The relevance of the regulation of the enzymatic complex branched-chain alfa-keto-acid dehydrogenase (BCKDH) in the Cell Biology and bioenergetics of Trypanosoma cruzi

• Grant number: 19/26106-6
• Support Opportunities: Scholarships in Brazil - Post-Doctorate
• Effective date (Start): October 01, 2020
• Effective date (End): September 30, 2022
• Field of knowledge: Biological Sciences - Parasitology - Protozoology of Parasites
• Principal Investigator: Ariel Mariano Silber
• Grantee: Higo Fernando Santos Souza
• 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

Abstract

Trypanosoma cruzi (T. cruzi) is the causative agent of Chagas Disease, also known as American Trypanosomiasis. The life cycle of pathogenic trypanosomatids is known to encompass different environments in their hosts. T. cruzi, for example, depends fundamentally on the intestinal tract of the insect vector, the bloodstream, and the cytoplasm of the mammalian host cell. In these environments the survival of the parasite is related to its ability to maintain intracellular homeostasis of ions and nutrients in a relatively constant state, besides catabolizing different substrates as a source of energy. Thus, several studies have described the importance of amino acids in the biology of different protozoa, among them T. cruzi. In fact, amino acids, besides acting on protein synthesis and energy metabolism, have several other functions in the parasite. Among them, it is worth mentioning the differentiation between replicative and infectious forms, the energetic sustenance of invasion of mammalian host cells, the establishment of infection and the resistance to different types of stress to which the parasite is naturally subjected throughout its biological cycle such as osmotic, thermal, oxidative and nutritional stress. In this regard, Branched-Chain Amino Acids (BCAAs): leucine, isoleucine and valine are among the essential amino acids, however, they are only synthesized by plants and some fungi. Previous studies indicate that BCAAs are integral to T. cruzi metabolism, which makes it curious that there are still few studies in the literature on the identification of relevant BCAA functions for parasite biology, as well as associated with other T. cruzi amino acids. . This project aims on the second step of the BCAA pathway, which is catalyzed by a complex called Branched-Chain ±-Ketoacid Dehydrogenase (BCKDH) dehydrogenase and it is composed of three enzymes (EC 1.2.4.4, EC 2.3.1.168, EC 1.8.1.4). In mammals, BCKDH is a highly regulated complex by phosphorylation (inactivation) and dephosphorylation (activation) and by product inhibition. Furthermore, it is believed that this complex is also controlled by the NADH/NAD+ ratio as well as acetyl-CoA/CoA. Given the above, this project aims to evaluate the relevance of the BCKDH complex in T. cruzi cell and bioenergetic biology, using the gene deletion technique CRISPR/Cas9 to knockout the complex-forming enzymes in T. cruzi. (AU)