Ether Phospholipids and Glycosylinositolphospholipids Are Not Required for Amastigote Virulence or for Inhibition of Macrophage Activation by Leishmania major

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 GPIanchored 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 (detergentresistant 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.

Leishmaniadisease development depends on the presence of apoptotic promastigotes in the virulent inoculum

“…Data on PS expression as the early sign of apoptosis on Leishmania are inconclusive. One study demonstrates that a parasite mutant lacking ether phospholipids (alkyl-acylbound phospholipids species) was still able to silence MF and survive intracellularly (19). This study does not, however, exclude the presence of other PS-containing lipids, such as lyso-acyl or diacyl-bound PS species on apoptotic Leishmania.…”

Leishmania major Expresses a Single Dihydroxyacetone Phosphate Acyltransferase Localized in the Glycosome, Important for Rapid Growth and Survival at High Cell Density and Essential for Virulence

“…Friedlin V1 strain (MHOM/IL/80/Friedlin) were maintained in liquid and semi-solid M199-derived medium (28). Transfection was performed according to Ngo et al (29), and selection was applied as appropriate in the presence of G418, nourseothricin, blasticidin, or puromycin (10, 150, 5, and 50 g/ml, respectively).…”

“…The 3Ј-untranslated region was excised from pCR-3ЈU-Lm-DAT after digestion with EcoRI and KpnI and ligated to the EcoRI and KpnI sites of pBS-5ЈU-LmDAT to give pBS.5Ј3ЈU-LmDAT (Ec220). The nourseothricin (SAT) and puromycin (PAC) cassettes were isolated from pXG.SAT (28) and pX63PAC (31), respectively, by digestion with EcoRI and SacI and ligated to the EcoRI and SacI sites of pBS.5Ј3ЈU-LmDAT yielding pBS.LmDAT:SAT (Ec231) and pBS.LmDAT:PAC (Ec225), respectively. All PCR-generated products were verified by sequencing.…”

“…pBEVY-L.LmDAT (Ec210) was obtained by subcloning the 4.3-kilobase BamHI (partial)-XbaI fragment of pUC19.LmDAT into the BamHI and XbaI sites of pBEVY-L (30). The plasmid pL-BSD (Ec221) was constructed as follows; the BSD cassette was PCR-amplified with the primers 5-XbaI-BSD (5Ј-GCTCTAGAT-GCCTTTGTCTCAAGAAGAATC-3Ј) and 3-XbaI-BSD (5Ј-GCTCT-AGATTAGCCCTCCCACACATAAC-3Ј) and pTEF1/BSD (Invitrogen) as template, digested with XbaI, and ligated into the SpeI sites of pXG.SAT (28) in sense orientation. To obtain the pL-BSD.HV-LmDAT (Ec247) plasmid, pL-BSD.LmDAT (Ec237) was first constructed by subcloning the 4.3 kilobases BamHI fragment of pUC19.LmDAT into the BamHI site of pL-BSD with the transcription orientation of the LmDAT gene similar to the BSD marker.…”

Regulated expression of the Leishmania major surface virulence factor lipophosphoglycan using conditionally destabilized fusion proteins

“…The precursor for Galf is UDP-Galf, which arises from the action of cytosolic UDP-galactopyranose mutase (UGM, encoded by the gene GLF) (16,23), and glf Ϫ knockouts confirm the requirement for this gene in Leishmania LPG biosynthesis (18). Although Galf occurs on other Leishmania glycoconjugates, including the abundant glycosylinositolphosphatidylinositols (GIPLs), previous studies establish that GIPLs play little role in L. major infectivity (24) and, accordingly, glf Ϫ knockouts show phenotypes identical to lpg1 Ϫ parasites lacking LPG alone (18,19,25). In this work, we used the dd fusion approach to regulate LPG expression through control of UGM levels, yielding parasites that when placed in the ''on'' and ''off'' states closely resemble WT and LPG-deficient mutants.…”