Sphingolipids are essential for differentiation but not growth in Leishmania

Abstract

Sphingolipids (SLs) play critical roles in eukaryotic cells in the formation of lipid rafts, membrane trafficking, and signal transduction. Here we created a SL null mutant in the protozoan parasite Leishmania major through targeted deletion of the key de novo biosynthetic enzyme serine palmitoyltransferase subunit 2 (SPT2). Although SLs are typically essential, spt2− Leishmania were viable, yet were completely deficient in de novo sphingolipid synthesis, and lacked inositol phosphorylceramides and other SLs. Remark ably, spt2− parasites maintained ‘lipid rafts’ as defined by Triton X‐100 detergent resistant membrane formation. Upon entry to stationary phase spt2− failed to differentiate to infective metacyclic parasites and died instead. Death occurred not by apoptosis or changes in metacyclic gene expression, but from catastrophic problems leading to accumulation of small vesicles characteristic of the multivesicular body/multivesicular tubule network. Stage specificity may reflect changes in membrane structure as well as elevated demands in vesicular trafficking required for parasite remodeling during differentiation. We suggest that SL‐deficient Leishmania provide a useful biological setting for tests of essential SL enzymes in other organisms where SL perturbation is lethal.

Characterization of inositol phosphorylceramides from Leishmania major by tandem mass spectrometry with electrospray ionization

“…Total lipids were extracted from L. major as previously described [19].°Briefly,°wild°type°L. major LV39°clone°5 (Rho/SU/59/P) cells were grown in M199 medium with supplements and 10% heat inactivated fetal bovine serum at 26°C.…”

“…Recent work indicates Leishmania parasites synthesize high levels of IPC as both promastigotes (the stage residing extracellularly in the midgut of sandfly vectors) and amastigotes (the stage residing intracellularly within phagolysosomes of mammalian macrophages), suggesting this lipid plays important roles in both stages [19 -21]. Mutants defective in the synthesis of sphingoid bases (via inactivation of serine palmitoyltransferase; spt2 -) or degradation of sphingosine-1-phosphate (via inactivation of sphingosine-1-phosphate lyase; spl -) exhibited severe defects in vesicular trafficking upon entry into stationary phase and failed to differentiate to infective metacyclic form, suggesting sphingolipid metabolism is crucial for the virulence of Leishmania parasites [19,21,22]. Remarkably, both mutants were rescued by ethanolamine, a downstream metabolite of the de novo SL pathway, a finding with profound implications to role of SLs in the promastigote stage [22].…”

“…However, despite disruptions in the de novo synthesis of SLs, spt2 -amastigotes retain high levels of IPC, suggesting that this stage salvages one or more IPC precursors from the mammalian host for conversion into the parasite specific IPC [20]. In this regard, the SL mutant amastigotes resemble WT amastigotes, which normally down-regulate the de novo SL and SPL (sphingosine-1-phosphate lyase)-dependent ethanolamine synthetic pathways [19,21,22]. Genetic analysis of the role of amastigote IPC thus awaits the development of parasite mutants deficient in IPC synthesis and/or SL salvage from the host.…”

“…and Trypanosoma spp.) have been previously analyzed by ESI tandem mass spectrometry in the negative-ion mode [6,7,19]. Here we report more detailed structural studies towards the identification of the long-chain base and the fatty acid substituent of IPC using tandem mass spectrometry.…”

Sphingolipids and membrane biology as determined from genetic models

“…Once it reaches stationary phase, it displays a progressively higher incidence of cell-shape abnormalities and loss of viability. In stationary phase, these parasites display defects in membrane structure such as increased vacuolation, alterations in flagellar surface, and the accumulation of small vesicles reminiscent of multivesicular bodies [104]. The stationary-phase parasites also fail to differentiate into the infective metacyclic stage.…”