Disruption of the lipid-transporting LdMT-LdRos3 complex in Leishmania donovani affects membrane lipid asymmetry but not host cell invasion

Abstract

Maintenance and regulation of the asymmetric lipid distribution across eukaryotic plasma membranes is governed by the concerted action of specific membrane proteins controlling lipid movement across the bilayer. Here, we show that the miltefosine transporter (LdMT), a member of the P4-ATPase subfamily in Leishmania donovani, and the Cdc50-like protein LdRos3 form a stable complex that plays an essential role in maintaining phospholipid asymmetry in the parasite plasma membrane. Loss of either LdMT or LdRos3 abolishes ATP-dependent transport of NBD-labelled phosphatidylethanolamine (PE) and phosphatidylcholine from the outer to the inner plasma membrane leaflet and results in an increased cell surface exposure of endogenous PE. We also find that promastigotes of L. donovani lack any detectable amount of phosphatidylserine (PS) but retain their infectivity in THP-1-derived macrophages. Likewise, infectivity was unchanged for parasites without LdMT-LdRos3 complexes. We conclude that exposure of PS and PE to the exoplasmic leaflet is not crucial for the infectivity of L. donovani promastigotes.

Figure 1. LdMT and LdRos3 form a stable complex in the Leishmania membrane.

(A, B) Native- and SDS-PAGE analysis of LdMT and LdRos3-GFP. LdRos3-GFP was expressed in ΔLdMT parasites (A) and in ΔLdRos3 parasites (B). Solubilised membrane proteins were separated by native PAGE and analyzed for GFP fluorescence. The membrane extract obtained from ΔLdRos3 parasites contains a prominent fast-migrating fluorescent band (band I). The extract obtained from ΔLdMT parasites also contains slow-migrating fluorescent protein bands (band II and III). Regions of the gel corresponding to the fluorescent bands were excised and loaded onto SDS-PAGE gels which were subsequently immunoblotted with polyclonal antibodies against LdRos3 (α-LdRos3) and LdMT (α-LdMT). Size markers indicate relative mobility of proteins in kDa. (C) Immunoblots from co-immunoprecipitation assays. LdMT-GFP was immunoprecipated from a detergent-solubilised membrane fraction (load) obtained from ΔLdMT parasites expressing LdMT-GFP as well as non-transfected wild-type parasites (wt) using anti-GFP-MicroBeads. Immunoprecipitates (IP) were subjected to immunoblot analysis using antibodies that recognize LdMT (α-LdMT), LdRos3 (α-LdRos3) and metalloprotease gp63 (α-gp63).

Figure 2. Inward translocation of NBD-PC and NBD-PE across the plasma membrane of Leishmania requires LdMT and LdRos3.

Promastigotes of wild type (wt), ΔLdMT and ΔLdRos3 lines were labelled with NBD-lipids for 30 min at 2°C and than washed and analysed by flow cytometry (A–C) or visualised by fluorescence microscopy (D). ATP depletion was achieved by preincubation with 5 mM 2-deoxyglucose and 20 mM sodium azide. To abolish the proton electrochemical gradient 50 µM of the protonophore CCCP was used. As a control LdMT-GFP and LdRos3-GFP on episomal Leishmania expression vectors were reintroduced in ΔLdMT and ΔLdRos3 mutants, respectively; control, non-labelled cells showing the intrinsic fluorescence of the GFP fusion proteins. Data are normalized to NBD-PC internalization of wild-type parasites; 100% corresponds to 468±96 a.u. NBD-PC. Data represent the means ± SE of at least three independent experiments. For statistical analysis Welch's test was performed. Significant differences in the lipid uptake of the mutants compared to the wild type are denoted by asterisks (** p = 0.05; * p = 0.01).

Figure 3. LdMT and LdRos3 are required to sustain plasma membrane PE asymmetry in Leishmania.

(A) Sensitivity of wild type (wt), ΔLdMT and ΔLdRos3 L. donovani parasites to the PE-binding peptide duramycin, the PS-binding peptide papuamide B and the sterol-binding amphotericin B. Parasites were diluted to 0.25×10⁶ cells/ml in medium containing duramycin, papuamide B or amphotericin B at the indicated concentrations. 72 h later viability was analysed as described in the Material and methods. As a control LdMT-GFP and LdRos3-GFP on episomal Leishmania expression vectors was reintroduced in ΔLdMT and ΔLdRos3 mutants, respectively. Means ± S.E. of at least three independent experiments are shown as percentage of untreated control parasites. (B) Endogenous PS and PE at the exoplasmic leaflet of the plasma membrane of these strains was visualised with annexin V-FITC and Biotin-Rho/Streptavidin-FITC, respectively. Cells were analyzed by phase contrast (DIC) and fluorescence microscopy (PI, FITC). Bar, 10 µm. Fluorescence intensity histograms were obtained by flow cytometry as described under “Materials and Methods.” Cells incubated in the absence of biotinylated Ro09-0198 peptide served as controls (dash line).

Figure 4. Total phospholipid composition of wild type, ΔLdMT and ΔLdRos3 L. donovani parasites.

Promastigote stages of wild type (wt), ΔLdMT and ΔLdRos3 lines were labelled for 48 h with ³²P-phosphate. Lipids were extracted, separated by two-dimensional thin layer chromatography, and then visualized by phosphorimager scanning (A) or ninhydrin staining (B). Representative two-dimensional TLC plates are shown. The location of individual species was verified by ESI-MS. Unidentified lipids are not marked. (C) Quantification of phospholipids in wild-type, ΔLdMT and ΔLdRos3 parasites. Data are expressed as the percentage of total phospholipids and represent the means ± S.E. of three independent experiments. PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; IPC, inositolphosphorylceramide.

Figure 5. Phagocytosis of ΔLdMT and ΔLdRos3 parasites by THP-1-derived macrophages is unchanged.

CellTrackerTM Green-labelled parasites were added (10∶1) to THP-1-derived macrophages prelabelled with CellTrackerTM Dil (red) and co-incubated for 16 h at 37°C. (A) Micrograph of double-fluorescence labelling of THP-1-derived macrophages phagocytosing Leishmaina parasites. Bar, 10 µm. (B) Percentage of phagocytosing THP-1-derived macrophages was determined by flow cytometry. Values are means ± SD of three independent experiments expressed as percentage of control (number of phagocytosed wild-type parasites by TPH-1-derived macrophages). The population of phagocytosing THP-1-derived macrophages ranged from 30 to 70% in control cultures.