D so far involve phosphatidylcholine (PtdCho), phosphatidylethanolamine (PtdEtn), phosphatidylserine (PtdSer), phosphatidylinositol (PtdIns), phosphatidylglycerol, and phosphatidate [4]. Others and we’ve shown that T. gondii contains frequent eukaryotic phospholipids as well as the pathways for autonomous synthesis [5]. Physiological functions of phospholipids inside the parasite are poorly understood nonetheless, and the majority of the underlying enzymes have not been characterized as however. In addition, despite a steadily rising interest in roles of lipids in host athogen interactions [9], the existence and biogenesis of divergent pathogenspecific lipids stay very a great deal underappreciated.Results T. gondii Contains an Exclusive Too As Important 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 next to PtdSer (Fig 1A). Other main lipids were PtdCho, PtdEtn, PtdIns, PtdSer, and phosphoethanolamineceramide (PEtnCer), confirming preceding reports [5,7]. To determine the precise identity of X1 fraction, we Methyl aminolevulinate Purity & Documentation executed mass spectrometry (MS) analysis, which revealed specific PEtnCer and PtdSer species, as expected (Fig 1B). The most prominent peak within this fraction with an m/z of 850.five, on the other hand, didn’t correspond to a PEtnCer or PtdSer species. Tandem MS with the indicated peak showed a neutral loss of 101 atomic mass units (m/z, 749.six) contrary towards the expected 87 for serine, or 141 for ethanolamine (Fig 1C). The m/z profile matched to threonine as the polar head group instead, which was also independently confirmed by HPLC analysis of amino acid derived from lipid hydrolysis (S1A Fig). The fatty acyl chains of this specific lipid, phosphatidylthreonine (PtdThr henceforth), had been identified as 20:1 and 20:four. Other detectable, but evidently minor, PtdThr species also contained comparably polyunsaturated and extended 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) were the important parasite lipids visualized by iodinevapor staining. PtdThr (X1), detected once more close to PtdSer, was authenticated by MS evaluation (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 uncommon and notably minor PtdSer analog in specific mammalian cells and selected prokaryotes [103]. It was also shown that the baseexchangePLOS Trilinolein medchemexpress Biology | DOI:10.1371/journal.pbio.November 13,two /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 instances and relative abundance of lipids isolated from extracellular tachyzoites (107). X1 represents a previously unknown lipid. (B) MS analysis of X1 fraction revealing PtdThr, PtdSer, and PEtnCer species. Individual lipids have been identified by their fragmentation patterns and m/z ratios within the damaging ionization mode. (C) MS/MS spectrum of X1derived main peak (m/z 850.five) from panel B. Note the neutralPLOS Biology | DOI:ten.1371/journal.pbio.November 13,three /Phosphatidylthreonine Is Essential for the Parasite Virulenceloss of 101 Da (transition from 850.five to 749.six). Acyl chains (sn1, 20:1; sn2, 20:four) had been identified.