Coordinated Action of Multiple Transporters in the Acquisition of Essential Cationic Amino Acids by the Intracellular Parasite Toxoplasma gondii (GC-MS Data set)
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 GC-MS data set of T.gondii extracellular parasites. Parasites cultured for two days in the absence or presence of the TgApiAT6-1 transporter in amino acid-free RPMI 1640 medium supplemented with [13C]-labelled amino acid mix. Polar metabolites were extracted and analysed by GC-MS. We compared the fractional abundance of [13C]-labelled amino acids to the total abundance of each amino acid following the 15 min uptake period. 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. This data set provides the raw and processed data files which accompanies Fig. 2A 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 GC-MS data set contains: 1. Sample Summary Table (Excel file) of including samples IDs (numbered) corresponding to the uploaded raw data files. 2. A compressed (.zip) folder containing raw data folders for each sample numbered with the same sample ID as in the summary spreadsheet. Each folder contains the .D format raw and processed results files.
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Freshly egressed WT (TATi/Tomato) or apiAT5-3Δ188–504 tachyzoites (108 parasites each sample) were incubated in 500 μl of amino acid-free RPMI 1640 medium supplemented with 2 mg/ml algal [ 13C]amino acid mix (Cambridge Isotope Laboratories) for 15 minutes (37˚C, 5% CO2). Labelling was terminated by rapid dilution in 14 ml of ice cold PBS. Parasite metabolites were extracted in chloroform:methanol:water (1:3:1 v/v/v) containing 1 nmol scyllo-inositol (Sigma). The aqueous phase metabolites were dried, methoxymated using 20 mg/ml methoxyamine in pyridine overnight, then trimethylsilylated by treatment with N,O-bis(trimethylsilyl)trifluoroacetamide containing 1% trimethylsilyl for 1 hr at room temperature. Samples were analyzed using GC-MS as described previously . The fractional labelling of all detected amino acids was estimated as the fraction of the metabolite pool containing one or more 13C-atoms after correction for natural abundance using the program DExSI . Total metabolite counts were normalized to scylloinositol as an internal standard. All samples were analyzed by GC/MS on a DB-5MS + DG column (J&W, Agilent, 30m × 0.25 mm, with 10 gap) column equipped Agilent 7890A-5975C gas chromatograph coupled mass spectrometer (GC/MS). Chromatograms were processed in MSD Chemstation D.01.02.16 software (Agilent). Chromatograms were processed in MSD Chemstation D.01.02.16 software (Agilent). The incorporation of 13C-atoms is estimated as percentage of the metabolite pool containing one or more 13C-atoms after correction for natural abundance. Total metabolite counts were normalized to scyllo-inositol as an internal standard and parasite numbers. The mass isotopomer distributions (MIDs) of individual metabolites were corrected for the occurrence of natural isotopes in both the metabolite and the derivitisation reagent. Metabolites were identified by comparison with the retention times and mass spectrum of authentic standards using Chemstation software (MSD Chemstation D.01.02.16, Agilent Technologies). GC‐MS chromatograms were aligned and processed with either AnalyzerPro (SpectralWorks) or PyMS (http://code.google.com/p/pyms/). Data normalization was performed by centering samples using the median values and scaled by inter‐quartile range (IQR). Missing values were imputed as half of the minimum abundance detected in other groups. PCAs were generated using Simca‐P 11 software (Umetrics) while Z‐transformation was used to scale metabolites in WT extracellular tachyzoites according to its corresponding intracellular value. References  Blume M, Nitzsche R, Sternberg U, Gerlic M, Masters SL, Gupta N, et al. Cell Host Microbe. 2015; 18(2):210–20. https://doi.org/10.1016/j.chom.2015.07.008 PMID: 26269956  Dagley MJ, McConville MJ. Bioinformatics. 2018; 34(11):1957–8. https://doi.org/10.1093/bioinformatics/ bty025 PMID: 29360933