D so far include phosphatidylcholine (PtdCho), phosphatidylethanolamine (PtdEtn), phosphatidylserine (PtdSer), phosphatidylinositol (PtdIns), phosphatidylglycerol, and phosphatidate [4]. Other people and we have shown that T. gondii consists of frequent eukaryotic phospholipids also because the pathways for autonomous synthesis [5]. Physiological functions of phospholipids inside the parasite are poorly understood having said that, and the majority of the Rankinidine Autophagy underlying enzymes have not been characterized as however. Moreover, in spite of a steadily rising interest in roles of lipids in host athogen interactions [9], the existence and biogenesis of divergent pathogenspecific lipids stay extremely a lot underappreciated.Final results 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 major lipid peak X1 eluting next to PtdSer (Fig 1A). Other major lipids were PtdCho, PtdEtn, PtdIns, PtdSer, and phosphoethanolamineceramide (PEtnCer), confirming preceding reports [5,7]. To ascertain the precise identity of X1 fraction, we executed mass spectrometry (MS) evaluation, which revealed particular PEtnCer and PtdSer species, as expected (Fig 1B). One of the most prominent peak in this fraction with an m/z of 850.5, nevertheless, did not correspond to a PEtnCer or PtdSer species. Tandem MS on the indicated peak showed a neutral loss of 101 atomic mass units (m/z, 749.6) 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), were identified as 20:1 and 20:4. Other detectable, but evidently minor, PtdThr species also contained comparably polyunsaturated and lengthy acyl chains (Fig 1B). Next, 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 (apart from PtdThr) have been the significant parasite lipids visualized by iodinevapor staining. PtdThr (X1), detected once more close to PtdSer, was authenticated by MS analysis (S1C Fig). PtdThr accounted for 20 nmol/108 parasites by lipid phosphorus quantification. It can be noteworthy that PtdThr has been 5 pdh Inhibitors medchemexpress previously reported as a rare and notably minor PtdSer analog in specific mammalian cells and chosen prokaryotes [103]. It was also shown that the baseexchangePLOS Biology | DOI:10.1371/journal.pbio.November 13,2 /Phosphatidylthreonine Is Required 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 analysis of X1 fraction revealing PtdThr, PtdSer, and PEtnCer species. Person lipids were identified by their fragmentation patterns and m/z ratios inside the adverse ionization mode. (C) MS/MS spectrum of X1derived big peak (m/z 850.five) from panel B. Note the neutralPLOS Biology | DOI:10.1371/journal.pbio.November 13,3 /Phosphatidylthreonine Is Essential for the Parasite Virulenceloss of 101 Da (transition from 850.five to 749.six). Acyl chains (sn1, 20:1; sn2, 20:4) were identified.
