Trypanosoma cruzi Mitochondrial Peroxiredoxin Promotes Infectivity in Macrophages and Attenuates Nifurtimox Toxicity.

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.

Cardiomyocyte diffusible redox mediators control Trypanosoma cruzi infection: role of parasite mitochondrial iron superoxide dismutase.

Chagas disease (CD), caused by the protozoa Trypanosoma cruzi, is a chronic illness in which parasites persist in the host-infected tissues for years. T. cruzi invasion in cardiomyocytes elicits the production of pro-inflammatory mediators [TNF-α, IL-1ß, IFN-γ; nitric oxide (·NO)], leading to mitochondrial dysfunction with increased superoxide radical (O2·-), hydrogen peroxide (H2O2) and peroxynitrite generation. We hypothesize that these redox mediators may control parasite proliferation through the induction of intracellular amastigote programmed cell death (PCD). In this work, we show that T. cruzi (CL-Brener strain) infection in primary cardiomyocytes produced an early (24 h post infection) mitochondrial dysfunction with H2O2 generation and the establishment of an oxidative stress evidenced by FoxO3 activation and target host mitochondrial protein expression (MnSOD and peroxiredoxin 3). TNF-α/IL-1ß-stimulated cardiomyocytes were able to control intracellular amastigote proliferation compared with unstimulated cardiomyocytes. In this condition leading to oxidant formation, an enhanced number of intracellular apoptotic amastigotes were detected. The ability of H2O2 to induce T. cruzi PCD was further confirmed in the epimastigote stage of the parasite. H2O2 treatment induced parasite mitochondrial dysfunction together with intra-mitochondrial O2·- generation. Importantly, parasites genetically engineered to overexpress mitochondrial Fe-superoxide dismutase (Fe-SODA) were more infective to TNF-α/IL-1ß-stimulated cardiomyocytes with less apoptotic amastigotes; this result underscores the role of this enzyme in parasite survival. Our results indicate that cardiomyocyte-derived diffusible mediators are able to control intracellular amastigote proliferation by triggering T. cruzi PCD and that parasite Fe-SODA tilts the process toward survival as part of an antioxidant-based immune evasion mechanism.