The Glycerol‐3‐Phosphate Acyltransferase TbGAT is Dispensable for Viability and the Synthesis of Glycerolipids in Trypanosoma brucei

Abstract

Glycerolipids are the main constituents of biological membranes in Trypanosoma brucei, which causes sleeping sickness in humans. Importantly, they occur as a structural component of the glycosylphosphatidylinositol lipid anchor of the abundant cell surface glycoproteins procyclin in procyclic forms and variant surface glycoprotein in bloodstream form, that play crucial roles for the development of the parasite in the insect vector and the mammalian host, respectively. The present work reports the characterization of the glycerol-3-phosphate acyltransferase TbGAT that initiates the biosynthesis of ester glycerolipids. TbGAT restored glycerol-3-phosphate acyltransferase activity when expressed in a Leishmania major deletion strain lacking this activity and exhibited preference for medium length, unsaturated fatty acyl-CoAs. TbGAT localized to the endoplasmic reticulum membrane with its N-terminal domain facing the cytosol. Despite that a TbGAT null mutant in T. brucei procyclic forms lacked glycerol-3-phosphate acyltransferase activity, it remained viable and exhibited similar growth rate as the wild type. TbGAT was dispensable for the biosynthesis of phosphatidylcholine, phosphatidylinositol, phosphatidylserine and GPI-anchored protein procyclin. However, the null mutant exhibited a slight decrease in phosphatidylethanolamine biosynthesis that was compensated with a modest increase in production of ether phosphatidylcholine. Our data suggest that an alternative initial acyltransferase takes over TbGAT’s function in its absence.

The Trypanosoma brucei dihydroxyacetonephosphate acyltransferase TbDAT is dispensable for normal growth but important for synthesis of ether glycerophospholipids

“…In contrast, Tb GAT acylates both DHAP and G3P but is implicated mainly in ester glycerophospholipid biosynthesis (solid arrow) [31]. …”

The glycosomal alkyl-dihydroxyacetonephosphate synthase TbADS is essential for the synthesis of ether glycerophospholipids in procyclic trypanosomes

“…The wild-type strain of procyclic forms of T. brucei used in this work is stock 427-60 and was cultivated in SDM-79 medium supplemented with 10 % heat inactivated fetal bovine serum as described in (Brun and Schonenberger 1979). Growth, transformation, and limiting dilution of parasites were carried out as previously described (Patel, Pirani et al 2016). Transgenic lines were selected in the presence of 5 μg/ml blasticidin, 2 μg/ml puromycin, and 3 μg/ml phleomycin as appropriate.…”

“…For complementation of the T. brucei null mutant, TbADS was inserted in sense orientation into the BamHI site of the plasmid pHD309PUR/BLEO.V5 to give pHD309BLEO.V5:TbADS (Ec871 (Patel, Pirani et al 2016)). The V5 encoding DNA was then deleted by cutting with HindIII and BamHI (partial) to create pHD309BLEO.TbADS (Ec884).…”

“…1 Patel, Pirani et al 2016, Zufferey, Pirani et al 2017). Tb DAT and Tb GAT exhibit slightly different specificities regarding the fatty acyl-CoA donor and localize in different subcellular compartments Tb GAT resides in the endoplasmic reticulum, while Tb DAT localizes to peroxisome-related organelles, called glycosomes in protozoan parasites of the family Trypanosomatidae (Opperdoes 1984, Zomer, Opperdoes et al 1995, Heise and Opperdoes 1997, Vertommen, Van Roy et al 2008, Patel, Pirani et al 2016). Both enzymes are dispensable for normal growth, but only Tb DAT is important for ether glycerophospholipid biogenesis.…”

Lipidomics and anti‐trypanosomatid chemotherapy

“…These analyses established that Lmj PEM1 added the first and second methyl group to PE, while Lmj PEM2 catalyzed all three methylation steps, although the addition of the first methyl group occurred very inefficiently. T. brucei possesses two initial acyltransferases, the glycerol‐3‐phosphate acyltransferase Tb GAT, which is dispensable for glycerolipid biosynthesis and growth of procyclic forms, and the dihydroxyacetonephosphate acyltransferase Tb DAT, which is essential for ether lipid production [62,63]. The role of T. brucei Tb PLA 1 in PC metabolism was investigated by global lipidomics investigation [64].…”