Glycosomal Phosphoenolpyruvate Carboxykinase <scp>CRISPR</scp> /Cas9‐Deletion and Its Role in <i>Trypanosoma cruzi</i> Metacyclogenesis and Infectivity in Mammalian Host

Trypanosoma cruzi, the causative agent of Chagas disease, possesses glycosomes-unique organelles that house key metabolic enzymes, several of which are promising therapeutic targets. Among them, phosphoenolpyruvate carboxykinase (PEPCK) plays a central role in succinic fermentation, the main pathway for NAD⁺ regeneration within the organelle. Using CRISPR/Cas9 editing, the PEPCK gene was disrupted in T. cruzi, producing single-allele knockout epimastigotes (TcPEPCK-sKO) with reduced PEPCK expression and enzyme activity. In a high glucose environment, PEPCK disruption impaired glucose consumption and mitochondrial respiration, particularly oxidative phosphorylation, reducing dependence on mitochondrial ATP production when glucose was supplied. To compensate, pyruvate phosphate dikinase was upregulated, increasing alanine production, possibly to maintain redox balance in glycosomes. Despite this metabolic adaptation, the growth of TcPEPCK-sKO epimastigotes was partially reduced compared with non-deleted parasites. In contrast, under low glucose conditions, PEPCK activity was not critical for mitochondrial bioenergetics, ATP production, or proliferation. Although TcPEPCK-sKO epimastigotes exhibited a minor reduction in growth in high glucose medium, their differentiation (metacyclogenesis) and invasion were severely compromised. However, once inside the host cell, TcPEPCK-sKO amastigotes increased their replication, leading to enhanced trypomastigote production. The same was observed in in vivo infection, where TcPEPCK-sKO infection in IFNγ-deficient mice caused uncontrolled parasitemia and severe pathology, highlighting the critical role of PEPCK in host-pathogen interactions. However, an intact immune system effectively contained TcPEPCK-sKO infection. Taken together, our findings demonstrate that glycosomal PEPCK is crucial for coupling glycolysis to mitochondrial bioenergetics, enabling the parasite differentiation within the insect vector and controlling the infection of mammalian host cells.