Papel da TcPI3K na expressão gênica e no metabolismo energético de Trypanosoma cruzi exposto à molécula de heme

Abstract

The epimastigote form of Trypanosoma cruzi, the causative agent of Chagas’ disease, faces environmental changes during its life cycle inside the digestive tract of the insect vector. Within this compartment, there are high amounts of carbohydrates, lipids, and amino acids of blood digestion, besides a high concentration of heme from hemoglobin degradation. Heme molecule triggers significant changes in epimastigotes physiology, including energy metabolism, gene expression, and cell proliferation. This molecule increases the gene expression of glycolysis and aerobic fermentation, and decreases oxidative phosphorylation producing reactive oxygen species, supporting the theory of glycosomal synthesis of ATP over mitochondrial respiration. However, the signaling pathways involved in the recognition of external and internal signals and their translation to such biological changes remain to be clear. In eukaryotes, phophatidylinositide signaling pathway is involved in several cell processes, including gene expression and metabolism. In T. cruzi, phosphatidylinositol-4,5-bisphosphate is a second messenger that can either be converted by TcPI3K on phosphatidylinositol-3,4,5-triphosphate, or on diacylglycerol and inositol-1,4,5-trifosfate by phospholipase C. In this work, the role of TcPI3K on proliferation, metabolism, and gene expression of T. cruzi were evaluated, aiming to find if those processes are regulated by phophatidylinositides like in other eukaryotes. Thus, epimastigote forms were cultivated for 7 days with and/or without heme and Ly294002, a PI3K inhibitor. Signaling pathway genes (TcPI3K, TcDAGK, TcCaM) and energy metabolism genes (TcPPDK, TcPEPCK, TcFRD, TcMDH, TcST e TcME) tested by qPCR were downregulated by the inhibitor, an effect heme was not able to revert. Mitochondrial respiration, tested by high-resolution respirometry, decreased with TcPI3K inhibition, but not intracellular ATP content and cell proliferation in either treatment group. Metabolomics of extracellular medium performed by magnetic nuclear resonance showed an increase in internalization of glucose, hexoses, glutamate, phenylalanine, and BCAA, but this profile was reversed by Ly294002, suggesting a role of TcPI3K on such uptake. The synthesis of alanine and succinate did not change in the presence of the drug. Collectively, the results suggest that a working TcPI3K is involved in mitochondrial respiration, on nutrient uptake, and on correct gene expression process.