Modulation of host central carbon metabolism and in situ glucose uptake by intracellular Trypanosoma cruzi amastigotes
Fig 4
Acquisition and metabolism of glucose by intracellular T. cruzi amastigotes.
Isolated T. cruzi amastigotes utilize exogenous substrates as determined by increased (A) oxygen consumption rate (OCR) and (B) extracellular acidification rate (ECAR). After establishing baseline rates, glucose (5 mM), glutamine (5 mM) or buffer were injected as substrates (subs), followed by 100 mM 2-DG to rapidly inhibit glycolysis, and 1 μM rotenone and antimycin A (R/A) to shut down mitochondrial respiration. Mean ± SD of 3 biological replicates. (C) Initial rate (V0) of [3H]-2-DG uptake by isolated T. cruzi amastigotes or trypomastigotes plotted for a range of substrate concentrations. Mean ± SD of two independent experiments with biological duplicates shown for each lifecycle stage. Inset shows Lineweaver-Burk plot. (D) Intracellular T. cruzi amastigotes access exogenous hexose in situ. T. cruzi-infected monolayers were incubated with 10 μCi [3H]-2-DG in the absence or presence of cytochalasin B (15 μM) for 20 minutes prior to isolation of intracellular amastigotes for scintillation counts. Mean ± SD of two independent experiments. Student’s t-test was applied (**p< 0.01). (E) [3H]-2-DG is internalized by intracellular amastigotes. Following isolation from monolayers pulsed with [3H]-2-DG, treatment of amastigotes with 0.05 mg/mL alamethicin released internalized, non-bound substrate. Mean ± SD of two independent experiments. (F) ATP levels measured in intracellular-derived amastigotes 24 hours after incubation with the indicated carbon substrate relative to initial ATP levels of freshly isolated parasites. Mean ± SD of 3 biological replicates per condition. One-way ANOVA with Dunnett’s multiple comparison test was applied for individual comparisons to the substrate deficient condition (***p< 0.001, ****p< 0.0001).