D polymer refine detection kit (Menarini/Leica, Germany). Tissue sections had been scanned at 230 nm resolution working with a MiraxMidi Scanner (Zeiss MicroImaging GmbH, Germany) [48].Supporting InformationS1 Data. 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. 2-Methoxy-4-vinylphenol MedChemExpress PtdThr is often a key phospholipid in T. gondii. (A) HPLC profile of threonine obtained by hydrolysis of X1lipid from extracellular tachyzoites (107). Detection and quantification was achieved by multiplereactionmonitoring (MRM) MS of threonine decarboxylation (transition, 120/74 Da). (B) Twodimensional TLC of lipids from tachyzoites (108) displaying key iodinestained phospholipids. Lipids had been identified by their migration patterns in comparison to genuine phospholipid requirements except for PtdThr, for which no industrial normal is accessible. (C) Chemical identity of PtdThr by MS analysis. TLCresolved X1 band from panel B was confirmed as PtdThr by fragmentation pattern and m/z ratios. (TIFF) S2 Fig. Human foreskin fibroblast cells don’t include detectable amounts of phosphatidylthreonine. (A) Liquid chromatographymass spectrometry (LCMS) elution profile showing the retention occasions and peak intensities of phospholipids isolated from human fibroblasts. (B) MS analysis in the indicated fraction revealing the prevalent occurrence of PtdSer species along with a total lack of detectable PtdThr species. Fibroblast lipids have been detected Akt kinase Inhibitors products within 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 with the orthologs of PTS and PSS from distinct organisms shows an early divergence of the two enzymes. TgPSS (ToxoDB: TGGT1_261480) clusters with all the mainstream PSS clade that also comprises other parasite orthologs. In contrast, TgPTS (ToxoDB: TGGT1_273540) segregates with selected parasitic (Eimeria, Neospora, Phytophtora) and freeliving (Perkinsus) chromalveolates. Colored circles signify bootstrap values. Sequences for performing phylogenetic analysis (www.phylogeny.fr) were obtained from the NCBI (www.ncbi.nlm.nih.gov) and parasite databases (www.ToxoDB. org). Accession numbers are indicated next towards the sequence. NCBI accession IDs for TgPTS and TgPSS are KJ026547 and KJ026548, respectively. (TIFF) S4 Fig. PtdThr synthase from T. gondii harbors multiple 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 Necessary 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 to the PSS activity and to substrate binding, respectively. Other conserved residues in PSS proteins show distinct substitutions in PTS orthologs (colored boxes). Gray bar below the alignment denotes the transmembrane domain. (TIFF) S5 Fig. Immunofluorescence costaining of TgPTSHA with organellespecific markers. Transgenic parasites ectopically expressing TgPTSHA beneath the control from the TgGRA1 promoter and 3’UTR at the UPRT locus had been generated by FUDR.
