Molecular and cellular characterization of glutamine synthetase and soluble pyrophosphatase putative genes from Trypanosoma cruzi by complementation of conditional mutants in Saccharomyces cerevisiae

• Grant number: 13/06293-0
• Support Opportunities: Research Grants - Visiting Researcher Grant - International
• Start date: September 16, 2013
• End date: December 15, 2013
• Field of knowledge: Biological Sciences - Parasitology - Protozoology of Parasites
• Principal Investigator: Ariel Mariano Silber
• Grantee: Ariel Mariano Silber
• Visiting researcher: Agustin Hernandez Lopez
• Visiting researcher institution: Universidad Pablo de Olavide (UPO), Spain
• Host Institution: Instituto de Ciências Biomédicas (ICB). Universidade de São Paulo (USP). São Paulo , SP, Brazil

Abstract

Trypanosoma cruzi is the etiological agent for Chagas disease. It is well known that this parasite uses amino acids as its main carbon and energy sources. Among the amino acids, glutamate can be singled out as responsible not only for being the major energy source to the cell but for being involved in the metacyclogenesis process. Noticeably, glutamine is known to regulate nitrogen assimilation through its interconversion with glutamate; however, this biosynthetic pathway has not been studied to date in T. cruzi. For example, glutamine synthesis from glutamate requires the presence of the enzyme glutamine synthetase, but the genes encoding this enzyme in T. cruzi have not yet been unequivocally identified. In order to widen and fill the gaps in our knowledge about the latter key biosynthetic reaction, we propose to characterize the putative genes coding for glutamine synthetase (GS) in T. cruzi making use of their capability to complement the growth defects observed in gln1- mutants in the yeast Saccharomyces cerevisiae. This characterization will encompass both molecular biology and cell biology. At the same time, the effectiveness of known inhibitors on these gene products will be assessed to evaluate the potential of this enzyme as a new therapeutic target. In any cell, inorganic pyrophosphatases are necessary for the anabolism to take place appropriately and not been inhibited by excess pyrophosphate, a common by-product in many reactions. Defects in the genes encoding these enzymes are considered to be incompatible with proliferation; however, the cellular effects of these mutations are still obscure. In S. cerevisiae, defects in the expression of IPP1, the gene encoding the major nucleo-cytoplasmic enzyme, induce cell death or cell cycle arrest depending on the choice of energy metabolism of the cell. On the other hand, defects in PPA2, coding for a mitochondrial pyrophosphatase, translate into inability to grow on respiratory carbon sources. In T. cruzi cells, two different types of pyrophosphatases co-exist: proton-pumping pyrophosphatases and soluble pyrophosphatases. In the genome of this parasite there are two sequences of the latter type. The sub-cellular localization and function of them are still unknown. The present project aims to validate these two sequences as bona fide soluble pyrophosphatases and characterize them partially by complementation of ipp1- and/or ppa2- yeast mutants affected in cytosolic and mitocondrial pyrophosphatases, respectively. (AU)