Analysis of nucleotide sugar pools in <i>L</i>. <i>major</i> wild type and <i>ugp</i>^-/-<i>usp</i>^-/c mutant.

<p>Nucleotide sugars were extracted from mid Log phase wild type (wt) and <i>ugp</i>^-/-<i>usp</i>^-/c promastigotes grown in presence of 1 μM (+) or 0.01 μM (-) FK506 and measured by liquid chromatography-electrospray ionisation-tandem mass spectrometry with multiple reaction monitoring. Values represent the mean ± SD from n = 3 independent cultures; for the <i>ugp</i>^-/-<i>usp</i>^-/c mutant, each culture represents a different clone. Significant differences (*p < 0.01, ** p < 0.005, *** p < 0.001), paired t-test.</p

Analysis of <i>in vitro</i> growth and cell viability of <i>L</i>. <i>major ugp</i>^-/-<i>usp</i>^-/c mutant.

<p>(A) <i>In vitro</i> growth of wild type and <i>ugp</i>^-/-<i>usp</i>^-/c promastigotes grown in absence or presence of various FK506 concentrations as indicated. Medium was inoculated with 10⁵ cells (upper panel). After 3 to 4 days, parasites were transferred to fresh medium containing the indicated FK506 concentration (lower panel). Error bars indicates the standard deviation. (B) Analysis of cell viability. Parasites grown in medium containing the indicated FK506 concentration were labelled with 7-AAD and analysed by flow cytometry. Mean percentages ± SEM of dead parasites (7-AAD⁺) were calculated from four independent experiments (n = 4). Significant difference (*p < 0.01, *** p < 0.001), One-way ANOVA with Tukey post test.</p

Depletion of USP in <i>L</i>. <i>major ugp</i>^-/-<i>usp</i>^-/c mutant and effect on glycoconjugates biosynthesis.

<p>(A) Western blot of Log phase wild type or <i>ugp</i>^-/-<i>usp</i>^-/c promastigotes lysates probed with an anti-USP antiserum (upper panel) or the anti-LPG monoclonal antibody WIC79.3 (middle panel). Parasites were cultivated in medium containing 1, 0.05, 0.005 µM or no FK506 as indicated. Loading was assessed by Coomassie staining of an identically loaded SDS-Page ran separately (lower panel). (B) <i>In vitro</i> conversion of Glc-1P (black bars) or Gal1P (white bars) into nucleotide sugars by cell lysates of wild type and <i>ugp</i>^-/-<i>usp</i>^-/c mutant cultivated in medium containing various FK506 concentrations as indicated. Values represent the mean ± SEM from n = 3 independent cultures; for the <i>ugp</i>^-/-<i>usp</i>^-/c mutant, each culture represents a different clone. Significant difference (*p < 0.01), One-way ANOVA with Tukey post test. (C) Negative-ion MALDI spectra of glycosylinositolphospholipids (GIPLs) isolated from wild type and <i>ugp</i>^-/-<i>usp</i>^-/c mutant grown in presence of 1 or 0.01 μM FK506. Each prominent peak is annotated with its m/z value, the letter a, b, c, d or e referring to the structure depicted above. The lipid moiety consists of a 1-alkyl-2-<i>lyso</i>-phosphatidylinositol or 1-alkyl-2-acyl-phosphatidylinositol. The length of the alkyl and acyl chains is indicated above each peak; C- indicates absence of an alkyl chain.</p

Generation of <i>Leishmania major ugp</i>^-/-<i>usp</i>^-/c mutant.

<p><i>L</i>. <i>major ugp</i>^-/-<i>usp</i>^-/c mutant was generated from a UGP deficient mutant [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004205#pntd.0004205.ref011" target="_blank">11</a>] by replacement of a USP allele with a construct encoding USP N-terminally fused to a mutated FK506 binding protein destabilizing domain (ddUSP). This system enables stabilisation of the fusion protein by addition of FK506 to the culture medium and its proteasomal degradation in absence of FK506. The second USP allele was replaced by transfection with a puromycin resistance gene (<i>PAC</i>). <i>BLE</i>, phleomycin resistance gene; <i>HYG</i>, hygromycin resistance gene; <i>SAT</i>, nourseothricin resistance gene.</p