Fig 1.
Glucose and glycerol aerobic metabolism of BSF trypanosomes.
Panel A shows a schematic representation of the glucose metabolism in BSF trypanosomes grown in glucose-rich medium (CMM_Glc), while panel B shows their glycerol metabolism in the absence of glucose (CMM_Glyc/GlcNAc). The approximately 10-fold higher metabolic flux in glycolysis (down to pyruvate) compared to that of pathways leading to succinate, acetate and alanine is illustrated by a proportional arrow thickness. End products excreted from metabolism of glucose and glycerol are shown in a grey rectangle. In Panel A, the GK step involved in glycerol production from glucose mainly under anaerobiosis is represented by dotted lines. The boxed number corresponds to the enzyme under investigation (GK) and the boxed ATP highlights the enzymatic step providing most of the cellular ATP. The circled glycosomal ATP, NAD+ and NADH highlight the main enzymatic steps involved in the maintenance of the glycosomal redox and ATP/ADP balances. The pathway used to maintain the glycosomal redox balance is highlighted by blue arrows, while the same pathway also used for conversion of glycerol-derived Gly3P into DHAP is shown with black arrows in panel B. The target of the SHAM metabolic drug (alternative oxidase) is indicated and the gluconeogenesis from glycerol-derived DHAP and G3P is shown in red. Abbreviations: DHAP, dihydroxyacetone phosphate; e-, electrons; F1,6BP fructose 1,6-bisphosphate; 1,3BPG, 1,3-bisphosphoglycerate; FUM, fumarate; F6P, fructose 6-phosphate; G3P, glyceraldehyde 3-phosphate; G6P, glucose 6-phosphate; Gly3P, glycerol 3-phosphate; MAL, malate; OAA, oxaloacetate; PEP, phosphoenolpyruvate; 3PG, 3-phosphoglycerate; PYR, pyruvate; SCoA, succinyl-CoA; SHAM, salicylhydroxamic acid; SUC, succinate. Indicated enzymes are: 1, hexokinase; 2, glucose-6-phosphate isomerase; 3a, phosphofructokinase; 3b, fructose-1,6-bisphosphatase (FBPase); 4, aldolase; 5, triose-phosphate isomerase; 6, glycerol-3-phosphate dehydrogenase; 7, glycerol kinase (GK); 8, glyceraldehyde-3-phosphate dehydrogenase; 9, phosphoglycerate kinase; 10, phosphoglycerate mutase; 11, enolase; 12, phosphoenolpyruvate carboxykinase; 13, glycosomal malate dehydrogenase; 14, fumarase; 15, NADH-dependent fumarate reductase; 16, pyruvate kinase; 17, L-alanine aminotransferase; 18, pyruvate dehydrogenase complex; 19a, acetyl-CoA thioesterase; 19b, acetate:succinate CoA-transferase; 20, succinyl-CoA synthetase; 21, mitochondrial FAD-dependent glycerol-3-phosphate dehydrogenase; 22, rotenone-sensitive NADH dehydrogenase (complex I of the respiratory chain); 23, rotenone-insensitive NADH dehydrogenase; 24, ubiquinone pool; 25, alternative oxidase (TAO); 26, mitochondrial FO/F1-ATP synthase; 27, mitochondrial ADP/ATP exchanger.
Fig 2.
Glycerol supports BSF growth in the absence of glucose.
Panel A shows growth curves of the 427 90–13 BSF parental strain in Creek's minimal medium (CMM) containing 10 mM glucose (CMM_Glc), 10 mM Glucose and 50 mM GlcNAc (CMM_Glc/GlcNAc), 10 mM glycerol and 50 mM GlcNAc (CMM_Glyc/GlcNAc), or only 50 mM GlcNAc without glucose and glycerol (CMM_GlcNAc). Filled squares and circles mean that the cells have been adapted during 3 months in CMM_Glc or CMM_Glyc/GlcNAc, respectively, before starting the growth monitoring. The crosses mean that all the cells were dead. Panel B shows glucose and glycerol consumption over time by the 427 90–13 BSF strain incubated in CMM and CMM_GlcNAc containing 0.5 mM glucose and no glycerol. Panels C and D show the same experiment as in panel B with cells incubated in CMM_Glc, CMM_Glyc/GlcNAc or CMM_Glc/Glyc (CMM containing equimolar amounts of glucose and glycerol) containing 5 mM glucose and/or glycerol (error bars indicate means ± SD of 3 biological replicates). Before the experiment, BSF have been grown for at least one month in CMM_Glc (panel C) or CMM_Glyc/GlcNAc (panel D).
Table 1.
Rate of glucose and glycerol consumption in BSF adapted to CMM_Glc or CMM_Glyc/GlcNAc.
Fig 3.
Excreted end products of glucose and glycerol metabolism by BSF parental and mutant cell lines.
a.i: RNAi cell line induced during 5 days by addition of tetracycline; .ni: non-induced RNAi cell line. b The cells have been grown in CMM_Glc or CMM_Glyc/GlcNAc before incubation in PBS containing one or two carbon sources. c Carbon source(s) added in the incubation medium (PBS). d Number of biological replicates. e The amount of glycerol excreted from glucose metabolism is not estimated because of overlapping resonances between glucose and glycerol. f,g Data not significantly different with p-value ≤0.05. *, **, *** Data significantly different with p-value ≤0.05, ≤0.01 and ≤0.001, respectively.
Fig 4.
Comparison of oxygen consumption and sensitivity to metabolic inhibitors.
Panel A shows oxygen consumption of parental BSF adapted to CMM_Glc or CMM_Glyc/GlcNAc growth conditions and resuspended in fresh CMM containing glucose (Glc, white) or glycerol/GlcNAc (Glyc, black) in the presence (+) or not (-) of 4 mM SHAM (error bars indicate means ± SD of 9 experiments, including 3 biological replicates). The SHAM EC50 values (μM) in BSF adapted to CMM_Glc (white), CMM_Glyc (grey) or CMM_Glyc/GlcNAc (black) are shown in panel B. Error bars indicate means ± SD of 3 biological replicates. The asterisks indicate data relevant for BSF adapted to CMM_Glc (white) and CMM_Glyc/GlcNAc (Black).
Fig 5.
Incorporation of [U-13C]-glycerol into BSF intracellular metabolites.
The 427 90–13 BSF parental cell line was incubated for 1 h in PBS containing 2 mM [U-13C]-glycerol alone or in the presence of 2 mM glucose (Panel A). The figure shows the 13C-enrichment of key metabolites of the intermediate metabolism at 0 to 6 carbon positions (m0 to m6, colour code indicated, "mx" denotes metabolites in which x of the atoms are 13C instead of 12C) with 13C expressed as percentage of all corresponding molecules (MID; Mass Isotopomer Distribution). Error bars indicate means ± SD of 3 biological replicates. The metabolic scheme in panel B represents the enzymatic reactions leading to production of the analysed metabolites from glycerol metabolism (bold faced and underlined ones). Gluconeogenesis and mannose 5-phosphate production are shown in red and part of the pentose phosphate pathway is in blue. The carbon sources incorporated in the metabolism network ([U-13C]-glycerol and CO2) are circled and the excreted end products are boxed. For abbreviations see Fig 1, other abbreviations are: Ala, alanine; M6P, mannose 6-phosphate; 2/3PG, 2- or 3-phosphoglycerate (these two metabolites are undistinguished by IC-MS/MS); 6PG, 6-phosphogluconate; 6PGL, 6-phosphogluconolactone; P5P, pentose 5P including ribose 5-phosphate (Ribo5P), ribulose 5-phosphate (Ribu5P) and xylulose 5-phosphate (Xylu5P), which are undistinguished by IC-MS/MS.
Fig 6.
Modulation of the glycerol and glucose metabolic pathways in BSF grown in CMM_Glyc/GlcNAc.
The expression of GK, TAO, ISG75 and FBPase in BSF grown in CMM_Glc and CMM_Glyc/GlcNAc, as well as FBPase in procyclic trypanosomes grown glucose-rich and glucose-depleted conditions was analysed by western blotting using the anti-hsp60 immune serum as control (Panel A). The arrow indicates a 55 kDa band corresponding to GK (calculated Mr: 56.4 kDa), above a non-specific signal produced by the anti-GK immune serum. To compare expression of FBPase/hsp60 the analysis of the BSF and PCF samples was performed on the same gel. Panel B shows the GK and HK enzymatic activities in the three growth conditions (error bars indicate means ± SD of 3 biological replicates). The proteomic data described in S3 Table is presented in Panel C by expressing the percentage of up- or down-regulation of the indicated enzymes in cells grown in CMM_Glyc/GlcNAc as compared to cells grown in CMM_Glc. The dashed line corresponds to two-fold up- or down-regulations. The numbers indicate the enzymatic steps described in Fig 1. The subunits of the pyruvate dehydrogenase complex and of the succinyl-CoA synthetase are annotated 18α-18δ and 20α-20β, respectively. Up- and down-expression of TAO and GK, respectively, are represented by grey bars, and that of glycolytic enzymes are shown with white bars. The asterisks mean that the proteome data have been confirmed by western blotting and/or enzymatic activity assay.
Fig 7.
Role of glycerol kinase in glycerol metabolism.
Panel A shows growth curves of the parental 427 90–13 BSF (WT) and RNAiGK cell lines incubated in CMM_Glc (Glc) or CMM_Glyc/GlcNAc (Glyc), in the presence (.i) or not (.ni) of tetracycline. The western blot controls with the anti-GK and anti-hsp60 (heat shock protein 60) immune sera are shown in the panel B and the GK and HK enzymatic activity of WT cells, as well as un-induced or 5-day induced RNAiGK cells are shown in panel C. The anti-GK immune serum reveals non-specific bands in addition to GK, that is indicated by an arrow. nd means not detected and error bars in panels A and C indicate the means ± SD of 3 biological replicates.