The glutamine synthetase of Trypanosoma cruzi is required for its resistance to ammonium accumulation and evasion of the parasitophorous vacuole during host-cell infection

• Published in: PLoS neglected tropical diseases Vol. 12; no. 1; p. e0006170
• Main Authors: Crispim, Marcell, Damasceno, Flávia Silva, Hernández, Agustín, Barisón, María Julia, Pretto Sauter, Ismael, Souza Pavani, Raphael, Santos Moura, Alexandre, Pral, Elizabeth Mieko Furusho, Cortez, Mauro, Elias, Maria Carolina, Silber, Ariel Mariano
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 01-01-2018; Public Library of Science (PLoS)
• Subjects: Accumulation; Amino acids; Ammonium; Ammonium compounds; ATP; Biochemistry; Biology and Life Sciences; Carbon sources; Care and treatment; Cations; Cell cycle; Chagas disease; Consumption; Diagnosis; Divalent cations; Enzymes; Etiology; Gene expression; Glutamate-ammonia ligase; Glutamine; Immunofluorescence; Immunology; Infections; Intracellular; Laboratories; Leishmania; Life cycle; Life cycle engineering; Life cycles; Localization; Mammals; Medicine and Health Sciences; Metabolism; Metabolites; Methionine; Methods; Mineral nutrients; Mitochondria; Niches; Nitrogen balance; Nutrient availability; Nutrient cycles; Parasites; Parasitology; Parasitophorous vacuole; Physical Sciences; Protozoa; Saccharomyces cerevisiae; Secretion; Supervision; Toxic wastes; Toxicity; Tropical diseases; Trypanosoma cruzi; Urea; Vector-borne diseases; Yeast; Yields; Brazil
• ISSN: 1935-2735; 1935-2727; 1935-2735
• DOI: 10.1371/journal.pntd.0006170

Trypanosoma cruzi, the etiological agent of Chagas disease, consumes glucose and amino acids depending on the environmental availability of each nutrient during its complex life cycle. For example, amino acids are the major energy and carbon sources in the intracellular stages of the T. cruzi parasite, but their consumption produces an accumulation of NH4+ in the environment, which is toxic. These parasites do not have a functional urea cycle to secrete excess nitrogen as low-toxicity waste. Glutamine synthetase (GS) plays a central role in regulating the carbon/nitrogen balance in the metabolism of most living organisms. We show here that the gene TcGS from T. cruzi encodes a functional glutamine synthetase; it can complement a defect in the GLN1 gene from Saccharomyces cerevisiae and utilizes ATP, glutamate and ammonium to yield glutamine in vitro. Overall, its kinetic characteristics are similar to other eukaryotic enzymes, and it is dependent on divalent cations. Its cytosolic/mitochondrial localization was confirmed by immunofluorescence. Inhibition by Methionine sulfoximine revealed that GS activity is indispensable under excess ammonium conditions. Coincidently, its expression levels are maximal in the amastigote stage of the life cycle, when amino acids are preferably consumed, and NH4+ production is predictable. During host-cell invasion, TcGS is required for the parasite to escape from the parasitophorous vacuole, a process sine qua non for the parasite to replicate and establish infection in host cells. These results are the first to establish a link between the activity of a metabolic enzyme and the ability of a parasite to reach its intracellular niche to replicate and establish host-cell infection.