Ergosterol isolated from the basidiomycete Pleurotus salmoneostramineus affects Trypanosoma cruzi plasma membrane and mitochondria

Abstract

Background: Major drawbacks of the available treatment against Chagas disease (American trypanosomiasis) include its toxicity and therapeutic inefficiency in the chronic phase of the infection, which makes it a concern among neglected diseases. Therefore, the discovery of alternative drugs for treating chronic Chagas disease requires immediate action. In this work, we evaluated the mushroom Pleurotus salmoneostramineus in the search for potential antiparasitic compounds.

Methods: Fruit bodies of the basidiomycete Pleurotus salmoneostramineus were triturated and submitted to organic solvent extraction. After liquid-liquid partition of the crude extract, three fractions were obtained and the bioguided fractionation study was conducted to isolate the active metabolites. The elucidation of the chemical structure was performed using GC-MS and NMR techniques. The biological assays for antiparasitic activity were carried out using trypomastigotes of Trypanosoma cruzi and murine macrophages for mammalian cytotoxicity. The mechanism of action of the isolated compound used different fluorescent probes to evaluate the plasma membrane permeability, the potential of the mitochondrial membrane and the intracellular levels of reactive oxygen species (ROS).

Results: The most abundant fraction showing the antiparasitic activity was isolated and chemically elucidated, confirming the presence of ergosterol. It showed anti-Trypanosoma cruzi activity against trypomastigotes, with an IC₅₀ value of 51.3 μg/mL. The compound demonstrated no cytotoxicity against mammalian cells to the maximal tested concentration of 200 μg/mL. The mechanism of action of ergosterol in Trypanosoma cruzi trypomastigotes resulted in permeabilization of the plasma membrane, as well as depolarization of mitochondrial membrane potential, leading to parasite death. Nevertheless, no increase in ROS levels could be observed, suggesting damages to plasma membrane rather than an induction of oxidative stress in the parasite.

Conclusions: The selection of naturally antiparasitic secondary metabolites in basidiomycetes, such as ergosterol, may provide potential scaffolds for drug design studies against neglected diseases.

Keywords: Ergosterol; Mechanism of action; Pleurotus salmoneostramineus; Trypanosoma cruzi.

Fig. 1

Structure of ergosterol confirmed by ¹³C, ¹H RMN and GC-EI-MS

Fig. 2

Effect of ergosterol on plasma membrane permeabilization of trypomastigotes of T. cruzi. Sytox Green (1 μM) fluorescence was measured spectrofluorimetrically every 20 min. Minimum and maximum permeabilization were obtained by non-treatment and Tx-100 0.5%, respectively. Fluorescence was quantified by calculating the mean percentages of untreated (0%) and Tx-100-treated (100%) trypomastigotes. *** p < 0.001 and **p < 0.002. A representative assay is shown

Fig. 3

Effect of ergosterol in the mitochondrial function of trypomastigotes. Fluorescence of Mitotracker Red CM-H2XROS dye (500 nM) was spectrofluorimetrically measured after 60 min of incubation. Maximal and minimal fluorescence was achieved by non- or FCCP (10 μM) treatments, respectively. Fluorescence was quantified by calculating the mean percentage of untreated parasites (100%). *p < 0.001. A representative assay is shown

Fig. 4

Effect of ergosterol in the ROS production of trypomastigotes. Fluorescence of H₂DCF-DA was measured after 60 min of incubation. Fluorescence was quantified by calculating the mean percentage of untreated (0%) and sodium azide-treated (100%) trypomastigotes. ***(p < 0.001). A representative assay is shown