D polymer refine detection kit (Menarini/Leica, Germany). Tissue sections have been scanned at 230 nm resolution working with 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 usually a big 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 major iodinestained phospholipids. Lipids were identified by their migration patterns in comparison to authentic phospholipid standards except for PtdThr, for which no industrial common 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 times and peak intensities of phospholipids isolated from human fibroblasts. (B) MS analysis of your indicated fraction revealing the prevalent occurrence of PtdSer species and a total lack of detectable PtdThr species. Fibroblast lipids had been detected within the negative ionization mode, as described for the parasite lipids. (TIFF) S3 Fig. Orthologs of PtdThr synthase are present in chosen freeliving and Tetrahydrozoline supplier parasitic protists but absent in most other organisms. Phylogenetic evaluation of your 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 chosen parasitic (Eimeria, Neospora, Phytophtora) and freeliving (Perkinsus) chromalveolates. Colored circles signify bootstrap values. Sequences for performing phylogenetic evaluation (www.phylogeny.fr) were obtained in the NCBI (www.ncbi.nlm.nih.gov) and parasite databases (www.ToxoDB. org). Accession numbers are indicated subsequent for the sequence. NCBI accession IDs for TgPTS and TgPSS are KJ026547 and KJ026548, (-)-Bicuculline methochloride Formula 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:ten.1371/journal.pbio.November 13,19 /Phosphatidylthreonine Is Needed for the Parasite Virulencestructure and membrane topology of TgPTS, as predicted by SOSUI system (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 under the alignment denotes the transmembrane domain. (TIFF) S5 Fig. Immunofluorescence costaining of TgPTSHA with organellespecific markers. Transgenic parasites ectopically expressing TgPTSHA beneath the control of the TgGRA1 promoter and 3’UTR in the UPRT locus were generated by FUDR.
