D so far incorporate phosphatidylcholine (PtdCho), phosphatidylethanolamine (PtdEtn), phosphatidylserine (PtdSer), phosphatidylinositol (PtdIns), phosphatidylglycerol, and phosphatidate [4]. Other people and we have shown that T. gondii includes common eukaryotic phospholipids as well as the pathways for autonomous synthesis [5]. Physiological functions of phospholipids in the parasite are poorly understood having said that, and a lot of the underlying enzymes haven’t been characterized as however. In addition, in spite of a steadily increasing interest in roles of lipids in host athogen interactions [9], the existence and biogenesis of divergent pathogenspecific lipids remain very a great deal underappreciated.Outcomes T. gondii Includes an Exclusive Too As Significant Phospholipid, PhosphatidylthreonineIn our expedition to characterize membrane biogenesis in T. gondii, we fractionated the parasite lipids by highperformance liquid chromatography (HPLC) and observed a significant lipid peak X1 eluting subsequent to PtdSer (Fig 1A). Other main lipids have been PtdCho, PtdEtn, PtdIns, PtdSer, and phosphoethanolamineceramide (PEtnCer), confirming preceding reports [5,7]. To figure out the precise identity of X1 fraction, we executed mass spectrometry (MS) analysis, which revealed specific PEtnCer and PtdSer species, as expected (Fig 1B). By far the most prominent peak within this fraction with an m/z of 850.5, even so, didn’t correspond to a PEtnCer or PtdSer species. Tandem MS of the indicated peak showed a neutral loss of 101 atomic mass units (m/z, 749.six) contrary to the anticipated 87 for serine, or 141 for ethanolamine (Fig 1C). The m/z profile matched to threonine because the polar head group rather, which was also independently confirmed by HPLC evaluation of amino acid derived from lipid hydrolysis (S1A Fig). The fatty acyl chains of this particular lipid, phosphatidylthreonine (PtdThr henceforth), have been identified as 20:1 and 20:4. Other detectable, but evidently minor, PtdThr species also contained comparably polyunsaturated and long acyl chains (Fig 1B). Subsequent, we resolved the parasite lipids by twodimensional thin layer chromatography (TLC). As apparent (S1B Fig), and also shown elsewhere [5], PtdCho, PtdEtn, PtdIns and PtdSer (in addition to PtdThr) had been the main parasite lipids visualized by iodinevapor staining. PtdThr (X1), detected once more close to PtdSer, was authenticated by MS A2e cathepsin Inhibitors Reagents analysis (S1C Fig). PtdThr accounted for 20 nmol/108 parasites by lipid phosphorus quantification. It truly is noteworthy that PtdThr has been previously reported as a rare and notably minor PtdSer analog in particular mammalian cells and selected prokaryotes [103]. It was also shown that the baseexchangePLOS Biology | DOI:ten.1371/journal.pbio.November 13,2 /Phosphatidylthreonine Is Necessary for the Parasite VirulenceFig 1. Lipidomics of T. gondii tachyzoites identifies a novel parasite lipid, PtdThr. (A) Elution profile showing the retention occasions and relative abundance of lipids isolated from extracellular tachyzoites (107). X1 represents a previously unknown lipid. (B) MS evaluation of X1 fraction revealing PtdThr, PtdSer, and PEtnCer species. Person lipids had been identified by their fragmentation patterns and m/z ratios in the unfavorable ionization mode. (C) MS/MS spectrum of Dimethoate Inhibitor X1derived main peak (m/z 850.5) from panel B. Note the neutralPLOS Biology | DOI:10.1371/journal.pbio.November 13,three /Phosphatidylthreonine Is Required for the Parasite Virulenceloss of 101 Da (transition from 850.5 to 749.six). Acyl chains (sn1, 20:1; sn2, 20:4) have been identified.
