Characterization and biological role of histidine catabolism in Trypanosoma cruzi

• Grant number: 17/12320-0
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• Start date: September 01, 2017
• End date: October 31, 2021
• Field of knowledge: Biological Sciences - Parasitology - Protozoology of Parasites
• Principal Investigator: Ariel Mariano Silber
• Grantee: Janaina de Freitas Nascimento
• Host Institution: Instituto de Ciências Biomédicas (ICB). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• Associated research grant: 16/06034-2 - The biological role of amino acids and their metabolites in Trypanosoma cruzi, AP.TEM
• Associated scholarship(s): 19/23180-0 - Development of an inducible RNA-targeting CRISPR-Cas system for Trypanosoma cruzi, BE.EP.PD

Abstract

Trypanosoma cruzi is the only trypanosomatid pathogenic to humans that contains the complete pathway that converts Histidine (His) to Glutamate (Glu). According to the classical literature, this pathway is composed of four enzymes: His ammonia lyase (HAL), Urocanate Hydratase (UH), Imidazolone Propionase (IP) and Formiminoglutamase. However, the literature has also shown that one of the intermediates of this pathway, the imidazolone propionate, could be converted by non-enzymatic oxidation to alpha-ketoglutarate (a-KG), an intermediate of the Tricarboxylic Acid Cycle (TCA). Thus, there is the possibility of an alternative route for the degradation of His and the consequent production of Proline (Pro) and/or Glutamine (Gln). However, it is not clear from literature data whether this conversion could occur in vivo. Finally, if the pathway of non-enzymatic conversion of urocanate to ±-KG is confirmed, and therefore the possibility of producing Glu through its amination via Glutamate dehydrogenase or Tyrosine aminotransferase (TAT), the role the enzymes Imidazolone Propionase and Formiminoglutamase in the metabolism of this amino acid as well as in the general biology of the parasite would be unclear. Thus, the present project has the following objectives: 1. Clone, express, characterize biochemically and obtain antibodies against the four enzymes that constitute the pathway of conversion of His to Glu; 2. Evaluate the possibility that the first two enzymes of the His degradation pathway (HAL and UH) are sufficient (under physiological conditions of the cell) to provide a-KG through the interruption of the pathway by using of IP inhibitors; 3. Obtain of T. cruzi strains with the deleted gene coding for the HAL or UH enzyme by CRISPR / Cas9, and subsequent phenotypic and metabolomic evaluation of these strains; 4. Obtain transfectants T. brucei cell lines (that naturally lack the His degradation pathway) expressing the genes encoding for the first two steps of the His degradation pathway (HAL and UH), and subsequent phenotypic and metabolomic evaluation of these cell lines.