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 data 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 actually a important 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) displaying big iodinestained phospholipids. Lipids were identified by their migration patterns in comparison to authentic phospholipid standards except for PtdThr, for which no industrial normal is offered. (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 contain 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 evaluation in the indicated fraction revealing the prevalent occurrence of PtdSer species as well as a full lack of detectable PtdThr species. Fibroblast lipids had been detected within the unfavorable ionization mode, as Linopirdine Neuronal Signaling described for the parasite lipids. (TIFF) S3 Fig. Orthologs of PtdThr synthase are present in chosen freeliving and parasitic protists but absent in most other organisms. Phylogenetic analysis of the orthologs of PTS and PSS from distinct organisms shows an early SKF-83566 Dopamine Receptor divergence of your 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 evaluation (www.phylogeny.fr) had been obtained from 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, respectively. (TIFF) S4 Fig. PtdThr synthase from T. gondii harbors a number of substitutions within the catalytic domain of an otherwise universal baseexchangetype PtdSer synthase. (A) SecondaryPLOS Biology | DOI:10.1371/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 beneath the alignment denotes the transmembrane domain. (TIFF) S5 Fig. Immunofluorescence costaining of TgPTSHA with organellespecific markers. Transgenic parasites ectopically expressing TgPTSHA under the handle from the TgGRA1 promoter and 3’UTR in the UPRT locus have been generated by FUDR.
