Abstract
Replication of Trypanosoma cruzi, the etiological agent of Chagas disease, displays peculiar features, such as absence of chromosome condensation and closed mitosis. Although previous proteome and subproteome analyses of T. cruzi have been carried out, the nuclear subproteome of this protozoan has not been described. Here, we report, for the first time to the best of our knowledge, the isolation and proteome analysis of T. cruzi nuclear fraction. For that, T. cruzi epimastigote cells were lysed and subjected to cell fractionation using two steps of sucrose density gradient centrifugation. The purity of the nuclear fraction was confirmed by phase contrast and fluorescence microscopy. Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) allowed the identification of 864 proteins. Among those, 272 proteins were annotated as putative uncharacterized, and 275 had not been previously reported on global T. cruzi proteome analysis. Additionally, to support our enrichment method, bioinformatics analysis in DAVID was carried out. It grouped the nuclear proteins in 65 gene clusters, wherein the clusters with the highest enrichment scores harbor members with chromatin organization and DNA binding functions.
Highlights
Trypanosoma cruzi, a protozoan that belongs to the Trypanosomatidae family, causes Chagas disease, the American trypanosomiasis, which affects approximately 7–8 million people worldwide, especially in Latin America [1]
One of them permitted the isolation of T. cruzi high density organelles [16], while the other was developed for the purification of T. brucei nucleus in order to produce samples suitable for proteomic analysis [17]
After ultracentrifugation, T. brucei nuclear fraction was concentrated in the interface between 2.30 M and 2.10 M sucrose[17], while T. cruzi nuclear fraction was found in the pellet (Fig 1)
Summary
Trypanosoma cruzi, a protozoan that belongs to the Trypanosomatidae family, causes Chagas disease, the American trypanosomiasis, which affects approximately 7–8 million people worldwide, especially in Latin America [1]. Chagas disease is mainly transmitted by triatomine bugs, but it can be disseminated through blood transfusion, organ transplant, vertical transmission from mother to offspring, ingestion of contaminated food and laboratory accidents [1]. T. cruzi possesses a digenetic life cycle, differentiating into four main life forms: epimastigote and metacyclic trypomastigote, which are found in the insect vectors, and trypomastigote and amastigote, which occur in mammalian hosts. The life form studied here, the kinetoplast (a mitochondrial complex array of DNA fibrils) is localized anterior to the nucleus [2].
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