Stress-Induced Proliferation and Cell Cycle Plasticity of Intracellular Trypanosoma cruzi Amastigotes

Abstract

The mammalian stages of the parasite Trypanosoma cruzi, the causative agent of Chagas disease, exhibit a wide host species range and extensive within-host tissue distribution. These features, coupled with the ability of the parasites to persist for the lifetime of the host, suggest an inherent capacity to tolerate changing environments. To examine this potential, we studied proliferation and cell cycle dynamics of intracellular T. cruzi amastigotes experiencing transient metabolic perturbation or drug pressure in the context of an infected mammalian host cell. Parasite growth plasticity was evident and characterized by rapid and reversible suppression of amastigote proliferation in response to exogenous nutrient restriction or exposure to metabolic inhibitors that target glucose metabolism or mitochondrial respiration. In most instances, reduced parasite proliferation was accompanied by the accumulation of amastigote populations in the G₁ phase of the cell cycle, in a manner that was rapidly and fully reversible upon release from the metabolic block. Acute amastigote cell cycle changes at the G₁ stage were similarly observed following exposure to sublethal concentrations of the first-line therapy drug, benznidazole, and yet, unlike the results seen with inhibitors of metabolism, recovery from exposure occurred at rates inversely proportional to the concentration of benznidazole. Our results show that T. cruzi amastigote growth plasticity is an important aspect of parasite adaptation to stress, including drug pressure, and is an important consideration for growth-based drug screening.IMPORTANCE Infection with the intracellular parasite Trypanosoma cruzi can cause debilitating and potentially life-threatening Chagas disease, where long-term parasite persistence is a critical determinant of clinical disease progression. Such tissue-resident T. cruzi amastigotes are refractory to immune-mediated clearance and to drug treatment, suggesting that in addition to exploiting immune avoidance mechanisms, amastigotes can facilitate their survival by adapting flexibly to diverse environmental stressors. We discovered that T. cruzi intracellular amastigotes exhibit growth plasticity as a strategy to adapt to and rebound from environmental stressors, including metabolic blockades, nutrient starvation, and sublethal exposure to the first-line therapy drug benznidazole. These findings have important implications for understanding parasite persistence, informing drug development, and interpreting drug efficacy.

Keywords: Chagas; amastigote; benznidazole; cell cycle; plasticity; stress.

FIG 1

T. cruzi amastigotes respond to exogenous nutrient availability by altering proliferation and cell cycle. (A) Schematic of unabated invasion, differentiation, and growth of T. cruzi Tula-βgal in vitro. (B) Experimental design used to measure responsiveness of T. cruzi amastigote growth in vitro. (C and D) Flow cytometry histograms of amastigotes (CFSE) isolated at 48 hpi (C) or 66 hpi (D) under the indicated growth conditions. Division indices are compared using one-way ANOVA and a post hoc Dunnett’s test for multiple comparisons (****, P < 0.0001; **, P < 0.01; ns, not significant). (E and F) Cell cycle profiles for T. cruzi amastigotes isolated from infected HFF at 48 hpi (E) or 66 hpi (F) under the indicated exogenous starvation conditions initiated at 18 hpi. Comparisons were made using a chi-squared test (G₁ versus S/G₂) and Bonferroni correction for multiple testing.

FIG 2

Rapid recovery of T. cruzi amastigote growth and cell cycle phase distribution following exposure to metabolic inhibitors. (A) Flow cytometry histograms of amastigotes (CFSE) isolated at 66 hpi. Cultures grown in 5.5 mM glucose are indicated with a solid line and filled histogram and cultures grown in medium without glucose are shown with a solid line and unfilled histograms. Cultures washed at 42 hpi are shown with a dotted line. Division indices (no glucose/5.5 mM glucose or not washed/washed) are compared using one-way ANOVA and a post hoc Dunnett’s multiple-comparison test where indicated (****, P < 0.0001). (B) Flow cytometry histograms of amastigotes (CFSE) isolated at 66 hpi. Histograms from cultures washed at 42 hpi are indicated with a dotted line. Division indices (not washed/washed) are compared using one-way ANOVA and a post hoc Dunnett’s multiple-comparison test (****, P < 0.0001). (C and D) Cell cycle distribution of amastigotes at 66 hpi under the indicated conditions with 2-DG (C) or GNF7686 (D). Comparisons were made using a chi-squared test (G₁ versus S/G₂) with Bonferroni correction for multiple testing.

FIG 3

Intracellular T. cruzi amastigotes tolerate prolonged complex III inhibition. (A) Amastigotes per infected cell from coverslips collected at the indicated time points following addition of GNF7686 (2.5 µM) or DMSO at 18 hpi. Medians are shown in red (n = 40 per condition). A Kruskal-Wallis test was used to determine significance, and a Dunn’s post hoc test was used for individual comparisons (****, P < 0.0001). (B) Representative images (DAPI staining) of amastigotes at the indicated time points (20 µM scale bar indicated). Inset: 4× zoomed sections where present (lower left). (C) Flow cytometry histograms of amastigotes (CFSE) isolated at 162 hpi following addition of 2.5 µM GNF7686 at 18 hpi. Histograms are from cultures with constant GNF7686 (solid line filled histogram) or washed at 138 hpi (dotted line no fill). Division indices (not wash/wash at 138 hpi) are compared using a t test (****, P < 0.0001). (D) Cell cycle distribution of amastigotes at 162 hpi under the indicated conditions. Comparisons were made using a chi-squared test (G₁ versus S/G₂) with Bonferroni correction for multiple testing.

FIG 4

Acute benznidazole exposure drives intracellular T. cruzi amastigotes to accumulate in G₁ and inhibits proliferation. (A and B) Flow cytometry histograms of isolated amastigotes (CFSE) at 42 hpi (A) and 66 hpi (B) following benznidazole treatment at 18 hpi. Division indices are compared to the results seen with DMSO using one-way ANOVA and a post hoc Dunnett’s multiple-comparison test for comparisons to DMSO data where indicated (****, P < 0.0001). (C and D) Amastigote cell cycle distributions at (C) 42 hpi and (D) 66 hpi following benznidazole treatment at 18 hpi. Comparisons were made using a chi-squared test (G₁ versus S/G₂) with Bonferroni correction for multiple testing.

FIG 5

Recovery from acute benznidazole treatment is inversely proportional to the concentration of drug present during exposure. (A) Concentration-response relationship for the inhibitory effects of acute exposure to Bz for 24 h (filled squares) or 48 h (filled circles) on intracellular T. cruzi (Tula-βgal) amastigote growth as measured by Beta-Glo luminescence at 66 hpi. Means ± standard deviations (SD) are shown (n = 4 per point). (B) Cell cycle distribution of amastigotes at 66 hpi. Comparisons between washed or constant exposure to Bz were made using a chi-squared test (G₁ versus S/G₂) with Bonferroni correction for multiple testing. (C) Flow cytometry histogram overlays of isolated amastigotes (CFSE) at 66 hpi from cultures washed at 42 hpi (nonfilled histograms, dotted line) or without removal (filled histograms, solid line) of Bz. Division indices (no wash/wash at 42 hpi) were compared using a one-way ANOVA and a post hoc Dunnett’s multiple-comparison test (****, P < 0.0001). (D) Amastigotes per cell from coverslips collected at the indicated time points following a pulse of benznidazole (18 hpi to 42 hpi). Medians are indicated in red. Coverslips were not collected following detection of the presence of visible extracellular trypomastigotes (as indicated), which occurred in all cases except at the concentration of 25 µM (nd = none detected).

FIG 6

LD₅₀ determination for benznidazole, 24-h pulse. (A) Clonal outgrowth (60 wells per plate, one plate per concentration for each independent replicate) following a 24-h pulse of Bz or DMSO followed by a 21-day recovery. Each experiment (indicated as squares, circles, triangles, or inverted triangles) is normalized to the number of clones seen under the DMSO condition from that given experiment (n = 4 experiments). Means ± SD are shown. (B) Clonal outgrowth determined on the basis of data from panel A (circles) graphed with the best-fit inhibitory curve (dotted line) and best-fit curve for clonal outgrowth (solid line). LD₅₀ values were calculated using a best-fit curve for clonal outgrowth. (C) Clonal outgrowth normalized to DMSO treatments following either 1 day or 5 days of exposure to GNF7686 at a concentration of 2.5 µM (n = 3, 60 wells per independent replicate). Outgrowth data were compared using a t test (P = 0.267). Means ± SD are shown.