Fig 1.
Schematic diagram of hexosamines biosynthesis and catabolism in Leishmania.
Exogenous sugars, such as glucose (Glc), glucosamine (GlcN) and N-acetylglucosamine (GlcNAc) are taken up via the hexose transporters and phosphorylated within glycosomes by hexokinase. Fructose 6-phosphate (Fru-6P) can be used for de novo hexosamine biosynthesis via cytosolic glutamine:fructose-6-phosphate amidotransferase (GFAT) and N-acetylglucosamine acetyltransferase (GNAT), which are essential for the synthesis of glycoconjugates. In contrast, catabolism of GlcNAc-6P depends on the glycosomal glucosamine deaminase (GND) and GlcNAc deacetylase (GNAD), allowing the utilization of GlcNAc as major carbon source.
Fig 2.
The L. major ∆gnat mutant is a GlcNAc auxotroph.
(A) Wild type (WT) and ∆gnat promastigotes were cultivated in M199 medium supplemented with 50 μg/ml GlcN or GlcNAc and parasite growth monitored by measuring optical density at 600nm. (B) ∆gnat growth in the absence of GlcNAc was rescued by ectopic expression of GNAT from pXG-PURO plasmid, ∆gnat [pX-PURO-GNAT], in M199. ∆gnat mutant was incubated in (C) limiting or (D) increasing concentrations of GlcNAc and growth monitored over time. Data represents mean from three biological repeats.
Fig 3.
∆gnat parasites lack GNAT activity and are defective in glycoconjugate biosynthesis.
(A) Cytosolic extracts of wild type (WT), ∆gnat and ∆gnat [pX-PURO-GNAT] cell lines were incubated with 1 mM GlcN6P for indicated times at 27°C. The relative abundances of GlcN6P and GlcNAc6P were determined by direct infusion-mass spectrometry in negative ion mode. (B) WT and Δgnat promastigotes were cultivated in hexosamine-free M199 medium for 24 hours and then pulse labelled with 3H-Glc for 30 min. Parasite extracts containing total cellular lipids were analysed by HPTLC and the major phospholipids phosphatidylethanolamine (PE), phosphatidylinositol (PI), inositolphosphatidylcholine (IPC), phosphatidylcholine (PC), phosphatidylinositolphosphate (PIP) and glycolipids (GIPL1, 2 and 3). (C) LPG and gp63 were detected by Western blotting using anti-phosphoglycan repeat antibody, LT15 (top panel) or anti-gp63 (middle panel) antibodies. The flagellar protein, SMP1, was used as a loading control (bottom panel). (D) The neutral glycan mannan contains increasing levels of mannose (M) and was resolved by HPTLC. (E) WT or ∆gnat parasites cell lysates were labeled with GDP-[3H]Man and dolichol-P-mannose (Dol-P-Man), the GIPL precursor, M1, and a lipid-linked oligosaccharide (LLO) precursors detected by HPTLC.
Fig 4.
Infectivity of the ∆gnat mutant in BALB/c mice and macrophages.
(A) WT and ∆gnat stationary phase promastigotes were incubated with peanut agglutinin (PNA) and the number of metacyclic (PNA negative) parasites determined. Error bars are SD from three biological repeats. (B) BALB/c mice were infected intradermally with promastigote stages (106 stationary phase) of L. major wild type (WT), ∆gnat and the complemented ∆gnat [pX-PURO-GNAT] parasite lines. Lesion formation and progression were monitored weekly. Error bars represent standard error of the mean (SEM), n = 5 mice. 1 of 2 independent experiments shown. (C) BALB/c mice were infected intradermally with lesion-derived WT and ∆gnat amastigotes (106) and lesion progression was scored weekly. Error bars represent SEM, n = 5 mice. (D) BALB/c bone marrow-derived macrophages were infected with stationary phase L. major WT and ∆gnat promastigotes. Intracellular parasite numbers at days 1, 2 and 4 post-infection were determined from three biological repeat experiments (more than 300 macrophages scored per experiment, error bars represent SEM and p-values are derived from the student’s t-test, two-tailored, unpaired). (E) RAW 264.7 macrophages were infected with lesion derived WT and ∆gnat amastigotes and number of intracellular parasites counted over time (mean and SEM from two biological repeat experiments, p-value was calculated by the student’s t-test).
Fig 5.
Intracellular ∆gnat parasites are rescued by exogenous GlcNAc sources.
(A) RAW 264.7 macrophages were infected with wild type (WT) and ∆gnat stationary promastigotes in the presence of either GlcNAc, hyaluronic acid (HA) or chitin. Intracellular growth was determined after fixation and staining with the DNA dye, Hoechst, and is expressed relative to parasite numbers at day four of untreated cells (whereby WT contained 67 +/- 8 parasites and ∆gnat 26 +/- 4 parasites). Error bars are the SD from three biological repeat experiments and p-values were calculated by the Student’s t-test. (B) WT and (C) ∆gnat promastigotes were cultured in the presence of GlcNAc, HA or chitin as the sole hexose source and growth was determined by OD600.
Fig 6.
High molecular weight hyaluronan rescue intracellular ∆gnat parasites.
RAW 264.7 macrophages were infected with (A) wild type (WT), (B) ∆gnat and (C) ∆gnd promastigotes in the absence of presence of different molecular weight HA. Intracellular parasites numbers at day 1 and 4 post-infection were determined by fluorescence microscopy after staining with Hoechst. Data represents mean and SD from three biological repeats and p-values were determined by the Student’s t-test.
Fig 7.
Hyaluronan localization to the Leishmania containing phagolysosome.
RAW 264.7 macrophages were infected with L. mexicana promastigotes and then labelled with FITC conjugated hyaluronan (HA::FITC, 10μg/ml) for 24 hours. Macrophage lysosomes were stained with LysoTracker. Live-macrophages were analysed by fluorescence microscopy. Arrow indicates two L. mexicana amastigotes. Scale bar = 10μm.