Disruption of the Lipid-Transporting LdMT-LdRos3 Complex in Leishmania donovani Affects Membrane Lipid Asymmetry but Not Host Cell Invasion

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

The plasma membrane lipid asymmetry of Leishmania donovani and its relevance for phagocytosis

In großen Teilen der Welt verursachen intrazelluläre Parasiten der Spezies Leishmania schwerwiegende Infektionen beim Menschen. Die Exposition eines Phospholipids (Phosphatidylserin, PS) steht unter Verdacht Fresszellen zur Aufnahme der Parasiten zu stimulieren. Bisher ist die Regulation der Phospholipidverteilung in der Plasmamembran dieser Parasiten kaum erforscht. In der vorliegenden Arbeit wurde ein lipidtransportierender Proteinkomplex identifiziert, der einen wesentlichen Beitrag zur asymmetrischen Lipidverteilung in der Plasmamembran von Leishmania donovani leistet. Die Zerstörung des Komplexes führte zum Verlust des einwärts gerichteten Lipidtransports und zur Anreicherung von Phosphatidylethanolamin (PE) auf der Zelloberfläche des Parasiten. Diese veränderte Lipidasymmetrie hatte jedoch keinen Einfluss auf die Phagozytose durch Makrophagen. Darüber hinaus brachte die Untersuchung des Insektenstadiums (Promastigote) verschiedener Leishmania Spezies zu Tage, dass die Menge an PS unterhalb des Detektionslimits modernster Nachweisverfahren liegt. Des Weiteren konnte gezeigt werden, dass der Parasit über einen Scramblase-Mechanismus verfügt, der durch intrazelluläres Kalzium stimulierbar ist. Die Scramblase-Aktivität ist, im Gegensatz zu dem zuvor beschriebenen einwärts gerichteten Lipidtransport, energieunabhängig und ermöglicht die bidirektionale Translokation von fluoreszenzmarkiertem Phosphatidylcholin (PC), PE, PS und Sphingomyelin (SM). Dementsprechend konnte nach Kalziumstimulierung endogenes PE auch in der äußeren Lipidschicht der Plasmamembran detektiert werden, wobei deren Barrierefunktion nicht beeinträchtigt wurde. Diese Ergebnisse geben neue Einblicke in die dynamische Regulation der Lipidverteilung über die Plasmamembran des Parasiten und verdeutlichen, dass die Exposition von PS und PE nicht essentiell für das Eindringen der Leishmanien in die Wirtszellen ist.The protozoan parasite Leishmania causes severe infections in humans throughout the world. Following the transmission via sand flies to its mammalian host the extracellular parasite has to gain entry into phagocytic cells to initiate a successful infection. Specific surface exposed phospholipids have been implicated in Leishmania macrophage-interaction, but the mecha-nisms controlling and regulating the plasma membrane lipid distribution remains to be eluci-dated. In the present work a lipid transporting protein complex was identified in Leishmania dono-vani which plays an essential role in maintaining an asymmetric lipid distribution across the plasma membrane. Loss of the protein complex abolishes the inward-directed lipid transport and thus e.g. to an increased cell surface exposure of phosphatidylethanolamine (PE). In spite of this altered lipid asymmetry the uptake by macrophages is unaffected. Moreover, Leishma-nia promastigotes of different species lack detectable amounts of phosphatidylserine (PS) although being infective. Furthermore, a scramblase activity following a cytosolic calcium signal was demonstrated. This scramblase mechanism facilitated, in contrast to the previous described inward directed lipid transport, the bidirectional movement of fluorescent lipid analogues of PC, PE, PS and SM in an energy-independent manner. In accordance with these findings endogenous PE was exposed to the outer plasma membrane leaflet following the Ca2+-signal, while the plasma membrane itself remained intact. These results provide novel insight into the dynamic regulation of the transbilayer lipid distri-bution across the parasite plasma membrane and reveal that exposure of PS and PE is not cru-cial for invasion of the host cell by Leishmania donovani promastigotes

LdMT and LdRos3 form a stable complex in the <i>Leishmania</i> membrane.

<p>(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).</p

Total phospholipid composition of wild type, ΔLdMT and ΔLdRos3 <i>L. donovani</i> parasites.

<p>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.</p

Inward translocation of NBD-PC and NBD-PE across the plasma membrane of <i>Leishmania</i> requires LdMT and LdRos3.

<p>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 <i>Leishmania</i> 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).</p

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

<p>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 <i>Leishmaina</i> 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.</p

Base peak ion chromatogram of HPLC/MS analysis of a lipid extract of <i>L. donovani</i>.

<p>Lipids were separated using a BioBasic-4-column as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042070#s4" target="_blank">Material and Methods</a>. Elution was performed at a flow rate of 50 µl/min by increase of solvent B (70% acetonitrile, 25% 2-propanol, 5% water) vs. solvent A (95% water, 5% acetonitrile). The HPLC gradient is indicated at the right of the chromatogram. The intervals of the retention times of the individual lipid classes are labeled and the assignment of the peaks to the individual lipid species is available in <b>Tab. 1</b>. Abbreviations: CL, cardiolipin; IPC, inositolphosphorylceramide; lyso-PL, lyso-phospholipids; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol.</p

Overview of various lipids from <i>L. donovani</i> and their annexin V-binding ability.

<p>(A) A total lipid extract from <i>L. donovani</i> promastigotes was separated by one-dimensional TLC using chloroform/methanol/water (65/25/4, v/v/v) as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042070#s4" target="_blank">Material and Methods</a>”. The individual lipid species were visualized by primuline staining, scraped off, extracted and subjected to MALDI-TOF and ESI mass spectrometry. Chromatograms shown are scanned by a Typhoon imager as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042070#s4" target="_blank">Material and Methods</a>. Regions (1–10) are marked and assignments are indicated. Abbreviations used in assignments: PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol; PS, phosphatidylserine; TAG, triacylglycerols; CL, cardiolipin, n.d., not determined. (B) Overlay assays. <i>Leishmania</i>: Individual lipids extracted from TLC region 1–10 were spotted onto nitrocellulose and incubated with annexin V-FITC in the presence and absence of Ca²⁺; an unloaded TLC region scraped off and treated exactly as regions 1–10 served as background control for the primuline signal (marked c). <i>Standard</i>: PC (18∶1/18∶1), PI, PA (18∶1/18∶1), PE (18∶1/18∶1), PG (18∶1/18∶1), PS (18∶1/18∶1), stearoyl-sphingomyelin (SM 18∶0), and cholesterol (Ch.) served as controls and emphasize the specificity of the assay. Location of spotted lipids is indicated with red broken circles. (C) Lipids extracted from TLC region 3 were subjected to one-dimensional TLC using chloroform/methanol/25% aqueous ammonium hydroxide (90/54/7, v/v/v) and stained with primuline. PC and PI were identified by MALDI-TOF mass spectrometry. Lipids from regions 3a and 3b were eluted, re-protonated and used for overlay assay with annexin V-FITC in the presence and absence of Ca²⁺.</p

Lipid analysis by MALDI–TOF mass spectrometry.

<p>Positive and negative ion MALDI-TOF mass spectra of a total lipid extract of <i>L. donovani</i>. Peaks are marked with their <i>m/z</i> values and assignments are summarized in <b>Tab. 2</b>. DHB was used as the matrix for positive ion detection, while 9-AA was used in the negative ion mode. As the focus of this study was to clarify the potential presence of PS, only the mass range where PS could be expected is shown.</p