Abstract
Ether phospholipids are major components of the membranes of humans and Leishmania. In protozoan parasites they occur separately or as part of the glycosylphosphatidylinositol (GPI) anchor of molecules implicated in virulence, such as lipophosphoglycan (LPG), smaller glycosylinositolphospholipids (GIPLs), and GPI-anchored proteins. We generated null mutants of the Leishmania major alkyldihydroxyacetonephosphate synthase (ADS), the first committed step of ether lipid synthesis. Enzymatic analysis and comprehensive mass spectrometric analysis showed that ads1- knock-outs lacked all ether phospholipids, including plasmalogens, LPG, and GIPLs. Leishmania ads1- thus represents the first ether lipid-synthesizing eukaryote for which a completely null mutant could be obtained. Remarkably ads1- grew well and maintained lipid rafts (detergent-resistant membranes). In virulence tests it closely resembled LPG-deficient L. major, including sensitivity to complement and an inability to survive the initial phase of macrophage infection. Likewise it retained the ability to inhibit host cell signaling and to form infectious amastigotes from the few parasites surviving the establishment defect. These findings counter current proposals that GIPLs are required for amastigote survival in the mammalian host or that parasite lyso-alkyl or alkylacyl-GPI anchors are solely responsible for inhibition of macrophage activation.
Highlights
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY328521
The surface of the Leishmania parasite is a major point of interaction with the host throughout the infectious cycle, which includes an extracellular promastigote form residing in the midgut of sand flies and an intracellular amastigote form adapted for survival within the phagolysosomes of vertebrate macrophages
Southern blot analysis showed that the Leishmania ADS1 gene was single copy (Fig. 1A), which was confirmed by targeted gene replacement
Summary
Parasite Culture—Leishmania major Friedlin strain V1 (MHOM/IL/ 80/Friedlin) promastigote forms were cultivated in M199 medium at 26 °C and transfected by electroporation [27]. For reverse transcriptase PCR, cDNA was prepared from RNA isolated from logarithmic phase promastigotes using random primers for the reverse transcription reaction (Invitrogen) and used as template for PCR with an oligonucleotide specific for the miniexon (SMB936, 5Ј-ACCGCTATATAAAGTATCAGTTCTGTACTTTA) and one specific for ADS1 (either SMB1012, 5Ј-ACTAGTGCTGTCCTGTGTTTTATCG, located in the 5Ј untranslated region, or SMB1017, 5Ј-ATCTGCATCTGGACATCC, located within the ADS1 coding region) These products were directly sequenced by the chain termination method. Cloning of ADS1 of L. major and Plasmid Construction—A 1.58-kb fragment was obtained following PCR with L. major genomic DNA template and the ADS1 degenerate primers SMB929 (5Ј-AAGTGGAAYGGNTGGGG) and SMB931 (5Ј-CCSATNCCGTGGTGGTGNGT). This was inserted into pCR2.1 (Invitrogen) to give pCRII.DHAP-PCR (strain B3772) and sequenced. The ADS1 BamHI fragment was ligated in the sense orientation into the BamHI site of pXG [33] or pXG-GFP2ϩ/ (B2952). pX63PAC-LdSAcP-1 (B4191) was obtained by inserting a sense blunt-
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