TrypanocidalActivity of Natural Sesquiterpenoids Involves Mitochondrial Dysfunction, ROS Production and Autophagic Phenotype in Trypanosomacruzi

Abstract

Chagas disease is a neglected tropical disease that is caused by the protozoan Trypanosomacruzi and represents a serious health problem, especially in Latin America. The clinical treatment of Chagas disease is based on two nitroderivatives that present severe side effects and important limitations. In folk medicine, natural products, including sesquiterpenoids, have been employed for the treatment of different parasitic diseases. In this study, the trypanocidal activity of compounds isolated from the Chilean plants Drimys winteri, Podanthus mitiquiand Maytenus boaria on three T. cruzi evolutive forms (epimastigote, trypomastigote and amastigote) was evaluated. Total extracts and seven isolated sesquiterpenoids were assayed on trypomastigotes and epimastigotes. Polygodial (Pgd) from D. winteri, total extract from P. mitiqui (PmTE) and the germacrane erioflorin (Efr) from P. mitiqui were the most bioactive substances. Pgd, Efr and PmTE also presented strong effects on intracellular amastigotes and low host toxicity. Many ultrastructural effects of these substances, including reservosome disruption, cytosolic vacuolization, autophagic phenotype and mitochondrial swelling (in the case of Pgd), were observed. Flow cytometric analysis demonstrated a reduction in mitochondrial membrane potential in treated epimastigotes and an increase in ROS production and high plasma membrane permeability after treatment with Pgd. The promising trypanocidal activity of these natural sesquiterpenoids may be a good starting point for the development of alternative treatmentsforChagas disease.

Keywords: Trypanosoma cruzi; autophagy; chagas disease; chemotherapy; mitochondria; sesquiterpenoids.

Figure 1

Molecular structures of sesquiterpenoids. Drimane sesquiterpenoids, including polygodial (Pgd), cinnamolide, dendocarbin A and isodrimeninol were purified from the bark of D. winteri. From P. mitiqui, the germacrane sesquiterpenoid erioflorin acetate (Efr) was isolated. The β-dihydroagarofuran compounds MB-16 and MB-22 were purified from M. boaria.

Figure 2

TEM analysis of untreated T. cruzi epimastigotes. (A–C) Control parasites showing normal ultrastructural aspects of nucleus (N), mitochondrion (M), kinetoplast (K), Golgi (G), endoplasmic reticulum (ER), flagellum (F) and reservosomes (R). Bars = 1 µm.

Figure 3

TEM analysis of T. cruzi epimastigotes treated with 42 µg/mL drimane sesquiterpenoid Pgd. (A–D) The treatment induced remarkable mitochondrial swelling (black asterisks), with dilation of the mitochondrial cristae (white arrows) and reservosome disorganization (black star). Endoplasmic reticulum profiles (black arrows) can be observed in treated parasites surrounding cytoplasmic portions and/or other cellular structures, including lipid droplets (L). Pgd also led to intense cytosolic vacuolization (V). N: nucleus, K: kinetoplast. Bars in A, B and D = 1 µm; bar in C = 0.5 µm.

Figure 4

TEM analysis of T. cruzi epimastigotes treated with Efr. The treatment with (A–C) 25 and (D–F) 50 µg/mL of this germacrane sesquiterpenoid induced the appearance of numerous autophagosomes (white stars) and the formation of endoplasmic reticulum profiles (black arrows) in close contact with subcellular structures such as lipid droplets (L). Parasites treated with Efr also showed reservosome disorganization (black stars) and cytosolic vacuolization (V). N: nucleus, M: mitochondrion, K: kinetoplast. Bars = 1 µm.

Figure 5

Flow cytometry analysis of ROS production in T. cruzi epimastigotes by DHE labelling. Pgd led to an increase in the percentage of parasites generating ROS. As a positive control, 10 µM menadione was employed. The graphs present the mean and standard deviation of least three independent experiments. * Asterisks represent the significant difference in relation to the untreated control group (p ≤ 0.02) by Mann-Whitney test.

Figure 6

Flow cytometry analysis of plasma membrane integrity in T. cruzi epimastigotes by PI labelling. Treatment with Pgd induced permeabilization of the parasite plasma membrane. As a positive control, epimastigotes were incubated with 0.1% saponin. The graphs present the mean and standard deviation of four independent experiments. *Asterisks indicatesignificant difference in relation to the untreated control group (p ≤ 0.03) by Mann-Whitney test.