Redirection of sphingolipid metabolism toward de novo synthesis of ethanolamine in Leishmania

Abstract

In most eukaryotes, sphingolipids (SLs) are critical membrane components and signaling molecules. However, mutants of the trypanosomatid protozoan Leishmania lacking serine palmitoyltransferase (spt2-) and SLs grow well, although they are defective in stationary phase differentiation and virulence. Similar phenotypes were observed in sphingolipid (SL) mutant lacking the degradatory enzyme sphingosine 1-phosphate lyase (spl-). This epistatic interaction suggested that a metabolite downstream of SLs was responsible. Here we show that unlike other organisms, the Leishmania SL pathway has evolved to be the major route for ethanolamine (EtN) synthesis, as EtN supplementation completely reversed the viability and differentiation defects of both mutants. Thus Leishmania has undergone two major metabolic shifts: first in de-emphasizing the metabolic roles of SLs themselves in growth, signaling, and maintenance of membrane microdomains, which may arise from the unique combination of abundant parasite lipids; Second, freed of typical SL functional constraints and a lack of alternative routes to produce EtN, Leishmania redirected SL metabolism toward bulk EtN synthesis. Our results thus reveal a striking example of remodeling of the SL metabolic pathway in Leishmania.

Figure 1

Synthesis of SLs and PtE in eukaryotes. Open block arrows represent pathways not present in Leishmania. Filled block arrows (gray) represent dominant metabolic routes in Leishmania promastigotes. ADS1: 1-alkyl dihydroxyacetonephosphate synthase; SDC: serine decarboxylase (not present in Leishmania); PSS: phosphatidylserine synthase (not present in Leishmania); BE: base exchange enzyme (phosphatidyl serine synthase 2; PSS2); PSD: phosphatidylserine decarboxylase; Smy: sphingomyelin; GSL: glycosylsphingolipid; DAG: diacylglycerol; DHAP: dihydroxyacetonephosphate.

Figure 2

spl⁻ and spt2⁻ mutants have similar defects. (A) Growth in vitro. Promastigotes were inoculated at 1 × 10⁵/ml and densities were measured. •: WT; ○: spt2⁻; ▾: spl⁻; ▿: spt2⁻/+SPT2; ▪: spl⁻/+SPL. (B) Stationary phase viability. (C) Metacyclogenesis (PNA lectin method). In (A–C), experiments were performed in duplicate or triplicate. Error bars represent s.d. (D) TLC separation of [3-³H]serine-labeled lipids from SPT2 and SPL mutants. The arrowhead indicates a species accumulated in the spl⁻ mutant probably corresponding to sphingosine-1-phosphate. Abbreviations: PtC, phosphatidylcholine; IPC, inositol phosphorylceramide; PtE, phosphatidylethanolamine; SB, sphingosine; S-1-P, sphingosine-1-phosphate. Note: the spot corresponding to PtC (now confirmed by MS analysis) was previously incorrectly labeled as PtS (Zhang et al, 2003). (E) SPL activity assay. The average and standard error of four determinations is shown. The difference between spl⁻ and the other lines is significant at the P<0.05 level. (F) Elevated levels of sphingosine occur in spl⁻ preparations. ESI/MS was performed in the positive-ion mode and the relative abundance of the m/z 274.2 d16:1 sphingosine peak was expressed as its ratio relative to the PtC peak (m/z 830.6). The identity of the sphingosine was confirmed by secondary collisions (not shown).

Figure 3

EtN restores the stationary-phase defects of spt2⁻ and spl⁻ mutants. (A–C) Exogenous EtN reverses the defects of spt2⁻ and spl⁻ mutants in growth and differentiation. WT (•), spt2⁻ (○), and spl⁻ (▾) promastigotes were inoculated at 1 × 10⁵/ml in media supplemented with EtN. Three days after entry into stationary phase, culture density, cell viability, and percentage of PNA metacyclics were determined. (D–I) Transmission EM images of WT (D, G), spt2⁻ (E, H), and spl⁻ (F, I) promastigotes in late stationary phase. Cells were grown in the absence (D–F) or presence (G–I) of 0.5 mM EtN. Arrows indicate flagellar pockets (D–I), arrowheads indicate acidocalcisomes (D, G–I), and asterisks indicate lipid inclusions accumulated in spt2⁻ (E) and spl⁻ promastigotes (F). Bars=1 μm.

Figure 4

EtN is essential for promastigote growth. Promastigotes (•: WT; ○: spt2⁻; ▾: spl⁻) were inoculated at 2 × 10⁵/ml in serum-free medium in the absence (A) or presence (B) of 1 mM EtN and densities were measured over time. Experiments were performed in duplicate and error bars represent s.d.

Figure 5

WT but not spl⁻ or spt2⁻ promastigotes contain increased levels of plasmalogen PtE during stationary phase. (A, B) Negative-ion ESI/MS spectra of plasmalogen PtE in promastigote lipids. WT, spl⁻ parasites or spt2⁻ parasites were grown in the absence of EtN. Total lipids from log- (A) or stationary-phase (B) parasites were extracted and analyzed by negative-ion ESI/MS. Before extraction, a PtE standard (14:0/14:0-PtE at m/z 634.5, indicated by open arrowheads) was added as an internal standard to each sample (15 μg per 10⁸ cells). Filled arrowheads indicate plasmalogen PtE species (at m/z 726.5 and 728.5). (C) Promastigotes were inoculated at 10⁵/ml in the absence or presence of 0.5 mM EtN. Cellular plasmalogen PtE levels were then determined. (D) WT promastigotes were inoculated at 10⁵/ml in the absence of EtN, and IPC and plasmalogen PtE levels were determined over time. Data were collected from duplicate experiments, and error bars represent s.d.

Figure 6

Virulence studies of spt2⁻ and spl⁻ mutants. (A–D) Mouse footpad infections with WT (•), spt2⁻ (○), and spl⁻ (▾) promastigotes. In (A) and (B), stationary phase promastigotes grown in the absence (A) or presence (B) of 0.5 mM EtN were inoculated. In (C) and (D), metacyclics grown in the presence of 0.5 mM EtN prepared by the PNA lectin method (C) or the density gradient method (D) were inoculated, and lesions sizes monitored. Error bars represent s.d. (E–H) Amastigotes of WT (E), spt2⁻ (F), and spl⁻ (G) were isolated from infected Balb/c mice and subjected to transmission EM analysis. Arrowheads indicate acidocalcisomes. Bars=1 μm. In (H), amastigotes were used to infect murine peritoneal macrophages (Mφs) at a ratio of two amastigotes per macrophage. Error bars represent s.d.

Figure 7

SLs are not required for the redistribution of LPG during promastigote development. Log (A) and metacyclic (B, isolated from stationary cultures grown in the presence of 0.5 mM EtN) promastigotes were extracted with 1% Triton and resubjected to Western blot analysis with the anti-LPG monoclonal antibody WIC79.3. I, insoluble; S, soluble.

Figure 8

EtN reverses the morphological defects of spl⁻ and spt2⁻ promastigotes in stationary but not in log phase. Images show WT, spt2⁻, spl⁻, spt2⁻/+SPT2, spl⁻/+SPL, spt2⁻ SSU∷SDC, and spl⁻ SSU∷SDC promastigotes in log or stationary phase (day 3). Percentages of round cells are indicated in each panel.