Phospholipid and sphingolipid metabolism in Leishmania

Abstract

In many eukaryotes, phospholipids (PLs) and sphingolipids (SLs) are abundant membrane components and reservoirs for important signaling molecules. In Leishmania, the composition, metabolism, and function of PLs and SLs differ significantly from those in mammalian cells. Although only a handful of enzymes have been experimentally characterized, available data suggest many steps of PL/SL metabolism are critical for Leishmania viability and/or virulence, and could be a source for new drug targets. Further studies of genes involved in the synthesis (de novo and salvage) and degradation of PLs and SLs will reveal their diverse effects on Leishmania pathogenesis.

Figure 1

PLs and SLs in L. major. (A) Lipid composition in L. major promastigotes based on the headgroups (left) or anchors (right) of PLs. Structures of each class of lipids were determined by ESI/MS/MS and abundance was estimated as previously described [22, 23, 26]. Other PLs include phosphatidylglycerol (PG), cardiolipin (CL), and phosphatidic acid. (B)–(F) Molecular species of PE and SLs in L. major. (B) A PLE: 1-O-octadec-1’-enyl 2-octadecenoyl sn-glycero-3-phosphoethanolamine (p18:0/18:1-PE). (C) A diacyl PE: 1,2-distearoyl sn-glycero-3-phosphoethanolamine (18:0/18:0-PE). (D) A lyso-PE: 1-O-octadec-1’-enyl 2-lyso sn-glycero-3-phosphoethanolamine (p18:0/lyso-PE). (E) A ceramide: N-stearoylhexadecesphing-4-enine (d16:1/18:0-Cer). (F) An IPC: phosphoryl inositol N-stearoylhexadecesphing-4-enine (d16:1/18:0-IPC).

Figure 2

Predicted metabolism of SLs and PLs in Leishmania. DAG: diacylglycerol; G-3-P: glycerol-3-phosphate; DHAP: dihydroxyacetonephosphate. Enzymes of which candidate genes have been identified in the L. major geneDB are indicated. Gray dotted arrows represent pathways where the putative enzymes have yet to be identified. Experimentally characterized enzymes/genes (circled) include DAT (dihydroxyacetone phosphate acyltransferase, system ID: LmjF34.1090), GAT (glycerol-3-phosphate acyltransferase, LmjF03.0080), ADS1 (1-alkyl dihydroxyacetone phosphate synthase 1, LmjF30.0120), G3PD (glycerol-3-phosphate dehydrogenase, LmjF10.0510), SPT (serine palmitoyltransferase, LmjF34.3740 and LmjF35.0320), SPL (sphingosine-1-phosphate lyase, LmjF30.2350), IPCS (IPC synthase, LmjF35.4990), and ISCL (inositol sphingolipidphospholipase C-like, LmjF08.0200). Other enzymes (that have yet to be characterized although their putative genes have been identified from L. major genome) include CS (ceramide synthase); CD (ceramidase); SK (sphingosine kinase); E/CK (ethanolamine/choline kinase); EPCT (ethanolamine-phosphate cytidylyltransferase); CPCT (cholinephosphate cytidylyltransferase); EPT (ethanolamine-specific phosphotransferase); C/EPT (choline/ethanolamine phosphotransferase); BE: (base exchange enzyme or PS synthase); and PSD (PS decarboxylase).

Figure 3

SL-null L. major generates normal DRM rafts. (A) TX100 insoluble (I) and soluble (S) fractions from log phase parasites (WT and spt2⁻) were analyzed by western-blot to assess the distribution of gp63, gp46, and glucose transporter (GT). (B) Log phase WT and spt2⁻ parasites were labeled with [1,2]¹⁴C-acetate and extracted with 1% TX100 at 4 °C or 37 °C. Detergent insoluble materials were separated from soluble materials by centrifugation. Lipids from both insoluble and soluble fractions were extracted and analyzed by thin layer chromatography. Ergo: ergosterol; PLs: phospholipids. (C) WT and spt2⁻ parasites with episomally expressed HASPB-GFP or FCaBP-HA were subjected to detergent extraction and western-blot analysis as described in (A), using anti-GFP and anti-HA antibodies. In addition, these parasites were subjected to fluorescence microscopy, as shown in D (GFP epifluorescence) and E (primary antibody: anti-HA mAb; secondary antibody: FITC-labeled goat-antimouse IgG). Leishmania culture, DRM isolation, and western-blot were performed as previously described [27]. To isolate metacyclics, spt2⁻ mutants were grown to stationary phase in the presence of 500 µM of EtN as previously described [23].

Figure 4

2D gel analysis of DRM fraction in L. major. DRM fractions were prepared from log phase WT and spt2⁻ parasites as described [27] and subjected to 2D gel electrophoresis. Among all detectable spots, only ~1.2% showed greater than 2.5-fold variation between WT and spt2⁻.