Figure 1.
Carbon source utilization by different L. mexicana developmental stages.
L. mexicana Prolog, Prostat and Amaaxenic were suspended in chemically defined medium (CDM) containing 13C-U-glucose (A) or 13C-glutamate (B) and the rate of utilization of the proffered 13C-carbon source (left hand panel) and production of overflow metabolites (right hand panel) assessed by 13C-NMR analysis of the conditioned medium. 13CO2 (detected as H13CO3−) was only quantified in the medium of promastigote stages, as it is not retained in the acidified amastigote medium. C. Proportion of internalized 13C-glucose that was secreted as partially oxidized end-products by Prolog, Prostat and Amaaxenic. Similar relative levels of secretion were observed in the two promastigote stages (∼50%), while negligible secretion was observed in Amaaxenic. D. Replication rates of different L. mexicana developmental stages. Prolog, Prostat and Amaaxenic were cultivated in RPMI supplemented with D2O (5% v/v, final) and the rate of DNA turnover determined by kinetic analysis of deuterium incorporation into adenosine deoxyribose. All data represent mean (n = 3) and standard deviation and are representative of several independent experiments.
Figure 2.
Isotopic enrichment of intracellular metabolite pools following cultivation with different 13C-labeled carbon sources.
A. L. mexicana Prolog, Prostat and Amaaxenic were suspended in CDM containing either 13C-U-glucose (Glc), a mixture of 13C-U-amino acids (AA), 13C-U-alanine (Ala), 13C-U-aspartate (Asp), or 13C-U-glutamate (Glu) for 3 h and 13C-enrichment (mol percent) in intracellular intermediates in central carbon metabolism determined by GC-MS. Data shown for 13C-Glc and individual amino acid labeling in Prolog panel was taken from [18]. Mean (n = 3) and SD for this data is provided in Tables S1–6 in Text S1. Abbreviations used are as follows G6P, glucose 6-phosphate; F6P, fructose 6-phosphate; S7P, seduheptulose 7-phosphate; Ru5P, ribulose 5-phosphate; 3PG, 3-phosphoglycerate; 2PG, 2-phosphoglycerate; PEP, phosphoenolpyruvate; Suc, succinate; Mal, malate; Fum, fumarate; Cit, citrate; Ala, alanine; Asp, aspartate; Glu, glutamate; Gly, glycine; Ser, serine; Thr, threonine; Pro, proline; Ile, isoleucine; Leu, leucine; Lys, lysine; Phe, phenylalanine; Val, valine; Put, putrescine; Orn, ornithine; MTA, 5-methylthioadenosine; Ura, uracil; CHO1, mannogen; I3P, inositol 3-phosphate; MI, myo-inositol; G3P, glycerol 3-phosphate.
Figure 3.
Stage-specific differences in TCA cycle fluxes.
A. Scheme showing how label from 13C-U-glucose can be incorporated into TCA cycle intermediates. 13C3-PEP (carbon skeletons in dark blue) can be carboxylated in glycosomes to generate 13C3-labeled OAA, malate, and succinate. These C4 dicarboxylic acids are used to top up the TCA cycle depleted during glutamate synthesis. Alternatively, PEP can be directly catabolized to pyruvate (carbon skeletons in light blue) and 13C-U-acetyl-CoA. B. Mass isotopomers of malate, citrate and glutamate in L. mexicana Prolog, ProStat and Amaaxenic labeled with 13C-U-glucose for 3 h. These data are taken from the labeling experiment shown in Fig. 2, and are representative of several other experiments. Data represent mean (n = 3) and SD.
Figure 4.
Fatty acid β-oxidation is increased in L. mexicana Amaaxenic.
A. L. mexicana promastigote and amastigote stages were suspended in CDM supplemented with 13C-U-fatty acids for 3 h and isotopic enrichment (mol percent) in intermediates of central carbon metabolism was determined by GC-MS. Abbreviations as for Figure 2. This data (mean (n = 3) and SD) is also presented in Tables S7 in Text S1. B. Mass isotopomers of citrate and glutamate in 13C-U-fatty acid fed (3 h) L. mexicana Amaaxenic. C. Wild type (WT) Prolog and Amaaxenic and glycocalyx deficient Δpmm-Prolog parasites were labeled with 13C-U-fatty acids for 48 h and isotopic enrichment in the total lipid fraction (exemplified by hexadecanoic acid, C18:0) and citrate was determined by GC-MS. D. Fatty acid β-oxidation is elevated during glucose starvation. L. mexicana Prolog, Prostat and Amaaxenic were labeled with 13C-U-fatty acids for 3 h in glucose-replete (+Glc) or glucose-free (−Glc) CDM and isotopic enrichment in intracellular pools of citrate and glutamate determined by GC-MS. Data represent mean (n = 3) and SD.
Figure 5.
Carbon source utilization by Amalesion.
A. L. mexicana amastigotes were harvested from susceptible Balb/c mice lesions and purified Amalesion immediately suspended in CDM containing either 13C-U-glucose (Glc), a mixture of 13C-U-amino acids (AA), 13C-U-alanine (Ala), 13C-U-aspartate (Asp), 13C-U-glutamate (Glu), or 13C-U- fatty acids (FFA). Isotopic enrichment in intracellular pools of metabolites in central carbon metabolism was determined by GC-MS (see also Tables S1–S7 in Text S1). B. The uptake of 13C-U-glucose and the secretion of labeled end products in Amalesion were determined by 13C-NMR analysis of culture supernantant after 24 h incubation. C. Mass isotopomer of citrate and glutamate in Amalesion labelled with 13C-U-fatty acids for 3 h. Data represent mean (n = 3) and SD.
Figure 6.
Inhibition of the TCA cycle in Amaaxenic is cytotoxic.
Sodium fluoroacetate (NaFAc) results in inhibition of the aconitase reaction in the TCA cycle. A. Amaaxenic were treated with 0.5 mM NaFAc and the steady-state levels of selected intermediates in central carbon metabolism determined by GC-MS. The fold-change in NaFAc-treated compared to sodium acetate-treated Amaaxenic are shown. For abbreviations see Figure 2 and R5P, ribose 5-phosphate and iCit, isocitrate. B. Prolog and Amaaxenic were cultivated in RPMI medium containing 10% iFCS supplemented with NaFAc (0.05 to 5 mM) for 48 h and viability assessed by labeling with propidium iodide. NaFAc toxicity in Amaaxenic was partially reversed by supplementation of the medium with 5 mM glutamate (background level of glutamate in the medium is 0.15 mM). Data represented as mean (n = 3–4) +/− SEM, *, P<0.05; **, P<0.00005 and #, P<0.005 C. Intracellular levels of key Amaaxenic metabolites following incubation in the presence or absence of NaFAc or glutamate. Exogenous glutamate did not lead to restoration of intracellular glutamate levels following NaFAc treatment. Data represent the mean (n = 3) and SD.
Figure 7.
Intracellular L. mexicana amastigotes are dependent on TCA cycle function and glutamate/glutamine synthesis.
RAW 264.7 macrophages were infected with L. mexicana Amaaxenic for 4 h and then incubated in RPMI supplemented with the metabolic inhibitors (A) sodium fluoroacetate (NaFAc, 10 mM) or (B) methionine sulphoximine (MSO, 5 mM). The medium of infected macrophages was also supplemented with either Glu or Gln (5 mM), as indicated. Percent infection and number of parasites per macrophage were determined by microscopy. Data represent the mean (n = 3) and SEM.
Figure 8.
Remodelling of L. mexicana central carbon metabolism during amastigote differentiation.
Key pathways of carbon utilization in L. mexicana promastigotes (A) and amastigotes (B) are represented. Major carbon sources (blue box) and overflow metabolites (open box) detected in this study are shown. Fluxes through dotted pathways are down-regulated relative to the other stage. Steps inhibited by NaFAc and MSO are indicated. Abbreviations used, αKG, α-ketoglutarate; AcCoA, acetyl-CoA; Ala, alanine; Asp, aspartate; Cit, citrate; Fum, fumarate; FA, fatty acids; G6P, glucose 6-phosphate; G3P, glyceraldehyde 3-phosphate; Gln, glutamine; Glu, glutamate; Mal, malate; OAA, oxaloacetate; OAc, acetate; PEP, phosphoenolpyruvate; PPP, pentose phosphate pathway; Pro, proline; Pyr, pyruvate; SCoA, succinyl-CoA; Suc, succinate; TCA, tricarboxylic acid cycle.