Synergistic effect and ultrastructural changes in Trypanosoma cruzi caused by isoobtusilactone A in short exposure of time

Abstract

Butanolides have shown a variety of biological effects including anti-inflammatory, antibacterial, and antiprotozoal effects against certain strains of Trypanosoma cruzi. Considering the lack of an effective drug to treat T. cruzi infections and the prominent results obtained in literature with this class of lactones, we investigated the anti-T. cruzi activity of five butanolides isolated from two species of Lauraceae, Aiouea trinervis and Mezilaurus crassiramea. Initially, the activity of these compounds was evaluated on epimastigote forms of the parasite, after a treatment period of 4 h, followed by testing on amastigotes, trypomastigotes, and mammalian cells. Next, the synergistic effect of active butanolides against amastigotes was evaluated. Further, metacyclogenesis inhibition and infectivity assays were performed for the most active compound, followed by ultrastructural analysis of the treated amastigotes and trypomastigotes. Among the five butanolides studied, majoranolide and isoobtusilactone A were active against all forms of the parasite, with good selectivity indexes in Vero cells. Both butanolides were more active than the control drug against trypomastigote and epimastigote forms and also had a synergic effect on amastigotes. The most active compound, isoobtusilactone A, which showed activity against all tested strains inhibited metacyclogenesis and infection of new host cells. In addition, ultrastructural analysis revealed that this butanolide caused extensive damage to the mitochondria of both amastigotes and trypomastigotes, resulting in severe morphological changes in the infective forms of the parasite. Altogether, our results highlight the potential of butanolides against the etiologic agent of Chagas disease and the relevance of isoobtusilactone A as a strong anti-T. cruzi drug, affecting different events of the life cycle and all evolutionary forms of parasite after a short period of exposure.

Fig 1. Chemical structures of the butanolides isolated from Aiouea trinervis (1–3) and Mezilaurus crassiramea (4 and 5).

Fig 2. Synergism effect of isoobtusilactone A and majoranolide against amastigotes.

A: Nonlinear regression with variable slope of each ratio (4:0-isoobtusilactone A alone; 0:4 majoranolide alone); B: Normalized isobologram of combination index of the ratios 3:1, 1:1 and 1:3 (■:IC₂₅, ♦:IC₅₀, ▲:IC₇₅, ●:IC₉₀); C: Drug reduction index plot (Dashed line represents the threshold of a favorable drug reduction).

Fig 3. Transmission electron micrographs showing the ultrastructure of intracellular amastigotes.

Nucleus (N); kinetoplast (K); *Swollen mitochondria. A-B: Control untreated; C-D: Parasites treated 4 hours with IC₅₀ of isoobtusilactone A.

Fig 4. Transmission electron micrographs showing the ultrastructure of trypomastigotes.

Nucleus (N); kinetoplast (K); swollen mitochondria (Black arrow). A: Control untreated; B: Treatment with isoobtusilactone A (LC₅₀) for 2 hours.

Fig 5. Scanning electron micrographs showing the morphology of trypomastigotes.

A: Control untreated; B: Treatment with isoobtusilactone A (LC₅₀) for 2 hours.

Fig 6. Evaluation of the interference of isoobtusilactone A on infection of host cells.

Nucleus (N); kinetoplast (K); intense cytoplasm disorganization (Black arrow). A: Percentage of infected cells by trypomastigotes (p<0.05 by Tukey); B: Number of amastigotes per infected cells (p<0.05 by Tukey); C: Transmission electron micrographs showing the morphology of infected cells with untreated trypomastigotes; D: Scanning electron micrographs showing the morphology of host cells after infection with treated trypomastigotes; E-F: Transmission electron micrographs showing the morphology of infected cells with treated trypomastigotes.