Abstract
Trypanosoma cruzi is the causative agent of Chagas disease which is currently treated by nifurtimox (NFX) and benznidazole (BZ). Nevertheless, the mechanism of action of NFX is not completely established. Herein, we show the protective effects of T. cruzi mitochondrial peroxiredoxin (MPX) in macrophage infections and in response to NFX toxicity. After a 3-day treatment of epimastigotes with NFX, MPX content increased (2.5-fold) with respect to control, and interestingly, an MPX-overexpressing strain was more resistant to the drug. The generation of mitochondrial reactive species and the redox status of the low molecular weight thiols of the parasite were not affected by NFX treatment indicating the absence of oxidative stress in this condition. Since MPX was shown to be protective and overexpressed in drug-challenged parasites, non-classical peroxiredoxin activity was studied. We found that recombinant MPX exhibits holdase activity independently of its redox state and that its overexpression was also observed in temperature-challenged parasites. Moreover, increased holdase activity (2-fold) together with an augmented protease activity (proteasome-related) and an enhancement in ubiquitinylated proteins was found in NFX-treated parasites. These results suggest a protective role of MPX holdase activity toward NFX toxicity. Trypanosoma cruzi has a complex life cycle, part of which involves the invasion of mammalian cells, where parasite replication inside the host occurs. In the early stages of the infection, macrophages recognize and engulf T. cruzi with the generation of reactive oxygen and nitrogen species toward the internalized parasite. Parasites overexpressing MPX produced higher macrophage infection yield compared with wild-type parasites. The relevance of peroxidase vs. holdase activity of MPX during macrophage infections was assessed using conoidin A (CA), a covalent, cell-permeable inhibitor of peroxiredoxin peroxidase activity. Covalent adducts of MPX were detected in CA-treated parasites, which proves its action in vivo. The pretreatment of parasites with CA led to a reduced infection index in macrophages revealing that the peroxidase activity of peroxiredoxin is crucial during this infection process. Our results confirm the importance of peroxidase activity during macrophage infection and provide insights for the relevance of MPX holdase activity in NFX resistance.
Highlights
Trypanosoma cruzi (T. cruzi) is the intracellular parasite that causes Chagas disease, which is considered a neglected disease and remains a public health problem in Latin America, estimated to affect approximately 6 million people and with 70 million at risk of infection (Pérez-Molina and Molina, 2018)
Taking into consideration the abovementioned results related to the peroxiredoxins of trypanosomatids and its different functions within the cell, we evaluated the ability of T. cruzi mitochondrial peroxiredoxin to act as holdase and studied the relative weight of both activities toward macrophage and NFX-derived toxicity
With the aim to evaluate the role of peroxidase vs. holdase activity of mitochondrial peroxiredoxin (MPX) during macrophage infections, we examined the effects of conoidin A (CA), a previously reported cell-permeable covalent inhibitor of the peroxidase activity of some peroxiredoxins, by alkylating or crosslinking the catalytic cysteines (Liu et al, 2010; Brizuela et al, 2014)
Summary
Trypanosoma cruzi (T. cruzi) is the intracellular parasite that causes Chagas disease, which is considered a neglected disease and remains a public health problem in Latin America, estimated to affect approximately 6 million people and with 70 million at risk of infection (Pérez-Molina and Molina, 2018). Trypanosoma cruzi has a complex life cycle that involves two hosts, the insect vector and the vertebrate host, and four stages of the parasite. The replicative, non-infective epimastigote stage is found in the insect midgut and, during its migration to the hindgut, differentiates to the infective, non-replicative metacyclic trypomastigotes that can invade different vertebrate cells. Trypomastigote transforms into the infective, replicative intracellular amastigote. Trypanosoma cruzi is a heterogeneous species, with large genetic variability and different strains that circulate between the vertebrate host and the insect vector. This heterogeneity may explain the variation in the clinical manifestation of Chagas disease and the geographical differences in morbidity and mortality (Laurent et al, 1997; Da Silva Manoel-Caetano and Silva, 2007; McCall and McKerrow, 2014)
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