direct involvement of any other co-substrates (e.

In addition to TgApiAT1 and TgApiAT5-3, two other T. Here, we identify TgApiAT6-1 as this second transporter. We elucidate the transport mechanism of TgApiAT6-1, as well as that of TgApiAT1, showing both to be bidirectional uniporters with the capacity to mediate amino acid exchange, and the capacity to facilitate the intracellular accumulation of these two essential cationic amino acids. In a previous study of the ApiAT family Fluoroplex (Fluorouracil Topical Cream)- Multum T.

Compared to parasites cultured in the absence of ATc, we observed a major defect in proliferation Fluoroplex (Fluorouracil Topical Cream)- Multum rTgApiAT6-1 parasites cultured in the presence of ATc (conditions under which TgApiAT6-1 is knocked down; Fig 1B).

ATc had no effect on the proliferation of wild type (WT) parasites under the same conditions (Fig 1C). These data indicate that the knockdown of rTgApiAT6-1 is associated with a severe impairment of parasite proliferation.

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. The presence of the constitutively-expressed TgApiAT6-1 fully restored parasite proliferation in the presence of ATc (Fig 1D). Together, these data indicate that TgApiAT6-1 is important for proliferation of the tachyzoite stage of T.

We compared the fractional abundance of 13C-labelled amino acids to the total abundance of each amino acid following the 15 min uptake period (Fig 2A). Of the 17 amino acids detected by GC-MS, only the uptake of 13C-Lys was significantly reduced when TgApiAT6-1 expression was knocked down. These data suggested that TgApiAT6-1 what are the indications be a Lys transporter, although it could also mediate the uptake of other amino acids not detected under johnson designs transport conditions of the experiments, or not detected by GC-MS, such as Arg.

Amino acids are represented by single letter codes; OxoP, 5-oxoproline. Uptake of a range of amino acids into oocytes expressing TgApiAT6-1. The uptake into uninjected oocytes (shown in S3A Fig) was subtracted for all substrates tested. Inhibition of Arg uptake into Fluoroplex (Fluorouracil Topical Cream)- Multum oocytes by a range of amino acids.

Amino acid substrates are represented by single letter codes. 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.

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. The uptake into uninjected oocytes has been subtracted for all substrate concentrations tested. After optimising its expression in oocytes (S2B and S2C Fig), we investigated the substrate specificity of TgApiAT6-1. We measured the uptake of a range of radiolabelled amino acids and amino acid derivatives in TgApiAT6-1-expressing oocytes, a selection of which are shown in Fig 2B.

Consistent with the metabolomics data, TgApiAT6-1 mediated Lys uptake (Fig 2B). Notably, TgApiAT6-1 also mediated uptake of Arg and some neutral amino acids including Met and Leu (Fig 2B). This may be because TgApiAT6-1 has a higher affinity for Lys than for the neutral amino acids, such that under the conditions of the 13C-labelled amino acid uptake experiment, the Lys in the medium excluded the other amino acids from the active site of the transporter.

To test whether this was the case, we measured TgApiAT6-1-mediated uptake of Arg in oocytes in the presence of a 10-fold (Fig 2C) or 100-fold (Fig 2D) higher concentration of other, unlabelled amino acids. At a 10-fold higher concentration of the unlabelled amino acid, only Lys inhibited Arg uptake (Fig 2C); however, at 100-fold higher concentrations, numerous neutral amino acids including Met, Leu, Phe and His partially inhibited Arg uptake (Fig 2D).

This is consistent with the transporter having a higher affinity for Lys than for the other unlabelled amino acids tested. To test the affinity of TgApiAT6-1 for Lys and Arg, we measured the uptake kinetics of these amino acids.



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