Impacto da deleção de genes envolvidos com a bioenergética por CRISPR/Cas9 no ciclo de vida do Trypanosoma cruzi

Abstract

Trypanosoma cruzi, the causative agent of Chagas disease, exhibits metabolic pathways and protein structures distinct from mammals, making it an attractive target for chemotherapy. In this study, we utilized the CRISPR/Cas9 genome-editing tool to delete genes involved in the parasite energy metabolism, focusing on key enzymes from pathways: glycolysis (hexokinase; HKg), succinate fermentation (phosphoenolpyruvate carboxykinase; PEPCKg, malate dehydrogenase; MDHg, and fumarate reductase; FRDg), and NADPH production (cytosolic malic enzyme; MEc). We generated mutants with partial deletions for HKg, MDHg, and PEPCKg and total deletions for FRDg and MEc. Glycolysis is the main pathway for glucose degradation. Partial deletion of HKg impaired the mitochondrial metabolism of the parasite, leading to a significant reduction in its proliferation and metacyclogenesis (differentiation from epimastigote into the infective metacyclic trypomastigote). Succinate fermentation is the principal pathway for NADH reoxidation within the glycosome. Partial deletion of PEPCKg (the first enzyme in this pathway) decreased its enzymatic activity, triggering a series of metabolic responses, including a reduction in glycolytic flux and a diminished reliance on mitochondrial metabolism for energy obtention. Although these alterations did not significantly affect proliferation, metacyclogenesis, and invasion capacity were severely compromised. Curiously, these mutants exhibited a significant increase in infection, parasitemia, and pathology in mammalian hosts, indicating that PEPCKg mutation promoted enhanced virulence. The roles of MDHg and FRDg in succinic fermentation are directly associated with NADH oxidation in the glycosome. Partial deletion of MDHg (the second enzyme in this pathway) did not affect parasite proliferation or differentiation but enhanced mitochondrial metabolic efficiency. Conversely, FRDg deletion (final enzyme) impaired growth, metacyclogenesis, and infectivity in vitro, although it did not abolish animal infection. Finally, we investigated the effects of MEc deletion, one of the enzymes responsible for NADPH generation in T. cruzi. Its absence impaired proliferation, metacyclogenesis, and infectivity, both in vitro and in vivo. Furthermore, the mutant parasites exhibited increased production of reactive oxygen species (ROS), losing their ability to maintain redox homeostasis when challenged with oxidant agents. Our results demonstrated that gene editing allowed us to elucidate the roles of various enzymes and their importance across the distinct life stages of Trypanosoma cruzi.