D polymer refine detection kit (Menarini/Leica, Germany). Tissue sections have been scanned at 230 nm resolution making use of a MiraxMidi Scanner (Zeiss MicroImaging GmbH, Germany) [48].Supporting InformationS1 Data. Excel spreadsheet containing underlying numerical data and statistical Aeroplysinin 1 site 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 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 have been identified by their migration patterns in comparison to genuine phospholipid standards except for PtdThr, for which no industrial standard 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 don’t include detectable amounts of phosphatidylthreonine. (A) Liquid chromatographymass spectrometry (LCMS) elution profile displaying the retention occasions and peak intensities of phospholipids isolated from human fibroblasts. (B) MS evaluation of the indicated fraction revealing the prevalent occurrence of PtdSer species and also a full lack of detectable PtdThr species. Fibroblast lipids had been detected in the damaging 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 analysis with the orthologs of PTS and PSS from distinct organisms shows an early divergence from 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) have been 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 various substitutions inside the catalytic domain of an otherwise universal baseexchangetype PtdSer synthase. (A) SecondaryPLOS Biology | DOI:10.1371/journal.pbio.November 13,19 /Phosphatidylthreonine Is Essential 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 signs 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 below the handle from the TgGRA1 promoter and 3’UTR at the UPRT locus had been generated by FUDR.
