Knocking out histidine ammonia-lyase by using CRISPR-Cas9 abolishes histidine role in the bioenergetics and the life cycle ofTrypanosoma cruzi

Trypanosoma cruzi,the causing agent of Chagas disease, is the only known trypanosomatid pathogenic to humans having a complete histidine to glutamate pathway, which involves a series of four enzymatic reactions that convert histidine into downstream metabolites, including urocanate, 4-imidazolone-5-propionate, N-formimino-L-glutamate and L-glutamate. Recent studies have highlighted the importance of this pathway in ATP production, redox balance, and the maintenance of cellular homeostasis inT. cruzi. In this work, we focus on the first step of the histidine degradation pathway, which is performed by the enzyme histidine ammonia lyase. Here we determined the kinetic and biochemical parameters of theT. cruzihistidine ammonia-lyase. By generating null mutants of this enzyme using CRISPR-Cas9 we observed that disruption of the first step of the histidine degradation pathway completely abolishes the capability of this parasite to metabolise histidine, compromising the use of this amino acid as an energy and carbon source. Additionally, we showed that the knockout of the histidine ammonia lyase affects metacyclogenesis when histidine is the only metabolizable source and diminishes trypomastigote infectionin vitro.