An Intracellular Ammonium Transporter Is Necessary for Replication, Differentiation, and Resistance to Starvation and Osmotic Stress in Trypanosoma cruzi

Abstract

Trypanosoma cruzi is an important human and animal pathogen and the etiologic agent of Chagas disease. The parasite undergoes drastic changes in its metabolism during its life cycle. Amino acid consumption becomes important in the infective stages and leads to the production of ammonia (NH 3 ), which needs to be detoxified. We report here the identification of an ammonium (NH 4 + ) transporter that localizes to acidic compartments and is important for replication, differentiation, and resistance to starvation and osmotic stress. Trypanosoma cruzi , the etiologic agent of Chagas disease, undergoes drastic metabolic changes when it transits between a vector and mammalian hosts. Amino acid catabolism leads to the production of ammonium (NH 4 + ), which needs to be detoxified. However, T. cruzi does not possess a urea cycle, and it is unknown how intracellular levels of ammonium are controlled. In this work, we identified an intracellular ammonium transporter of T. cruzi (TcAMT) that localizes to acidic compartments (reservosomes, lysosomes). TcAMT has 11 transmembrane domains and possesses all conserved and functionally important amino acid residues that form the pore in other ammonium transporters. Functional expression in Xenopus oocytes followed by a two-electrode voltage clamp showed an inward current that is NH 4 + dependent at a resting membrane potential ( V h ) lower than −120 mV and is not pH dependent, suggesting that TcAMT is not an NH 4 + /H + cotransporter but an NH 4 + or NH 3 /H + transporter. Ablation of TcAMT by clustered regularly interspaced short palindromic repeat analysis with Cas9 (CRISPR-Cas9) resulted in significant defects in epimastigote and amastigote replication, differentiation, and resistance to starvation and osmotic stress. IMPORTANCE Trypanosoma cruzi is an important human and animal pathogen and the etiologic agent of Chagas disease. The parasite undergoes drastic changes in its metabolism during its life cycle. Amino acid consumption becomes important in the infective stages and leads to the production of ammonia (NH 3 ), which needs to be detoxified. We report here the identification of an ammonium (NH 4 + ) transporter that localizes to acidic compartments and is important for replication, differentiation, and resistance to starvation and osmotic stress.