D polymer refine detection kit (Menarini/Leica, Germany). Tissue sections have been scanned at 230 nm resolution applying a MiraxMidi Scanner (Zeiss MicroImaging GmbH, Germany) [48].Supporting InformationS1 Information. Excel spreadsheet containing underlying numerical information and statistical analyses for Figs 1A, 5BE, 6B and 6C, 7B and 7C, 8AC, S1A, S7, S8B, S9A and S9B and S12A and S12B Figs. (XLSX) S1 Fig. PtdThr is really a main phospholipid in T. gondii. (A) HPLC profile of threonine obtained by hydrolysis of X1lipid from extracellular tachyzoites (107). Detection and quantification was accomplished by multiplereactionmonitoring (MRM) MS of threonine decarboxylation (transition, 120/74 Da). (B) Twodimensional TLC of lipids from tachyzoites (108) showing key iodinestained phospholipids. Lipids were identified by their migration patterns in comparison to genuine phospholipid requirements except for PtdThr, for which no commercial typical is out there. (C) Chemical identity of PtdThr by MS evaluation. TLCresolved X1 band from panel B was confirmed as PtdThr by fragmentation pattern and m/z ratios. (TIFF) S2 Fig. Human foreskin fibroblast cells do not contain detectable amounts of phosphatidylthreonine. (A) Liquid chromatographymass spectrometry (LCMS) elution profile showing the retention times and peak intensities of phospholipids Toloxatone MedChemExpress isolated from human fibroblasts. (B) MS Anthraquinone-2-carboxylic acid custom synthesis analysis on the indicated fraction revealing the prevalent occurrence of PtdSer species as well as a full lack of detectable PtdThr species. Fibroblast lipids have been detected inside the unfavorable ionization mode, as described for the parasite lipids. (TIFF) S3 Fig. Orthologs of PtdThr synthase are present in selected freeliving and parasitic protists but absent in most other organisms. Phylogenetic evaluation of the orthologs of PTS and PSS from distinct organisms shows an early divergence with the two enzymes. TgPSS (ToxoDB: TGGT1_261480) clusters using the mainstream PSS clade that also comprises other parasite orthologs. In contrast, TgPTS (ToxoDB: TGGT1_273540) segregates with chosen parasitic (Eimeria, Neospora, Phytophtora) and freeliving (Perkinsus) chromalveolates. Colored circles signify bootstrap values. Sequences for performing phylogenetic analysis (www.phylogeny.fr) were obtained in the NCBI (www.ncbi.nlm.nih.gov) and parasite databases (www.ToxoDB. org). Accession numbers are indicated subsequent towards the sequence. NCBI accession IDs for TgPTS and TgPSS are KJ026547 and KJ026548, respectively. (TIFF) S4 Fig. PtdThr synthase from T. gondii harbors numerous substitutions in the catalytic domain of an otherwise universal baseexchangetype PtdSer synthase. (A) SecondaryPLOS Biology | DOI:10.1371/journal.pbio.November 13,19 /Phosphatidylthreonine Is Required for the Parasite Virulencestructure and membrane topology of TgPTS, as predicted by SOSUI program (http://bp.nuap. nagoyau.ac.jp/sosui). (B) Amino acid sequence alignment of PSS and PTS from T. gondii with orthologs from indicated organisms. The diamond and arrow indicators specify the residues contributing for the PSS activity and to substrate binding, respectively. Other conserved residues in PSS proteins show distinct substitutions in PTS orthologs (colored boxes). Gray bar beneath the alignment denotes the transmembrane domain. (TIFF) S5 Fig. Immunofluorescence costaining of TgPTSHA with organellespecific markers. Transgenic parasites ectopically expressing TgPTSHA below the control with the TgGRA1 promoter and 3’UTR at the UPRT locus were generated by FUDR.