Leishmania major Expresses a Single Dihydroxyacetone Phosphate Acyltransferase Localized in the Glycosome, Important for Rapid Growth and Survival at High Cell Density and Essential for Virulence

Abstract

Despite major advances in the understanding of pathogenesis of the human protozoan parasite Leishmania major, little is known about the enzymes and the primary precursors involved in the initial steps of synthesis of its major glycerolipids including those involved in virulence. We have previously demonstrated that the initial step of acylation of the precursor glycerol 3-phosphate is not essential for the synthesis of ester and ether phospholipids in this parasite. Here we show that Leishmania expresses a single acyltransferase with high specificity for the precursor dihydroxyacetone phosphate and shows the best activity in the presence of palmitoylCoA. We have identified and characterized the LmDAT gene encoding this activity. LmDAT complements the lethality resulting from the loss of both dihydroxyacetone phosphate and glycerol-3-phosphate acyltransferase activities in yeast. Recombinant LmDAT exhibits biochemical properties similar to those of the native enzyme of the promastigote stage parasites. We show that LmDAT is a glycosomal enzyme and its loss in a ⌬lmdat/⌬lmdat null mutant results in complete abrogation of the parasite dihydroxyacetone phosphate acyltransferase activity. Furthermore, lack of LmDAT causes a major alteration in parasite division during the logarithmic phase of growth, an accelerated cell death during stationary phase, and loss of virulence. Together, our results demonstrate that LmDAT is the only dihydroxyacetone phosphate acyltransferase of the L. major localized in the peroxisome, important for growth and survival and essential for virulence.Worldwide, protozoan parasites of the genus Leishmania cause a large spectrum of important human diseases collectively named leishmaniasis. These parasites develop within the digestive tract of the sand fly vector as flagellated, mobile promastigotes and differentiate into and multiply as non-motile amastigotes within the phagolysosomal compartment of vertebrate host macrophages.Glycerolipids constitute 70% of total lipids in the protozoan parasite Leishmania (1-3). They are classified into ester and ether lipids depending on the substitution at position 1 of the glycerol backbone. Ester lipids harbor an acyl group, whereas ether lipids carry a fatty alcohol moiety. Lipids of Leishmania parasites have been a focus of extensive studies because some of their derivatives, such as lipophosphoglycan and glycosylphosphatidylinositol-anchored protease gp63, were shown to be important for parasite virulence and development (for review, see . Lipids are also essential cell constituents and, therefore, must be constantly synthesized to allow multiplication of the parasite. This suggests that the pathways leading to their synthesis are essential for parasite proliferation and pathogenesis and, thus, offer a reasonable target for rational design of new anti-leishmanial drugs. In fact, a lipidbased drug, miltefosine, is a potent antileishmanial compound that inhibits parasite growth in vitro and in vivo and is currently used for treatment of visceral and muc...

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

“…The latter encodes a protein of 1242 amino acids with a calculated molecular mass of 137 kDa. Tb DAT is much larger than the orthologs of higher eukaryotes such as mouse or fly [25]. The N-terminal domain does not show any similarity to any known proteins as the leishmanial ortholog while the C-terminal domain contains the four typical conserved motifs that are likely involved in catalysis and substrate recognition [25].…”

“…In the related parasite Leishmania major , the two enzymes Lm GAT and Lm DAT are the only initial acyltransferases [18, 24, 25]. Lm GAT specifically acylates G3P, contributing to ester glycerophospholipid biosynthesis only, and is dispensable for parasite growth and pathogenesis [18].…”

“…Lm GAT specifically acylates G3P, contributing to ester glycerophospholipid biosynthesis only, and is dispensable for parasite growth and pathogenesis [18]. In contrast, Lm DAT preferentially adds acyl groups to DHAP, participates in ester as well as ether glycerophospholipid biosynthesis, which is important for normal growth, survival during stationary phase and virulence [25]. …”

The Bifunctional Protein TtFARAT from Tetrahymena thermophila Catalyzes the Formation of both Precursors Required to Initiate Ether Lipid Biosynthesis

“…14 C]fructose-1,6-bisphosphate), 0.25 unit of aldolase, 10 units of triose phosphate isomerase, 25 M acyl-CoA (palmitoyl-, palmitoleyl-, stearyl-and oleyl-CoAs), 120 mM KCl, 0.1% (w/v) CHAPS, 4 mM MgCl 2 , 8 mM NaF, 1 mM DTT, and 4 mg/ml fatty acid-deficient BSA, and it was preincubated for 16 min at 30°C before adding the enzyme source (23). Both reactions were stopped by adding 600 l of 1% HClO 4 , and lipid extraction was performed as described previously by Athenstaedt et al (24).…”

Gene expression analysis of wild Leishmania major isolates: identification of genes preferentially expressed in amastigotes

“…However, we still need better strategies for the rapid identification of such genes. Over the past decades, several molecules playing a key role in the pathogencity of the Leishmania parasite have been identified (Zufferey and Ben Mamoun 2006;Joshi et al 2005;Zhang and Matlashewski 2004;Descoteaux et al 2002;Spath et al 2000;Beverley and Turco 1998;Mottram et al 1998;Mukhopadhyay et al 1998;Zhang and Matlashewski 1997;Chakrabarty et al 1996;Descoteaux et al 1995). The majority of Leishmania virulence studies are based on laboratory clones; it is therefore unknown whether the conclusions drawn from these studies are relevant to the natural pathogenicity of the parasite in the field.…”