Fig 1.
Glucose is phosphorylated into glucose 6-phosphate (G6P) by hexokinase (HK) and utilized further in glycolysis, shaded in blue, or channelled into the pentose phosphate pathway (PPP). In the oxidative branch of the PPP, shaded in green, G6P is converted via three subsequent steps comprising glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconolactonase (6-PGL), and 6-phosphogluocnate dehydrogenase (6-PGDH), resulting in the production of ribulose 5-phosphate, reduced NADPH and CO2. In the non-oxidative branch, shaded in lilac, ribulose 5-phosphate is converted by ribulose-5-phosphate epimerase (RuPE) to xylulose 5-phosphate or by ribose-5-phosphate isomerase (RPI) to ribose 5-phosphate. These and other sugar phosphates can be utilized by transketolase (TKT) and transaldolase (TAL) in reactions shuffling two or three carbons, respectively. Adapted from [35].
Fig 2.
Growth of Δtkt cells in vitro and in vivo.
(A) Growth of WT, Δtkt, and Δtkt + TKT promastigote cells in Homem medium over 5 days, n = 3. The inset shows Western blot analysis of WT, Δtkt, and Δtkt + TKT cell lines probed with α-TKT antibody. (B) Infection of THP-1 macrophages with Leishmania. After 4 hours of incubation the parasites were washed off and infection assessed at time points indicated. 10:1 parasite to host ratio, n = 4, * = p < 0.005. (C) Number of parasites per each infected macrophage was counted, n = 4, * = p < 0.005. (D) Development of foot pad lesions in BALB/c mice infected with WT, Δtkt, and Δtkt + TKT cells, n = 2, four and five mice were used per each group in the two respective replicates.
Fig 3.
Measurement of reactive oxygen species by DCFDA.
Reactive oxygen species inside cells were assessed directly by measuring DCFDA fluorescence. Whereas in untreated cells, ROS levels were slightly but significantly higher in Δtkt than WT, the difference was much more prominent after 8 h treatment with 200 μM potassium antimonyl tartrate, n = 6, * p < 0.0001.
Table 1.
Sensitivity to oxidative stress inducing agents and other drugs.
Fig 4.
Relative changes in glycolysis and the PPP observed in metabolomic analyses.
(A) Scheme of glycolysis and the PPP with indicated changes of metabolites detected by LC-MS analysis. Numbers are relative values in Δtkt compared to WT, red numbers and arrows indicate the fold increase, whereas blue indicates the fold decrease. Text and arrows in grey indicate steps missing after TKT depletion. Metabolites highlighted in bold were identified based on matches with respective standards, the rest are annotated based on mass and predicted retention time. (B) Similar scheme as in A, but showing GC-MS analysis. * p < 0.05.
Table 2.
Intensity of respective metabolites detected by metabolomic analyses.
Values listed represent fold changes relative to WT.
Fig 5.
Central carbon flux is decreased in Δtkt.
(A) Glucose consumption by WT and Δtkt cell lines. The graph indicates the concentration of glucose detected in spent media of the respective cultures, starting in Homem medium containing 1 mM glucose, n = 4. (B) Metabolic end products as detected in spent media of WT and Δtkt cultures by LC-MS, all three metabolites were identified based on matches with the respective standards. Growth rates were not significantly different. FM, fresh medium.
Fig 6.
Scheme of the flow of carbon atoms from glucose through glycolysis and the PPP.
If 1,2-13C2-glucose is used, products from glycolysis contain two or no carbons labelled, whereas in the PPP one carbon is cleaved as CO2, hence the three carbon products contain one or no carbons labelled. Based on Lee et al. [28]. Glyceraldehyde 3-phosphate (GA3P) used for the quantification is highlighted.
Fig 7.
Mannogen is decreased in Δtkt.
(A) Intermediates of glycolysis and the PPP as detected by LC-MS, when WT and Δtkt cells were grown in Homem medium containing either glucose (Glu), or fructose (Fru) as the main carbon source. Values in the first column (WT in glucose) are considered reference and the following values indicate relative change in intensity detected, n = 4. #—metabolites identified based on matches with respective standards, all the other annotations are predicted. * p < 0.05. (B) Levels of sugar oligomers detected in WT and Δtkt cells grown in the presence of glucose or fructose, n = 4. Identity of these metabolites is predicted based on mass and retention time, however, the mass is very specific for sugar oligomers. (C) Mannogen as detected by HPTLC. Shown is one of three replicates.
Fig 8.
Relative shift in mRNA levels detected for the respective enzymes in glycolysis and the PPP by RNAseq analysis.
Relative values detected in Δtkt when compared to WT are indicated. mRNA increased more than two fold is indicated in red, and decreases of more than two fold in blue, respectively. Red and blue arrows accompanying metabolites represent results of the GC-MS analysis, as depicted in Fig 4B. * p < 0.001.
Table 3.
Activities of selected enzymes as measured in WT and Δtkt cell lysates.
Fig 9.
Difference in glucose utilisation in WT and Δtkt cell lines.
Results of LC-MS metabolomics analysis when U-13C-glucose was used, 50% of glucose in the medium contained all carbons labelled, U-13C-glucose, and 50% was unlabelled glucose. Bars represent intensity of each respective metabolite detected. The part coloured grey is the non-labelled proportion, that labelled blue is one carbon, violet two carbons, green three carbons, orange four carbons and red six carbons labelled. (A) Metabolites of glycolysis and the PPP detected. (B) Intermediates of the TCA cycle and connected metabolites.
Fig 10.
Relative levels of different amino acids measured in WT and Δtkt cells and their growth media.
(A) Selected amino acids in fresh and spent media of WT and Δtkt cultures over four days of cultivation as detected by LC-MS analysis, FM—fresh medium, n = 4. (B) Intracellular levels of amino acids in WT, Δtkt, and Δtkt + TKT cultures detected by LC-MS. Intensity detected in WT is considered reference and the other values indicate relative abundance, n = 4. All the amino acids were identified based on matches with respective standards.
Fig 11.
Scheme of mannogen biosynthesis.
Blue and red colours indicate enzymes and their relative mRNA abundance in Δtkt when compared to WT. GlcNAc, N-acetylglucosamine; GlcN, glucosamine; Glc, glucose; GlcNAc6P, N-acetylglucosamine 6-phosphate; GlcN6P, glucosamine 6-phosphate; G6P, glucose 6-phosphate; F6P, fructose 6-phosphate; Man6P, mannose 6-phosphate; UDP-GlcNAc, uridine diphosphate N-acetylglucosamine; GDP-Man, guanosine diphosphate mannose; HK, hexokinase; PGI, glucose-6-phosphate isomerase; NAGD, N-acetylglucosamine-6-phosphate deacetylase; GND, glucosamine-6-phosphate deaminase; GNAT, glucosamine-6-phosphate acetylase; GFAT, glutamine:Fru6P aminotransferase; PMM, phosphomannomutase; MPGT, mannose-1-phosphate guanylyltransferase; dashed arrows indicate multiple enzymatic steps; based om Naderer, et al. [36] and Garami, et al. [70].
Fig 12.
Immunofluorescence microscopy of cells transfected with all variants of altered GFP-TKT constructs.
Shown from the longest at the top to the shortest at the bottom. Antibody against triosephosphate isomerase (TIM) was used as a glycosomal marker. The GFP signal corresponds to GFP-TKT complexes as verified by a Western blot analysis (S5C Fig).