Modulation of host central carbon metabolism and in situ glucose uptake by intracellular Trypanosoma cruzi amastigotes

Abstract

Obligate intracellular pathogens satisfy their nutrient requirements by coupling to host metabolic processes, often modulating these pathways to facilitate access to key metabolites. Such metabolic dependencies represent potential targets for pathogen control, but remain largely uncharacterized for the intracellular protozoan parasite and causative agent of Chagas disease, Trypanosoma cruzi. Perturbations in host central carbon and energy metabolism have been reported in mammalian T. cruzi infection, with no information regarding the impact of host metabolic changes on the intracellular amastigote life stage. Here, we performed cell-based studies to elucidate the interplay between infection with intracellular T. cruzi amastigotes and host cellular energy metabolism. T. cruzi infection of non-phagocytic cells was characterized by increased glucose uptake into infected cells and increased mitochondrial respiration and mitochondrial biogenesis. While intracellular amastigote growth was unaffected by decreased host respiratory capacity, restriction of extracellular glucose impaired amastigote proliferation and sensitized parasites to further growth inhibition by 2-deoxyglucose. These observations led us to consider whether intracellular T. cruzi amastigotes utilize glucose directly as a substrate to fuel metabolism. Consistent with this prediction, isolated T. cruzi amastigotes transport extracellular glucose with kinetics similar to trypomastigotes, with subsequent metabolism as demonstrated in 13C-glucose labeling and substrate utilization assays. Metabolic labeling of T. cruzi-infected cells further demonstrated the ability of intracellular parasites to access host hexose pools in situ. These findings are consistent with a model in which intracellular T. cruzi amastigotes capitalize on the host metabolic response to parasite infection, including the increase in glucose uptake, to fuel their own metabolism and replication in the host cytosol. Our findings enrich current views regarding available carbon sources for intracellular T. cruzi amastigotes and underscore the metabolic flexibility of this pathogen, a feature predicted to underlie successful colonization of tissues with distinct metabolic profiles in the mammalian host.

Fig 1. T. cruzi infection increases glucose uptake by host cells.

(A) Uptake of [³H]-2-deoxyglucose ([³H]-2-DG) into uninfected or T. cruzi-infected NHDF monolayers at 48 hours post infection (48 hpi), in which infection was established with a varying multiplicity of infection (MOI). Mean ± SD shown for 3 biological replicates per MOI. One-way ANOVA with Dunnett’s multiple comparison test was applied for individual comparisons to the uninfected control group (*p<0.05, ****p<0.0001). (B) Cytochalasin B (10 μM) blocks uptake of [³H]-2-DG in uninfected and infected NHDF monolayers (48 hpi). Mean ± SD shown for 3 biological replicates. Two-way ANOVA with Tukey’s multiple comparisons test was applied (****p<. 0001). (C) [³H]-2-DG uptake by NHDF or (D) C2C12 myoblasts following a 48 h infection with T. cruzi Tulahuén, CL Brener or CL-14 strains. NHDF were infected for 2 hours with MOI 40 for Tulahuén strain and MOI 150 for CL Brener and CL-14 strains. C2C12 were infected for 2 hours with MOI 80 for Tulahuén strain and MOI 150 for CL Brener and CL-14 strains. Mean ± SD for 3 biological replicates. One-way ANOVA with Dunnett’s multiple comparison test was applied for individual comparisons to the uninfected control group (**p< 0.01, ***p< 0.001, ****p< 0.0001).

Fig 2. T. cruzi infection increases host mitochondrial content and respiration.

(A) Extracellular lactate measured in culture supernatants of uninfected and T. cruzi-infected NHDF monolayers (48 hpi). Mean ± SD shown for 2 biological replicates each. Student’s t-test was applied. (B) Oxygen consumption rate (OCR) in uninfected and T. cruzi-infected NHDF monolayers (48hpi) before and after injection of oligomycin (O), FCCP (F), and rotenone and antimycin A (R/A). Mean ± SD shown for 4 biological replicates. (C) T. cruzi-infected NHDF monolayers were treated with 1 μM ELQ300 to selectively remove amastigote respiration from the total OCR signal (Infected, ±ELQ300). Increased host respiration during T. cruzi infection (Infected, +ELQ300). Mean ± SD shown for 4 biological replicates per condition. Two-way ANOVA with Tukey’s multiple comparisons test was applied (*p<0.05, ***p<0.001, ****p<0.0001). (D) Geometric mean fluorescence intensity of mitochondrial mCherry signal for each condition relative to uninfected controls in NHDF and C2C12 myoblast. Infected cells were discriminated based on T. cruzi GFP expression (S2D Fig), and the geometric mean mCherry fluorescence was determined from each subpopulation (S2E Fig). Mean ± SD for 2 independent experiments. Two-way ANOVA with Dunnett’s multiple comparisons test was applied (*p< 0.05, **p< 0.01).

Fig 3. Intracellular T. cruzi replication is sensitive to exogenous glucose but not host mitochondrial electron transport chain activity.

(A) Proliferation of T. cruzi amastigotes in human dermal fibroblasts with ETC complex III deficiency (CIII mutant) or two independent control fibroblast lines (Normal 1 and 2) derived from flow cytometric data (as detailed in Methods). Data are normalized to represent the percentage of initial amastigotes (18 hpi) that divided the indicated number of times by 48 hpi. Mean ± SD of 2 independent experiments. Dotted lines represent average number of complete amastigote divisions achieved by 48 hpi in each condition. (B) Proliferation of T. cruzi amastigotes in NHDF cultured in medium with varying glucose concentrations. Dotted lines represent average number of amastigote divisions achieved by 48 hpi as determined by flow cytometry of CFSE-labeled parasites. (C) Dose-dependent inhibition of T. cruzi growth in NHDF by 2-deoxyglucose (2-DG) in varying glucose concentrations. Relative number of T. cruzi-ß-galactosidase parasites assessed by Beta-Glo luminescence at 66 hpi shown with nonlinear fit using log(inhibitor) vs. response with variable slope. Mean ± SD of 4 biological replicates per point. (D) Arrest of T. cruzi amastigote proliferation in NHDF in the presence of 2 mM 2-DG under conditions of glucose depletion. Dotted lines represent average number of amastigote divisions achieved by 48 hpi as determined by flow cytometry of CFSE-labeled parasites. (E) Fluorescence micrographs of aldehyde-fixed, DAPI-stained NHDF monolayers corresponding to conditions used in panel D, in which host cell nuclei (large) and parasite DNA (smaller dots) are readily observed. Arrows point to 2 intracellular amastigotes that persist after severe growth restriction caused by glucose withdrawal and 2-DG treatment.

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).