Coordinated Action of Multiple Transporters in the Acquisition of Essential Cationic Amino Acids by the Intracellular Parasite Toxoplasma gondii (LC-MS Repeat Data set 3)
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. Here we provide an LC-MS data set of Xenopus laevis oocytes expressing the TgApiAT6-1 transporter in the presence of Arg or Lys and the TgApiAT1 transporter in the presence of Arg. This data set measures the change in intracellular oocyte metabolism over a time course of up to 54 hours, charting the uptake of Lys or Arg by the transporters in addition to fluctuations in endogenous oocyte metabolite levels. In addition, samples are also present that demonstrate the efflux of the loaded substrates at 30min, 3hr, and 12hr following the removal of either the Arg or Lys incubating solution. This data set provides the .raw data files which accompanies Fig. 6 in the publication: Coordinated Action of Multiple Transporters in the Acquisition of Essential Cationic Amino Acids by the Intracellular Parasite Toxoplasma gondii Stephen J. Fairweather, Esther Rajendran, Martin Blume, Kiran Javed, Birte Steinhöfel, Malcolm J. McConville, Kiaran Kirk, Stefan Bröer, Giel G. van Dooren bioRxiv 2021.06.25.450001; doi: https://doi.org/10.1101/2021.06.25.450001 This is the data set for the 3rd repeat LC-MS experiment, containing: 1. Sample Summary Table (Excel file) of including samples IDs (numbered) corresponding to the uploaded raw data files. 2. A compressed (zipped) folder containing individual raw data files numbered with the same sample ID as in the summary spreadsheet. The folder contains a .raw output file of the Total Ion Chromatograms (TIC) from each sample in the Sample Summary Table. Processed .mzml files converted form each sample .raw file can be provided upon request of the authors from the publication above. They are not provided directly here due to file size constraints. Raw peak height was used for the quantification of metabolites. Intracellular Arg and Lys oocyte concentrations were quantified using the LC-MS calibration curves with known Arg/Lys standards (in Fig. S9 of the cited publication).
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For each condition 12 oocytes were washed × 3 with 1 × ND96 solution (96 mM NaCl, 2 mM KCl, 1 mM MgCl2, 5 mM HEPES, pH 7.4) and incubated in the same buffer containing 1 mM Lys or Arg substrate for 48 hrs. Incubations were quenched by placing oocyte-containing tubes on ice and washing × 4 with 1 ml of ice-cold MilliQ H2O. Polar metabolites were extracted in chloroform:water:methanol (1:1:3) to isolate aqueous metabolites followed by centrifugation at 13,000 × g for 5 min to remove cell debris and unsolved material. The second extraction involved adding 1:5 H2O:mixture. The supernatant was centrifuged again at 13,000 × g for 5 min and the upper aqueous phase was removed Samples were desiccated on a vacuum centrifuge, then re-solved with acetonitrile/H2O (80%/20% v/v). Chromatographic separation was performed on an Ultimate 3000 RSLC nano Ultra high performance liquid chromatography (UHPLC) system (Dionex) by using a ZIC cHILIC column (3.0 μm polymeric, 2.1x150mm; Sequant, Merck). The gradient started with 80% mobile phase B (Acetonitrile; 0.1% v/v Formic acid) and 20% mobile phase A (10mM ammonium formate; 0.1% v/v formic acid) at a flow rate of 300 µl/min, followed by a linear gradient to 20% mobile phase B over 18 min. The column was re-equilibrated for 12 mins using 80% mobile phase B with the same flow rate. The column was maintained at 40°C and the injection sample volume was 4µl. The mass detection was conducted by Q-Exactive Plus Orbitrap mass spectrometer (Thermo Scientific) in positive electrospray mode with the following settings: resolution 70,00, m/z range 60–900, AGC target 3 × 106 counts, sheath gas 40 l min-1, auxiliary gas 10 l min-1, sweep gas 2 l min-1, and capillary temperature 250°C, spray voltage +3.5kV (full-scan mode). The MS/MS data was collected through data dependent top 5 scan mode using high-energy c-trap dissociation (HCD) with resolution 17,500, AGC target 1 × 105 counts and normalized collision energy (NCE) 30. A pooled sample of all extracts was used as a quality control (QC) sample to monitor signal reproducibility and stability of analytes. Blanks and QC samples were run within the batch to ensure reproducibility of the data. Arg and Lys were calibrated as an external standard by first making serial concentration and then aliquoting the same volume from each (8 μl) to give the following amounts (pmol): 80, 200, 400, 800, 2000, 4800. Calibration curves for both metabolites were freshly determined with each new batch of LC-MS/MS run and example curves. The acquired raw metabolite data were converted into mzXML format and processed through an open source software MS-DIAL. Identification of metabolites was performed by first using publicly available MS/MS libraries matching exact mass (MS tolerance 0.01 Da) and mass fragmentation pattern (MS tolerance 0.05 Da), and then further confirmed by standards through retention time where possible.