Caracterização do perfil protéico e da atividade mitocondrial de Trypanosoma cruzi sob o estresse de radiação gama

Abstract

The Trypanosoma cruzi is considered as one of the eukaryotes most resistant to ionizing radiation, enduring sub-lethal doses of up to 1.0 kGy. The ionizing radiation by gamma rays promotes double-strand and single-strand breaks on the DNA and, additionally, generates reactive oxygen species (ROS). After irradiation the parasites cease growing for as long as 96 hours, further resuming growth and reaching the stationary phase no later than 240 hours after irradiation. The pattern of chromosomal bands which are extremely fragmented by radiation exposure is restored approximately 48 hours after the stress. To enable the parasite to tolerate such damage condition, an efficient apparatus to recognize and repair lesions is necessary. In the present work we investigate T. cruzi radioresistance by analyzing its protein expression patterns and mitochondrial activity in different time points after a dose of 500 Gy of gamma rays. Using the 2DTDIGE methodology, 543 spots were identified as significantly differentially expressed after T. cruzi exposure to radiation. From these 543 spots, 53 different proteins were identified by mass spectrometry (MS/MS) and classified according to biological function. When the functional annotation of these 53 proteins was analyzed, a tendency of overexpression of proteins with molecular weight lower than predicted was observed. This tendency suggests the presence of post-translational modifications and/or processing mechanisms acting upon the identified proteins, leading to the emergence of peptides which are shorter than predicted in response to gamma radiation exposure. Other tendencies observed on proteomic analysis include alterations on the following biological processes: positive regulation of the protein synthesis process, repression of proteins involved with folding processes (except for two endoplasmic reticulum chaperones, which are induced), repression of ATP generation through oxidative phosphorylation, of glycolysis, and of the amino acids metabolism. Our results also indicate that the de novo protein synthesis is essential for T. cruzi recovery from gamma radiation exposure, since the treatment with translation inhibitors drastically interferes with the growth resumption on irradiated parasites. When the mitochondrial activity of T. cruzi xviii was evaluated, we verified that irradiated parasites generate higher levels of ATP than their non-irradiated counterparts, with an associated higher O2 consumption by the former. Additionally, the gene expression analysis of irradiated T. cruzi using qRT-PCR showed that genes from the mitochondrial maxicircle were induced after radiation exposure. Both of these results point to a higher mitochondrial activity after irradiation-caused stress. Nevertheless, we have also observed a lower H2O2 production by irradiated parasites, thus indicating the existence of an efficient mechanism for free radicals clearance in the organism. The present study unravels the peculiar response of T. cruzi to ionizing radiation and evokes questions about the manner in which this organism is capable of performing drastic changes on its protein expression profile and mitochondrial activity to enable its survival in such adverse conditions.