Leishmania Promastigotes Lack Phosphatidylserine but Bind Annexin V upon Permeabilization or Miltefosine Treatment

The protozoan parasite Leishmania is an intracellular pathogen infecting and replicating inside vertebrate host macrophages. A recent model suggests that promastigote and amastigote forms of the parasite mimic mammalian apoptotic cells by exposing phosphatidylserine (PS) at the cell surface to trigger their phagocytic uptake into host macrophages. PS presentation at the cell surface is typically analyzed using fluorescence-labeled annexin V. Here we show that Leishmania promastigotes can be stained by fluorescence-labeled annexin V upon permeabilization or miltefosine treatment. However, combined lipid analysis by thin-layer chromatography, mass spectrometry and 31 P nuclear magnetic resonance (NMR) spectroscopy revealed that Leishmania promastigotes lack any detectable amount of PS. Instead, we identified several other phospholipid classes such phosphatidic acid, phosphatidylethanolamine; phosphatidylglycerol and phosphatidylinositol as candidate lipids enabling annexin V staining.FAZIT (AW)Research Training Group 1121 of the German Research FoundationCarlsberg FoundationCenter for Synthetic Biology at Copenhagen UniversityUNIK research initiative of the Danish Ministry of Science, Technology and Innovatio

Analysis of [3-³H]serine-labeled lipids from <i>L. donovani</i> parasites.

<p>Promastigotes were labelled for 16 h with [3-³H]serine. Lipids from 10⁸ parasites were extracted, separated by two-dimensional thin layer chromatography, and then visualized by fluorography. The location of individual lipid species was verified by ESI and MALDI-TOF mass spectrometry. Unidentified lipids are not marked. Cer, ceramide, PC, phosphatidylcholine; PE, phosphatidylethanolamine; IPC, inositolphosphorylceramide; SB, sphingosine. The position of the phosphatidylserine standard (PS) is enclosed by a dotted circle.</p